cytisine study data a1

Br. J. Pharmac. (1969), 35, 161-174.

Some studies on cytisine and itsmethylated derivativesR. B. BARLOW AND L. J. McLEOD*

Department of Pharmacology, University of Edinburgh

1. In mice cytisine hydrochloride is less toxic intravenously than nicotinehydrogen tartrate, but more toxic by intraperitoneal or oral administration.Compared with cytisine, caulophylline hydrogen iodide is one-fifth to one-tenthas toxic and caulophylline methiodide is less than one-thirtieth as toxic.2. The surprising low oral toxicity of cytisine and nicotine may be ascribedto the method of administration; if the drug is placed directly in the stomachthere is no possibility of absorption through buccal mucous membranes.3. The peripheral effects of nicotine, cytisine and caulophylline are similar,though on some preparations those of nicotine last longer. In most testscytisine is active in doses from a quarter to three-quarters of those of nicotine,caulophylline in doses from 10 to 20 times those of cytisine. Caulophyllinemethiodide is virtually inactive.4. Cytisine and caulophylline may differ from nicotine in their central effects.5. Cytisine and caulophylline are active as the cations. The pKa of cytisineis 7.92 and that of caulophylline is 7.04; the difference accounts, in part, forthe weaker activity of caulophylline. The caulophylline ion is generally one-sixth to one-third as active as the cytisine ion.6. The introduction of the second methyl group to form the quaternary saltdoes not appear to cause a dramatic change in the conformation of the molecule.Caulophylline methiodide appears to be feebly active because it has feebleaffinity.

The alkaloid cytisine occurs in a number of plants of the leguminosae familyand was considered by Dale & Laidlaw (1912) to be the toxic principle of thecommon laburnum. After tests in cats, rabbits and fowls, they described its peri-pheral actions as being " qualitatively indistinguishable from nicotine " though theyobserved that it differed from nicotine in that it did not produce an ear-twitch incats. Zachowski (1938) confirmed its pharmacological resemblance to nicotinebut, from experiments on the blood pressure of the cat, concluded that it had greateractivity in stimulating sympathetic ganglia than in blocking them.The chemical structure of cytisine was worked out by Ing (1931, 1932) and its

absolute configuration is now known (Okudu & Katauku, 1961). It contains a

* Present address: School of Pharmacy, Hobart Technical College, Tasmania, Australia.G

R. B. Barlow and L. J. McLeod

secondary amino group (Fig. 1) and the tertiary base, N-methyl cytisine, occurs asthe alkaloid caulophylline, together with cytisine, in several plants. The toxicityof caulophylline was examined by Kalaschnikow & Kusnetzow (1938) and itspharmacological properties briefly reported by Scott & Chen (1943). Although itsperipheral actions resembled those of nicotine, the convulsions produced by caulo-phylline in mice differed from those produced by nicotine. The quaternary com-pound, caulophylline methiodide, was prepared by Ing (1931) but does not seemto have been tested pharmacologically.

This paper describes a comparison of the pharmacological properties of nicotine,cytisine, caulophylline and caulophylline methiodide. These compounds form aninteresting chemical series because cytisine is a relatively rigid structure, and conse-quently the structural effects of methylation are limited to one particular part andunlikely to lead to changes in conformation elsewhere. The work also includesa study of the effects of changes in pH on the activity of cytisine and caulophyllineon the frog rectus, to see whether these are active as the ions or as the unchargedbase.

MethodsChemical

Melting points were determined with a Mettler FP 1 instrument; analyses forhalide are gravimetric with samples of 50-100 mg.

WvN\ ,Me

N

0(-)-7R:9S cytisi'ne ( --)-S nicotine

Me/Me ItMe

0~~~~~(-)-7R:9S caulophylline (-)-79:9S caulophylline

methiodide

FIG. 1. Albsolute configurations of (-)-cytisine, (-)-caulophylline and (-)-caulophyllinemethiodide (Okudu & Katauku, 1961) compared with (-)-nicotine (Hudson & Neuberger, 1950).Note the hydrogen atom attached to the basic nitrogen in ring C of cytisine can be in eitherof two conformations, but the methyl group attached to this atom in caulophylline is morelikely to be as shown, than in the alternative arrangement (shown for the hydrogen atom incytisine).

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Some studies on cytisine and its methylated derivatives

Cytisine melting point 1520 C, [a]D -1190 in water, was bought from Fluka A.G.201'

Caulophylline hydrogen iodide was prepared by the method of Ing (1931) butwith ethanol instead of methanol as solvent. Recrystallized material had m.p.276.5° C; found; 1-, 38.08; calculated for C12H170N2J; I-, 38.30%. Partheil (1892)recorded m.p. 2700 C; Ing (1931) recorded m.p. 2800 C. The free base was preparedby treating the hydrogen iodide with alkali and extracting with chloroform, dryingthe extract with magnesium sulphate, and distilling off the solvent. The residualoil would not crystallize and was heated under reduced pressure in a cold-fingerapparatus. The base was deposited on the condenser as a solid, m.p. 1280 C;[a]2D, -219° in water. Ing (1931) recorded m.p. 134°. Power & Salway (1913)recorded [a]D, - 221.60 in water, temperature not specified.

Caulophylline methiodide was prepared by heating the base under reflux inethanol with an excess of methyl iodide. Recrystallized material had m.p. 265.50 C;[a]% -137.9° in water; found; I-, 36.75; calculated for Cl3H1,0N2J; I-, 36.65%.Ing (1931) recorded m.p. 2760 (dec).

All the recrystallized material used for biological testing appeared to be homo-geneous when chromatographed on paper in a solvent system consisting of butanol,ethanol and water (5:5:2) and developed with a modified Dragendorff reagent(Thies & Reuther, 1954). The materials also all showed an ultraviolet absorptionmaximum in water at 303 mg, with log. e=3.81, and in the infrared, absorptionmaxima corresponding to the a-pyridone group occurred at 1650, 1555 and1567 cm`.The nicotine used was the (- )-isomer supplied as the hydrogen tartrate by

British Drug Houses Ltd.Dissociation constants. The pKa values were determined by the method of

Albert & Goldacre (1943). The procedure was the same as that of Barlow &Hamilton (1962) but with a stream of nitrogen in place of a stirrer driven by com-pressed air and with a Pye Dynacap instrument in place of the Marconi pHmeter.

BiologicalToxicity. The acute toxicity of the compounds by intravenous, intraperitoneal

and oral administration was studied in female albino mice, strain CS1, weighingbetween 17 and 24 g. These were divided into groups of ten and the animals ineach group all received the same dose of the same drug by the same route, the dosebeing expressed as ,moles/kg, based on the average weight of the group. By eachroute each drug was tested using at least five different dose-levels; in other words,at least five groups of mice were used for each drug given by one particular route.The mice were observed for 1 hr after dosing, and from the number which diedin this period, the LD50 was calculated by the method of Litchfield & Wilcoxon(1949). The dose-levels were chosen so that, as far as possible, they were uniformlyabove and below the LD50 and, in all, about 50% of the animals died in the testswith one particular drug given by one particular route.

In these experiments nicotine was tested as the hydrogen tartrate, cytisine as thehydrochloride, and caulophylline as the hydrogen iodide, made up in 0.9% saline.

Ganglionic preparations. The superior cervical ganglion preparation of the cat,anaesthetized with chloralose, was set up as described by Paton & Perry (1953).

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The preganglionic sympathetic nerve was stimulated with rectangular wave pulsesof 0.7 msec duration at a rate of 10 shocks/sec. Usually a stimulus of 3-5 V wasnecessary to produce a maximum contracture of the nictitating membrane. Injec-tions were made retrogradely into the external carotid artery, though in one instancethe lingual artery was used. The blood pressure was recorded from a femoralartery. The relative blocking activity of the compounds was estimated by com-paring the doses which produced roughly comparable block of the responses of thenictitating membrane to continuous stimulation of the preganglionic nerve. Therelative stimulant activity of the compounds was estimated in a 2+1 assay bycomparing the doses which produced comparable contractions of the nictitatingmembrane (in the absence of stimulation of the preganglionic nerve).The relative ability of compounds to raise arterial blood pressure was studied

in rats anaesthetized with urethane and cats anaesthetized with chloralose. Theblood pressure was recorded from a carotid artery and the drugs were injected intoa femoral vein, in a volume not exceeding 0.2 ml. and washed in with 0.9% saline.The isolated guinea-pig ileum was set up in Tyrode solution at 370 C and bubbled

with air. The volume of the organ bath was approximately 10 ml. and the drugswere added by pipette in a volume not exceeding 0.4 ml., allowed to act for 30 secand then washed out. The interval between doses was 2 min.

Striated muscle. The chick biventer-cervicis preparation was set up, as describedby Ginsborg & Warriner (1960), in Krebs-Henseleit solution (Krebs & Henseleit,1932) at 370 C bubbled with 95% oxygen and 5% carbon dioxide. The nerve inthe tendon was stimulated with rectangular wave shocks, which produced maximaltwitches, of 0.7 msec duration at 6-8 shocks/min. The volume of the bath was

approximately 30 ml. and the drugs were added by pipette in a volume not exceed-ing 0.4 ml., allowed to act until the contracture had reached a maximum (usuallyfor about 10 min) and then washed out. Recovery usually occurred rapidly so theinterval between doses was about 15 min. The compounds had a marked effecton the slow fibres, which masked any effects they may have had on the twitchresponses. Their relative activities on this preparation were therefore estimated bycomparing the doses which produced roughly the same degree of contracture.

The rectus abdominis muscle of the frog (Rana pipiens) was set up at room

temperature in a modified Ringer solution bubbled with air. This solution had thefollowing composition (in 1 1.): sodium chloride, 7.50 g (128 m-equiv); potassiumchloride, 0.14 g (1.9 m-equiv); calcium chloride, 0.12 g (2.2 m-equiv); Tris(2-amino-2(hydroxymethyl)propane-1: 3-diol), 1.92 g (15.9 m-equiv); to which was

added 0.1 N hydrochloric acid; 14 ml. produced a pH of 7.10; 10 ml. produceda pH of 7.90; 9.4 ml. produced a pH of 8.20. The pH of the fluid in contact withthe tissue differed slightly from these values and in every experiment samples were

removed after contact for 4.5 min and their pH measured with a Pye Dynacap pHmeter. Although there were differences of about 0.2 units between the pH of a

particular buffer in different experiments, the variation did not exceed 0.02 unitsduring any one experiment.

Equipotent molar ratios for the compounds relative to the quaternary salt,p-pyridylmethyltrimethylammonium, were obtained by 2+1 and 2 + 2 assay tech-niques using an automatic apparatus (Barlow, Scott & Stephenson, 1967). Thedrugs, made up in the desired concentration, were allowed to act on the preparationfor 4.5 min and the interval between doses was 30 min. When an assay had been

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completed, the pH was altered and a concentration of the quaternary compound wasapplied repeatedly until the responses were regular, indicating that the tissue hadbecome adjusted to the new medium. This usually took less than an hour-that is,the second and third responses to the concentration of quaternary compound afterthe change were usually consistent. The assay was then repeated at the new pH.The rat diaphragm preparation was set up exactly as described by Bulbring

(1946), in Tyrode solution at 370 C bubbled with 95% oxygen and 5% carbondioxide. The phrenic nerve was stimulated with rectangular wave shocks, whichproduced maximal twitches, of 0.75 msec duration at 5 shocks/min. The volume ofthe bath was approximately 25 ml. and the drugs were added by pipette in a volumenot exceeding 0.4 ml., allowed to act until the block was fully developed and thenwashed out. The interval between doses was between 5 and 20 min. The relativeactivity was estimated in a 2+1 assay by comparing the doses which producedcomparable degrees of block.The anterior tibialis muscle preparation of the cat, anaesthetized with chloralose,

was set up as described by Brown (1938). The peroneal nerve was stimulatedwith rectangular wave pulses, which produced maximal contractions of the muscle,of 0.7 msec duration at a rate of 6-8 shocks/min. The drugs were injected, in avolume not exceeding 0.1 ml., retrogradely into the anterior tibial artery. The bloodpressure was recorded from a carotid artery. Measurement of blocking activity wasusually by a 2+ 1 assay method, but in one experiment only approximate estimateswere obtained by comparing the doses which produced roughly 50% block.

Respiration. Rabbits were anaesthetized with urethane and a cannula insertedinto the trachea. This was connected to a respirometer (Gaddum, 1941) so thatboth the rate of respiration and changes in the volume of inspired air could berecorded. The blood pressure was recorded from a carotid artery. The drugs wereinjected in a volume not exceeding 0.2 ml. into a femoral vein and washed in with0.9% saline. The interval between doses was 5 min and approximate estimatesof relative activity were made by comparing the doses which produced roughly thesame increase in the rate of respiration.

Acetylcholinesterase. Purified acetylcholinesterase, from ox red cells, was ob-tained from Nutritional Biochemicals Corporation. The effects of the compoundson the hydrolysis of acetylcholine (10-3M) by this enzyme were studied by mano-metric methods as described by Barlow & Zoller (1964).

Results

Estimates of the LD50 of nicotine, cytisine and the methylated derivatives ofcytisine, are shown in Table 1. Those mice which did not die within 1 hr werecounted as survivors. Occasionally an animal treated with nicotine died manyhours afterwards, but this never happened with the other drugs. The time afteradministration at which death occurred was noted and the average time of deathfor the animals killed in each group is indicated.With nicotine the onset of symptoms was much faster than with the other com-

pounds and the convulsions were qualitatively different. The tail became erect,rather like the Straub reaction, breathing appeared to be difficult, and there wasfrothing at the mouth, which was held wide open. The head was initially held

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high, but subsequently forced down upon the chest. A few seconds before deaththe body became rigid, except for rapid trembling motions of the limbs.With cytisine, caulophylline and caulophylline methiodide the convulsions were

much slower in onset and much less severe, but there were more pronounced tonicand clonic contractions, particularly of the hind limbs. The body did not becomeas rigid as with nicotine and, just before death, the hind legs were stretchedright back. All the animals which survived remained sedated for between 15 minand 1 hr afterwards.The results of the tests on the other preparations are summarized in Table 2.

Cytisine was invariably more active than caulophylline and usually more active thannicotine. Caulophylline methiodide was almost inactive and, in most tests, noeffects were observed even with very large doses.

TABLE 1. Acute toxicityRoute of administration

i.v. i.p. OralNicotine LD5O ,umoles/kg 1.92 59.0 1,425

limits 1.75-2.12 53.6-65.0 1,370-1,486LD50 mg/kg 0-3 9-5 230

(6) (7) (9)Time of death 32 0±0.8 2-41 +0 1 2.86±0r1

sec (28) min (38) min (45)

e.p.m.r. 1 1 1

Cytisine LD50 ,umoles/kg 9.10 49.5 535limits 7.9-10.5 466-57.5 411-696LD50 mg/kg 1-73 9 4 101

(6) (6) (7)Time of death 372-4-31 5-32±04 12.7 ±0.6

sec (36) min (28) min (46)

e.p.m.r. 475 0.84 0 37

LD5O gmoles/kglimitsLD50 mg/kg

Time of death

e.p.m.r.

LD50 ,umoles/kglimitsLD50 mg/kg

Time of death

e.p.m.r.

10389-9-118

21(6)

252210-302

51(5)

41.3±1*4 6.92+40.4sec (31) min (26)

53.6 4-27

280239-328

61(6)

79.5+0*3sec (32)

146

>2,000

The table shows the LDSO in umoles/kg and the fiducial limits (P=0 05). The value in mg/l g isshown below. The number of dose levels tested-that is, the number of groups of mice used-isshown in parentheses. The mean time after administration at which death occurred is shown±thestandard error with the total number of animals which died shown in parentheses. The lowestentry shows the equipotent molar ratio (e.p.m.r.) for the compound relative to nicotine,calculatedfrom the values of LD50.

Caulophylline >2,500

Caulophyllinemethiodide

>8,000

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The effects produced by cytisine and caulophylline were similar to each other butwere not always exactly the same as those produced by nicotine. Consequentlyalthough a quantitative comparison of cytisine with caulophylline was possible, aquantitative comparison with nicotine is of doubtful value in some tests. Thedifference was most noticeable with the cat tibialis preparation, where the blockingaction of nicotine lasted much longer than that of cytisine and caulophylline (Fig. 2).To a lesser extent this was true of the blocking action of the compounds on the catsuperior cervical ganglion (Fig. 3). Caulophylline methiodide, in high doses, causeda block on this preparation but this was never accompanied by an initial stimula-tion. It is probably the result of a different type of action and so this compoundcannot really be compared with the others. After the block there was an increase

TABLE 2. Equipotent molar ratios for the compounds relative to (-)-nicotine in the various tests

PreparationCat superior

cervicalganglion

BloodpressureGuinea-pigileum

Chick biventerFrog rectusRat diaphragmCat tibialisRabbitrespiration

Cytisine Caulophylline

StimulationBlockCat (rise)Rat (rise)

0.75±0.15 (6)1174±007 (4)0.54±011 (4)041±014 (10)

(Contraction) 0.25±0.02 (3)

(Contracture) 6.2540.48 (4)(Contracture) 0.64 (6)(Block) 0'66 (2)(Block) *0.23 ±t0.02 (3)

(Increase) 3 05±0t33 (4)

14.2±2.8 (6)20.1±2.2 (4)5-60O±081 (4)0.84±024 (10)

2.80±0-18 (3)

25-14+7.2 (4)2.7 (6)15.4 (2)

*2.40±0.19 (3)

Effectivedose/animal

orconcentration

Caulophylline ofmethiodide (-)-nicotine

27.0+3.9 (4)

Very feeble (2)

Feeble

27.7±2.8 (4)

10 n-moles100 n-moles300 n-moles50 n-moles

10-5M

10-6M10-5M

2x 10-4M300 n-moles

1 zmoleThe mean is given+the standard error, with the number of experiments shown in parentheses.Figures for the cat tibialis, marked with an asterisk, are of doubtful value because of the differencein the time-course of the effects (see text), though they give a fair comparison of cytisine and caulo-phylline. Only a rough comparison was possible on the rat diaphragm preparation because thecompounds were not very active and large amounts of material were needed. The figures for thefrog rectus have been calculated from separate comparisons of the compounds with ,-pyridylmethyl-trimethylammonium and of this compound with (-)-nicotine. The final column shows theapproximate dose or concentration which was effective in these experiments.

S S

Suxamethonium Cytisine

5 min

. * 0S

Caulophylline Nicotine

1 hr

FIG. 2. Effects on the cat tibialis muscle. The record shows contractions of the tibialismuscle in response to stimulation of the peroneal nerve. Compare the transient blockadeproduced by cytisine (200 n-moles), caulophylline (2,000 n-moles) with the longer effects ofsuxamethonium (5 n-moles) and the prolonged effects of nicotine (400 n-moles). S indicatesan injection of 0.9% sodium chloride (0.2 ml.).

S

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in the size of the contracture; this supramaximal response could well be the resultof inhibition of cholinesterase by the high concentrations of caulophylline methio-dide used (see later).The results of the comparisons on the rat blood pressure were very variable but

this variation can probably be ascribed to the preparation rather than to differences

0 -S

* aS

* 0*S S S

0S

0

Caulophylline Caulophylline Cytisine Nicotinemeth3iodidem

3 min

FIG. 3. Effects on the cat superior cervical ganglion. The upper record shows the bloodpressure and the lower record the contracture of the nictitating membrane in response tostimulation of the preganglionic nerve. Cytisine (75 n-moles), caulophylline (1,000 n-moles).caulophylline methiodide (1,000 n-moles) and nicotine (75 n-moles) all produced comparabledegrees of block. Note that cytisine, caulophylline and nicotine increased contracture beforethe onset of block and that the effects of nicotine lasted longer than those of cytisine andcaulophylline. Caulophylline methiodide did not cause an increase in contracture before theonset of block, but did so afterwards. Note the effects of the compounds on the bloodpressure. S indicates an injection of 0.9, sodium chloride (0.2 ml.).

TABLE 3. Effects ofpH on the activity of cytisine on the frog rectusEquipotent molar

ratio at

More Moreacid alkalinepH pH(a) (b)

8 356.00

5.90

5.45

3-76

5 18

3-80

7-20

9.45

7.65

7-65

5.00

Ratioa/b(c)139

1 22

1.73

2-04

1 .48

1-31

Proportion of

(d) ion (e) basepresent

at more acid pH

(d) (e)1-53 0-25

1.53

1.53

1-57

1.52

1.54

0 25

025

0-20

0-32

0.26Equipotent molar ratios for cytisine relative to ,B-pyridyl-methyltrimethylammonium are shown(a, b), together with the pH in the experiments at which they were obtained. The ratio (c) of theseequipotent molar ratios can be compared with the effect of the changes in the pH on the proportionof ion (d) and base (e) present.The average of the values in column c is 1.53, and in column d is 154.

pH7-887-10

7-107-88

7-887-10

6-987-88

7-927-22

7 127.9

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between the actions of the drugs, because it did not occur when the compounds werecompared on the cat blood pressure.

Cytisine and caulophylline were without effect on the hydrolysis of acetylcholineby acetylcholinesterase in concentrations as high as 4 x 10-3M, but caulophyllinemethiodide was weakly active. Estimates of the p150 were, 2.82, 2.82, 2.65 (mean2.76; substrate concentration 10-3M).The pKa values at 250 C were 7.92 for cytisine and 7.04 for caulophylline; three

estimates were made with each compound and gave identical results.

Effects of pH on activity on the frog rectus

Table 3 shows the equipotent molar ratios for cytisine relative to the quaternarycompound, f8-pyridylmethyltrimethylammonium, at different pH values. The in-creased activity in the more acid pH suggests that it is the cytisine ion which is theactive species and, quantitatively, the effects on the equipotent molar ratio are ingood agreement with the effects on ionisation, calculated from the pH and pK,.On average the equipotent molar ratio at the more acid pH is 1/1.53 times that atthe more alkaline pH, and the proportion ionized at the more acid pH is 1.54 timesthat at the more alkaline. In these experiments it did not seem to matter whethercytisine was tested in the more acid medium first or in the more alkaline.

Table 4 shows the results of similar experiments with caulophylline. Like cytisinethis compound is clearly more active at the more acid pH, but the quantitative

TABLE 4. Effects ofpH an the activity ofcaulophylline on the frog rectus

Equipotent molar Proportion ofratio at

(d) ion (e) baseMore More presentacid alkaline Ratio at more acid pHpH pH a/b

pH (a) (b) (c) (d) (e)7.43 338.03 79*6 2-42 2-93 0-74

7.13 22.88.20 130 4.84 6.15 0.53

7.13 25.98.20 125 5.70 6.15 0.53

8.13 797-15 45 1.75 5.20 0.53

8.13 81-37.08 39.3 2.07 5.61 050

8.20 1686.87 49.7 3.38 7-16 043

8.14 1006-94 32-3 3.07 6.59 0.41

Equipotent molar ratios for caulophylline relative to P-pyridylmethyltrimethylammonium are shown(a, b) together with the pH in the experiments at which they were obtained. The ratio (c) of theseequipotent molar ratios can be compared with the effect of the changes in the pH on the proportionof ion (d) and base (e) present. The experiments in which caulophylline was tested at the moreacid pH first are shown in the upper section of the table.

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agreement between the observed and calculated changes is much better in the experi-ments where the more acid pH was used before the more alkaline. In similarexperiments with nicotine tested at different pH values on the frog rectus, Hamilton(1963) observed exactly the same phenomenon; the quantitative agreement wasmuch better when the more acid pH was used first.

Discusion

The results of the toxicity experiments indicate that cytisine is less toxic thannicotine intravenously but more toxic by intraperitoneal or oral administration. Byany of these routes, however, it does not appear to be particularly lethal in mice.The intravenous LD50 of cytisine, 1.73 mg/kg, corresponds to 120 mg/70 kg and theequivalent of the oral LD5O would be 7 g/70 kg. In two experiments, doses ofcytisine 1.2 times the LD50 in mice were tested on guinea-pigs and rats and did notcause death. The animals appeared to be sedated, but not distressed, so thereseems to be no reason to believe that mice are particularly resistant to cytisinecompared with other rodents.The likely toxic dose in man, of course, is difficult to predict, especially as cytisine

can cause vomiting. The doses used in many of the pharmacological tests arp low,so quite small amounts might produce effects which were unpleasant, but the experi-ments with animals suggest that these are not likely to be lethal. Instances of deathfrom laburnum poisoning were reported in the last century (Radziwillowicz, 1888)but although there have been many cases of poisoning this century, none of theseseems to have been fatal (Mitchell, 1951). In forty-four enquiries about laburnumpoisoning to the Scottish Poisons Information Bureau between May 1963 and May1968 there was complete and uneventful recovery in all instances.

The high LD50 values for nicotine by the oral route were surprising in view, forexample, of Gaddum's statement (1953) that " if a couple of drops of pure nicotineare placed on a dog's tongue the dog drops down dead in a few seconds." Estimatesof the LD50 for nicotine, however, vary very considerably (Larson, Haag &Silvette, 1961) and it seems that, by the oral route, solutions of salts of nicotine areless toxic than solutions of the base. We may have obtained high values becausewe used a salt and also because we placed the dose directly into the stomach. Theacid environment will greatly delay absorption and the use of a stomach-tube toadminister the drug will prevent any absorption through the mucous membranesof the buccal cavity, which might otherwise occur during its passage from the mouthto the stomach. Absorption in this way, between the mouth and the stomach, mightwell account for the discrepancy between values of the oral LD50 for nicotine (seeabove). At normal body pH, there is rapid absorption of nicotine base acrossmembranes and, if the dose is not placed directly in the stomach, a considerableproportion may be absorbed before it reaches an acid environment. There shouldbe much less absorption of nicotine from solutions of the salts, especially if theseare acid (such as the hydrogen tartrate). The toxicity of cytisine may be similarlydependent on whether it is tested as the base or as the salt.

From the results of the experiments in mice it seems that death from cytisineeither occurs rapidly or not at all (see Table 1) which suggests that the body is ableto tolerate or detoxicate the drug if it is slowly absorbed. Larson, Finnegan &Haag (1949) have, in fact, shown that when nicotine is given intravenously by infu-sion over a period of 8 hr, some animals are able to tolerate up to 11 times the

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amount which would be lethal in a single injection. In view of this it could beargued that it is unnecessary to wash out the stomach of children who have eatenlaburnum, or other plants containing cytisine, especially as the alkaloid itself islikely to cause vomiting. It might be much more important to wash out the mouthwith an acidic buffer.Our results with the various tests confirm the resemblance between the pharmaco-

logical properties of cytisine and nicotine observed by Dale & Laidlaw (1912). Theyalso confirm the conclusions of Zachowski (1938), that cytisine is more powerfulas a ganglion stimulant than as a ganglion blocking agent. The resemblance betweenthe peripheral effects of cytisine and nicotine is striking and our results show thatthis resemblance is quantitative as well as qualitative. On the cat and rat bloodpressure, the guinea-pig ileum, the frog rectus and the rat diaphragm, comparableeffects are produced by doses of cytisine from one-quarter to two-thirds of those ofnicotine. The results with the cat tibialis are not really comparable, because theeffects of cytisine are only transient. The results with the chick biventer arenoticeably different but it is possible that these indicate marked sensitivity of chickmuscle to nicotine, rather than an insensitivity to cytisine.From the differences between the types of convulsion produced by the two

alkaloids, as well as from the absence of ear-twitches after cytisine noted by Dale& Laidlaw (1912), it seems that the similarity of the effects may not be so great inthe central nervous system. The action of cytisine on the respiration of anaesthe-tized rabbits is certainly weaker than would be expected from its effects on theperipheral nervous system.From the experiments at different pH with the frog rectus it appears that cytisine

also resembles nicotine in being active as the ion. Equipotent ionic ratios shouldtherefore be compared, rather than equipotent molar ratios. These are shown inTable 5, in which the mean values of the equipotent molar ratio from Tables 1 and2 have been corrected for the degree of ionization of nicotine and of the compound.With cytisine this makes little difference, because the pKa, 7.92, is only slightly lessthan that of nicotine, 8.01 (Barlow & Hamilton, 1962). Caulophylline, however, isa much weaker base, pK, 7.04, and it is clear that part of the decline in pharmaco-logical activity produced by methylating cytisine is due to the effect of methylationon basicity. The decline in basic strength is likely to be caused by steric hindrance;

TABLE 5. Equipotent ionic rctiosPreparation Cytisine Caulophylline

(a) (a) (b)Cat superior Stimulation 072 4.36 605

cervical ganglion Block 113 6.27 5.55

Blood pressure Cat (rise) 053 1-72 3.25Rat (rise) 0O40 026 065

Guinea-pig ileum (Contraction) 0-24 070 2.92Chick biventer (Contracture) 5.90 5.63 096Frog rectus (Contracture) 064 2.67 4-17Rat diaphragm (Block) 063 3'85 612Cat tibialis (Block) 0O22 074 3.36Rabbit respiration (Increase) 2.94 8.52 2.90The equipotent molar ratios in Tables 1 and 2 have been corrected for the degree of ionization ofthe compounds. Figures in columns a are relative to the univalent nicotinium ion; those in b arefor the caulophylline ion relative to the cytisine ion.

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the methyl group, which is electron-releasing and therefore usually base-strengthening(see, for example, Clark & Perrin, 1964), restricts the access of hydrogen ions to thenitrogen atom and this is already limited by the bulk of the bridged-ring system(Fig. 1).Even allowing for the difference in basicity, however, it is clear that the caulo-

phylline ion is weaker than the cytisine ion. In most tests it has between one-halfand one-sixth of the activity, though on the chick biventer it has the same activityas the cytisine ion and on the rat blood pressure it is even more active. Apart fromthese quantitative differences, however, the pharmacological effects of cytisine andcaulophylline are indistinguishable.The feeble activity of caulophylline methiodide is surprising. Apart from its

weak effects as an inhibitor of acetylcholinesterase and as a ganglion-blocking agent,it seems to be inactive. It is toxic to mice only when very large doses are givenintravenously, but the symptoms produced are similar to those of cytisine andcaulophylline. We thought it possible that the introduction of the second methyIgroup into caulophylline might have altered the conformation of ring C, making theboat form preferable to the chair form shown in Fig. 1. Normally the boat formis thermodynamically less favoured, but it seemed that the change might occur

30

20

10

0

-10 -

-20

-3050 40 30 20 17 10

Wave numbers (kv) D line sodiumFIG. 4. Optical rotatory dispersion curves. Wave numbers (kv; 1 kv=1,000 cm-) areplotted against the molar rotations. Measurements were made with 2 x 10-4M concentrationsin distilled water. Similar results were obtained with the hydrogen iodides as with the basesexcept that above 40,000 wave numbers (250 m,u) the iodide absorption was so strong that therotation could not be measured. The Cotton effect between 31,000 and 35,000 wave numberscorresponds to the peak absorption due to the pyridone chromophore, and is the same shapefor all three compounds. The measurements were made with a Bellingham and StanleyPolarmatic 62 instrument, which scans continuously, but does not measure rotation directly.The absolute values were calculated for the points shown ( x - - x, caulophylline;O ,cytisine; A- - -A caulophylline methiodide) and the intervening parts of the curves weredrawn from the traces of the relative rotations recorded by the instrument.

172

Some studies on cytisine and its methylated derivatives 173

because the basic nitrogen atom in ring C and the pyridine nitrogen atom in ringsA and B are fairly close together. The introduction of a second methyl group,which must be placed between these two atoms, might tend to force them furtherapart but, to offset this there would then be considerable steric interference betweenthe other methyl group on the nitrogen atom in ring C and an axial hydrogen atom.These changes in conformation involve the movement of groups close to thechromophore, the pyridone system (X max, 303 mM), and consequently might beexpected to produce changes in the optical rotatory dispersion curves. These wereexamined, however, and found all to have the same shape (Fig. 4), and consequentlythe inactivity of caulophylline methiodide, either as an agonist or an antagonist,does not seem to be caused by any fundamental change in preferred conformation.

It seems, then, that the progressive methylation of cytisine decreases activitybecause it decreases the ability of the onium group, the nitrogen atom in ring C,to fit the receptor. This is likely to be a steric effect, due simply to the increasein size. Chemical support for this comes from the decreased basicity of caulophyl-line compared with cytisine, and also from the difficulty of preparing caulophyllinemethiodide, which is only obtained after heating caulophylline with methyl iodidefor 48 hr. It is, however, possible that cytisine produces effects when only a smallproportion of receptors is occupied and consequently has only a low affinity. Thedecline in activity with methylation would then indicate a decline in efficacy ratherthan in affinity. The absence of agonist activity in caulophylline methiodide clearlyindicates a decline in efficacy, but the absence also of appreciable antagonist activitysuggests that methylation has also decreased affinity unless the affinity of cytisineis very low indeed-that is, unless cytisine has a particularly high efficacy.From molecular orbital calculations, Kier (1968) has deduced the preferred con-

formations of nicotine and acetylcholine and has suggested that the presence in amolecule of a quaternary nitrogen atom and a negatively charged atom situated4.85 + 0.1 A away are key features for nicotine-like activity. In models the nitrogenatom in ring C of cytisine appears to be 4.8-9 A away from the oxygen atom of thepyridone group, which will be partially negatively charged. This agreement, how-ever, may be fortuitous. It is not clear whether the key features confer affinity orefficacy on a molecule or, more probably, a particularly desirable combination ofthe two properties. They cannot be the only criteria for activity, however, becausethough they are present in cytisine and caulophylline, they are also present incaulophylline methiodide, which is virtually inactive.

We wish to thank Dr. J. C. P. Schwarz and Mr. F. Rutherford of the Chemistry Departmentfor the optical rotatory dispersion measurements and the Faculty of Medicine for the awardof a Fellowship to one of us (L. J. McL.). The cytisine was purchased with part of a grantfrom the Tobacco Research Council.

REFERENCESALBERT, A. & GOLDACRE, R. (1943). The nature of the amino-group in aminoacridines. Part 1.

Evidence from electrometric studies. J. chem. Soc., 454-462.BARLOW, R. B. & HAMILTON, J. T. (1962). Effects of some isomers and analogues of nicotine on

junctional transmission. Br. J. Pharmac. Chemother., 18, 510-542.BARLOW, R. B., SCOTT, N. C. & STEPHENSON, R. P. (1967). The affinity and efficacy of onium salts

on the frog rectus abdominis. Br. J. Pharmac. Chemother., 31, 188-196.BARLow, R. B. & ZOLLER, A. (1964). Some effects of long chain polymethylene bis-onium salts

on junctional transmission in the peripheral nervous system. Brit. J. Pharmac. Chemother., 23,131-150.

174 R. B. Barlow and L. J. McLeod

BROWN, G. L. (1938). The preparation of the tibialis anterior (cat) for close arterial injection.J. Physiol., Lond., 92, 22-23P.

BULBRING, E. (1946). Observations on the isolated phrenic nerve diaphragm preparation of the rat.Br. J. Pharmac. Chemother., 1, 38-61.

CLARK, J. & PERRIN, D. D. (1964). Prediction of the strengths of organic bases. Q. Rev. chenm.Soc., 18, 295-320.

DALE, H. H. & LAIDLAW, P. P. (1912). The physiological action of cytisine, the active alkaloid oflaburnum (Cytisus laburnum). J. Pharmac. exp. Ther., 3, 205-221.

GADDUM, J. H. (1941). A method of recording respiration. J. Physiol., Lond., 99, 257-264.GADDUM, J. H. (1953). Pharmacology, 4th ed., p. 183. Oxford Medical Publications: Oxford

University Press.GINSBORG, B. L. & WARRINER, J. (1960). The isolated chick biventer cervicis nerve-muscle prepara-

tion. Br. J. Pharmac. Chemother., 15, 410-411.HAMILTON, J. T. (1963). The influence of pH on the activity of nicotine at the neuromuscular

junction. Can. J. Biochem., 41, 283-289.HUDSON, C. S. & NEUBERGER, A. (1950). The stereochemical formulas of the hydroxyproline and

allohydroxyproline enantiomorphs and some related substances. J. org. Chem., 15, 24-34.ING, H. R. (1931). Cytisine. Part 1. J. he/m. Soc., 2195-2203.ING, H. R. (1932). Cytisine. Part 11. J. chem. Soc., 2778-2780.KALASCHNIKOW, W. P. & KUSNETZOW, A. I. (1938). Pharmako-chemische und pharmako-logische

Untersuchung einiger reizender Expektorantien. Chem. Zent.B!., 1, 932.KIER, L. B. (1968). A molecular orbital calculation of the preferred conformation of nicotine.

Molec. Pharma-., 4, 70-76.KREBS, H. A. & HENSELEIT, K. (1932). Untersuchungen uber die Harnstoffbildung im Tierkorper.

Hoppe Seyvler's Z. physiol. Chem., 210, 33-66.LARSON, P. S., FINNEGAN, J. K. & HAAG, H. B. (1949). Studies on the fate of nicotine in the body.

J. Pharmac. exp. Ther., 95, 506-508.LARSON, P. S., HAAG, H. B. & SILVETTE, H. (1961). Tobacco. Experimental and Clinical Studies.

pp. 439-440. Baltimore: The Williams and Wilkins Company.LITCIIFIELD, J. T. & WILCOXON, F. W. (1949). A simplified method of evaluating dose-effect

experiments. J. Pharmac. exp. Ther., 96, 99-113.MITCHELL, R. G. (1951). Laburnum poisoning in children. Lancet, 2, 57-58.OKUDU, S. & KATAUKU, H. (1961). Absolute configuration of (-)-anagyrine and of related C 15

lupin alkaloids. Chem. Ind. Lond., 29, 1115-1116.PARTHEIL, A. (1892). Ueber Cytisin und Ulexin. Arch. Pharm., 230, 448-498.PATON, W. D. & PERRY, W. L. M. (1953). The relationship between depolarization and block in

the cat's superior cervical ganglion. J. Physio!., Lond., 119, 43-57.POWER, F. B. & SALWAY, A. H. (1913). The constituents of the rhizome and roots of Caulophylluml

thalicoides. J. chem. Soc., 103, 191-210.RADzIWILLOWICZ, R. (1888). Ueber Cytisin. Arb. Pharmak. Inst. Dorpat., 11, 56-101.SCOTT, C. C. & CHEN, K. K. (1943). The pharmacological action of N-methylcytisine. J. Pha!-nmac.

exp. Ther., 79, 334-339.THIES, H. & REUrHER, F. W. (1954). Ein Reagens zum Nachweis von Alkaloiden auf Papier-

chromatogrammen. Naturwissenschafteni, 41, 230-231.ZACHOWSKI, J. (1938). Zur pharmakologie des Cytisins. Arch. exp. Path. Pharmak., 189, 327-344.

(Received A'igitst 29, 1968)

Molecular Determinants of Subtype-selective Efficacies ofCytisine and the Novel Compound NS3861 at HeteromericNicotinic Acetylcholine Receptors*

Received for publication, November 13, 2012, and in revised form, December 6, 2012 Published, JBC Papers in Press, December 10, 2012, DOI 10.1074/jbc.M112.436337

Kasper Harpsøe‡1, Helle Hald‡¶1, Daniel B. Timmermann¶1, Marianne L. Jensen¶, Tino Dyhring¶, Elsebet Ø. Nielsen¶,Dan Peters¶, Thomas Balle�, Michael Gajhede‡, Jette S. Kastrup‡, and Philip K. Ahring¶2

From the ‡Faculty of Health and Medical Sciences, University of Copenhagen, Denmark, ¶Aniona ApS, 93 Pederstrupvej,DK-2750 Ballerup, Denmark, and the �Faculty of Pharmacy, The University of Sydney, Australia

Background: The contribution of �-subunits to agonist efficacy in nicotinic receptors is incompletely understood.Results: Two nicotinic agonists displayed opposing efficacy profiles at receptors containing �2- or �4-subunits and maximalefficacy was determined by both ligand-binding and transmembrane �-subunit domains.Conclusion: The �-subunit is an important determinant of agonist efficacy.Significance: These findings provide support to structure-guided nicotinic receptor drug design.

Deciphering which specific agonist-receptor interactionsaffect efficacy levels is of high importance, because this will ulti-mately aid in designing selective drugs. The novel compoundNS3861 and cytisine are agonists of nicotinic acetylcholinereceptors (nAChRs) and both bind with high affinity to hetero-meric �3�4 and �4�2 nAChRs. However, initial data revealedthat the activation patterns of the two compounds show verydistinct maximal efficacy readouts at various heteromericnAChRs. To investigate the molecular determinants behindthese observations, we performed in-depth patch clamp electro-physiological measurements of efficacy levels at heteromericcombinations of �3- and �4-, with �2- and �4-subunits, andvarious chimeric constructs thereof. Compared with cytisine,which selectively activates receptors containing �4- but not�2-subunits, NS3861 displays the opposite �-subunit prefer-ence and a complete lack of activation at �4-containing recep-tors. The maximal efficacy of NS3861 appeared solely depend-ent on the nature of the ligand-binding domain, whereasefficacy of cytisine was additionally affected by the nature of the�-subunit transmembrane domain. Molecular docking tonAChR subtype homology models suggests agonist specificinteractions to two different residues on the complementarysubunits as responsible for the �-subunit preference of bothcompounds. Furthermore, a principal subunit serine to threo-nine substitution may explain the lack of NS3861 activation at�4-containing receptors. In conclusion, our results are consist-ent with a hypothesis where agonist interactions with the prin-cipal subunit (�) primarily determine binding affinity, whereasinteractionswith key amino acids at the complementary subunit(�) affect agonist efficacy.

Activation of the neuronal nicotinic acetylcholine receptors(nAChRs)3 is known to involve agonist binding in a site con-taining a set of highly conserved aromatic amino acid residuesin the extracellular ligand-binding domain (LBD) (1). In hetero-meric neuronal nAChRs, this agonist binding site has beenshown to be located in the interface between an�- and a�-sub-unit, described as the principal and complementary binding sitecomponents, respectively (2). Consistent with these findings,functional studies have identified segments of the LBD of both�- and�-subunits as important determinants of agonist affinity(3, 4). A significant part of the binding site is made up by theflexible C-loop that changes spatial location upon agonist bind-ing (5). Thismovement is subsequently translated into the poreopening by a not yet fully elucidated mechanism thought toinvolve rigid body rotation of the inner �-sheets, movements ofloops in the interface between the LBD and the transmembranedomain (TMD), and a tilt of helix M2 (6).Agonists can display strong subtype selectivity, primarily in

terms of efficacy, for heteromeric neuronal nAChRs containingparticular �- or �-subunits (7, 8), despite a very high sequenceidentity of the residues involved in agonist binding (9). By anal-ogy to the findings that agonist efficacy in glutamate receptorsappears tightly linked to domain closure (10), C-loop closurehas been suggested to be directly coupled to agonist efficacy inCys-loop receptors (11–13). However, in a recent study, it wasdemonstrated that a structurally related series of agonists withsimilar binding affinities but divergent efficacies all resulted insimilar C-loop closure in co-crystallization experiments withacetylcholine-binding protein (AChBP) (14). Hence, it appearsthat neither the binding determinants nor the degree of C-loopclosure are sufficient to explain how efficiently an agonist gatesa receptor.

* This work was supported in part by grants from the Drug Research Acad-emy, Faculty of Pharmaceutical Sciences, University of Copenhagen (toK. H.), the Danish Ministry of Science, Innovation and Higher Education (toH. H.), the Carlsberg Foundation (to T. B. and K. H.), and the LundbeckFoundation (to T. B.).

1 These authors contributed equally to this work.2 To whom correspondence should be addressed. Tel.: 45-4460-8223; Fax:

45-4460-8080; E-mail: [email protected].

3 The abbreviations used are: nAChR, nicotinic acethylcholine receptor; ACh,acetylcholine; DH�E, dihydro-�-erythroidine hydrobromide; CRR, concen-tration-response relationship; LBD, ligand-binding domain; NS3861, 3-(3-bromo-thiophen-2-yl)-8-methyl-8-aza-bicyclo[3.2.1]oct-2-ene; TMD,transmembrane domain; AChBP, acetylcholine-binding protein.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 4, pp. 2559 –2570, January 25, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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Despite having a large range of efficacies, the above men-tioned series of partial agonists all contained the same corescaffold, which explains the similar binding affinities (14). Themajor differences were observed in the interactions with thecomplementary subunit. Based on this, it was hypothesized thatagonist efficacy is dependent on the strength with which theagonist can mediate interactions between the principal andcomplementary subunits. From the current knowledge it islikely that this inter-subunit interaction is an integral first stepin triggering the movements or stabilizing the conformationthat eventually lead to channel gating, however, it also promptsnew questions. The first question is whether the principal sub-unit only delivers the framework to which an agonist binds,whereas determinants important for ligand efficacy are locatedon the complementary subunit? Furthermore, irrespective ofthe exact mechanism, translation of binding into ion-channelgating involves transmission of conformational changes in theLBD to the pore-forming TMD. The next question is then,whether the LBD fully determines agonist efficacy and theTMD consequently acts purely as a mechanical channel-gatingswitch, i.e. would any TMD that interlinks well with a givenLBD result in similar efficacy levels of specific agonists?As part of a drug discovery program, novel nAChR agonists

of various selectivity profiles were designed. One such agonist,NS3861, was found to selectively activate �3- but not �4-con-taining nAChRs and it displays higher efficacy at the �3�2receptor comparedwith the�3�4 receptor (present report). Tothe best of our knowledge, such a selectivity pattern has notpreviously been reported. Interestingly, this selectivity profilereciprocates that of the nAChR agonist cytisine, which isknown to selectively activate �4- but not �2-containingnAChRs (8, 15). These two compounds were therefore used aspharmacological tools to explore the relative contributions tomaximum agonist efficacy of the principal (�) and complemen-tary (�) subunits as well as the LBDs versus the TMDs. Thestudies entailed patch clamp electrophysiological measure-ments of heteromeric combinations of �3- and �4- with �2-and �4-subunits, and various chimeric constructs thereof.Receptor homology models and molecular docking were usedto interpret the experimental data in terms of agonist bindingmodes and interactions to the binding site residues of the prin-cipal and complementary subunits.In essence, we found the identity of the �-subunit to contrib-

ute to agonist efficacy in an “either/or” fashion. However, thedeterminants responsible for “fine tuning” agonist efficacywereparticularly associated with the �-subunit LBD, although the�-subunit TMD was also observed to have some impact. Ourfindings from the molecular docking approach show that theobserved subunit selectivities of NS3861 and cytisine can beattributed to a few residues in the binding sites that differbetween �3- and �4- as well as �2- and �4-subunits.

EXPERIMENTAL PROCEDURES

Materials—(�/�)-NS3861 was synthesized at NeuroSearchA/S. (�)-Cytisine (C2899), (�)-nicotine (N5260), acetylcholine(ACh) (A9101), and dihydro-�-erythroidine hydrobromide(D149) were purchased from Sigma. All other chemicalswere purchased from Sigma or Merck and were analytical

grade.Molecular biology kits were fromQiagen (QIAquick col-umns,MaxiprepDNA, andGel Extraction kits). T4DNA ligase,T7 DNA polymerase, SalI, NotI, and NruI enzymes were fromNew England Biolabs. Oligonucleotides as well as sequencingservices were from MWG Biotech. Escherichia coli strainRZ1032 was from Quantum Biotechnologies and strain XL1-Blue from Stratagene. The rat pituitary carcinoma cell lineGH4Cl (CCL-82.2) and the human embryonic kidney 293 cellline HEK293 (CRL-1573) were from the American Type Cul-ture Collection. Cell culture flasks were fromNunc, Dulbecco’smodified Eagle’s medium (DMEM) from Lonza (BE12–604/U1), Ham’s F-10 medium from Invitrogen (31550-023), fetalbovine serum (FBS) from Invitrogen (10270-106), horse serumfrom Invitrogen (26050-088), poly-D-lysine from Sigma(P7405), Lipofectamine PlusTM and trypsin/EDTA from Invit-rogen, Geneticin G418 from Sigma (A1720), and Zeocin fromInvitrogen (450430). The calcium indicator fluo-4/AM and[3H]epibatidine (55 Ci/mmol) were from Invitrogen andPerkinElmer Life Sciences, respectively. Borosilicate capillarytubes were from Vitrex (155710).Molecular Biology—Cloning of the human nAChRs �3-, �4-,

�2-, and �4-subunits was as described previously (16). nAChR�-subunits and �-chimeras were in the pNS3n vector, whereasnAChR �-subunits and �-chimeras were in the pNS3z vector.pNS3n and pNS3z are custom designed vectors derived frompcDNA3 (Invitrogen) where “n” and “z” denotes selection usingthe NeoR or ZeoR genes, respectively. Both vectors use theCMV promoter to drive expression of the insert in mammaliancells. To generate�3/�4-,�4/�3-,�2/�4-, and�4/�2-chimerasa unique SalI restriction site was introduced close to the begin-ning of TM1 in each subunit by site-directed mutagenesis asdescribed by Slilaty et al. (17). Briefly, uracilated plasmids con-taining nAChR subunits were isolated from E. coli strainRZ1032, linearized with NruI, and used as template inmutagenesis reactions using a mutagenesis oligo, a closingoligo, T7 DNA polymerase, and T4 DNA ligase. Sequences ofthe mutagenesis oligonucleotides were: �3-SalI, CTCACTGT-ACATCCGTCGACTGCCCTTGTTCTACAC; �4-SalI, GCC-TTCGTCATCCGTCGACTGCCGCTCTTCTACAC;�2-SalI,GACTTCA-TCATTCGTCGACGGCCGCTCTTCTACAC;and �4-SalI, GACTTCATCATCCGTCGACGGCCTCTGTT-CTACAC. Correct introduction of SalI sites were verified byrestriction enzyme digestion and further sequencing. ChimericnAChRs were made by restriction digestion of the plasmidswith SalI and NotI (polylinker site), gel electrophoresis purifi-cation of fragments, and re-ligation of relevant nAChR combi-nations. The resulting chimeric constructs were verified byrestriction enzyme digestion and sequencing. To obtain an�4-subunit with a Thr183 to Ser mutation as well as a �2-sub-unit with Val136 to Ile and Phe144 to Leumutations,�4- and�2-LBDs, including themutations, were purchased fromGenScriptand ligated with the respective TMDs using the SalI site.Cell Culture and Transfection—HEK293 cells were propa-

gated at 37 °C in culture flasks in a humidified atmosphere con-taining 5% CO2. The growthmedium consisted of DMEM sup-plemented with 10% FBS. For generation of clones stablyexpressing�3�2,�3�4,�4�2, and�4�4nAChRs,HEK293 cellswere seeded in T12.5 culture flasks, cultured to 50–70% con-

Determinants of Nicotinic Receptor Agonist Efficacy

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fluence, and transfected with a total of 1 �g of expression plas-mids using Lipofectamine PlusTM according to the manufac-turer’s protocol. Twenty-four hours post-transfection, the cellswere detached using trypsin/EDTA and seeded in T75 cultureflasks with a tear-off lid. To select stably expressing cells theculturemediumwas supplementedwith 0.5mg/ml ofG418 and0.125 mg/ml of Zeocin. Following selection, single clones werepicked and propagated in selection media until sufficient cellsfor freezing were available. Thereafter the cells were propa-gated in regular culture media.GH4Cl cells were cultured in tissue culture flasks at 37 °C in

a humidified atmosphere containing 5% CO2. The culturemedium consisted of Ham’s F-10 medium supplemented with15% horse serum and 2.5% FBS. On the day before transfection,cells were seeded in 35-mm Petri dishes containing poly-D-ly-sine-coated round glass coverslips (Ø 3.5 mm, custom made atVWR International). The GH4Cl cells were then transfectedusing Lipofectamine PlusTM according to manufacturer’s pro-tocol. Each Petri dish was transfected with a mixture of plas-mids containing cDNAs coding for relevant nAChRs and greenfluorescent protein (Clontech); the latter to identify transfectedcells in the patch clamp set-up. After transfection, the culturemediumwas supplementedwith 50mMKCl, as elevatedK� haspreviously been shown to facilitate expression of nAChRs in theGH4Cl cell line (18).Xenopus laevis oocyte preparation and electrophysiological

experiments were performed as described previously (19).Briefly, to obtain isolated oocytes lobes from ovaries of femaleadult X. laeviswere removed and defolliculated using collagen-ase. Oocytes were injected with a total of �25 ng of cRNAencoding human wild-type or concatenated subunits and incu-bated for 2–7 days at 15–18 °C in modified Barth’s solution (90mM NaCl, 1.0 mM KCl, 0.66 mM NaNO3, 2.4 mM NaHCO3, 10mM HEPES, 2.5 mM sodium pyruvate, 0.74 mM CaCl2, 0.82 mM

MgCl2, 100 �g/ml of gentamycin, and pH adjusted to 7.5).Radioligand Binding Experiments—HEK293 cells stably

expressing human�3�2,�3�4,�4�2, and�4�4 receptors wereharvested, washed once with 50 mM Tris-HCl (pH 7.4), andstored at �80 °C until the day of experiment. Thawed mem-brane pellets were re-suspended in 15 ml of ice-cold Tris-HClbuffer and centrifuged for 10min (27,000� g) at 4 °C. The finalpellets were re-suspended inTris-HCl buffer and used for bind-ing experiments. The conditions for receptor binding assayswere as described previously for [3H]epibatidine binding to ratbrain (20). Briefly, binding to �3�4 receptors was performedusing 0.3 nM [3H]epibatidine at 14 to 27 �g of protein/assay,whereas �3�2, �4�2, and �4�4 receptors were labeled with0.03 nM [3H]epibatidine at 32 to 300 �g of protein/assay. Thesamples were incubated in a final volume of 1050 �l (for �3�4)and 8400 �l for 1 and 4 h, respectively, at room temperature.Nonspecific binding was determined in the presence of 30 �M

(�)-nicotine, and binding was terminated by rapid filtration.Radioactivity was determined by conventional liquid scintilla-tion counting. Compounds were tested at 8 concentrationsranging from 0.03 nM to 30 �M. Estimates of IC50 values inbinding experiments were analyzed using the nonlinear curve-fitting programGraphPad Prism, and Ki values were calculatedfrom IC50 values using the Cheng and Prusoff equation: Ki �

IC50/(1/(liter/Kd)). The Kd values for each receptor type areshown in Table 3.Patch Clamp Electrophysiology—Electrophysiological mea-

surements in GH4Cl cells were performed in voltage clampusing conventional whole cell patch clamp techniques. All datawere obtained with an EPC-9 amplifier (HEKA). The holdingpotential was �60 mV in all experiments. Coverslips with cellswere placed in a diamond-shaped polycarbonate recordingchamber (Warner Instruments) fixed at the stage of an invertedmicroscope (Olympus). Throughout the experiment, the cellswere perfused with extracellular buffer (140 mM NaCl, 4 mM

KCl, 2mMCaCl2, 1mMMgCl2, and 10mMHEPES, pH adjustedto 7.4 using NaOH). Micropipettes were made from borosili-cate capillary tubes by means of a horizontal micropipettepuller (Zeitz Instruments). The pipettes were filled with intra-cellular buffer (120 mM potassium gluconate, 6 mM KCl, 5 mM

NaCl, 2 mM MgCl2, 10 mM HEPES, 0.5 mM EGTA, 2 mM ATP,and 0.2 mM GTP (ATP and GTP were added immediatelybefore use), pH adjusted to 7.4 using KOH). Initial pipetteresistance was �2 M�. Agonist/antagonists were deliveredwith an ultra-fast application system using a double barreledapplication pipette (the so-called �-tube) controlled via a piezo-ceramic device (Burleigh Instruments) as described previously(16). Data were sampled at 20 kHz, low-pass filtered at 6.7 kHzand only accepted if the series resistance was �10 M�. Seriesresistance was compensated by 80%. Agonist application pulseslasted 1 s and the stimulation frequency was 1 pulse per 30 s, toensure full recovery of the nAChRs from agonist-induceddesensitization between pulses. The responses of the patchclamp electrophysiological experiments were quantified bymeasuring peak current amplitude and relating this to 1 mM

ACh control responses. Concentration-response curves wereanalyzed using GraphPad Prism. Figures displaying currenttraces were made using SigmaPlot.X. laevis Oocyte Electrophysiology—Oocytes were subjected

to two-electrode voltage-clamp electrophysiological testingusing a custom-built system as described previously (19). Drugswere dissolved inOR2 (90mMNaCl, 2.5mMKCl, 2.5mMCaCl2,1.0 mM MgCl2, 5.0 mM HEPES, and pH adjusted to 7.5), andsolutions were applied directly to the oocytes via a glass capil-lary tube placed in the vicinity of the cell to ensure rapid solu-tion exchange (few seconds). Each application lasted �1 minand peak current amplitudes were measured. To ensure fullrecovery between applications at high agonist concentrations,the protocol included dynamic adjustments of washing periods.Homology Modeling and Induced-fit Docking—Homology

models of the �3�2, �3�4, and �4�4 dimer interfaces werebuilt as previously described for our�4�2 homologymodel (21)using the same template structures. The amino acid sequencesof the other three subunit combinations were aligned to thetemplates by use of the alignment for the �4�2 homologymodel and substitution of the �4 sequence with that of �3and/or the �2 sequence with that of �4 using T-Coffee, version9.01 (22). The amino acid sequences of the LBDs of �3, �4, �2,and �4 (Protein Knowledgebase entries P32297, P43681,P17787, and P30926 from www.uniprot.org (23)) and residuenumbers are listed accordingly. The rotamer library in PyMOL(24) was used to select side chain conformations of Val136,




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Phe144, andLeu146 in�2 aswell as of Ile134, Leu142, andLeu144 in�4, which point away from the agonist binding site to allowmaximal available space for ligand docking. Subsequently, theprotein structures were prepared for docking with the ProteinPreparation Wizard workflow (25) using default settings.(�/�)NS3861 and (�)-cytisine were built in Maestro (26) andenergy minimized using MacroModel (27) with default set-tings. Both compounds were used as input for induced fit dock-ing (28) in each of the four homologymodels using the centroidof (�)-nicotine included in themodels to define the box center.Ligand and receptor van der Waals scaling was set to 0.8 and1.0, respectively, maximum number of poses to 10, only theresidues for which we previously altered conformations wereoptimized during refinement and otherwise default settingswere used. The best scoring of each receptor-ligand combina-tion according to IFDScore were selected for analysis.

RESULTS

Activation patterns of cytisine and the novel compoundNS3861 (Fig. 1) were investigated to address the relative con-tribution(s) to maximal agonist efficacy of the LBD and theTMD in heteromeric neuronal nAChRs. The compounds weretested at various combinations of wild-type subunits and sub-unit chimeras, which were generated by fusing the LBD of onesubunit to the TMD of another subunit (denoted “LDB/TMD”,i.e. �3/�4, �4/�3, �2/�4, and �4/�2). Because agonist proper-ties are known to vary between heterologous expression sys-tems (29), it was chosen to address agonist efficacy using anultra-fast solution exchange methodology in whole cell patchclamp analysis of receptors transiently expressed in the ratGH4C1 cell line.Current densities for cells expressing chimeric subunits

were, on average, less than for the comparable wild-type sub-unit combinations when the LBD contained�3, whereas recep-torswith�4 expressedwell (Table 1). Lower expression levels ofchimeric constructs versus wild-type can possibly reflectreduced efficiency of chimeric subunit assembly. However, inall the receptor combinations used here, current densities weremore than sufficient to ensure reliable observations of partialagonists.A prerequisite for using LBD/TMD chimeric constructs for

evaluating agonist efficacy is conservation of basic biophysicalproperties such as expression level, kinetic properties, and ago-nist or antagonist sensitivity relative to wild-type receptorcounterparts. To validate that the constructs used in this studyadhere to this premise, two “fully” chimeric subunit combina-tions (�3/�4 � �4/�2 and �4/�3 � �2/�4) were thoroughlyevaluated initially and compared with wild-type heteromericreceptors.

Characterization of Wild-type and Chimeric nAChR Cons-tructs—The potency of ACh for activating wild-type �3�4 and�4�2 was in the range of �80 �M (Fig. 2), which is comparablewith previous studies (15, 30–32). This shows that the �4�2nAChRspredominantly assembled into the (�4)3(�2)2 stoichio-metry in these experiments (21). Expression of the fully chime-ric subunit combinations �4/�3 � �2/�4 and �3/�4 � �4/�2yielded functional receptors with potencies of ACh in the samerange as for the wild-type receptors (Fig. 2), indicating normalresponsiveness of chimeric receptors to ACh.When evaluating efficacies of agonists at heteromeric

nAChRs, a further complication relates to the fact that thesecan assemble in different stoichiometries of �- and �-subunits(30, 31, 33). Because recent results have shown that acetylcho-line and other agonists can activate an additional binding site inthe �4�4 interface (21), it is always necessary to considerwhether agonists have stoichiometry dependent activationpatterns.ACh concentration-response relationships (CRR) for the

fully chimeric andwild-type receptors were therefore evaluatedin X. laevis oocytes. Under conditions favoring the 3�:2� stoi-chiometry bothwild-type and chimeric receptorswith�4 in theLBD displayed ACh potencies in the same range as obtained inpatch clamp, whereas conditions favoring 2�:3� resulted inlower EC50 values, in particular for �4�2 (Table 2). Receptorscontaining the �3-LBD in 3�:2� stoichiometries appearedslightly (�3-fold) less potent compared with the patch clampexperiments, but all data for wild-type receptors are in goodagreement with previously reported results (15, 30–32).Kinetic properties of ACh-activated currents at wild-type

receptors containing the �2-subunit clearly displayed fasterdesensitization kinetics (Tau: �50 ms) relative to those con-taining �4-subunits (Tau: �250 ms) (Fig. 3 and Table 1). Amore slowly desensitizing/steady-state current, particularly in�2-containing receptors often followed initial rapid desensiti-zation. These findings are in good support of earlier studies onnAChR kinetic properties (7, 34–36). With respect to desensi-

FIGURE 1. Chemical structures of (�)-cytisine and (�/�)-NS3861.

TABLE 1Time constants and current densities of wild-type and chimericnAChRsPatch clamp data were obtained as described in the legend to Fig. 2. Time constantsfor current-decay of 1mMACh-evoked responseswere calculated using a one-phaseexponential decay nonlinear regression curve fitting routine. Data used for fittingwere from the peak recorded value and 200 ms forward in time. The current densi-ties are calculated as peak current of 1 mMACh (pA) divided by the cell capacitance(pF) for n � 10 experiments and values are presented � S.E.M.

Receptor Tau Current density

ms pA/pF�3 � �2 35 � 2 584 � 220�3 � �4 273 � 15 761 � 278�3 � �2/�4 106 � 7 163 � 59�3 � �4/�2 102 � 7 191 � 37�3/�4 � �2 51 � 3 111 � 39�3/�4 � �4 568 � 28 429 � 103�3/�4 � �2/�4 90 � 5 125 � 20�3/�4 � �4/�2 93 � 6 70 � 15�4 � �2 68 � 4 1236 � 372�4 � �4 213 � 12 345 � 76�4 � �2/�4 92 � 7 407 � 133�4 � �4/�2 71 � 4 183 � 46�4/�3 � �2 56 � 2 1350 � 429�4/�3 � �4 60 � 3 565 � 138�4/�3 � �2/�4 71 � 3 689 � 212�4/�3 � �4/�2 87 � 5 673 � 213




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tization kinetics for receptors containing chimeric subunits, allturned out to have desensitization time constants intermediatebetween those of wild-type receptors, except �3/�4 � �4 forwhich the value was slightly (�2-fold) higher.Finally, conservation of basal pharmacological properties of

the orthosteric site in the fully chimeric receptors was tested bydetermining the ability of the �2-selective competitive antago-nist DH�E to block wild-type and fully chimeric receptors. Theamino acid determinants responsible for binding of DH�E areknown to be distributed across both�- and�-subunits (37–39).The potency of DH�E for inhibition of �4�2 exceeded that forinhibition of �3�4 by more than 3 orders of magnitude (Fig. 2).Replacing the TMD of �4�2 with that of �3�4 (i.e. �4/�3 ��2/�4) did not affect high affinity DH�E inhibition, whereassubstitution of the LBDof�4�2with�3�4 (i.e.�3/�4� �4/�2)reduced DH�E potency to the level of the wild-type �3�4nAChR (Fig. 2). This shows that the antagonist potency ofDH�E is fully defined by the LBD and that the structural integ-rity of the LBD is preserved in the chimeric receptors.

Based on these data, it is highly likely that all combinations ofwild-type and chimeric constructs result in receptors, whichexpress reliably in GH4C1 cells. Comparisons of ACh EC50 val-ues, desensitization time constants, and DH�E IC50 values forthe fully chimeric receptors show that basic biophysical andligand-binding properties were well preserved and generallymatch those of wild-type receptors.Cytisine—Initially, cytisine was characterized with respect to

its binding affinity for heteromeric nAChRs in radioligand dis-placement experiments, using membrane preparations fromHEK293 cells stably expressing human �3�2, �3�4, �4�2, or�4�4 nAChRs. Cytisine was observed to displace [3H]epiba-tidine from �4�2 and �4�4 nAChRs with subnanomolar affin-ity (Table 3). Approximately 20-fold lower affinitywas observedat the �3�2 receptors and a further �20-fold affinity reductionwas seen at �3�4 receptors.Using patch clamp electrophysiology, the CRR of cytisine

was subsequently determined at wild-type �3�4 and �4�2nAChRs as well as fully chimeric receptors having the sameLBD interfaces. Cytisine behaved as a lowpotency full agonist atthe wild-type �3�4 nAChR (Figs. 3B and 4) but produced nosignificant activation of the �4�2 nAChR (Emax � 10%) at con-centrations up to 1 mM (Figs. 3I and 4). At the fully chimericreceptors, cytisine had low efficacy at �3/�4 � �4/�2 withpotency roughly matching that at �3�4 receptors (Figs. 3H and4), whereas no appreciable efficacy was seen at �4/�3 � �2/�4receptors (Figs. 3O and 4). Judging from the appearance of cur-rent traces for �3�4 and �4�4 (Fig. 3, B and J), desensitizationkinetics of cytisine resembled that of ACh. These results cor-roborate previous reports suggesting that cytisine bindspotently to �4�2 but primarily activates �4-containing recep-tors (7, 8, 15, 40).To further probe the importance of different LBD and TMD

combinations, maximal efficacy of cytisine was determined atthe remaining permutations of wild-type and chimeric con-structs (Fig. 3,A,C–G,K–N, andP). Combinations between any�-subunit (chimeric or wild-type) with wild-type �4 yieldedhighmaximum cytisine efficacy (Emax � 76–113%), whereas allcombinationswith awild-type�2-subunit resulted in an almost

FIGURE 2. CRRs for ACh and DH�E at �3�4 and �4�2 nAChRs as well as chimeras thereof. Functional receptor activation was measured in whole cell patchclamp experiments using transiently transfected GH4C1 cells in a setup equipped with an ultra-fast application system. Agonist was applied for 1 s and peakcurrent amplitudes were baseline subtracted and normalized to a 1 mM ACh response recorded in the same cell. A, ACh CRRs obtained in patch clampexperiments at �3�4, �4�2, �3/�4 � �4/�2, and �4/�3 � �2/�4 nAChRs (symbol identification as in panel C). Data points are plotted as mean � S.D. of n �4 –12 experiments as a function of the ACh concentration and fitted to the Hill equation by nonlinear regression. The data point for 3160 �M ACh at �3�4 wasomitted from the fit as this yielded a low submaximal response. B, DH�E CRRs obtained in patch clamp experiments at �3�4, �4�2, �3/�4 � �4/�2, and�4/�3 � �2/�4 nAChRs (symbol identification as in panel C). Experiments were largely conducted as described above except cells were preincubated withDH�E for 30 s before a co-application of 1 mM ACh and DH�E. Data points are plotted as mean � S.D. of n � 5–9 experiments as a function of the DH�Econcentration and fitted to the Hill equation by nonlinear regression. C, table of ACh agonist potencies (EC50 values) and DH�E inhibitor potencies (IC50 values)at the four receptor subtypes. All EC50 values have overlapping 95% confidence intervals, whereas pairwise overlap was observed for IC50 values.

TABLE 2Concentration-response relationships (CRRs) for ACh, cytisine, andNS3861 recorded in X. laevis oocytesOocytes were injected with nAChR subunits in 1:4 or 4:1 ratios as indicated toobtain receptors in (�*)2(�*)3 or (�*)3(�*)2 stoichiometries, respectively. CRRs forcytisine andNS3861 were recorded paired with an ACh-CRR, i.e. two 5-point CRRsfor each oocyte, the first ACh and the second cytisine or NS3861. Peak currentamplitudes were baseline subtracted and normalized to themaximal value obtainedby fittingACh-CRRs from each oocyte to theHill equation by non-linear regression.Normalized valueswere then fitted to theHill equation to establishmaximal efficacy(Emax) and concentration of half maximal activation (EC50). Data are presented asEmax in %, and EC50 in �M from n � 6–14 experiments.

Receptor�:�ratio ACh Cytisine NS3861

Emax EC50 Emax EC50 Emax EC50

% �M % �M % �M

�3�2 4:1 100 290 23 280 160 1.6�3�4 4:1 97 330 48 180 65 1�3/�4 � �4/�2 4:1 100 350 35 150 71 1.4�4�2 1:4 100 1.2 NEa NE�4�2 4:1 98 93 NE NE�4�4 1:4 100 20 37 0.12 17 1.9�4�4 4:1 99 71 22 0.72 6.3 1.2�4/�3 � �2/�4 4:1 98 100 3.8 13 9.0 0.64

a NE, no effect.




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complete loss (Emax � 10%) of efficacy (Fig. 5A). Replacing theTMDof�4with that of�2 gave a substantial, but not complete,loss of efficacy (Emax �13–30% for �3, �4, �3/�4, or �4/�3 incombination with �4/�2). On the other hand, irrespective ofthe �-subunit, replacing the TMD of �2 with that of �4 did notincrease the efficacy levels above that observed for receptorscontaining the wild-type �2-subunit (Emax � 10%).Finally, to investigate whether subunit stoichiometry of

�4-containing receptors could affect the observed maximalefficacies of cytisine, CRRs were recorded on nAChRsexpressed in oocytes (Table 2). These experiments qualitativelyconfirm the subtype-selective efficacy of cytisine for �4-con-taining receptors observed from patch clamp studies in combi-nations with �4. Independent of the subunit ratio, cytisine didnot activate �4�2 receptors but activated �4�4 receptors withsubmicromolar potencies (Table 2).NS3861—Characterization of NS3861 with respect to

[3H]epibatidine displacement binding showed this compoundto bind potently at all tested nAChR subtypes (Table 3).NS3861was �10-fold affinity selective for �3�4, relative to the �4�4nAChR and in both cases a�10–40-fold affinity reductionwasobserved by substituting �4 with �2. Thus, in sharp contrast tocytisine and nicotine, NS3861 has higher binding affinities at�4- versus �2-containing receptors.By analogy to the experiments reported for cytisine, the CRR

of NS3861 at �3�2, �3�4, and �4�2, and two fully chimericreceptors was investigated using patch clamp electrophysiol-ogy. NS3861 was found to activate wild-type �3�4 nAChRs(Figs. 3R and 4) but did not activate wild-type �4�2 (Figs. 3Yand 4). Interestingly, whereas NS3861 was a partial agonist at�3�4, it proved to be a full agonist at �3�2 albeit with�10-foldlower potency consistent with the binding affinities (Figs. 3Qand 4). Efficacy at the �3/�4 � �4/�2 receptor was almostidentical to that of wild-type �3�4 (Figs. 3X and 4), whereas noefficacy could be seen at �4/�3 � �2/�4 (Figs. 3AA and 4). Theactivation and desensitization properties of currents evoked byNS3861 were generally reminiscent of the currents evoked by

ACh, as judged from the appearance of current traces (Fig. 3,Qand R).Maximal efficacy of NS3861 was next investigated in exper-

iments involving the remaining permutations of wild-type andchimeric constructs (Fig. 3, S–W and Z). Unlike the efficacyprofile of cytisine, which was independent of the �-subunit,NS3861 was only capable of activating subunit combinationscontaining the LBD of �3, as no appreciable efficacy (Emax �10%) was detected in subunit combinations involving the LBDof �4 (Fig. 5B). However, substituting wild-type �3 for the�3/�4 chimera resulted in indistinguishable maximum effica-cies. It is further evident, that in combination with an �3-LBDthe identity of the �-subunit is highly important for efficacy. Inthese experiments, NS3861 acted as a full agonist whenever a�2-LBD was present (Emax � 97–129%), whereas it was only apartial agonist when a �4-LBD was present (Emax � 30–55%).By analogy to the findings made for the �-subunit, the �-sub-unit influence on efficacy was also confined to the extracellularLBD.The subtype-selective efficacy of NS3861 was qualitatively

reproduced in oocytes (Table 2). Independent of subunit ratios,NS3861 displayed no efficacy at �4�2 receptors. �3�2 and�3�4 receptors expressed in 3�:2� stoichiometries were, how-ever, activated with maximal efficacies significantly above orpartial relative to ACh, respectively. Because both combina-tions contain an �3�3 interface the observed �2 subtype-selec-tive efficacy appears independent of this interface.Binding Modes of Cytisine and NS3861 in nAChR Homology

Models—To obtain possible binding modes for cytisine andNS3861 the compounds were docked into homology models of�3�2, �3�4, �4�2, and �4�4 using an induced-fit protocol.Thus, side chain conformations of selected residues are sam-pled during the docking calculation with the ligand present inthe binding site and different ligand-receptor complexes aregiven as output. The best scoring complexes of cytisine in eachreceptor model display the same binding mode matching theinteractions of classical nicotinic agonists (5, 41), with hydro-gen bonds to the backbone carbonyl of a tryptophan and awatermolecule (Fig. 6B). Ile134 and Leu142 in �4 adopt side chainconformations, which display better van derWaals interactionsto cytisine compared with the corresponding Val136 and Phe144of �2; the only residues in the binding site that differ betweenthe �2- and �4-subunits (Fig. 6A).Contrary to cytisine, the obtained binding modes of NS3861

in all four homology models only display the hydrogen bond tothe tryptophan and has the thiophene ring located perpendic-ular to the pyridine of, for example, nicotine (41), with the bro-mine atom located near Phe144 and Leu142 of�2 and�4, respec-tively (Fig. 6C). Furthermore, because of Thr183 in�4 compared

FIGURE 3. Representative traces of ACh-, cytisine-, and NS3861-evoked currents in patch-clamp experiments at the indicated nAChRs. Data wereobtained as described in the legend to Fig. 2 and agonist application is shown as a solid bar above all current traces. The cytisine or NS3861 concentration givingrise to maximal efficacy at each receptor combination was identified by testing a range of concentrations. Traces shown are from compound concentrationsthat yielded maximal responses. Note that in many cases, the maximal response was not obtained with the highest concentration tested (see e.g. 100 �M

NS3861 at �3�2 and 10 �M NS3861 at �3�4 in Fig. 4B). There is no obvious technical reason for this but from previous similar observations it was speculatedthat it could be due to nonspecific ion-channel blockage at high ligand concentrations (46). A–P, cytisine-evoked currents shown alongside 1 mM ACh-evokedcurrents recorded in the same cells. Cytisine concentrations were 100 �M in traces F, J, L, N, and P, 316 �M in trace B, and 1 mM in the remaining traces (from 5,000to 100,000 times the highest observed binding Ki value). Q-AA, NS3861-evoked currents shown alongside 1 mM ACh-evoked currents recorded in the same cells.NS3861 concentrations were 3.16 �M in traces R and S, 10 �M in traces Q, T, U, V, W, and X, and 100 �M in the remaining traces (from 2,000 to 5,000 times thehighest observed binding Ki value).

TABLE 3Radioligand displacement by cytisine, NS3861, and nicotine at �3�2,�3�4, �4�2, and �4�4 nAChRsRadioligand binding was determined as the potency for displacement of [3H]epiba-tidine-binding in membrane preparations fromHEK293 cell lines stably expressinghuman nAChRs. Saturation data were fitted using a hyperbolic function assuming asingle binding site for [3H]epibatidine (Kd values). Ki values are reported as mean �S.E.M. in nM, n � 3–4 determinations, each conducted in triplicate.

Ki

Compound �3�2 �3�4 �4�2 �4�4

Epibatidine Kd 0.025 0.082 0.012 0.022Cytisine 8.7 � 1.3 196 � 19 0.43 � 0.09 0.77 � 0.09NS3861 25 � 4 0.62 � 0.09 55 � 11 7.8 � 0.7Nicotine 23 � 2 250 � 12 1.7 � 0.2 13 � 1




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with Ser181 in �3 the binding modes of NS3861 obtained in�4-containing receptors display a �1 Å shift of the thiophenering toward the �-subunit as measured on the sulfur and bro-mine atoms. This brings the bromine atom within 3.4 Å of oneof the sulfur atoms in the disulfide bond of the C-loop andwithin 3.1 Å of one of the methyl groups of Leu144/146 in the�-subunits (Fig. 6D).Agonistic Behavior of NS3861 and Cytisine on Point Mutated

Receptors—To investigate if Ile134 and Leu142 of �4 wereresponsible for the selective cytisine activation of �4- versus�2-containing receptors, a mutant �2-subunit was constructedwith two point mutations substituting the correspondingVal136 and Phe144 with Ile and Leu. Cytisine was then tested at�4�2(V136I/F144L) receptors expressed in oocytes. Here itdisplayed clear agonistic behavior resulting in activation of themutant receptor with an efficacy level (Emax � 18%; n � 9; 95%confidence interval, 16–19%) comparable with what isobserved for �4�4 receptors in this system (Table 2). Likewise,NS3861 was applied to �4(T183S)�2 mutant receptors inoocytes to test if Thr183 was responsible for the lack of activa-tion of �4-containing receptors. Indeed, NS3861 activates themutant receptor acting as a partial agonist (Emax � 36%; n � 9;

95% confidence interval, 33–40%). Hence, the oocyte experi-ments qualitatively demonstrate the importance of Ile134 andLeu142 in �4 as well as Ser181 in �3 for agonistic behavior ofcytisine and NS3861, respectively.

DISCUSSION

Early studies have shown that both �- and �-subunits innAChRs take part in defining the pharmacology of agonists atthe macroscopic level (7, 8). Recently, it was shown that at themicroscopic level very specific agonist-receptor interactions atthe complementary subunit determine efficacy of a series ofcompounds (14). The present report extends this knowledge tofunctional studies of two agonists (cytisine and NS3861) withparticularly pronounced profiles of efficacy selectivity (in thefollowing the term “efficacy selectivity” is used to describe ago-nist subtype-selective efficacies). Furthermore, it was investi-gated whether efficacy is solely determined by the LBD orwhether the TMD also plays a role.Despite a high binding selectivity for �2- versus �4-contain-

ing nAChRs, our patch clamp data show that cytisine is a fullagonist on �4-containing receptors and at best a very weakpartial agonist when �2 is present, in line with previous reports

FIGURE 4. CRRs for cytisine and NS3861 at wild-type and chimeric nAChRs. Patch clamp data were obtained as described in the legend to Fig. 2. A, cytisineis a full agonist at �3�4 and a partial agonist at �3/�4 � �4/�2 nAChRs (symbol identification as in panel C). Data points are plotted as mean � S.D. of n � 3–11experiments as a function of the cytisine concentration and fitted to the Hill equation by nonlinear regression. B, NS3861 is a full agonist at �3�2 and a partialagonist at �3�4 and �3/�4 � �4/�2 nAChRs (symbol identification as in panel C). Data points are plotted as mean � S.D. of n � 3–10 cells as a function of theNS3861 concentration and fitted to the Hill equation by nonlinear regression. The data points for 100 �M NS3861 on �3�2 and 10 �M NS3861 on �3�4 wereomitted from the fitting routine as these concentrations yielded low submaximal responses. C, table of fitted maximal efficacy (Emax) and half-maximalactivation concentration (EC50) at the five receptor subtypes. � indicates Emax � 10% set as minimum efficacy for reliable observations and fitting. EC50 valuesfor the two cytisine as well as the three NS3861 fitted graphs have overlapping 95% confidence intervals, respectively.

FIGURE 5. Maximal efficacies of cytisine and NS3861 at wild-type and chimeric nAChRs. Patch clamp data for cytisine and NS3861 were obtained asdescribed in the legends to Figs. 2– 4 and maximal compound efficacy values are reported from the most efficacious concentrations as described in the legendto Fig. 3. The horizontal dotted lines indicate the 10% level set as minimum for reliable efficacy detection. A, cytisine is a full agonist at receptors containingfull-length �4-subunits, a partial agonist with �4/�2-subunits, and has no efficacy at receptors containing a �2-LBD. B, NS3861 is a full agonist at receptorscontaining the LBD of �3�2, a partial agonist with the LBD of �3/�4, and has no efficacy at �4-containing receptors. Values are expressed as mean � S.D. of n �3–14 cells for each subunit combination.




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(7, 8, 15). Furthermore, even though binding to �4�4 occurswith �250-fold higher affinity compared with �3�4, the effi-cacy selectivity of cytisine is independent of the identity of the�-subunit. Importantly, cytisine displayed partial efficacyresulting in peak currents up to �30% at receptors containingthe �4/�2-chimera clearly indicating a role of the TMD indetermining maximal efficacy of this compound.Maximal efficacy of agonists at �4�2 receptors have pre-

viously been shown to be dependent on receptor stoichiom-etry (21). However, concentration-response data obtained inoocytes expressing uniform populations of either (�4)3(�2)2or (�4)2(�2)3 receptors lead to the conclusion that the pres-ence of an �4�4 interface has no or very limited effect on cyti-sine receptor activation (Table 2).In agreement with our previous observations on determi-

nants of efficacy at nAChRs (14), induced-fit docking of cytisineto homologymodels of the�4�2 and�4�4 interfaces suggests alarger van der Waals contact area and thus better interactionsto the �4- versus �2-subunit as an explanation for the �4-selec-tive activation. The only differences in the agonist binding sitebetween�2- and�4-subunits areVal136 andPhe144 versus Ile134and Leu142, respectively (Fig. 6A). The longer side chain of Ileversus Val and the nonplanar nature of Leu versus Phe allow alarger van der Waals contact surface between the complemen-

tary subunit and cytisine in �4- versus �2-containing receptors(Fig. 6B). Because cytisine is relatively short compared withmany other agonists, e.g. NS3861 and epibatidine, it may beimperative specifically for cytisine that the binding site residuesof the complementary subunit are long, flexible and hydropho-bic to make intimate van der Waals contacts and result inreceptor activation. This hypothesis is supported by oocyte datain a receptor containing a mutant �2-subunit with Val136 andPhe144 point mutated to Ile and Leu, respectively. In thisexpression system, cytisine efficacy approaches the levelobserved for �4�4 and although not comparable with the fullactivation observed in patch clamp, it is in sharp contrast to thelack of activation of wild-type �4�2 in oocytes.The novel nAChR agonist NS3861 presents a selectivity pro-

file distinct from that of cytisine. Our patch clamp experimentsshow the nature of the�-subunit to be crucial forNS3861 activ-ity. In addition, efficacy levels were determined by the nature ofthe �-subunit in a manner reciprocal to that of cytisine (i.e. �2over �4). Full activation was seen at �3�2 receptors, partialefficacy at �3�4 receptors, and only insignificant peak currentsat �4�2 receptors. As NS3861 displays a binding affinity in thenanomolar range on �4�2 receptors, the lack of activation at�4-containing receptors clearly does not reflect lack of binding.Furthermore, it is also independent of the presence of an �4�4

FIGURE 6. Comparison of binding site residues and binding modes of cytisine and NS3861 as obtained from induced-fit docking in the nAChRhomology models. A, sequence alignment of binding site residues (on red background) in �3-, �4-, �2-, and �4-subunits as defined by residues within 5 Å ofthe agonist included in the models and with side chains pointing toward it. B, comparison of obtained binding modes of cytisine in �4�2 (green carbons) and�4�4 (cyan carbons). C, comparison of obtained binding modes of NS3861 in �3�2 (purple carbons) and �3�4 (magenta carbons). D, comparison of the obtainedbinding modes of NS3861 in �3�2 (purple carbons) and �4�2 (orange carbons). The overall structure of the homology models and residues that are identical inthe compared models are represented as a white cartoon and sticks, respectively. Yellow dashed lines indicate hydrogen bonds and red dashed lines indicatesteric clashes. The water molecule is represented as a small red sphere.




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interface. Irrespective of �- and �-subunit stoichiometry,NS3861 shows no efficacy at �4�2 receptors and only marginalefficacy at �4�4 receptors expressed in oocytes. Efficacy selec-tivity for �3�2 versus �3�4 was also qualitatively confirmed inoocytes under conditions where both populations contain an�3�3 interface, which indicates that the maximal efficacy isindependent of this interface. Additionally, contrary to cytisinethe results from chimeric receptor combinations show that theefficacy of NS3861 was independent of TMD identity in thesestudies.Structurally, the observed �3-selectivity of NS3861 might

appear surprising as the only difference in the agonist bindingsite between the �3- and �4-subunit is Ser181 versus Thr183,respectively (Fig. 6A). However, in our obtained bindingmodesthe orientation of the thiophene ring of NS3861 is almost per-pendicular to the pyridine ring of, for example, NS3531 ornicotine in co-crystal structures with a homologous AChBP(14, 41), presumably due to the presence of the bromo substit-uent. This puts the agonist under the influence of Thr183 in �4as evident by the�1Å shift in bindingmode in�4�2 comparedwith �3�2 as measured on the thiophene sulfur and the bro-mine atoms (Fig. 6D). Because the docking protocol used canonly account for side chain flexibility, the full consequences interms of backbone conformational changes in the receptor areuncertain. However, the shift in binding mode of NS3861,caused by Thr183 of �4, brings the bromine within 3.4 Å of oneof the sulfur atoms in the disulfide bond of the C-loop andwithin 3.1 Å of one of themethyl groups of Leu146 and Leu144 inthe �2- and �4-subunits, respectively (Fig. 6D). Such short dis-tances correspond to steric clashes between agonist and recep-tor. This could indicate that the shifted binding mode ofNS3861 in �4-containing receptors opposes channel activationby hindering a conformational change of the interface into theactivated state, by preventing either a necessary agonist-medi-ated intimate contact between the �4- and the �-subunitand/or preventing full closure of the C-loop. This is backed bythe observation that, whereas NS3861 was unable to activatewild-type �4�2 receptors it displays agonistic behavior at�4(T183S)�2 mutant receptors in oocytes. Although theobtained efficacy level in these experiments did not match thatof wild-type �3�2 it still demonstrates that Thr183 is, at leastpartly, responsible for the lack of NS3861 efficacy on �4-con-taining receptors.Consistent with the poor hydrogen bond acceptor ability of

the thiophene sulfur atom we do not observe an interaction tothe water molecule, as we do for cytisine (Fig. 6B) and, whichhas been reported for other agonists (12, 14, 41). The fact thatNS3861 is a full agonist at �3�2 indicates that, in contrast to�4�2 agonists (42), a hydrogen bond acceptor is not essentialfor full activation of �3�2 receptors. This seems to be consist-ent with the situation in �7 nAChRs where a co-crystal struc-ture of AChBP with an �7-selective agonist displays a bindingmode, which does not involve interaction with the water mol-ecule (12). This is also consistent with superior binding affinityat �3- versus �4-containing receptors.

Aswas the case for cytisine, the�-subunit plays an importantrole for NS3861 efficacy but we observe almost the reverse�-subunit selectivity profile with preference for �2. To the best

of our knowledge, NS3861 represents the first agonist reportedto possess efficacy selectivity for the �3�2 nAChR subtype.Based on the obtained binding modes at the �3�2 and �3�4interfaces we do not observe a specific interaction as an obviousreason for the �2-efficacy selectivity of NS3861. Given thatbinding affinity at �2-containing receptors is �10-fold lowerthan that at �4-containing receptors, this is perhaps not sur-prising. From the proximity of the bromine atom of NS3861and Phe144 of �2 (Fig. 6C) we propose an electrostatic interac-tion between the negative electrostatic potential around thecircumference of the bromine and the positive electrostaticpotential at the edge of the phenyl group in the phenylalanine(43). Because polarization of aromatic rings is not explicitlyhandled in forcefield-based methods, such an interaction can-not be expected as part of the output from the induced-fit dock-ing. However, our binding model of NS3861 in �3�2 requiresonly a small conformational change of Phe144 to establish thiselectrostatic interaction, which is not possible with Leu142 inthe �4-subunit and could be the reason for the selectiveactivation.The results obtained with cytisine and NS3861 corroborate

previous reports that both the �- and �-subunits take part indetermining the binding affinity in heteromeric receptors (3, 4).The data are also consistent with previous observations thatboth subunits can affect agonist efficacy (7, 8). However, byevaluating specific binding site determinants that make con-tacts to these agonists, a more detailed picture emerges. Thedata presented here for cytisine are perfectly consistent with ahypothesis stating that the �-subunit mostly plays a requisiterole for agonist efficacy, in the sense that it provides a set of keydeterminants essential for agonist binding. Positioning of thepositive charge of the ligand inside the aromatic nest deliveredmostly by the �-subunit provides an excessive binding poten-tial, which is necessary for high affinity binding, but not suffi-cient for gating. Ligand interactionswith residues on the�-sub-unit are much weaker and thus play a less important role inagonist binding. However, because inter-subunit contacts aresuggested to determine agonist efficacy (14) these weaker�-subunit interactions have a decisive role in fine-tuning ago-nist efficacy.As illustrated by our homology models and binding modes

derived from docking, both �3- and �4-subunits are capable ofbinding cytisine in a manner permissive for gating. However,despite a large difference in binding affinity, maximal efficacyends up being fully determined by the �-subunit. Comparedwith other nicotine-like agonists, such as epibatidine andNS3920 (14), cytisine is �2 and �3 Å shorter, respectively,measured from the cationic nitrogen to the most distal atom,and only the presence of Ile and Leu in �4 allows for sufficientvan der Waals contact surface when it is docked in the model.Likewise, the�3-subunit is capable of bindingNS3861 in a “cor-rect” manner, but partial versus full agonism is determined bythe �-subunit possibly through interactions with Phe144 in �2.

The fact that NS3861 does not activate �4-containing recep-tors may seem contradictory to the hypothesis of a requisiterole for the �-subunit. However, the fact that Thr183 in �4 pre-vents activation can be explained by the shift inNS3861 bindingmode observed from our docking results, which may prevent




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NS3861 from binding in a manner permissive for gating. Thus,despite high affinity binding through the key �-determinants,no �-subunit can adapt sufficiently to give efficacy.Recent publications have claimed that partial agonist efficacy

is due to prevention of full C-loop closure (11–13) and thusoriginates from interactions with the principal component ofthe binding site, i.e. the �-subunit. Although the �3 efficacyselectivity of NS3861 could potentially also be explained bychanges in C-loop closure, the remaining results obtained inthis work are inconsistent with this hypothesis. Cytisine fullyactivates both �3�4 and �4�4 receptors but results in virtuallyno efficacy at�3�2 and�4�2 receptors (Fig. 5). As seen, this canbe rationalized by specific interactions between cytisine andresidues from the �-subunit. In contrast, the C-loop closurehypothesis would imply that the specific combination of cyti-sine and �2 should prevent closure of the �3- and �4- C-loops.However, docking of cytisine to both�3�2 and�4�2 homologymodels show that in both cases a fully closedC-loop can accom-modate this relatively small agonist without significant stericclashes. Furthermore, a very recent co-crystal structure of aAChBP with cytisine confirms our obtained binding mode andshows a fully closed C-loop (44). In fact, two additional crystalstructures of AChBPs in complex with partial agonists, lobeline(13) and varenicline (44), also display full C-loop closure. Thus,the present data and thementioned three AChBP crystal struc-tures support the study by Rohde et al. (14) in which it is con-cluded that C-loop closure is not enough to explain differencesin maximum efficacies and that agonist interactions with keybinding site residues at the �-subunit plays a decisive role incontrolling efficacy levels.Another inference from the present data is thatmaximal effi-

cacy can be affected by the nature of the �-subunit TMD.Although that may seem trivial, the influence is clearly highlyagonist dependent. All the data obtained here were normalizedto maximal ACh-evoked currents. Given that the maximal effi-cacy levels of NS3861 at receptors with the same �3- and�-LBD (e.g. �3�2 and �3 � �2/�4) were virtually identical,maximal efficacy of ACh and NS3861 do not differ relative toeach other. ACh and NS3861 thus have either identical or no�-subunit TMDdependence. Cytisine on the other hand clearlywent from a full to a partial agonist when a wild-type �4-sub-unit was replaced with a �4/�2-subunit. Although this under-scores the importance of the�-subunit for determining efficacyit also suggests that �2-TMD containing receptors may bemore difficult to gate and require particularly strong interac-tions to the complementary subunit, which cytisine may not becapable of even with a �4-LBD. The observations that �2-con-taining receptors generally desensitize more rapidly than�4-containing receptors is also consistent with such basic gat-ing differences. No effects of LBD/TMDchimeraswere seen forthe �-subunits as long as the LBD remained constant, which infact support the hypothesis of the requisite role of thesesubunits.The gatingmechanismof nAChRs is still not fully elucidated,

but the data presented here points toward an important role ofthe �-subunit, which potentially involves global changes of�-subunit conformation. Such changes may be linked to the

F-loop movement in the complementary subunit previouslysuggested as part of the activation mechanism (45).

Acknowledgments—We thank Anne B. Fisher, Kirsten V. Hauge-gaard, Lene G. Larsen, and Camilla Irlind for expert technicalassistance.

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Kastrup and Philip K. AhringThomas Balle, Michael Gajhede, Jette S. Dyhring, Elsebet Ø. Nielsen, Dan Peters,Timmermann, Marianne L. Jensen, Tino Kasper Harpsøe, Helle Hald, Daniel B. ReceptorsHeteromeric Nicotinic Acetylcholine the Novel Compound NS3861 atSubtype-selective Efficacies of Cytisine and Molecular Determinants ofProtein Structure and Folding:

doi: 10.1074/jbc.M112.436337 originally published online December 10, 20122013, 288:2559-2570.J. Biol. Chem.

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ВІСНИК ФАРМАЦІЇ 2(78)201468 ISSN 1562-7241

Recommended by Doctor of Pharmacy, professor O.A.Ruban

UDC 339.13:658.628:178.7 (477)

ANALYSIS OF THE DRUG ASSORTMENT FOR TREATING NICOTINE DEPENDENCE PRESENTED AT THE PHARMACEUTICAL MARKET OF UKRAINEM.M.Kobets, Yu.M.КоbetsNational University of PharmacyKey words: marketing research; аassortment; medicines for treating nicotine dependence; nicotine addiction

The analysis of the drug assortment for treating nicotine dependence presented at the Ukrainian pharmaceutical market has been carried out according to the ATC-classification and countries-producers with the purpose of providing the population with effective, qualitative and safe medicines. Two types of therapy are used for nicotine addiction: nicotine replacement therapy and therapy with the use of medicines, which do not contain nicotine. In the process of our investigations it has been found that 11 trade names (TD) of medicines for treating nicotine dependence (MTND) have been registered in Ukraine. Among them there are 8 nicotine-containing drugs, 1 medicine contains cytisine, 1 includes varenicline and 1 is a homeopathic medicine produced by 5 countries. It has been determined that the majority of medicines for treating nicotine dependence are presented by foreign producers at the Ukrainian market. The majority of the drug assortment for treating nicotine dependence is imported drugs of such pharmaceutical companies as McNeil, AB (Sweden); Novartis Consumer Health, S.A. (Switzerland); Sopharma (Bulgaria), Pfizer (the USA). The analysis of medicines for treating nicotine dependence has been carried out by medicinal forms. Medicines for treating nicotine dependence are presented at the market of Ukraine in such forms as chewing gums, tablets, transdermal plasters, granules. According to the maximum indexes received the assortment contour of the target segment of the Ukrainian pharmaceutical market of MTND (macrocontour) has been developed. The macrocontour of the target market segment can be used for studying the assortment of a particular chemist’s shop with the purpose of possibility of the assortment extension.

Harmful consequences of tobacco use have been al-ready proven. But there are a lot of people, who either do not know about them or do not want to give up this bad habit or can not do it. Sociological investigations testify that the major part of smokers (60-70%) are try-ing to give up this bad habit. However, according to the narcologists’ opinion, nicotine addiction may be as strong as heroin addiction [2, 15].

Smoking consequences represent a great danger to humanity even more than AIDS, tuberculosis, maternal mortality, traffic accidents, suicides and murders taken together [5, 9].

Due to predictions of scientists at the University of Edinburgh (Britain) if nicotine consumption does not change the present rate, this figure will reach 10 million by 2030. Totally nicotine will kill about one billion of people in the XXI-st century [3].

According to the World Health Organization, Ukraine sets the 17-th place among all countries as to nicotine consumption. It is 1.5% of all cigarettes in the world, while the population of Ukraine is not more than 0.85% of the Earth population. There are about 9 million of active smokers in Ukraine. They are the third of all population capable to work. This is a great number of people, their health can be improved without material costs involvement by having influence only on one fac-tor – smoking. Therefore, nicotine control is one of the major problems in our country [8, 14, 15].

One of the ways to help smokers to get rid of nico-tine dependence is taking medicines for treating nico-tine dependence (MTND). It is reasonable to conduct the marketing research of the MTND market segment. The results of these investigations give the opportunity to determine availability and the level of providing with such medicines, as well as the tendency for market de-velopment of the medicines under research.

Among the scientific works closely connected with the research direction, there are significant works of such scientists as Z.M.Mnushko [7], I.V.Pestun [7], V.K.Smir- nov [11], O.I.Yermolova [11], O.I.Speranska [11], S.K.Zy- ryanov [15], Yu.B.Belousov [15] and others.

The aim of this work is to research the domestic mar-ket of MTND for further study of availability of these medicines and possibility of demand satisfaction in nico-tine dependence treatment.

Materials and MethodsMonitoring over the situation at the market has been

conducted on the basis of official sources content analysis as to drugs, which help to give up smoking: the State Register of medicines of Ukraine, Compendium 2013, Rx-Index classifier of drugs.

Results and DiscussionNicotine is the major smoking pathological factor.

It is a colourless and odourless alkaloid, which is part of Solanaceous family, mainly tobacco [4]. The name “nicotine” comes from the Latin word “nicotina tabacum”

NEWS OF PHARMACY 2(78)2014 69ISSN 1562-7241

received its name from the family name of a French am- bassador Jean Nicot, who brought tobacco seeds and leaves to France in the middle of the ХVI-th century [6, 13].

At present the Ukrainian drug market is presented by domestic and foreign producers of MTND. But the choice must be determined by the convenience of a me-dicinal form, economic availability and efficiency for a specific patient. Two types of therapy are used for nico-tine addiction: nicotine replacement therapy and therapy with the use of medicines, which do not contain nicotine.

The main conception based on the stage-by-stage analysis of the MTND assortment has been used ac-cording to the following criteria: the ATC-classification (anatomic-therapeutic-chemical classification), medicinal forms, country and company-producer [1, 10]. Ac-cording to the results of analysis the assortment mac-rocontour of the target market segment has been com-posed in order to satisfy the demand in medicines for

treating nicotine dependence. The period of analysis was 2013.

In total 11 trade names (TD) of medicines for treating nicotine dependence have been selected during the con-tent analysis [10, 12]. Among them there are 8 nicotine-containing drugs (72.7%), 1 medicine contains cytisine (9.1%), 1 includes varenicline (9.1%) and 1 is a homeo-pathic medicine (9.1%) produced by 5 countries (Fig. 1).

About 3/4 of medicines at the market are nicotine-containing drugs, which are represented by foreign pro-ducers and manufactured in two medicinal forms: 75% nicotine-containing drugs in the form of a chewing gum, 25% – as a transdermal plaster.

During the analysis it has been found that the most MTND at the Ukrainian market are presented by foreign producers (91%).

The majority of the drug assortment for treating nicotine dependence is imported drugs of such pharmaceu-

Fig. 1. The Ukrainian market of medicines for treating nicotine dependence by active substances.

TableThe firm structure of the Ukrainian market of drugs for treating nicotine dependence

Сountry Manufacturer Trade name of the drugThe number of trade

names Medicinal formАbs. Share, %

Nicotine replacement therapySweden McNeil, AB Nicorette with a mint flavour

6 54.5

chewing gumNicorette with the taste of fresh mint chewing gumNicorette, winter mint chewing gumNicorette with the taste of fresh fruit chewing gumNicorette, flavoured mint chewing gumNicorette transdermal patch transder-mal patch

Switzerland Novartis Consumer Health, S.A.

Nicotinell with a mint flavour2 18.2

chewing gumNicotinell transdermal patch transder-mal patch

Medicines that contain no nicotineBulgaria Sopharma Tabex (cytisine) 1 9.1 tabletsUSA Pfizer (USA) Champix (varenicline) 1 9.1 tabletsUkraine “National Homoeo-

pathic Society” JSCTabacum-Plus (varenicline)

1 9.1 granules

Total: 11


tical companies as McNeil, AB (Sweden) – 54.5%; No- vartis Consumer Health, S.A. (Switzerland) – 18.2%; So- pharma (Bulgaria) and Pfizer (the USA) – 9.1% (Table).

Distribution of medicines registered for treating nicotine dependence among the countries-producers is shown in Fig. 2. Unfortunately, the share of a domestic pro-ducer is only 9.1%.

Medicines for treating nicotine dependence are presented at the Ukrainian market in such medicinal forms as tablets, chewing gums, transdermal plasters, granules. Nicotine-containing drugs are produced in the form of chewing gums (54.5%) and transdermal plasters (18.2%) and provide modified nicotine release. Other medicines are produced in the form of tablets (18.2%) and granules (9.1%) (Table).

According to results of the analysis the assortment contour (macrocontour) of the target segment of the Ukrainian pharmaceutical market has been developed taking into account the maximum index-characteristics [1] (Fig. 3).

Information received on the basis of the macrocon-tour allows to form the chemist’s assortment policy de-pending on the available proposal and demand for these medicines.

Thus, in the process of our research it has been found that foreign MTND are 91%, the majority of foreign medi-cines are nicotine-containing drugs (72.7%), among them 54.5% of medicines are presented in the form of a chewing gum and are produced by Swedish companies-producers.

The marketing data of the domestic market analysis of medicines for treating nicotine dependence are the basis for further prediction and can be used by chemist’s and pharmaceutical companies with the purpose of develop-ment of variants for their strategy of development, goods assortment and the influence on the consumers’ behaviour.

CONCLUSIONS1. The assortment of MTND registered at the Uk-

rainian market has been analyzed according to the ATC- classification. Eleven trade names of medicines for treating nicotine dependence have been registered in Ukraine.

Fig. 2. The structure of the commodity market of drugs treating nicotine dependence registered in Ukraine by countries-producers, %.

Fig. 3. The assortment macrocontour of the target segment of the pharmaceutical market of MTND.

NEWS OF PHARMACY 2(78)2014 71ISSN 1562-7241

2. The assortment of MTND available in Ukraine has been investigated by countries-producers. The ma-jority of MTND at the market of Ukraine is presented by foreign producers (91%).

3. Analysis of MTND has been carried out by me-dicinal forms. Medicines for treating nicotine dependence are presented at the market of Ukraine in such forms as chewing gums (54.5%), tablets (18.2%), trans-dermal plasters (18.2%), granules (9.1%).

4. According to the maximum indexes received the assortment contour of the target segment of the Ukrainian pharmaceutical market of MTND (macrocontour) has been developed. The macrocontour of the target market segment can be used for studying the assortment of a particular chemist’s shop with the purpose of possibility of the assortment extension.

It is reasonable to carry out further investigations for studying MTND availability.

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2. Golichenko M. Canadian HIV/AIDS Legal Network. – 2011. – P. 8-13.3. Holtfreter B. // J. of Clin. Periodontol. – 2010. – Vol. 37. – Р. 211-219.4. Kolassa E. // J. of Pharmac. Markеting and Management. – 2002. – Vol. 14, №3-4. – P. 101-108.5. Malhotra S. // J. of Pharmac. Marketing and Management. – 2004. – Vol. 16, №4. – P. 97-106.6. Martsevich S.U., Lukina U.V. // [Electronic resource]: – Режим доступа: http://elibrary.ru/item.asp?id=187471557. Mnushko Z.N., Pestun I.V. Theory and practice of marketing investigations in pharmacy. – Kharkov: publ.

NPhaU, 2008. – P. 15-22.8. Niklaus P. Lang // J. of Clin. Periodontol. – 2009. – Vol. 36. – Р. 3-8.9. Philip M. Preshaw // J. of Clin. Periodontol. – 2009. – Vol. 36. – Р. 1-2.10. Rx-index. Classifier of medical preparations.[Electronic resource]: – Regime of access :http://www.rxindex.

com.ua/atc#N07BA11. Smirnov V.K., Ermolova O.I., Speranskaya O.I. // Narcol. – 2010. – №6. – P. 36-39.12. State register of medicines [Electronic resource]: – Regime of access: http://www.drlz.Kiev.ua/13. Tonnesen P. // JAMA. – 2003. – Vol. 269. – P. 1268-1271.14. Velitchka D., Weitz B. // J. of Marketing. – 2006. – Vol. 70, №1. – P. 48-56.15. Ziryanov S.K., Belousov U.B. // Preventive Medicine. – 2010. – Vol. 13, №6. – P. 21-23.

АНАЛІЗ АСОРТИМЕНТУ ПРЕПАРАТІВ, ЩО СПРИЯЮТЬ ВІДМОВІ ВІД ТЮТЮНОПАЛІННЯ, ПРЕДСТАВЛЕНИХ НА ФАРМАЦЕВТИЧНОМУ РИНКУ УКРАЇНИМ.М.Кобець, Ю.М.КобецьКлючові слова: маркетингові дослідження; асортимент; препарати, що сприяють відмові від тютюнопаління; нікотинова залежність З метою забезпечення доступності населення до ефективних, якісних та безпечних лікарсь- ких засобів проведено аналіз асортименту представлених на фармацевтичному ринку Украї- ни препаратів, що сприяють відмові від тютюнопаління, за АТС-класифікацією та країна-ми-виробниками. При нікотиновій залежності використовують 2 види терапії: нікотинозамісну терапію і терапію з використанням лікарських засобів, що не містять нікотину. У ході до-сліджень встановлено, що в Україні зареєстровано 11 торгових назв препаратів для лікуван-ня нікотинової залежності, серед яких 8 нікотиновмісних препаратів, (1 препарат містить цитизин, 1 – вареніклін та 1 гомеопатичний препарат), що випускаються 5 країнами-ви-робниками. Встановлено, що більшість препаратів, які сприяють відмові від тютюнопа-ління, на ринку України представлені іноземними виробниками. Більшість асортименту лі-карських препаратів для лікування нікотинової залежності складають імпортні препарати фармацевтичних компаній McNeil, AB (Швеція), Novartis Consumer Health, S.A. (Швейцарія), Sopharma (Болгарія), Pfizer (США). Проведено аналіз препаратів, що сприяють відмові від тютюнопаління, за лікарськими формами. Лікарські засоби для лікування нікотинової залеж-ності представлені на ринку України у вигляді таких лікарських форм, як гумки жувальні, таблетки, пластирі трансдермальні та гранули. За отриманими максимальними показни-ками розроблений асортиментний контур цільового сегменту фармацевтичного ринку пре-паратів, що сприяють відмові від тютюнопаління (макроконтур). Макроконтур цільового сегменту ринку може бути використаний для вивчення асортименту окремої аптеки (мікро-контур) з метою можливості поповнення асортиментних портфелів.


АНАЛИЗ АССОРТИМЕНТА ПРЕПАРАТОВ, СПОСОБСТВУЮЩИХ ОТКАЗУ ОТ ТАБАКОКУРЕНИЯ, ПРЕДСТАВЛЕННЫХ НА ФАРМАЦЕВТИЧЕСКОМ РЫНКЕ УКРАИНЫМ.Н.Кобец, Ю.Н.Кобец Ключевые слова: маркетинговые исследования; ассортимент; препараты, способствующие отказу от табакокурения; никотиновая зависимостьС целью обеспечения доступности населения к эффективным, качественным и безопас-ным лекарственным средствам проведен анализ ассортимента препаратов, способствую-щих отказу от табакокурения, представленных на фармацевтическом рынке Украины, по АТС-классификации и странам-производителям. При никотиновой зависимости использу-ют 2 вида терапии: никотинозаместительную терапию и терапию с использованием ле-карственных средств, не содержащих никотина. В ходе исследования установлено, что в Украине зарегистрировано 11 торговых названий препаратов для лечения никотиновой за-висимости, среди которых 8 никотинсодержащих препаратов (1 препарат содержит ци- тизин, 1 – варениклин и 1 гомеопатический препарат), которые выпускаются 5 страна-ми-производителями. Установлено, что большинство препаратов, которые способствуют отказу от табакокурения, на рынке Украины представлены иностранными производите-лями. Большинство ассортимента лекарственных препаратов для лечения никотиновой зависимости составляют импортные препараты фармацевтических компаний Mcneіl, AB (Швеция), Novartіs Consumer Health, S.A. (Швейцария), Sopharma (Болгария), Pfіzer (США). Проведен анализ препаратов, которые способствуют отказу от табакокурения, по лекар-ственным формам. Лекарственные средства для лечения никотиновой зависимости пред-ставлены на рынке Украины в виде таких лекарственных форм, как резинки жевательные, таблетки, пластыри трансдермальные и гранулы. По полученным максимальным показа-телям разработан ассортиментный контур целевого сегмента фармацевтического рынка препаратов, способствующих отказу от табакокурения (макроконтур). Макроконтур це-левого сегмента рынка может быть использован для изучения ассортимента отдельной аптеки (микроконтур) с целью возможности пополнения ассортиментных портфелей.

Accepted Article Preview: Published ahead of advance online publication

Varenicline and Cytisine Diminish the Dysphoric-Like State

Associated with Spontaneous Nicotine Withdrawal in Rats

Moe Igari, Jon C Alexander, Yue Ji, Xiaoli Qi, Roger LPapke, Adrie W Bruijnzeel

Cite this article as: Moe Igari, Jon C Alexander, Yue Ji, Xiaoli Qi, Roger L Papke,

Adrie W Bruijnzeel, Varenicline and Cytisine Diminish the Dysphoric-Like State

Associated with Spontaneous Nicotine Withdrawal in Rats, Neuropsychopharma-

cology accepted article preview 21 August 2013; doi: 10.1038/npp.2013.216.

This is a PDF file of an unedited peer-reviewed manuscript that has been accepted

for publication. NPG are providing this early version of the manuscript as a service

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Received 27 June 2013; revised 7 August 2013; accepted 11 August 2013;Accepted article preview online 21 August 2013

© 2013 Macmillan Publishers Limited. All rights reserved.

1

Varenicline and cytisine diminish the dysphoric-like state associated with

spontaneous nicotine withdrawal in rats

Moe Igari1, Jon C. Alexander

1, Yue Ji

1, Xiaoli Qi

1, Roger L. Papke

2, Adrie W. Bruijnzeel

1

1 Department of Psychiatry,

2 Department of Pharmacology and Therapeutics, University of

Florida, Gainesville, Florida 32611, USA

Running title: Varenicline and cytisine reduce nicotine withdrawal

Keywords: Nicotine, varenicline, cytisine, withdrawal, ICSS, rats

Correspondence should be addressed to:

Adrie W. Bruijnzeel, PhD, University of Florida, Department of Psychiatry, McKnight Brain

Institute, 1149 Newell Dr, Gainesville, FL 32611, Email: [email protected]. Phone: (352) 294-

4931, Fax: (352) 392-8217


2

Abstract

Tobacco addiction is characterized by a negative mood state upon smoking cessation and relapse

after periods of abstinence. Clinical studies indicate that negative mood states lead to craving and

relapse. The partial α4/α6/β2* nicotinic acetylcholine receptor (nAChR) agonists varenicline and

cytisine are widely used as smoking cessation treatments. Varenicline has been approved in the

US for smoking cessation and cytisine is used in Eastern European countries. Despite the

widespread use of these compounds very little is known about their effects on mood states. These

studies investigated the effects of varenicline, cytisine, and the cytisine-derivative 3-(pyridin-3’-

yl)-cytisine (3-pyr-Cyt) on brain reward function in nicotine-naïve and nicotine withdrawing rats.

The cytisine-derivative 3-pyr-Cyt is a very weak α4β2* nAChR partial agonist and like cytisine

and varenicline has antidepressant-like effects in animal models. The intracranial self-stimulation

(ICSS) procedure was used to investigate the effects of these compounds on brain reward

function. Elevations in ICSS thresholds reflect a dysphoric state and a lowering of thresholds is

indicative of a potentiation of brain reward function. It was shown that acute administration of

nicotine and varenicline lowered ICSS thresholds. Acute administration of cytisine or 3-pyr-Cyt

did not affect ICSS thresholds. Discontinuation of chronic, 14-days, nicotine administration led

to elevations in ICSS thresholds that lasted for about 2 days. Varenicline and cytisine, but not 3-

pyr-Cyt, diminished the nicotine withdrawal-induced elevations in ICSS thresholds. In

conclusion, these studies indicate that varenicline and cytisine diminish the dysphoric-like state

associated with nicotine withdrawal and may thereby prevent relapse to smoking in humans.


3

INTRODUCTION

Tobacco products are highly addictive and smoking cessation leads to dysphoria, craving, and

impaired cognitive function (American Psychiatric Association 2000; Bruijnzeel 2012).

Extensive evidence indicates that nicotine is the main psychoactive component of tobacco that

leads to addiction (Stolerman and Jarvis 1995). Nicotine mediates its psychoactive effects by

stimulating nicotinic acetylcholine receptors (nAChRs) in the brain. The nAChRs are pentameric

ligand-gated ion channels. In the mammalian brain, the heteromeric nAChRs consist of α2-6 and

β2-4 subunits and the homomeric nAChRs consist of α7 subunits (Dani and Bertrand 2007;

Leslie et al. 2013). Nicotine mediates its rewarding effects at least partly by activating

α4/α6/β2*, α3β4*, and α7 nAChRs (Liu et al. 2012; Picciotto and Kenny 2013; Jackson et al.

2013; Toll et al. 2012; Markou and Paterson 2001). Cessation of nicotine administration leads to

negative affective (dysphoric- and anxiety-like behavior) and somatic withdrawal signs (Epping-

Jordan et al. 1998). Clinical and preclinical studies suggest that relapse to smoking is mainly due

to craving and negative affective withdrawal signs (Bruijnzeel 2012; Koob and Volkow 2010).

There is extensive evidence for a role of α4β2* nAChRs in dysphoric- and anxiety-like behavior

associated with nicotine withdrawal (Picciotto and Kenny 2013; Changeux 2010).

During the last decade, a great deal of progress has been made towards the development

of treatments for tobacco addiction. Smoking cessation treatments target non-cholinergic

receptors (e.g., clonidine), a combination of cholinergic and non-cholinergic receptors (e.g.,

bupropion), or only cholinergic receptors (e.g., varenicline and cytisine)(Gourlay et al. 2004;

Slemmer et al. 2000; Jorenby et al. 2006). In the present studies, we evaluated the effects of three

structurally related cholinergic receptor agonists (cytisine, varenicline, and 3-(pyridin-3’-yl)-


4

cytisine [3-pyr-Cyt]) on nicotine withdrawal in rats. Both varenicline and 3-pyr-Cyt were

developed by modifying the chemical structure of cytisine. Varenicline was discovered while

searching for more potent and efficacious α4β2* nAChR partial agonists and 3-pyr-Cyt was

discovered during a search for more selective α4β2* nAChR partial agonists (Coe et al. 2005;

Mineur et al. 2009). Cytisine has been used as a smoking cessation aid in Eastern European

countries since the 1960s (Etter 2006; West et al. 2011). Cytisine is a partial agonist at α4β2*

and α6β2* nAChRs and a full agonist at α3β4* and α7 nAChRs (Salminen et al. 2004; Grady et

al. 2010). The cytisine derivative varenicline is a US FDA approved smoking cessation treatment

and activates the same nAChRs as cytisine (Grady et al. 2010; Mihalak et al. 2006). Cytisine and

varenicline have a similar efficacy at α4β2*, α3β4*, and α7 rodent nAChRs but cytisine is more

efficacious at α6β2* nAChRs (Grady et al. 2010; Salminen et al. 2004).

Some preclinical studies suggest that cytisine has rewarding effects. Cytisine is self-

administered by mice, induces conditioned place preference (CPP), and repeated administration

of cytisine leads to a sensitized locomotor response in rats (Museo and Wise 1994a; Museo and

Wise 1994b; Rasmussen and Swedberg 1998). Drug discrimination studies indicate that cytisine

partially generalizes (i.e., substitutes) for nicotine (Stolerman et al. 1984; LeSage et al. 2009).

Few studies have investigated the rewarding effects of varenicline. One study showed that low,

but not high, doses of varenicline induce a leftward shift in a rate-frequency curve-shift ICSS

procedure, which suggests that varenicline has rewarding effects (Spiller et al. 2009). In contrast,

varenicline does not induce CPP, which would suggest that varenicline does not have rewarding

properties (Biala et al. 2010).

Despite that cytisine and varenicline are widely used as treatments for smoking cessation;

it has not been investigated if varenicline and cytisine diminish the dysphoria associated with


5

nicotine withdrawal. The goal of these studies was to investigate the effects of cytisine and

varenicline on brain reward function in nicotine-naïve and nicotine withdrawing rats by using a

discrete-trial intracranial self-stimulation (ICSS) procedure. The acute administration of drugs of

abuse lowers ICSS thresholds which is indicative of a potentiation of brain reward function. In

contrast, drug withdrawal leads to elevations in ICSS thresholds, which reflects a dysphoric-like

state (Barr et al. 2002). In addition to the effects of cytisine and varenicline, the effects of the

cytisine derivative 3-pyr-Cyt on brain reward function was evaluated. This compound is a very

weak partial agonist at α4β2* nAChRs and inhibits acetylcholine-mediated responses on α4β2*

nAChRs expressed in oocytes (Mineur et al. 2009; Papke et al. 2010). In contrast to varenicline

and cytisine, 3-pyr-Cyt does not bind to α3β4* or α7 nAChRs and might therefore have a better

side effect profile (Mineur et al. 2009). Like varenicline and cytisine, 3-pyr-Cyt has

antidepressant-like effects in a variety of mouse models (Mineur et al. 2009).

MATERIAL AND METHODS

Subjects

Male Wistar rats (200-225 g, Charles River, Raleigh, NC) were used for the experiments. The

animals were housed (2 per cage) in a temperature and humidity-controlled vivarium and

maintained on a 12-h light-dark cycle (light off at 8 AM). All experiments were conducted

during the dark phase. All subjects were treated in accordance with the National Institute of

Health guidelines regarding the principles of animal care. Animal facilities and experimental

protocols were in accordance with the Association for the Assessment and Accreditation of


6

Laboratory Animal Care (AAALAC) and approved by the University of Florida Institutional

Animal Care and Use Committee.

Drugs

Nicotine and mecamylamine were purchased from Sigma-Aldrich (St. Louis, MO). Varenicline,

cytisine, and 3-pyr-Cyt were purchased from Tocris (Ellisville, MO). All drugs were dissolved in

sterile saline. The pH of the nicotine solution was adjusted to 7.0 immediately before the

subcutaneous (sc) injections. The drug 3-pyr-Cyt was dissolved by heating the mixture to 40°C.

All drugs were injected (sc) in a volume of 1 ml/kg of body weight. Nicotine doses are expressed

as free base and other drug doses are expressed as salt.

Surgical Procedures

The surgical procedures were conducted as described previously by our group (Bruijnzeel et al.

2012; Marcinkiewcz et al. 2009; Bruijnzeel et al. 2009). Electrode implantations. The rats were

anesthetized with isoflurane and prepared with electrodes (Plastic One, Roanoke, VA) in the

medial forebrain bundle (anterior-posterior -0.5 mm; medial lateral ±1.7 mm; dorsal-ventral -8.3

mm from dura). Osmotic minipump implantations and removal. Anesthetized rats were prepared

with minipumps (28-day, Durect Corporation, Cupertino, CA) that were filled with either saline

or nicotine. The nicotine concentration was adjusted for body weight and to deliver 3.16 mg/kg

of nicotine base per day.

Intracranial Self-Stimulation Procedure


7

Rats were trained on a modified discrete-trial ICSS procedure (Kornetsky and Esposito 1979), as

described previously (Markou and Koob 1992; Bruijnzeel et al. 2007). The operant conditioning

chambers were housed in sound-attenuating chambers (Med Associates, Georgia, VT). The

operant conditioning chambers had a response wheel centered on a sidewall and a photobeam

detector recorded the rotations. Brain stimulation was delivered by constant current stimulators

(Stimtek, Acton, MA). After the rats were trained, each test session provided an ICSS threshold

and response latency. The ICSS threshold was defined as the midpoint between stimulation

intensities that supported responding and current intensities that failed to support responding.

The response latency was defined as the time interval between the beginning of the non-

contingent stimulus and a positive response.

Experimental design

Experiments 1.1-1.4. Acute effect of nicotine (1.1), varenicline (1.2), cytisine (1.3), and 3-

pyr-Cyt (1.4) on brain reward function. Drug naïve rats were used for all experiments. Rats

were prepared with electrodes and trained on the ICSS procedure. When the ICSS thresholds

were stable (< 10% variation over 5-day period) the rats received nicotine (0, 0.03, 0.1, 0.3, 0.6

mg/kg, n=12), varenicline (0, 0.1, 0.3, 1, 3 mg/kg, n=9), cytisine (0, 0.1, 0.3, 1, 3, 5 mg/kg,

n=12) , or 3-pyr-Cyt (0, 0.3, 0.6, 0.9 mg/kg, n=13). Varenicline and 3-pyr-Cyt were

administrated 30 min before testing and cytisine and nicotine 15 min before testing. The drugs

were administered according to a Latin-square design. One dose of cytisine (5 mg/kg) and one

dose of varenicline (0.1 mg/kg) were added after the Latin-square to complete the dose response

curves. It was also investigated if the effects of varenicline on ICSS thresholds were mediated by

the activation of nAChRs. Therefore, the nAChR antagonist mecamylamine (3 mg/kg) was


8

injected 15 min before the administration of 0.3 mg/kg of varenicline. In all the experiments, the

minimum time interval between the drug injections was at least 72 h. All the tested drugs have

short half-lives in rats (nicotine t1/2=1.3 h; varenicline t1/2=4 h; cytisine t1/2=1.5 h;

mecamylamine t1/2=1.2 h)(Kyerematen et al. 1988; Rollema et al. 2010; Obach et al. 2006;

Debruyne et al. 2003). The half-life of 3-pyr-Cyt has not been reported. However, in previous

studies a wash-out period of 48 h (> 72 h in present study) was used between injections (Mineur

et al. 2009).

Experiments 2.1-2.3. Effect of varenicline (2.1), cytisine (2.2), and 3-pyr-Cyt (2.3) on brain

reward function in nicotine withdrawing rats. The rats were trained on the ICSS procedure

and when the ICSS thresholds were stable, the rats were prepared with nicotine or saline pumps.

The minipumps were removed after 14 days. The ICSS thresholds were assessed 6, 12, 24, 36,

48, 72, 83, 96, 120, 144 and 168 h after minipump removal. Varenicline (0.3 mg/kg, sc; saline-

pump/varenicline-injection [inj], n = 9; nicotine-pump/varenicline-inj, n = 13), 3-pyr-Cyt (0.3

mg/kg, sc; saline-pump/3-pyr-Cyt-inj, n = 9; nicotine-pump/3-pyr-Cyt-inj, n = 12) or saline

(saline-pump/saline-inj, n = 9; nicotine-pump/saline-inj, n = 14) were injected 30 min before

ICSS testing at the 12-72 h time points. Cytisine (3 mg/kg, sc; saline-pump/cytisine-inj, n = 10;

nicotine-pump/cytisine-inj, n = 12) was administrated 15 min before ICSS testing. The same

saline-pump/saline-inj and nicotine-pump/saline-inj groups served as control for experiments

2.1-2.3. The ICSS thresholds were assessed for an additional 4 days after discontinuing

varenicline, cytisine, or 3-pyr-Cyt administration.

Statistical analyses


9

The ICSS parameters were expressed as a percentage of the 3-day baseline before the first drug

injection (Expt.1) or before pump removal (Expt.2). For experiment 1, one-way repeated-

measures analyses of variance (ANOVA) were used to analyze the effects of nicotine,

varenicline, cytisine, and 3-pyr-Cyt on ICSS parameters. When the ANOVAs revealed

significant effects then Newman-Keuls post hoc tests were conducted. For experiment 2, paired

t-tests were conducted to investigate the effect of chronic (14 days) administration of nicotine or

saline on ICSS parameters (pre-pump implantation vs. pre-pump removal). Bonferroni corrected

t-tests were conducted to compare ICSS parameters between groups. Three-way ANOVAs (6-

168 h time points) were conducted to investigate the effects of varenicline, cytisine, and 3-pyr-

Cyt on spontaneous nicotine withdrawal with time as within-subjects factor and pump content

and drug as between-subjects factors. To investigate the effects of the drugs on nicotine

withdrawing rats or control rats (12-72 h) additional 2-way ANOVAs were conducted. Area

Under the Curves (AUCs) were analyzed with a 2-way ANOVA with pump content and drug as

between-subjects factors. After the ANOVA, all the saline groups were compared to the saline-

saline group and all nicotine groups to the nicotine-nicotine group by using the Dunnett’s

multiple comparison procedure (Dunnett 1955). Statistical analyses were performed using IBM

SPSS (version 21) for Windows software.

RESULTS

Experiments 1.1-1.4. Effect of nicotine, varenicline, cytisine, and 3-pyr-Cyt on ICSS

thresholds. The mean (±S.E.M.) absolute ICSS thresholds and response latencies (3-day

averages) before the administration of nicotine, varenicline, cytisine, and 3-pyr-Cyt are reported


10

in Table 1. Repeated administration of the drugs did not alter the pre-test day ICSS thresholds or

response latencies (data not shown). Nicotine (Expt. 1.1). There was a main effect of nicotine on

ICSS thresholds (F4,44=15.14, p<0.001, Figure 1a) and response latencies (F4,44=2.91, p< 0.05,

Figure 1b). Post hoc analyses indicated that the effect of nicotine on ICSS thresholds is

bidirectional, low doses of nicotine (0.1, 0.3 mg/kg) lowered ICSS thresholds and a high doses of

nicotine (0.6 mg/kg) elevated ICSS thresholds. In addition, nicotine (0.3 mg/kg) decreased the

response latencies, which is indicative of a stimulant-like effect. Varenicline (Expt. 1.2). The

ANOVA analysis indicated that there was a main effect of varenicline on ICSS thresholds

(F4,32=3.60, p< 0.05, Figure 2a) and response latencies (F4,32=3.38, p<0.05, Figure 2b). The

post hoc comparisons revealed that low doses of varenicline lowered ICSS thresholds (0.1, 0.3, 1

mg/kg) and decreased the response latencies (0.1, 0.3 mg/kg). Pretreatment with the nAChR

antagonist mecamylamine prevented the varenicline-induced lowering of ICSS thresholds and

the decrease in response latencies. Cytisine (Expt. 1.3). Cytisine did not affect ICSS thresholds

(F5, 55=1.80, n.s., Table 2) or response latencies (F5,55=0.91, n.s., Table 2). 3-pyr-Cyt (Expt.

1.4). The cytisine derivative 3-pyr-Cyt did not affect ICSS thresholds (F3,36=1.69, n.s., Table 2)

or response latencies (F3,36=1.44, n.s., Table 2).

Experiments 2.1-2.3. Effect of varenicline, cytisine, and 3-pyr-Cyt on brain reward function

in nicotine withdrawing rats. Mean (±) absolute ICSS thresholds and response latencies before

minipump implantation and before minipump removal are shown in Table 3. Chronic

administration of nicotine (n = 51) or saline (n = 37) did not affect ICSS thresholds. Chronic

administration of nicotine (n = 51, all groups combined) led to a small, but significant, decrease

in the response latencies (pre-pump implantation vs. pre-pump removal; t(50)=2.03, p<0.05).


11

There were no significant differences in ICSS thresholds or latencies between the various

nicotine and saline groups before minipump removal (Table 3).

Experiment 2.1. Effect of varenicline on brain reward function in nicotine withdrawing

rats. The removal of the nicotine pumps, but not saline pumps, led to elevations in ICSS

thresholds (Pump: F1,41=41.62, p<0.001; Pump x Time: F10,410=3.47, p<0.001, Figure 3a).

Varenicline lowered ICSS thresholds and this effect was dependent on pump content (nicotine or

saline)(Drug x Time: F10,410=5.72, P<0.001; Drug x Pump x Time: F10,410=1.99, p< 0.05).

Additional two-way ANOVAs were conducted for the varenicline treatment period (12-72 h).

These analyses showed that varenicline lowered the ICSS thresholds of the nicotine withdrawing

rats (Drug: F1,25=14.97, p<0.001). There was a trend toward a decrease in ICSS thresholds in

the saline-control rats (post saline-pump removal) treated with varenicline but this effect did not

reach statistical significance (Drug: F1,16=4.31, P=0.054).

Post hoc analyses indicated that in the nicotine withdrawing rats treated with saline the

ICSS thresholds were elevated from 6 to 48 h post pump removal. In the nicotine-withdrawing

rats treated with varenicline the ICSS thresholds were only elevated at one time point (36 h).

Varenicline diminished the elevations in ICSS thresholds associated with nicotine withdrawal. At

the 12, 24, 36 h time points, the ICSS thresholds were lower in the nicotine withdrawing rats

treated with varenicline than in the nicotine withdrawing rats treated with saline. After the

cessation of varenicline administration, there was an increase in the ICSS thresholds in the rats

that had been exposed to the nicotine pumps (96, 120, 144 h time points). This suggests that

cessation of varenicline administration leads to a dysphoric-like state in animals with a history of

nicotine dependence.


12

There were no main effects of Pump content or Drug treatment on the response latencies

(Figure 3a). There was, however, a significant effect of Time (F10, 410=4.13, p<0.001), Pump x

Time interaction (F10, 410=3.49, p< 0.001) and Drug x Time interaction (F10, 410=3.31,

p<0.001). The post hoc comparisons revealed that varenicline decreased the response latencies in

the saline-pump (12 h) and nicotine-pump rats (72 h). Furthermore, the latencies of the nicotine-

withdrawing rats (nicotine-pump/saline-inj) were slightly increased (~10%), but this did not

reach statistical significance. Previous studies have also shown that cessation of nicotine

administration leads to a small increase in response latencies (Bruijnzeel et al. 2007).

Experiment 2.2. Effect of cytisine on brain reward function in nicotine withdrawing rats.

The removal of the nicotine pumps, but not saline pumps, led to elevations in the ICSS

thresholds (Pump: F1,41=13.75, p<0.001; Time: F10,410=8.13, p<0.001; Pump x Time:

F10,410=2.49, p<0.01, Figure 4a). The administration of cytisine lowered ICSS thresholds and

this effect was dependent on pump content (Pump x Drug x Time: F10,410=4.95, p < 0.001).

The two-way ANOVA (12-72 h period) indicated that cytisine lowered the ICSS thresholds of

the nicotine withdrawing rats (Drug: F1,24=7.50, p<0.05) but did not affect the ICSS thresholds

of the saline-pump control rats (Drug: F1,17=3.62, n.s.). The post hoc analyses indicated that at

the 24 h time point the ICSS thresholds of the nicotine withdrawing animals treated with cytisine

were lower than those of the nicotine withdrawing rats treated with saline. Furthermore, cytisine

reduced the duration of the elevations in ICSS thresholds in the nicotine withdrawing rats (36 vs.

48 h). Nicotine withdrawal or pretreatment with cytisine did not affect the response latencies

(Figure 4b). There was, however, a main effect of Time on response latencies (Time:

F10,410=6.21, P<0.001). This might have been caused by the removal of the minipumps


13

(surgery effect). This led to a very small increase in response latencies and the latencies

gradually returned to baseline levels. The post hoc analyses did not detect any differences in

response latencies between the experimental groups.

Experiment 2.3. Effect of 3-pyr-Cyt on brain reward function in nicotine withdrawing rats.

Statistical analyses indicated that removal of the nicotine pumps led to an elevation in ICSS

thresholds (Pump: F1,40=49.00, p<0.001; Time: F10,400=5.17, p<0.001; Pump x Time:

F10,400=8.61, p<0.001, Figure 5a). The overall three-way ANOVA indicated that 3-pyr-Cyt did

not affect the ICSS thresholds. The 3-way ANOVA analyses indicated that the latencies of the

nicotine withdrawing rats were longer than the latencies of the saline control rats (Time:

F10,400=7.77, p<0.001; Pump x Time: F10,400=3.58, p<0.001, Figure 5b). The statistical

analyses also revealed a trend towards a Pump x Drug interaction (F1,40=3.48, p=0.07), which

suggests that the effects of 3-pyr-Cyt on the latencies depend on the treatment history (chronic

nicotine or saline). The post hoc test indicated that 3-pyr-Cyt increased the latencies in the

nicotine withdrawing rats (36 h time point) but not in the control rats.

An additional statistical analysis was conducted to compare the effects of all three drugs

(varenicline, cytisine, and 3-pyr-Cyt) on nicotine withdrawal (12-72 h). This study showed that

the drug treatments affected the ICSS thresholds of the nicotine withdrawing rats and that the

thresholds of these rats returned to baseline levels over time (Drug: F3,47=14.04, p<0.001; Time:

F4,188=10.05, p<0.001). The post hoc comparisons indicated that there was no difference in

ICSS thresholds between the nicotine withdrawing rats treated with varenicline or cytisine (12-

72 h). Furthermore, the ICSS thresholds of the nicotine withdrawing rats treated with varenicline

(P<0.01) or cytisine (p<0.05) were lower than those of the nicotine withdrawing rats treated with


14

saline. The ICSS thresholds of the nicotine withdrawing rats treated with varenicline (P<0.001)

or cytisine (p<0.001) were also lower than those of the nicotine withdrawing rats treated with 3-

pyr-Cyt.

An AUC analysis was conducted to compare the cumulative effects of the various

treatments on ICSS thresholds during the treatment period (12-72 h). The AUC was calculated

using the following formula: (Pruessner et

al. 2003). In this formula, ti indicates the time distance between the points at which ICSS

thresholds were assessed, mi indicates the ICSS thresholds at a specific time point, n the number

of time points, the baseline is 100 (see Figures 3-5), and time is the treatment period (60 h). The

ANOVA analysis indicated that ICSS thresholds were elevated in the nicotine withdrawing rats

and that the drug treatments (varenicline, cytisine, and 3-pyr-Cyt) differentially affected the

nicotine withdrawing and the control rats (Pump: F1,80=59.50, p<0.001; Drug: F3,80=7.50, p<

0.001; Pump x Drug interaction: F3, 80=8.38, p<0.001, Figure 6). Dunnett t-tests comparisons

indicated that varenicline and cytisine lowered the ICSS thresholds of the nicotine withdrawing

rats. Furthermore, cytisine elevated the ICSS thresholds of the saline-treated control rats and 3-

pyr-Cyt did not affect the ICSS thresholds of the nicotine withdrawing rats or the saline-pump

control rats.

DISCUSSION

The present studies investigated the effects of varenicline, cytisine, and 3-pyr-Cyt on ICSS

thresholds in nicotine-naïve and nicotine withdrawing rats. The first series of experiments

showed that nicotine and varenicline, but not cytisine or 3-pyr-Cyt, lowered ICSS thresholds in


15

nicotine-naïve rats. The second series of experiments showed that varenicline and cytisine, but

not 3-pyr-Cyt, diminishes the elevations in ICSS thresholds associated with nicotine withdrawal.

These findings indicate that varenicline potentiates brain reward function and diminishes the

dysphoric-like state associated with nicotine withdrawal. Cytisine does not potentiate brain

reward function but, like varenicline, also diminishes the dysphoric-like state associated with

nicotine withdrawal.

The first set of experiments showed that the effects of nicotine and varenicline on ICSS

thresholds in nicotine-naïve rats are somewhat similar. Both nicotine and varenicline produced

an inverted U-shaped dose-effect curve. Low doses of nicotine and varenicline did not affect

ICSS thresholds and medium doses lowered ICSS thresholds. A high dose of nicotine (0.6 mg/kg

nicotine base) elevated the thresholds above baseline levels while a high dose of varenicline (1.8

mg/kg base or 3 mg/kg salt; MW varenicline salt: 361.35 Da, MW varenicline base: 211.27 Da)

did not affect ICSS thresholds. The effects of nicotine on ICSS thresholds are in line with a

previous study that showed that low, but not high, doses of nicotine lower ICSS thresholds

(Kenny et al. 2009). In this study by Kenny et al., (2009) the highest dose of nicotine (0.5 mg/kg

base) did not elevate ICSS thresholds above baseline levels. This nicotine dose was slightly

lower than the highest nicotine dose in the present study (0.6 mg/kg base) and therefore it is

likely that a slightly higher dose would have elevated the ICSS thresholds above baseline levels.

Place conditioning studies have also shown that low doses of nicotine are rewarding (i.e., place

preference) and that high doses of nicotine are aversive (i.e., place aversion)(Le Foll and

Goldberg 2005; Fudala et al. 1985). It should be noted that one study has reported that a high

dose of nicotine (0.5 mg/kg base) can lower ICSS thresholds (Harrison et al. 2002). This study is

not in line with the present ICSS study (Figure 1a) or another ICSS study by the same research


16

group (Kenny et al. 2009). At this point it is not clear what caused this discrepancy between

these studies, but it should be noted that Harrison et al. (2002) also reported a much greater

nicotine-induced decrease in ICSS thresholds. The present study and Kenny et al. (2009) showed

that nicotine lowers the ICSS thresholds by 10-15% while Harrison et al. (2002) reported a 25-

30% decrease in ICSS thresholds. It is unlikely that these differences were due to differences in

rat strains or test procedures because male Wistar rats were used for all these studies and the

same test procedure was used to assess ICSS thresholds.

In the present study, one noteworthy difference between the nicotine and the varenicline

dose-effect curves was the dose-range that lowered ICSS thresholds. In the nicotine study, low

doses of nicotine lowered ICSS thresholds but the highest dose (0.6 mg/kg of nicotine base)

elevated ICSS thresholds. In contrast, low doses of varenicline and a relatively high dose (0.6

mg/kg of varenicline base, equivalent to 1 mg/kg varenicline salt) lowered ICSS thresholds.

Thus, 0.6 mg/kg of nicotine base has aversive effects and the same dose of varenicline has

rewarding effects. This difference between nicotine and varenicline might be due to the fact that

varenicline has a lower efficacy for a variety of nAChRs (Papke et al. 2011). The maximal

efficacy of varenicline for the α4β2 nAChR receptor is only 24% of that of nicotine (Coe et al.

2005). More importantly, a recent study showed that the efficacy of nicotine for the α4β2α5

nAChR was 35% that of acetylcholine, while the efficacy of varenicline was only 9% of that of

acetylcholine (Papke et al. 2011). The activation of α5* nAChRs in the medial habenula plays an

important role in the aversive effects of nicotine (Fowler et al. 2011). Therefore, it could be

speculated that the low efficacy of varenicline for the α5* nAChR explains the fact that a

relatively high dose of varenicline (0.6 mg/kg base) has rewarding effects while a similar dose of

nicotine (0.6 mg/kg base) has aversive effects.


17

In the present study, cytisine did not affect the ICSS thresholds of nicotine-naïve rats.

This would suggest that cytisine does not potentiate brain reward function. Very few studies

have compared the effects of nicotine, cytisine, and varenicline on the brain reward system.

Cytisine and varenicline have a similar affinity and efficacy for the nAChRs (α4β2*, α6β2*) that

mediate the rewarding effects of nicotine (Papke et al. 2010; Grady et al. 2010; Picciotto and

Kenny 2013). Therefore, it is unlikely that the differences between varenicline and cytisine on

ICSS thresholds are due to differences in nAChR binding and efficacy. One study compared the

maximum nicotine, cytisine, and varenicline-induced increase in dopamine turnover in the

nucleus accumbens (Coe et al. 2005). Nicotine (sc) induced a 177% increase in dopamine

turnover in the nucleus accumbens and the effect of cytisine (sc) and varenicline (sc) were 40%

and 32% of those of nicotine. In a similar study, nicotine (sc) induced a 180% increase in

dopamine turnover in the nucleus accumbens and orally administered varenicline and cytisine

induced a 130-140% increase in dopamine turnover (Rollema et al. 2010). Importantly, the same

study showed that orally administered varenicline and cytisine are rapidly and completely

absorbed into the circulation (Rollema et al. 2010). Taken together, these studies suggest that

cytisine and varenicline induce a similar increase in dopamine levels, but both these drugs are

less efficacious than nicotine. In the same study it was shown that varenicline was 20-times more

potent in increasing dopamine turn-over than cytisine. This is most likely because systemic

administration of cytisine leads to relatively low levels of this drug in the brain (Rollema et al.

2010). Oral administration of cytisine and varenicline leads to similar plasma levels, but the

plasma / brain ratio is 3.9 for varenicline and 0.11 for cytisine (Rollema et al. 2010). It has been

suggested that varenicline and cytisine readily enter the brain but that cytisine is removed via an

active efflux mechanism (Rollema et al. 2010). Therefore, it might be possible that acute cytisine


18

does not lower ICSS thresholds in nicotine-naïve rats because it is rapidly removed from the

brain. In the present study, the cytisine derivative 3-pyr-Cyt did affect ICSS thresholds. Low

doses did not affect ICSS thresholds and higher doses tended to elevate the ICSS thresholds. This

is mostly likely due to the fact that this compound is a very weak partial agonist at α4β2*

nAChRs and at high doses diminishes acetylcholine transmission (Mineur et al. 2009; Papke et

al. 2010).

The second set of experiments showed that varenicline and cytisine, but not 3-pyr-Cyt,

diminishes the elevations in ICSS thresholds associated with nicotine withdrawal. A close look at

the data indicates that the ICSS thresholds in the nicotine withdrawing rats treated with saline

were elevated for 2 days (6-72 h). In the nicotine withdrawing rats treated with varenicline the

ICSS thresholds were only elevated at one time point (36 h), and in the nicotine withdrawing rats

treated with cytisine the ICSS thresholds were elevated at two time points (12 and 36 h). This

pattern of results would suggest that varenicline is slightly more effective than cytisine in

diminishing the dysphoric-like state associated with nicotine withdrawal. However, an additional

ANOVA analysis indicated that there was no significant difference in ICSS thresholds between

the nicotine withdrawing rats treated with varenicline or cytisine. These findings suggest that

both varenicline and cytisine diminish the dysphoric-like state associated with nicotine

withdrawal. The administration of 3-pyr-Cyt did not diminish the elevations in ICSS thresholds

associated with nicotine withdrawal. At all time points, the ICSS thresholds of the nicotine

withdrawing rats treated with 3-pyr-Cyt were higher than those of nicotine withdrawing rats

treated with saline. There were, however, no significant differences between the nicotine

withdrawing rats treated with saline or 3-pyr-Cyt. It has been reported that 3-pyr-Cyt is a very

weak partial agonist at α4β2* nAChRs (Mineur et al. 2009; Papke et al. 2011). The present data


19

suggests that a very weak partial agonist at α4β2* nAChRs does not attenuate the elevations in

ICSS thresholds associated with nicotine withdrawal and might therefore not diminish the

negative mood state associated with smoking cessation.

After the cessation of varenicline, cytisine, and 3-pyr-Cyt administration the ICSS

thresholds were assessed for an additional 4 days. The ICSS thresholds of the nicotine-

withdrawing rats treated with saline, cytisine, or 3-pyr-Cyt were elevated at one time point (96

h). However, the ICSS thresholds of the nicotine-withdrawing rats treated with varenicline were

elevated for an additional 2 days (96 -114 h). Cessation of varenicline administration did not lead

to elevations in ICSS thresholds in the rats that were chronically treated with saline (saline-pump

group). One possible explanation for the elevations in ICSS thresholds after the cessation of

varenicline administration is that nicotine addiction is at least partly mediated by a nicotine-

induced upregulation and desensitization of nAChRs (Dani and Balfour 2011). Varenicline might

maintain this state and therefore cessation of varenicline administration might lead to delayed

nicotine withdrawal signs (Hussmann et al. 2012). The present finding would suggest that

cessation of varenicline use in former smokers could lead to a dysphoric state. To our

knowledge, the effect of varenicline withdrawal on mood states has not been thoroughly

investigated in large clinical trials. There are, however, some case reports that indicate that

cessation of varenicline intake in former smokers can lead to agitation, anxiety, and

hallucinations and these symptoms gradually resolve over time (May and Rose 2010; Laine et al.

2009).

The present study showed that cytisine is as effective as varenicline in diminishing

nicotine withdrawal and there are no delayed withdrawal signs. Cytisine might also be given

additional consideration as smoking cessation aid because cytisine has a lower sensitivity than


20

varenicline for human α3β4* nAChRs (ganglionic receptor)(Stokes and Papke 2012). The α3β4*

nAChR is highly expressed in brain stem areas involved in cardiovascular control and

stimulation of these receptors may increase the risk for cardiovascular disorders (Sobieraj et al.

2013; Perry et al. 2002).

At the end of the studies, the area under the curve was calculated for the various nicotine

and saline groups. This was done in order to investigate the cumulative effects of the various

treatments on ICSS thresholds during the drug-treatment period (12-72 h). These data confirm

that varenicline and cytisine diminish the elevations in ICSS thresholds associated with nicotine

withdrawal to a similar degree. It was interesting to note that the ICSS thresholds in the saline-

pump/cytisine-inj group were elevated. This indicates that repeated administration of a high dose

of cytisine lowers ICSS thresholds in the nicotine withdrawing rats but elevates ICSS thresholds

in the drug-naïve control rats. This is in line with the observation that high doses of nicotinic

receptor agonists have aversive effects (Spiller et al. 2009; Le Foll and Goldberg 2005; Fudala et

al. 1985). However, as can be seen in figure 4a, the rats rapidly develop tolerance to the aversive

effects of cytisine while cytisine continues to prevent the elevations in ICSS thresholds

associated with nicotine withdrawal.

It has been suggested that drugs that lower ICSS thresholds are more likely to be abused

than drugs that do not affect ICSS thresholds or elevate ICSS thresholds (Kornetsky et al. 1979).

Indeed, widely abused drugs such as cocaine and amphetamine lower ICSS thresholds

(Kornetsky and Esposito 1979; Cryan et al. 2003). However, despite the fact that varenicline

lowers ICSS thresholds it has been suggested that its abuse liability is extremely low (McColl et

al. 2008). This might be because the dose-response window for nicotinic receptor agonists to

potentiate brain reward function in humans is very narrow and higher doses induce negative side


21

effects such as nausea (McColl et al. 2008). Although the abuse liability of varenicline is low

there is evidence that varenicline can have unintended side effects in certain psychiatric patients.

For example, clinical evidence indicates that varenicline can precipitate manic episodes in people

with bipolar disorder (Hussain et al. 2011; Alhatem and Black 2009; Francois et al. 2011; Knibbs

and Tsoi 2011). Therefore, some caution with the use of varenicline in psychiatric patients is

warranted.

In conclusion, the present studies indicate that varenicline and cytisine diminish the

dysphoric-like state associated with spontaneous nicotine withdrawal in rats. It is suggested that

these smoking cessation aids may prevent relapse to smoking in humans by diminishing the

negative mood state associated with smoking cessation.

Funding and Disclosure:

The authors declare no conflict of interest.

Acknowledgements: This work was funded by a James & Esther King Biomedical Research

Program grant (1KG12) to RP and AB, and a National Institute on Drug Abuse (DA023575) and

Flight Attendant Medical Research Institute (Grant 52312) to AB.


22

Legends

Figure 1: Nicotine potentiates brain reward function. (a) Acute nicotine lowers ICSS thresholds

and (b) decreases response latencies. Asterisks (*p < 0.05, **p < 0.01) indicate lower ICSS

thresholds or shorter response latencies compared to saline-treated rats. Data are expressed as

means ± SEM (n = 12 / group).

Figure 2: Varenicline potentiates brain reward function. (a) Acute varenicline lowers ICSS

thresholds and (b) decreases response latencies. Asterisks (*p < 0.05) indicate lower ICSS

thresholds or shorter response latencies compared to saline. Abbreviations: M3, mecamylamine 3

mg/kg; V0.3 varenicline 0.3 mg/kg. Data are expressed as means ± SEM (n = 9 / group).

Figure 3: Varenicline diminishes the dysphoric-like state associated with nicotine withdrawal.

(a) Effect of varenicline on the elevations in ICSS threshold associated with spontaneous

nicotine withdrawal. (b) Effect of varenicline on response latencies. The number of animals per

group was: saline-saline, n = 9; saline-varenicline, n = 9; nicotine-saline, n = 14, nicotine-

varenicline, n = 13. (a) At the 6 h time-point, the ICSS thresholds of the nicotine-saline rats and

the nicotine-varenicline rats were similar (117% of baseline). Therefore, only one data-point is

visible for these two groups. Asterisks (*p < 0.05, **p < 0.01) indicate elevated ICSS thresholds

compared to the saline-saline group. Plus signs (+p < 0.05, ++p < 0.01) indicate lower ICSS

thresholds compared to nicotine-saline group. (b) Asterisks (*p < 0.05, **p < 0.01) indicate

shorter latencies compared to saline-saline group. Three specific periods are depicted in the

figures (before, during, and after varenicline-treatment) and these periods are separated by line


23

breaks. The bar at the bottom of each figure indicates the varenicline-treatment period. The rats

received varenicline or saline before each time point (12-72 h). Abbreviations: Sal, saline; Nic,

nicotine; Var, varenicline. Data are expressed as means ± SEM.

Figure 4: Cytisine diminishes the dysphoric-like state associated with nicotine withdrawal. (a)

Effect of cytisine on the elevation in ICSS thresholds associated with spontaneous nicotine

withdrawal. (b) Effect of cytisine on response latencies. The number of animals per group was:

saline-saline, n = 9, saline-cytisine, n = 10; nicotine-saline, n = 14, nicotine-cytisine, n = 12.

Asterisks (*p < 0.05, **p < 0.01) indicate elevated ICSS thresholds compared to the saline-saline

group. Plus sign (+p < 0.05) indicates lower ICSS thresholds compared to nicotine-saline group.

Three specific periods are depicted in the figures (before, during, and after cytisine-treatment)

and these periods are separated by line breaks. The bar at the bottom of each figure indicates the

cytisine-treatment period. The rats received cytisine or saline before each time point (12-72 h).

Abbreviations: Sal, saline; Nic, nicotine, Cyt, cytisine. Data are expressed as means ± SEM.

Figure 5: The cytisine derivative 3-pyr-Cyt does not affect the dysphoric-like state associated

with nicotine withdrawal. (a) Effect of 3-pyr-Cyt on the elevation in ICSS thresholds associated

with spontaneous nicotine withdrawal. (b) Effect of 3-pyr-Cyt on response latencies. The number

of animals per group was: saline-saline, n = 9; saline-3-pyr-Cyt, n = 9; nicotine-saline, n = 14;

nicotine-3-pyr-Cyt, n = 12. Asterisks (*p < 0.05, **p < 0.01) indicate elevated ICSS thresholds

or increased response latencies compared to the saline-saline group. Three specific periods are

depicted in the figures (before, during, and after 3-pyr-Cyt-treatment) and these periods are

separated by line breaks. The bar at the bottom of each figure indicates the 3-pyr-Cyt-treatment


24

period. The rats received 3-pyr-Cyt or saline before each time point (12-72 h). Abbreviations:

Sal, saline; Nic, nicotine; 3pC, 3-pyr-Cyt. Data are expressed as means ± SEM.

Figure 6: Area under the curve analyses for the effects of varenicline, cytisine, and 3-pyr-Cyt on

nicotine withdrawal-induced elevations in ICSS thresholds. The treatment period was from 12-72

h after removal of the minipumps. The number of animals per group was: saline-saline, n = 9;

saline-varenicline, n = 9; saline-cytisine, n = 10; saline-3-pyr-Cyt, n = 9; nicotine-saline, n = 14;

nicotine-varenicline, n = 13; nicotine-cytisine, n = 12; nicotine-3-pyr-Cyt, n = 12. Asterisks (*p

< 0.05, *** p<0.001) indicate higher ICSS thresholds compared to saline-saline group. Plus signs

(++ p < 0.01) indicate lower ICSS thresholds compared to nicotine-saline group. Abbreviations:

AUC, area under the curve; Sal, saline; Nic, nicotine; Var, varenicline; Cyt, cytisine; 3pC, 3-pyr-

Cyt. Data are expressed as means ± SEM.


25

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Table 1. Absolute baseline ICSS thresholds and response latencies prior to first drug injection.

Compound N Thresholds (µA) Latencies (s)

Nicotine (Expt. 1.1) 12 91.1 ± 4.9 3.1 ± 0.1

Varenicline (Expt. 1.2) 9 124.1 ± 13.2 3.4 ± 0.1

Cytisine (Expt. 1.3) 12 126.8 ± 8.4 3.3 ± 0.1

3-pyr-Cyt (Expt. 1.4) 13 102.1 ± 5.4 3.4 ± 0.1

The baselines are averages of thresholds and latencies that were obtained on three consecutive

days before the first drug injection.


Table 2. Acute effects of cytisine and 3-pyr-Cyt on ICSS thresholds and response latencies.

Cytisine (Expt. 1.3, n=12)

Dose (mg/kg) 0 0.1 0.3 1 3 5

Thresh. (µA) 97.6 ± 3.0 101.6 ± 2.5 102.2 ± 1.3 98.8 ± 1.6 103.4 ± 2.9 105.9 ± 4.3

Latency (s) 97.6 ± 2.5 102.0 ± 2.3 98.4 ± 2.6 98.5 ± 3.1 96.7 ± 3.4 99.1 ± 3.5

3-pyr-Cyt (Expt. 1.4, n=13)

Dose (mg/kg) 0 0.3 0.6 0.9

Thresh. (µA) 99.4 ± 2.9 100.0 ± 2.6 102.2 ± 3.1 106.5 ± 4.2

Latency (s) 101.3 ± 1.7 98.2±1.8 100.8 ± 2.0 96.9 ± 1.2

Drugs were administered subcutaneously and data are expressed as a percentage of 3-day

baselines prior to first drug injection. Abbreviations: Thresh., ICSS thresholds.


Table 3. Absolute baseline ICSS thresholds and response latencies prior to minpump

implantation and removal.

Pre pump-implantation Pre pump-removal

Pump Drug N Threshold (µA) Latencies (s) Threshold (µA) Latencies (s)

Saline (all groups) 37 105.4 ± 3.8 3.3 ± 0.1 106.7 ± 3.7 3.3 ± 0.1

Nicotine (all groups) 51 101.6 ± 3.5 3.4 ± 0.0 102.6 ± 3.5 3.3 ± 0.1*

Saline Saline 9 115.8 ± 11.8 3.4 ± 0.1 118.8 ± 12.2 3.3 ± 0.1

Saline Varenicline 9 105.3 ± 3.5 3.2 ± 0.1 107.0 ± 2.4 3.3 ± 0.1

Saline Cytisine 10 106.1 ± 7.7 3.4 ± 0.1 105.6 ± 6.2 3.3 ± 0.1*

Saline 3-pyr-Cyt 9 94.3 ± 4.0 3.3 ± 0.1 95.6 ± 3.6 3.3 ± 0.1

Nicotine Saline 14 96.7 ± 5.3 3.2 ± 0.1 98.9 ± 5.2 3.2 ± 0.1

Nicotine Varenicline 13 106.9 ± 8.5 3.3 ± 0.1 107.4 ± 8.6 3.3 ± 0.1

Nicotine Cytisine 12 101.2 ± 8.0 3.4 ± 0.1 102.4 ± 8.4 3.2 ± 0.1

Nicotine 3-pyr-Cyt 12 102.1 ± 6.1 3.6 ± 0.1++

105.0 ± 5.5 3.4 ± 0.1*

The ICSS thresholds and latencies are 3-day baselines that were obtained on consecutive days

before pump-implantation or pump-removal. Asterisks (*p < 0.05) indicate difference between

pre pump-implantation and pre pump-removal baselines within the same group. Plus signs (++p

< 0.01) indicate significant difference compared to saline-pump/3-pyr-Cyt-inj group.


Br. J. Pharmac. (1969), 35, 161-174.

Some studies on cytisine and itsmethylated derivativesR. B. BARLOW AND L. J. McLEOD*

Department of Pharmacology, University of Edinburgh

1. In mice cytisine hydrochloride is less toxic intravenously than nicotinehydrogen tartrate, but more toxic by intraperitoneal or oral administration.Compared with cytisine, caulophylline hydrogen iodide is one-fifth to one-tenthas toxic and caulophylline methiodide is less than one-thirtieth as toxic.2. The surprising low oral toxicity of cytisine and nicotine may be ascribedto the method of administration; if the drug is placed directly in the stomachthere is no possibility of absorption through buccal mucous membranes.3. The peripheral effects of nicotine, cytisine and caulophylline are similar,though on some preparations those of nicotine last longer. In most testscytisine is active in doses from a quarter to three-quarters of those of nicotine,caulophylline in doses from 10 to 20 times those of cytisine. Caulophyllinemethiodide is virtually inactive.4. Cytisine and caulophylline may differ from nicotine in their central effects.5. Cytisine and caulophylline are active as the cations. The pKa of cytisineis 7.92 and that of caulophylline is 7.04; the difference accounts, in part, forthe weaker activity of caulophylline. The caulophylline ion is generally one-sixth to one-third as active as the cytisine ion.6. The introduction of the second methyl group to form the quaternary saltdoes not appear to cause a dramatic change in the conformation of the molecule.Caulophylline methiodide appears to be feebly active because it has feebleaffinity.

The alkaloid cytisine occurs in a number of plants of the leguminosae familyand was considered by Dale & Laidlaw (1912) to be the toxic principle of thecommon laburnum. After tests in cats, rabbits and fowls, they described its peri-pheral actions as being " qualitatively indistinguishable from nicotine " though theyobserved that it differed from nicotine in that it did not produce an ear-twitch incats. Zachowski (1938) confirmed its pharmacological resemblance to nicotinebut, from experiments on the blood pressure of the cat, concluded that it had greateractivity in stimulating sympathetic ganglia than in blocking them.The chemical structure of cytisine was worked out by Ing (1931, 1932) and its

absolute configuration is now known (Okudu & Katauku, 1961). It contains a

* Present address: School of Pharmacy, Hobart Technical College, Tasmania, Australia.G


secondary amino group (Fig. 1) and the tertiary base, N-methyl cytisine, occurs asthe alkaloid caulophylline, together with cytisine, in several plants. The toxicityof caulophylline was examined by Kalaschnikow & Kusnetzow (1938) and itspharmacological properties briefly reported by Scott & Chen (1943). Although itsperipheral actions resembled those of nicotine, the convulsions produced by caulo-phylline in mice differed from those produced by nicotine. The quaternary com-pound, caulophylline methiodide, was prepared by Ing (1931) but does not seemto have been tested pharmacologically.

This paper describes a comparison of the pharmacological properties of nicotine,cytisine, caulophylline and caulophylline methiodide. These compounds form aninteresting chemical series because cytisine is a relatively rigid structure, and conse-quently the structural effects of methylation are limited to one particular part andunlikely to lead to changes in conformation elsewhere. The work also includesa study of the effects of changes in pH on the activity of cytisine and caulophyllineon the frog rectus, to see whether these are active as the ions or as the unchargedbase.

MethodsChemical

Melting points were determined with a Mettler FP 1 instrument; analyses forhalide are gravimetric with samples of 50-100 mg.

WvN\ ,Me

N

0(-)-7R:9S cytisi'ne ( --)-S nicotine

Me/Me ItMe

0~~~~~(-)-7R:9S caulophylline (-)-79:9S caulophylline

methiodide

FIG. 1. Albsolute configurations of (-)-cytisine, (-)-caulophylline and (-)-caulophyllinemethiodide (Okudu & Katauku, 1961) compared with (-)-nicotine (Hudson & Neuberger, 1950).Note the hydrogen atom attached to the basic nitrogen in ring C of cytisine can be in eitherof two conformations, but the methyl group attached to this atom in caulophylline is morelikely to be as shown, than in the alternative arrangement (shown for the hydrogen atom incytisine).

162


Cytisine melting point 1520 C, [a]D -1190 in water, was bought from Fluka A.G.201'

Caulophylline hydrogen iodide was prepared by the method of Ing (1931) butwith ethanol instead of methanol as solvent. Recrystallized material had m.p.276.5° C; found; 1-, 38.08; calculated for C12H170N2J; I-, 38.30%. Partheil (1892)recorded m.p. 2700 C; Ing (1931) recorded m.p. 2800 C. The free base was preparedby treating the hydrogen iodide with alkali and extracting with chloroform, dryingthe extract with magnesium sulphate, and distilling off the solvent. The residualoil would not crystallize and was heated under reduced pressure in a cold-fingerapparatus. The base was deposited on the condenser as a solid, m.p. 1280 C;[a]2D, -219° in water. Ing (1931) recorded m.p. 134°. Power & Salway (1913)recorded [a]D, - 221.60 in water, temperature not specified.

Caulophylline methiodide was prepared by heating the base under reflux inethanol with an excess of methyl iodide. Recrystallized material had m.p. 265.50 C;[a]% -137.9° in water; found; I-, 36.75; calculated for Cl3H1,0N2J; I-, 36.65%.Ing (1931) recorded m.p. 2760 (dec).

All the recrystallized material used for biological testing appeared to be homo-geneous when chromatographed on paper in a solvent system consisting of butanol,ethanol and water (5:5:2) and developed with a modified Dragendorff reagent(Thies & Reuther, 1954). The materials also all showed an ultraviolet absorptionmaximum in water at 303 mg, with log. e=3.81, and in the infrared, absorptionmaxima corresponding to the a-pyridone group occurred at 1650, 1555 and1567 cm`.The nicotine used was the (- )-isomer supplied as the hydrogen tartrate by

British Drug Houses Ltd.Dissociation constants. The pKa values were determined by the method of

Albert & Goldacre (1943). The procedure was the same as that of Barlow &Hamilton (1962) but with a stream of nitrogen in place of a stirrer driven by com-pressed air and with a Pye Dynacap instrument in place of the Marconi pHmeter.

BiologicalToxicity. The acute toxicity of the compounds by intravenous, intraperitoneal

and oral administration was studied in female albino mice, strain CS1, weighingbetween 17 and 24 g. These were divided into groups of ten and the animals ineach group all received the same dose of the same drug by the same route, the dosebeing expressed as ,moles/kg, based on the average weight of the group. By eachroute each drug was tested using at least five different dose-levels; in other words,at least five groups of mice were used for each drug given by one particular route.The mice were observed for 1 hr after dosing, and from the number which diedin this period, the LD50 was calculated by the method of Litchfield & Wilcoxon(1949). The dose-levels were chosen so that, as far as possible, they were uniformlyabove and below the LD50 and, in all, about 50% of the animals died in the testswith one particular drug given by one particular route.

In these experiments nicotine was tested as the hydrogen tartrate, cytisine as thehydrochloride, and caulophylline as the hydrogen iodide, made up in 0.9% saline.

Ganglionic preparations. The superior cervical ganglion preparation of the cat,anaesthetized with chloralose, was set up as described by Paton & Perry (1953).

163


The preganglionic sympathetic nerve was stimulated with rectangular wave pulsesof 0.7 msec duration at a rate of 10 shocks/sec. Usually a stimulus of 3-5 V wasnecessary to produce a maximum contracture of the nictitating membrane. Injec-tions were made retrogradely into the external carotid artery, though in one instancethe lingual artery was used. The blood pressure was recorded from a femoralartery. The relative blocking activity of the compounds was estimated by com-paring the doses which produced roughly comparable block of the responses of thenictitating membrane to continuous stimulation of the preganglionic nerve. Therelative stimulant activity of the compounds was estimated in a 2+1 assay bycomparing the doses which produced comparable contractions of the nictitatingmembrane (in the absence of stimulation of the preganglionic nerve).The relative ability of compounds to raise arterial blood pressure was studied

in rats anaesthetized with urethane and cats anaesthetized with chloralose. Theblood pressure was recorded from a carotid artery and the drugs were injected intoa femoral vein, in a volume not exceeding 0.2 ml. and washed in with 0.9% saline.The isolated guinea-pig ileum was set up in Tyrode solution at 370 C and bubbled

with air. The volume of the organ bath was approximately 10 ml. and the drugswere added by pipette in a volume not exceeding 0.4 ml., allowed to act for 30 secand then washed out. The interval between doses was 2 min.

Striated muscle. The chick biventer-cervicis preparation was set up, as describedby Ginsborg & Warriner (1960), in Krebs-Henseleit solution (Krebs & Henseleit,1932) at 370 C bubbled with 95% oxygen and 5% carbon dioxide. The nerve inthe tendon was stimulated with rectangular wave shocks, which produced maximaltwitches, of 0.7 msec duration at 6-8 shocks/min. The volume of the bath was

approximately 30 ml. and the drugs were added by pipette in a volume not exceed-ing 0.4 ml., allowed to act until the contracture had reached a maximum (usuallyfor about 10 min) and then washed out. Recovery usually occurred rapidly so theinterval between doses was about 15 min. The compounds had a marked effecton the slow fibres, which masked any effects they may have had on the twitchresponses. Their relative activities on this preparation were therefore estimated bycomparing the doses which produced roughly the same degree of contracture.

The rectus abdominis muscle of the frog (Rana pipiens) was set up at room

temperature in a modified Ringer solution bubbled with air. This solution had thefollowing composition (in 1 1.): sodium chloride, 7.50 g (128 m-equiv); potassiumchloride, 0.14 g (1.9 m-equiv); calcium chloride, 0.12 g (2.2 m-equiv); Tris(2-amino-2(hydroxymethyl)propane-1: 3-diol), 1.92 g (15.9 m-equiv); to which was

added 0.1 N hydrochloric acid; 14 ml. produced a pH of 7.10; 10 ml. produceda pH of 7.90; 9.4 ml. produced a pH of 8.20. The pH of the fluid in contact withthe tissue differed slightly from these values and in every experiment samples were

removed after contact for 4.5 min and their pH measured with a Pye Dynacap pHmeter. Although there were differences of about 0.2 units between the pH of a

particular buffer in different experiments, the variation did not exceed 0.02 unitsduring any one experiment.

Equipotent molar ratios for the compounds relative to the quaternary salt,p-pyridylmethyltrimethylammonium, were obtained by 2+1 and 2 + 2 assay tech-niques using an automatic apparatus (Barlow, Scott & Stephenson, 1967). Thedrugs, made up in the desired concentration, were allowed to act on the preparationfor 4.5 min and the interval between doses was 30 min. When an assay had been

164


completed, the pH was altered and a concentration of the quaternary compound wasapplied repeatedly until the responses were regular, indicating that the tissue hadbecome adjusted to the new medium. This usually took less than an hour-that is,the second and third responses to the concentration of quaternary compound afterthe change were usually consistent. The assay was then repeated at the new pH.The rat diaphragm preparation was set up exactly as described by Bulbring

(1946), in Tyrode solution at 370 C bubbled with 95% oxygen and 5% carbondioxide. The phrenic nerve was stimulated with rectangular wave shocks, whichproduced maximal twitches, of 0.75 msec duration at 5 shocks/min. The volume ofthe bath was approximately 25 ml. and the drugs were added by pipette in a volumenot exceeding 0.4 ml., allowed to act until the block was fully developed and thenwashed out. The interval between doses was between 5 and 20 min. The relativeactivity was estimated in a 2+1 assay by comparing the doses which producedcomparable degrees of block.The anterior tibialis muscle preparation of the cat, anaesthetized with chloralose,

was set up as described by Brown (1938). The peroneal nerve was stimulatedwith rectangular wave pulses, which produced maximal contractions of the muscle,of 0.7 msec duration at a rate of 6-8 shocks/min. The drugs were injected, in avolume not exceeding 0.1 ml., retrogradely into the anterior tibial artery. The bloodpressure was recorded from a carotid artery. Measurement of blocking activity wasusually by a 2+ 1 assay method, but in one experiment only approximate estimateswere obtained by comparing the doses which produced roughly 50% block.

Respiration. Rabbits were anaesthetized with urethane and a cannula insertedinto the trachea. This was connected to a respirometer (Gaddum, 1941) so thatboth the rate of respiration and changes in the volume of inspired air could berecorded. The blood pressure was recorded from a carotid artery. The drugs wereinjected in a volume not exceeding 0.2 ml. into a femoral vein and washed in with0.9% saline. The interval between doses was 5 min and approximate estimatesof relative activity were made by comparing the doses which produced roughly thesame increase in the rate of respiration.

Acetylcholinesterase. Purified acetylcholinesterase, from ox red cells, was ob-tained from Nutritional Biochemicals Corporation. The effects of the compoundson the hydrolysis of acetylcholine (10-3M) by this enzyme were studied by mano-metric methods as described by Barlow & Zoller (1964).

Results

Estimates of the LD50 of nicotine, cytisine and the methylated derivatives ofcytisine, are shown in Table 1. Those mice which did not die within 1 hr werecounted as survivors. Occasionally an animal treated with nicotine died manyhours afterwards, but this never happened with the other drugs. The time afteradministration at which death occurred was noted and the average time of deathfor the animals killed in each group is indicated.With nicotine the onset of symptoms was much faster than with the other com-

pounds and the convulsions were qualitatively different. The tail became erect,rather like the Straub reaction, breathing appeared to be difficult, and there wasfrothing at the mouth, which was held wide open. The head was initially held

165


high, but subsequently forced down upon the chest. A few seconds before deaththe body became rigid, except for rapid trembling motions of the limbs.With cytisine, caulophylline and caulophylline methiodide the convulsions were

much slower in onset and much less severe, but there were more pronounced tonicand clonic contractions, particularly of the hind limbs. The body did not becomeas rigid as with nicotine and, just before death, the hind legs were stretchedright back. All the animals which survived remained sedated for between 15 minand 1 hr afterwards.The results of the tests on the other preparations are summarized in Table 2.

Cytisine was invariably more active than caulophylline and usually more active thannicotine. Caulophylline methiodide was almost inactive and, in most tests, noeffects were observed even with very large doses.

TABLE 1. Acute toxicityRoute of administration

i.v. i.p. OralNicotine LD5O ,umoles/kg 1.92 59.0 1,425

limits 1.75-2.12 53.6-65.0 1,370-1,486LD50 mg/kg 0-3 9-5 230

(6) (7) (9)Time of death 32 0±0.8 2-41 +0 1 2.86±0r1

sec (28) min (38) min (45)

e.p.m.r. 1 1 1

Cytisine LD50 ,umoles/kg 9.10 49.5 535limits 7.9-10.5 466-57.5 411-696LD50 mg/kg 1-73 9 4 101

(6) (6) (7)Time of death 372-4-31 5-32±04 12.7 ±0.6

sec (36) min (28) min (46)

e.p.m.r. 475 0.84 0 37

LD5O gmoles/kglimitsLD50 mg/kg

Time of death

e.p.m.r.

LD50 ,umoles/kglimitsLD50 mg/kg

Time of death

e.p.m.r.

10389-9-118

21(6)

252210-302

51(5)

41.3±1*4 6.92+40.4sec (31) min (26)

53.6 4-27

280239-328

61(6)

79.5+0*3sec (32)

146

>2,000

The table shows the LDSO in umoles/kg and the fiducial limits (P=0 05). The value in mg/l g isshown below. The number of dose levels tested-that is, the number of groups of mice used-isshown in parentheses. The mean time after administration at which death occurred is shown±thestandard error with the total number of animals which died shown in parentheses. The lowestentry shows the equipotent molar ratio (e.p.m.r.) for the compound relative to nicotine,calculatedfrom the values of LD50.

Caulophylline >2,500

Caulophyllinemethiodide

>8,000

166


The effects produced by cytisine and caulophylline were similar to each other butwere not always exactly the same as those produced by nicotine. Consequentlyalthough a quantitative comparison of cytisine with caulophylline was possible, aquantitative comparison with nicotine is of doubtful value in some tests. Thedifference was most noticeable with the cat tibialis preparation, where the blockingaction of nicotine lasted much longer than that of cytisine and caulophylline (Fig. 2).To a lesser extent this was true of the blocking action of the compounds on the catsuperior cervical ganglion (Fig. 3). Caulophylline methiodide, in high doses, causeda block on this preparation but this was never accompanied by an initial stimula-tion. It is probably the result of a different type of action and so this compoundcannot really be compared with the others. After the block there was an increase

TABLE 2. Equipotent molar ratios for the compounds relative to (-)-nicotine in the various tests

PreparationCat superior

cervicalganglion

BloodpressureGuinea-pigileum

Chick biventerFrog rectusRat diaphragmCat tibialisRabbitrespiration

Cytisine Caulophylline

StimulationBlockCat (rise)Rat (rise)

0.75±0.15 (6)1174±007 (4)0.54±011 (4)041±014 (10)

(Contraction) 0.25±0.02 (3)

(Contracture) 6.2540.48 (4)(Contracture) 0.64 (6)(Block) 0'66 (2)(Block) *0.23 ±t0.02 (3)

(Increase) 3 05±0t33 (4)

14.2±2.8 (6)20.1±2.2 (4)5-60O±081 (4)0.84±024 (10)

2.80±0-18 (3)

25-14+7.2 (4)2.7 (6)15.4 (2)

*2.40±0.19 (3)

Effectivedose/animal

orconcentration

Caulophylline ofmethiodide (-)-nicotine

27.0+3.9 (4)

Very feeble (2)

Feeble

27.7±2.8 (4)

10 n-moles100 n-moles300 n-moles50 n-moles

10-5M

10-6M10-5M

2x 10-4M300 n-moles

1 zmoleThe mean is given+the standard error, with the number of experiments shown in parentheses.Figures for the cat tibialis, marked with an asterisk, are of doubtful value because of the differencein the time-course of the effects (see text), though they give a fair comparison of cytisine and caulo-phylline. Only a rough comparison was possible on the rat diaphragm preparation because thecompounds were not very active and large amounts of material were needed. The figures for thefrog rectus have been calculated from separate comparisons of the compounds with ,-pyridylmethyl-trimethylammonium and of this compound with (-)-nicotine. The final column shows theapproximate dose or concentration which was effective in these experiments.

S S

Suxamethonium Cytisine

5 min

. * 0S

Caulophylline Nicotine

1 hr

FIG. 2. Effects on the cat tibialis muscle. The record shows contractions of the tibialismuscle in response to stimulation of the peroneal nerve. Compare the transient blockadeproduced by cytisine (200 n-moles), caulophylline (2,000 n-moles) with the longer effects ofsuxamethonium (5 n-moles) and the prolonged effects of nicotine (400 n-moles). S indicatesan injection of 0.9% sodium chloride (0.2 ml.).

S

167


in the size of the contracture; this supramaximal response could well be the resultof inhibition of cholinesterase by the high concentrations of caulophylline methio-dide used (see later).The results of the comparisons on the rat blood pressure were very variable but

this variation can probably be ascribed to the preparation rather than to differences

0 -S

* aS

* 0*S S S

0S

0

Caulophylline Caulophylline Cytisine Nicotinemeth3iodidem

3 min

FIG. 3. Effects on the cat superior cervical ganglion. The upper record shows the bloodpressure and the lower record the contracture of the nictitating membrane in response tostimulation of the preganglionic nerve. Cytisine (75 n-moles), caulophylline (1,000 n-moles).caulophylline methiodide (1,000 n-moles) and nicotine (75 n-moles) all produced comparabledegrees of block. Note that cytisine, caulophylline and nicotine increased contracture beforethe onset of block and that the effects of nicotine lasted longer than those of cytisine andcaulophylline. Caulophylline methiodide did not cause an increase in contracture before theonset of block, but did so afterwards. Note the effects of the compounds on the bloodpressure. S indicates an injection of 0.9, sodium chloride (0.2 ml.).

TABLE 3. Effects ofpH on the activity of cytisine on the frog rectusEquipotent molar

ratio at

More Moreacid alkalinepH pH(a) (b)

8 356.00

5.90

5.45

3-76

5 18

3-80

7-20

9.45

7.65

7-65

5.00

Ratioa/b(c)139

1 22

1.73

2-04

1 .48

1-31

Proportion of

(d) ion (e) basepresent

at more acid pH

(d) (e)1-53 0-25

1.53

1.53

1-57

1.52

1.54

0 25

025

0-20

0-32

0.26Equipotent molar ratios for cytisine relative to ,B-pyridyl-methyltrimethylammonium are shown(a, b), together with the pH in the experiments at which they were obtained. The ratio (c) of theseequipotent molar ratios can be compared with the effect of the changes in the pH on the proportionof ion (d) and base (e) present.The average of the values in column c is 1.53, and in column d is 154.

pH7-887-10

7-107-88

7-887-10

6-987-88

7-927-22

7 127.9

168


between the actions of the drugs, because it did not occur when the compounds werecompared on the cat blood pressure.

Cytisine and caulophylline were without effect on the hydrolysis of acetylcholineby acetylcholinesterase in concentrations as high as 4 x 10-3M, but caulophyllinemethiodide was weakly active. Estimates of the p150 were, 2.82, 2.82, 2.65 (mean2.76; substrate concentration 10-3M).The pKa values at 250 C were 7.92 for cytisine and 7.04 for caulophylline; three

estimates were made with each compound and gave identical results.

Effects of pH on activity on the frog rectus

Table 3 shows the equipotent molar ratios for cytisine relative to the quaternarycompound, f8-pyridylmethyltrimethylammonium, at different pH values. The in-creased activity in the more acid pH suggests that it is the cytisine ion which is theactive species and, quantitatively, the effects on the equipotent molar ratio are ingood agreement with the effects on ionisation, calculated from the pH and pK,.On average the equipotent molar ratio at the more acid pH is 1/1.53 times that atthe more alkaline pH, and the proportion ionized at the more acid pH is 1.54 timesthat at the more alkaline. In these experiments it did not seem to matter whethercytisine was tested in the more acid medium first or in the more alkaline.

Table 4 shows the results of similar experiments with caulophylline. Like cytisinethis compound is clearly more active at the more acid pH, but the quantitative

TABLE 4. Effects ofpH an the activity ofcaulophylline on the frog rectus

Equipotent molar Proportion ofratio at

(d) ion (e) baseMore More presentacid alkaline Ratio at more acid pHpH pH a/b

pH (a) (b) (c) (d) (e)7.43 338.03 79*6 2-42 2-93 0-74

7.13 22.88.20 130 4.84 6.15 0.53

7.13 25.98.20 125 5.70 6.15 0.53

8.13 797-15 45 1.75 5.20 0.53

8.13 81-37.08 39.3 2.07 5.61 050

8.20 1686.87 49.7 3.38 7-16 043

8.14 1006-94 32-3 3.07 6.59 0.41

Equipotent molar ratios for caulophylline relative to P-pyridylmethyltrimethylammonium are shown(a, b) together with the pH in the experiments at which they were obtained. The ratio (c) of theseequipotent molar ratios can be compared with the effect of the changes in the pH on the proportionof ion (d) and base (e) present. The experiments in which caulophylline was tested at the moreacid pH first are shown in the upper section of the table.

169


agreement between the observed and calculated changes is much better in the experi-ments where the more acid pH was used before the more alkaline. In similarexperiments with nicotine tested at different pH values on the frog rectus, Hamilton(1963) observed exactly the same phenomenon; the quantitative agreement wasmuch better when the more acid pH was used first.

Discusion

The results of the toxicity experiments indicate that cytisine is less toxic thannicotine intravenously but more toxic by intraperitoneal or oral administration. Byany of these routes, however, it does not appear to be particularly lethal in mice.The intravenous LD50 of cytisine, 1.73 mg/kg, corresponds to 120 mg/70 kg and theequivalent of the oral LD5O would be 7 g/70 kg. In two experiments, doses ofcytisine 1.2 times the LD50 in mice were tested on guinea-pigs and rats and did notcause death. The animals appeared to be sedated, but not distressed, so thereseems to be no reason to believe that mice are particularly resistant to cytisinecompared with other rodents.The likely toxic dose in man, of course, is difficult to predict, especially as cytisine

can cause vomiting. The doses used in many of the pharmacological tests arp low,so quite small amounts might produce effects which were unpleasant, but the experi-ments with animals suggest that these are not likely to be lethal. Instances of deathfrom laburnum poisoning were reported in the last century (Radziwillowicz, 1888)but although there have been many cases of poisoning this century, none of theseseems to have been fatal (Mitchell, 1951). In forty-four enquiries about laburnumpoisoning to the Scottish Poisons Information Bureau between May 1963 and May1968 there was complete and uneventful recovery in all instances.

The high LD50 values for nicotine by the oral route were surprising in view, forexample, of Gaddum's statement (1953) that " if a couple of drops of pure nicotineare placed on a dog's tongue the dog drops down dead in a few seconds." Estimatesof the LD50 for nicotine, however, vary very considerably (Larson, Haag &Silvette, 1961) and it seems that, by the oral route, solutions of salts of nicotine areless toxic than solutions of the base. We may have obtained high values becausewe used a salt and also because we placed the dose directly into the stomach. Theacid environment will greatly delay absorption and the use of a stomach-tube toadminister the drug will prevent any absorption through the mucous membranesof the buccal cavity, which might otherwise occur during its passage from the mouthto the stomach. Absorption in this way, between the mouth and the stomach, mightwell account for the discrepancy between values of the oral LD50 for nicotine (seeabove). At normal body pH, there is rapid absorption of nicotine base acrossmembranes and, if the dose is not placed directly in the stomach, a considerableproportion may be absorbed before it reaches an acid environment. There shouldbe much less absorption of nicotine from solutions of the salts, especially if theseare acid (such as the hydrogen tartrate). The toxicity of cytisine may be similarlydependent on whether it is tested as the base or as the salt.

From the results of the experiments in mice it seems that death from cytisineeither occurs rapidly or not at all (see Table 1) which suggests that the body is ableto tolerate or detoxicate the drug if it is slowly absorbed. Larson, Finnegan &Haag (1949) have, in fact, shown that when nicotine is given intravenously by infu-sion over a period of 8 hr, some animals are able to tolerate up to 11 times the

170


amount which would be lethal in a single injection. In view of this it could beargued that it is unnecessary to wash out the stomach of children who have eatenlaburnum, or other plants containing cytisine, especially as the alkaloid itself islikely to cause vomiting. It might be much more important to wash out the mouthwith an acidic buffer.Our results with the various tests confirm the resemblance between the pharmaco-

logical properties of cytisine and nicotine observed by Dale & Laidlaw (1912). Theyalso confirm the conclusions of Zachowski (1938), that cytisine is more powerfulas a ganglion stimulant than as a ganglion blocking agent. The resemblance betweenthe peripheral effects of cytisine and nicotine is striking and our results show thatthis resemblance is quantitative as well as qualitative. On the cat and rat bloodpressure, the guinea-pig ileum, the frog rectus and the rat diaphragm, comparableeffects are produced by doses of cytisine from one-quarter to two-thirds of those ofnicotine. The results with the cat tibialis are not really comparable, because theeffects of cytisine are only transient. The results with the chick biventer arenoticeably different but it is possible that these indicate marked sensitivity of chickmuscle to nicotine, rather than an insensitivity to cytisine.From the differences between the types of convulsion produced by the two

alkaloids, as well as from the absence of ear-twitches after cytisine noted by Dale& Laidlaw (1912), it seems that the similarity of the effects may not be so great inthe central nervous system. The action of cytisine on the respiration of anaesthe-tized rabbits is certainly weaker than would be expected from its effects on theperipheral nervous system.From the experiments at different pH with the frog rectus it appears that cytisine

also resembles nicotine in being active as the ion. Equipotent ionic ratios shouldtherefore be compared, rather than equipotent molar ratios. These are shown inTable 5, in which the mean values of the equipotent molar ratio from Tables 1 and2 have been corrected for the degree of ionization of nicotine and of the compound.With cytisine this makes little difference, because the pKa, 7.92, is only slightly lessthan that of nicotine, 8.01 (Barlow & Hamilton, 1962). Caulophylline, however, isa much weaker base, pK, 7.04, and it is clear that part of the decline in pharmaco-logical activity produced by methylating cytisine is due to the effect of methylationon basicity. The decline in basic strength is likely to be caused by steric hindrance;

TABLE 5. Equipotent ionic rctiosPreparation Cytisine Caulophylline

(a) (a) (b)Cat superior Stimulation 072 4.36 605

cervical ganglion Block 113 6.27 5.55

Blood pressure Cat (rise) 053 1-72 3.25Rat (rise) 0O40 026 065

Guinea-pig ileum (Contraction) 0-24 070 2.92Chick biventer (Contracture) 5.90 5.63 096Frog rectus (Contracture) 064 2.67 4-17Rat diaphragm (Block) 063 3'85 612Cat tibialis (Block) 0O22 074 3.36Rabbit respiration (Increase) 2.94 8.52 2.90The equipotent molar ratios in Tables 1 and 2 have been corrected for the degree of ionization ofthe compounds. Figures in columns a are relative to the univalent nicotinium ion; those in b arefor the caulophylline ion relative to the cytisine ion.

171


the methyl group, which is electron-releasing and therefore usually base-strengthening(see, for example, Clark & Perrin, 1964), restricts the access of hydrogen ions to thenitrogen atom and this is already limited by the bulk of the bridged-ring system(Fig. 1).Even allowing for the difference in basicity, however, it is clear that the caulo-

phylline ion is weaker than the cytisine ion. In most tests it has between one-halfand one-sixth of the activity, though on the chick biventer it has the same activityas the cytisine ion and on the rat blood pressure it is even more active. Apart fromthese quantitative differences, however, the pharmacological effects of cytisine andcaulophylline are indistinguishable.The feeble activity of caulophylline methiodide is surprising. Apart from its

weak effects as an inhibitor of acetylcholinesterase and as a ganglion-blocking agent,it seems to be inactive. It is toxic to mice only when very large doses are givenintravenously, but the symptoms produced are similar to those of cytisine andcaulophylline. We thought it possible that the introduction of the second methyIgroup into caulophylline might have altered the conformation of ring C, making theboat form preferable to the chair form shown in Fig. 1. Normally the boat formis thermodynamically less favoured, but it seemed that the change might occur

30

20

10

0

-10 -

-20

-3050 40 30 20 17 10

Wave numbers (kv) D line sodiumFIG. 4. Optical rotatory dispersion curves. Wave numbers (kv; 1 kv=1,000 cm-) areplotted against the molar rotations. Measurements were made with 2 x 10-4M concentrationsin distilled water. Similar results were obtained with the hydrogen iodides as with the basesexcept that above 40,000 wave numbers (250 m,u) the iodide absorption was so strong that therotation could not be measured. The Cotton effect between 31,000 and 35,000 wave numberscorresponds to the peak absorption due to the pyridone chromophore, and is the same shapefor all three compounds. The measurements were made with a Bellingham and StanleyPolarmatic 62 instrument, which scans continuously, but does not measure rotation directly.The absolute values were calculated for the points shown ( x - - x, caulophylline;O ,cytisine; A- - -A caulophylline methiodide) and the intervening parts of the curves weredrawn from the traces of the relative rotations recorded by the instrument.

172

Some studies on cytisine and its methylated derivatives 173

because the basic nitrogen atom in ring C and the pyridine nitrogen atom in ringsA and B are fairly close together. The introduction of a second methyl group,which must be placed between these two atoms, might tend to force them furtherapart but, to offset this there would then be considerable steric interference betweenthe other methyl group on the nitrogen atom in ring C and an axial hydrogen atom.These changes in conformation involve the movement of groups close to thechromophore, the pyridone system (X max, 303 mM), and consequently might beexpected to produce changes in the optical rotatory dispersion curves. These wereexamined, however, and found all to have the same shape (Fig. 4), and consequentlythe inactivity of caulophylline methiodide, either as an agonist or an antagonist,does not seem to be caused by any fundamental change in preferred conformation.

It seems, then, that the progressive methylation of cytisine decreases activitybecause it decreases the ability of the onium group, the nitrogen atom in ring C,to fit the receptor. This is likely to be a steric effect, due simply to the increasein size. Chemical support for this comes from the decreased basicity of caulophyl-line compared with cytisine, and also from the difficulty of preparing caulophyllinemethiodide, which is only obtained after heating caulophylline with methyl iodidefor 48 hr. It is, however, possible that cytisine produces effects when only a smallproportion of receptors is occupied and consequently has only a low affinity. Thedecline in activity with methylation would then indicate a decline in efficacy ratherthan in affinity. The absence of agonist activity in caulophylline methiodide clearlyindicates a decline in efficacy, but the absence also of appreciable antagonist activitysuggests that methylation has also decreased affinity unless the affinity of cytisineis very low indeed-that is, unless cytisine has a particularly high efficacy.From molecular orbital calculations, Kier (1968) has deduced the preferred con-

formations of nicotine and acetylcholine and has suggested that the presence in amolecule of a quaternary nitrogen atom and a negatively charged atom situated4.85 + 0.1 A away are key features for nicotine-like activity. In models the nitrogenatom in ring C of cytisine appears to be 4.8-9 A away from the oxygen atom of thepyridone group, which will be partially negatively charged. This agreement, how-ever, may be fortuitous. It is not clear whether the key features confer affinity orefficacy on a molecule or, more probably, a particularly desirable combination ofthe two properties. They cannot be the only criteria for activity, however, becausethough they are present in cytisine and caulophylline, they are also present incaulophylline methiodide, which is virtually inactive.

We wish to thank Dr. J. C. P. Schwarz and Mr. F. Rutherford of the Chemistry Departmentfor the optical rotatory dispersion measurements and the Faculty of Medicine for the awardof a Fellowship to one of us (L. J. McL.). The cytisine was purchased with part of a grantfrom the Tobacco Research Council.

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on the frog rectus abdominis. Br. J. Pharmac. Chemother., 31, 188-196.BARLow, R. B. & ZOLLER, A. (1964). Some effects of long chain polymethylene bis-onium salts

on junctional transmission in the peripheral nervous system. Brit. J. Pharmac. Chemother., 23,131-150.

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BROWN, G. L. (1938). The preparation of the tibialis anterior (cat) for close arterial injection.J. Physiol., Lond., 92, 22-23P.

BULBRING, E. (1946). Observations on the isolated phrenic nerve diaphragm preparation of the rat.Br. J. Pharmac. Chemother., 1, 38-61.

CLARK, J. & PERRIN, D. D. (1964). Prediction of the strengths of organic bases. Q. Rev. chenm.Soc., 18, 295-320.

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(Received A'igitst 29, 1968)

In vivo modulation of dopaminergic nigrostriatal pathways by cytisine derivatives:Implications for Parkinson's Disease

J. Andrés Abin-Carriquiry a,⁎, Gustavo Costa a, Jessika Urbanavicius a, Bruce K. Cassels b,Marco Rebolledo-Fuentes b, Susan Wonnacott c, Federico Dajas a

a Department of Neurochemistry, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguayb Department of Chemistry, Faculty of Sciences, University of Chile, Santiago, Chilec Department of Biology and Biochemistry, University of Bath, Bath, UK

A B S T R A C T

Keywords:CytisineDopamineNeuroprotectionParkinson's DiseaseNicotinic acetylcholine receptor6-Hydroxydopamine

Nicotinic acetylcholine receptor agonists are considered potential pharmacological agents for Parkinson's Diseasetreatment, due to their ability to improve experimental Parkinson symptomatology, reduce 3,4-dihydroxy-L-phenylalanine-induced dyskinesias and stop the neurodegenerative process at an experimental level. In thepresent work, the ability of the nicotinic agonist cytisine and two halogenated derivatives (3-bromocytisine and5-bromocytisine) to induce striatal dopamine release was characterized in vivo by microdialysis. Cytisine,5-bromocytisine and nicotine were much more efficacious than 3-bromocytisine in eliciting dopaminerelease in response to their local application through the microdialysis probe. Moreover, the agonists wereintermittently administered before and after an intranigral injection of 6-hydroxydopamine (6-OHDA), andstriatal dopamine tissue levels were assessed 8 days after the lesion. Both cytisine and its 5-bromo derivative(but not the 3-bromo derivative) significantly prevented the decrease of striatal dopamine tissue levels inducedby 6-OHDA. These results suggest that the efficacy of nicotinic agonists to stimulate dopamine release in vivothrough presynaptic nicotinic receptors could be related to their potential to induce striatal protection.

1. Introduction

The motor symptoms that are at the core of Parkinson's Disease –

rigidity, bradykinesia and postural instability – are linked to the loss ofnigrostriatal dopaminergic neurons (Singh et al., 2007). However, theadministration of 3,4-dihydroxy-L-phenylalanine (L-DOPA) or dopa-mine receptor agonists, the key therapeutic strategies in use today toimprove the dopaminergic functions in Parkinson's Disease, areunable to stop the neurodegenerative process (Singh et al., 2007).

Acetylcholine modulates the function of the nigrostriatal dopa-mine system through multiple subtypes of nicotinic acetylcholinereceptors located postsynaptically on the neuronal cell bodies of thesubstantia nigra and presynaptically on terminals at the corpusstriatum. Because nicotinic receptor activation increases the fre-quency of firing at the neuronal bodies (Lichtensteiger et al., 1982;Clarke et al., 1985) or causes neurotransmitter release at the dopa-minergic terminals (Lichtensteiger et al., 1982; Clarke et al., 1985;Kaiser and Wonnacott, 2000; Wonnacott et al., 2000; Zhou et al.,2001; Zoli et al., 2002), nicotinic receptors are considered to be po-

tential therapeutic targets for the treatment of Parkinson's Diseasesymptoms (Quik et al., 2007a, b). In addition, epidemiological studieshave shown that smokers have a lower incidence of Parkinson'sDisease and nicotine, a non-selective nicotinic receptor agonist, hasbeen postulated to be responsible for this effect (Baron, 1986; 1996;Gorell et al., 1999).

Previously, we have shown that systemic administration of nicotinepartially prevents the decrease of dopamine levels in the corpusstriatum following administration of 6-hydroxydopamine (6-OHDA)(Costa et al., 2001; Abin-Carriquiry et al., 2002; Urbanavicius et al.,2007). As Quik et al. recently reviewed, other studies have confirmedthese results in different experimental models of Parkinson's Disease(Visanji et al., 2006; Quik et al., 2006a, b, 2007c; Khwaja et al., 2007;Urbanavicius et al., 2007).

Depending on the experimental paradigm, both α7 and/or α4β2subtypes of nicotinic receptors have been described as mediatingprotection by nicotine against various toxic insults in cell cultures(Kihara et al., 1997, 1998; Hejmadi et al., 2003; Stevens et al., 2003),consistent with a requirement for stimulation of more than onereceptor subtype for protection (O'Neill et al., 2002; Dajas-Bailadorand Wonnacott, 2004; Wonnacott et al., 2006). In vivo, althoughnicotine-induced protection against experimental parkinsonianlesions has been shown to be mediated by nicotinic receptor (Costaet al., 2001), identification of particular receptor subtypes has not

⁎ Corresponding author. Department of Neurochemistry, Instituto de InvestigacionesBiológicas Clemente Estable, Avenida Italia 3318, 11600 Montevideo, Uruguay. Tel./fax:+598 2 4872603.

E-mail addresses: [email protected], [email protected] (J.A. Abin-Carriquiry).

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http://dx.doi.org/10.1016/j.ejphar.2008.05.013

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been reported yet. Visanji et al. recently demonstrated that in contrastto the non-selective agonist nicotine, subtype-selective nicotinic ago-nists did not protect striatal dopamine terminals from a 6-OHDA insultin rats (Visanji et al., 2006), suggesting that interaction with multiplenicotinic receptor subtypesmay be necessary to afford neuroprotection.

Cytisine, an alkaloid present in many plants of the Leguminosaefamilyandabroad-specific nicotinic receptoragonist, has been shown toprotect cells in culture against toxic agents (Kihara et al., 1998; Jonnalaand Buccafusco, 2001). In this context, we recently studied a series ofcytisine derivatives in striatal slices, showing that halogenation inpositions 3 and 5 could increase or reduce respectively their potency toinduce dopamine releasewithout significant changes in their selectivityfor nicotinic receptors subtypes (Abin-Carriquiry et al., 2006).

Given the potential of nicotinic receptor agonists as therapeuticagents in Parkinson's Disease it appeared meaningful to extend thecharacterization of cytisine and its bromo derivatives, studying theirpharmacological profile in vivo. In the present study we exploredthe ability of cytisine, 3-bromocytisine (3-BrCy) and 5-bromocytisine(5-BrCy) to induce dopamine release in vivo by microdialysis, as wellas their capacity to prevent the decline in dopamine tissue levels inexperimental Parkinson's Disease.

2. Materials and methods

2.1. Animals

Experiments were carried out using male Sprague–Dawley rats(230–260 g). Animals had access to food and water ad libitum, andwere housed in groups of six in a temperature-controlled environ-ment on a 12-h light/dark cycle.

Experimental procedureswere approvedby theCommitteeonEthicalCare and Use of Laboratory Animals of the Instituto de InvestigacionesBiológicas Clemente Estable in accordancewith the National Institutes ofHealth guide for the care and use of laboratory animals.

2.2. Materials

Chemicals for high performance liquid chromatography (HPLC)analysis, artificial cerebrospinal fluid and saline were purchased fromBaker (Phillipsburg, PA, USA). Dopamine (hydrochloride), 3,4-dihy-droxyphenylacetic acid, 6-OHDA, (−)-nicotine tartrate, and L-ascorbicacid were obtained from Sigma (St. Louis, MO, USA). Chlorisondaminewas donated by Novartis Pharmaceuticals (NJ, USA).

2.3. Cytisinoids

Cytisine and its derivatives were obtained as previously described(Houlihan et al., 2001; Slater et al., 2003) and used as the hydrochloridesalts. Briefly, cytisine was purified from the seeds of the Mexican plantSophora secundiflora using standard methodology. Bromination ofcytisine with a slight excess of molecular bromine in acetic acid ledto the formation of a mixture of products containing a small amount of3,5-dibromocytisineandmainly3-BrCyand5-BrCy. Thesewere separatedby column chromatography on silica gel, crystallized to homogeneity, andcharacterized by 1H and 13C NMR and HREIMS. Definitive structure as-signments were based on 1H–1H COSY experiments.

2.4. Microdialysis

Animals were anaesthetized with ketamine (90 mg/kg)/xylazine(5 mg/kg) and placed in a D. Kopf stereotaxic frame. Through a skullhole, a guide cannula (BAS, MD-2250) was implanted into the dorsalstriatum (caudate–putamen) and secured to the skull with steelscrews and dental cement. The coordinates for cannula implantationwere A/P=+0.6, L/M=+3.2, D/V=−3.2 from Bregma according to theatlas of Paxinos and Watson (Paxinos and Watson, 1986). After sur-

gery, the rats were housed individually into test cages and allowed torecover for 24–48 h. On the experimental day, a microdialysis probe(BAS MD-2204, membrane length=4.0 mm) was inserted into thecannula. The location of the tip of the microdialysis probe was −7.2.The probe was connected to a microperfusion pump and continuouslyperfused with artificial cerebrospinal fluid (147 mM NaCl, 3.4 mMCaCl2, 4.0 mM KCl) at a flow rate of 2.0 μl/min. The sample collection(30 μl sample every 15 min) was started after a 2-h equilibrationperiod. Concentrations of dopamine were determined immediatelyusing an Epsilon Electrochemical Detector e5P with oxidationpotential set at +0.650 V (glassy carbon working electrode versus aAg/AgCl reference electrode). The column (Phenomenex Luna, 5 μm,C18, 4.6×100 mm) was kept at room temperature. The mobile phaseconsisted of citric acid (0.15 M), sodium octylsulphate (0.6 mM), 4%acetonitrile and 1.6% tetrahydrofuran at pH 3.0; with a flow rate of1.0 ml/min. The average concentration of the first three dialysissamples was determined as baseline and defined as 100%. Nicotinicreceptor agonist solutions were prepared in artificial cerebrospinalfluid adjusting the pH to 7.0 with HCl/NaOH and perfused through thedialysis probe during 15 min. Doses of nicotine utilized wereaccording to the literature (Marshall et al., 1997; Quarta et al., 2007).At the end of each experiment, the probewas removed and the animalsacrificed. The brain was immediately removed, dissected and frozenfor confirmation of probe localization (Abin-Carriquiry et al., 2002).

The in vitro recovery of each nicotinic agonist wasmeasured from astandard 1-mM solution by HPLC-UV. Eluate concentrations of nico-tine, cytisine, 3-BrCyand 5-BrCyweredetermined immediately using aGilson UV–Visible 118 Detector at 255 nm. The column (PhenomenexLuna, 5 μm, C18, 4.6×100 mm) was kept at room temperature. Themobile phase consisted of acetic acid (50 mM), sodium octylsulphate(0.6mM) and 15%methanol at pH 3.7; with a flow rate of 1.4 ml/min. Asimilar recovery of 17% was found for the different nicotinic agonists.

2.5. Intranigral injection of 6-OHDA

6-OHDA lesionswere provoked by the injection of 6-OHDA into thesubstantia nigra as previously described (Costa et al., 2001). Briefly,animals were anaesthetized with halothane (Halocarbon Laboratories,River Edge, NJ, USA) and placed in a D. Kopf stereotaxic frame. Througha skull hole, the needle (0.022 mm o.d., 0.013 mm i.d.) of a Hamiltonsyringe (5 μl) was attached to a micro-injection unit (D. Kopf), and waslowered to the right substantia nigra (H, −4.8; L, −2.2; V, −7.2 frombregma, according to the atlas of Paxinos andWatson). A total of 2.0 μlof a 6-OHDA solution (3mg/ml) prepared immediately before use, wasinjected over 1 min and the needle was slowly withdrawn, allowingthe drug to diffuse for another 4 min. Body temperature was main-tained at 37 °C using a temperature control system (Costa et al., 2001).Control rats for 6-OHDA lesions were injected with 2 μl vehicle(artificial cerebrospinal fluid with 0.2% ascorbic acid).

2.6. Agonists administration schedule

Rats (groups of 6–8) that had been injected with 6-OHDA (6 μg) inthe right substantia nigra received nicotinic receptor agonist or salinesubcutaneously, according to the following protocols: (1) 4 h before,and 20, 44 and 68 h after 6-OHDA; (2) the same administrationschedule following treatment with the long-lasting nicotinic antago-nist chlorisondamine (10 mg/kg s.c.) given 30 min before the firstapplication of nicotinic receptor agonist or saline. After chlorisonda-mine administration, the motor activity of the rats decreasedshowing also bilateral palpebral ptosis. These effects lasted a fewhours, with complete recovery afterwards. No other symptoms wereobserved.

The starting dose (1 mg/kg) for cytisine and 5-BrCy protectionstudies was similar to the one shown to be effective for nicotine in vivo(Costa et al., 2001). In the case of 3-BrCy, a dose of 1 mg/kg induced

tonic–clonic convulsions that lasted approximately 5 min. These ef-fects limited the concentrations that could be evaluated in vivo.

Since even after receiving 0.3 mg/kg 3-BrCy, animals still showed astrong depressed motor activity and ptosis for approximately 1 h, thedoses studied were lowered (0.01, 0.05 and 0.10 mg/kg).

2.7. Neurochemical analysis

To measure dopamine tissue levels, rats were decapitated 8 daysafter 6-OHDA injection, the brains rapidly removedand the left and rightcorpus striatum dissected and stored at −70 °C. On the next day tissuesamples were weighed, sonicated in 1000 μl of perchloric acid (0.1 M)and centrifuged (15,000 g) for 15 min. Samples were then injected intoan HPLC system (PM-80 BAS, West Lafayette, IN, USA) equipped with aC18 column (5 μm particles, 220 mm×34.6 mm; BAS, USA) and anelectrochemical detector (LC-4C BAS) with oxidation potential set at+0.75 V (glassy carbon working electrode versus a Ag/AgCl referenceelectrode). The mobile phase was composed of citric acid (0.15 M),sodiumoctylsulphate (0.6mM), 4%acetonitrile and1.6% tetrahydrofuranat pH 3.0; with a flow rate of 1.0 ml/min (Costa et al., 2001).

2.8. Statistical analysis

2.8.1. MicrodialysisDopamine levels were calculated from a standard solution and

values were expressed as a percentage of basal levels (individual means

of three pre-drug fractions). Statistical analysis was carried out usingone- or two-way analysis of variance (ANOVA) for repeatedmeasures ofthe dopamine values followed by post-hoc t-test when appropriate.

2.8.2. Tissue levelsDopamine levels in the lesioned hemisphere of each animal were

expressed as percent of the level in the unlesioned side. Comparison ofthe means was performed by ANOVA followed by Tukey–KramerMultiple Comparison test.

3. Results

3.1. Striatal dopamine release evoked by cytisine and bromocytisines

The nicotinic agonists were evaluated for their ability to evokedopamine release in the striatum by in vivo microdialysis followinglocal application via the dialysis probe. Basal striatal extracellulardopamine levels of dialysate (1.91±0.34 nM) were similar to thoseobtained in our previous studies (Abin-Carriquiry et al., 2002). Localperfusion of nicotine, cytisine and 5-BrCy increased extracellularlevels of dopamine in a concentration-dependent way (Fig. 1). 3-BrCywas the most potent and release was obtained already with 0.33 mMconcentrations, showing little concentration-dependence over anextended concentration range.

The comparison of dopamine release induced by cytisine and itsderivatives at10mMconcentrations showed that 5-BrCywas significantly

Fig. 1. Extracellular dopamine assessed by microdialysis in awake rats 24 h after the implantation of a cannula in the corpus striatum. Drugs were applied locally through the probeand dopamine release was monitored after the application of nicotine, cytisine, 5-BrCy and 3-BrCy at different doses after three stable basal samples. Data are expressed as percentover the basal levels (mean±S.E.M.). For each dose n=4–6. ⁎ denotes significant difference against controls (artificial cerebrospinal fluid, aCSF) (⁎Pb0.05). Δ ⁎ denotes significantdifference against agonist+chlorisondamine (Chl) 1 mM (ΔPb0.05).

more efficacious than cytisine and 3-BrCy, being 3-BrCy the less effi-cacious (Fig. 1).

Perfusion of chlorisondamine (1 mM), a non-specific nicotinicreceptor antagonist, prevented the increase in dopamine releaseinduced by every agonist (Fig. 1). Chlorisondamine alone did not affectsignificantly the basal levels of dopamine (1.61±0.60 nM).

3.2. Prevention of striatal dopamine decrease after 6-OHDA injection

Eight days after intranigral injection of 6 μg of 6-OHDA, there was asignificant decrease in dopamine tissue levels in the ipsilateral stria-tum (lesioned side) when compared with the contralateral (non-lesioned side), (Fig. 2). Control rats injected in the substantia nigrawith 2 μl vehicle did not show any difference in dopamine levelsbetween ipsilateral and contralateral striatum (17.5±1.7 and 17.6±2.8 ng/mg of wet tissue weight, respectively).

As reported previously (Costa et al., 2001), intermittent nicotineadministration (1 mg/kg, nicotine tartrate salt); significantly attenu-ated the 6-OHDA-induced decrease in striatal dopamine tissue levels(Fig. 2). Cytisine (2 mg/kg) and 5-BrCy (1 mg/kg), administered ac-cording to the same schedule, prevented similarly the decrease ofstriatal dopamine levels. In contrast, 3-BrCy (0.10 mg/kg) was unableto prevent the decrease of striatal dopamine tissue levels induced by6-OHDA (Fig. 2). Lower doses of 3-BrCy (0.01, 0.05 mg/kg) did notprevent the dopamine decrease either (data not shown).

The protection afforded by cytisine and 5-BrCy was preventedby the prior administration of the long-acting non-specific nicotinicreceptor antagonist chlorisondamine (10 mg/kg). Dopamine levels(expressed as percent of lesioned versus intact corpus striatum) were19.0±3.9%, 28.3±8.7% and 20.5±8.0% for saline, cytisine and 5-BrCytreatments, respectively.

4. Discussion

Our data showed that local application of cytisine and its bromoderivatives induced dopamine release in the corpus striatum, beingcytisine and 5-BrCy, as well as nicotine significantly more efficaciousthan 3-BrCy.

Besides, cytisine and 5-BrCy prevented the decrease of striataldopamine tissue levels 8 days after the intranigral injection of 6-OHDAin rats. To the extent of our knowledge, this is the first experimentalevidence of in vivo protection by nicotinic receptor agonists other thannicotine. This protection was mediated by nicotinic receptor, since it

was blocked by chlorisondamine administration. However, pharma-cological equivalent doses of 3-BrCy (taking into account that itspotency to induce dopamine release is more than one order of mag-nitude higher than cytisine and 5-BrCy) did not have protective effectagainst the 6-OHDA lesion.

In a previous work we compared the effects of cytisine halogena-tion at C3 and C5 onα4β2 and α7 nicotinic receptor binding, showingthat both 3-BrCy and 5-BrCy presented higher affinity for heteromericα4β2 than for homomeric α7 nicotinic receptors. Additionally weevaluated their ability to evoke [3H]dopamine and [3H]noradrenalinerelease in striatal and hippocampal slices, respectively, showing thatboth agonists presented greater potency as releasers of [3H]dopaminethan of [3H]noradrenaline. Nevertheless, both derivatives do not differsignificantly in subtype specificity between themselves or withcytisine or nicotine (Cassels et al., 2005; Abin-Carriquiry et al., 2006).

In recent years an important line of experimental evidence hasshown the protective capacity of nicotine in in vivo experimentalmodels of Parkinson's Disease, an effect mediated by nicotinicreceptor agonism (Costa et al., 2001; Visanji et al., 2006; Quik et al.,2006b, 2007a,c). Our results suggest that the prevention of striataldopamine decrease is not an exclusive property of nicotine but also ofother non-subtype specific nicotinic receptor agonists. Moreover weshowed that protection afforded by nicotinic agonists is blocked bychlorisondamine, a non-specific nicotinic receptor blocker (Costaet al., 2001). In this sense Visanji et al. showed that neither themodulation of α4β2 nor α7 subtypes alone appears to provideprotection in vivo (Visanji et al., 2006). Taken together these resultswould suggest that broad nicotinic receptor subtype specificity wouldbe necessary for prevention of dopaminergic terminal degeneration.

Nicotine, cytisine and 5-BrCy, which attenuated dopamine lossafter 6-OHDA were also the most efficacious in evoking dopaminerelease. This latter effect was clearly dose-dependent and was blockedby chlorisondamine. 3-BrCy, which failed to induce protective effectsin a wide concentration range, was also the least efficacious inducer ofdopamine release. In this sense, the agonist dopamine releasingefficacy (reflecting efficacy at nicotinic receptors) appears to be morerelevant than potency itself to induce the plastic changes on thedopaminergic pathway.

As an explanatory hypothesis, the experimental evidence suggeststhat activation of nicotinic receptor leads to Ca2+ entry into theterminal (Rapier et al., 1990; Grady et al., 1992; Puttfarcken et al.,2000). The greater efficacy shown by cytisine and 5-BrCy inducingdopamine release, could be associated to a major Ca2+ entry to the cell,leading to activation of several steps in the synaptic vesicle cycle(Smith et al., 1998; Stevens and Wesseling, 1998; Turner, 2004) andtriggering specific intracellular signalling cascades related to plasticchanges in dopamine metabolism and other key protective pathways(Dajas-Bailador and Wonnacott, 2004).

However an indirect pathway, involving the activation of dopami-nergic autoreceptors (Bozzi and Borrelli, 2006; Scheller et al., 2007)could also explain the relationship between efficacy and protection.

Evidence based largely on experimental in vitro and in vivo rodentstudies is showing that dopamine agonists may have neuroprotectiveproperties in addition to their symptomatic effects (Le and Jankovic,2001; Schapira, 2002). In this sense, ropinirole has been shown toprotect mouse striatal neurons against 6-OHDA toxicity, by stimulat-ing the increase of glutathione, catalase and superoxide dismutaseantioxidant activities in the striatum, and this effect was mediatedthrough dopamine D2 receptor (Iida et al., 1999). Nevertheless, furtherexperiments are required in order to discriminate between bothmechanisms.

The results obtained in the present work show for the first time invivo, that efficacious non-selective nicotinic receptor agonists, otherthan nicotine, are able to reduce striatal dopamine depletion inducedby 6-OHDA injection in the substantia nigra and efficaciously evokedopamine release in vivo.

Fig. 2. Striatal dopamine assessed 8 days after the injection of 6-OHDA (6 μg) in thesubstantia nigra. Agonists (nicotine, cytisine, 5-BrCy and 3-BrCy) were subcutaneouslyadministered 4 h before, and 20, 44 and 68 h after 6-OHDA injection. Data are expressedas percent (mean±S.E.M.) of lesioned versus intact corpus striatum. For each treatmentn=6–8. ⁎ denotes significant difference against the control group (⁎Pb0.05). Δ denotessignificant difference against the saline group (ΔPb0.05).

The protective effect of cytisine and 5-BrCy is a challenging resultthat provides a new lead for understanding the central nervous sys-tem plasticity mediated by nicotinic receptors, suggesting their po-tential for Parkinson's Disease treatment.

Acknowledgements

This work was supported by the Wellcome Trust CollaborativeResearch Initiative Grant 073295/Z/03/Z and FONDECYT Grant 1040776.

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Stevens, C.F., Wesseling, J.F., 1998. Activity-dependent modulation of the rate at whichsynaptic vesicles become available to undergo exocytosis. Neuron 21, 415–424.

Stevens, T.R., Krueger, S.R., Fitzsimonds, R.M., Picciotto, M.R., 2003. Neuroprotectionby nicotine in mouse primary cortical cultures involves activation of calcineurinand L-type calcium channel inactivation. J. Neurosci. 23, 10093–10099.

Turner, T.J., 2004. Nicotine enhancement of dopamine release by a calcium-dependentincrease in the size of the readily releasable pool of synaptic vesicles. J. Neurosci. 24,11328–11336.

Urbanavicius, J., Ferreira, M., Costa, G., Abin-Carriquiry, J.A., Wonnacott, S., Dajas, F.,2007. Nicotine induces tyrosine hydroxylase plasticity in the neurodegeneratingstriatum. J. Neurochem. 102, 723–730.

Visanji, N.P., O'Neill, M.J., Duty, S., 2006. Nicotine, but neither the alpha4beta2 ligandRJR2403 nor an alpha7 nAChR subtype selective agonist, protects against a partial6-hydroxydopamine lesion of the rat median forebrain bundle. Neuropharmacol-ogy 51, 506–516.

Wonnacott, S., Kaiser, S., Mogg, A., Soliakov, L., Jones, I.W., 2000. Presynaptic nicotinicreceptors modulating dopamine release in the rat striatum. Eur. J. Pharmacol. 393,51–58.

Wonnacott, S., Barik, J., Dickinson, J., Jones, I.W., 2006. Nicotinic receptors modulatetransmitter cross talk in the CNS: nicotinic modulation of transmitters. J. Mol.Neurosci. 30, 137–140.

Zhou, F.M., Liang, Y., Dani, J.A., 2001. Endogenous nicotinic cholinergic activity regulatesdopamine release in the striatum. Nat. Neurosci. 4, 1224–1229.

Zoli, M., Moretti, M., Zanardi, A., McIntosh, J.M., Clementi, F., Gotti, C., 2002.Identification of the nicotinic receptor subtypes expressed on dopaminergicterminals in the rat striatum. J. Neurosci. 22, 8785–8789.

611

Augmented Responses to Intrathecal NicotinicAgonists in Spontaneous Hypertension

Imran M. Khan, Morton P. Printz, Tony L. Yaksh, Palmer Taylor

Abstract Abnormal central cholinergic activity has beenreported to be responsible in part for the pathogenesis of highblood pressure in spontaneously hypertensive rats (SHR).Administration of cholinergic agonists in brain and spinal cordresults in exaggerated pressor responses in SHR. Studies todate have focused largely on the muscarinic cholinergic system.Recently, we demonstrated that intrathecal administration ofnicotinic agonists results in pressor, tachycardic, and irritationresponses. In the present study we examine the cardiovascularand behavioral responses to nicotine and cytisine administeredintrathecally in La Jolla strain (LJ) SHRLJ and age-matchedWistar-Kyoto (WKYLJ) rats. Nicotinic agonists produced aug-mented pressor, heart rate, and irritation responses in SHRLJcompared with normotensive rats. In both SHRLJ and WKYLJrats, cytisine elicited a greater nociceptive response and greaterspinobulbar component to the pressor response than nicotine.SHRLJ and WKYLJ rats also differ in that the SHRU strainshows a diminished tendency for desensitization to cytisine. As

The central cholinergic nervous system has beenimplicated in the pathogenesis of hypertensionin several rat models, including the spontane-

ously hypertensive rat (SHR).14 Central administrationof cholinergic agonists leads to increases in blood pres-sure and heart rate in SHR and normotensive rats;however, the responses are augmented in SHR.3-6 Inhi-bition of the central cholinergic system in young SHRcan delay the onset of hypertension1-7 or cause a fall inblood pressure in mature hypertensive rats.8 The exag-gerated cardiovascular responses to cholinergic agonistsin SHR compared with normotensive rats can be ob-served at both spinal6 and supraspinal1-4 levels. In thebrain, a role of muscarinic receptors in the maintenanceof high blood pressure in hypertensive rats is evi-dent. 2A10 On the other hand, several studies have shownthat nicotinic receptor stimulation in lower brain stemleads to a depressor response; also, the number ofnicotinic binding sites in these regions is reported to belower in SHR compared with normotensive rats.11-12

However, in a recent study Tseng et al5 demonstratedthat microinjections of nicotine in the rostral ventrolat-eral medulla produce an augmented pressor and tachy-cardic response in SHR compared with Wistar-Kyoto(WKY) or Sprague-Dawley (SD) normotensive rats.

Received March 22, 1994; accepted in revised form June 15,1994.

From the Departments of Pharmacology (I.M.K., M.P.P.,T.L.Y., P.T.) and Anesthesiology (T.L.Y.), University of Califor-nia, San Diego, La Jolla, Calif.

Correspondence to Dr Palmer Taylor, Department of Pharma-cology 0636, University of California, San Diego, La Jolla, CA92093.

© 1994 American Heart Association, Inc.

in Sprague-Dawley rats, in SHRU and WKYLJ rats the cardio-vascular and behavioral responses to intrathecal nicotine weresignificantly inhibited by mecamylamine, dihydro-/3-erthyroi-dine, and methyllycaconitine. However, methyllycaconitine,which effectively blocked cytisine-elicited cardiovascular andbehavioral responses in Sprague-Dawley and WKYU rats, wasunable to inhibit the maximal rise in cytisine-elicited bloodpressure, heart rate, and irritation responses in SHRU. Incontrast to the heightened cardiovascular and behavioral re-sponses, the number of nicotinic binding sites in spinal cordmembranes was significantly decreased in the hypertensive rats.The exaggerated responses to spinal nicotinic agonists in thepresence of lower receptor number and the lower propensity todesensitize to cytisine-elicited irritation responses in SHRLJsuggest that amplification of postcoupling events is enhanced inthe hypertensive rats. (Hypertension. 1994;24:611-619.)

Key Words • nicotine • receptors, nicotinic • receptors,cholinergic • rats, inbred SHR

Similar to their role in brain, a role of muscarinicreceptors in mediating an exaggerated pressor responsein SHR has been demonstrated in the spinal cord.6

However, detailed studies of spinal nicotinic acetylcho-line receptors have not been conducted. We previouslyobserved that intrathecal administration of nicotinicagonists causes an increase in blood pressure and heartrate and results in nociceptive behavior.13 The cardio-vascular and behavioral responses appear to be causedby a localized spinal action of the nicotinic agents, andthe cardiovascular response results from an increase inspinal sympathetic outflow.14 Moreover, nicotinic ago-nist-elicited blood pressure and behavioral responsesoccur via independent receptor-linked pathways. Finally,nicotinic receptor binding can be identified in regionalareas of the spinal cord, and the locations of bindingsites correlated with functional pharmacological endpoints.15

In the present study we document the cardiovascularand behavioral responses to spinal administration ofnicotinic agonists in La Jolla strain (LJ) SHRLJ andcompare them with those found in WKYLJ rats. Addi-tionally, we compare the pharmacological specificity ofthe spinal nicotinic receptors in both SHRLJ and WKYLJrats. Finally, we ascertain ligand binding to nicotinicreceptors in the spinal cord membranes of both ratstrains to determine whether the number or affinity ofthe nicotinic receptors differs between SHRLJ andWKYLJ rats.

MethodsExperimental Animals

Male rats used for cardiovascular and behavioral testingwere age matched (12 to 16 weeks old). All animals were bred

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612 Hypertension Vol 24, No 5 November 1994

and maintained (12-hour diurnal cycle) within our breedingcolony located within the animal care facility at the Universityof California, San Diego (UCSD). WKY rats were from stockoriginally obtained from Charles River Laboratories (Wilming-ton, Mass) and were brother-sister mated through 18 or moregenerations. SHR were also derived from Charles River Lab-oratories stock and maintained by brother-sister mating. Sincethese animals have been bred in La Jolla since 1981, they arereferred to as SHRLJ and WKYLJ. All studies were carried outaccording to protocols approved by the UCSD InstitutionalAnimal Care Committee.

Cardiovascular and Behavioral StudiesFor spinal drug delivery, intrathecal catheters were im-

planted as described previously.16 Briefly, rats were anesthe-tized with halothane (2% to 3%), and 9-cm saline-filled PE-10tubing was placed into the intrathecal space through thecisterna membrane. The catheter tip resided at the rostral endof the lumbar enlargement.16 The catheter was externalized ontop of the skull, and the incision was closed. Rats were allowedto recover for at least 4 days before further study, and onlyanimals exhibiting normal motor behavior were used in thestudy.

Rats with intrathecal implants were catheterized in the tailartery with PE-50 tubing under halothane (2% to 3%) anes-thesia as described previously.13 Heart rates were measuredwith a cardiotachometer triggered from the pressure pulses.Blood pressure and heart rate were recorded on a polygraph(model 7, Grass Instrument Co).

Behavioral responses were measured according to a scoringprotocol described earlier.13 Briefly, a score of 1 was given foreach of the following behavioral responses: movement of thelimbs, twisting and turning, tail erection, and high-pitchedsqueaking. The maximum assignable score was 4.

Drugs were dissolved in sterile saline solutions and admin-istered with a hand-driven syringe pump over a period of 15 to25 seconds. All drug solutions were prepared to achieve a10-JJ.L final delivery volume. Drug administration was followedby 10 /xL of saline flush within 15 seconds to clear the catheterof drugs.

Experimental Paradigms for Drug Administration(1) Agonist Dose-Response Relations. Dose-response curves

for agonists were developed by administration of agonists at10-fold increases in dose every 25 to 35 minutes. During thistime the three parameters measured always returned tobaseline.

(2) Agonist Desensitization. After the administration of thehighest dose in paradigm 1, desensitization to agonists for thethree parameters was evaluated by repeated administration ofthe highest dose at 25- to 35-minute intervals.

(3) Antagonist Specificity to Spinal Nicotinic Agonist-ElicitedResponses. The effect of a near maximal dose (50 /j,g IT) of theantagonist, determined previously,13 was used to block theresponse elicited by 5 /xg of agonist. Since cytisine showeddesensitization, antagonism of cytisine responses was deter-mined with separate rats and compared with vehicle-treatedrats receiving cytisine only.

Spinal Cord Membrane PreparationMembranes were prepared according to the procedure de-

scribed earlier.15 Briefly, a 5-cm (1 cm from the sacral end)segment of the spinal cord was dissected and placed in apolypropylene tube. For measurement of [3H]cytisine bindingin the dorsal lumbar and ventral lumbar regions and theintermediolateral spinal cord, the 5-cm spinal cord segmentwas dissected into a 2-cm segment of lumbarosacral and 3-cmsegment of intermediolateral spinal cord. The lumbarosacralspinal cord was dissected into dorsal and ventral portions. Alltissue sections were stored at — 70°C.

Spinal cord sections were homogenized in ice-cold 50mmol/L Tris-HCl buffer, pH 7.4.15 The homogenate was cen-trifuged at 48 OOOg for 10 minutes; the pellet was resuspendedin fresh buffer and centrifuged a second time; and the finalpellet was resuspended in fresh buffer.

Equilibrium Binding AssaysEquilibrium binding assays were conducted according to the

procedures described earlier.15 Briefly, the assay mixture con-sisted of 400 to 600 /xg of membrane protein in a finalincubation volume of 125 /AL. Final concentrations of[3H]cytisine varied between 0.05 and 10 nmol/L (250 to 50 000cpm); stock solutions were prepared in assay buffer. Incuba-tions were carried out in a cold room (4°C) with gentle shakingfor 60 minutes. Assays were initiated by the addition of themembrane suspension with rapid mixing to the [3H]cytisinesolutions in a polypropylene tube. The incubations were termi-nated by dilution with 3 mL ice-cold assay buffer immediatelyfollowed by rapid filtration under vacuum through WhatmanGF/C filter papers previously equilibrated with 0.5% polyeth-yleneimine at 4°C. Filters were then rinsed three times with 3mL of ice-cold buffer. Specific binding was determined as thedifference in binding between samples containing excess unla-beled /-nicotine (40 /xmol/L) and those containing only [3H)cy-tisine. Protein was assayed by the bicinchoninic acid proteinassay.

DrugsThe following chemicals were obtained from Sigma Chemi-

cal Co: /-nicotine, cytisine, atropine sulfate, and mecamyl-amine. Methyllycaconitine (MLA) and dihydro-/3-erythroidine(D/3E) were from Research Biochemicals International.

StatisticsAll values presented are mean±SEM. Student's t test for

unpaired data was used to determine differences between twotreatment groups. Differences between multiple groups werecompared using ANOVA.

ResultsBasal Systolic Blood Pressure and Heart Rate inSHRU and WKYLJ Rats

Basal systolic blood pressure before each pharmaco-logical intervention was significantly higher in SHRLJ

(194±2 mmHg, n=54) than in age-matched WKYLJ

littermates (139±1 mmHg, n=49, /><.0001) over thecourse of this study. Similarly, basal heart rate wasslightly but significantly elevated in SHRLJ (464±5 beatsper minute [bpm]) compared with WKYLJ rats (446 ±4bpm, P<.007). In the animal subgroups used in thedifferent experimental paradigms, systolic blood pres-sure was always significantly higher in SHRLJ than inage-matched WKYLJ rats. However, heart rate was notsignificantly elevated in SHRLJ over age-matchedWKYLJ rats; greater significance might be achieved witha larger sample size.

Cardiovascular and Behavioral Responses to SpinalNicotinic Agonists in SHRLJ and WKYLJ Rats

Basal systolic blood pressures and heart rates inSHRU (n = 19) and WKYLJ rats (n = 17) used for thisprotocol were 190±2 and 136±2 mm Hg (P<.001) and478±8 and 453±7 bpm (P<.02), respectively.

Intrathecal nicotine produced a dose-dependent in-crease in blood pressure, heart rate, and irritationresponses in both SHRU and WKYLJ rats; however, thenicotine-elicited rises in blood pressure and heart rate



Khan et al SHR and Spinal Nicotinic Receptors 613

1 10Nicotine (nmoles, i.t.)

FIG 1. Line graphs show changes in systolic blood pressure(SBP), heart rate, and irritation index after intrathecal injection of0.05, 0.5, and 5.0 ^g (-)-nicotine in spontaneously hypertensive(O, n=10) and Wistar-Kyoto (A, n=8) rats. Responses of SBPand heart rate to 5.0 /xg IT nicotine and irritation responses to 0.5fig IT nicotine, respectively, in Sprague-Dawley rats (o, n=6) arealso shown.13 Each value represents mean±SEM. *P<.001,tP<.01, §P<.05.

were greater in SHRLj compared with WKYLJ rats, withthe largest difference observed at 11 nmol (5 /xg) ofadministered nicotine (Fig 1A and IB). The pressor andheart rate responses elicited by nicotine were also sig-nificantly higher than those found in SD rats (also at 5pig nicotine).13

In contrast to the cardiovascular responses, the irrita-tion response to spinal nicotine did not differ betweenthe three strains at higher doses of the agonist (Fig 1C).However, SHRLJ were more sensitive than the normo-tensive controls in producing nicotine-elicited behav-ioral responses at the lowest dose of 0.05 pig. Althoughall three rat strains exhibited maximal irritation scores of4 at the highest drug dose, the irritation response inSHRLJ appeared most intense to the evaluator.

Similar to nicotine, intrathecal cytisine also elicitedaugmented pressor responses in SHRLJ compared withnormotensive rats (Fig 2A); however, the largest differ-ence was observed between SHRLJ and normotensiverats at 2.6 nmol (0.5 /xg) cytisine. Although in ourprevious study a leveling off of the dose dependence inpressor response in SD rats was not observed, in thepresent study the maximal attainable increase in systolicblood pressure was observed by 0.5 jag cytisine in bothSHRLJ and WKYLJ rats (Fig 2A). The heart rate re-sponse to cytisine in SHRLJ was not greater than in

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FIG 2. Line graphs show changes in systolic blood pressure(SBP), heart rate, and irritation index after intrathecal injection of0.05, 0.5, and 5.0 fig cytisine in spontaneously hypertensive (•,n=9) and Wistar-Kyoto (A, n=9) rats. Effects of 0.5 and 5.0 j ig ITcytisine on SBP and 0.05 ixg IT cytisine on irritation responses inSprague-Dawley rats (•, n=5) are also shown.13 Each valuerepresents mean±SEM. *P<.001, fP<-002, §P<.005, HP<.01,#P<.02, "P<.05.

WKYLJ rats (Fig 2B). SHRLJ were more sensitive to thecytisine-elicited nociceptive response, similar to the nic-otine responses, than either WKY or SD rats at thelowest dose (0.05 jug) (Fig 2C).

Effects of Repeated Intrathecal Administration ofNicotine and Cytisine on Cardiovascular andBehavioral Responses in SHRLJ and WKYLJ Rats

We previously demonstrated13 that in SD rats sequen-tial administrations of nicotine did not cause appreciabledesensitization to the agonist-elicited responses. As inSD rats, repeated administrations of intrathecal nicotinein both SHRLJ and WKYLJ rats did not cause a signifi-cant decrease in the magnitude of any of the responses(data not shown). However, repeated administrations ofcytisine revealed a marked decrease in the magnitude ofthe responses in SD rats,13 and in the present study bothSHRLJ and WKYLJ rats exhibited desensitization of allthree responses to repeated dosing of cytisine. Thepropensity for desensitization of the three responses,particularly the irritation response, was diminished inthe SHRLJ strain compared with normotensive strains.There was a marked reduction in the magnitude of thethree responses in all rat groups with the second dose of26 nmol (5 /xg) of intrathecal cytisine (Fig 3A through3C). However, on the third dose, the two normotensive




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FIG 3. Bar graphs show effects of repeated intrathecal admin-istration of 5.0 ;u.g cytisine on systolic blood pressure (SBP),heart rate, and behavioral responses in spontaneously hyperten-sive (open bars, n=9), Wistar-Kyoto (hatched bars, n=8), andSprague-Dawley (shaded bars, n=4)13 rats. Each value repre-sents mean±SEM. Cytisine was administered at intervals of 25to 30 minutes. *P<.002, tP<.02, §P<.03, HP<.04, #P<.05.

strains showed further blunting of the cytisine-elicitedirritation, whereas the nociceptive response in SHRLJ

did not show a further significant decrease. The irrita-tion response to cytisine on the third dose was signifi-cantly higher in SHRLJ than in the other two rat groups(Fig 3C). Similarly, the pressor and heart rate responseswere also significantly higher in SHRLJ (Fig 3A and 3B).

Effect of Nicotinic Receptor Antagonists onNicotine-Elicited Cardiovascular and BehavioralResponses in SHRLJ and WKYLJ Rats

Basal systolic blood pressures and heart rates inSHRLJ (n=17) and WKYLJ rats (n=14) used for thisprotocol were 194±3 and 141 ±2 mm Hg (P<.001) and464±9 and 451 ±6 bpm (P>.05), respectively. The ionchannel blocker mecamylamine almost completely abol-ished the three responses to intrathecal nicotine in bothSHRLJ and WKYLJ rats (Table 1). Similarly, the com-petitive nicotinic antagonist D/3E also significantly inhib-ited the responses to nicotine in both rat strains (Table1). Likewise, MLA, which appeared to exhibit mixedcompetitive and noncompetitive (or channel) block-ade,1315 also significantly blocked the cardiovascular andbehavioral responses to nicotine in the two rat groups(Table 1). The data were essentially similar to thoseobtained in SD rats13 for nicotine and the three nicotinicreceptor antagonists. None of the antagonists lowered

blood pressure or heart rate in SHRLJ or WKYLJ rats(data not shown).

D/3E and MLA Antagonism of Intrathecal Cytisine-Elicited Responses in SHRLJ and WKYLJ Rats

Basal systolic blood pressures and heart rates inSHRLJ (n=17) and WKYLJ (n = 17) rats used for thisprotocol were 197±3 and 140±2 mm Hg (i><.001) and449±9 and 434±7 bpm (P>.05), respectively.

Prior treatment with D/3E did not affect the maximalcytisine-elicited responses (within 1 to 4 minutes aftercytisine administration) in SHRLJ or WKYLJ rats (Table2). However, similar to the situation observed in SDrats,13 this competitive antagonist significantly blockedthe pressor and heart rate responses to intrathecalcytisine for the first minute of the response after agonistadministration (Fig 4A and 4B) in SHRLJ. In addition,complete manifestation of cytisine-induced nociceptiveresponse after D/3E was also delayed to the third minutecompared with untreated SHRLJ (Fig 4C). In SHRLJwithout antagonist, the pattern of cytisine-elicited rise inblood pressure and heart rate was similar to that in SDrats; however, the score of 4 for the irritation responsewas already evident by the first minute (Fig 4A through4C). Thus, in SHRLJ the irritation response, by virtue ofits more rapid onset, may also contribute to the in-creased pressor response in the first minute.

In WKYLJ rats, D/3E blocked the tachycardia only inthe second minute after cytisine (Fig 4E). The onset ofthe irritation response, similar to that in SD rats (un-published observations), was delayed until the secondminute (Fig 4F), and D/3E blocked only the rise inirritation occurring in the second minute. Dj3E had noeffect on the pressor response to intrathecal cytisine inWKYLJ rats (Fig 4D). It appears that the initial pressorresponse to cytisine in WKYLJ rats is less sensitive toD/3E than in SHRLJ and SD rats.

Similar to its effect on cytisine-elicited responses inSD rats, MLA significantly inhibited the cardiovascularand behavioral responses to cytisine in WKYLJ rats(Table 2, Fig 5D through 5F). However, cytisine-elicitedmaximal increases in pressor, heart rate, and irritationresponses in SHRLJ were not blocked by MLA at thisdose. Although the maximal rises in blood pressure,heart rate, and irritation responses to intrathecal cytisinewere not blocked in SHRLJ, MLA significantly inhibitedthe rise in blood pressure up until the second minute andthe rise in heart rate and the irritation response up untilthe third minute after cytisine administration (Fig 5Athrough 5C). The data indicate that MLA exhibits aslightly different pharmacological specificity in blockingspinal nicotinic receptors in SHRLJ compared with SD orWKYLJ rats.

[3H] Cytisine Binding in Spinal Cord Membranes ofSHRU and WKYLJ Rats

As shown in Fig 6, [3H]cytisine showed saturablebinding to a single class of binding sites with no coop-erativity in spinal cord membranes of both SHRLJ andWKYLJ rats. Although the affinity of [3H]cytisine for thebinding sites did not differ between the two rat strains,the total number of sites was higher in the normotensiveWKYLJ rats. The total number of binding sites in thewhole spinal cord (5-cm segment) in WKYU rats did notdiffer significantly from that in SD rats.15




TABLE 1. Effects of Mecamylamine, Dihydro-/3-erythroidine, and Methyllycaconitine onMaximal Pressor, Heart Rate, and Behavioral Responses Induced by 5 /*g IT Nicotine inConscious SHRLJ and WKYLJ Rats

Parameter

Systolic blood pressure

Mecamylamine

Dihydro-/3-erythroidine

Methyllycaconitine

Heart rate

Mecamylamine


Methyllycaconitine

Irritation index

Mecamylamine


Methyllycaconitine

Response Remainingin SHRLJ, %

7±3 (n = 5)*

22±10 (n=7)t

24±13(n=5)§

21±14§

21±14||

19±8||

5±5*

27±10t

35±13±

Response Remainingin WKYLJ Rats, %

8±5 (n=5)t

26±11 (n=5)t

13±4(n=4)§

11 ±8 *

6±9$

36±13[|

20±12t

13±10f

44±12§

SHRLj indicates spontaneously hypertensive rats, La Jolla strain; WKYLJ, Wistar-Kyoto rats, La Jollastrain. Each value represents percentage of response compared with responses before antagonistadministration (each 50 /xg IT).

*P<.0001, fP<-0003, $P<001, §P<.004, ||P<.03 compared with saline-pretreated rats.

The total number and affinity of the nicotinic recep-tors in various regions of the spinal cord were alsodetermined in SHRLJ and WKYLJ rats (Table 3). Theaffinity of the receptors did not differ significantly be-tween strains. The larger number of binding sites wasfound in the dorsal lumbarosacral region followed by theintermediolateral region in both SHRLJ and WKYLJ rats.The lowest number of binding sites was observed in theventral lumbarosacral region (Table 3). Similar to thewhole spinal cord, fewer binding sites were found inSHRLJ than in WKYLJ rats in all three regions. Nodifference in protein concentration in any of the spinalcord regions was observed between the two rat groups(data not shown).

DiscussionIntrathecal administration of nicotine and cytisine

elicits dose-dependent increases in blood pressure and

heart rate and results in the manifestation of nociceptiveresponses in both SHRLJ and age-matched normotensiveWKYLJ rats. These qualitative observations are similarto those we reported for SD rats.13 The pressor andheart rate responses to nicotine and the pressor re-sponse to cytisine were exaggerated in SHRLJ comparedwith either WKYLJ or SD normotensive rats. AlthoughSHRLJ did not show a potentiated nociceptive responseto either nicotine or cytisine at the highest dose used (5fig), the behavioral response was greater in SHRLJ at a0.05-/xg dose of either agonist compared with bothnormotensive rat strains. As in SD rats, the duration ofaction of cytisine was longer than nicotine in both SHRLJ

and WKYLJ rats.Cholinergic components of the central nervous system

have been implicated in the regulation of cardiovascularand behavioral responses. Hyperactivity in this compo-

TABLE 2. Effects of Dihydro-/3-erythroidine and Methyllycaconitine on Maximal Pressor,Heart Rate, and Behavioral Responses Induced by 5 M 9 IT Cytisine in Conscious SHRLJ

and WKYLJ Rats

Parameter

Systolic blood pressure


Methyllycaconitine

Heart rate


Methyllycaconitine

Irritation index

Dihydro-0-erythroidine

Methyllycaconitine

Response Remainingin SHRLJ, %

79±9 (n=5)

90±9 (n=5)

91 ±19

72±14

100

100

Response Remainingin WKYLJ Rats, %

88±11 (n=6)

51 ±11 (n=5)*

84±11

54±13f

100

53 ±8$

Definitions are as in Table 1. Each value represents percentage of response compared withresponses in the absence of antagonist (each 50 yjg IT).

*P<.01, tP<-015, tP<.001 compared with saline-pretreated rats.




SHR, WKY,

FIG. 4. Line graphs show dihydro-/3-erythroidineantagonism of cytisine-elicited systolic blood pres-sure (SBP) (A and D), heart rate (B and E), andbehavioral (C and F) responses in spontaneouslyhypertensive rats (SHRLJ) and Wister-Kyoto(WKYLJ) rats, respectively. Closed and open circlesrepresent control (n=7) and treated (n=5) SHRLJ

rats; closed and open triangles represent control(n=5) and treated (n=6) WKYLJ rats, respectively.Horizontal axis represents values measured duringthe first, second, and third minutes after a singleintrathecal administration of cytisine (5 /xg). Eachvalue represents mean±SEM. *P<.006, tP<-008,§P<.02, HP<.05 compared with control responsesat the respective time points.

1st min 2nd min 3rd minTime

1st min 2nd min 3rd minTime

FIG 5. Line graphs show methyllycaconitine an-tagonism of cytisine-elicited systolic blood pres-sure (SBP) (A and D), heart rate (B and E), andbehavioral (C and F) responses in spontaneouslyhypertensive rats (SHRLJ) and Wistar-Kyoto(WKYLJ) rats, respectively. Closed and open circlesrepresent control (n=7) and treated (n=5) SHRLJ

rats; closed and open triangles represent control(n=5) and treated (n=6) WKYLJ rats, respectively.Horizontal axis represents values measured duringthe first, second, and third minutes after a singleintrathecal administration of cytisine (5 jig). Eachvalue represents mean±SEM. *P<.002, tP<-003,§P<.001, HP<.01, #P<.05 compared with controlresponses at the respective time points.

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20

FIG 6. Line graph shows Scatchard analysis of saturationbinding of [3H]cytisine to spinal cord membrane preparationsfrom spontaneously hypertensive (o) and Wistar-Kyoto (•) rats.Each analysis of saturation binding was done with pooled spinalcord membranes from five or six rats. These binding profiles arerepresentative of the four tabulated in Table 3.

nent has been seen in several rat models of hyperten-sion.128 Central administration of cholinergic agonists inboth the brain and spinal intrathecal space has led toexaggerated pressor responses in SHR compared withnormotensive rats.4-6 Most of the studies have focusedon the role of muscarinic cholinergic receptors. Ourstudy shows that nicotinic cholinergic receptor stimula-tion in the spinal cord also results in an augmentedcardiovascular response in a hypertensive rat model. Inaddition, our study establishes that spinal nicotinic re-ceptor stimulation leads to a potentiated nociceptiveresponse in the SHR. This finding may relate to theinvolvement of environmental factors and stress in thepathogenesis of hypertension in SHR.1719

We have shown previously that the pressor and be-havioral responses elicited by nicotinic agonists aremediated through two distinctly located and indepen-dent receptor-linked pathways.13-14 Nicotine and cytisineshow differential stimulatory capacities for pressor andnociceptive responses. The major portion of the pressorresponse to nicotine is mediated by nicotinic receptorstimulation in the thoracic spinal cord of sympatheticoutput, whereas the nociceptive response to spinal nic-otine requires an intact bulbospinal pathway.14 Thecytisine-elicited pressor response, on the other hand, hastwo components: an initial transient fast desensitizingresponse (probably resulting from nicotinic receptorstimulation in the intermediolateral region) and a sec-ondary delayed pressor response of longer durationassociated with the onset of the irritation or nociceptiveresponse.1314 This component is mediated through thespinobulbar pathway.14 Moreover, the irritation re-

sponse to spinal cytisine, in contrast to nicotine, is notsensitive to competitive nicotinic receptor antagonists.Although the heart rate response is variable, in mostexperimental conditions the increased heart rate ap-pears to correlate with the irritation response.

As in SD rats, repetitive intrathecal administration ofnicotine in both SHRLJ and WKYLJ rats did not revealdesensitization of the pressor, heart rate, and nocicep-tive responses. However, with repetitive cytisine admin-istration, the three rat strains exhibited desensitization.Of the three strains, the SHRLJ showed a diminishedpropensity to desensitize. Reduced desensitization wasmost evident with the irritation response.

In addition to the slow rate of desensitization tocytisine-elicited responses, SHRLJ showed a very rapidonset of cardiovascular and behavioral responses tointrathecal cytisine. The maximal rise in blood pressure,heart rate, and nociceptive responses occurred withinthe first minute after cytisine administration (Fig 4Athrough 4C). However, in WKY rats, the onsets of themaximal rise in blood pressure, heart rate, and irritationresponses were delayed (Fig 4D through 4F). Interest-ingly, SD rats showed onset profiles to cytisine-elicitedpressor and heart rate responses similar to those inSHRLJ but showed a delayed onset of the irritationresponse.13 Thus, SHRLJ show different rates of onsetand desensitization of responses to cytisine than SD orWKYLJ rats.

Nicotinic receptor antagonists exhibited inhibitoryprofiles to nicotine in SHRLJ and WKYLJ rats resem-bling those in SD rats.13 However, the competitivenicotinic antagonists D/3E and MLA produced interest-ing differences for antagonism of cytisine-elicited re-sponses in these two rat groups. In contrast to theresponses in SD and SHRLJ rats, in WKYLJ rats D|3E didnot block the initial rise in blood pressure after intra-thecal cytisine administration. This may indicate thatD/3E sensitivity for spinal nicotinic receptors may differin WKY rats. Alternatively, as shown in the dose-response curve for cytisine in WKYLJ rats (Fig 2A),the maximal pressor response may be saturated by the0.5-/xg dose of cytisine. This may also be true forthe initial pressor response to intrathecal cytisine. Thus,the inhibitory effect of Dj3E may not be as apparent witha 5-/xg dose of cytisine, because its response lies near themaximal plateau of the cytisine dose-response curve.D/3E blocked the initial rise in irritation response in bothSHRLJ and WKYLJ rats, suggesting that cytisine and

TABLE 3. Equilibrium Dissociation Constants (KJ and Total Saturable Sites (Smax) of fHJCytisine Binding toMembrane Preparations From Various Spinal Cord Regions of SHRLJ and WKYLJ Rats

Spinal Cord Region

Whole (5-cm) segment

Dorsal lumbarosacral

Ventral lumbarosacral

Intermediolateral cell column

Ka, nmol/L

SHRLJ

0.70±0.14

1.08±0.34

0.85±0.10

0.82±0.30

WKYLJ

0.59±0.12

1.05 ±0.34

0.70±0.20

0.66±0.20

SHRLJ

13.1 ±0.1

18.1+0.9

11.5+0.5

12.9+0.4

, fmol/mg Protein

WKYLJ

17.2±1.2*

22.4±0.9+

14.1+0.6+

16.0±0.7t

Definitions are as in Table 1. Standard error represents three to four measurements on separate preparations. Each preparationwas made from pooled spinal cord membranes from six to nine rats.

*P<.01, tP<02 , +P<.04 compared with SHRLJ for the same spinal cord region.




nicotine share a common mechanism for eliciting theinitial portion of the irritation response.

In contrast to D/3E, MLA blocked significantly allthree responses to intrathecal cytisine in WKYLJ rats. InSHRLJ rats, MLA did not block the maximal rise inblood pressure, heart rate, and irritation responses.However, it significantly inhibited the initial rise in bloodpressure and heart rate responses after cytisine admin-istration. In addition, it delayed manifestation of theirritation response in the first 2 minutes after cytisineadministration. Thus, MLA antagonizes a different com-plement of nicotinic receptors in the spinal cord.

Interestingly, none of the nicotinic receptor antago-nists had any effect on basal heart rate and bloodpressure in SHRLJ or normotensive rats, suggesting thatspinal nicotinic receptors may not have a dominant rolein maintaining higher basal blood pressure in SHRLJ. Itis well documented that sympathetic nerve activity iselevated in SHR compared with WKY rats.2022 Inaddition, the baroreceptor reflex is blunted in SHR.23

Although altered sympathetic activity of central nervoussystem origin is known to be involved in the pathogen-esis of hypertension in SHR, the blunted baroreceptoractivity in SHR compared with WKY rats is evident afterthe onset of hypertension in SHR.2122 As already men-tioned, intrathecal nicotine administration elicits pressorand tachycardic responses by increasing the spinal sym-pathetic outflow. As such, in both SHR and WKY rats,the nicotinic agonists would lead to increased bloodpressure and heart rate. A blunted baroreceptor reflex,enhanced sympathetic output, and vascular reactivity inSHR may also contribute to exaggerated responses inhypertensive versus normotensive rats. Enhanced pres-sor responses to central administration of several pep-tide agonists are also observed in SHR compared withnormotensive rats.2426 Also, enhanced depressor re-sponses to amino acids administered in particular brainregions have been documented in SHR relative to WKYrats.27-28

We found a lower density of nicotinic receptors inspinal cord membranes in SHR than in SD or WKY rats.Yamada et al12 observed similar decreases in nicotinicreceptor number in various brain regions, including themedulla oblongata, of stroke-prone SHR compared withWKY rats. Microinjections of nicotine or acetylcholinein various regions of the medulla, including the dorsalmedulla, nucleus of the solitary tract, and area postrema,result in a decrease in blood pressure and heartrate.541'29 No differences in the responses were observedbetween SHR and normotensive rats.5 However, Tsenget al5 demonstrated that microinjections of nicotine inthe rostral ventrolateral medulla produce a dose-depen-dent increase in blood pressure and heart rate in SHRand WKY and SD rats that is blocked by hexametho-nium. Moreover, the pressor and tachycardic responsesto nicotine were augmented in SHR compared with thenormotensive rats. Thus, similar to our observations inspinal cord, an augmented pressor and heart rate re-sponse to nicotinic receptor stimulation in the presenceof a decreased number of nicotinic receptors could beobserved in a different region of the rat central nervoussystem. The fact that the rostral ventrolateral medulla issensitive to nicotinic agonists that elicit cardiovascularresponses is noteworthy because this region of the brainhas been implicated in the tonic and reflex regulation of

blood pressure.3031 Moreover, direct innervation fromthis brain region to the intermediolateral region in thespinal cord has been documented.30-32 Thus, it appearsthat the two separate sites in the brain and spinal cordinvolved in the neuronal circuitry of regulating tonic andreflex cardiovascular responses are sensitive to nicotine.Moreover, nicotinic receptor stimulation in the two siteselicits similar augmented cardiovascular responses inSHR.

Increased cholinergic activity in various brain regionshas been demonstrated in SHR.3334 Moreover, inhibi-tion of the cholinergic system by intracerebroventricularhemicholinium-3 treatment led to a fall in blood pres-sure in hypertensive rats.1'7 Thus, the lower nicotinicreceptor numbers in the brain regions of hypertensiveversus normotensive rats could be explained by a feed-back mechanism whereby SHR compensate for theincreased cholinergic sensitivity in the central nervoussystem by downregulating cholinergic receptors duringthe onset of hypertension.

Compared with higher central nervous system centers,little information is available for the cholinergic activityand receptors in spinal cord of SHR. The densities of thenicotinic receptors in the dorsal lumbarosacral, ventrallumbarosacral, and intermediolateral regions, althoughlower in SHRLJ, have a regional distribution similar tothat in WKYLJ rats. Nicotinic receptor number is highestin the dorsal lumbarosacral region, followed by theintermediolateral and ventral lumbarosacral sections ofthe spinal cord in both rat strains (Table 3). Moreover,we have shown previously that the intermediolateralregion is most sensitive to intrathecal nicotinic agonistsin eliciting the pressor response, whereas the entirelength of the thoracolumbar spinal column mediates theirritation response to intrathecal nicotinic agonists.14

The fact that the SHRLJ is the strain most sensitive to thenicotinic agonist-elicited irritation response suggests anenhanced amplification process of the postcouplingevents in the dorsal lumbar spinal cord to increasesympathetic activity in the SHRLJ. It will be of interest toascertain whether the decreased receptor number pre-cedes the hypertension or is a feedback manifestation ofthe apparent enhanced amplification of spinal cholin-ergic stimulation.

AcknowledgmentsThis work was supported by the University of California

Tobacco Related Disease Research Program and grant HL-35018 from the US Public Health Service to P. Taylor.

References1. Giuliano R, Brezenoff HE. Increased central cholinergic activity

in rat models of hypertension. J Cardiovasc Pharmacol.1987;10:113-122.

2. McCaughran JA, Murph D, Schechter N, Friedman R. Partici-pation of the central cholinergic system in blood pressure regu-lation in the Dahl rat model of essential hypertension. / CardiovascPharmacol. 1983;5:1005-1009.

3. Buccafusco JJ, Spector S. Role of central cholinergic neurons in exper-imental hypertension. J Cardiovasc Pharmacol. 1980;2:347-355.

4. Hoffman WE, Schmid PG, Phillips MI. Central cholinergic andnoradrenergic stimulation in spontaneously hypertensive rats. /Pharmacol Exp Ther. 1978;206:644-651.

5. Tseng C-J, Appalsamy M, Robertson D, Mosqueda-Garcia R.Effects of nicotine on brain stem mechanisms of cardiovascularcontrol. J Pharmacol Exp Ther. 1993;265:1511-1518.




6. Buccafusco JJ, Magri V. The pressor response to spinal cholinergicstimulation in spontaneously hypertensive rats. Brain Res Bull.1990;25:69-74.

7. Vargas HM, Brezenoff HE. Suppression of hypertension duringchronic reduction of brain acetylcholine in spontaneously hyper-tensive rats. J Hypertens. 1988;6:739-745.

8. Brezenoff HE, Caputi AP. Intracerebroventricular injection ofhemicholinium-3 lowers blood pressure in conscious spontaneouslyhypertensive rats but not in normotensive rats. Life Sci.1980;26:1037-1045.

9. Caputi AP, Camilleri BH, Brezenoff HE. Age-related hypotensiveeffect of atropine in unanesthetized spontaneously hypertensiverats. Eur J Pharmacol. 1980;66:103-109.

10. Brezenoff HE, Vargas H, Xaio Y-I. Blockade of brain M2-muscarinicreceptors lowers blood pressure in spontaneously hypertensive rats.Pharmacology. 1988;36:101-105.

11. Kubo T, Misu Y. Changes in arterial blood pressure after micro-injections of nicotine into the dorsal area of the medulla oblongataof the rat. Neuropharmacology. 1981;20:521-524.

12. Yamada S, Kagawa Y, Ushijima H, Takayanagi N, Tomita T,Hayashi E. Brain nicotinic cholinoreceptor binding in spontaneoushypertension. Brain Res. 1987;410:212-218.

13. Khan IM, Taylor P, Yaksh TL. Cardiovascular and behavioralresponses to nicotinic agents administered intrathecally. JPharmacol Exp Ther. 1994;270:150-158.

14. Khan IM, Taylor P, Yaksh TL. Stimulatory pathways and sites ofactions of intrathecally administered nicotinic agents. / PharmacolExp Ther. In press.

15. Khan IM, Yaksh TL, Taylor P. Ligand specificity of nicotinic ace-tylcholine receptors in rat spinal cord: studies with nicotine andcy'tisine. J Pharmacol Exp Ther. 1994;270:159-166.

16. Yaksh TL, Rudy AT. Chronic catheterization of the spinal sub-arachnoid space. Physiol Behav. 1976;17:1031-1036.

17. Brody MJ, Haywood JR, Touw KB. Neural mechanisms in hyper-tension. Annu Rev Physiol. 1980;42:441-453.

18. Hallback M. Interaction between central neurogenic mechanismsand changes in cardiovascular design in primary hypertension. AdaPhysiol Scand Suppl. 1975;424:l-59.

19. Yamori Y, Matsumoto M, Yamabe H, Okamoto K. Augmentationof spontaneous hypertension by chronic stress in rats. Jpn Circ J.1969;33:399-409.

20. Judy WV, Watanabe AM, Henry DP, Besch HR, Murphy WR,Hockel GM. Sympathetic nerve activity: role in regulation of bloodpressure in the spontaneously hypertensive rat. Circ Res. 1976;38(suppl II):II-21-II-29.

21. Judy WV, Watanabe AM, Murphy WR, Aprison BS, Yu P-L.Sympathetic nerve activity and blood pressure in normotensivebackcross rats genetically related to the spontaneously hypertensiverat. Hypertension. 1979;l:598-604.

22. Judy WV, Farrell SK. Arterial baroreceptor reflex control of sym-pathetic nerve activity in spontaneously hypertensive rat.Hypertension. 1979;l:605-614.

23. Sapru HN, Wang SC. Modification of aortic baroreceptor resetting inthe spontaneously hypertensive rat. Am J Physiol. 1976;230:664-674.

24. Martins DTO, Fior DR, Nakaie CR, Lindsey CJ. Kinin receptors ofthe central nervous system of spontaneously hypertensive ratsrelated to the pressor response to bradykinin. Br J Pharmacol.1991;103:1851-1856.

25. Lindsey CJ, Fujita K, Martins TO. The central pressor effect ofbradykinin in normotensive and hypertensive rats. Hypertension.1988;ll(suppl I):I-126-I-129.

26. Lowes VL, Ferguson AV. Modified cardiovascular sensitivity of thearea postrema to vasopressin in spontaneously hypertensive rats.Brain Res. 1994;636:165-168.

27. Smith JK, Barron KW. GABAergic responses in ventrolateral medullain spontaneously hypertensive rats. Am J Physiol. 1990;258:R450-R456.

28. Smith JK, Barron KW. Cardiovascular effects of 1-glutamate andtetrodotoxin microinjected into the rostral and caudal ventrolateralmedulla in normotensive and spontaneously hypertensive rats.Brain Res. 1990;506:l-8.

29. Criscione L, Reis DJ, Talman WT. Cholinergic mechanism in thenucleus tractus solitari and the cardiovascular regulation in rat. EurJ Pharmacol. 1983;88:47-55.

30. Reis DJ, Morrison S, Ruggiero DA. The C, area of the brainstemin tonic and reflex control of blood pressure. State of the art lecture.Hypertension. 1988;ll(suppl I):I-8-I-13.

31. Sun M-K, Guyenet PG. Hypothalamic glutamatergic input to med-ullary sympathoexcitatory neurons in rats. Am J Physiol.1986;251:R798-R810.

32. Granata AR, Ruggiero DA, Park DH, Joh TH, Reis DJ. Brain stemarea with C, epinephrine neurones mediates baroreflex vasode-pressor responses. Am J Physiol. 1985;248:H547-H567.

33. Trimarchi GR, Buccafusco JJ. Changes in regional brain synap-tosomal high affinity choline uptake during the development ofhypertension in spontaneously hypertensive rats. Neurochem Res.1987;12:247-254.

34. Helke CJ, Muth EA, Jacobowitz DM. Changes in central cho-linergic neurons in the spontaneously hypertensive rat. Brain Res.1980;188:425-436.



I M Khan, M P Printz, T L Yaksh and P TaylorAugmented responses to intrathecal nicotinic agonists in spontaneous hypertension.

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 1994 American Heart Association, Inc. All rights reserved.

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JPET #211235

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Title Page

METHAMPHETAMINE-LIKE DISCRIMINATIVE-STIMULUS EFFECTS OF NICOTINIC AGONISTS

Rajeev I. Desai and Jack Bergman

Preclinical Pharmacology Laboratory (RID, JB)

McLean Hospital/Harvard Medical School

115 Mill Street, Belmont, MA 02478, USA

JPET Fast Forward. Published on January 3, 2014 as DOI:10.1124/jpet.113.211235

Copyright 2014 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on January 3, 2014 as DOI: 10.1124/jpet.113.211235

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Running Title Page

Running title: Stimulant-like effects of nicotinic agonists

Correspondence:

Rajeev I Desai Preclinical Pharmacology Laboratory, McLean Hospital/Harvard Medical School 115 Mill Street, Belmont, MA 02478, USA E-mail: [email protected] Phone: 617-855-3303 FAX: 617-855-2417

Document statistics:

Pages: 41

Tables: 3

Figures: 6

References: 56

Abstract: 245 words

Introduction: 738 words

Discussion: 1495 words

Non-standard abbreviations: ANOVA, Analysis of Variance; DA, dopamine; DHβE, dihydro-β-erythroidine hydrobromide; ED50, Effective Dose50; FR, Fixed-ratio; LEDs, Light-emitting diodes; MA, d-methamphetamine; TO, Time-out; nAChR, nicotinic acetylcholine receptors


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ABSTRACT Nicotine recently was shown to engender methamphetamine (MA)-like discriminative-stimulus

effects in rats, which may be indicative of shared psychomotor stimulant properties. To further

investigate such overlapping discriminative-stimulus effects, nicotinic agonists varying in

efficacy and selectivity were studied in squirrel monkeys that discriminated a moderate i.m. dose

of MA (0.1 mg/kg) from vehicle. These included α4β2-selective ligands that may vary in

efficacy from relatively high [nicotine, (+)- and (-)-epibatidine] to relatively low [isoarecolone,

varenicline, (-)-cytisine, (-)-lobeline] and the α7-selective ligands anabaseine and anabasine.

Results show that nicotine, (+)-epibatidine, and (-)-epibatidine substituted fully for MA, whereas

the highest doses of other nicotinic agonists produced intermediate levels of MA-like effects

(isoarecolone, anabaseine, anabasine, and varenicline) or did not [(-)-cytisine and (-)-lobeline]

substitute for MA. The relative potencies of nicotinic agonists, based on ED50 values,

corresponded better with their relative affinities at α4β2 than α7 receptors. Regardless of

selectivity or efficacy, nicotinic agonists also were observed to produce untoward effects

including salivation and emesis during or after experimental sessions. In pretreatment studies,

the α4β2-selective antagonist dihydro-β-erythroidine hydrobromide (DHβE: 0.032 and 0.1

mg/kg) and the partial agonists varenicline (0.0032–0.1 mg/kg) and (-)-cytisine (0.032 and 0.1

mg/kg) surmountably antagonized (>10-fold rightward shift) nicotine’s MA-like effects but were

ineffective in blocking nicotine’s emetic effects. Overall, our results show that: 1) MA-like

discriminative-stimulus effects of nicotinic agonists likely are mediated through α4β2 nAChR

actions and 2) nicotinic α4β2 partial agonists, like the nicotinic antagonist DHβE, can reduce

MA-like behavioral effects of nicotine.


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INTRODUCTION

The discriminative-stimulus effects of nicotine have been widely characterized in

laboratory animals (e.g., Jutkiewicz, 2011; Cunningham et al., 2012; see Smith and Stolerman,

2009 for review), and have been related to subjective effects that promote its persistent

consumption among users of tobacco or other nicotine delivery devices (e.g., Smith and

Stolerman, 2009; Benowitz, 2010). Pharmacological studies with selective agonists and

antagonists additionally have identified likely mechanisms of action mediating the

discriminative-stimulus effects of nicotine. For example, such effects of nicotine are readily

mimicked by centrally acting nicotinic agonists with high affinity for the α4β2 nicotinic

acetylcholine receptor (nAChR) subtype but not by drugs that act selectively at other subtypes of

nAChR (e.g., α3β4 or α7) or by drugs from other pharmacological classes (e.g., muscarinic

agents; see Smith and Stolerman, 2009 for review). Moreover, non-competitive

(mecamylamine) and competitive (dihydro-β-erythroidine hydrobromide; DHβE)] antagonists

that block α4β2 nAChRs in CNS attenuate nicotine’s discriminative-stimulus effects, whereas

peripherally-restricted antagonists or antagonists at other nAChR subtypes (e.g., nicotinic α7,

muscarinic) are ineffective. In conjunction, such evidence strongly suggests that the

discriminative-stimulus effects of nicotine are centrally-mediated, primarily via α4β2 nAChRs

(e.g., Smith and Stolerman, 2009).

A growing body of evidence also indicates that, as with monoaminergic psychomotor

stimulant drugs [e.g., cocaine, d-amphetamine, methamphetamine (MA)], the projection from

ventral tegmental area (VTA) to the nucleus accumbens in the mesocorticolimbic dopamine

(DA) system is a key element in brain circuitry that mediates the neurochemical and behavioral


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effects of nicotine. Accordingly, increases in DA neurotransmission have been proposed to

mediate the reinforcing effects of nicotine and its consumption (e.g., Di Chiara, 2000; Smith and

Stolerman, 2009). The involvement of common neural substrates also has led to the suggestion

that nicotine and monoaminergic psychomotor stimulant drugs might engender overlapping

subjective effects and, in laboratory animals, discriminative-stimulus effects (e.g., Smith and

Stolerman, 2009). Data from some, but not all, previous studies in nicotine- and stimulant

(cocaine- or d-amphetamine)-trained subjects have supported this suggestion (see Smith and

Stolerman, 2009 for review), and have profitably advanced our understanding of the stimulant-

like effects of nicotine and other nicotinic ligands. For example, using the psychomotor

stimulant MA as a discriminative-stimulus in rats, our recent studies in rats suggest that: a)

nicotinic agonists may vary in the extent to which they produce MA-like stimulant effects; b)

α4β2 nAChR-mediated actions may play an important role in the MA-like stimulant effects of

nicotinic agonists; and c) varenicline dose-dependently antagonized the MA-like stimulant

actions of nicotine, consistent with the view that nicotinic partial agonists may help manage

nicotine addiction and tobacco consumption (Rollema et al., 2007; Desai and Bergman, 2010).

The present research was conducted to further investigate the discriminative-stimulus

effects of nicotine and related compounds. The goals of this work were to determine whether the

discriminative-stimulus effects of MA and nicotine overlap in primate species and, if so, to

examine the pharmacology of that overlap with a range of nAChR ligands. Using standard drug

discrimination procedures, squirrel monkeys first were trained to distinguish a moderate dose of

0.1 mg/kg MA from saline. Next, the effects of monoamine uptake inhibitors (MA, cocaine),

DA D1- and D2-like agonists [SKF82958, R-(-)-NPA], and a selective serotonin reuptake

inhibitor (citalopram) were tested to confirm the role of dopaminergic mechanisms in these


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effects of MA (Tidey and Bergman, 1998). This provided a pharmacologically empirical basis

for characterizing the effects of nicotinic ligands in MA-trained subjects. Subsequently,

substitution tests with a wide range of nicotinic agonists were conducted to evaluate their ability

to mimic MA’s discriminative-stimulus effects. Drugs studied included α4β2 nAChR subtype-

selective ligands previously characterized as either full agonists [nicotine, (+)-epibatidine, (-)-

epibatidine] or partial agonists [(isoarecolone, varenicline, (-)-cytisine, (-)-lobeline); Anderson

and Arenric, 1994; Baido and Daly, 1994; Hahn et al., 2003; Rollema et al., 2007]. Substitution

tests also were conducted with the α7 nAChR subtype-selective agonists, anabasine and

anabaseine (de Fiebre et al., 1995; Kem et al., 1997). Finally, drug interaction studies were

conducted to compare modulation of the MA-like discriminative-stimulus effects of nicotine by

the α4β2 competitive antagonist DHβE (Williams and Robinson, 1984) and the partial agonists

varenicline and (-)-cytisine. Overall, results show overlap in the discriminative-stimulus effects

of nicotinic agonists and MA in nonhuman primates and provide further support for the views

that: a) MA-like stimulant effects of nicotinic agonists are primarily mediated through actions at

α4β2 nAChRs; and b) nicotinic partial agonists that attenuate nicotine’s stimulant-like

discriminable effects may be useful pharmacotherapeutic adjuncts in the management of nicotine

addiction.


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METHODS

Subjects Four experimentally naïve adult male squirrel monkeys (Saimiri sciureus), weighing 650 to 900

g were subjects in the present studies. All subjects were individually housed in a climate-

controlled vivarium under an automated 12-hr light/dark cycle. Except during testing, monkeys

had unlimited access to water and were fed a daily allotment of high protein monkey chow

(Purina Monkey Chow, St. Louis, MO), supplemented with fruit and multivitamins in the home

cage. All monkeys were weighed daily; food intake was not restricted, and diets were adjusted

as needed to maintain recommended body weights. Behavioral experiments were conducted

daily (Monday–Friday) between 08:00 AM and 06:00 PM, under protocols that were approved

by the Institutional Animal Care and Use Committee at McLean Hospital. Subjects were

maintained in the McLean Animal Care Facility in accordance with guidelines provided by the

Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animals

Resources, Commission on Life Sciences, National Research Council (2011). This facility is

licensed by the U.S. Department of Agriculture.

Apparatus

The apparatus and methodology were comparable to those employed previously (Kangas et al.

2013; Tidey and Bergman, 1998). During experimental sessions, monkeys sat in customized

Plexiglas chairs (Kelleher and Morse, 1968) that were enclosed in ventilated, sound-attenuating

chambers provided with white noise at all times to mask extraneous sounds. While seated,

monkeys faced a panel containing two sets of colored stimulus lights. Two response levers

extended into the chamber, one below each set of stimulus lights, and were comfortably within


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the subject’s reach. The two response levers were set 15 cm apart. Depression of either lever

with a force greater than 0.2 N produced an audible click and was recorded as a response. Prior

to each behavioral session, a shaved portion of the monkey’s tail was secured under brass

electrodes by a small stock and was coated with electrode paste to ensure a low-resistance

electrical contact between electrodes and tail. Brief, low-intensity stimuli (200 msec; 3 mA)

could be delivered to the electrodes from a 60 Hz transformer. Experimental variables and data

collection were controlled by PC computers with Med Associates interfacing equipment and

operating software (MED-PC, MedState Notation, Med Associates Inc., St. Albans, VT, USA).

MA Discrimination

Subjects first were trained to press each of the two response levers under a 10-response

fixed-ratio (FR10) schedule of stimulus-termination. Under this schedule, a brief, mild electric

stimulus (200 ms; 3 mA) was programmed for delivery to the tail every 10 s during the

illumination of red lights on the front panel. The completion of ten consecutive lever-press

responses (FR10) on one lever within 10 s turned off the red lights and the associated program of

current delivery. The completion of each FR10 also initiated a 50-sec timeout (TO) period,

during which all lights in the chamber were extinguished and responding had no programmed

consequences. The delivery of four electric stimuli prior to completion of the FR requirement

also turned off all lights, terminated the program of stimulus delivery, and initiated the 50-sec

TO period. Once performance was stable on both levers under the FR10 response requirement,

subjects were trained to discriminate i.m. injections of 0.1 mg/kg MA from i.m. injections of

saline (Tidey and Bergman, 1998). Previous studies have indicated that the generalization

profiles for many drugs greatly depend on training dose, and that higher doses generally are more


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pharmacologically restrictive than lower doses. For example, this type of relationship previously

has been exploited by Spealman and colleagues to study the role of different monoaminergic

mechanisms in the discriminative-stimulus effects of cocaine (Spealman, 1995). Thus, in the

present study, we lowered the training dose of MA from the highly restrictive dose of 0.32 mg/kg

to 0.1 mg/kg to assess the possibility of overlapping discriminative-stimulus effects among

compounds from different pharmacological classes. After MA injection, only responses on one

lever were reinforced; after saline injection, only responses on the other lever were reinforced.

The assignment of MA-associated and saline-associated levers was counterbalanced across

monkeys. During all training sessions, responses on the inappropriate lever reset the FR

response requirement on the injection-appropriate lever.

When discrimination performance was stable from day to day at or above criterion (90%

accuracy), daily training sessions were extended to comprise one to four components. Each

component, which consisted of 10 presentations of the FR10;TO 50-s schedule, was preceded by

a 10-min TO period during which vehicle or MA could be administered. The number of

components in daily training sessions varied in a pseudo-random manner, with the stipulation

that MA was injected only before the final component of the session. Additionally, sessions in

which only saline was administered in all components occurred periodically to avoid an invariant

association between injection of MA and the final session component.

Drug testing was initiated when >90% of responses occurred on the injection-appropriate

lever during the preceding training session and four of the last five training sessions. Test

sessions comprised four components during which all schedule parameters and contingencies

were identical to those in the training sessions, with the exception that 10 consecutive responses

on either lever extinguished the red lights and terminated the associated program of current


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delivery. Testing was conducted once or twice per week with training sessions on intervening

days. During test sessions, incremental doses of a test drug were administered at the beginning

of the 10-min TO periods preceding components of the test session (cumulative dosing). This

procedure allowed determination of the effects of up to four cumulative doses during a single test

session.

After all drugs were studied for their ability to substitute for MA, drug interaction studies

were conducted to determine how pretreatment with selected compounds [varenicline, (-)-

cytisine, and DHβE] modified the MA-like effects of nicotine. Pretreatment times were based on

data from preliminary experiments and published reports (Stolerman et al., 1995; 1997; Rollema

et al., 2007; Desai and Bergman, 2010). Studies were conducted by administering single doses

of varenicline (0.0032–0.1 mg/kg), (-)-cytisine (0.032–0.1 mg/kg) or DHβE (0.032–0.1 mg/kg) 5

min prior to re-determination of the cumulative dose-response function for nicotine (0.032–1.0

mg/kg), i.e., 15 min prior to the first session component. Cumulative doses of nicotinic ligands

in substitution studies and pretreatment doses in drug interaction studies were selected on the

basis of preliminary dose-ranging experiments. Doses ranged from those without effect to those

that fully substituted for nicotine or produced untoward physiological effects that precluded

further increase in either cumulative dose or, in drug interaction studies, pretreatment dose. In

the latter case, observed effects were tabulated for presentation in a table.

Drugs

Methamphetamine (MA) hydrochloride, cocaine hydrochloride, ± SKF82958

hydrobromide [(±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-

benzazepine hydrobromide], R-(-)-NPA [R(−)-10,11-dihydroxy-N-n-propylnoraporphine


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hydrochloride], (-)-nicotine hydrogen tartrate, and (-) lobeline hydrochloride were obtained from

Sigma-Aldrich (St. Louis, MO). Citalopram hydrobromide was generously supplied by

Lundbeck (Valby, Denmark). (-)-Cytisine [(1R,5S)-1,2,3,4,5,6-hexahydro-1,5-methano-8H-

pyrido[1,2-a][1,5]diazocin-8-one], and anabasine hydrochloride [(S)-(+)-3-(2-

piperidinyl)pyridine hydrochloride] were obtained from Tocris (Minneapolis, MN). Anabaseine

dihydrochloride (3,4,5,6-tetrahydro-2,3′-bipyridine dihydrochloride), (+)-epibatidine [(2R)-2-(6-

chloro-3-pyridinyl)-7-azabicyclo[2.2.1]heptane monohydrochloride], (-)-epibatidine [(2R)-2-(6-

chloro-3-pyridinyl)-7-azabicyclo[2.2.1]heptane monohydrochloride], isoarecolone hydrochloride

(1-methyl-4-acetyl-1,2,3,6-tetrahydropyridine hydrochloride), and dihydro-β-erythroidine

hydrobromide (DHβE) [(2S,13bS)-2,3,5,6,8,9,10,13- octahydro-2-methoxy-1H,12H-

benzo[i]pyrano[3,4-g]indolizin-12-one hydrobromide] were obtained from the National Institute

on Drug Abuse (Bethesda, MD). Varenicline [6,7,8,9-tetrahydro-6,10-methano-6H-

pyrazino[2,3-h][3]benzazepine] was generously donated by Dr. Hans Rollema (Pfizer Global

Research and Development). All drugs were dissolved in 0.9% saline or water, and were

injected by the intramuscular route of administration. The pH of nicotine and varenicline was

adjusted as needed to 7.0 with 0.1N sodium hydroxide. Doses of each drug are expressed in

terms of the free base.

Data Analysis

Data from the test component immediately following injection were used to express the

effects of the administered cumulative dose. The percentage MA-associated lever responding in

each component of the session was calculated by dividing the number of responses on the

injection lever by the total number of responses on both levers. Response rates were calculated


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for each component by dividing the total number of responses by the duration of the component

minus timeout periods. If the mean response rate in a component was less than 0.2 responses per

sec, data from that component were excluded from further analysis. Mean results for vehicle and

each dose of a drug were calculated by averaging data for the four subjects. Complete

substitution with a dose of test drug alone or after pretreatment was defined for individual

subjects and for the group of subjects as the allocation of ≥90% of total responses to the MA-

associated lever. An intermediate level of responding (31-89%) on the MA-lever was defined as

incomplete substitution, whereas the allocation of ≤30% of responses to the MA-lever was

defined as no substitution.

Data were further analyzed to compare potency and maximum effects among drugs, to

evaluate drug interactions, and to examine correspondence between the effects of nicotinic drugs

in the present experiments and their published affinities for different subtypes of the nicotinic

receptor. As appropriate, ANOVA followed by Dunnett’s t-test or a paired t-test was used to

evaluate statistical significance of averaged data (defined at the 95% level of confidence; p <

0.05). When appropriate, interpolation or linear regression using Bioassay Software (Bioassay

version Beta 6.2; MED Associates Inc.) was used to calculate ED50 values (S.E.M. for

interpolation and 95% confidence limits for regression) from data points on the linear portions of

the dose-response functions (Snedecor and Cochran, 1967). In experiments to evaluate

varenicline-, (-)-cytisine-, and DHβE-nicotine interactions, ED50 values were determined for

nicotine alone and in the presence of each drug; pairs of ED50 values were considered to be

significantly different if their 95% confidence limits did not overlap. When significant

differences in ED50 values were obtained, relative potency estimates were calculated using

standard parallel-line bioassay techniques described by Finney (1964). Finally, correspondence


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between the effects of drugs in drug discrimination and receptor binding studies was examined

by comparing the relative potency of nicotinic drugs in the present experiments (i.e., ED50 values

divided by the ED50 value for nicotine alone) and their published relative affinities for binding

α4β2 and α7 nicotinic receptors in vitro (Ki values divided by the Ki value for nicotine alone).

Relative affinity values for each drug were obtained from previously published radioligand

binding experiments in rat brain. Data were taken from experiments using [3H]-nicotine binding

for the α4β2 receptor subtype and [125I]-α-bungarotoxin (Bgt) binding for the α7 nicotinic

receptor subtype, and affinities relative to nicotine were averaged across studies (see Table 1).


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RESULTS

MA Discrimination. The 0.1 mg/kg training dose of MA maintained reliable discriminative-

stimulus control in all four subjects throughout the present studies (>18 months). During testing

sessions, maximum responding on the MA-associated lever in all subjects occurred following a

cumulative dose of 0.1 mg/kg MA (Fig. 1; top panel). MA also produced a dose-related

elevation in response rate, which increased to approximately 200% of vehicle control values

following the training dose or the cumulative test dose of 0.1 mg/kg; however, differences in the

magnitude of effect among individual subjects precluded statistical significance for the averaged

data (F4,15 = 2.17; P > 0.05). Neither the position and slope of the cumulative dose-response

function for MA discrimination (0.0032–0.1 mg/kg) nor its apparent rate-increasing effects

varied significantly over the course of the present studies. Consequently, discrimination and

response rate data for MA at the beginning and end of the present studies were averaged for

control values and graphic presentation.

Dopaminergic drugs. The administration of MA (0.0032–0.1 mg/kg) in test sessions produced

dose-dependent substitution for the training dose of MA, with full substitution following the

cumulative dose of 0.1 mg/kg MA (Fig. 1; top panel). Like MA, the nonselective monoamine

transport blocker cocaine and the DA D1-like and D2-like agonists SKF82958 and R-(-)-NPA,

respectively, produced dose-dependent and full substitution for 0.1 mg/kg MA, with maxima of

90–94% responding on the MA-associated lever following cumulative doses of 0.32 mg/kg

cocaine, 0.1 mg/kg SKF82958, and 0.01 mg/kg R-(-)-NPA (Fig. 1; top panel). In contrast to

MA, cocaine, and the DA D1- and D2-like agonists, the serotonin-selective reuptake inhibitor

citalopram did not substitute for the training dose of MA (maximum: 2% drug-lever responding


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after a cumulative dose of 10 mg/kg; Fig. 1, top panel). Higher cumulative doses of citalopram

were not studied to avoid untoward effects, e.g. convulsions, previously observed with high

doses of serotonin-selective reuptake inhibitors (Spealman, 1995). Although cocaine, like MA,

increased responding in a dose-related manner, response rates were not significantly changed

from vehicle values by any of the drugs studied (Fs4,15 ≤ 1.23; Ps > 0.05; Fig. 1; bottom panels).

Nicotinic agonists. Nicotine produced dose-dependent increases in responding on the MA-

associated lever and fully substituted for the 0.1 mg/kg training dose of MA following

cumulative doses of 0.1 (90%) and 0.32 mg/kg (100%) nicotine (Fig. 2, top left panel). The (+)-

and (-)-enantiomers of epibatidine also produced dose-dependent increases on the MA-associated

lever, and both isomers produced >85% responding on the MA-associated lever following the

cumulative dose of 0.001 mg/kg [mean ± SEM: 86 ±14.3% for (+)-epibatidine and 96.8 ±3.33

for (-)-epibatidine; Fig. 2, top middle and right panels, respectively]. Neither nicotine nor the

enantiomers of epibatidine significantly altered rates of responding from control values in the

group of four monkeys (Fig. 2; bottom panels). However, the highest doses of each drug

produced observable effects, including profuse salivation and emesis (see below and Table 2).

The nicotinic agonists isoarecolone, anabaseine, anabasine, and varenicline also produced

dose-dependent increases in responding on the MA-associated lever (Fig. 3; top left panel).

However, substitution was incomplete following the highest cumulative doses of these agonists,

with mean values ranging from approximately 50 to 65% for responding on the MA-associated

lever. Among these ligands, a clear plateau in MA-like effects indicative of partial agonist

actions was observed only with varenicline: the mean MA-like effects of the highest cumulative

pretreatment dose, 0.32 mg/kg, did not exceed those of the immediately preceding cumulative


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dose (0.18 mg/kg) and rates of responding were comparable to control values. The two highest

doses of isoarecolone also produced comparable levels of responding on the MA-associated

lever; however, the 0.5 log unit increase in dose from 3.2 to 10 mg/kg produced a small, albeit

non-significant, increase in responding on the MA-associated lever and a decrease in response

rate to 60% of control values, precluding a more definitive characterization of its efficacy. No

indication of a plateau in MA-like effects was apparent with either anabasine or anabaseine.

Anabasine did not alter mean rates of responding over the range of doses studied, whereas

anabaseine reduced response rate in a dose-dependent manner and, following the highest

cumulative dose (3.2 mg/kg) nearly or completely eliminated responding. As with nicotine and

regardless of the presence or absence of effects on response rates, the highest cumulative doses

of each of these nicotinic agonists produced untoward physiological signs that precluded further

testing (see below and Table 2).

The rank order of potency with which nicotinic agonists produced MA-like effects was:

(-)-epibatidine ≈ (+)-epibatidine > nicotine ≈ varenicline > anabaseine > anabasine >

isoarecolone (Table 1). Based on ED50 estimates, isoarecolone and the two enantiomers of

epibatidine were, respectively, the least and most potent nicotinic ligands in the present studies,

and approximately 85- to 80-fold less and more potent than nicotine, respectively. The

remaining drugs were approximately 3-fold (varenicline) and 10 to 20-fold (anabaseine,

anabasine) less potent than nicotine in producing MA-like discriminative-stimulus effects (Table

1).

In contrast to the above ligands, (-)-cytisine and (-)-lobeline failed to substitute for the

training dose of MA, producing ≤30% responding on the MA-associated lever over the range of

doses that could be studied (Fig. 3; top right panel). Although response rates were not


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significantly altered following the highest cumulative doses of (-)-cytisine and (-)-lobeline,

untoward observable effects precluded the administration of higher doses (see below; Table 2).

Observable effects of nicotinic agonists. The observable effects of nicotinic agonists in the

present study included untoward physiological signs following one or more cumulative doses of

each drug (Table 2). Regardless of the presence or absence of MA-like discriminative-stimulus

effects, the highest cumulative doses of each agonist produced profuse salivation in all subjects

that often was followed by emesis. In addition to producing excessive salivation and emesis, the

highest cumulative doses of isoarecolone, anabasine, (-)-cytisine, and (-)-lobeline produced

tremor in individual subjects (Table 2). Convulsions after cumulatively administered doses of

3.2 or 10 mg/kg lobeline also were noted in two subjects; these signs were quickly and

completely attenuated by i.m. diazepam (1.0 mg/kg).

Antagonism of nicotine’s MA-like effects. Pretreatment with the selective α4β2 nicotinic

antagonist, DHβE (0.032 or 0.1 mg/kg) antagonized the MA-like discriminative-stimulus effects

of cumulatively administered nicotine (0.01–1.0 mg/kg; Fig. 4, top panel). Both doses of DHβE

produced a comparable level of antagonism, evident as an approximately 10-fold rightward shift

in the dose-response curve and yielding an approximately 10-fold increase in the ED50 value for

nicotine’s MA-like effects (Fig. 4, top panel; Table 3). The highest cumulative dose of nicotine

after treatment with DHβE (1.0 mg/kg) produced approximately 70% responding on the MA-

associated lever and an approximately 30-40% decrease in response rates (Fig. 4; bottom right

panel).


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Pretreatment with the nicotinic α4β2 partial agonist varenicline (0.0032, 0.032, or 0.1

mg/kg) also antagonized MA-like discriminative-stimulus effects engendered by cumulatively-

administered nicotine (0.01–1.0 mg/kg). Like DHβE, all doses of varenicline shifted the nicotine

dose-response curve rightward to a similar extent (Fig. 5, top left panel). Based on estimated

ED50 values, the potency of nicotine for producing MA-associated responding decreased

approximately 12-fold in the presence of 0.032 mg/kg of varenicline, and approximately 16-fold

following a 30-fold higher pretreatment dose of varenicline (0.1 mg/kg; Table 3). As in

experiments with DHβE, the highest cumulative dose of nicotine (1.0 mg/kg) after treatment

with the two highest doses of varenicline (0.032 and 0.1 mg/kg) produced a moderate

(approximately 30-40%) but statistically non-significant decrease in response rates (Fig. 5;

bottom left panel).

Like DHβE and varenicline, the nicotinic α4β2 partial agonist (-)-cytisine (0.032 or 0.1

mg/kg) antagonized the MA-like discriminative-stimulus effects of cumulatively-administered

nicotine (0.01–1.0 mg/kg) by shifting its dose-effect curve rightward (Fig. 5, top right panel).

Based on ED50 values, the effects of nicotine were displaced approximately 7- and 14-fold

rightward by pretreatment with 0.032 mg/kg and 0.1 mg/kg (-)-cytisine, respectively (Table 3).

Like varenicline, pretreatment doses of (-)-cytisine did not substantively alter nicotine’s effects

on response rates, and only moderate (<50%) decreases from vehicle-control rates of responding

were observed following their combination (Fig. 5, bottom right panel).

Although not studied separately, neither the α4β2 antagonist DHβE nor the α4β2 partial

agonists varenicline and (-)-cytisine appeared to attenuate the emetic effects of cumulative doses

of nicotine that produced full substitution for MA. As described above, tremor or frank

convulsions following treatment with several nicotinic ligands was evident in the present studies,


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and higher doses of DHβE, like α4β2 full and partial agonists, previously have been reported to

produce seizure activity (Damaj et al. 1999; Dobelis et al. 2003). Consequently, higher

pretreatment doses of DHβE, varenicline, or (-)-cytisine or higher cumulative doses of nicotine

after pretreatment with these drugs were not studied so as to avoid further untoward effects in the

present studies.


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DISCUSSION

The main objective of the present studies was to characterize discriminative-stimulus

effects of nicotine and nicotinic ligands in monkeys that discriminated a moderate training dose

of the monoaminergic stimulant MA (0.1 mg/kg). Initial experiments indicated that, as in

previous studies with a higher MA training dose (0.3 mg/kg), indirect monoaminergic agonists

(MA, cocaine) and direct DA D1- and D2-like receptor agonists (SKF82958 and R-(-)-NPA,

respectively) engendered dose-dependent and full substitution for MA (Tidey and Bergman,

1998). These results, supporting the view that DA-related mechanisms play a prominent role in

the discriminative-stimulus effects of MA, provide a pharmacologically empirical basis for

evaluating behavioral overlap in the effects of drugs that act via different (dopaminergic vs.

nicotinic) receptor mechanisms.

Nicotine and the enantiomers of epibatidine also produced dose-dependent increases in

MA-associated responding, and fully (or, in the case of (+)-epibatidine, nearly fully) substituted

for MA without greatly altering response rates. The comparable effects of these nicotinic full

agonists are consistent with their similar nicotinic α4β2 subtype selectivity (see Table 1) and

with previous drug discrimination data from nicotine-trained rodents (Reavill et al., 1987; Damaj

et al., 1994). They contrast somewhat with data from MA-trained rats in which only nicotine

fully substituted for the training dose of MA (Desai and Bergman, 2010). In those studies, 0.001

mg/kg of both (+)-epibatidine and (-)-epibatidine produced approximately 60-70% responding on

the MA-associated lever, whereas a 3-fold increase in the dose of both enantiomers completely

eliminated responding, precluding further testing. Differences in the two studies may reflect

species-related differences in vulnerability to the rate-decreasing effects of the epibatidine

enantiomers or, alternatively, differences in the resistance of responding maintained by stimulus-


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termination (present studies) and food presentation (previous studies) to their behaviorally

disruptive effects. Notwithstanding these considerations, the present findings clearly show that

the discriminative-stimulus effects of nicotine and the enantiomers of epibatidine overlap

substantively with those of monoaminergic stimulants like MA in primate species.

The nicotinic receptor ligand varenicline also engendered dose-related MA-like effects

but, in contrast to nicotine and epibatidine, the highest doses produced only intermediate levels

of substitution. Varenicline previously has been characterized as a nicotinic partial agonist at the

α4β2 receptor subtype (Rollema et al., 2007; 2010) and, depending on experimental conditions,

may substitute partially or fully for nicotine in nicotine-trained rats and monkeys (Rollema et al.,

2007; Smith et al., 2007; LeSage et al., 2009; Jutkiewicz et al., 2011; Cunningham et al., 2012).

The plateau in the dose-effect function for varenicline at an intermediate level of responding on

the MA-associated lever in the present experiments, in conjunction with its ability to antagonize

the stimulant effects of nicotine in MA-trained rodents (Desai and Bergman, 2010), is consistent

with its characterization as a nicotinic partial agonist.

Like varenicline, isoarecolone is characterized as an α4β2-selective ligand and can fully

reproduce the discriminative-stimulus effects of nicotine in nicotine-trained rats (Reavill et al.,

1987; Damaj et al., 1994). Based upon other behavioral and biochemical findings, however,

isoarecolone has been forwarded as a nicotinic partial agonist (Reavill et al., 1987; Mirza et al.

1996; Whiteaker et al., 1995, Hahn et al. 2003; Shoaib, 2006). The present findings that the

highest doses of isoarecolone produced only an intermediate level of substitution for MA might

be considered supporting evidence for that view. However, this interpretation remains

speculative in the absence of a more definitive characterization of isoarecolone’s efficacy, e.g.,

varenicline-like antagonism of nicotine’s behavioral effects.


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The minor tobacco alkaloids anabasine and anabaseine produced dose-related increases in

responding on the MA-associated lever without nicotine-like full substitution or a varenicline-

like plateau in MA-like effects. Both drugs previously have displayed α7 and, with lower

efficacy, α4β2 receptor-mediated agonist actions (Arendash et al., 1995; Kem et al., 1997;

Stevens et al., 1998). Possibly, the limited nicotine-like effects of anabasine and anabaseine in

the present experiments reflect relatively low efficacy at the α4β2 receptor (e.g., Takada et al.,

1989; Brioni et al., 1994; de Fiebre et al., 1995; Stolerman et al., 1995; Desai and Bergman,

2010). Alternatively, α7-mediated actions of anabasine and anabaseine may have obscured the

full expression of their MA-like effects. Although such explanations are speculative in the

absence of further information, the present and previous findings in monkeys and rats (Desai and

Bergman, 2010) show that anabasine and anabaseine can produce MA-like effects. Although

limited, such stimulant-like effects of minor tobacco alkaloids, like those of nicotine, may

contribute to the maintenance of tobacco consumption (Clemens et al., 2009; see Hoffman and

Evans, 2012 for review).

(-)-Cytisine and (-)-lobeline, which have high nAChR affinity and α4β2 subtype-

selectivity (see Table 1), failed to engender MA-like discriminative-stimulus effects in the

present studies. Previously, (-)-cytisine was shown to both partially substitute for nicotine in rats

and block its discriminative-stimulus effects, consistent with its characterization as a nicotinic

partial agonist (Stolerman et al., 1984; Reavill et al., 1990; Brioni et al., 1994; Jutkiewicz et al.,

2011; Cunningham et al., 2012). The absence of MA-like effects in the present studies suggest

that (-)-cytisine may have less of a stimulant action in primate species than other α4β2 partial

agonists such as varenicline. (-)-Lobeline, like (-)-cytisine, is considered a partial agonist at the

α4β2 nAChR but, in addition, appears to act through multiple mechanisms, including


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monoamine uptake inhibition (Damaj et al., 1997; Dwoskin and Crooks, 2002). Thus, (-)-

lobeline has been shown to substitute for a low training dose of cocaine, yet attenuate the effects

of a higher training dose of cocaine or methamphetamine (Miller et al., 2001; Harrod et al., 2003;

Desai et al., 2003; Cunningham et al., 2006). Further indicative of its poorly-understood actions,

(-)-lobeline has been reported to produce nicotine-like effects in studies of locomotor activity,

but not in place conditioning, self-administration, or drug discrimination studies (Fudala and

Iwamoto, 1986; Corrigall and Coen, 1989; Reavill et al., 1990; Stolerman et al., 1995; Harrod et

al., 2003). Although the absence of MA-like effects in the present study is not inconsistent with

its characterization as a nicotinic partial agonist, additional data showing that (-)-lobeline, like

varenicline, can antagonize such effects of nicotine would strengthen this categorization.

Although only isoarecolone and anabaseine decreased response rates, the highest

cumulative doses of all nicotinic agonists produced untoward physiological signs (emesis,

tremor, or convulsions) that precluded the study of higher doses (see Table 2). However, profuse

salivation and emesis alone did not appear to interfere with discrimination behavior, e.g.,

nicotine and epibatidine fully substituted for MA despite profuse salivation and emesis in all

subjects. Although the precise mechanism responsible for these adverse physiological signs

remains unclear, it is notable that they can be produced by both α4β2 nAChR agonists and

antagonists as well as by α7-selective ligands (Damaj et al. 1999; Dobelis et al. 2003). Thus, it is

unlikely that these signs reflect actions at a single subtype of nicotinic receptor.

Pretreatment with the competitive antagonist DHβE and the α4β2-selective partial

agonists varenicline and (-)-cytisine shifted the dose-effect function for nicotine’s MA-like

effects rightward, complementing similar results in nicotine-trained rats (Stolerman et al., 1997;

Jutkiewicz et al., 2011). Although each drug served as a surmountable antagonist, the range of


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antagonism was surprisingly limited, i.e., an approximately 1-1.25 log unit rightward shift in the

nicotine dose-effect function. The limited range of antagonist actions of DHβE and (-)-cytisine

might reflect the use of two antagonist doses spanning only a 0.5 log unit range; higher

pretreatment doses might have led to additional antagonism. In the case of varenicline, however,

the lowest pretreatment dose (0.003 mg/kg) increased nicotine’s ED50 value approximately 10-

fold, whereas 10- and 30-fold increases in pretreatment dose produced only a <2-fold further

increase in nicotine’s ED50 value. The reasons for such limited dose dependence in the nicotine-

antagonist effects of varenicline are uncertain but may be partly related to training dose. In

previous studies of the same three antagonists in nicotine-trained rats, dose dependence was

more evident in subjects that discriminated a low, rather than high, dose of nicotine (Jutkiewicz

et al., 2011). Possibly, a lower training dose of MA and a concomitant increase in nicotine’s

potency might also have revealed greater antagonist dose dependence in the present studies.

Comparison of the potencies of nicotinic agonists with their reported binding affinities at

α4β2 and α7 nicotinic receptor subtypes reveals a good correspondence between their relative

behavioral potencies and their relative potencies for inhibiting [3H]-nicotine binding at the α4β2

receptors (r2 = 0.83, p = 0.005) but not [125I]-α-Bgt binding at α7 receptors (r2 = 0.01, p = 0.83;

Fig. 6; Table 1). These observations parallel a similar analysis in MA-trained rats, and are

consistent with the ability of the α4β2 receptor blocker DHβE, but not the α7 receptor blocker

MLA, to antagonize nicotine’s discriminative-stimulus effects (Brioni et al., 1996; Desai and

Bergman, 2010). In concert with the antagonist effects of DHβE and the partial agonists

varenicline and cytisine in the present experiments, such correspondence provides added support

for the idea that the stimulant-like effects of nicotine and other nicotinic agonists are


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predominantly mediated by their actions at the α4β2 nAChR (Reavill et al., 1987; 1988;

Stolerman et al., 1995; Desai and Bergman, 2010).


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ACKNOWLEDGMENTS

We thank Elise Trowel for expert technical support, Dr. Hans Rollema (Pfizer Inc.) for providing

varenicline for the present studies, and Drs. B. Kangas and C.A. Paronis for their comments on

an earlier version of this manuscript. We thank the NIDA Drug Supply Program for providing

the enantiomers of epibatidine, isoarecolone, anabaseine, and DHβE for the present studies.


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AUTHORSHIP CONTRIBUTION

Participated in research design: Desai, Bergman

Conducted experiments: Desai

Performed data analysis: Desai

Wrote or contributed to the writing of the manuscript: Desai, Bergman

Other: Desai acquired funding for the research.


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REFERENCES

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[3H]methylcarbamylcholine in rat brain. Eur.J.Pharmacol. 253:261-267.

Arendash GW, Sengstock GJ, Sanberg PR, and Kem WR (1995) Improved learning and memory

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FOOTNOTES This research was supported by the National Institutes of Health/National Institute on Drug

Abuse [grant R21 - DA026548].


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LEGENDS FOR FIGURES Figure 1. Effects of cumulative administration of the indirect monoamine agonists MA and

cocaine, the direct DA D1- and D2-like agonists SKF82958 and R-(-)-NPA, respectively, and the

selective serotonin reuptake inhibitor, citalopram, in squirrel monkeys trained to discriminate 0.1

mg/kg MA from saline under a fixed-ratio schedule of stimulus termination. Ordinates:

percentage MA-associated responding (top panel), response rates (bottom panel). Abscissae:

cumulative drug dose in mg/kg (log scale). Each data point represents all four subjects tested at

each dose. The percentage of responses emitted on the MA-associated lever was not plotted if

fewer than half of the subjects responded at that dose. During control sessions, the training dose

of MA produced 97.3% ± 1.8 (S.E.M.) responding on the MA-associated lever and injections of

saline produced 1.1% ± 0.9 (S.E.M.) responding on the saline-associated.

Figure 2. Effects of the cumulatively administered nicotinic agonists, nicotine and the

enantiomers of epibatidine, in squirrel monkeys trained to discriminate 0.1 mg/kg MA from

saline. Ordinates for the top and bottom panels and abscissae are as in Fig. 1. See Figure 1 for

other details. Each data point represents all four subjects tested (n = 4) at each dose.

Figure 3. Left panels: Effects of the cumulatively administered nicotinic agonists, isoarecolone,

anabaseine, anabasine, and varenicline in squirrel monkeys trained to discriminate 0.1 mg/kg

MA from saline. Right panels: Effects of the cumulatively administered nicotinic agonists, (-)-

cytisine and (-)-lobeline in squirrel monkeys trained to discriminate 0.1 mg/kg MA from saline.

Ordinates for the top and bottom panels and abscissae are as in Fig. 1. See Figure 1 for other

details. Each data point represents all four subjects tested (n = 4) at each dose.


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Figure 4. Effects of pretreatment with the selective competitive α4β2 nicotinic antagonist,

DHβE on MA-like responding produced by nicotine in squirrel monkeys trained to discriminate

0.1 mg/kg MA from saline. Ordinates for top and bottom panels as in Fig. 1. Abscissae: dose

of cumulatively administered nicotine (mg/kg; log scale). See Fig. 1 for other details. Each data

point represents all subjects tested (n = 4) at each dose.

Figure 5. Effects of pretreatment with the partial nicotinic agonists varenicline and (-)-cytisine

on MA-like responding produced by nicotine in squirrel monkeys trained to discriminate 0.1

mg/kg MA from saline. Ordinates for top and bottom panels as in Fig. 1. Abscissae: dose of

cumulatively administered nicotine (mg/kg; log scale). See Fig. 1 for other details. Each data

point represents all subjects tested (n = 4) at each dose.

Figure 6. Relationship between the relative potencies of nicotinic drugs in the present MA-

discrimination studies and their relative affinities at α4β2 and α7 nicotinic receptors in

radioligand binding studies (see Methods). Abscissa: affinity relative to nicotine for inhibiting

binding of radioligand to α4β2 (top panel) and α7 (bottom panel) nicotinic receptors; ordinates:

potency of nicotinic drugs relative to nicotine, based on ED50 values, for engendering MA-

associated lever responding (from Table 1). Isoarecolone was excluded from this correlation

analysis at the α7 nicotinic receptor subtypes because affinity values obtained at this site are not

clearly defined (see Table 1).


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TABLES Table 1: ED50 values (± S.E.M) and relative potencies with which nicotinic agonists produce MA-like effects in squirrel monkeys (nicotine=1) and their affinity and relative affinity at α4β2 and α7 nACh receptors (nicotine=1). The in vitro values for inhibiting [3H] nicotine binding at α4β2 nicotinic receptors and [125I]-α-Bgt binding at α7 nicotinic receptors are the average of values taken from the references cited below.

a Jensen et al., 2003, b Anderson & Arenric, 1994, c Xiao and Kellar, 2004, d, e Marks et al., 1986, 1996, f Sullivan et al., 1994, g Decker et al., 1995, h Badio and Daly, 1994, i Hahn et al., 2003, j Damaj et al., 1994, k,l Rollema et al., 2007, 2010, m Kem et al., 1997; n de Fiebre et al., 1995; o Damaj et al., 1997; p Miller et al., 2003 ND = Not Determined

Drug

Doses mg/kg

ED50 ± S.E.M mg/kg

(μmol/kg)

Relative Potency

(MA-like Sd)

In Vitro Affinity

(Ki values) at α4β2 (nM)

Relative Affinity at

α4β2 receptors

In Vitro Affinity (Ki values) at α7

(nM)

Relative Affinity at

α7 receptors

Nicotine 0.01–0.32

0.032 ± 0.014

(0.198) 1

3.4 a-e, g-j, l

1

4895 i

1

(+)-Epibatidine 0.0001–0.001

0.0005 ± 0.00003

(0.0024)

0.015

0.05 h, j

0.015

255 f

0.052

(-)-Epibatidine 0.0001–0.001

0.0005 ± 0.00002

(0.0024)

0.015

0.06 h, j

0.018

109 f

0.022

Isoarecolone 0.32–10

2.8 ± 0.73

(19.3)

87.5

611 i

179.7

>100,000 i

20.43

Anabaseine 0.1–1.0

0.53 ± 0.17

(3.31)

16.6

32 m

9.41

58 m

0.012

Anabasine 0.1–1.0

0.74 ± 0.09

(4.31)

23.1

260 m

76.5

58 m

0.012

Varenicline 0.032–0.18

0.11 ± 0.025

(0.52)

3.44

0.17 k, l

0.05

620 k

0.127

(-)-Cytisine 0.032–1.0

ND

ND

0.012–1.5 a-e

0.004–0.44

260–15000 a, d, n

0.05–3.06

(-)-Lobeline 0.1–3.2

ND

ND

1.5–16 b, o, p

0.44– 4.71

11600–13100 n, p

2.37–2.68

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

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Table 2: Cumulative i.m. doses (mg/kg) of nicotinic agonist that produced observable untoward effects (excessive salivation, emesis, tremor, or convulsions) during test sessions (n=4 Ss). One or more effects were observed following injection of the listed cumulative dose either prior to or following the session component in which that dose was studied. + represents the observation of each effect in an individual subject.

Drug Doses (mg/kg)

Salivation/Foam Emesis Tremor Convulsions

Vehicle 0 - - - - Nicotine 0.32 + + + + + + + + - -

(+)-Epibatidine 0.001 + + + + + + + + - - (-)-Epibatidine 0.001 + + + + + + + + - -

Isoarecolone

1.0 3.2

10.0

+ + + +

+ + + +

+ + + +

+ + + +

- -

+ + +

- - -

Anabaseine 1.0 3.2

+ + + + + + +

+ + + + + + +

- -

- -

Anabasine 1.0 + + + + + + -

Varenicline

0.1 0.18 0.32

+ + + + + + + + + + + +

+ + + + + +

+ + +

- - -

- - -

(-)-Cytisine 0.32 1.0

+ + + + + + + +

+ + + + + + +

- + + +

- -

(-)-Lobeline 3.2

10.0 + + + +

+ + + +

+ +

+ +




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Table 3: Doses of nicotine alone and after pretreatment with DHβE, varenicline, or (-)-cytisine that are calculated to produce 50% responding on the MA-associated lever (ED50 values with 95% confidence intervals) and relative potencies of nicotine after pretreatment [ED50 nicotine / ED50 nicotine after pretreatment). Data were obtained in squirrel monkeys trained to discriminate i.m. injections of 0.1 mg/kg (0.67 μmol/kg) MA from saline.

Pretreatment Drug

Doses (mg/kg)

ED50 (95% CL) mg/kg

(μmol/kg)

Relative Potency

Nicotine

0.01–0.32

0.03 (0.02–0.05) (0.20)

1

DHβE

0.032

0.1

0.39 (0.19–1.90) (2.39)

0.30 (0.12–2.76)

(1.85)

0.08 (0.03–0.20)

0.11 (0.03–0.27)

Varenicline

0.0032

0.032

0.1

0.39 (0.19–2.55)

(2.40)

0.75 (0.45–1.69) (4.63)a

0.53 (0.26–3.02)

(3.25)a

0.09 (0.02–0.23)

0.06 (0.01–0.14)

0.07 (0.02–0.16)a

(-)-Cytisine

0.032

0.1

0.23 (-0.19–0.02)b

(1.39)

0.44 (0–0.13)b (2.72)

0.13 (0.03–0.36)

0.08 (0.02–0.23)

a Significant deviation from linearity; b Estimate due to non-significant regression




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Figure 1

0

10

20

30

40

50

60

70

80

90

100

MACocaineCitalopramSKF82958R-(-)-NPA

% M

A-a

sso

cia

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re

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on

din

g

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1

2

3

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0032

0

Cumulative Dos

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sp

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te (

resp

/se

c)

032

0.01

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20.

10.

32 1.0

3.2 10 32

ose mg/kg (i.m.)




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Figure 2

0102030405060708090

100

% M

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sso

cia

ted

re

sp

on

din

g

0102030405060708090

100

0

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6

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0.00

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0.32 1.

0

Nicotine

Cumulative Dose mg/kg (i.m.)

Re

sp

on

se

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te (

resp

/se

c)

0

1

2

3

4

5

6

V MA

0.00

0

0.00

0032

Cumulative D

0102030405060708090

100

0.00

01

0.00

032

0.00

1

0.00

32

(+)-Epibatidine

Dose mg/kg (i.m.)

0

1

2

3

4

5

6

V MA

0.00

01

0.00

032

0.00

1

0.00

32

(-)-Epibatidine0.

0000

32

Cumulative Dose mg/kg (i.m.)




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Figure 3T

his article has not been copyedited and formatted. T

he final version may differ from

this version.JPE

T Fast Forw

ard. Published on January 3, 2014 as DO

I: 10.1124/jpet.113.211235 at ASPET Journals on June 19, 2015 jpet.aspetjournals.org Downloaded from


Figure 4

010

20

30

4050

60

70

8090

100

% M

A-a

sso

cia

ted

re

sp

on

din

g

0

1

2

3

4

5

6

V MA

0.00

32

0.01

0.03

2

Nicotine Dose m

Re

sp

on

se

Ra

te (

resp

/se

c)

.032 0.

1

0.32 1.

0

Nicotine+ 0.032 DHβE+ 0.1 DHβE

3.2

e mg/kg (i.m.)




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Figure 5

0

10

20

30

40

50

60

70

80

90

100

% M

A-a

sso

cia

ted

re

sp

on

din

g

0

1

2

3

4

5

6

V MA

0.00

32

0.01

0.03

2

0.1

0.32 1.

0

Nicotine+ 0.0032 Varenicline+ 0.032 Varenicline+ 0.1 Varenicline

3.2

Nicotine Dose mg/kg (i.m.)

Re

sp

on

se

Ra

te (

resp

/se

c)

0

10

20

30

40

50

60

70

80

90

100

% M

A-a

sso

cia

ted

re

sp

on

din

g

.2

0

1

2

3

4

5

6

V MA

0.00

32

0.01

0.03

2

0.1

0.32 1.

0

Nicotine+ 0.032 (-)-Cytisine+ 0.1 (-)-Cytisine

3.2

Nicotine Dose mg/kg (i.m.)

Re

sp

on

se

Ra

te (

resp

/se

c)




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Figure 6

α4β2 Binding(Log Relative Affinity)

-3 -2 -1 0 1 2 3

MA

-ass

oci

ated

res

po

nd

ing

(Lo

g R

elat

ive

Po

ten

cy)

-4

-3

-2

-1

0

1

2

r2 = 0.83p = 0.005

(+)-Epibatidine

(-)-Epibatidine

Varenicline

Nicotine

α7 Binding(Log Relative Affinity)

-3 -2 -1 0 1 2 3

MA

-ass

oci

ated

res

po

nd

ing

(Lo

g R

elat

ive

Po

ten

cy)

-4

-3

-2

-1

0

1

2

r2 = 0.01p = 0.83

Anabaseine

Anabasine

Isoarecolone




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10 Laidlaw: Laburnum Poisoningawd Cytisine

Laburnum Poisoning and Cytisine.

By P. P. LAIDLAW, B.C.

THE common laburnum tree, Cytisus laburnuont, was shown by Gray,in 1862 [1], to contain an alkaloid which he named "cytisine." Thisalkaloid has been found in a number of other plants, and has since beeninvestigated from a chemical point of view by Husemiiann and Marme [2],Partheil [3], and others. Its physiological action has been examinedand described by Gray, Husemann and Marme, Cornevin [4], Prevostand Binet [5], and Radziwillowicz [6]. The poisonous effects oflaburnum are usually attributed to the presence of cytisine in the plant.As' Dr. Dale and I have recently been engaged in re-investigating theaction of cytisine, and have been able to determine its pharmacologicaleffects with greater accuracy and fuller detail than previous workers,I thought it might be interesting to analyse a number of poisoning casesin man, and show that such new facts as we have discovered confirmthe view that the alkaloid is responsible for the poisonous properties ofthe plant.

A large number of cases of accidental poisoning are recorded.Radziwillowicz collected 181 cases in 1888, and since that date addi-tional cases have been described. The great majority of these occur inchildren, and are for the most part miild; fatal cases are rare. Threeare described in the British Medical Journal [7 and 8] and one in theLancet [9]; Radziwillowicz records others. The milder cases [10] runa course somewhat as follows: A child swallows a few laburnum seedsunder the impression that they are peas; or he eats the flowers, pods,or leaves, possibly in search for a new sensation in flavours. About anhour afterwards the child feels unwell, complains of being unable towalk, or seems weak and helpless. Some complain of headache, giddi-ness, and stomach-ache. Shortly after the onset of these symptoms hevomits and appears to be very ill. The skin of the face in particular ispale, cold, and moist, the pulse rapid and thin. The pupils may becontracted at first, but dilate in the later stages; they react to light inmild cases. Sickness continues, and purgation may occur.

The recorded cases among adults are not severe, and usually havetheir origin in the mistaken use of laburnum flowers to flavour dishes.

Therapeutical and Pharmacological Section1Their use for this purpose is due to the cook mistaking the flowers forthose of Robiniapseudacacia. Vallette [11] records one instance of thisaccident. A household of four members, three females and one male,partook of some poisoned fritters. The three women suffered frommild laburnum poisoning, the man developed no symptoms. Theearliest symptom in one of these cases was a sensation of numbness inthe hands and inability to play the piano. She was assisted to bed,where the symptoms were very similar to those already described.

The synmptoimis in a severe case are well illustrated in the followingabstract from the Lancet [12]

J. W., aged 6, ate a hearty tea at 6 p.m.; at 8 p.m. he swallowed somelaburnum seeds, saying they were peas. At 9 p.m. he appeared to be very ill,and vomited. Seen shortly afterwards, he was very pale; skin cold and clammyto the touch; pupils contracted; drowsy at times, but could be roused readily.There was no pain. Pulse, 108; axilla temperature, 97.50 F.; respiration 22.At 10.15 p.m. the drowsiness was more marked; the pupils dilated; pulse 130and very weak; respiration, 25; rectal temperature, 960 F. At 10.30 p.m. thepatient could only be roused with difficulty. The skin was very cold and bathedin perspiration; the pupils were widely dilated and insensitive to light. Caffeinewas administered hypodermically, and a hot bath ordered. A mixture ofammonia and ether was given by the mouth at short intervals. After the hotbath improvement was noticed, and the symptoms gradually subsided. At2 a.m., the child was sleeping peacefully, and next day was comparatively well,though the pupils remained dilated for twenty-four hours longer.

In fatal cases the synmptoms are similar; the drowsiness beconmesmore pronounced as intoxication progresses, and the patient ultimatelybecomes comatose, with widely dilated pupils, which are insensitive tolight; the respiration becomes stertorous, and cyanosis of the lipsdevelops; the pulse becomes very rapid and the blood-pressure low.Death is due to respiratory failure, with or without conyulsions [13].

In a limited number of cases symptoms of acute enteritis appear tobe superimposed on the usual series. A striking example of this typeof case is recorded by Wheelhouse [14]

A child, aged 5 years and 7 months, had been unwell for two days beforebeing seen, and admitted having swallowed some laburnum seeds. On thethird day more seeds were eaten, and the usual symptoms of laburnumpoisoning developed. Vomiting and diarrhbea were, however, marked symptomsand could not be controlled. The patient was drowsy, then restless andirritable by turns. The pressure of the bedclothes appeared to be irritating,and they were repeatedly thrown off. For three days these symptoms were

11

12 Laidlaw: Laburnum Poisoni'ng and Cytisine

present, and very little fluid was retained, although the patient was very thirsty.Temporary improvement was noticed on the fourth day, and fluids were retainedbetter, but the progress was not maintained, and death ensued on the sixth day.

It has been suggested that the symptoms of acute enteritis, whichare sometimes seen in cases of laburnum poisonifig, are due to someunknown, highly irritating substance in the plant. They do not appearregularly, however, and may be examples of idiosyncrasy in response tocytisine. As far as I am aware they have no parallel in animal experi-ments with the alkaloid.

Post mortem, no positive signs of laburnum poisoning can be dis-covered apart from the isolation of the alkaloid from the viscera [13],and as this is present in other plants the evidence is inconclusive.In a few cases the mucous membrane of the alimentary canal isinjected.

The treatment of laburnum poisoning resolves itself into removal ofthe poison by emetics or stomach-tube and the treatment of symptomsas they arise. Radziwillowicz has shown that cytisine is readilyexcreted in the urine; diuretics, therefore, seem to be indicated. In anumber of cases hot baths seem to have been beneficial.

The action of cytisine on intact animals reproduces with fair accuracythe symptoms of laburnum poisoning in man. The herbivora are lesssusceptible than the carnivora. The goat is very resistant: slugs areimmune. Shortly after a hypodermic injection of cytisine has beengiven to a dog, symptoms of nausea (salivation and uneasiness) develop,and vomiting follows. The pupils dilate and the nictitating membraneprolapses. With large doses or with intravenous administration therespiration first becomes hurried and deep, and later becomles slow, andmay ultimately cease.

The respiratory and emetic effects are central in origin and are dueto a stimulating action on the respective centres. The respiratorycentre is readily paralysed with large doses. Muscular tremors areperceptible and weakness of the limbs is obvious. Large doses inanesthetized or pithed animals under artificial respiration cause acomplete paralysis of the nerve-endings in voluntary muscle (curareeffect); about 6 mg. are required to produce this effect in a fair-sized cat.The muscular weakness may be'the expression of a mild stage in thisprocess. The pupil dilates in the cat under the influence of cytisine, andthe nictitating membrane at first is withdrawn and later prolapses. Ifrepeated doses of cytisine are given to a pithed cat, it is observable thatthe effect on the eye becomes less and less, until further doses produce

Therapeutical and Pharmacological Section

no effect. When this stage is reached it is found that the superiorcervical ganglion is paralysed and that electrical stimulation of thecervical sympathetic trunk is without action on the eye, but stimulationof the branches from the ganglion to the periphery still causes normal

FIG. 1.

Cat, pithed. Artificial respiration; blocd-pressure base line and signal; timein ten seconds. First curve, effect of 0-2 mgm. nicotine; second curve, effectof 0-2 mgm. cytisine.

responses. Once the ganglion system is paralysed with cytisine, nicotineis inicapable of producing any effect. A flow of saliva is observablefrom the submaxillary gland of the dog and cat on administration of

13

14 Laidlaw: Laburnum Poisoning and Cytisine

cytisine. L'arge doses cause paralysis of the chorda tympani and renderthis ganglion system insensitive to nicotine.

On the blood-pressure cytisine exerts a series of effects, through its

FIG. 2.Cat, as in fig. 1. Effect of 1-5 mgm. nicotine; effect of 0 5 mngm. cytisine, and

1-5 mgm. nicotine.

stimulant action on ganglion cells. Thus the heart at first is slowedfrom a weak stimulant effect on the cardio-inhibitory apparatus, laterit becomes very rapid, probably in part from stimulation of the stellate

Therapeutical and Pharmacological Section 15

FIG. 3.Cat, pithed. Artifitlial respiration, upper tracing bladder volume; blood-pressure

base line and signal; time in ten seconds. Effect of a mngm. cytisine.


ganglion and partly from escape from vagus control. The arteriolesare primarily constricted throughout the body, from stimulation of thesympathetic ganglia. These factors bring about a large rise of blood-pressure; with large doses the secondary paralytic action of the alkaloidbecomes very marked, and the normal tone of the vessels cannot bemaintained, and so the blood-pressure falls.

The intestinal movements in the cat are primarily inhibited, butsubsequently become larger and more frequent. The bladder isthrown into a powerful and prolonged contraction, owing to thestimulating action of the alkaloid upon the sacral autonomic ganglia.

(At this point tracings illustrating the action of cytisine were shownby means of the epidiascope. Tracings showing the action of nicotineunder similar conditions of experiment were also shown. Comparisonsbetween these were made and the similarity in action emphasized.)

It will have been observed that these actions of cytisine which I havequoted are typical of the alkaloid nicotine. It is unnecessary to multiplyexamples from further experiments which were carried out in con-junction with Dr. Dale. I could quote many others which show theclose similarity in action between nicotine and cytisine. Their resen-blance in action is so close that by their action on animals alone it wouldbe difficult to say whether a given solution was cytisine or nicotine.Edmunds [15] has shown that lobeline, the chief alkaloid of lobelia, hasan action almost identical with that of nicotine. Apart, therefore, froinisolation of the alkaloids themselves it would be difficult to determinewhether one was dealing with nicotine, lobeline, or cytisine.

In conclusion, I should like to point out the similarity between casesof laburnum poisoning and nicotine poisoning. The man who canremember, as I do, his first overdose of nicotine through excessivesmoking in the days of his youth has an excellent picture of laburnumpoisoning. The restlessness, giddiness, tremors, muscular weakness, thenausea and salivation, the cold, clammy sweat, and vomiting, are alltypical. In severe cases the widely dilated pupil, the drowsiness andcoma, and the modes of death in both cases are very similar.

REFERENCES.

[1] GRAY. Edinb. Med. Journ., 1862, vii, 2, pp. 908, 1025.[2] HuSEMANN und MARME. Zeitschr. f. Chem., 1865, i, p. 161.[3] PARTHEIL. Ber. d. deutsch. chem. Gesellsch., 1890, xxxiii, 2, p. 3201.[4] CORNEVIN. Comptes rend., 1886, p. 777.[5] PREVOST et BINET. Rev. mned. de la Suisse romande, 1887, vii, pp. 516, 553; 1888,

viii, p. 670.[6] RADZIWILLOWICz. Arb. d. Pharin. Inst. z. Dorpat, 1888, ii, p. 56.


[7] Brit. Med. Journ., 1870, i, p. 79.[8] Brit. Med. Journ., 1882, p. 199.[9] Lancet, 1868, i, p. 55.

[10] RADZIWILLOWICZ. Loc. cit.; also Lancet, 1877, ii, p. 414; 1901, ii, p. 491; 1905, ii,p. 635; Brit. Med. Journ., 1883, i, p. 1117.

[11] VALETTE. Rev. med. de la Suisse romande, Geneve, 1908, xxviii, p. 366.[12] Lancet, 1877, ii, p. 341.[13J Brit. Med. Journ., 1882, i, p. 199; also RADZIWILLOWICZ.[14] WHEELHOUSE. Brit. Med. Journ., 1870, i, p. 79; see also RADZIWILLOWICZ, 10c. cit.[15] EDMUNDS. Amer. Journ. Phys., Bost., 1904, xi, p. 78.

DISCUSSION.

Professor CUSHNY, F.R.S., said he would like to hear whether there wasa laburnum habit. The reuson- why Edmunds investigated the question oflobeline was that it had been discovered that lobelia was used by the NorthAmerican Indians, particularly those of the northern part of the continent,instead of tobacco. In fact, lobelia was known as Indian tobacco, and thereseemed every reason to suppose that this substance was used by Indians toa considerable extent. Langley showed, years ago, that pituri, from whichanother alkaloid, piturine, was derived, was used in Australia by the blacks,who were in the habit of chewing it, much in the same way as some people inthis country use tobacco. So that those three alkaloids, which were practi-cally identical in action, were used by various aboriginal races. Langley foundthat piturine acted in the same way as nicotine, and Edmunds could notdistinguish the symptoms of lobelia poisoning from those produced fromnicotine. There was found to be the same difficulty in obtaining satisfactorytolerance of lobeline as there was in the case of nicotine.

Dr. H. H. DALE desired in the first place to associate himself with thecaution of his colleague as to attributing all the symptoms, which had beendescribed as the result of laburnum poisoning, to the action of the alkaloidcytisine. He did not think there was any doubt that in the majority of casesthe effect of laburnum corresponded closely to the effect of cytisine; but, asDr. Laidlaw said, it looked as if, in a minority of cases, there were some otherpoison playing a part. The occurrence of enteritis, for example, was far moresuggestive of the possible presence of some toxalbumin. One naturallysuggested that, perhaps, because of the association with Robinia psedtdacacia, theflowers of which had been confused with those of laburnum, with the resultthat some of the accidental cases of laburnum poisoning had arisen. In thesecond place, he had also had in mind the point which occurred to ProfessorCushny. It was rather curious that mankind seemed to have an instinct forseeking out, and using for their enjoyment, alkaloids which had the particularaction which had been described. It would be interesting to hear of theexistence of a laburnum habit. As far as the records appeared to show, therewas no indication of such a thing. The only other point which might beworth suggesting was the possibility of the therapeutic application of the

N-22a


substance. It had been tried by two different people. Rose Bradford, manyyears ago, did some physiological experiments on the action of an alkaloid,which was described as ulexine, being isolated from the seeds of common gorse.This had since been identified with cytisine. Bradford, finding it causeddiuresis when used experimentally in animals, tried it, he believed, in a fewpatients as a diuretic. What success it had in that direction did not appear tohave been recorded. Radziwillowicz was struck, in his investigations, by itspowerful effect in producing a rise of blood-pressure, and therefore tried it oncertain patients who were suffering from migraine associated with low blood-pressure. He appeared to be under the impression that a favourable effectresulted. Now that the type of its physiological action seemed clearly estab-lished, the only reasonable suggestion of a therapeutic application was onesimilar to that for which lobelia had been used, and it would be interesting toknow whether cigarettes made from laburnum leaves might have some value incases of asthma, such as had been attributed to the use of lobelia.

Dr. W. MURRELL said he considered that the suggestion just made byDr. Dale about using laburnum seeds in the treatment of asthma was a verygood one. The idea occurred to him while the paper was being read.

Dr. T. R. ELLIOTT said an interesting inquiry would be to attempt todistinguish between the various nicotine-like bodies. The paper by thePresident analysed the power of the rabbit's liver, when the animal was madetolerant of the poison, to destroy nicotine itself to some extent; would it becapable of dealing in a similar way with the allied cytisine ?

The PRESIDENT (Professor H. E. Dixon, F.R.S.) said the Section wouldwish to thank Mr. Laidlaw for his interesting paper. He (the President)desired to make only one remark, namely, that in acute cases of nicotinepoisoning the symptoms came on with the fall of blood-pressure. He spokeof cigar smoking by young people. While the blood-pressure was going up,during say the first fifteen minutes, the smoker had a feeling of well-being;then the poisonous symptoms came on suddenly, the blood-pressure arrived atits maximum, and then dropped rapidly, and the smoker turned pale andshowed the usual symptoms of collapse. In the case of three boys, he hadfound that the first symptom complained of was a rumbling in the abdomen,obviously associated with peristalsis, not vomiting; in one case there wasdefinite diarrhaca. He suggested that that was possibly due to depression ofthe inhibitory sympathetic ganglia. By depressing those one cut off theinhibition, and the vagus was allowed to have all its own way for the timebeing, and there was increased peristalsis as a result. He mentioned this,because it was conceivable that those related alkaloids might have a differentdegree of effect. Cytisine alkaloid, for example, might have a more drasticeffect on the alimentary canal than had nicotine, as the result of a more pro-found depression of ganglion cells, so the effect might conceivably be physiological, and not due to another substance.

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-- . --- in seinem Schreibenyom 24. 6. 1965 "An aIle Bezirks- und Kreisarzte,an aIle arztlichén Direktoren" auf "die zunehmen-den Zahlen der Tabakschadigungen, besonders diein den letzten Jahren standig ansteigenden Ziffernder Todesfalle an Bronchialkrebs und Herzinfarkt"hin und empfahl u. a::

"Starke Raucher sollten sowohl psychologisch beein-fluBt aIs auch medikamentos unterstützt werden, dasRauchen dauernd einzustellen oder es zumindest nachder Entlassung zu reduzieren...Geeignete Arzte sollten zunehmend für Tabakentwoh-nungssprechstunden im Rahmen ihrer Tiitigkeit gewon-Den und ihnen sollte jede Moglichkeit eiDer Unter-stützung gewiihrt werden . . ."

- - haben Entwéih-nungssprechstunden eingerichtet, die Gruppenbe-handlung entwéihnungswilliger Raucher ~ufgenom-men und verschiedene Tabakentwéihnungsmedika-mente des In- und Auslands erprobt.

Die besten Erfahrungen haben wir übereinstim-mend mit den cytisinhaltigen bulgarischen "Tabex"-Tabletten von Pharmachim Sofia gemacht.

Die frühere -Forschungsstelle für experimentelle

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eine "Tabex"-Versuchsreihe gemeinsam mit uns,parallel zu einer ausgedehnten Plazeboreihe durch,deren Ergebnisse uns dankenswerterweise zur Ver-fügung gestellt wurden.F. Lickint (1955/56) schrieb über das Cytisin fol-

gendes:

"Hier hat einmai der bekannte Pharmakologe FiLhnervorgeschiagen, den Goldregen (Cytisus Iaburnum) oderden Stechginster (Ulex europaeus) aIs Ersatzmittei zurauchen, da das darin enthaitene Cytisin bzw. Ulexineinen nikotinahnlichen Effekt entfaitet, aber ohne of-fenbar zur Sucht zu verieiten 0 . 0"

Der bulgarische Apotheker Straschimir Ingilisow

Der Hersteller gibt bekannt:

"Es stimuliert die Atmung, hauptsiichlich auf refiekto-rischem Wege - es steigert die Adrenalin-Ausschüt-

, tung aus dem Nebennierenmark und steigert den Blut-! druck Sein Wirkungsmechanismus iihnelt dem des

Nikotins. Jedoch ist die therapeutische Breite im Ge-l gensatz zum Nikotin vi el groBer...\ Bei überdosierung iiuBert sich der toxische Effekt

beider Alkaloide in Brechreiz, Erbrechen, Erweiterungder Pupillen, Tachycardie, allgemeinem Schwiiche-gefühl, Atmungsliihmung u. a.""Relative Kontraindikationen: Hoher Blutdruck. undAtherosklerose, in welchen Fiillen die Behandlungunter iirztlicher Aufsicht durchgeführt werden sollte."Bei Siittigung des Organismus mit Cytisin empfindet, ..)

der Raucher keinen Nikotinmangel.. ." , .., ;i Unsere Patienten wurden in Gruppendiskussionen ' ~

zunachst aufgefordert, zu versuchen, das Rauchenohne ein Medikament einzustellen. Diejenigen, diebei einer zweiten Vorstellung noch raùchten undglaubten, auf eine medikament6se Unterstützungnicht verzichten zu k6nnen, bekamen "Tabex" ver-ordnet. lm Regelfall wurde ihnen folgendes Be-handlungsschema übergeben, gleichzeitig mit demdringenden ~at, das Rauchen sofort mit Beginn derTablettenkur ganzlich zu unterlassen:1. bis 3. Tag: 6mal 1 Tablette,4. bis 8. Tag: 5mal 1 Tablette,9. bis 13. Tag: 4mal 1 Tablette,

14. bis 17. Tag: 3mal 1 Tablette.Die - - -kommt Patienten, die - oit unbewuBt - zur An-hebung ihres erniedrigten Blutdrucks rauchen, sehrzugute. Bei Hypertonikern wenden wir "Tabex" nuran, wenn der Blutdruck 2mal w6chentlich gemessenwird. Auch bei Patienten jenseits des 50. Lebens-, ;'jahres und bei allen übrigen, bei denen eine ',~Atherosklerose festgestellt oder verrnutet wird,verrnindern wir die Dosierung wie folgt:1. Tag: 6mal 1 Tablette,2. Tag: 5mal 1 Tablette,3. Tag: 4mal 1 Tablette,ab 4. Tag: 3mal 1 Tablette.ln solchen Fallen erteilen wir den Patienten diestrenge Weisung, vom ersten Tag der Tablettenkuran keinerlei Tabakwaren mehr zu rauchen undempfehlen, die Dosierung baldm6glichst wei ter zuverrnindern und - wenn kein starkes Verlangennach Tabak mehr auftritt, das Praparat nur nochgelegentlich zu nehmen, wenn die Gefahr einesRückfalls zu drohen scheint, z. B. in Belastungs-situationen.Wahrend der Zeit der Tabletteneinnahme überprü-fen wir bei Hypertonikern 2mal w6chentlich denBlutdruck. Bei diesem Vorgehen haben wir keine

- .wickelt. Eine Tablette enthiilt 1,5 mg des lobelin-und nikotiniihnlichen atemanregenden AlkaloidsCytisin. '

S. Moeschlin (1964) aIs Toxikologe schreibt überdas Cytisin u. a.:

"Pharmako1ogie: Cytisin wirkt iihnlich wie Nikotin,aber stiirker erregend, Sympathicomimeticum, zentralvor allem auf die MeduIIa obiongata (vor allem Vaso-motoren- und Brechzentrum) zuerst erregend, dannliihmend. Blutdruck teils zentral, teils peripher gestei-gert (GefiiJ3verengung). Tachycardie, Mydriasis, Schwin-deI, CephaIaea...u"Vergiftungserscheinungen: Diese gleichen weitgehendder Nikotinvergiftung.. ."

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gieich der Entwohnungsergebnisse mit psychothera...J1 peutischen Methoden, mit LobeIin-Injektionen und -1 mit "Tabex". Die "Tabex"-Ergebnisse sind hier -i zumindest nach 4 und 8 Wochen - besser aIs die

i mit anderen Methoden erreichten, die Kurve Pl(vorwiegend Rückfailige) nicht gerechnet.

Wenn auch zahIreiche Dauererfoige mit "Tabex"-Behandlung nach individuellen Beratungen erreichtwurden, so sind doch die Spaterfolge besser, wenndie Patienten regelmaBig an der Gruppenbehand-

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--oder keinen Dauererfolg erzielt hatten und groB~tenteils telefonisch zur kostenlosen Erprobung desneuen Priiparats eingeladen worden waren. ("Ta-bex" war 1965 bis Mitte 1966 noch nicht in genü-gender Menge erhiiltlich.)Von diesen 100 Patienten waren nach 4 Wochen 46,nach 8 Wochen 36, ~ach 13 Wochen noch 31 undnach 26 Wochen noch 21 Nichtraucher.Wird diese Gruppe Pl, die eine andere Zusammen-setzung aIs die Gruppen F (Franze), P2 (Paun, 2.Gruppe) und R (Rehbrücke) aufweist, in die Be-rechnung einbezogen, vermindert sich die Erfolgs-rate der "Tabex"-Behandlung (Ta belle 2). Das Er-

Tabelle2 Ge.amtheit der mit Erralg hebandelten PatienLen nacb der Ze;Ldauerde. Nicbt..aueben. (Erliiuterung .iebe Text)

i( Von den behandelten Rauchcrn waren NichtraucherBach Kurbeginu noch minde.'en.4 Wochen 8 Wochen 13 ~ ochen 26 Wochen'ab.. % ab.. '!tG ab.. % ab.. %

Gruppe Pro-bBOdco

Blutdrucksteigerungen und auch keine sonstigenKomplikationen beobachtet.ln den folgenden Wochen wurden Einzel- undGruppengesprache mit den Patienten geführt. Nach3 Monaten und nach 6 Monaten wurden sie noch-mals über den Erfolg der "Tabex"-Kur, ihr Befin-den, den Tablettenverbrauch und etwaige Neben-wirkungen befragt.Es zeigte si ch, daB der Tablettenverbrauch in derRegel unter dem des empfohlenen Schemas blieb.Von den Patienten der Gruppe F, die mindestenseine Woche nach AbschluB der Tabletteneinnahmenoch nicht wieder geraucht hatten, verbrauchtennur 18 0/0 mehr aIs 50 Tabletten, 66 0/0 zwischen 21und 50 Tabletten und 16 Ofo bis zu 20 Tabletten.

~

Auch St. Stojanov u. M. Janatschkova (1965) be-richten, daB die Mehrzahl der Patienten auf dasRauchen verzichtete, ohne das ganze Behandlungs-schema durchzuführen.Die Forschungsstelle für experimentelle Onkologie, 36 36

187 57

21 21107 37v.289

-PI 46 46F + Pl + P, 241 73

F+PI +P.+RPlaccbo

31 31161 49

100

330

..tell Nichtraucher und 239 Probanden mit einem

~,~lacebo-Praparat - davon waren nur 340/0 nach2 bis 4 Monaten Nichtraucher (Gruppe R). Zusam-men mit den gemeinsam mit der Forschungsstellemit "Tabex" behandelten Patienten ergibt sich eineGruppe von insgesamt 266 Patienten zum Vergleichmit den 239 mit dem Placebo-Praparat behandelten(Tabelle 1).

202 SS80 34

366

239

. Noch nicht aIle Probandcn konntcn bcf.agt wcrdcn.

TaJ..II. 1 AuCgliederung der mit Erfolg bebandelten Patienten naeb der Minde.t-.ejtdauer de. Njcbtraucben. (Erliiuterung .jebe Text)

Von den behandelten Rauchern wareo NichlraucherBach Kurbegino ooch miode.teo.4 Wocbeo" 8 Wocheo 13 Wocheo 26 Wocheo'ab.. % ab.. % ab.. % ab.. %

Pro-banden

Gruppe

III 86

84 84

195 85

83

68

151

1516680

68 52

62 62

130 57

51 47v.l0835 43

v.8186 46

...189

130

100

230

36266

239

F

P,

F+P,

R

F + P, + R

Placebo

~ . Nocb nicbt aile Probanden konnten beirast werden.

Auswahl, BehandIungsart sowie Erhebung der Be-handlungsergebnisse gleichen sich in wesentlichenKriterien, so daE die Zusammenfassung in einerGruppe berechtigt erscheint. Das Patientengut setztsich RUS Rauchern verschiedenen Alters, verschiede-nen Raucheralters, verschiedener sozialer Stellungund verschiedenen Tabakverbrauchs zusammen unddari so aIs reprasentativ für eine Raucherbera-tungsstelle angesehen werden.Der Vergleich zwischen den "Tabex"- und Plazebo-werten, der allerdings nur für die Patienten, die8 Wochen nach Kurbeginn noch Nichtraucherwaren, getroften werden kann, fallt signifikant(nach den KoUerschen TafeIn) zugunsten des"Tabex" RUS. Die ersten 100 von Paun mit "Tabex"behandelten Patienten (Gruppe Pl) sind in derTabeIIe 1 nicht enthalten. Es waren ausgesuchtschwere Falle von Tabakabhangigkeit, vorwiegendsol che, die mit Lobelinbehandlung keinen Erfolg

gebnis bleibt jedoch signifikant besser ais das mitPlazebo erreichte. Unter diesen erschwerten Bedin-gungen waren also 55 0/0 aller mit " Tabex" Behan-delten 8 Wochen nach Kurbeginn noch Nichtrau-cher.Die rückfalligen Raucher verbrauchten zum Befra-gungszeitpunkt im allgemeinen geringere Tabak-mengen aIs var der " Tabex"-Kur. ln der Gruppe Fz. B. rauchten bis zu 6 Wochen nach dem RückfaIl53 0/0 der Rückfalligen weniger aIs die Halfte ihrerursprünglichen taglichen Zigarettenanzahl. Hierausmochten wir jedoch keine voreiligen optimistischenSchlüsse ziehen. Hinsichtlich etwaiger Nebenwir-kungen wurde in der Gruppe F jeder einzelnePatient ausdrücklich befragt, in den Gruppen Piund P2 geschah das nicht.lm folgenden sind die von den Patienten angege-benen Erscheinungen kommentarlos zusammenge-stellt, ohne Rücksicht auf Klarung der Frage, obes sich. dabei um Nikotinentziehungssymptomeoder Nebenwirkungen des Cytisins oder auch umZufaIlsbeobachtungen handelt.Die Patienten wurden hierzu in zwei Kategorieneingeteilt:1. erfolgreich behandelte (4 Wochen nach Kurbeginnnoch nicht rauchend),2. mit nur kurzzeitigem oder ohne Erfolg behan-delte (4 Wochen nach Kurbeginn noch oder wiederrauchend).Diejenigen Patienten, die die Kur erfolgreich durch-führlen, klagten wesentlich seltener über Be-schwerden aIs diejenigen, die sich nicht entschlie-Ben konnten, das Rauchen vôllig aufzugeben. Emst-hait Entwôhnungswillige haben bei Auftretenirgendwelcher Beschwerden oftmals die Dosis ver-mindert, aber das Rauchen aufgegeben, weniger'test Entschlossene brachen die Tablettenkur ab undrauchten mehr oder weniger stark weiter. Irgend-

64

68

66

426234

Summary366 patients were treated with "Tabex" (cytisin) withina tobacco detoxication cure. The results achieved weresignificantly better than with 239 patients treated witha placebo compound. For patients suffering from hy-pertonia and arteriosclerosis the dose was reduced.Most patients did not even need the whole cure-package.Side effects were bath minor and rare. "Tabex" ranksfirst as compai'ed to other compounds on an inter-national level. The therapeutic success achieved-aboveaIl, the permanent success-can be essentially improvedby group treatment.

lung (1 Monat lang wochentlich) - auch in derNachbetreuung (1 Jahr lang monatlich, dann vier-teljiihrlich) - teilgenommen haben.

Zahlreiche Teilnehmer der Gruppenbehandlunghaben sich - abgeschreckt von dem derzeit rechthohen Abgabepreis (35,40 M für 100 Tabletten) -giinzlich oh ne Medikation das Rauchen abgewohnt.Die Rezepte wurden nur in der Hiilfte der Fiilleeingelost. Andererseits ist es einem GroBteil derPatienten, die schon vergebliche Entwohnungsver-su che mit oder ohne Medikamente hinter sich hat-ten, erst mit Hilfe von "Tabex" gelungen, ihreRauchgewohnheit zu überwinden. Auf Grund die-ser Erfahrungen halten wir das Priiparat "Tabex"für das gegenwiirtig wirksamste Adjuvans bei derTabakentwohnung und empfehlen, es in jedem Fallanzuwenden, wo die ehrliche Absicht des Patienten,Nichtraucher zu werden, vorliegt und psychothera-peutische und physiotherapeutische MaBnahmenallein nicht zum Ziel führten.

Literatur

1. BaU, K. P., B. J. Kirby u. C. Bogen: Brit. med. J., 1(1965), 1651-1653. - 2. Benndorf, S., G. Kempe, G.Scharfenberg, R. Wendekamm u. E. Winkelvoss: Dtsch.Ges.wesen 23/44 (1968), 2092-2096. - 3. Best, E. W. R., u.Mitarb.: Canad. med. Ass. J. 96 (1967), 1104-1108. -4. Borbély, F.: in: Die Toxikologie des Tabaks. Her-ausg. v. K. Biittig, H. Huber Verlag, Bern und Stutt-gart 1962. - 5. Ejrup, B.: Svenska Liikartidningen 53(1956), 2634 u. 56 (1959), 1899. - 6. Ders.: Brit. Columbiamed. J. 2 (1960), 411. - 7. Ders.: CA (N.Y.) 13 (1963),183-186. - 8. Ford, St. Jr., u. F. Ederer: J. Amer. med.Ass. 194 (1965), 139-142. - 9. Fühner: zit. n. Lickint1955/1956. - 10. Hammond, E. C.: Amer. J. publ. Hth.55 (1965), 682-691. - Il. Henke, M.: Med. heute, 14(1965), 302-303. - 12. Hochbaum, G. M.: Amer. J. pub1.Hlth. 55 (1965), 692-697. - 13. Hoffstaedt, E. G. W.: Prac-titioner 195 (1965), 794-798. - 14. Lickint, F.: Dtsch. Ges.wesen 4 (1949), 1403. - 15. Ders.: Therapiewoche 6 (1955/56), 444-448. - 16. Moeschlin, S.: Klinik und Therapieder Vergiftungen. 4. Aufl;' Stuttgart 1964, S. 556-557. -17. Paun, D.: Z. iirztl. Fortbild. 58 (1964), 690-693. - 18.Ders.: Dtsch. Ges.wesen 21 (1966), 520-523. - 19. Paun,D., u. J. Franze: Erfassung und Betreuung der Rauchermit chronischer Bronchitis in der Raucherberatungs-stelle Berlin. Vortrag, gehalten auf dem Symposion derGesellschaft für Bronchologie der DDR am 25. 3. 1968in Magdeburg. - 20. Plakun, A. L., u. Mitarb.: Amer.J. publ. Hth. "56 (1966), 434-441. - 21. Rassegna inter-nazionale di clinica e terapia, Napoli, XLIV /17 (1964).- 22. Stojanov, St., u. M. Janatschkova: Chimpharm 2(1965), 13-16 (bulgar.). - 23. Stojko, A. G.: Chro-nitscheskij Nikotinism (Tabakokurenie) i jego Letsche-nie, Medgis, Moskau 1958 (russ.). - 24. Thompson, D. S.,u. Th. R. Wilson: J. Amer. med. Ass. 196 (1966),1048-1052. - 25. The Health Consequences of Smoking.US Department of Health, Education, and Welfare,Washington 1967, Public Health Service PublicationNo. 1696. - 26. van Proosdy, C.: Smoking..., Amster-dam, London, New York, Princeton; Elsevier 1960.

Das Praparat wird ab 1. Quartal in der DDR er-haltlich sein.

~ ZusammenfassungEs wurden 366 Patienten mit" Tabex" (Cytisin) aIsTabakentw6hnungsmedikament behandelt. Die Er-gebnisse waren signifikant besser aIs bei 239 miteinem Plazebopriiparat Behandelten. Bei Hyperto-nie und Atherosklerose wurde die Dosis vermindert.Die meisten Patienten ben6tigten nicllt die ganzeKurpackung. ~ebenwirkungen waren recllt gering~nd seIten:...Das Priiparat scllneidet im internatio~a-Ien Vergleicll am besten ab. Eine Gruppenbehand-lungkann--diè--Erfolge -: insbesondere die Dauer-erfolge - wesentIicll verbessern.

Pe31OMe

y 366 60JIbHbIX c qeJIblO OTBbIKaHHSl HX OT KypeHHJI npH-MeHSlJICSl npenapaT Ta6eKc (qHTH3HH). llOJIyqeHHble pe-3YJIbTaTbI B 3TOt:x rpynne 6bIJIH AOCTOBepHO JIyqwe, qeMy 239 JIeqeHHbIX nJIaqe6o-npenapaToM. llpH rHnepTo-HHH H aTepOCKJIepO3e AO3Y YMeHbWaJIH. B 60JIbWHHCTBeCJIyqaeB He 6bIJIO Heo6xOAHMOCTM B npHeMe Bcex Ta-6JIeTOK, npeAYCMOTpeHHbIX KypCOM JIeqeHHSl. llo6oqHbleSlBJIeHHSl 6bIJIH He3HaqHTeJIbHbIMH H peAKHMM. CpaBHe-HHe YKa3aHHoro npenapaTa C APyrHMH npenapaTaMH,HMelOxqMMHCSl Ha Me2KAYHapOAHOM pbIHKe, nOKa3aJIO,qTO OH SlBJISleTCSl HaMJIyqWHM. rpynnoBoe JIeqeHHe MO-2KeT cnoc06CTBOBaTb nOJIyqeHHIO exqe JIyqWHX AaHHbIX,oco6eHHO B OTHoweHHH OTAaJIeHHbIX pe3YJIbTaTOB.

Manuskripteingang: 30. Mai 1968i~1

Anschrift: Dr. D. Paun und Dr. J. Franze, Raucher-beratungsstelle der Poliklinik am Krankenhaus imFriedrichshain, 1017 Berlin, Leninallee 171

interdisciplinary

Effect of cytisine on some brain and hepatic biochemical parameters in spontaneously hypertensive rats

Rumyana SIMEONOVA, Vessela VITCHEVA, Mitka MITCHEVA

Laboratory of “Drug metabolism and drug toxicity”, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Medical University, Sofia, Bulgaria

ITX030110A04 • Received: 11 December 2009 • Revised: 22 January 2010 • Accepted: 25 January 2010

ABSTRACTTobacco smoking is a risk factor for variety of cardio-vascular diseases, such as hypertension, myocardial infarction, stroke and many

others. It is of great importance for hypertensive patients to stop smoking. One of the medicines widely used for smoking cessation

in Bulgaria is the original Bulgarian product Tabex®, which is developed on the basis of natural plant alkaloid cytisine. The aim of the

following study was to ivestigate the effects of cytisine on some brain and hepatic biochemical parameters in spontaneously hyper-

tensive rats (SHR), an widely used rodent model for human essential hypertension, and to compare the obtained results with their

age-matched normotensive controls Wistar Kyoto (WKY). Multiple cytisine administration did not affect the activity of ethylmorphine-

N-demethylase (EMND) and anylinehydroxylase (AH), as well as the quantity of cytochrome P 450, nor in WKY neither in SHR In the liver

cytisine increased the MDA quantity both in SHR and in WKY, by 25% (p<0.05) and by 29% (p<0.05) respectively, while the GSH level

was not significantly changed by the compound in both strains. In contrast, on the brain level, cytisine administration to SHR caused

more prominent toxicity, resulted in GSH depletion and increased MDA quantity, while in WKY strain did not exert any toxicity. Cytisine

did not significantly affect ALAT and ASAT activity in both strains. In conclusion, the results of our study suggest higher brain toxicity

of cytisine in spontaneously hypertensive rats, that might be due to their pathophysiological characteristics.

KEY WORDS: SHR; cytisine; toxicity; metabolism

Correspondence address:

Vessela Vitcheva

Department of Pharmacology and Toxicology, Faculty of Pharmacy

“Dunav” str. № 2; Sofia – 1000; BULGARIA

TEL.: +359 2 9236 548 • FAX: +359 2 987 987 4

E-MAIL: [email protected]

cytisine. Cytisine is an alkaloid from the plant Laburnum

anagyroides Med., (Cytisus laburnum L., Fabaceae) which

if widely distributed in the Sought part of Central and

Eastern Europe. All parts of this plant contain the alka-

loid cytisine, but the largest quantity (up to 3%) is found

in seeds. (Tzankova and Danchev, 2007). In Bulgaria,

cytisine, as a smoking cessation aid, has been used since

the 1960s and has been manufactured and marketed since

1964 as Tabex® (Sopharma, Bulgaria).

Taking into consideration a number of hypertensive

patients who smoke and the necessity to stop this vice,

paucity of available data on drug metabolism and drug

toxicity of cytisine on this pathological state, it is impor-

tant to characterize the effects of cytisine on some liver

and brain biochemical parameters in spontaneously

hypertensive rats. SHRs are a suitable model for investiga-

tion not only of the cardio-vascular diseases, but also of

drug metabolism and drug toxicity in this pathological

condition. At the same time, it is well known that SHRs

are more prone to liver and brain injury, provoked by

some compounds.

The aim of the following study was to investigate the

effects of cytisine, administered to SHR for 14 days on

some brain and hepatic biochemical parameters.

Introduction

According to the World Health Organisation, one third

of the adult population smokes. Epidemiological data

show that tobacco smoking provokes many diseases in

cardio-vascular, pulmonary and other systems and it is

one of the major causes for premature death in the world.

Tobacco may aggravate the hypertensive state in patients

with cardio-vascular diseases. That is why smoking

cessation is obligatory condition for them. Because of

the great importance of tobacco dependence, there are

many approaches to resolve this problem. The current

most effective method for treatment of nicotine addiction

is nicotine replacement therapy. In Bulgaria, the most

used method is the therapy with the original Bulgarian

drug Tabex®. The product is developed on the basis of

Interdisc Toxicol. 2010; Vol. 3(1): 21–25.

doi: 10.2478/v10102-010-0004-4

Published online in:

www.setox.eu/intertox & www.versita.com/science/medicine/it/

Copyright©2009 Slovak Toxicology Society SETOX

ORIGINAL ARTICLE

22Rumyana Simeonova, Vessela Vitcheva, Mitka Mitcheva

Eff ect of cytisine on some brain and hepatic biochemical parameters

ISSN: 1337-6853 (print version) | 1337-9569 (electronic version)

Materials and methods

Reagents and chemicalsAll reagents used were of analytical grade. Cytisine was

provided by Sopharma Pharmaceuticals, Sofia, Bulgaria.

The other chemicals: NaHCO3, KH2PO4, Trichloracetic

acid, 2-Thyobarbituric acid, CH3COOH, Glucoso-6-

phosphate, Semicarbazide, Nicotinamide, Ba(OH)2,

ZnSO4, Ethylmorphine, Anyline, Na2S2O5, NADP, Phenol

were purchased form Sigma Chemical Co. (Germany).

2,2 -dinitro-5,5`dithiodibenzoic acid (DTNB), K2HPO4

and NaH2PO4 × 2H2O were obtained from MERCK

(Germany).

For the aim of the experiments, cytisine was dissolved

ex tempore in distilled water in quantity for receiving

working solutions. The solutions were administered once

daily via stomach tube (1ml/100 g b.w.).

AnimalsExperiments were performed in 12 male SHR (body

weight 180–230 g) and 12 WKY (body weight 200–250 g),

obtained from Charles River Laboratories (Sulzfeld,

Germany). The animals were housed in Plexiglas cages (3

per cage) at 20±2 °C and 12-h light : 12-h dark cycle. Food

and water were provided ad libitum.

Blood pressure was measured in conscious animals

using a semi-automated tail-cuff device (LE5002, Letica

S/A, Spain). Before the experimental period, the rats

were conditioned to the restraining cylinders and blood

pressure measurement. Rats were pre-warmed for 10 min

using a temperature – controlled warming holder (37 °C)

to facilitate tail blood flow before their blood pressure

was measured. The mean of tree tail-cuff readings was

used as the systolic and diastolic blood pressure value.

Body weight and organ weight (livers and brains) were

measured with a standard laboratory scale.

All procedures were approved by the Institutional

Animal Care Committee and performed strictly follow-

ing the principles stated in the European Convention

for the Protection of Vertebrate Animals used for

Experimental and other Scientific Purposes (ETS 123)

(1991).

Design of the experimentThe animals were divided into four groups (n=6 each).

The first group was SHR receiving cytisine at dose 5

mg/kg (Tabex. Product monograph, Sopharma, 2006)

p.o., once daily for 14 days. The second group was WKY,

receiving cytisine 5 mg/kg po once a day for 14 days. The

third and fourth groups included respective controls,

untreated animals from two rat strains, which were

involved in the experiment from the very beginning and

housed under the same standard laboratory conditions as

treated animals.

All animals were fasted overnight before euthanasia.

24 hours after the last administered dose of cytisine,

the animals of all groups was weighed, euthanized by

decapitation and blood, livers and brains were taken for

biochemical assessment.

Preparation of liver microsomes for biochemical assay (Guengerich, 1987) Rats were decapitated and the livers were excised,

perfused with 0.15 M KCl and minced. The latter was

homogenized with 3 volumes of 1.17% KCl solution in

a glass homogenizer. The liver homogenates were then

centrifugated at 10,000 × g for 30 min. The supernatant

fractions were centrifugated at 105,000 × g for 60 min.

The resulting microsomal pellets were stored at –20 °C

until assayed.

Evaluation of Phase I of biotransformationAssay of aniline 4-hydroxilase activity (Cohen et al., 2000)

4-hydroxilation of aniline to 4-aminophenol, that is

chemically converted to a phenol-indophenol complex

with an absorption maximum at 630 nm. Enzyme activity

is expressed as nmol/min/mg.

Assay of EMND activity (Cohen et al., 2000)

The enzyme activity was evaluated by the formation of

formaldehyde, trapped in the solution as semicarbazone

and measured by the colorimetric procedure of Nash, at

415 nm. Enzyme activity is expressed as nmol/min/mg.

Assessment of cytochrome P450 quantity (Omura and Sato, 1964)

At the day of assay the microsomal pellets were resus-

pended and diluted in phosphate buffer + EDTA (pH=7.4).

Liver protein concentration was mesured, using the

method of Lowry (1951) and was adjusted to 10 mg/ml.

Cyt P450 quantity was quantified spectrophotometrically

as a complex with CO, at 450 nm

Preparation of brain and liver homogenate for MDA assessment (Deby and Goutier, 1990)Samples of liver and brain tissue were homogenized in

25% trichloracetic acid (TCA) and 0.67% thiobarbituric

acid (TBA) in Glass homogenizer (PX-OX 2000). The

samples were then mixed thoroughly, heated for 20 min in

a boiling water bath, cooled and centrifuged at 4,000 rpm

for 20 min. The absorbance of supernatant was measured

at 535 nm against a blank that contained all the reagents

except the tissue homogenate. MDA level was expressed

as nmol/g wet tissue.

Preparation of brain and liver homogenate for GSH assessment (Fau et al., 1994)Samples of liver and brain tissue were homogenated in 5%

trichloracetic acid (TCA) in Glass homogenizer (PX-OX

2000) and centrifugated for 20 minutes at 4,000 rpm.

GSH was assessed by measuring non-protein sulfhy-

dryls after precipitation of proteins with TCA, followed

by measurement of thiols in the supernatant by the DTNB

reagent (Bump et al., 1983). GSH level was expressed as

nmol/g wet tissue.

Evaluation of transaminase activity (ALAT and ASAT) in serumThe blood was taken into a tube containing EGTA. Serum

was separated by centrifugation in bench centrifuge

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Interdisciplinary Toxicology. 2010; Vol. 3(1): 21–25

Copyright © 2010 Slovak Toxicology Society SETOX

(Eppendorf, MiniPlus) at 10,000 rpm for 10 min, 4 °C and

serum activity of aspartate aminotransferase (ASAT) and

alanine aminotransferase (ALAT), were measured using

automated, optimized spectrophotometrical method

(COBOS Integra 400 plus, Roche Diagnostics).

Statistical analysisThe results were presented as ± SD of 6 animals in each

group. Student’s t-test was used. Probability values less

than 0.05 were considered significant.

Results

Eff ect of cytisine on P 450 quantity and enzyme activities (Table 1).Untreated SHRs presented higher (p<0.05) activity of

EMND than untreated WKY. Cytisine treatment did

not affect the activity of EMND and AH, as well as the

quantity of P 450, nor in WKY neither in SHR.

Eff ect of cytisine on hepatic GSH and MDA levels (Table 2)Untreated SHRs showed lower (by 26% (p<0.05) GSH level

and higher (by 38% (p<0.05) MDA quantity than WKY

untreated group. Cytisine increased the MDA quantity

both in SHR and in WKY, by 25% (p<0.05) and by 29%

(p<0.05) respectively, while the GSH level was not signifi-

cantly changed by the compound in both strains.

Eff ect of cytisine on brain GSH and MDA levels (Table 3)Brains from control SHR group displayed lower GSH level

by 30% (p<0.05) and higher MDA level by 25% (p<0.05),

compared to the control WKY group. In brain homogenate

from SHR, cytisine decreased GSH level by 25% (p<0.05)

and increased MDA quantity by 22% (p<0.05). The results

were compared to the unntreated SHRs. Cytisine did not

change these parameters in WKY.

Eff ect of cytisine on transaminases (Table 4)Cytisine did not affect significantly ALAT and ASAT

activity in both strains.

Discussion

Cytisine, a natural plant alkaloid, has been used in

Bulgaria for 40 years in the clinical management of smok-

ing cessation and has shown similar to nicotine pharma-

cological characteristics. Being a competitive blocker of

α4β2* nAChRs, cytisine behaves as an antagonist in the

presence of nicotine and therefore would decrease craving

and attenuate nicotine withdrawal symptoms in humans

(Lukas, 2006).

Cytisine is considered to be efficient and safe drug

for smoking cessation (Etter, 2008). However there is not

sufficient clinical experience with Tabex® in patients with

chronic diseases, like hypertension, diabetes etc.

Regarding the lack of experimental data about

cytisine metabolism and toxicity, in the present study,

Table 1. Effect of cytisine on cytochrome P450 quantity, AH and EMND

activity after multiple administration in male SHR, compared to male

WKY.

WKY SHR

Parameter Control Cytisine Control Cytisine

P-450 quantity (nmol/mg)

0.336 ± 0.002 0.356 ± 0.004 0.365 ± 0.01 0.350 ± 0.009

AH activity (nmol/mg/min)

0.043 ± 0.002 0.044 ± 0.003 0.041 ± 0.004 0.036 ± 0.005

EMND activity (nmol/mg/min)

0.332 ± 0.007 0.305 ± 0.011 0.472 ± 0.04* 0.511 ± 0.02

Data are expressed as mean ± SD of 6 rats, * p<0.05 vs control WKY

Table 2. Effect of cytisine multiple administrations on hepatic GSH level

and MDA quantity in male SHR, compared to male WKY.

WKY SHR


GSH (nmol/g)

6.34 ± 0.16 5.74 ± 0.16 4.69 ± 0.14* 4.47 ± 0.16

MDA

(nmol/g)1.05 ± 0.03 1.35 ± 0.11* 1.45 ± 0.04* 1.81 ± 0.12+

Data are expressed as mean ± SD of 6 rats,

* p<0.05 vs control WKY; +p<0.05 vs control SHR

Table 3. Effect of cytisine multiple administrations on brain GSH level

and MDA quantity in male SHR, compared to male WKY.

WKY SHR


GSH (nmol/g)

1.28 ± 0.03 1.16 ± 0.08 0.89 ± 0.05* 0.67 ± 0.07+

MDA

(nmol/g)3.89 ± 0.05 3.82 ± 0.09 4.86 ± 0.04* 5.95 ± 0.07+

Data are expressed as mean ± SD of 6 rats,

* p<0.05 vs control WKY; +p<0.05 vs control SHR

Table 4. Effect of multiple administration of cytisine on serum ALAT and

ASAT activity in male SHR, compared to male WKY.

WKY SHR


ALAT UI/L

66.2 ± 6.2 61.3 ± 19.1 82.6 ± 4.2 92.7 ± 23.2

ASAT UI/L

234.2 ± 12.5 228.3 ± 9.3 329.3 ± 9.8* 289.2 ± 22.5

Data are expressed as mean ± SD of 6 rats, * p<0.05 vs control WKY

we investigated the effect of cytisine on some hepatic

and brain biochemical parameters, using spontaneously

hypertensive rats (SHR), that are considered to be a suit-

able pathophysiological model for essential hypertension

in human (Yoshimoto et al. 2003)

Our results on the level of drug metabolizing enzyme

systems reveiled higher EMND activity, marker of CYP

24Rumyana Simeonova, Vessela Vitcheva, Mitka Mitcheva

Eff ect of cytisine on some brain and hepatic biochemical parameters

ISSN: 1337-6853 (print version) | 1337-9569 (electronic version)

3A (Amacher and Schomaker, 1998) in SHR with no dif-

ference in cytochrome P 450 quantity and AH activity,

marker of CYP 2E1 (Monostory et al, 2004), compared

to control WKY rats (Table 1). These results are in good

correlation with the studies, carried out by Merrick et

a. (1985) that showed a significant increase in EMND

activity in SHR with only slight increases in cytochrome

P450 quantity and AH activity, compared to WKY rats.

Few years latter Basu et al. (1994) proved that this elevated

CYP 3A activity in SHR is related to the augmentation of

the arterial blood pressure.

14 days oral administration of cytisine did not change

the activity of EMND and the activity of AH, as well as

the quantity of cytochrome P 450 nor in SHR, neither in

WKY. These results are supported by data in the literature

that discuss cytisine minimal hepatic biotransformation

in humans and 90–95% excretion of the unchanged

compound in the urine (Tabex. Product monograph,

Sopharma, 2006).

One of the essential factors for hypertension is the

oxidative stress, which is characterized by low level of

the intracellular protector GSH and high level of MDA,

a product of lipid peroxidation and marker of oxidative

stress. In their study Cediel et al. (2003) proved that in

liver homogenates from SHR, MDA levels were higher and

the ratio reduced/oxidized glutathione (GSH/GSSG) and

glutathione peroxidase activity (GPx), were lower, com-

pare to WKY. Our results support these findings. Both

in liver and brain the quantity of GSH was significantly

lower and the MDA level was significantly higher in SHR,

compared to the control WKY (Table 2 and 3).

On the hepatic level, cytisine treatment did not affect

the GSH quantity nor in SHR, neither in WKY, while

the quantity of MDA was significantly increased in both

strains. The lack of toxicity on GSH might be due to the

lack of bioactivation of cytisine to metabolites that might

conugate with GSH and cause its depletion. On the other

hand, on the basis of our results we could suggest that

the entire molecule of cytisine might have the ability to

induce a process of lipid peroxidation, manifestated by the

observed increase in MDA quantity.

There are a number of in vivo studies on the potential

hepatotoxic effect of cytisine in different animal species.

The study of Angelova (1971) determined that chronic

administration of cytisine to rats, at a dose of 1.35 mg/kg

during 90 days caused a 2-fold increase in blood glutamate

pyruvate transaminase (GPT) concentration, without

significant changes in blood glutamic oxaloacetic trans-

ferase (GOT) and alkaline phosphatase. Such changes

were not observed when cytisine was administered dur-

ing 45 days to mice (3.3 mg/kg) and 180 days to rats (0.45

and 0.9 mg/kg) or dogs (0.46 mg/kg). In our experiment

cytisine, administered orally 5 mg/kg for 14 days did not

significantly change the values of serum transaminase

activity, ASAT and ALAT, in any of the treated strains.

These results might be due to the shorter period of admin-

istration (14 days) and to the large individual variations.

On the brain level, multiple cytisine administration

caused more prominent toxicity in SHRs, resulted in GSH

depletion and increased MDA quantity, while in WKY

strain did not exert any toxic effect. Reavill et al. (1990),

in their studies in rats found out that cytisine crosses the

blood-brain barrier less readily than nicotine. This might

be one of the possible explanations for the observed

lack of brain toxicity in WKY rats. On the other hand,

it is proved that in chronic hypertension the blood brain

barrier is characterised with an increased permeability

due to disrupted tight junctions caused by endothelial

dysfunctions (Lippoldt et al., 2000). The better perme-

ability of cytisine through the BBB in hypertensive rats,

slower blood brain circulation in this state (Kishi et al.,

2004) could explain the higher brain toxicity of cytisine

in hypertensive animals.

In conclusion, the results of our study suggest higher

brain toxicity of cytisine in spontaneously hypertensive

rats, that might be due to their pathophysiological

characteristics.

Acknowledgement

This work was supported by Sopharma Trading, Bulgaria.

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REPRINT FROM

DAS DEUTSCHE

GESUNDHEITSWESEN

Organ of the German Association for Clinical Medicine

VOLUME 23/68 ISSUE 44 PAGES 2088-2091 2092-2096 2111-2112

__________________________________________________________________________

Smoking Cessation with “Tabex” Tablets Containing Cytisine

By D. PAUN and J. FRANZE

Results of Pharmacological Smoking Cessation

using Cytisine (Tabex®)

By S.BENNDORF, G.KEMPE, G. SCHARFENBERG, R.WENDEKAMM, and E. WINKELVOSS

Question Forum

HEALTH PROTECTION From the Smoker Counseling Desk (Director: Dr. Paun) of the polyclinic at Krankenhaus im Friedrichshain, Berlin (Medical Director: Prof. K. Scheidler)

Smoking Cessation using “Tabex” Tablets Containing Cytisine By D. PAUN and J. FRANZE

The Minister of Health of the Democratic Republic of Germany pointed to the “rising amount of

impairment from tobacco, particularly the increasing number of deaths from bronchial cancer and heart attacks in recent years” in his memo of 24 June 1965 to “all regional and district physicians and all medical directors” and recommended among other things:

“Heavy smokers should be influenced psychologically and supported with medication to give up smoking permanently or to at least reduce smoking after discharge… Qualified physicians should be enlisted increasingly to conduct smoking cessation clinics and they should be offered every possible support for this undertaking…”

We followed up on this appeal and set up smoking cessation clinics which used group therapy for smokers who wished to quit, and tested several domestic and foreign medications for smoking cessation.

We achieved the best results using the Bulgarian “Tabex” tablets containing cytisine from Pharmachim in Sofia.

The former Research Center for Experimental Oncology of the German Academy of Science in Potsdam-Rehbrücke also collaborated with us on a “Tabex” study, parallel to an extensive placebo

study, the results of which they kindly made available to us. F. Link int (1955/56) wrote the following about cytisine: “The well-known pharmacologist Fühner has suggested smoking laburnum (Cytisus laburnum) or golden gorse (Ulex europaeus) instead, since the cytisine or ulexine contained in them has an effect similar to nicotine, but apparently does not lead to addiction…”

The Bulgarian pharmacist Strashimir Ingilisov developed “Tabex” from laburnum. One tablet contains 1.5 mg of the respiratory stimulant alkaloid cytisine which is similar to lobeline and nicotine.

The toxicologist S. Moeschlin (1964) wrote about cytisine: “Pharmacology: Cytisine has an effect similar to nicotine, but is more stimulating, a sympathicomimetic compound, initially centrally stimulating especially the medulla oblongata (in particular the vasomotoric and vomiting centers), then paralyzing them. Blood pressure is elevated centrally and peripherally (vascular stenosis). Tachycardia, mydriasis, vertigo, cephalea…” “Symptoms of poisoning: These are quite similar to nicotine poisoning…”

The manufacturer announces: “It stimulates respiration, mainly reflectory – it increases the distribution of adrenaline in the adrenal medulla, and raises blood pressure… Its effective mechanism is similar to that of nicotine. However, in contrast to nicotine, its therapeutic range is much broader… In case of an overdose, the toxic effect of both alkaloids causes nausea, vomiting, widening of the pupils, tachycardia, general weakness, respiratory paralysis, etc.” “Relative counter indications: High blood pressure and arteriosclerosis, in which case treatment should be conducted under a physician’s supervision.”

If his organism is saturated with cytisine, a smoker does not experience nicotine withdrawal…”

In group discussions, our patients were first asked to attempt to quit smoking without any medication. Those who were still smoking at a second meeting and believed that they could not quit without pharmacological support were prescribed “Tabex”. They usually received the following treatment plan,

together with the urgent advice to quit smoking completely at the beginning of the medication therapy: 1st to 3rd day: 1 tablet 6 times daily

4th to 8th day: 1 tablet 5 times daily 9th to 13th day: 1 tablet 4 times daily 14th to 17th day: 1 tablet 3 times daily

The elevation of blood pressure caused by this compound was often quite positive for patients who – often subconsciously – smoked to raise their low blood pressure. We used “Tabex” in persons with

hypertension only if blood pressure was measured twice weekly. For patients above age 50 and for all others with diagnosed or suspected arteriosclerosis, the dosage was reduced as follows:

1st day: 1 tablet 6 times daily 2nd day: 1 tablet 5 times daily 3rd day: 1 tablet 4 times daily

4th day and after: 1 tablet 3 times daily In these cases, we advised the patients urgently not to smoke any tobacco products after the first day

of the treatment and recommended that they further reduce the dosage as soon as possible and – if no strong urge for tobacco was present – to take the tablets only occasionally, when a relapse was imminent, e.g. in stress situations.

During the period of treatment, we checked the blood pressure in patients with hypertension twice weekly. During this process, we did not observe any elevated blood pressures or any complications of

any kind. In the following weeks, individual and group sessions were held with the patients. After 3 months and again after 6 months, they were questioned as to the success of the “Tabex” therapy, their condition,

and any side effects. It was shown that the most subjects consumed fewer tablets than the recommended amount in the treatment plan. Of the patients in group F who had not smoked again for at least a week after

completing the treatment plan, only 18% consumed more than 50 tablets, 66% between 21 and 50 tablets, and 16% up to 20 tablets. St. Stoianov and M. Janachkova (1965) also reported that the majority of patients gave up smoking

without having to complete the entire treatment plan. The Research Center for Experimental Oncology in Potsdam-Rehbrücke treated 36 subjects with “Tabex” – 42% of whom were non-smokers after 4 to 5 months – and 239 subjects with a placebo –

of these, only 34% were non-smokers after 2 to 4 months (Group R). With the patients treated jointly with the Research Center there is a total of 266 patients in comparison with the 239 who were given a placebo.(Table 1).

Table 1 Classif ication of the patients w hose treatment w as successful according to the minimum period of smoking cessation (for explanations, see text) ___________________________________________________________________________________________ Group After the beginning of treatment, at least ,,,,, of the smokers treated w ere nonsmokers for at least: 4 w eeks

abs. % 8 w eeks abs. %

13 w eeks abs. %

26 w eeks1 abs. %

Subjects

__________________________________________________________________________

_____________________________________________________________________________________________ F 111 86 83 64 68 52 51

of 108

47 130

P2 84 84 68 68 62 62 35 of 81

43 100

F + P2 195 85 151 66 130 57 86

of 189

46 230

R 15 42 36 F + P2 + R 166 62 266 Placebo 80 34 239

1 Not all subjects have been questioned.

The selection, type of treatment, and compilation of treatment results are identical in essential criteria, so that summarizing them in one group appears to be justifiable. The patient group comprises

smokers of various ages, various smoking durations, varying social status, and various levels of consumption, and may thus be considered representative for a smoking counseling office. The comparison between “Tabex” and placebo levels, which can be made only for subjects who were

still non-smokers 8 weeks after the treatment began, is significantly (according to Koller’s tables) in “Tabex’” favor. The first 100 of Paun’s patients treated with “Tabex” (Group P1), are not included in Table 1. These included hand-picked cases of severe tobacco dependency, mostly those who were

not successfully treated or did not achieve lasting success with lobeline and who for the most part were contacted by telephone and invited to join a free trial of the new compound. (In 1965 until the middle of 1966, “Tabex” was not yet available in sufficient amounts.)

Of these 100 patients, 46 were non-smokers after 4 weeks, 36 after 8 weeks, 31 after 13 weeks, and 21 after 26 weeks. If this group P1, which has a different composition than groups F (Franze), P2 (Paun, 2nd group), and R

(Rehbrücke), is included in the calculation, the success rate of “Tabex” treatment is lowered (Table 2). The result remains, however, significantly better than that achieved with the placebo. Under these difficult conditions, 55% of all subjects treated with “Tabex” were still non-smokers 8 weeks after the

beginning of treatment. Table 2 Total of successfully treated patients according to the duration of nonsmoking (for explanation see text)

Group After the beginning of treatment, at least ,,,,, of the smokers treated w ere

nonsmokers for at least:

Subjects

4 w eeks 8 w eeks 13 w eeks 26 w eeks1 abs. % abs. % abs. % abs. % _____________________________________________________________________________________________________

P1 46 46 36 36 31 31 21 21 100 F + P1 + P2 241 73 187 57 161 49 107

of 289 37 330

F + P1 +

P2 + R

202

55

366

Placebo 80 34 239 _____________________________________________________________________________________________________ 1 Not all subjects could be questioned

At the time of questioning, the relapsed smokers generally consumed smaller amounts of tobacco

than before the “Tabex” treatment. In group F, for example, the relapsed smokers smoked less than half of their previous consumption up to 6 weeks after relapse. But we should not draw any premature optimistic conclusions from this fact. Every single patient in group F was expressly asked about side

effects; this was not the case for groups P1 and P2.

In the following, the side effects mentioned by the patients are listed without any remarks, irrespective of whether they are nicotine withdrawal symptoms, side effects of cytisine, or coincidental

observances. For this the patients were divided into two categories:

1. successful treatment (non-smokers 4 weeks after beginning treatment)

2. only temporary success or unsuccessful treatment (still smoking or smoking again 4 weeks after beginning treatment)

The patients who successfully completed treatment had significantly fewer complaints than those who

could not be convinced to quit smoking completely. Subjects who seriously wanted to quit often lowered the dosage when any side effects appeared, but still quit smoking; subjects who were less serious about quitting simply stopped taking the tablets and resumed smoking more or less as before.

Groups P1 and P2 (200 patients) _____________________________________________________________________________________________________

Symptoms Category 1 Category 2 Total ______________________________________________________________________________________________________ Vomiting ---- 2 2 Abdominal complaints 1 ---- 1

Queasiness 2 ---- 2 Nausea ---- 1 1

Headache ---- 1 1 Heart pain, or unpleasant sensation around the heart 1 2 3

Tendency to faint 1 ---- 1 Dizziness 1 ---- 1 Fatigue 1 ---- 1 Missing a period ---- 1 1

Slight urticaria ---- 1 1 Shortness of breath 1 ---- 1 Unclear symptoms ---- 2 2 _____________________________________________________________________________________________________

8 of 130 (6%)

10 of 70 (14%)

18 of 200 (9%)

Group F (130 patients)

_____________________________________________________________________________________________ Symptoms Category 1 Category 2 Total _____________________________________________________________________________________________ Constipation 5 ---- 5

Diarrhea ---- 1 1 Nausea ---- 1 1 Abdominal complaints 3 2 5 Loss of appetite ---- 1 1

“Unclean saliva” 1 ---- 1 Gum bleeding ---- 1 1 Headaches ---- 1 1

General nervousness 4 ---- 4 Depression 1 ---- 1 General fatigue ---- 1 1 Heart pressure 2 ---- 2

_____________________________________________________________________________________________ 16 of 111

(15%) 8 of 19 (42%)

24 of 130 (17%)

Fig. Results of treatment from several authors according to various methods. The lines represent the percentage of patients treated (ordinates) w ho w ere non-smokers at the time of questioning (abscissa = w eeks after treatment began) ____________________________________________________________________________________________________

Author No. of subjects Method of treatment Sign ____________________________________________________________________________________________________ Ball, Kirby, & Bogen (1965)

92 Mainly psychotherapy in groups, medication only in individual cases

-------------------------

Ejrup (1965) 903 Medication (lobeline injections, meprobamate, cholinergic drugs) and intensive individual care

— — — — — —

Franze 130 “Tabex” ______________

Paun 100 (mainly previously relapsed patients)

“Tabex” — · · · — · · · —

Paun 100

(regular patients)

“Tabex” _ _ _ _ _ _

Stoiko 200 Psychotherapy in groups of a social unit — · — · — · — · Thompson & Wilson (1966)

201 Predominantly psychotherapy in groups using a “5 day plan” ____

_____________________________________________________________________________________________________

[Table showing results of different methods]

There were no complications or cases of subjects not being fit for work during the “Tabex” treatment. The graph shows an international comparison of the results of smoking cessation using

psychotherapy, lobeline injections, and “Tabex”. The “Tabex” results here are – at least after 4 and 8 weeks – better than the results achieved using other methods, not including line P1 (predominantly relapses).

Even though numerous long-term successes were achieved with the “Tabex” treatment and individual counseling, the long-term success rate was better for the patients who participated regularly in group

therapy sessions (weekly for 1 month) – as well as in post-treatment counseling (monthly for one year, then every three months). Numerous participants of group therapy – deterred by the relatively high price (35.40 East German

marks for 100 tablets) – quit smoking without any medication at all. The prescriptions were used by only half of the subjects. On the other hand, a large proportion of subjects who had already made many unsuccessful attempts to quit smoking with or without medication were successful for the first

time using “Tabex” to curb the smoking habit. On the basis of this experience, we consider “Tabex” to be the most effective adjuvant for smoking cessation currently available and recommend using it in every case where the patient sincerely desires to quit and psychotherapy and physiotherapy alone

were not enough to achieve this goal. The compound will be available in the German Democratic Republic (East Germany) in the first

quarter. Summary

We treated 366 patients with “Tabex” (cytisine) to quit smoking. The results were significantly better than for 239 subjects treated with a placebo. The dosage was reduced for patients with hypertension

and arteriosclerosis. Most of the patients did not require all of the tablets in the package. Side effects were slight and rare. This compound achieved the best results in an international comparison. The results – particularly long-term results – can be improved significantly by group therapy.

Manuscript submitted: 30 May 1968 Address: Dr. D. Paun and Dr. J. Franze, Raucherberatungsstelle der Poliklinik am Krankenhaus im

Friedrichshain (Smoker Counseling Desk of the polyclinic at the Krankenhaus im Friedrichshain), 1017 Berlin, Leninallee 171

STRESZCZENIECytyzyna jest roślinnym alkaloidem, znanym w lecznictwie od ponad pięćdziesięciu lat, o właściwościach agoni-

stycznych w odniesieniu do nikotynowych receptorów acetylocholinergicznych. W ostatnich latach opublikowano kilka interesujących wyników badań farmakologicznych cytyzyny, co spowodowało wzrost zainteresowania tym lekiem wśród naukowców i lekarzy praktyków. Praca, w swej pierwszej części przedstawia nowe dane, głównie dotyczące farmakodynamiki cytyzyny, które stanowią silną przesłankę przemawiającą za jej zastosowaniem w leczeniu uzależ-nienia od nikotyny, a – być może – także innych zaburzeń i chorób. W dalszej części pracy opisano najbardziej charak-terystyczne efekty ośrodkowego i obwodowego działania cytyzyny. Przedstawiono wstępne wyniki badań klinicznych, które potwierdzają terapeutyczną skuteczność cytyzyny, porównywalną do skuteczności bupropionu, leku stosowane-go z wyboru w leczeniu nikotynizmu. Zwrócono uwagę na cytyzynę jako modelowy związek do poszukiwania innych terapeutycznie skutecznych i bezpiecznych analogów.

SUMMARYCytisine is an alkaloid of plant origin known in the health service for over fifty years, having agonistic properties

to cholinergic nicotinic receptors. In the recent years, a few interesting results of studies on cytisine that aroused scientists and physicans interest in the drug have been published. The first part of the paper describes the new data, particulary concerning the pharmacodynamics of cytisine, which constitutes a strong indication to the treatment of nicotine addiction and maybe other disorders and diseases. In the further part of the paper the most characteristic results of central and peripheral action of cytisine are discussed. The initial results of clinical studies confirm the ef-ficacy of cytisine comparable with the efficacy of bupropion, the drug used as first-line pharmacotherapy for tobacco dependence. Attention is drawn to cytisine as a paternal substance to the search for other effective therapeutically and safe analogues.

Słowa kluczowe: cytyzyna, nikotyna, palenie papierosów, receptory nikotynowe, uzależnienie od nikotyny Key words: cytisine, nicotine, cigarette smoking, nicotinic receptors, nicotine addiction

Praca poglądowaReview

PIOTR TUTKA1, KATARZYNA MRÓZ1, WITOLD ZATOŃSKI2

Cytyzyna – renesans znanego alkaloidu. Aspekty farmakologiczne zastosowania w leczeniu uzależnienia od nikotyny

Cytisine – renaissance of well known alkaloid. Pharmacological aspects of efficacy in the treatment of tobacco dependence

1Katedra i Zakład Farmakologii i Toksykologii Akademii Medycznej w Lublinie2 Zakład Epidemiologii i Prewencji Nowotworów, Centrum Onkologii – Instytut im. Marii Curie Skłodowskiej w Warszawie

WSTĘP

Inhalowanie dymu tytoniowego (aktywne pale-nie tytoniu) zawierającego 4000 związków chemicz-nych stanowi główny czynnik zagrożenia ludzkiego zdrowia. Na początku XXI wieku używa tytoniu po-

nad miliard osób. Światowa Organizacja Zdrowia szacuje, że każdego roku przedwcześnie umiera z tego powodu ponad 5 mln osób. W Europie (także w Polsce) i Ameryce Północnej 80% deklaruje chęć rzucania palenia. Wyprowadzenie palenia tytoniu z ludzkiego zwyczaju wymaga udziału medycyny.

FARMAKOTERAPIA W PSYCHIATRII I NEUROLOGII, 2006, 1, 33–39

PIOTR TUTKA, KATARZYNA MRÓZ, WITOLD ZATOŃSKI34

Ważnym elementem postępowania medycznego jest obecność na rynku farmaceutycznym skutecz-nych leków. W ostatnich latach wzrasta zaintere-sowanie cytyzyną jako potencjalnie ważnym lekiem w tym zakresie.

Terapeutyczne zastosowanie cytyzyny znane jest od dawna. Przed ponad pięćdziesięciu laty była sto-sowana w krajach zachodniej Europy jako środek moczopędny (21), a w dawnym Związku Radziec-kim jako środek stymulujący ośrodek oddechowy i działający na chemoreceptory w ciałach aortal-nych i zatoki szyjnej (12). Po raz pierwszy została zastosowana w leczeniu uzależnienia od nikotyny w latach 60. w Bułgarii (43, 44).

Cytyzyna dostępna jest od wielu lat na rynku medycznym w Polsce w formie tabletek w prepara-cie Tabex (Sopharma, Sofia, Bułgaria), zalecanym przez producenta jako skuteczny środek farma-kologiczny w leczeniu uzależnienia od nikotyny. Ostatnio, po latach zapomnienia, cytyzyna skupia na sobie ponownie szczególną uwagę klinicystów jako atrakcyjny lek przeciw paleniu tytoniu. Stała się także obiektem zainteresowania farmakologów i na jej bazie syntetyzowane są nowe pochodne o obiecujących zastosowaniach terapeutycznych (3, 4, 5, 9, 10, 16).

POCHODZENIE I BUDOWA CHEMICZNA

Cytyzyna jest alkaloidem chinolizydynowym, występującym u roślin z rodziny Leguminosae. Na skalę przemysłową otrzymywana jest z nasion, niekiedy z liści i kwiatów złotokapu zwyczajnego (Laburnum anagyroides, syn.: L. Vulgare, Cytisus laburnum). Obecna jest również w organach roślin z rodzaju Sophora, Baptisia, Genista i Ulex.

Chociaż cytyzyna była wyizolowana już w poło-wie XIX wieku (31), jej strukturę określono dopiero w latach 30. XX wieku (19)( ryc. 1.).

MECHANIZM DZIAŁANIA

Chociaż molekularne mechanizmy uzależnienia od nikotyny nie są dobrze poznane, to główną rolę w uzależnieniu przypisuje się neuroadaptacji sys-temów neuroprzekaźnikowych, przede wszystkim układowi cholinergicznemu (13, 14). Powszechnie przyjmuje się, że działanie uzależniające nikotyny wynika, przynajmniej w części, z jej oddziaływania na system acetylocholinergicznych receptorów ni-kotynowych (nAChR), zwłaszcza receptorów pod-typu α4β2 (46). Podobnie, jak to jest w przypadku większości substancji uzależniających, aktywacja receptorów α4β2 powoduje wzrost wydzielania do-paminy w jądrze półleżącym i korze przedczołowej (10, 14). Wzrost stężenia dopaminy w układzie mezolimbicznym jest odpowiedzialny za uczucie przyjemności, obniżenie apetytu, zmiany nastroju, zmniejszenie lęku i napięcia, pobudzenie oraz po-prawę pamięci. Zaprzestanie palenia papierosów przez uzależnionego od nikotyny powoduje obniże-nie stężenia dopaminy w układzie mezolimbicznym, a to z kolei prowadzi do objawów zespołu odstawie-nia. Teoretycznie, skuteczny w leczeniu nikotyni-zmu środek farmakologiczny powinien umiarkowa-nie podwyższać stężenie dopaminy w układzie me-zolimbicznym, zapobiegając lub łagodząc objawy z odstawienia, a jednocześnie powinien blokować dostęp nikotyny do receptorów α4β2. Zatem, efek-tywnym związkiem może być częściowy agonista receptorów α4β2, jakim jest cytyzyna.

Badania powinowactwa receptorowego cytyzyny przeprowadzono z zastosowaniem metod in vitro i wielu modeli zwierzęcych.

Biorąc pod uwagę powinowactwo do nAChR w całym mózgu (bez wyróżniania podtypów i bez uwzględniania czynników farmakokinetycznych), które badane było w preparatach mózgu szczurów in vitro, cytyzyna wykazuje większe powinowactwo do nAChR niż nikotyna (1). Jednocześnie należy

Rycina 1. Budowa chemiczna cytyzyny

Wzór strukturalny

cytyzyna (C11H14ON2)

Wzór stereoizomeryczny

(-)-7R:9S cytyzyna

CYTYZYNA – RENESANS ZNANEGO ALKALOIDU. ASPEKTY FARMAKOLOGICZNE ZASTOSOWANIA W... 35

zauważyć, że w badaniach in vitro homogenatów mózgu szczurów, cytyzyna łączyła się z niemal iden-tyczną populacją receptorów co nikotyna (z wysoką specyficznością sięgającą 60-90%), wykazując przy tym największe powinowactwo do receptorów we wzgórzu, prążkowiu i korze, większe niż do recep-torów w hipokampie, móżdżku i podwzgórzu (28). Również w preparatach ludzkich mózgów, najwięk-sza gęstość miejsc, z którymi łączyła się cytyzyna wykazywana była we wzgórzu (17). Cytyzyna jest milion razy bardziej selektywna w stosunku do re-ceptorów nikotynowych niż muskarynowych (1).

Cytyzyna różni się w swej aktywności wewnętrznej i powinowactwie w stosunku do poszczególnych pod-typów nAChR. Najpełniejsze dane zgromadzono na temat powinowactwa leku do podtypu α4β2 nAChR.

Liczne badania z użyciem znakowanych ligandów (16, 17, 24, 28, 30, 50) i badania czynnościowe (23, 29, 35) demonstrują, że cytyzyna jest kompetycyj-nym, częściowym agonistą receptorów α4β2. Z badań wynika też, że cytyzyna jest jednym z najsilniejszych związków wiążących się z α4β2 nAChR. U szczurów wykazuje powinowactwo do mózgowych receptorów α4β2 w stężeniach nanomolowych (9, 26, 41). Siła działania 1 mM cytyzyny na receptory α4β2 oocytów szczura wynosi 14,7% siły działania 1 mM endogen-nego neuroprzekaźnika, acetylocholiny (29).

Z drugiej jednak strony, cytyzyna podawana łącznie z acetylocholiną redukuje odpowiedź re-ceptorów α4β2 na acetylocholinę, a więc działa wo-bec niej antagonistycznie (29).

Chociaż stwierdzono, że cytyzyna wiąże się w stężeniach nanomolowych z nAChR zawierającymi podjednostkę β2 i β4 (30), to równocześnie stwier-dzono, że skuteczność jej działania jest niższa w sto-sunku do receptorów zawierających podjednostkę β2 (α2β2, α3β2, α4β2) niż tych, które zawierają podjed-nostkę β4 (α2β4, α3β4, α4β4)(7, 29), odwrotnie niż to jest w przypadku nikotyny i acetylocholiny.

Oprócz działania agonistycznego wobec recep-torów α4β2, cytyzyna jest również agonistą recep-torów α3β4 (5, 9). Dane pochodzące z badań nad receptorami nikotynowymi oocytów żaby szponia-stej (Xenopus laevis), które oddają właściwości far-makologiczne receptorów nikotynowych w mózgu szczurów, wskazują, że powinowactwo cytyzyny do receptorów α3β4 jest 47 razy mniejsze niż do re-ceptorów α4β4 i 11 razy mniejsze niż do recepto-rów α2β4 (30).

Wykazano m.in. w badaniach z zastosowaniem rekombinowanych receptorów ludzkich, że cytyzy-na jest także pełnym agonistą o względnie niskiej aktywności wewnętrznej receptorów zawierających

podjednostkę α7 (7). Jej powinowactwo do receptora α7 zachodzi w stężeniach mikromolowych (9, 41).

Skuteczność cytyzyny jako agonisty poszcze-gólnych podtypów nAChR determinuje jej efekty czynnościowe. Badania czynnościowe potwierdza-ją kompetycyjną naturę tego leku w stosunku do nAChR (18, 23, 29). Coe i wsp. (9) oceniali wpływ cytyzyny na obrót dopaminy w układzie mezolim-bicznym u samców szczurów Sprague Dawley. Jak wcześniej wspomniano, dopamina pełni niezwykle istotną rolę w procesach doprowadzających do uza-leżnienia od nikotyny. Obrót dopaminy jest miarą jej syntezy i utylizacji. W badanich Coe i wsp. (9) pomiary obrotu dopaminy dokonywane w jądrze półleżącym po 60 min. od podskórnego podania 5,6 mg/kg cytyzyny wykazały, że jej wpływ na obrót do-paminy stanowi 40% wpływu nikotyny podawanej podskórnie w dawce 1 mg/kg. Wyniki tych badań dowodzą potencjalnej korzyści cytyzyny jako środka umiarkowanie podwyższającego poziom dopaminy dla łagodzenia lub znoszenia objawów związanych z zaprzestaniem palenia papierosów.

Drugą niezwykle istotną właściwością cytyzyny jest jej zdolność do antagonizowania efektów rów-nocześnie podawanej nikotyny. Ocena odpowiedzi w oocytach Xenopus w obecności znanego stężenia nikotyny pozwala na określenie właściwości anta-gonistycznych i czynnościowych efektów różnych związków. Wyniki uzyskane przez Coe i wsp. (9) wskazują, że cytyzyna antagonizuje czynnościowe efekty nikotyny zastosowanej w stężeniu 10 µM. Ci sami badacze wykazali, że cytyzyna w podskór-nej dawce 5,6 mg/kg redukuje wywołany nikotyną (1 mg/kg podskórnie) wzrost obrotu dopaminy w ją-drze półleżącym o 36%. Świadczy to o niezwykle ko-rzystnym profilu antagonistycznym cytyzyny, która, zapobiegając dostępności nikotyny do jej recepto-rów, w rezultacie zapobiega dalszemu uzależnianiu od nikotyny. Prowadzi to do stopniowego zmniej-szania i zanikania istniejącej u palaczy zależności od nikotyny. Należy jednak podkreślić, że badania in vivo wykazujące antagonizowanie efektów niko-tyny przez cytyzynę są nieliczne i konieczny jest ich dalszy rozwój.

OŚRODKOWE EFEKTY DZIAŁANIA

Pierwsze dane na temat właściwości farmako-logicznych cytyzyny pochodzą z początków XX wie-ku (11). Z farmakologicznego punktu widzenia, działania cytyzyny są podobne do działań nikotyny. Występujące różnice w tych działaniach są raczej


ilościowe niż jakościowe. Na przykład, jak już wspo-mniano, cytyzyna silnie łączy się z receptorami dla nikotyny w preparatach mózgu szczurów, ale dzie-sięciokrotnie słabiej od nikotyny wywołuje efekty behawioralne wynikające z pobudzenia tych recep-torów (6, 42).

Cytyzyna słabo penetruje do mózgu. Po podaniu podskórnym cytyzyny samcom szczurów w dawce 1 mg/kg jej stężenie we krwi wynosi 516 ng/ml, a stężenie w mózgu 145 ng/ml. Zatem, stężenie w mózgu stanowi niespełna 30% stężenia we krwi (37, 38). Słabsze działanie ośrodkowe cytyzyny, i co za tym idzie, mniej wyrażone efekty behawio-ralne, są przynajmniej częściowo wynikiem jej sła-bej penetracji do ośrodkowego układu nerwowego, co może wynikać z jej małej lipofilności i niskiego współczynnika rozdziału pomiędzy rozpuszczalni-kami organicznymi a wodą.

Nie jest to jednak jedyne wytłumaczenie tego faktu, gdyż oprócz czynników farmakokinetycz-nych, znaczenie mogą mieć również czynniki far-makodynamiczne. Cytyzyna, po podaniu szczurom dawek uważanych w badaniach farmakologicznych za umiarkowane (tzn. 1 mg/kg), osiąga stężenia w mózgu, które powinny, jak się wydaje, wywołać efekty nie różniące się od tych osiąganych po poda-niu nikotyny. Tak się jednak nie dzieje. Wyjaśnie-niem tego interesującego zjawiska może być to, że cytyzyna działa na więcej niż jedną klasę recepto-rów nikotynowych lub ich podtypów lub też istnie-nie różnic w działaniu leku na ten sam typ recepto-ra, o czym pisano już powyżej.

Podanie agonistów nAchR szczurom wywołuje u nich wzrost aktywności ruchowej, a także przy-spiesza uczenie się oraz poprawia pamięć zwierząt (8, 22). Nikotyna podawana obwodowo szczurom powoduje u nich spadek aktywności ruchowej przez pierwsze 20 min., a po tym okresie czasu wyraźnie nasila tę aktywność. Efekt wzrostu aktywności ru-chowej jest jeszcze bardziej zaznaczony u szczu-rów poddanych uprzednio przewlekłej ekspozycji na nikotynę. Z kolei cytyzyna, słabiej niż nikotyna, powoduje spadek aktywności ruchowej szczurów przez początkowe 20 min. i nie wykazuje wpływu na aktywność ruchową szczurów uprzednio prze-wlekle poddawanych ekspozycji na nikotynę (45). Należy jednak odnotować, że po podawaniu nikoty-ny i cytyzyny bezpośrednio do nakrywki w brzusznej części śródmózgowia szczurów, cytyzyna wykazuje większą od nikotyny siłę działania w wywoływaniu aktywności ruchowej zwierząt (25, 36).

W badaniu behawioralnym Reavill i wsp. (37) obserwowali zachowanie szczura i jego odpowiedź

na podaną nikotynę bądź cytyzynę. Zwierzęta zo-stały tak nauczone, że jedzenie otrzymają tylko wtedy, gdy po podaniu nikotyny nacisną odpowied-ni przycisk. Po podaniu cytyzyny szczury odpowia-dały naciśnięciem przycisku w maksymalnie 65% przypadków, podczas gdy po nikotynie w 95% przy-padków. Brak było przy tym zależności pomiędzy rosnącą dawką cytyzyny, a ilością oczekiwanych odpowiedzi. Cytyzyna podana razem z nikotyną nie osłabiała przy tym w sposób istotny odpowiedzi na nikotynę, nie mając wpływu na zachowanie szczu-rów, które nie różniło się po podaniu samej nikotyny, jak i po podaniu nikotyny z cytyzyną. Jednocześnie istnieją dane mówiące o tym, że w doświadczeniu przeprowadzonym wg podobnego schematu szczu-ry przyzwyczajone do cytyzyny, odpowiadają w 93% na podanie nikotyny tym samym zachowaniem, na-tomiast te przyzwyczajone do nikotyny odpowiadają jedynie w 54% na podanie cytyzyny (cross-genera-lization). Efekt ten stanowi kolejny dowód na to, że cytyzyna jedynie częściowo pobudza receptory nikotynowe, nie wykazując pełnej aktywności we-wnętrznej (6).

Dopamina, oprócz kluczowej roli w mechani-zmie uzależnienia od nikotyny, ma także istotne znaczenie w wielu innych procesach patologicznych ośrodkowego układu nerwowego. Wzrost obrotu do-paminy jest mechanizmem kompensacyjnym, który normalizuje zaburzone przekaźnictwo dopaminer-giczne, do czego dochodzi w procesach neurodege-neracyjnych, na przykład w chorobie Parkinsona. Istnieje kilka modeli zwierzęcych naśladujących zmiany neuropatologiczne obserwowane w chorobie Parkinsona u ludzi. Jeden z takich modeli stosuje neurotoksynę określaną jako MPTP, która zmniejsza stężenie dopaminy w prążkowiu. In vivo, cytyzyna częściowo zapobiega indukowanej przez MPTP re-dukcji stężenia dopaminy (15). Dodatkowym ko-rzystnym działaniem cytyzyny może być, wynikające z tworzenia przez cytyzynę kompleksów z żelazem, zmniejszanie produkcji wolnych rodników, które przyczyniają się do rozwoju zmian degeneracyjnych tkanki nerwowej (15). Zatem, cytyzyna ma poten-cjalne właściwości neuroprotekcyjne.

U gryzoni cytyzyna wykazuje również pewne działanie przeciwbólowe (34, 40).

OBWODOWE EFEKTY DZIAŁANIA

Cytyzyna wykazuje działania obwodowe podob-ne do nikotyny. Obwodowe efekty działania cytyzy-ny wywoływane są zwykle przez dawki stanowiące


od ¼ do 2/3 dawek nikotyny potrzebnych do wywo-łania tego samego efektu (2).

Obwodowe efekty cytyzyny wynikają z jej oddzia-ływania na receptory nikotynowe zlokalizowane w błonach postsynaptycznych zwojów wegetatyw-nych i rdzeniu nadnerczy. Powinowactwo leku do receptorów zwojowych zawierających podjednost-kę α3 jest mniejsze niż do ośrodkowych receptorów α4β2. Cytyzyna ma zdolność stymulowania zwojów nerwowych (działanie gangiostymulujące), jak i ich blokowania (działanie ganglioblokujące). Działanie gangliostymulujące cytyzyny jest jednak silniejsze niż działanie ganglioblokujące (48). Działanie gan-gliostymulujące przejawia się m.in. wzrostem ci-śnienia tętniczego krwi. Zgodnie z badaniami Bar-lowa i McLeoda (2) przeprowadzonymi na szczu-rach, cytyzyna podwyższa ciśnienie krwi około 2,4 razy silniej niż nikotyna. Powoduje również silniej-szy skurcz przepony szczura, mięśni jelita świnki morskiej, mięśnia prostego brzucha żaby, silniejsze pobudzenie zwoju szyjnego górnego kota (2) oraz silniejszy skurcz mięśni jelita szczura (33).

W średnich i dużych dawkach cytyzyna może powodować desensytyzację receptorów zwojowych oraz blok kanału jonowego w ich obrębie (7). Sloan i wsp. (42) przeprowadzili doświadczenia nad gan-glioblokującymi właściwościami cytyzyny, w których badali wpływ dożylnie podanego leku na częstość akcji serca, częstość oddechów, ciśnienie tętnicze krwi, objętość wyrzutową i minutową serca u szczu-rów poddanych znieczuleniu przy użyciu uretanu i pentobarbitalu. Wyniki tych doświadczeń wskazu-ją, że cytyzyna 1,4 razy silniej niż nikotyna obniża ciśnienie krwi, ale wykazuje ¼ –1/8 siły działania ni-kotyny w zwalnianiu częstości akcji serca. Cytyzyna, w mniejszym stopniu niż nikotyna, wywołuje wzrost objętości wyrzutowej i minutowej serca.

Cytyzyna, poza działaniem na zwoje nerwowe, pobudza w sposób zależny od stężenia jonów Ca2, wrażliwe na nikotynę komórki chromochłonne w części rdzeniowej nadnerczy. Efektem takiego działania jest wzrost wydzielania katecholamin, a to z kolei powoduje wzrost ciśnienia tętniczego krwi. Podczas palenia pierwszego papierosa, u ludzi z ni-skim ciśnieniem krwi bądź niskim stężeniem cu-kru we krwi, już po 20 min. nikotyna ujawnia swoje działanie poprzez wyrzut katecholamin, głównie adrenaliny do krwi. W ten sposób zarówno ciśnie-nie krwi, jak i stężenie cukru zostają podwyższone. Przerwanie palenia prowadzi do obniżenia ciśnie-nia i stężenia cukru we krwi. Mogą one odpowia-dać, przynajmniej częściowo, za kliniczne objawy zespołu odstawienia. Zespół odstawienia może być

leczony przywróceniem prawidłowego ciśnienia oraz stężenia cukru we krwi poprzez zastosowa-nie analeptyków, trankwilizatorów i specyficznie działających substytutów nikotyny, właśnie takich jak cytyzyna.

Cytyzyna pobudza ośrodek oddechowy, głównie drogą odruchową poprzez pobudzanie receptorów nikotynowych w zatoce tętnicy szyjnej. Działanie cytyzyny na ośrodek oddechowy jest słabsze od działania nikotyny. Wykazano to m.in. u królików poddanych znieczuleniu, u których słabiej od niko-tyny przyspieszała oddech (2). W celu osiągnięcia takiej samej siły działania, dawka cytyzyny musi być trzykrotnie większa niż dawka nikotyny. Cytyzyna, w przeciwieństwie do nikotyny, nie powoduje pra-wie żadnych zmian w objętości oddechowej płuc.

Działanie cytyzyny podanej systemowo jest blo-kowane poprzez mekamylaminę, pempidynę i hek-sametonium (38). Przy bezpośrednim podaniu cyty-zyny do rdzenia kręgowego, trimetafan, silny anta-gonista receptorów nikotynowych w zwojach nerwo-wych oraz d-tubokuraryna, antagonista receptorów ośrodkowych i w obrębie płytki nerwowo-mięśnio-wej, wykazują bardzo słabą aktywność w blokowaniu łączenia się cytyzyny z receptorami (20).

CYTYZYNA W LECZENIU ZESPOŁU UZALEŻNIENIA OD TYTONIU

Opisane powyżej szczególne właściwości farma-kologiczne cytyzyny, związku o podobnym działaniu do nikotyny, przemawiają za kontynuacją badań nad jej skutecznością kliniczną w leczeniu zespołu uzależnienia od tytoniu.

Cytyzyna w postaci wyciągu z nasion rośliny zło-tokap zwyczajny jako preparat Tabex jest obecna na polskim rynku od ponad 40 lat. Skuteczność kliniczna tego leku została potwierdzona w wielu badaniach przeprowadzonych w latach 60. i 70., głównie na terenie byłego NRD (32, 39). Badania te (chociaż nie odpowiadają obowiązującym obec-nie standardom) wyraźnie wskazują na znaczącą skuteczność leku w rzucaniu palenia, także w przy-padku pacjentów z poważnymi schorzeniami (32, 39). Ze względu na długoletnią obecność prepara-tu na polskim rynku i jego popularność wśród pa-cjentów zostały niedawno podjęte badania nad jego skutecznością w leczeniu zespołu uzależnienia od tytoniu. Jako pierwszą, z cyklu planowanych ba-dań, przeprowadzono otwartą obserwację kliniczną w 2003 roku, w której oceniano skuteczność i bez-pieczeństwo podawania cytyzyny palącym pacjen-


tom. Wyniki obserwacji potwierdziły zarówno sku-teczność cytyzyny – 27% pacjentów zaprzestało pa-lenia i utrzymywało abstynencję po 12 tygodniach od rozpoczęcia leczenia, jak i bezpieczeństwo jej stosowania – nie stwierdzono poważnych objawów ubocznych (49). Obecnie przygotowywane jest przeprowadzenie klasycznego badania klinicznego odpowiadającego zasadom dobrej praktyki klinicz-nej (Good Clinical Practice).

Korzystne z terapeutycznego punktu widzenia właściwości cytyzyny jako skutecznego środka le-czącego uzależnienie od tytoniu stały się podstawą do badań jej pochodnych. Jedną z pochodnych jest wareniklina (6,7,8,9-tetrahydro-6,10-metano-6H--pyrazino[2,3-h][3]benzazepina), intensywnie ba-dana pod kątem zastosowania w leczeniu uzależ-nienia od tytoniu (9). Pierwsze opublikowane do-niesienia z badań klinicznych wskazują na wysoką skuteczność warenikliny, przewyższającą dotych-czas stosowane preparaty stosowane w leczeniu uzależnienia od nikotyny (27, 47).

Warto zwrócić uwagę, że ostatnio przeprowa-dzone porównawcze badania warenikliny i cytyzyny wykazały porównywalne powinowactwo do recepto-rów α4β2 oraz porównywalny profil dopaminergicz-ny (9). Dostępność i niska cena cytyzyny sprawiają, że wydaje się być ona bardzo atrakcyjnym lekiem w leczeniu zespołu uzależnienia od tytoniu.

Ostatnio dostarczono nowych danych, które ilu-strują inne interesujące działania farmakologiczne pochodnych cytyzyny (m.in. przeciwbólowe, hipo-tensyjne i neuroprotekcyjne) (3, 4, 5, 16). Wyda-je się, że związki powstałe w wyniku modyfikacji cząsteczki cytyzyny mogą być wartościowymi li-gandami dla nAChR i stanowić interesującą per-spektywę terapeutyczną. Należy przypuszczać, że w najbliższej przyszłości zainteresowanie cytyzyną jako skutecznym środkiem leczącym uzależnienie od tytoniu i modelową substancją do poszukiwania efektywnych terapeutycznie i bezpiecznych analo-gów będzie rosło.

PIŚMIENNICTWO

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various pharmacological activities. Farmaco. 2003, 58, 265-277.

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6. Chandler CJ, Stolerman IP. Discriminative stimulus pro-perties of the nicotinic agonist cytosine. Psychopharmaco-logy (Berl). 1997, 129, 257-64.

7. Chavez-Noriega LE, Crona JH, Washburn MS, Urrutia A, Elliott KJ, Johnson EC. Pharmacological characterization of recombinant human neuronal nicotinic acetylcholine receptors h alpha 2 beta 2, h alpha 2 beta 4, h alpha 3 beta 2, h alpha 3 beta 4, h alpha 4 beta 2, h alpha 4 beta 4 and h alpha 7 expressed in Xenopus oocytes. J. Pharmacol. Exp. Ther. 1997, 280, 346-56.

8. Clarke PB, Kumar R. The effects of nicotine on locomotor activity in non-tolerant and tolerant rats. Br. J. Pharmacol. 1983, 78, 329-37.

9. Coe JW, Brooks PR, Vetelino MG et al. Varenicline: an al-pha4beta2 nicotinic receptor partial agonist for smoking cessation. J. Med. Chem. 2005, 48, 3474-3477.

10. Cohen C, Bergis OE, Galli F et al. SSR591813, a novel selective and partial alpha4beta2 nicotinic receptor agonist with potential as an aid to smoking cessation. J. Pharma-col. Exp. Ther. 2003, 306, 407-20.

11. Dale HH, Laidlaw PP. The physiological action of cytisi-ne, the active alkaloid of laburnum (Cytisus laburnum). J. Pharmac. Exp. Ther. 1912, 3, 205-221.

12. Dallemagne MJ, Heymans CH. Respiratory stimulants. In: Manske R.H.F. (red.): The alkaloids, Academic Press, New York, 1955, 109-139.

13. Dani JA, De Biasi M. Cellular mechanisms of nicotine ad-diction. Pharmacol. Biochem. Behav. 2001, 70, 439-446.

14. Di Chiara G. Role of dopamine in the behavioural actions of nicotine related to addiction. Eur. J. Pharmacol. 2000, 393, 295-314.

15. Ferger B, Spratt C, Teismann P, Seitz G, Kuschinsky K. Effects of cytisine on hydroxyl radicals in vitro and MPTP--induced dopamine depletion in vivo. Eur. J. Pharmacol. 1998, 360, 155-163.

16. Fitch RW, Kaneko Y, Klaperski P, Daly JW, Seitz G, Gun-disch D. Halogenated and isosteric cytisine derivatives with increased affinity and functional activity at nicotinic acetylcholine receptors. Bioorg. Med. Chem. Lett. 2005, 15, 1221-1224.

17. Hall M, Zerbe L, Leonard S, Freedman R. Characteriza-tion of [3H] cytisine binding to human brain membrane preparations. Brain. Res. 1993, 600, 127-133.

18. Houlihan LM, Slater Y, Guerra DL et al. Activity of cytisine and its brominated isosteres on recombinant human α7, α4β2 and α4β4 nicotinic acetylcholine receptors. J. Neuro-chem. 2001, 78, 1029-1043.

19. Ing HR, Patel RP. Synthesis of local anesthetic from cytisi-ne. J. Chem. Soc. 1936, 1774-1775.

20. Khan IM, Yaksh TL, Taylor P. Ligand specificity of nicotinic acetylocholine receptors in rat spinal cord: studies with ni-cotine and cytisine. J. Pharmacol. Exp. Ther. 1994, 270, 159-166.

21. Lebeau P, Janot MM. Traite de Pharmacie Chimique, tome IV, Masson, Paris, 1955, 2859.

22. Levin ED, Christopher NC, Briggs SJ, Rose JE. Chronic ni-cotine reverses working memory deficits caused by lesions of the fimbria or medial basalocortical projection. Brain Res. Cogn. Brain. Res. 1993, 1, 137-43.

23. Luetje CW, Patrick J. Both α and β subunits contribute to the agonist sensitivity of neuronal nicotinic acetylocholine receptor. J. Neurosci. 1991, 11, 837-845.

24. Monteggia LM, Gopalakirshnan M, Touma E et al. Cloning and transient expression of genes encoding the human al-


pha 4 and beta 2 neuronal nicotinic acetylcholine receptor (nAChR) subunits. Gene. 1995, 155, 189-193.

25. Museo E, Wise RA. Cytisine-induced behavioral activation: delineation of neuroanatomical locus of action. Brain Res. 1995, 670, 257-263.

26. Nguyen HN, Rasmussen BA, Perry DC. Subtype-selective up-regulation by chronic nicotine of high-affinity nicotinic receptors in rat brain demonstrated by receptor autoradio-graphy. J. Pharmacol. Exp. Ther. 2003, 307, 1090-1097.

27. Oncken C, Wartsky E, Reeves K, Anziano R. Varenicline is efficacious and well tolerated in promoting smoking cessa-tion: results from a 7-week, randomized, placebo and bu-propion-controlled trial. W: Materials of 7th Annual SRNT European Conference, Prague, 2005.

28. Pabreza LA, Dhawan S, Kellar KJ. [3H] cytisine binding to nicotinic cholinergic receptors in brain. Mol. Pharmacol. 1991, 39, 9-12.

29. Papke RL, Heinemann SF. Partial agonist properties of cy-tisine on neuronal nicotonic receptors containing the beta 2 subunit. Mol. Pharmacol. 1994, 45, 142-149.

30. Parker MJ, Beck A, Luetje CW. Neuronal nicotinic recep-tor β2 and β4 subunits confer large differences in agonist binding affinity. Mol. Pharmacol. 1998, 54, 1132-1139.

31. Partheil A. Isolation. Arch. Pharm. 1894, 232. 32. Paun D, Franze J. Breaking the smoking habit using cytisin

containing “Tabex” tablets. Dtsch Gesundheitsw. 1968, 23, 2088-2091.

33. Penchev B, Ivanov D, Dimitrov M. Pharmacological and to-xicological effects of nicotine and cytisine in experimental animals. Experimental study at the Department of Phar-macology and Toxicology, Medical University, Sofia, 2002.

34. Rao TS, Correa LD, Reid RT, Lloyd GK. Evaluation of anti-nociceptive effects of neuronal nicotinic acetylocholine re-ceptors ligands in rat tail-flick assay. Neuropharmacology. 1996, 35, 393-405.

35. Rapier C, Lunt GG, Wonnacott S. Nicotinic modulation of [3H]dopamine release from striatal synaptosomes: phar-macological characterization. J. Neurochem. 1990, 54, 937-945.

36. Reavill C, Stolerman IP. Locomotor activity in rats after administration of nicotinic agonists intracerebrally. Br. J. Pharmacol. 1990, 99, 273-278.

37. Reavill C, Walther B, Stolerman IP, Testa B. Behavioural and pharmacokinetics studies on nicotine, cytisine and lo-beline. Neuropharmacology. 1990, 29, 619-624.

38. Romano C, Goldstein A, Jewell NP. Characterization of the receptor mediating the nicotine dicriminative stimulus. Psychopharmacology(Berl). 1981, 74, 310-315.

39. Schmidt F. Medical support of nicotine withdrawal. Report on a double blind trail in over 5000 smokers (author’s transl). MMW Munch. Med. Wochenschr. 1974, 116, 557-564.

40. Seale TW, Singh S, Basmadjan G. Inherited selective hypo-analgesic response to cytisine in the tail-flick test in CF-1 mice. NeuroReport. 1998, 9, 201-205.

41. Slater YE, Houlihan LM, Maskell PD et al. Halogenated cytisine derivatives as agonists at human neuronal nicoti-nic acetylocholine receptor subtypes. Neuropharmacology 2003, 44, 503-515.

42. Sloan JW, Martin WR, Bostwick M, Hook R, Wala E. The comparative binding characteristics of nicotinic ligands and their pharmacology. Pharmacol. Biochem. Behav. 1988, 30, 255-267.

43. Stojanow S. Treatment of nicotinism with the Bulgarian drug Tabex. Med. Biol. Inform. 1967, 1.

44. Stojanow S, Janaczkowa M. Treatment of nicotinism with the Bulgarian drug Tabex. Chimpharm. 1965, 2, 13.

45. Stolerman IP, Garcha HS, Mirza NR. Dissociations betwe-en the locomotor stimulant and depressant effects of ni-cotinic agonists in rats. Psychopharmacology (Berl). 1995, 117, 430-437.

46. Tapper AR, McKinney SL, Nashmi R et al. Nicotine activa-tion of alpha4* receptors: sufficient for reward, tolerance, and sensitization. Science. 2004, 306, 1029-1032.

47. Tonstad S, Hays JT, Jorenby DE, Reeves K, Billing CB, Gong J, Azoulay S. On behalf of the Varenicline Phase 3 Investigators. Smoking cessation efficacy and safety of an α4β2 nicotinic receptor partial agonist: optimizing results. W: Materials of American Heart Association Scientific Ses-sions, Dallas, 2005.

48. Zachowski J. Zur pharmakolgie des Cytisins. Arch. Exp. Path. Pharmak. 1938, 189, 327-344.

49. Zatoński W, Cedzyńska M, Przewoźniak K, Karpińska E, Lewandowska D, Bobek-Pastucha E, Jońska J, Surowińska J, Wojciechowska U, Jaworski M. An open label observatio-nal study of herbal cytisine (Tabex) as an aid to smoking cessation. W: Materials of 7th Annual SRNT European Conference, Prague, 2005.

50. Zhang J, Steinbach JH. Cytisine binds with similar affinity to nicotinic alpha4beta2 receptors on the cell surface and in homogenates. Brain Res. 2003, 959, 98-102.

Adres korespondencyjny: Piotr Tutka, Katedra i Zakład Farmakologii i Toksykologii,

ul. Jaczewskiego 8, 20-090 Lublin; tel: 0 81 7425654, e-mail: [email protected]

10 Laidlaw: Laburnum Poisoningawd Cytisine

Laburnum Poisoning and Cytisine.

By P. P. LAIDLAW, B.C.

THE common laburnum tree, Cytisus laburnuont, was shown by Gray,in 1862 [1], to contain an alkaloid which he named "cytisine." Thisalkaloid has been found in a number of other plants, and has since beeninvestigated from a chemical point of view by Husemiiann and Marme [2],Partheil [3], and others. Its physiological action has been examinedand described by Gray, Husemann and Marme, Cornevin [4], Prevostand Binet [5], and Radziwillowicz [6]. The poisonous effects oflaburnum are usually attributed to the presence of cytisine in the plant.As' Dr. Dale and I have recently been engaged in re-investigating theaction of cytisine, and have been able to determine its pharmacologicaleffects with greater accuracy and fuller detail than previous workers,I thought it might be interesting to analyse a number of poisoning casesin man, and show that such new facts as we have discovered confirmthe view that the alkaloid is responsible for the poisonous properties ofthe plant.

A large number of cases of accidental poisoning are recorded.Radziwillowicz collected 181 cases in 1888, and since that date addi-tional cases have been described. The great majority of these occur inchildren, and are for the most part miild; fatal cases are rare. Threeare described in the British Medical Journal [7 and 8] and one in theLancet [9]; Radziwillowicz records others. The milder cases [10] runa course somewhat as follows: A child swallows a few laburnum seedsunder the impression that they are peas; or he eats the flowers, pods,or leaves, possibly in search for a new sensation in flavours. About anhour afterwards the child feels unwell, complains of being unable towalk, or seems weak and helpless. Some complain of headache, giddi-ness, and stomach-ache. Shortly after the onset of these symptoms hevomits and appears to be very ill. The skin of the face in particular ispale, cold, and moist, the pulse rapid and thin. The pupils may becontracted at first, but dilate in the later stages; they react to light inmild cases. Sickness continues, and purgation may occur.

The recorded cases among adults are not severe, and usually havetheir origin in the mistaken use of laburnum flowers to flavour dishes.

Therapeutical and Pharmacological Section1Their use for this purpose is due to the cook mistaking the flowers forthose of Robiniapseudacacia. Vallette [11] records one instance of thisaccident. A household of four members, three females and one male,partook of some poisoned fritters. The three women suffered frommild laburnum poisoning, the man developed no symptoms. Theearliest symptom in one of these cases was a sensation of numbness inthe hands and inability to play the piano. She was assisted to bed,where the symptoms were very similar to those already described.

The synmptoimis in a severe case are well illustrated in the followingabstract from the Lancet [12]

J. W., aged 6, ate a hearty tea at 6 p.m.; at 8 p.m. he swallowed somelaburnum seeds, saying they were peas. At 9 p.m. he appeared to be very ill,and vomited. Seen shortly afterwards, he was very pale; skin cold and clammyto the touch; pupils contracted; drowsy at times, but could be roused readily.There was no pain. Pulse, 108; axilla temperature, 97.50 F.; respiration 22.At 10.15 p.m. the drowsiness was more marked; the pupils dilated; pulse 130and very weak; respiration, 25; rectal temperature, 960 F. At 10.30 p.m. thepatient could only be roused with difficulty. The skin was very cold and bathedin perspiration; the pupils were widely dilated and insensitive to light. Caffeinewas administered hypodermically, and a hot bath ordered. A mixture ofammonia and ether was given by the mouth at short intervals. After the hotbath improvement was noticed, and the symptoms gradually subsided. At2 a.m., the child was sleeping peacefully, and next day was comparatively well,though the pupils remained dilated for twenty-four hours longer.

In fatal cases the synmptoms are similar; the drowsiness beconmesmore pronounced as intoxication progresses, and the patient ultimatelybecomes comatose, with widely dilated pupils, which are insensitive tolight; the respiration becomes stertorous, and cyanosis of the lipsdevelops; the pulse becomes very rapid and the blood-pressure low.Death is due to respiratory failure, with or without conyulsions [13].

In a limited number of cases symptoms of acute enteritis appear tobe superimposed on the usual series. A striking example of this typeof case is recorded by Wheelhouse [14]

A child, aged 5 years and 7 months, had been unwell for two days beforebeing seen, and admitted having swallowed some laburnum seeds. On thethird day more seeds were eaten, and the usual symptoms of laburnumpoisoning developed. Vomiting and diarrhbea were, however, marked symptomsand could not be controlled. The patient was drowsy, then restless andirritable by turns. The pressure of the bedclothes appeared to be irritating,and they were repeatedly thrown off. For three days these symptoms were

11

12 Laidlaw: Laburnum Poisoni'ng and Cytisine

present, and very little fluid was retained, although the patient was very thirsty.Temporary improvement was noticed on the fourth day, and fluids were retainedbetter, but the progress was not maintained, and death ensued on the sixth day.

It has been suggested that the symptoms of acute enteritis, whichare sometimes seen in cases of laburnum poisonifig, are due to someunknown, highly irritating substance in the plant. They do not appearregularly, however, and may be examples of idiosyncrasy in response tocytisine. As far as I am aware they have no parallel in animal experi-ments with the alkaloid.

Post mortem, no positive signs of laburnum poisoning can be dis-covered apart from the isolation of the alkaloid from the viscera [13],and as this is present in other plants the evidence is inconclusive.In a few cases the mucous membrane of the alimentary canal isinjected.

The treatment of laburnum poisoning resolves itself into removal ofthe poison by emetics or stomach-tube and the treatment of symptomsas they arise. Radziwillowicz has shown that cytisine is readilyexcreted in the urine; diuretics, therefore, seem to be indicated. In anumber of cases hot baths seem to have been beneficial.

The action of cytisine on intact animals reproduces with fair accuracythe symptoms of laburnum poisoning in man. The herbivora are lesssusceptible than the carnivora. The goat is very resistant: slugs areimmune. Shortly after a hypodermic injection of cytisine has beengiven to a dog, symptoms of nausea (salivation and uneasiness) develop,and vomiting follows. The pupils dilate and the nictitating membraneprolapses. With large doses or with intravenous administration therespiration first becomes hurried and deep, and later becomles slow, andmay ultimately cease.

The respiratory and emetic effects are central in origin and are dueto a stimulating action on the respective centres. The respiratorycentre is readily paralysed with large doses. Muscular tremors areperceptible and weakness of the limbs is obvious. Large doses inanesthetized or pithed animals under artificial respiration cause acomplete paralysis of the nerve-endings in voluntary muscle (curareeffect); about 6 mg. are required to produce this effect in a fair-sized cat.The muscular weakness may be'the expression of a mild stage in thisprocess. The pupil dilates in the cat under the influence of cytisine, andthe nictitating membrane at first is withdrawn and later prolapses. Ifrepeated doses of cytisine are given to a pithed cat, it is observable thatthe effect on the eye becomes less and less, until further doses produce

Therapeutical and Pharmacological Section

no effect. When this stage is reached it is found that the superiorcervical ganglion is paralysed and that electrical stimulation of thecervical sympathetic trunk is without action on the eye, but stimulationof the branches from the ganglion to the periphery still causes normal

FIG. 1.

Cat, pithed. Artificial respiration; blocd-pressure base line and signal; timein ten seconds. First curve, effect of 0-2 mgm. nicotine; second curve, effectof 0-2 mgm. cytisine.

responses. Once the ganglion system is paralysed with cytisine, nicotineis inicapable of producing any effect. A flow of saliva is observablefrom the submaxillary gland of the dog and cat on administration of

13


cytisine. L'arge doses cause paralysis of the chorda tympani and renderthis ganglion system insensitive to nicotine.

On the blood-pressure cytisine exerts a series of effects, through its

FIG. 2.Cat, as in fig. 1. Effect of 1-5 mgm. nicotine; effect of 0 5 mngm. cytisine, and

1-5 mgm. nicotine.

stimulant action on ganglion cells. Thus the heart at first is slowedfrom a weak stimulant effect on the cardio-inhibitory apparatus, laterit becomes very rapid, probably in part from stimulation of the stellate


FIG. 3.Cat, pithed. Artifitlial respiration, upper tracing bladder volume; blood-pressure

base line and signal; time in ten seconds. Effect of a mngm. cytisine.


ganglion and partly from escape from vagus control. The arteriolesare primarily constricted throughout the body, from stimulation of thesympathetic ganglia. These factors bring about a large rise of blood-pressure; with large doses the secondary paralytic action of the alkaloidbecomes very marked, and the normal tone of the vessels cannot bemaintained, and so the blood-pressure falls.

The intestinal movements in the cat are primarily inhibited, butsubsequently become larger and more frequent. The bladder isthrown into a powerful and prolonged contraction, owing to thestimulating action of the alkaloid upon the sacral autonomic ganglia.

(At this point tracings illustrating the action of cytisine were shownby means of the epidiascope. Tracings showing the action of nicotineunder similar conditions of experiment were also shown. Comparisonsbetween these were made and the similarity in action emphasized.)

It will have been observed that these actions of cytisine which I havequoted are typical of the alkaloid nicotine. It is unnecessary to multiplyexamples from further experiments which were carried out in con-junction with Dr. Dale. I could quote many others which show theclose similarity in action between nicotine and cytisine. Their resen-blance in action is so close that by their action on animals alone it wouldbe difficult to say whether a given solution was cytisine or nicotine.Edmunds [15] has shown that lobeline, the chief alkaloid of lobelia, hasan action almost identical with that of nicotine. Apart, therefore, froinisolation of the alkaloids themselves it would be difficult to determinewhether one was dealing with nicotine, lobeline, or cytisine.

In conclusion, I should like to point out the similarity between casesof laburnum poisoning and nicotine poisoning. The man who canremember, as I do, his first overdose of nicotine through excessivesmoking in the days of his youth has an excellent picture of laburnumpoisoning. The restlessness, giddiness, tremors, muscular weakness, thenausea and salivation, the cold, clammy sweat, and vomiting, are alltypical. In severe cases the widely dilated pupil, the drowsiness andcoma, and the modes of death in both cases are very similar.

REFERENCES.

[1] GRAY. Edinb. Med. Journ., 1862, vii, 2, pp. 908, 1025.[2] HuSEMANN und MARME. Zeitschr. f. Chem., 1865, i, p. 161.[3] PARTHEIL. Ber. d. deutsch. chem. Gesellsch., 1890, xxxiii, 2, p. 3201.[4] CORNEVIN. Comptes rend., 1886, p. 777.[5] PREVOST et BINET. Rev. mned. de la Suisse romande, 1887, vii, pp. 516, 553; 1888,

viii, p. 670.[6] RADZIWILLOWICz. Arb. d. Pharin. Inst. z. Dorpat, 1888, ii, p. 56.


[7] Brit. Med. Journ., 1870, i, p. 79.[8] Brit. Med. Journ., 1882, p. 199.[9] Lancet, 1868, i, p. 55.

[10] RADZIWILLOWICZ. Loc. cit.; also Lancet, 1877, ii, p. 414; 1901, ii, p. 491; 1905, ii,p. 635; Brit. Med. Journ., 1883, i, p. 1117.

[11] VALETTE. Rev. med. de la Suisse romande, Geneve, 1908, xxviii, p. 366.[12] Lancet, 1877, ii, p. 341.[13J Brit. Med. Journ., 1882, i, p. 199; also RADZIWILLOWICZ.[14] WHEELHOUSE. Brit. Med. Journ., 1870, i, p. 79; see also RADZIWILLOWICZ, 10c. cit.[15] EDMUNDS. Amer. Journ. Phys., Bost., 1904, xi, p. 78.

DISCUSSION.

Professor CUSHNY, F.R.S., said he would like to hear whether there wasa laburnum habit. The reuson- why Edmunds investigated the question oflobeline was that it had been discovered that lobelia was used by the NorthAmerican Indians, particularly those of the northern part of the continent,instead of tobacco. In fact, lobelia was known as Indian tobacco, and thereseemed every reason to suppose that this substance was used by Indians toa considerable extent. Langley showed, years ago, that pituri, from whichanother alkaloid, piturine, was derived, was used in Australia by the blacks,who were in the habit of chewing it, much in the same way as some people inthis country use tobacco. So that those three alkaloids, which were practi-cally identical in action, were used by various aboriginal races. Langley foundthat piturine acted in the same way as nicotine, and Edmunds could notdistinguish the symptoms of lobelia poisoning from those produced fromnicotine. There was found to be the same difficulty in obtaining satisfactorytolerance of lobeline as there was in the case of nicotine.

Dr. H. H. DALE desired in the first place to associate himself with thecaution of his colleague as to attributing all the symptoms, which had beendescribed as the result of laburnum poisoning, to the action of the alkaloidcytisine. He did not think there was any doubt that in the majority of casesthe effect of laburnum corresponded closely to the effect of cytisine; but, asDr. Laidlaw said, it looked as if, in a minority of cases, there were some otherpoison playing a part. The occurrence of enteritis, for example, was far moresuggestive of the possible presence of some toxalbumin. One naturallysuggested that, perhaps, because of the association with Robinia psedtdacacia, theflowers of which had been confused with those of laburnum, with the resultthat some of the accidental cases of laburnum poisoning had arisen. In thesecond place, he had also had in mind the point which occurred to ProfessorCushny. It was rather curious that mankind seemed to have an instinct forseeking out, and using for their enjoyment, alkaloids which had the particularaction which had been described. It would be interesting to hear of theexistence of a laburnum habit. As far as the records appeared to show, therewas no indication of such a thing. The only other point which might beworth suggesting was the possibility of the therapeutic application of the

N-22a


substance. It had been tried by two different people. Rose Bradford, manyyears ago, did some physiological experiments on the action of an alkaloid,which was described as ulexine, being isolated from the seeds of common gorse.This had since been identified with cytisine. Bradford, finding it causeddiuresis when used experimentally in animals, tried it, he believed, in a fewpatients as a diuretic. What success it had in that direction did not appear tohave been recorded. Radziwillowicz was struck, in his investigations, by itspowerful effect in producing a rise of blood-pressure, and therefore tried it oncertain patients who were suffering from migraine associated with low blood-pressure. He appeared to be under the impression that a favourable effectresulted. Now that the type of its physiological action seemed clearly estab-lished, the only reasonable suggestion of a therapeutic application was onesimilar to that for which lobelia had been used, and it would be interesting toknow whether cigarettes made from laburnum leaves might have some value incases of asthma, such as had been attributed to the use of lobelia.

Dr. W. MURRELL said he considered that the suggestion just made byDr. Dale about using laburnum seeds in the treatment of asthma was a verygood one. The idea occurred to him while the paper was being read.

Dr. T. R. ELLIOTT said an interesting inquiry would be to attempt todistinguish between the various nicotine-like bodies. The paper by thePresident analysed the power of the rabbit's liver, when the animal was madetolerant of the poison, to destroy nicotine itself to some extent; would it becapable of dealing in a similar way with the allied cytisine ?

The PRESIDENT (Professor H. E. Dixon, F.R.S.) said the Section wouldwish to thank Mr. Laidlaw for his interesting paper. He (the President)desired to make only one remark, namely, that in acute cases of nicotinepoisoning the symptoms came on with the fall of blood-pressure. He spokeof cigar smoking by young people. While the blood-pressure was going up,during say the first fifteen minutes, the smoker had a feeling of well-being;then the poisonous symptoms came on suddenly, the blood-pressure arrived atits maximum, and then dropped rapidly, and the smoker turned pale andshowed the usual symptoms of collapse. In the case of three boys, he hadfound that the first symptom complained of was a rumbling in the abdomen,obviously associated with peristalsis, not vomiting; in one case there wasdefinite diarrhaca. He suggested that that was possibly due to depression ofthe inhibitory sympathetic ganglia. By depressing those one cut off theinhibition, and the vagus was allowed to have all its own way for the timebeing, and there was increased peristalsis as a result. He mentioned this,because it was conceivable that those related alkaloids might have a differentdegree of effect. Cytisine alkaloid, for example, might have a more drasticeffect on the alimentary canal than had nicotine, as the result of a more pro-found depression of ganglion cells, so the effect might conceivably be physiological, and not due to another substance.

Review

Cytisine for the treatment of nicotine addiction:

from a molecule to therapeutic efficacy

Piotr Tutka1, Witold Zatoñski2

�Department of Experimental and Clinical Pharmacology, Medical University of Lublin, Jaczewskiego 8,

PL 20-090 Lublin, Poland

�Department of Epidemiology and Cancer Prevention, The M. Sklodowska-Curie Memorial Cancer Center

and Institute of Oncology, Roentgena 4, PL 02-781 Warszawa, Poland

Correspondence: Piotr Tutka, e-mail: [email protected]

Abstract:

Cytisine, a natural plant alkaloid, has been marketed in Central and Eastern Europe for over 40 years for the clinical management of

smoking cessation. Despite the fact that cytisine has been used by millions of smokers, its characteristics have not been reviewed in

scientific literature in English, and presently existing clinical studies on its effectiveness and safety are insufficient to warrant

licensing by modern standards. Understanding of the mechanism of cytisine action as a smoking cessation aid provides a necessary

basis for conducting clinical trials to confirm its efficacy as an optimal antismoking therapy. Hereafter, we present a review of

current knowledge about the pharmacokinetics, pharmacodynamics, toxicity, therapeutic efficacy and safety of cytisine, and about

its derivatives that are under development. Recent pharmacological research has elucidated that the drug is a low efficacy partial

agonist of �4�2 nicotinic acetylcholine receptors, which are believed to be central to the effect of nicotine (NIC) on the reward

pathway. The drug reduces the effects of NIC on dopamine release in the mesolimbic system when given alone, while

simultaneously attenuating NIC withdrawal symptoms that accompany cessation attempts. Clinical studies on cytisine as a smoking

cessation aid have demonstrated that the drug is effective and safe. Our recent uncontrolled trial has shown that a 12-month carbon

monoxide-verified continuous abstinence rate following a standard course of treatment with cytisine with minimal behavioral

support is similar (13.8%; N = 436) to that observed following treatment with NIC replacement therapy. Since cytisine exhibits

a desirable pharmacological profile which makes it an attractive smoking cessation drug, it should be advanced to randomized

clinical trials. However, more detailed preclinical studies on its pharmacokinetics and safety profile are required.

Key words:

cytisine, nicotine addiction, smoking cessation, nicotine, dopamine, tobacco dependence, varenicline

Abbreviations: ACh – acetylcholine, CEE – Central and East-

ern Europe, CNS – central nervous system, CO – carbon mon-

oxide, CYP – cytochrome P450, DA – dopamine, ip – intrape-

ritoneal, iv – intravenous, nAChRs – nicotinic acetylcholine re-

ceptors, NE – norepinephrine, NIC – nicotine, NRT – nicotine

replacement therapy, po – per os, sc – subcutaneous

Introduction

Tobacco smoking is one of the main threats to human

health and is the biggest single factor of premature

mortality in Poland and worldwide. It is estimated that

in XXI century 1 million smokers will die because of

their smoking [115]. Despite attempts to control tobacco

use covering a broad spectrum of interventions, break-

ing the nicotine (NIC) habit remains difficult.

The best results in the treatment of NIC addiction

are achieved when a combination of pharmacotherapy

and non-pharmacological treatments, including addi-

tional behavioral support, is applied. The current most

effective pharmacological tools are nicotine replace-

ment therapy (NRT) and bupropion [101], and in

some countries recently approved varenicline. NRT

�� 777

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increases 12-month continuous abstinence rate in

smokers about 1.5 to 2 times in comparison to pla-

cebo [134]. Bupropion doubles the chances of success

of quit attempts [64]. Despite their advantages, cur-

rent pharmacotherapies are too expensive for many

smokers, especially in developing countries, and are

not widely disseminated to the general population of

smokers. It is supposed that new medicines, like vare-

nicline, rimonabant, and NIC vaccines will be expen-

sive and unaffordable for many smokers. Thus, there

is an urgent need for identification and evaluation of

other forms of pharmacotherapy which would be ef-

fective, safe and less expensive for health care sys-

tems and smokers. Modern development of such

therapies should be based on the conclusions from

preclinical studies explaining neurobiological mecha-

nism(s) underlying NIC addiction and identifying tar-

gets for pharmacological treatments. Data derived

from these studies suggest that an optimal antismok-

ing agent should mimic the behavioral and biochemi-

cal effects of NIC but should be devoid of addictive

liability or positive reward [46].

These guidelines of mimicking the biochemical ef-

fects of NIC yet lacking addictive or positive reward-

ing properties led us to focus on cytisine, an alkaloid

of plant origin marketed for over 40 years in Central

and Eastern Europe (CEE) [146, 148, 149, 159, 160].

In addition, our recent trial confirming its efficacy and

safety and potential low cost of the therapy encouraged

us to propose cytisine as an attractive drug for smoking

cessation that should receive wider awareness.

Background

Origin of cytisine

Cytisine is a quinolizidine alkaloid originating from

seeds and many other parts of plants of the Legu-

minosae (Fabaceae) family, including Laburnum,

Sophora, Baptisia and Ulex spp. [69, 93]. The greatest

amount of the alkaloid is found in the seeds of the

common garden decorative plant Laburnum anagyroi-

des (Cytisus laburnum; Golden Rain accacia) (about

1–5%). In 1912, Dale and Laidlaw [37] have de-

scribed cytisine to be the toxic component of this

plant.

History of the use in medicine

The extracts from the Laburnum seeds and flowers

have been used in traditional medicine for hundreds

of years. However, a historical clock for cytisine

started thousands years ago in America where Indians

have consumed the seeds for their emetic and purga-

tive effects during rites and magical practices [42, 94,

156]. In Europe, traditional medicine has recom-

mended alcoholic extracts containing cytisine for con-

stipation, migraine, insomnia, cough and neuralgias.

About 100 years ago, cytisine was used as an antiasth-

matic agent and an insecticide. During the Second

World War the leaves of Laburnum anagyroides were

used as a tobacco substitute [132]. There are also re-

ports indicating that cytisine or cytisine-containing

plants have been administered as a diuretic in Western

Europe [82], an analeptic in the former Soviet Union

[39, 158] as well as an agent replacing NIC in smokers

making a quit attempt in CEE [55, 86].

Cytisine as a smoking cessation aid has been used

since the 1960s in Bulgaria. The first clinical study

using cytisine for smoking cessation was carried out

by Stoyanov and Yanachkova in 1965 [141]. In the

next 10 years, other pharmacological and clinical

studies in Bulgaria, Poland, Russia, East and West

Germany were performed, demonstrating good effi-

cacy and safety of the drug [11–14, 58, 76, 91, 112,

113, 129, 130, 141, 142]. Since the results of those

studies were promising, cytisine was developed, and

has been manufactured and marketed from 1964 as

Tabex® (Sopharma, Bulgaria), and has been widely

distributed in CEE.

Poland is one of the countries where cytisine has

been widely used. During the last 40 years probably

hundreds of thousands of smokers have been using

cytisine while quitting. An important factor that de-

cides about a wide use of the drug is its low cost. The

cost of a full course of the treatment in Poland is ap-

proximately the equivalent of 10 $ or 8.9 C (for more

information see: www.bpg.bg/tabex); thus, much less

than the cost of NRT or bupropion. However, despite

its 40+ years on the market, there have been no re-

ports of clinical observations or studies published in

scientific literature in English. Consequently, cytisine

is unknown outside of CEE. At least to our knowl-

edge, cytisine has not been used for therapeutic pur-

poses in Western countries. Very recently, a review of

the efficacy data confirming the therapeutic potential

of cytisine has been published in English [44].

778 ��

�

Cytisine appears to be the oldest medication used

for smoking cessation. After many years of oblivion,

cytisine is presently a medication interesting to chem-

ists, pharmacologists and clinicians because of its po-

tential to be an inexpensive, safe and effective medi-

cation for smoking cessation. Recent clinical data

have demonstrated the effectiveness of cytisine for

smoking cessation. Cytisine also has intrigued re-

searchers because of the clinical success of one of its

synthetic derivatives – varenicline.

Chemistry

Although cytisine was isolated already in 1863 by

Husemann and Marme, its chemical structure was de-

scribed only in the 1930s [66–68].

Chemically, cytisine is (1R-cis)-1,2,3,4,5,6-hexa-

hydro-1,5-methano-8H-pyrido[1,2-a][1,5]diazocin-8-one

(C11H14ON2). The structural formula is depicted in

Figure 1. X-ray crystal structure analysis indicates that

its chemical structure closely resembles that of NIC

[9]. The quasi-aromatic ring in cytisine and the pyri-

dine ring of NIC are associated in a similar way in re-

lationship to the nitrogen atom in the bispidine ring

and the nitrogen atom in the pyrrolidine ring, respec-

tively [9].

Cytisine crystallizes as big colorless crystals, easily

soluble in water, chloroform and ethanol, less soluble

in benzene, ethyl acetate and acetone, and insoluble in

ether [30].

Cytisine is a compound with a relatively rigid con-

formation. The rigidity of the molecule makes it an at-

tractive template for structure-activity studies. In re-

cent years, the studies on the structural modification

of cytisine have led to the development of novel com-

pounds of potential therapeutic interest [1, 18, 19, 21,

31, 34, 50]. One of such compounds, varenicline has

recently been approved by the US Food and Drug Ad-

ministration for the treatment of NIC addiction

[31–33, 54, 73, 102, 145].

Pharmacology

Pharmacokinetics: preclinical animal studies

The available data on the disposition kinetics and me-

tabolism of cytisine derive from animal studies. Phar-

macokinetics was investigated after a single per os (po)

or intravenous (iv) administration of the drug at a dose

of 2 mg/kg in mice [79], and after a single po (5 mg/kg)

or iv (1 mg/kg) administration in rabbits [143]. The re-

sults of the latter study are shown in Table 1.

Absorption

Cytisine given po was readily absorbed in the gastro-

intestinal tract [81]. In mice, approximately 42% of

the drug administered po was absorbed with the time

to reach maximum blood concentration of 120 min

[79]. Oral bioavailability in rabbits was 34% and

�� 779

Cytisine for smoking cessation��

Tab. 1. Pharmacokinetic parameters of cytisine after oral or intravenous administration in rabbits

Route of administration Bioavailability % C�� µg/l T�� min Volume ofdistribution l/kg

Eliminationhalf-life min

Renal clearanceml/min

Oral (5 mg/kg; N = 8) 34 388.9 35 6.2 52 167.2

Intravenous (1 mg/kg; N = 10) – – – 1 37 43

Adapted from [143] with permission of Sopharma

Nicotine Cytisine

N CH3

N N

NH

O

Fig. 1. Chemical structures of cytisine and nicotine. Reprinted from [30]

maximum plasma concentration was observed after

35 min [143].

Transdermal application in rabbits resulted in

a steady state that was reached in two phases [127].

The first phase lasted 24 h, and the second phase con-

tinued through the next 3 days. In the first phase, drug

absorption capacity and concentration in blood were

twice higher than in the second phase [127].

Distribution

After a single po or iv administration in mice, the

highest concentration of cytisine was found in the

liver, adrenal glands and kidneys. Following iv ad-

ministration, the highest concentration of the drug in

the bile was 200 times that in the plasma [79].

The ability of a drug to cause dependence is related

to its ability to penetrate into the central nervous sys-

tem (CNS). The lipophilic characteristics of a drug

are most frequently used to indicate the probable

penetration of the drug through the blood-brain bar-

rier. The partition coefficient among organic solvents

and water for cytisine is lower than for NIC and is ex-

pressed as the logw of approximately 0.21 for cytisine

and 1.24 for NIC at pH = 7.4 [122]. When cytisine

was administered to rats at a dose of 1 mg/kg, its con-

centration in plasma was 516 ng/ml, and in the brain

145 ng/ml. Thus, the brain concentration was not

more than 30% of the plasma concentration. For com-

parison, administration of the 0.1 mg/kg dose of NIC

resulted in a mean plasma NIC concentration of

62 ng/ml and the concentration in the brain was 65%

of that in plasma. These data show that cytisine

weakly penetrates the blood-brain barrier as com-

pared to NIC [122, 124]. Therefore, trials of drug

modification to improve the penetration of the blood-

brain barrier have been performed [18].

Metabolism and elimination

The half-life assessed after iv single dose of cytisine

was 200 min in mice and was longer than the half-life

of NIC [79, 147]. Approximately 32% of the dose

could be recovered from the urine over 24 h and 3%

from feces over 6 h. After po application, 18% of the

applied dose was excreted within 24 h in the urine [79].

In rabbits, the elimination half-life was 37 and 52 min

after iv or po administration, respectively [143].

In contrast to NIC, cytisine undergoes minimal me-

tabolism and 90–95% of its dose is excreted un-

changed in the urine [147]. The clearance rates after

po and iv application in rabbits were 167 and

43 ml/min, respectively [143].

So far, the pharmacokinetics of the drug in humans

has not been established. It is critical considering

regulatory requirements, that the pharmacokinetics

and pharmacodynamics in humans is established. In

addition, pharmacokinetics is essential in special pa-

tient populations, e.g. in patients with renal insuffi-

ciency, geriatric and pediatric patients. Also, sex- or

smoking status-related differences in kinetics should

be defined. Studies of drug metabolism exploring the

potential influence on hepatic P450 enzymes should

also be performed.

Pharmacodynamics

Receptor binding

The evidence that NIC is responsible for the depend-

ence on tobacco has been reviewed many times [15].

It is commonly accepted that NIC addiction results, at

least partially, from its interaction with neuronal nico-

tinic acetylcholine receptors (nAChRs). NAChRs are

ligand-gated cation channels widely expressed in the

brain, autonomic ganglia, adrenal medulla and neuro-

muscular junctions, and on numerous other cell types

[126]. Postsynaptic nAChRs exert a stimulatory effect

on neurons, presynaptic nAChRs modulate while syn-

aptic transmission by the release of many neurotrans-

mitters [36, 71].

NAChRs types are pentamers formed from differ-

ent combinations of genetically distinct subunits

�(1–10), �(1–4), �, �, and � [74, 88]. Although a large

number of neuronal subtypes have been identified,

�4�2*, �3�4, and �7 predominate in the brain [88].

So far, the studies on cytisine have focused on target-

ing these three most prevalent subtypes of nAChRs.

The presynaptic �4�2* (where * denotes the possi-

ble inclusion of an additional, unspecified subunits

[88]) nAChRs modulate dopamine (DA) release and

overflow from dopaminergic terminals in the meso-

limbic system, which are believed to be central to the

effect of NIC on the reward pathway [22, 125]. The

development of new subtype-selective �4�2* ligands

has been shown to be successful for treating NIC ad-

diction [54, 73, 145].

Affinity. A substantial amount of information on

pharmacodynamics of cytisine has come from studies

on neuroblastoma cell lines which naturally express

780 ��

nAChRs and from rat or human recombinant nAChRs

expressed in Xenopus laevis oocytes. In vitro binding

studies showed that cytisine had a million-fold higher

selectivity for nAChRs over muscarinic acetylcholine

receptors [3]. In the rat whole-brain membrane prepa-

rations, cytisine exhibited higher affinity for nAChRs

(however, no specific subunit distinction was deter-

mined) than NIC [3]. On the other hand, there is

a study in which no significant differences between

cytisine or NIC were demonstrated [107]. In rats, the

affinity of cytisine for nAChRs was the highest in the

thalamus, striatum and cortex [107]. In human brain

membrane preparations, the highest density of

cytisine binding sites was shown in the thalamus [59].

An autoradiographic study by Hall et al. [60] in

rhesus monkey demonstrated the localization of bind-

ing sites for [(3)H]cytisine and [(3)H]NIC in layer IV

of some cortical areas, with mostly the thalamic nu-

clei, and presubiculum displaying high levels of label-

ing for cytisine. Moderate levels of binding were dem-

onstrated in the subiculum, septum, and mesencepha-

lon, and low levels were demonstrated in layers I, II

and VI of the cortex, dentate gyrus, and amygdala [60].

Cytisine differs in the affinity for different sub-

types of nAChRs. The alkaloid binds with high affin-

ity predominantly to �4�2* subtype [31, 50, 59, 63,

98, 107, 110, 135, 137, 161]. The experiments com-

paring the binding of cytisine and nicotine to �4�2*

nAChRs demonstrated that the affinity of cytisine was

about 7-fold higher than that of NIC [31, 65]. The

autoradiographic study of rat brains demonstrated that

cytisine bound to �4�2* nAChRs at nanomolar con-

centration (Ki = 0.45 nM, [107]; Ki = 2.4 nM, [19]).

These data are similar to results of experiments with

rat receptors expressed in Xenopus oocytes (Ki = 1.03 nM,

[110]; Ki = 0.17 nM, [31]) or cell lines (Ki = 0.12 nM,

[50]) and data from the studies using recombinant hu-

man receptors expressed in Xenopus oocytes or cell

lines (Ki = 1.07 nM, [63]; Ki = 1.2 nM, [135]).

It has been reported that the affinity of �4�2*

nAChRs for many ligands differs significantly de-

pending on whether receptors are examined in intact

cells (Xenopus oocytes) or in cell homogenates [47].

Zhang and Steinbach [161] compared binding of

cytisine and NIC to specific sites in homogenates pre-

pared from HEK 293 cells which stably express hu-

man neuronal �4�2* receptors or to surface receptors

on intact cells. They found that cytisine bound to sur-

face receptors in intact cells, and showed an insignifi-

cant difference in affinity between these receptors and

the receptors in cell homogenates. The binding of

NIC to receptors on intact cells was about 4-fold

higher than binding of cytisine. When NIC and

cytisine competed for binding sites of intact cells,

NIC fully blocked cytisine binding but cytisine only

partially inhibited NIC binding [161].

Cytisine binds to �3�2* nAChRs with relatively

low affinity [108]. The potency of cytisine was lower

than those of epibatidine and NIC, and similar to the

potency of acetylcholine (ACh)[25].

The �3�4* nAChRs are present in autonomic gan-

glia where they mediate synaptic transmission, and

represent a minority population of NIC receptors in

the brain. As demonstrated in cultured cells or

Xenopus oocytes, cytisine bound and activated �3�4*

receptors at low micromolar concentrations (Ki = 840 nM,

[31]; Ki = 220 nM, [50]) with an affinity slightly lower

than that of NIC. The affinity of cytisine for �3�4* re-

ceptors was 11-fold and 47-fold lower than the affinity for

�2�4* and �4�4* receptors, respectively [110].

Homomeric �7 receptors are relatively widespread

in the brain, have the highest Ca2+ permeability and

the fastest desensitization kinetics of the nAChRS,

and are involved in glutamate release in the hippo-

campus [118, 133]. According to the study on recom-

binant human receptors by Chavez-Noriega et al.

[24], cytisine is a full agonist of �7 nAChRs, display-

ing an affinity which is > 24000-fold and 5-fold lower

than the affinities for �4�2* and �3�4* subtypes, re-

spectively [31]. The affinity for �7 receptors was at

low micromolar concentrations with the values of Ki

ranging from 4.2 to 8.4 µM [31, 63, 135]. In compara-

tive binding assays reported by Imming et al. [65], the

affinity of cytisine for �7 receptors was 2-fold lower

than that of NIC.

The rank order of cytisine affinity derived from

experiments on receptors expressed in Xenopus oo-

cytes or cell cultures is the following: �4�2* > �4�4*

> �2�4* > �3�2* > �3�4* > �7 [31, 110].

Cytisine exhibited very limited binding to receptors

for cholecystokinin (A), histamine (H3), kainic acid,

N-methyl-D-aspartic acid (NMDA), phencyclidine,

serotonin (5-HT3), thyrotropin-releasing hormone

(TRH), vasoactive intestinal peptide (VIP), and had

no affinity for receptors for adenosine (A1 and A2),

angiotensin II, galanin, insulin, interleukin 1�, leuko-

triene B4, neurokinin 1, neuropeptide Y, platelet-

activating factor, serotonin (5-HT1A), tromboxane

(A2), tumor necrosis factor as well as muscarinic (M1

and M2) and sigma receptors [19].

�� 781


Efficacy. The functional effects of cytisine are mark-

edly determined by its agonist efficacy at each nico-

tinic ACh receptor. The first electrophysiological

studies on the alkaloid’s agonist/antagonist activity

were performed by Luetje and Patrick [87] as well as

Papke and Heinemann [108]. In their experiments, the

responses of �4�2*- and �3�2*-injected oocytes to

the application of 1 mM cytisine amounted only to

15% and 2.5% of the responses to 1 mM ACh, respec-

tively. The coadministration of cytisine and ACh re-

sulted in the reduction of the responses to ACh. Thus,

they suggested that cytisine was a partial agonist of

�2-containing nAChRs. The study by Coe et al. [31]

confirmed this suggestion. Using 10 µM concentra-

tion of cytisine and measuring the current evoked at

�4�2* nAChRs, Coe et al. [31] estimated agonistic

activity of the drug at 56% of the response to 10 µM

of NIC. When both agents were co-applied, cytisine

partially blocked (30% inhibition) effect of NIC. So,

it clearly indicated the partial antagonistic profile of

cytisine. In the comparative studies, the efficacy of

the drug was similar at both rat and human �4�2*

nAChRs [21, 24, 109].

Cytisine shows a wide variation in functional effi-

cacy at different subtypes of nAChRs. Efficacy at

�4�2* was much lower than at various �4*-contai-

ning and �7 receptors [21, 63, 109, 135].

Taken together, the above-described findings from

in vitro and in vivo assays indicate that cytisine has

high affinity for rat, rhesus monkey and human cere-

bral nAChRs, displaying selectivity especially to

�4�2* subtype. The affinity of cytisine for �4�2* re-

ceptors seems to be superior to that of NIC and many

other nAChRs agonists. The drug has negligible affin-

ity for many neurotransmitter and hormone receptors.

Cytisine displays low efficacy at �4�2* and other

�2*-containing receptors. In other words, cytisine is

a low-efficacy partial �4�2* agonist and a full agonist

at �7 and �3�4* nAChRs.

Pharmacological action

Based on our understanding of cytisine pharmacol-

ogy, it appears to be of potential therapeutic utility in

aiding smoking cessation and possibly in treating

other diseases. The first data on pharmacological ac-

tion of cytisine were reported at the beginning of 20th

century [37, 157]. Paskov and Dobrev [111], continu-

ing the investigations begun in the former Soviet Un-

ion, compared the pharmacological action of cytisine

and NIC. Ten years later, Daleva and Sheykova [38]

published an additional study on pharmacological

properties of cytisine and its derivatives.

The alkaloid exerts central and peripheral effects

that are similar to the effects of NIC. The differences

in effects between NIC and cytisine are related to the

concentration of the drug rather than to sites of its ac-

tion or response.

Central nervous system effects: clinical targets

for cytisine

The central effects of cytisine resemble the effects of

NIC at the nAChRs. Its central activity results from

the stimulation of nAChRs resulting in the modula-

tion of the release of neurotransmitters, such as DA

and norepinephrine (NE).

Mechanism of antismoking action. It is widely

accepted that the development of NIC addiction and

the neurobiological mechanisms explaining NIC rein-

forcement, withdrawal syndrome and relapse depend

on its action as an agonist on �4�2* nAChRs. The �4�2*

nAChRs stimulation results in molecular changes in the

nucleus accumbens and prefrontal cortex entailing do-

paminergic activation [40, 41, 46]. The absence of

striatal DA release in NIC-treated �2 subunit knock-

out mice indicates that �4�2* nAChRs play a key role

in the DA-releasing effects of NIC [116]. An increase

in the DA level is responsible for pharmacological re-

ward whereas lower DA levels are associated with the

withdrawal symptoms experienced by smokers during

cessation attempts (Fig. 2).

Modulation of [3H]DA release by cytisine via

�2*-containing nAChRs was shown in the studies by

Grady et al. [57] and Abin-Carriquiry et al. [1]. The

latter study used native rat nicotinic receptors and

showed that cytisine elicited the [3H]DA release from

striatal slices with maximum release that was approxi-

mately 50% of that seen with NIC.

The partial agonist effect of cytisine at �4�2*

nAChRs was also demonstrated in vivo by measure-

ment of the DA turnover (a measure of the utilization

and synthesis of DA) in the nucleus accumbens of

conscious Sprague-Dawley rats [31]. When given

alone sc at a dose of 5.6 mg/kg, the drug increased the

DA turnover with an efficacy amounting to 40% of

the maximal NIC effect. In animals treated concur-

rently with NIC, cytisine inhibited the NIC response

by 36% [31]. In the comparative study, the effects of

782 ��

cytisine on central dopaminergic response were simi-

lar to the effects of its derivative, varenicline that in-

creased the DA turnover with an efficacy 32% of the

maximal NIC response and inhibited NIC’s effect by

66% [31].

In conclusion, in vitro and in vivo results suggest

that in NIC addiction, cytisine would moderately in-

crease the DA level in the mesolimbic system, attenu-

ating the withdrawal symptoms, and on the other

hand, it should minimize the addictive effects of NIC

by decreasing the DA level (Fig. 2). The lower effi-

cacy of cytisine in causing DA release compared with

NIC suggests that cytisine should be significantly less

addictive. Thus, from therapeutic point of view,

cytisine shows a pharmacological profile which

makes it an attractive smoking cessation drug.

Other central nervous system effects. Apart from

its action on dopaminergic neurons in the mesolimbic

system, cytisine modulates the release of NE. The NE

release is governed by �4*-containing receptors [89].

The compound elicited [3H]NE release from hippo-

campal preparations in a concentration-dependent

manner with a potency approximately 50-fold lower

than in the [3H]DA release assay [1]. Both the potency

and efficacy of cytisine in the modulation of the NE

release were comparable with those of NIC [29]. The

role of cytisine in the release of other neurotransmit-

ters has not been fully determined. It is only known

that cytisine appears to have no effect on the release

of serotonin [119].

As mentioned above, cytisine exerts NIC-like phar-

macological effects but its effects are weaker than

those of NIC. Besides the mechanisms mentioned

above, weaker effects of cytisine are possibly due to

its poorer penetration into the CNS because of

a weaker lipophilic profile and low partition coeffi-

cient between organic solvent and water [122, 124].

Cytisine’s lower efficacy as compared to NIC in reso-

lution of withdrawal symptoms is probably related to

pharmacokinetic effects in addition to pharmacody-

namic effects. Cytisine administered to rats at a mod-

erate dose (1 mg/kg) achieves a concentration in the

brain, which should induce similar effects as those

elicited by NIC. However, this does not occur. The in-

duction of a similar response to NIC only occurs if the

dose was adjusted for the known difference in the

blood-brain barrier permeability and the difference in

the receptor affinity.

Locomotor activity. The systemic application of

NIC or other agonists of nAChRs can increase the lo-

comotor activity and this effect is more prominent

with a prolonged exposure [28, 80, 100, 121, 139].

The application of NIC into the peripheral nervous

system of rats resulted in a decrease in their kinetic

activity during the first 20 min. After 20 min, the ki-

netic activity increased. In rats chronically exposed to

NIC, cytisine caused neither a decrease in kinetic ac-

tivity in the first 20 min nor an increase in kinetic ac-

tivity [139]. It is noteworthy that in another behav-

ioral model based upon locomotor activity, cytisine

was more potent than NIC, when it was administered

by direct injection into the central tegmental area of

the brain [100, 121]. The locomotor-activating effects

associated with the tegmental area injections of

cytisine seem to be mediated by the mesolimbic DA

system [100].

Drug discrimination. Drug discrimination is a method

for assessing the behavioral actions of drugs. NIC

readily acts as a potent discriminative stimulus [117].

Reavill et al. [122] compared the discriminative

stimulus effects of cytisine with those of NIC. They

examined rats’ behavior and their reaction to transder-

mal application of NIC or cytisine. Animals were

positively rewarded with food after administration of

NIC. Cytisine-appropriate response rate was 65%,

while corresponding value for NIC was 95%. There

was no correlation between increasing cytisine dose

and the number of proper answers. Cytisine applied

together with NIC did not impair significantly the re-

action to NIC: rats’ behavior after applying only NIC

�� 783


Fig. 2. Mechanism of antismoking effect of cytisine. DA – dopamine

or after applying cytisine and NIC together did not

differ. In another study, in rats trained to discriminate

cytisine from saline, NIC produced full dose-

dependent generalization of up to 93%. In rats trained

to discriminate NIC, cytisine also increased drug-

appropriate responses; however, this effect was of

smaller magnitude (54%). These results showed that

cytisine, like NIC could serve as a robust discrimina-

tive stimulus, but it was much less potent than NIC in

the behavioral studies. There are some differences be-

tween both drugs, like the asymmetrical cross-

generalizations and differences in susceptibility to an-

tagonism by mecamylamine. These studies confirm

that cytisine only partially activates nicotine recep-

tors, and does not provide equal CNS response [23].

Analgesia. The role of NIC and other nAChRs

agonists in analgesia has been well documented [61].

In the hot-plate test, cytisine exhibited an anti-

nociceptive effect, which was weaker than the effect

of epibatidine, but more potent than the effect of NIC

[120, 131].

Cognitive functions. Epidemiological studies indi-

cate a negative correlation between smoking and an

incidence of Parkinson’s disease and, to a lesser ex-

tent, Alzheimer’s disease [52]. NIC and some its ana-

logues were shown to enhance cognitive function and

are considered as a potential therapy for patients with

Alzheimer’s disease [70]. The question to be resolved

by future research is whether cytisine can imitate NIC

and would offer therapeutic benefits in patients with

these diseases. Animal studies showed that cytisine,

similarly to NIC, facilitated retention of avoidance

training and improved memory and learning [20, 83,

84]. The beneficial action of cytisine on cognitive pro-

cesses was blocked by flupentixol, an antagonist of DA

D1/D2 receptors. It suggests that such effects of

cytisine involve dopaminergic neurotransmission [20].

Neuroprotection. An increase in DA turnover is

believed to compensate for impaired transmission in

neurodegenerative processes, for example in Parkin-

son’s disease. NIC facilitates the DA release from

neurons in the nigrostriatal pathway, the area that is

depleted of DA-containing neurons in Parkinson’s

disease [56]. Therefore, NIC is being investigated as

a treatment for Parkinson’s disease. Several models

are used to mimic the neuropathology of Parkinson’s

disease. One model uses a neurotoxin 1-methyl-4-

phenyl-1,2,3,6-tetrahydropyridine (MPTP) that causes

degeneration of nigrostriatal dopaminergic neurons,

decreases the DA concentration in the striatum and

produces parkinsonian symptoms. In vivo, cytisine

partially prevented the MPTP-induced reduction of

the striatal DA concentration and the increase in the

DA turnover [48]. Some investigators reported that

the neuroprotective effects of cytisine resulted from

its interaction with �7 subtype and, maybe, �2*-con-

taining subtypes of nAChRs [72, 138]. On the basis of

suggestions that an increased iron release from ferritin

is involved in the pathogenesis of Parkinson’s disease

[16], Ferger et al. [48] proposed an interesting hy-

pothesis that cytisine might form complexes with iron

and may thus lead to a decrease in hydroxyl radical

production.

Therefore, the beneficial effects on cognitive func-

tion and neuroprotective properties make cytisine

a possible therapeutic tool for therapy Parkinson’s and

Alzheimer’s disease but further detailed studies are

required. A few studies available to date suggest that

the alkaloid might be a promising substance for study-

ing neuroprotective effects on nigrostriatal dopamin-

ergic neurons.

Mood. Many smokers report that smoking helps

them relieve depression, and some smokers become

depressed after smoking cessation. Of interest was the

observation that cytisine exhibited antidepressive ac-

tivity in rats by overcoming immobility in the Por-

solt’s test [6]. Neurochemical effects of cytisine resem-

ble effects of some antidepressant drugs, so it is rea-

sonable to consider cytisine as a potential therapy in

smokers with depression and other affective disorders.

Peripheral effects

Systemic administration of cytisine affects the auto-

nomic ganglia, the adrenal medulla, and carotid sinus

via nAChRs localized in these tissues. Usually, pe-

ripheral effects are manifested after application of the

drug at doses that are 1/4 to 2/3 lower than the doses

of NIC required to cause the same effect [10].

Cytisine can have both excitatory and inhibitory ef-

fects in autonomic ganglia; however, the stimulating

effect is more potent than its blocking effect [157].

The study of Barlow and McLeod [10] showed that

cytisine stimulated cat superior cervical ganglion at

concentrations 25% lower, and blocked the ganglion

at concentrations 17% higher than respective concen-

trations of NIC causing the same effects. The stimula-

tion of sympathetic ganglia results in an increase in

blood pressure. The elevation of blood pressure was

over 2-fold greater than after administration of NIC.

784 ��

Also, the effects of cytisine on contraction of the guinea

and rat pig ileum, the frog rectus as well as on block of

the rat diaphragm were also more potent than those of

NIC [10, 111, 114]. The stimulant effect on guinea pig

ileum was antagonized by hexamethonium [8].

Cytisine at high doses caused desensitization of

ganglionic and/or channel blockade of nAChRs [24].

As a consequence, a decrease in blood pressure and

heart rate, an increase in cardiac output as well as an

increase in respiration rate was observed. Animal

studies showed that cytisine was 1.4 times more po-

tent than NIC in decreasing blood pressure but was

1/4 to 1/8 as potent as NIC in decreasing heart rate,

respiration rate and enhancing minute volume. Cytisi-

ne’s ability to affect tidal volume was weaker than

that of NIC [136].

Apart from its action on the autonomic ganglia,

cytisine stimulates, in a Ca2+-dependent manner,

chromaffin cells in the adrenal medulla. The affinity

of the alkaloid for different subtypes of nAChRs in

the adrenal medulla was similar to the affinity for the

ganglionic receptors [17]. Cytisine was somewhat less

efficacious than NIC in adrenal chromafin cells [153].

A consequence of the activation of adrenal nAChRs is

an enhanced release of catecholamines, which elevate

blood pressure and blood glucose level. It is well

known that stopping smoking leads to a decrease in

blood pressure and blood glucose values. Cytisine, by

mimicking the effects of NIC, can restore, at least par-

tially, normal values of blood pressure and prevent the

reduction of the blood glucose level and in this way it

can attenuate some symptoms of NIC withdrawal.

Cytisine at low doses stimulates the respiratory

center, mainly by the activation of nAChRs in carotid

sinus; however, it has been suggested that cytisine can

also exert a direct action on the respiratory center

[111]. In anesthetized rats, the stimulating action of

cytisine was less potent than that of NIC. In order to

reach the same response, the dose of cytisine must be

3-fold higher than that of NIC [10].

Toxicity

Data demonstrating the toxicity of cytisine derive

mainly from the studies in rodents [4, 5, 111], dogs [4,

85], cats [157] and horses [62, 123]. Moreover, sev-

eral cases of accidental poisoning with Laburnum

seeds and a case of an intentional overdose of cytisine

(Tabex®) in humans have been reported [123, 142].

Animal toxicity

Acute and chronic toxicity

The therapeutic index of cytisine is wide [111]. In the

acute experiment, the LD50 values (i.e. doses of the

drug causing death in 50% of animals) obtained after

iv, sc, and po administration of the drug were 2.3, 13,

and 13 mg/kg for male, and 3.1, 13, and 29 mg/kg for

female mice, respectively. In rats (of both sexes), the

LD50s were 9, 11, and 38 mg/kg after ip, sc, and po

administration, respectively. When cytisine was in-

jected sc in dogs, the LD50 value was 4 mg/kg [4].

The most frequent symptoms of toxicity in animals

are from the gastrointestinal tract. The first symptom

is almost always vomiting. The onset of symptoms is

quick, with vomiting and other gastrointestinal distur-

bances appearing 45 min to 4 h after administration

[4, 75, 77]. The effects of toxic doses include initially

weakness, dizziness, impairment of motor coordina-

tion, atactic gait, slow movements, muscular facial

twitches, increased reflex sensitivity, mydriasis, rapid,

shallow breathing, occasional urinary retention, and

ultimately, tonic-clonic convulsions followed by col-

lapse and coma with respiration cessation, mainly

caused by paralysis of respiratory muscles [4, 75, 77,

111, 157]. Thus, these symptoms are the same as the

symptoms of NIC overdose.

Organ toxicity

Hepatotoxicity in vitro. The data about toxicity of

cytisine for individual organs are fragmentary. Hepa-

totoxicity has been studied the most. A recent in vitro

study assessed cellular function by determination of

hepatocyte viability and the amount of reduced glu-

tathione, which characterized the possible toxic he-

patic metabolism of xenobiotics [143]. Cellular vi-

ability of hepatocytes treated with cytisine at 5 nM

was reduced by 12% vs. untreated controls while NIC

lowered it by 35% (p < 0.001). Cytisine (5 nM)

decreased the level of reduced glutathione by 17%

(p < 0.01) while NIC caused a 41% (p < 0.001) reduc-

tion in comparison to control. The effect of both com-

pounds was dose-related. Taken together, the results

indicated that cytisine exhibited lower cytotoxicity,

more weakly affecting hepatocyte viability and re-

�� 785


duced glutathione levels than NIC at equivalent con-

centrations (5 nM–250 µM).

The same study demonstrated higher toxicity of

cytisine compared to NIC in the assay of malondial-

dehyde, a product of lipid peroxidation, which gives

information on the possible cytotoxicity associated

with the formation of free radicals and unlocking of

lipid peroxidation processes. A possible explanation

of higher toxic effects of cytisine in the malondialde-

hyde test could be characteristic of cytisine which, in

contrast to NIC, does not undergo biotransformation

in hepatocytes (approximately 90–95% of cytisine is

excreted unchanged in the urine) [143].

Lactate dehydrogenase is a marker of membrane

integrity, commonly used in in vitro experiments on

isolated heptocytes. The increased lactate dehydroge-

nase leakage into the medium indicates cell injury.

Both cytisine and NIC significantly increased, in

a concentration-dependent manner, the release of lac-

tate dehydrogenase into the medium. The toxic effect

of cytisine on this parameter was comparable to that

of NIC [143].

Hepatotoxicity in vivo. The potential hepatotoxic

effect of cytisine was studied in vivo in different ani-

mal species. In rats, chronic administration of the

drug at a dose of 1.35 mg/kg during 90 days caused

a 2-fold increase in blood glutamic pyruvic transfe-

rase (GPT) concentration, without significant changes

in blood glutamic oxaloacetic transferase (GOT) and

alkaline phosphatase. Such changes were not ob-

served when cytisine was administered during 45 days

to mice (3.3 mg/kg) and 180 days to rats (0.45 and

0.9 mg/kg) or dogs (0.46 mg/kg) [5].

Toxicity for other organs and tissues. Cytisine ad-

ministered po during 5 days in rats at doses of 1 or

5 mg/kg did not show ulcerogenic activity evaluated

by macroscopic and histological analysis. Cytisine

had more favorable safety profile with respect to the

stomach mucosa in comparison with NIC given po at

equal doses [150].

In experiments assessing the effects of cytisine (Ta-

bex®) on the human kidney epithelial cell line HEK-

293T, the drug displayed marginal cytotoxic potential

[43]. Changes in transepithelial electrical resistance

are indicative of toxic effects. A measurement of trans-

epithelial electrical resistance showed no significant

alterations of this parameter upon continuous expo-

sure of HEK-293T monolayers to cytisine. Moreover,

a morphological analysis showed that the cell cultures

treated with cytisine at the concentrations up to

200 µM did not differ morphologically from the un-

treated controls. It has been concluded that cytisine is

devoid of cytotoxic effects on human kidney cells [43].

In contrast to hepatocytes, cytisine did not exhibit

toxic effect on P-diploid human embryonal pulmo-

nary cells, human larynx cancer cell lines HEp-2 or

human epitheliod cancer cell lines HeLa [144].

There were no other recorded alterations in the

clinical laboratory parameters and histomorphological

changes in experiments with the chronic application

cytisine to mice (7.6 mg/kg within 30 days) and rats

(up to 1.35 mg/kg for 90 days).

Genotoxicity

Cytisine was not genotoxic in the test for cytogenetic

aberrations in vivo in mice bone marrow cells. When

applied orally at doses of 1 mg/kg or 5 mg/kg, it did

not induce statistically significant increase in fre-

quency of cells with abnormalities as compared with

the control. Only at 10 mg/kg, cytisine induced mini-

mal, non-specific chromosomal abnormalities. In

comparison with NIC that caused damage to DNA in

epithelial cells [128], cytisine had much lesser clasto-

genic activity. Higher frequency of chromosomal ab-

errations was seen even at the lowest dose of NIC

(0.1 mg/kg) as compared with the 100-fold higher

dose of cytisine (10 mg/kg) [143]. An analysis of the

genotoxicity study did not indicate any clastogenic

activity of cytisine to be risky for humans [143].

Teratogenicity and embryotoxicity

Potential toxic effects of the alkaloid on reproduction

were studied in rats and chicken. Administration of

cytisine at doses of 3–4 mg/kg to pregnant rats did not

result in visible skeletal and visceral abnormalities in

fetuses [78]. However, the more complex study by

Todorov et al. [144] showed some adverse effects of

cytisine on the fetus. The alkaloid at a dose of 9 mg,

equal to the maximum human daily therapeutic dose,

reduced hen fetal weights by 11%. The doses of 4.5 mg

(1/2 the maximum human daily therapeutic dose) and

45 mg (5 times the maximum human daily therapeutic

dose) reduced the weights by 4.5 and 52%, respec-

tively. Those percentages of retardation in the weight

gain were correlated with the percentages of lethality

of hen fetuses which were 8, 32, and 44% for 4.5, 9,

and 45 mg/kg, respectively. Experimental data indi-

786 ��

cate that cytisine shows a tendency to higher toxicity

during the first half of embryo development [144].

In addition, cytisine impaired closure of abdominal

and thoracic walls that can be possibly explained by

the toxic effect of cytisine on the proliferating cells

during the early development of the embryos. There is

no evidence for skeletal malformations caused by the

drug, although curved limbs in fetuses were observed

[144]. Furthermore, the histomorphological examina-

tion showed dose-dependent dystrophy (from mini-

mal to moderate) of the cells in the heart muscle, liver,

stomach, and spleen [144]. There are no studies con-

cerning the potential toxic effects of cytisine in preg-

nant humans.

Human toxicity

The available human data indicate that cytisine is gen-

erally well-tolerated [12, 91, 142, 160]. However, the

high doses of the alkaloid can be strongly toxic. It has

been known since 1877 [156] that the consumption of

the seeds and flowers of Laburnum anagyroides or

drinking a tea made from the flowers of this plant can

cause toxic effects in children or the elderly [35, 51,

53, 97, 123, 155]. Cytisine seems to be the chemical

responsible for these responses [53]. The symptoms

of cytisine poisoning resemble NIC poisoning symp-

toms [123].

Although the lethal doses of cytisine in humans

were not recorded, there was the report of one fatal

case of Laburnum seed poisoning in a 50-year-old

schizophrenic man who had swallowed 23 pods

[123]. The most likely absorbed dose of cytisine was

about 50 mg. It has been stated that the lethal dose of

cytisine for a dog or cat is about 3–4 mg/kg [27].

Thus, assuming that the man was 75 kg in weight, it

would suggest that the lethal dose was about 0.5

mg/kg.

One report identified the toxic effects of cytisine

after overdose of Tabex® taken as a suicidal attempt

[142]. This report described a case of a 48-year-old

woman who took large quantities of the drug. The

first time she took 40–50 tablets (1.5 mg/tablet) and

experienced initial vomiting, followed by a loss of

consciousness and clonic seizures. After admission to

an emergency department and recovering conscious-

ness, she experienced muscle spasms, headache, diz-

ziness, weakness, double vision, dysarthric speech,

and hypotension. During the hospitalization, she was

treated with infusions of sodium chloride and glucose

as well as with analeptics, vitamins and phenobarbi-

tal. After 5 days, she was discharged from the hospital

without any symptoms. Subsequently, she took the

second overdose of about 90 tablets of the drug, to-

gether with unknown amount of scopolamine and

hyoscamine. She fainted again but soon recovered

consciousness. No seizures were noted and her symp-

toms were less severe and after 7 days she was again

discharged from the hospital. Noteworthy, no clinical

and laboratory features of liver or other internal organ

damage were observed.

Cytisine as a medication for nicotine

addiction – clinical studies

A literature review identified 12 studies described in

19 papers reporting efficacy and safety of cytisine in-

vestigated as a smoking cessation aid. All studies, ex-

cept for two Russian studies, used cytisine in the mar-

keted form Tabex®, containing 1.5 mg of the com-

pound per tablet. Among all studies, only 3 studies

were placebo-controlled [11, 112, 130].

The majority of studies were performed in 1960s

and 1970s. Unfortunately, these studies had several

design and analysis shortcomings. Their documenta-

tion and design would not be considered sufficient to

support registration in most European countries be-

cause of the lack of appropriate follow-up, and inade-

quate verification of abstinence criteria. However, the

data do suggest that the drug is safe and efficacious at

the doses used in the trials. Since 2000, two trials

were performed, but only that by Zatonski et al. [160]

has been published in scientific literature in English.

Efficacy

During the past 40 years, Tabex® has been used in

CEE by millions of smokers trying to stop smoking.

More than 4000 subjects have taken Tabex® in effi-

cacy trials.

In the first Bulgarian study reported in 3 papers

[140, 141, 142], the treatment groups consisted of

a small number of smokers (N = 70 for group I con-

sisting of healthy and psychiatric patients, and N = 17

for group II consisting of psychiatric patients). The

dosing regimen of cytisine (Tabex®) followed recom-

mendation of the manufacturer (Tab. 4). The subjects

�� 787


noticed a marked therapeutic effect occurring as early

as the 1st to the 4th day of treatment. After the 20-day

treatment course, the successful abstinence rates were

56% and 29% in I and II group as declared by self-

report, respectively. However, no longer-term absti-

nence was assessed and the study lacked adequate

definition of abstinence criteria.

The placebo-controlled studies on cytisine (Ta-

bex®) by Paun and Franze [112, 113] adopted a 17-

day treatment period with similar dosing regimen.

They examined the effect of cytisine in 366 patients

vs. 239 control patients. Within the cytisine-treated

patients, 230 suffered from concomitant chronic bron-

chitis. The abstinence from smoking was determined

by patients’ self-report. Among all patients receiving

cytisine, 55% were abstinent at the 8th week from the

beginning of therapy. At the 26th week, the continu-

ous abstinence rate decreased to 21% patients. In the

placebo-controlled part of the study, the abstinence

rate in cytisine group (N = 42) was 42% in the 8th

week, compared to 33.5% patients in the placebo

group (N = 239). However, in contrast to the placebo

group, the cytisine-treated patients had additional

psychological support (group psychotherapy), which

could have affected the results in this group of pa-

tients. Similarly to the Bulgarian studies, the cessation

was observed most often during the first 5 days of the

treatment. Although the treatment groups in the stud-

ies by Paun and Franze [112, 113] were larger than

those in the Bulgarian studies, the design and docu-

mentation of these studies would also not be consid-

ered sufficient to support registration of the drug in

many European countries.

Other German studies by Benndorf et al. [11–14]

and Scharfenberg et al. [129] enrolled 1452 chronic

smokers. In the former placebo-controlled, double-

blind study (N = 157 in the cytisine-treated group and

N = 157 in the placebo group), the abstinence rates

were 76% and 70% at the 4th and 16th weeks, respec-

tively. In that study, it was noteworthy that the absti-

nence rate in the placebo group was significantly

higher than usually obtained in other studies and it

was 31% at the 16th week [11]. Independently of ab-

solute values of figures in both groups, it should be

underlined that smokers treated with Tabex® for 4

weeks had over 2-fold superior abstinence rate com-

pared to subjects treated with placebo. The double-

blind study carried out by Scharfenberg et al. [129]

compared effectiveness of Tabex® in 607 patients

with placebo in the same number of patients. Sixty

five and 30.5% of all patients treated with the drug

were abstinent at the 4th week and 6th month, respec-

tively, from the beginning of therapy, as compared to

40.5% and 16% in the placebo group, respectively.

Two years later, 21% of the patients treated with Ta-

bex® were still non-smokers and this result was sig-

nificantly higher than that in the placebo group

(13%)(p < 0.001).

Other results examining the efficacy of cytisine in

smoking cessation came from a double-blind,

placebo-controlled trial carried out by Schmidt [130].

In that study, about 1975 smokers were allocated to

receive one of 12 drugs of whom 250 were treated

with cytisine (Tabex®). The drug demonstrated an ef-

ficacy of 41% after the end of treatment (25-day treat-

ment period at the recommended dosing) vs. 31% in

placebo group. After 12 weeks this percentage

dropped to 27% which was still higher than for pla-

cebo (21%), and all the other drugs tested, including

Atabakko (caffeine + theobromine), Citotal, Nicobre-

vin, Nicocortyl, Ni-Perlen, Pempidil, Potassium, Ra-

dix Levistici, Raucherstop 5-HT, Targophagin, Unilo-

bin, and Viotil. However, because of the reported effi-

cacy rates for several other drugs, it is questionable

whether reliable assessments of efficacy were used.

No adverse events were observed or reported in the

paper.

In one study, buccal films containing cytisine alone

(1.5 mg), anabasine alone (1.5 mg) or cytisine (0.75 mg)

and anabasine (0.75 mg) were applied for 15 days in

three groups consisting of 23, 23, and 16 chronic

smokers. Forty seven percent of patients receiving

films, including patients with coronary heart disease,

hypertension and diabetes mellitus, were reported ab-

stinent at 15 days [105, 106]. Results by group were

not indicated but it was stated that the films contain-

ing cytisine alone or cytisine in combination with ana-

basine were more effective than the films containing

anabasine alone. The abstinence rate after 6 to 14

months was about 23%. No adverse events were ob-

served/reported in the paper. The data from another

study using the same regimen of the treatment, collected

after 6 months in 201 smokers (including some psychi-

atric patients), demonstrated the abstinence rate of 50%,

however, the results by groups were not given [95].

A very small but interesting clinical study by Vlaev

[152] investigated the possibility of parallel treatment

of nicotine addiction and depression. Five patients di-

agnosed with depression, 3 of them with psychogenic

depression and 2 with intermittent depression, were

788 ��

treated with ascending doses of cytisine (Tabex®)

increased every day to a maximum daily dose of

22.5 mg. As a result, a quick reduction of the depressive

symptoms was observed at the end of the 1st week in

patients with psychogenic depression, and at the 2nd

week in patients with intermittent depression. In addi-

tion, the smokers reported a decreased desire to

smoke but no detailed data are available. A slight de-

crease in arterial blood pressure was noted in some

patients.

�� 789


Tab. 2. Sample characteristics and outcome of an open-label studyby Zatonski et al. [160]

Total number of patients attending clinic 438

Number of patients excluded 2

Total of patients enrolled 436

Percentage of (n) males 43.8 (191)

Mean (SD) age (years) 44.4 (13.1)

Percentage of patients (n) smoking more than 10cigarettes per day

95.0 (414)

Percentage of patients (n) smoking more than 20cigarettes per day

51.6 (225)

Mean (SD) FTND score (dependence) 6.1 (2.2)

Percentage of patients (n) with post-secondaryeducation

26.8 (117)

Mean (SD) age of starting to smoke regularly 18.9 (4.5)

Percentage of patients (n) having tried to quitbefore

70.2 (306)

Percentage of patients (n) attending one session 54.1 (236)

Mean (SD) number of visits to clinic 1.7 (0.9)

Number of patients followed up at 12 weeks 342

Number of those followed up who reported taking� 1 dose of medication

315

Percentage of patients (n) abstinentat 12 weeks

27.5 (120/436)

Percentage of patients (n) reporting gastricdisturbance/nausea

10.4 (33/315*)

Percent pf patients (N) stopping medication due toadverse events

15.5 (49/315*)

Number of patients attempted to follow up at 12months

120

Number of patients followed for up at 12 months 112

Number of patients reporting abstinence 68

Percentage of patients (n) confirmedabstinent at 12 months by CO

13.8 (60/436)

*The number reporting having taken medication; CO – exhaled-aircarbon monoxide concentration; FTND – Fagerström Test for NicotineDependence

Tab. 3. Adverse effects reported by patients treated with cytisine (Ta-bex®) in some clinical trials [91, 105, 130, 143, 160]

System organSigns and symptoms

Frequency(%)

CARDIOVASCULAR

tachycardiaincrease in blood pressurebradycardiacold fingers

< 1–14711

EYES

lacrimation < 1

GASTROINTESTINAL

dry mouth and throatabdominal pain (mainly upper)nauseaconstipationtaste changes (mainly bitter taste)vomitingdiarrheaflatulenceburning tongueheartburnsalivationelevation of aminotransferases

350–20

1–11.5< 1–81–4

< 1–42

11

< 1–2< 1< 1

GENERAL

weaknessmalaisefatigue

71

< 1

METABOLISM/NUTRITION

appetite changes (mainly increased appetite)weight gain

4721

NERVOUS

Neurological

headachevertigoheaviness in the head

< 1–172–4< 1

Psychiatric

irritabilitysleep disturbances (insomnia, drowsiness, abnormaldreams, nightmares)mood changesanxietyloss of concentrationdizzinessloss of sexual interest

361–21

1511

1–62

< 1

RESPIRATORY/THORACIC

dyspneaincreased expectoration

< 1< 1

SKELETOMUSCULAR

muscular pain < 1–10

SKIN/SUBCUTANEOUS TISSUE

rashincreased perspirationsagging skin

2< 1< 1

In another study on small number of heavy smok-

ers (N = 14), 50% of patients receiving cytisine (Ta-

bex®) were abstinent at 2 weeks after 25-day treat-

ment [91]. Granatowicz [58] reported that 70% of

smoking cessation clinic patients treated with cytisine

for 27 days (N = 1968) were abstinent at 6-month

follow-up. Another study by Marakulin et al. [92]

showed very high percentage of abstinent patients at

the end of 21-day treatment with Tabex® (70%, N = 388).

However, a high percentage of abstinence was also

noted in the control group (53%, N = 232). Notewor-

thy, both groups of patients had 12–14 sessions of

autogenic training.

A meta-analysis was recently published based on

trials of cytisine for smoking cessation [44]. The

meta-analysis of 3 placebo-controlled trials [112, 129,

130] gave a pooled odds ratio of 1.93 (95% confi-

dence interval (CI:1.21–3.06) after up to 8 weeks. For

two double blind, placebo-controlled trials with

longer follow-up [129, 130], the pooled odds ratio af-

ter 3–6 months was 1.83 (CI:1.12–2.99). For one

double-blind, placebo-controlled study with follow-

up after 2 years [129], the odds ratio was 1.77

(CI:1.29–2.43). For comparison, the odds ratios for

different forms of NRT after 12 months was 1.99

(CI:1.5–2.64), and for bupropion 2.06 (CI:1.74–2.4)

[45, 64]. The author concluded that cytisine was very

probably effective for smoking cessation.

Recently, encouraging results have been seen in an

uncontrolled, open-label trial in Poland [160] (Tab. 2).

A total of 436 subjects were provided with Tabex®

administered as recommended by the manufacturer

(Tab. 4) for 25 days with minimal behavioral support.

The participants were followed up for up to 12 weeks

and those who reported being abstinent by self-report

were additionally assessed at 12 months. Their

12-month abstinence, defined by the Russell Standard

[154] (up to 5 cigarettes/12 months), was verified by

an exhaled carbon monoxide (CO) concentration

(<10 ppm). The success rate at 12 weeks was 27.5%.

A total of 13.8% of those attending the 12-month

smokers’ clinic reported being abstinent. This long-

term abstinence rate is similar to that noted following

NRT [49, 134]. Cytisine reduced craving and with-

drawal signs and, for those who smoked while receiv-

ing study drug, diminishes smoking satisfaction, e.g.

it caused changes in the taste of cigarette in 53% of

patients. Although it is impossible to conclude defini-

tively about the efficacy of the drug on the basis of an

open-label study, the results of this study confirm pre-

vious suggestions that cytisine is useful for smoking

cessation. Furthermore, the results of the study sup-

ported the argument for randomized controlled study

on efficacy of cytisine. A double-blind, placebo-

controlled, randomized trial (N = 370/arm) is being

planned.

Safety and tolerance

A literature review indicates that during over 40 years

of cytisine marketing as a smoking cessation aid, no

severe adverse reactions have been reported at thera-

peutic doses [11, 91, 142, 160]. In general, the drug is

believed to be well tolerated; however, some adverse

effects which can result in discontinuation of the drug

were observed. In the available studies, the rate of dis-

continuation varied from 6% [143] to 15.5% [91, 160].

According to the first study on cytisine safety, 61%

of patients treated with the drug had no adverse reac-

tions [141, 142]. Another study reported that in 29%

of patients no adverse effects were observed [91]. Ad-

verse effects were usually transient with mild to mod-

erate manifestation. Most often they occurred during

the initial phase of therapy and did not result in seri-

ous health complications [91, 160].

All adverse events that were reported in currently

reviewed research are listed in Table 3. Because

safety has been variably and sporadically reported in

different historical trials, actual incidence and preva-

lence of adverse events is difficult to assess. Also,

some adverse events may have been due to smoking

cessation, and may or may not be attributable to

cytisine. It is very likely that clinical manifestations,

which were reported as side effects, like irritability, an

increase in appetite, weight gain, insomnia or fa-

tigue/malaise, can result from NIC deprivation and

were not adverse events of cytisine.

790 ��

Tab. 4. Recommended therapeutic schedule of cytisine (Ta-bex®)(Sopharma, Sofia, Bulgaria)

Days Dose per day (mg)

1–3 9 (6 � 1.5)

4–12 7.5 (5 � 1.5)

13–16 6 (4 � 1.5)

17–20 4.5 (3 � 1.5)

21–25 3 (2 � 1.5)

Tabex® – 1 tabl. contains 1.5 mg of cytisine

The most common adverse events were gastric dis-

tress which occurred in the first days of therapy and

usually decreased during the course of treatment. The

mechanism of the gastrointestinal adverse effects of

cytisine was not fully elucidated. Most likely they re-

sult from sympathetic neural stimulation mediated by

the drug. The gastrointestinal adverse effects, except

for constipation, occurred with similar frequency in

patients who stopped and did not stop smoking [160].

Nausea was the most frequent reason of discontinua-

tion of treatment and occurred in about 2/3 of the peo-

ple who stopped taking the drug. The drop-out rate

due to nausea was not higher for cytisine (10%) [160]

compared with bupropion (14%) [104]. The reduction

of a maximal recommended dose from 9 to 4.5 mg per

day and/or administration of H2-blockers or proton

pump inhibitors always brought relief. Granatowicz

[58] reported that patients with stomach or duodenum

lesions showed no change after taking cytisine. How-

ever, since there are no data to support clinical deci-

sion, the use of cytisine in patients suffering from an

active peptic ulcer disease or gastroesophageal reflux

disorder should be very cautious. Also, a particular

caution should be taken in patients who had these dis-

eases in the past.

In some cases, a mild elevation of both systolic and

diastolic blood pressure as well as a transient mild

tachycardia could be observed but usually these

symptoms did not cause withdrawal of the treatment.

Importantly, the recent study demonstrated that

cytisine seems to be safe in hypertensive patients

whose blood pressure was sufficiently controlled with

the standard antihypertensive drugs [160].

Among all psychiatric symptoms observed after

administration of the drug, irritability, insomnia and

mood changes were relatively common. Headache

was the most frequent neurological adverse effect (up

to 17% patients). It occurred with the frequency similar

to that observed in patients treated with bupropion

(14%) or varenicline (15.5%) [54]. The discontinuation

rate due to this symptom was not higher than 3% [160].

Smoking cessation is very often associated with the

weight gain. An analysis of the changes in body

weight in patients effectively treated with Tabex®,

measured before the start of therapy and after 12

months, demonstrated the weight gain of an average

of 7 kg in 82% and weight reduction in 17% patients

[160]. These results are not consistent with an earlier

study which did not find statistically significant

changes in body weight of subjects prior to and after

the course of treatment [113].

Other adverse reactions listed in Table 3 were rela-

tively rare and did not cause persistent or serious

health complications.

Interactions with nicotine and other drugs

Cigarette smoking can affect drug therapy by both

pharmacokinetic and pharmacodynamic mechanisms.

For example, in animal studies, NIC induced the ac-

tivity of several cytochrome P450 (CYP) enzymes re-

sponsible for the metabolism of a number of drugs

[2]. There is a question whether cytisine like NIC can

affect the CYP enzymes. So far, any data on this issue

have not been identified.

Theoretically, the treatment with cytisine in pa-

tients who continue smoking could cause an enhance-

ment of some effects of NIC. There are no reports de-

scribing clinically significant reactions that could re-

sult from the interaction between NIC and cytisine,

and studies investigating this issue are needed.

An influence of cytisine on the action of other

drugs has not been established yet. There are only

some clinical observations of co-administration of

cytisine with other drugs. In one study, cytisine was

given to 17 patients treated with neuroleptics due to

various psychiatric diseases [142]. Any changes in the

efficacy of the treatment of the basic disease were not

observed during the course of treatment with cytisine.

In addition, 29% of patients stopped smoking but, un-

fortunately, for a very short period of time. Consider-

ing the hepatotoxic potential of some neuroleptics, the

authors monitored the liver function in cytisine-

treated patients and no abnormal results were found.

In the same study, no unfavorable interactions with in-

sulin and antidepressants were observed.

Theoretically, the cardiovascular action of cytisine,

i.e. an elevation of blood pressure and heart rate,

could be associated with a smaller decrease in blood

pressure and heart rate during therapy with �-adrenoly-

tics. Moreover, the antinociceptive activity of cytisine

may have clinical consequence by changing the effects

of some analgesics. Product information states that

cytisine should not be co-applied with tuberculostatic

drugs [143] but a reason for this is not given.

�� 791


Cytisine derivatives and their pharmacology

Cytisine has been a starting point for the studies

aimed to search novel compounds of potential thera-

peutic interest from many years. Some of them were

obtained from the natural material, e.g. N-methyl-

cytisine, also known as caulophylline. Initially, the

structural modifications of cytisine were restricted to

improving its respiratory analeptic profile or obtain-

ing local anesthetics [68, 90] but no pharmacological

results were published for many obtained compounds.

More recently, the studies were mainly aimed at creat-

ing derivatives with improved binding affinity and ef-

ficacy and minimal side effects. The chemical modifi-

cations of cytisine would be expected to increase its

lipophilicity, thus improving the ability to pass the

blood-brain barrier, to reduce the affinity for gan-

glionic receptors and to alter its selectivity for differ-

ent subtypes of central nAChRs.

Cytisine can be structurally modified at the secon-

dary amino groups, conjugated double bonds and car-

bonyl groups [18]. Substitution of the basic nitrogen

atom by a methyl group decreased the activity of

cytisine as an nAChRs agonist [10]. N-methylcytisine

(caulophylline) in vitro showed diminished affinity

and functional potency at all nicotinic receptor sub-

types [135]. The introduction of a nitro group at the

3-position of the pyridone nucleus enhanced the affin-

ity for nAChRs while the introduction of substituents

at the basic nitrogen, though reducing to different de-

grees the affinity, gave rise to compounds with

a higher selectivity for central (�4�2*) versus gan-

glionic (�3-containing) receptor subtype [19]. In the

study on recombinant human receptors expressed in

clonal cell lines and Xenopus oocytes, bromination of

cytisine at the 5-position of the pyridone ring caused

a modest decrease in both affinity and efficacy while

iodination caused a decrease in affinity and different

effects on efficacy, ranging from a decrease (�7,

�4�4* nAChRs) to a marked increase (�4�2*

nAChRs) [135]. Bromination of cytisine at the 3-position

increased potency in binding assays by about 10-fold

at �4�2*, 40-fold at �7 and more than 100-fold at

�4�4* nAChRs [63]. With regard to efficacy, the

bromo-isosteres of cytisine were more efficacious

agonists at �4�4* than at �4�2* nAChRs, mirroring

the pattern of efficacy of cytisine [33, 50, 63]. Thus,

cytisine and its bromo-isosteres can be useful tools for

research purposes to distinguish between different

subtypes of nAChRs [151].

Abin-Carriquiry et al. [1] compared the effect of

bromination and iodination of cytisine on [3H]DA re-

lease from rat striatal preparations. Both 3-bromo-

cytisine and 3-iodocytisine exhibited an increased

binding affinity for �4�2* and �7 nAChRs and were

more potent than cytisine in evoking [3H]DA release

from the rat striatal slices.

Another structural modification of cytisine consists

in replacing the pyridone oxygen by a sulfur atom.

Thiocytisine, the result of such modification, showed

a modest partial agonism at human �4�2* subtype

and was inactive at rat �3�4* subtype. Thiocytisine

showed an extremely weak, low-efficacy partial ago-

nism at the neuromuscular type of nAChRs [50, 65].

Chellappan et al. [26] found that novel 9-vinyl

cytisine derivative had a very similar agonist activity

profile to that of cytisine.

Recently, many cytisine derivatives were investi-

gated for biological activities. Some interesting in

vivo responses were obtained concerning DA antago-

nism, analgesia, anaphylaxis, an inhibition of stress-

induced ulcers, antiinflammmatory and antihyperten-

sive action [18]. For example, 2-methoxyphenyl-

piperazinylpropyl-cytisine exhibited the strong anal-

gesic activity in the writhing test and formalin test in

animals [18]. The same derivative had a strong and

long-lasting antihypertensive action in rats. Of inter-

est, the antihypertensive activity was related neither

to ganglionic blockade nor �1- and �2-adrenoceptor

blockade and remains somewhat intriguing. The hy-

poglycemic activity of an N-methyl derivative, real-

ized via an increase in insulin release, is also worth men-

tioning [99]. In vitro experiments demonstrated some

other biological activities of cytisine analogues, including

a positive cardio-inotropic activity, �-antagonism, �1- and

�2-antagonism, and an inhibition of platelet activating

factor-induced platelet aggregation [18].

Despite the synthesis of a number of novel cytisine

derivatives, few have succeeded in clinical trials, em-

phasizing the limitations in translation from screening

models to clinical applications. The most thoroughly

studied compound, chemically and pharmacologically

related to cytisine, is varenicline. Its structure derived

from 3-substituted cystinoids [31]. Varenicline, exam-

ined at a variety of rat nAChRs expressed in Xenopus

oocytes, displayed high affinity for �4�2* nAChRs

and potent partial agonism at these receptors [31, 33].

Varenicline had lower potency and higher efficacy at

792 ��

�3�4* receptors and seems to be a weak partial ago-

nist at �3�2* and �6-containing nAChRs, and a full

agonist at �7 nAChRs [96]. The pharmacological ef-

fects of varenicline on nAChRs are similar to the ef-

fects of cytisine. Varenicline mimics the effects of

NIC on DA release in the nucleus accumbens when

given alone but suppresses this response to a subse-

quent NIC challenge and reduces NIC self-

administration [31, 46]. It should be underlined that its

dopaminergic profile is very similar to that of cytisine.

Varenicline has undergone full-scale clinical devel-

opment and has been recently approved by the U.S.

Food and Drug Administration for the treatment of

NIC addiction. The good efficacy of the drug in smok-

ing cessation was demonstrated in 6 clinical studies, in-

cluding 2 comparative trials with bupropion. The CO-

confirmed continuous abstinence rate during the last 4

weeks of the therapy with varenicline 1 mg b.i.d. for 12

weeks was 31–45% and it was significantly better than

for sustained-release bupropion (29–30%) or placebo

(18%) [54, 73, 103, 104]. The continuous abstinence

rate from week 9 through 52 across different studies

was 19–23% vs. 14–16% for bupropion and 4–10% for

placebo [54, 73, 145]. The adverse effects were rela-

tively common but did not result in significantly higher

discontinuation rate than with placebo [54, 104]. Taken

together, the reported results suggest that varenicline

represents a promising alternative to agents currently

used for the therapy of NIC addiction.

SSR591813 (Sanofi-Synthelabo, France) is another

nAChRs ligand chemically and pharmacologically re-

lated to cytisine [34]. In in vitro and in vivo assays,

SSR591813 displayed functionally selective partial

�4�2* agonist activity. Similarly to cytisine, it be-

haved as an agonist with lower efficacy than NIC as

for its capacity to release DA, and as an antagonist in

the presence of NIC. Interestingly, SSR591813 lacked

affinity for ganglionic �3�4* nAChRs and lacked

cardiovascular effects in animal models of NIC de-

pendence. Like varenicline, SSR591813 may have

therapeutic potential in the management of smoking

cessation [34] but it needs to be further assessed in

clinical trials which ultimately will determine the use-

fulness of this compound as a smoking cessation aid.

Conclusions

Cytisine, a natural plant alkaloid, used in CEE for 40

years in the clinical management of smoking cessa-

tion, has been shown to have pharmacological charac-

teristics similar to NIC. Recent advances in cytisine

pharmacological research have elucidated that the

drug is a low efficacy partial agonist of �4�2*

nAChRs. Cytisine binding to �4�2* receptors can at-

tenuate the consequences of both NIC exposure and

its withdrawal. Because of a competitive blockade of

�4�2* receptors, cytisine behaves as an antagonist in

the presence of NIC; it reduces the DA-releasing and

discriminative stimulus effects of NIC. Consequently,

it limits the psychogenic reward from NIC obtained

through smoking, a key component of tobacco depend-

ence. However, once attached to �4�2* nAChRs, its

effect is much weaker than that of NIC. Thus, cytisine

would decrease craving and attenuate NIC withdrawal

symptoms that often precipitate relapses.

Many clinical studies on cytisine as a smoking ces-

sation aid have suggested that the drug is efficacious

and safe; however, these studies do not conform to

modern standards in conducting and reporting drug

trials, and should be interpreted with caution. Our re-

cent uncontrolled trial conducted in 436 smokers con-

firmed the previously reported efficacy of cytisine.

The 12-month CO verified continuous abstinence rate

was similar (13.8%) to that observed following treat-

ment with NRT. In addition to being efficacious,

cytisine seems to be well-tolerated. The most fre-

quently reported adverse effects are gastrointestinal in

origin. The obvious advantage of the drug is its low

cost, which could make it an effective treatment avail-

able to millions of smokers.

Since cytisine exhibits a desirable in vitro and in

vivo profile, it should be advanced to randomized con-

trolled trials. Before that, more information on its phar-

macokinetics and safety profile in humans for dosages

recommended by the manufacturer is required.

Acknowledgments:

We would like to express our thanks to Dr. Carolyn Dresler for her

critical review of the manuscript. We also thank Sopharma and

Professors Rumen Nikolov and Nikolai Danchev for valuable

collaboration. The editorial assistance of Dr. Tomasz Mróz,

Katarzyna Mróz and Magdalena Cedzynska is also acknowledged.

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�� 793


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Received:

October 13, 2006; in revised form November 15, 2006.

798 ��

57Vinnikov, D., Brimkulov, N., & Burjubaeva, A. (Year). A double-blind, randomised, placebo-controlled trial of cytisine forsmoking cessation in medium-dependent workers. Journal of Smoking Cessation, 3(1), 57–62. DOI 10.1375/jsc.3.1.57

Address for correspondence: Denis Vinnikov, Public Association ‘Lung Health’, Togolok Moldo street, 1, Bishkek, Kyrgyzstan. E-mail: [email protected]

In order to prevent lung cancer, chronic obstructive pul-monary disease and other smoking-related illnesses,comprehensive tobacco control should comprise theprocess of successful quitting by current smokers. Self-aid has been shown to have a very little effect, butbehavioural support, along with one of the medicationalternatives, could help up to 30% of quitters abstainfrom smoking at least for the first 6 months (Silagy etal., 2004). Various forms of nicotine-replacementtherapy (NRT) are available, and more than 100 double-blind, randomised controlled trials have been publishedshowing that NRT doubles success rate of quitters (Westet al., 2007). New medications with evident effect havebeen introduced, and their potential for smoking cessa-tion has been demonstrated in large studies.

However, individual counseling along with NRT orvarenicline still remain an expensive choice for peopleleaving in the low-income countries. In a study done by theauthors (unpublished) up to 83% of current smokers saidthey would like to cease smoking within the next 6 months,but 88.5% admitted they would only rely on their

willpower because of very poor awareness and unavailabil-ity of the NRT and varenicline. For a low-income countrythe absolute cost of medication for smoking cessation is animportant matter for the majority of smokers. That is whycytisine has been in use for several years in some countriesof Central Asia. Its 25-day course is 5 to 15 times cheaperthan the 25-day treatment with NRT (Etter, 2006).

This medication is the active substance in ‘Tabex’. Itis a natural extract from the seeds of Cytisus Laburnumtree, and a partial agonist of α 4β 2-nicotinic acetyl-choline receptors, which are responsible for reinforcingeffects of nicotine. Being an agonist of these receptors,it reduces negative withdrawal symptoms of cigaretteabstinence and cravings. Additionally, by preventingthe nicotine binding to these receptors, it decreases itsreinforcing effects. In Bulgaria, this medication hasbeen used for about 40 years, but it has never beenincluded into any recommendations or guidelinesbecause of very few publications describing the use ofthis medication to treat tobacco dependence in English(Karam-Hage et al., 2007).

A Double-Blind, Randomised, Placebo-Controlled Trial of Cytisine for SmokingCessation in Medium-Dependent Workers

Denis Vinnikov,1,2 Nurlan Brimkulov,2 and Aichurek Burjubaeva1

1 Public Association ‘Lung Health’, Bishkek, Kyrgyzstan2 Kyrgyz State Medical Academy, Bishkek, Kyrgyzstan

Among many studies on cytisine only a few have been controlled trials, and the aim of this studywas to assess the efficacy of cytisine in a randomized controlled double-blind trial compared to

placebo in medium-dependent smoking men working in mining industry. Materials and methods: 171middle-aged smokers were randomised to either cytisine (25-days regimen) or placebo; both groupsreceived individual counseling with brochure. Self-reported continuous abstinence was assessed at 8and 26 weeks. Results: At the end of week 8 there were no differences in number of abstinent sub-jects, but at 26 weeks 10.6% of subjects were abstinent in cytisine group compared to 1.2% inplacebo (p = .01). In both groups, we did not find any weight increase, but quality of life improved inboth groups, and physical and social functioning improved in cytisine group. Conclusions: Cytisinemay be an effective medication to help smokers quit even for those working in difficult working condi-tions with high relapse rate.

ARTICLE AVAILABLE ONLINE

Journal of Smoking Cessation

Among many studies on cytisine only a few havebeen controlled, and the majority of these were donelong time ago (Etter et al., 2007). In a recent uncon-trolled trial (Zatonski et al., 2006), authors reported thatof 315 patients, 27.5% had reported they did not smokeat 6 months and 13.8% — at 12 months, verified byexhaled CO. The pooled OR from two double-blind,placebo-controlled trials was 1.83 at 3 and 6 monthscompared with placebo, and in another trial theyreported OR 1.77 at 12 months (Etter, 2006).

The efficacy of this medication is still disputable dueto limitations in the studies undertaken. The otherproblem with smoking cessation treatment is that poordata are available on real-life efficacy of medications andinterventions, because the studies have been done inclinics with more compliant patients and excellent readi-ness to quit. With the current study, we aimed toinvestigate the efficacy of cytisine in a randomised con-trolled double-blind trial compared to placebo inmedium-dependent smoking men working in themining industry.

Materials and MethodsPatients

Participants were drawn from current daily smokerswithout any serious chronic diseases. Subjects were 20years old and older, smoked at least 15 cigarettes a dayduring the year prior to inclusion into the trial, hadclaimed high motivation to quit smoking and readinessto do so immediately and had not had any experience ofcytisine use before.

We recruited all our patients from the staff of themining company in Kyrgyzstan, mainly men, who eitherhad taken part in group cessation programs at the work-place before, but unsuccessfully, or were invited into thistrial as the first attempt to stop smoking. They wererecruited via an advertisement at their workplace, and350 subjects expressed their wish to participate. First,they were screened if they met the inclusion criteria;those who did not were counselled individually for 15minutes, received a targeted brochure on how to quitsmoking and left. Otherwise people were subjected toinitial testing using their smoking history, anthropomet-ric measurements, Fagerstrom test for nicotinedependence (FTND ; Fagerstrom et al., 1991), health-related quality of life (QL), exhaled carbon monoxide(CO) measurement and lung function testing. Data wereverified with exhaled carbon monoxide.

We excluded patients having serious or unstable dis-orders, and the data were checked using their annualscreening data profiles. Usually, they all have to undergoannual screening, chest X-ray, electrocardiogram,cardiac ultrasonography upon indications, lung functiontesting, blood cell count, and blood biochemical assay.Patients contra-indicated for cytisine use were excluded.Those contraindications were ischemic heart disease,

58 JOURNAL OF SMOKING CESSATION

Denis Vinnikov, Nurlan Brimkulov and Aichurek Burjubaeva

severe arrhythmias, severe atherosclerosis, schizophre-nia, tumors, pregnancy and breastfeeding. Patients wereadvised to abstain from the use of other medications, ifno acute or chronic diseases required their use.

Study Design

We planned this study in parallel groups as randomised,double-blind and placebo-controlled. It consisted of 2-weeks screening and counselling of patients, followed byinitial administration of medication, with 6 monthsfollow-up. We included all the requirements of theRussell standard for smoking cessation trials (West et al.,2005), and the study design was approved by the EthicalCommittee of Ministry of Health of Kyrgyz Republicand was done in accordance with Declaration ofHelsinki. All patients signed an informed consent to par-ticipate in this trial in their native language.

Within the screening phase, patients were randomlydivided into two groups. One group received cytisinetablets according to the manufacturer’s instructions: (1)first 3 days smoking should be reduced by half, and eachtablet should be taken every 2 hours (6 tablets a day); (2)days 4–12 — smoking must be discontinued, and eachtablet should be taken every 2.5 hours (5 tablets a day);(3) days 13–16 — each tablet must be taken every 3hours (4 tablets a day); (4) days 17–20 — each tabletmust be taken every 4 hours (3 tablets a day); (5) days21–22 — each tablet must be taken every 6 hours (2tablets a day); and (6) days 23–25 — one tablet a day.The second group was taking placebo tablets using thesame regimen. Randomisation was done using randomi-sation code made by a sided statistical scientist, and thecode was kept with him.

Subjects were instructed not to enrol in any otherconsultations or treatment programs, and stop smokingon the fifth day of cytisine use, and the day of treatmentinception was chosen individually. Participants werescreened at the end of week 8 and week 26. At each visit,we measured continuous abstinence since the quit date,measured their bodyweight, asked about the reasons toresume smoking and side effects of the medication.Then we measured their exhaled CO level with the useof piCO Smokerlyzer (Bedfont, United Kingdom), andasked patients to fill in the SF-8 questionnaire. All thoseprocedures were undertaken by a trained physician.

The primary efficacy measures were continuousabstinence since the smoking discontinuation from day5 to the end of week 8 and from day 5 to end of week 26.That was defined as no cigarettes at all and verified withexhaled CO level. Those patients that reported continu-ous abstinence but had exhaled CO level 9 ppm or morealong with those who missed at least one visit were con-sidered smokers. As secondary efficacy measures we usedexhaled CO and change in health-related QL.

To follow the Russell standard, we defined (1) dura-tion of abstinence 6 months (26 weeks); (2) full

abstinence at examination points. The question we usedwas ‘Have you smoked even a single cigarette during thelast 4/22 weeks since you discontinued the use of themedication?’; (3) biochemical verification was done withexhaled CO monitor; (4) we did intention-to-treatanalysis — patients who did not take medication at allwere excluded from the analysis, and those patients whowithdrew from the trial and moved to an unreachableplace (totally 13 subjects) were excluded from the analy-sis; (5) protocol violators that did not come to follow-upor were abstinent with CO higher or equal to 9 ppmwere considered smokers; (6) follow-up was blind.

Statistical Analysis

Data were processed using Statistica 6.0 (StatSoft) andNCSS 2002. Data are shown as percentages or means ±standard deviation. Within the groups, changes in con-tinuous variables (CO level, number of cigarettes, etc.)were compared using the Wilcoxon test or analysis ofvariance. The primary outcome measures were evaluatedusing a logistic regression analysis.

ResultsOf all 350 patients initially screened, 171 were enrolledand took at least one dose of medication (figure 1).Patients of both groups were similar to each other intheir smoking histories, and there were mostly men par-ticipating, smoking one pack of cigarettes a day onaverage (table 1). Only a quarter of our patients hadused any medication previously to quit smoking, but themajority tried to quit a few times before. Our smokershad medium tobacco dependence.

The rates of continuous abstinence at the end of week8 of treatment did not differ between the groups (p = .38)

59JOURNAL OF SMOKING CESSATION

A Randomised Trial of Cytisine for Smoking Cessation

and generally were low. Nine patients in the cytisinegroup remained abstinent at this time compared with fivepatients in the placebo group (see Table 2). However, bythe end of the 26th week of treatment only one patient inplacebo group was a non-smoker with low exhaled COlevel, whereas those nine abstinent patients in cytisinegroup were still nonsmokers (p = .01).

The main effects logistic regression was used in theanalysis to study the probability of continuous absti-nence at weeks 5 to 26. We analysed the effect of cytisineuse, age, cigarettes smoked, smoking duration, previousattempts to quit, FTND score and exhaled carbonmonoxide, and the frequencies of the variables areshown in Table 3. Previous attempts to stop smokingand the use of medications to stop smoking before hadno association with the abstinence, and other analysedvariables are shown in Table 3. We also analysed ifanswers to FTND questions could have association withabstinence. We found that OR of abstinence for subjectswho find it not difficult to abstain from smoking inplaces where smoking was prohibited was 2.01(0.50–8.07).

In general, we saw a reduction in exhaled CO levelin the entire group from 26.4 ± 10.5 to 20.8 ± 12.0 ppm(p < .001). In the cytisine group, CO reduced from 26.7± 8.7 to 19.3 ± 11.0 ppm (p < .001), but in the placebogroup we did not find a statistically significant lesseningof CO (from 26.1 ± 12.1 to 22.5 ± 12.7 ppm).

Table 1

Baseline Characteristics of Subjects

Cytisine group Placebo group(n = 85) (n = 86)

Characteristics of patients

Male/female 84 (99%) / 1 (1%) 82 (95%) / 4 (5%)

Mean (SD) age, years 38.3 (7.7) 39.4 (9.5)

Smoking status

Mean (SD) cig per day 21.7 (6.9) 21.9 (7.0)

Mean (SD) smoking years 19.8 (7.7) 17.7 (7.5)

Attempted to quit previously, % 87.7 84.0

Mean (SD) number of attempts 3.4 (3.3) 3.2 (3.2)

Used medications to quit, % 27.1 27.3

Mean (SD) FTND score (0–10) 5.3 (1.4) 5.3 (1.7)

Mean (SD) exhaled CO, ppm 26.7 (8.7) 26.1 (12.1)

171 included

Tabex group

N = 85

8 weeks

79 assessed

26 weeks

75 assessed

4 moved away

2 lost to follow-up

5 moved away

2 lost to follow-up

4 lost to follow-up 5 moved away

4 lost to follow-up

Placebo group

N = 86

8 weeks

79 assessed

26 weeks

70 assessed

350 recruited

initial counseling

Figure 1

Trial profile.

Table 2

Continuous Abstinence Rates*During Treatment and Follow-Up

Weeks Cytisine Placebo p

5–8 9 (10.6%) 5 (5.7%) 0.36

5–26 9 (10.6%) 1 (1.2%) 0.01

Note: *No cigarettes at all, verified by exhaled CO.

Treatment with both cytisine and placebo did notchange the subjects’ bodyweight. Initially our patientsweighed 73.4 ± 10.2, and 26 weeks after their weight was73.1 ± 10.2 kg. Cytisine use did not change bodyweight(74.6±10.5 at baseline and 73.8 ± 10.3 kg in the end) asdid placebo use (72.2 ± 9.7 vs. 72.4 ± 10.2 kg). In nineabstinent subjects in cytisine group we did not see anyweight gain — 70.7 ± 6.3 kg at baseline and 69.4 ± 6.4kg at 26 weeks.

All patients were asked about their health-relatedquality of life using eight questions from the general SF-8 questionnaire. Initially, there were no differences in QLbetween those receiving cytisine and those receivingplacebo. Treatment with cytisine led to improvement ofphysical activity (PA), social activity (SA), role physical(RP), mental health (MH), physical pain (PP) andgeneral health (GH) scores (from 47.5 ± 6.3 to 50.2 ±5.0; from 48.2 ± 5.6 to 49.9 ± 4.1; from 49.0 ± 5.7 to 51.6± 5.0; from 48.9 ± 7.3 to 51.5 ± 6.0; from 55.8 ± 5.8 to57.8 ± 5.2 and from 46.0 ± 7.0 to 48.8 ± 6.0 respec-tively). But treatment with placebo also led toimprovement of four scores — MH, VT, PP and GH (seeFigure 2). In verified abstinent subjects of both groups(N = 10), there was a significant improvement of onlytwo scores — RP and GH, and the latter increased from45.5 ± 6.8 to 53.0 ± 5.3.

Eight patients from the entire group experienced sideeffects and stopped using either cytisine or placebobecause of side effects — 4 in the cytisine group (4.7%)and 4 (4.7%) in the placebo group. The most commonadverse effects were dyspepsia (in one cytisine and twoplacebo patients), nausea (in two cytisine and oneplacebo patient), headache in one cytisine and oneplacebo patient, and other effects (in one cytisine andone placebo). There was not enough evidence to relatethese effects to study medication in both groups.

DiscussionOur study showed that cytisine was effective in smokingcessation when delivered as part of individual coun-selling compared to placebo in medium-dependentsmoking men. In this study it increased the cessationrate by 8 times compared to placebo. Meanwhile, the

overall cessation rate for this medication in this studywas low compared with other trials.

In our study we invited smokers who might have beendifferent from the subjects of other studies on cytisine. Westudied the medication with medium- dependent menworking in the mining industry, and not attending spe-cialised clinic for smoking cessation. The workingconditions of these people predisposed them to heaviersmoking in a stressful environment, where relapse wasmore probable. In conditions of heavy physical load inreal life, craving would be the most probable reason forrelapse, as it is in a usual setting (Killen et al., 1997). Themajority of subjects in our study, both on medication andplacebo, explained their relapse as a strong craving forsmoking, which in their working conditions might play agreater role. This may explain why the abstinence rate inthe placebo group was so unexpectedly low — only onepatient, even after the individual counselling, remainedabstinent after 6 months.

This study may shed more light on the real-worldefficacy of cytisine. In the previous studies, the medica-tion doubled the cessation rate, but the placebo grouppatients showed only two times lower abstinence rates,and it was around 10%. In our study we got an OR ofsmoking cessation of 8.9 with cytisine because of verylow cessation rate in placebo group. Apart from the highplacebo relapse rate, the other fact possibly explainingsuch low abstinence in placebo group could be theabsolute prevalence of working men in the study group.In spite of the fact women are generally harder to quit(Bjornson et al., 1995), working men in the miningindustry are still a very difficult subpopulation ofsmokers. This was a limitation of our study, and weassume abstinence rates in the active group could be dif-ferent if more women were included in the group.

Another limitation of this study could be the shorttiming of for the follow-up period. Although theRussell Standard recommends 6 months for a follow-up period, it could be valuable to observe patients after12 months and even longer.

In this trial we measured health-related quality of lifeas an indicator of the impact of smoking cessation orreduction. We used short questionnaire, and we foundthat certain indices changed in both groups. In general,both interventions led to improvement of QL, and thiswas due to counseling offered to subjects of both groups.So even individual counselling was sufficient to improvemental and general health and some other indices. Butonly cytisine group showed significant improvement ofphysical and social activities.

Overall, this study showed that cytisine was an effec-tive medication to help smokers quit, even for thoseworking in difficult working conditions with highrelapse rate. This medication, probably, has less efficacycompared to other medication options, but due to lowcost it may become a medication of choice for low-



Table 3

Logistic Regression Model for the Abstinence Predictors

Variable OR 95% CI

Cytisine use 8.93 1.06–75.28

Age 1.11 0.91–1.37

Weight 1.02 0.96–1.1

Number of cigarettes 1.06 0.89–1.26

Smoking duration 0.95 0.77–1.18

FTND score 1.13 0.60–2.14

Exhaled CO level 0.99 0.91–1.09

income smokers willing to quit. It is well tolerated andcould be a good alternative for medication-aid cessationin countries where the high cost of smoking cessationmedication hampers the wide use of other options.

Acknowledgments

The authors would like to thank Drs Elena Ryzhkovaand Ludmila Erejepova for their tremendous input tothe data collection, along with Drs Hans LeRoux andFrancois DuToit for their persistent contribution intothe study organisation in the company clinic.

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Pope, F. et al. (1995). Gender differences in smoking cessa-

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(2008). Cytisine for smoking cessation: a research agenda.

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Karam-Hage M., & Cinciripini P.M. (2007). Pharmacotherapy

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40

42

44

46

48

50

52

54

56

58

60

PA SA RP MH PP GH

(a)

Figure 2

Change in QL of patients treated with cytisine (a) and placebo (b)Note: PA – physical activity, SA – social activity, RP – role physical, MH – mental health, PP – physical pain, GH – general health, VT – vitality.

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Silagy C., Lancaster T., Stead L., et al. (2004). Nicotine replace-ment therapy for smoking cessation. Cochrane Database ofSystemic Reviews, 4, CD000146.

West R., Hajek P., Stead L., & Stapleton J. (2005). Outcome cri-teria in smoking cessation trials: Proposal for a commonstandard. Addiction, 100, 209–303.

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smoking cessation effective in the ‘real world’? Findings

from a prospective multinational cohort study. Thorax, 62,

998–1002.

Zatonski W., Cedzynska M., Tutka P., & West R. (2006). An

uncontrolled trial of cytisine (Tabex) for smoking cessa-

tion. Tobacco Control, 15, 481–484.



Rationale, pharmacology and clinicalefficacy of partial agonists of a4b2

nACh receptors for smoking cessationHans Rollema1, Jotham W. Coe2, Leslie K. Chambers1, Raymond S. Hurst1,Stephen M. Stahl4 and Kathryn E. Williams3

1Department of Neuroscience Biology, Pfizer Global Research and Development, Groton, CT 06340, USA2Department of Chemistry, Pfizer Global Research and Development, Groton, CT 06340, USA3Department of Clinical Development, Pfizer Global Research and Development, Groton, CT 06340, USA4Department of Psychiatry, University of California at San Diego, 1930 Palomar Point Way, Suite 103 Carlsbad, CA 92008, USA

Most smokers repeatedly fail in their attempts to stopsmoking because of the addictive nature of the nicotinein tobacco products. Nicotine dependence is probablymediated through the activation of multiple subtypesof neuronal nicotinic acetylcholine receptor (nAChR),among which the mesolimbic a4b2 subtype has a pivotalrole. Here, we discuss the rationale for and the design ofa4b2 nAChR partial agonists as novel treatments fortobacco addiction. Such agents are expected to exhibita dual action by sufficiently stimulating a4b2-nAChR-mediated dopamine release to reduce craving whenquitting and by inhibiting nicotine reinforcement whensmoking. Potent and selective a4b2 nAChR partial ago-nists that exhibit dual agonist and antagonist activity inpreclinical models can be identified. The validity of thisapproach is demonstrated by the clinical efficacy ofthe a4b2 nAChR partial agonist varenicline, which hassignificantly better quit rates than do other treatmentsand offers a new option for smoking cessation pharma-cotherapy.

Smoking and tobacco addictionDespite broad awareness of the health risks to individuals,tobacco smoking is the leading cause of preventable mor-tality in industrialized countries [1]. Globally, tobacco-attributable mortality is projected to increase to 6.4 millionper annum in 2015, and account for 10% of all deaths [2].Currently available treatments for nicotine addiction, in-cluding nicotine replacement therapy (NRT) and bupropion(Zyban1), only double the placebo quit rate in clinical trials[3,4], highlighting the unmet need for more-effective thera-pies. Even a marginal improvement in quit rate comparedwith existing therapies will have a significant impact onboth individualwellbeingand thepublichealthburden[5,6].

It is well recognized that smoking is one of the mostdifficult addictions to overcome: a result of the combinationof the reinforcing effects of nicotine, and possibly othersubstances in tobacco, and the strong behavioral com-ponents and environmental cues associated with smoking

[7–9]. Cigarettes are particularly addictive because theyare readily available and are extremely efficient at deliver-ing the neuroactive components of tobacco to the brain. Inessence, cigarettes provide, with each inhalation, indivi-dualized control over the amount and frequency of nicotinethat is delivered to the brain and, thus, over mesolimbicdopamine (DA) neurotransmission – a crucial element ofthe response to addictive substances. The rapid and tran-sient increases in DA release in the nucleus accumbensthat inhaled nicotine produces will initiate and sustaincompulsive substance-seeking behavior and drug depen-dence in humans, as described for other drugs of abuse.Several studies using genetically modified mice in whichthe a4 and/or b2 subunit of the neuronal nicotinic acetyl-choline receptor (nAChR) (Box 1) is deleted provide strongevidence that the nAChR containing these subunits is akey mediator of these nicotinic effects. Consequently, thea4b2 nAChR has become a molecular target for the designof novel smoking-cessation agents, which are the subject ofthis article.

Although the central role of nicotine in tobacco addictionis not in debate, it is important to emphasize that thisaddiction is the result of changes in multiple neurotrans-mitter and receptor systems, coupled with environmentaland behavioral secondary reinforcers that support contin-ued smoking. Despite significant progress in knowledge ofthe dynamics of nAChRs, including receptor activation,desensitization, reactivation and upregulation, the exactrole and complex interactions of these neurobiologicalsubstrates have not yet been satisfactorily unraveled. Acritical evaluation of these underlying mechanisms isbeyond the scope of this discussion (for recent reviews,see Refs [10–13]).

In this article, we focus on the hypothesis that activationof a4b2 nAChRs is central to nicotine dependence and wediscuss the a4b2 nAChR as a drug target, addressing therationale, design and development of partial agonists of thisreceptor subtype as a smoking cessation pharmacotherapy.

Role of a4b2 nAChRs in nicotine dependenceRecent insights into the molecular mechanismsthat underlie nicotine dependence have revealed unique

Opinion TRENDS in Pharmacological Sciences Vol.28 No.7

Corresponding author: Rollema, H. ([email protected]).Available online 18 June 2007.

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opportunities fordevelopingmore-efficacious treatments fortobacco addiction. Upon smoking, inhaled nicotine entersthe brain within seconds, reaching maximal concentrationswithin 2 min [14,15], and acts onnAChRs located onDAandGABA neurons in the mesolimbic system in the ventraltegmental area (VTA) (Figure 1). Nicotine can both activateand desensitize these receptors, depending on the concen-tration and duration of exposure (Box 1). Activation ofnAChRs on mesolimbic DA neurons leads to DA release,whereas the desensitization of nAChRs onGABAneurons isthought to attenuate the GABA-mediated inhibitory drive.In addition, nicotine interacts with nAChRs on glutamateneurons that regulate the activity of DA andGABAneuronsin the VTA. The differential activation and desensitizationof nAChR subtypes on these neurons result in stimulatedDA release in the nucleus accumbens (Figure 1), whichinitiates a physiological response that strongly contributesto the reinforcing effects of nicotine [11,12,16,17].

Although nicotine can interact with several nAChRsubtypes in the mesolimbic pathway, such as a4-, a6-and a7-containing nAChRs [18], there is convincing evi-dence that activation of mesolimbic nAChRs containing a4

and/or b2 subunits – either as a4b2 or more complex (a4b2*)combinations – has a pivotal role in the reinforcing effectsof nicotine. For instance, preclinical studies in transgenicmice have shown that elimination of either the a4 or b2

subunit attenuates the pharmacological and behavioraleffects of nicotine [19,20], and targeted expression of b2

subunits in the VTA of b2-knockout mice reinstatesnicotine-seeking behavior and nicotine-induced DA release[21]. Recent clinical evidence that selective partial agonistsof a4b2 nAChRs are efficacious smoking-cessation agentsprovides further validation of the role of a4b2 nAChRs intobacco addiction.

Between cigarettes, brain nicotine levels graduallydecrease, triggering several processes that contribute tothe cycle of craving and urge to smoke that maintainsnicotinedependence. It isbelieved that the rapidly recurringand transitory increases in mesolimbic DA levels followingrepeated exposure to, and withdrawal from, nicotine trans-mit salient reward and aversive signals to higher corticalcenters, facilitating the learning and associations thatlead to physical dependence, which is characterized byboth somatic and psychoactive symptoms [7,22–24]. This

Box 1. Neuronal nAChRs: activation and desensitization

Neuronal nAChRs are pentameric ligand-gated ion channels as-sembled from five of the 11 different a and b subunits that havebeen identified in mammals. They consist of either five a subunits or acombination of a and b subunits (e.g. a4b2, the predominant subtypeform in the mammalian brain), forming a central ion-conductingchannel. According to the allosteric theory [49,50], these channels canbe open (‘activated’), closed (‘resting’) or desensitized to regulate thepassage of cations across the cell membrane. The transition into andout of the open mode can be monitored at the level of the individualreceptors or populations of receptors (Figure I), using a variety ofelectrophysiology, ion flux or imaging methods.

The proportion of receptors in each mode is determined by theconcentration and intrinsic activity of the agonist and by theduration of agonist binding. In the absence of agonist, the receptorsstrongly favor the closed mode, whereas agonist binding shifts theequilibrium from the closed to the open and desensitized modes. Inthe open mode, cations flow down their respective electrochemicalgradients, depolarize the membrane and elicit an excitatory signal

in neurons. However, agonist-bound receptors prefer the desensi-tized mode; in the continued presence of agonists such as nicotine,an increasingly greater fraction of receptors will become desensi-tized over time. For most ligand-gated ion channels, the binding of afull agonist favors the open mode more strongly than does thebinding of a partial agonist. Thus, at a given level of receptoroccupancy, partial agonists enable fewer ions to cross the cellmembrane, resulting in a smaller excitatory signal and less DArelease than are caused by full agonists (Figure II). It has been wellestablished that long-term exposure to nicotine can modify thefunction and expression of a4b2 nAChRs through diverse mechan-isms, including receptor desensitization, posttranslational modifi-cations and receptor upregulation, all of which could have a role innicotine addiction [51,52]. For instance, prolonged exposure to lowlevels of agonist can desensitize nAChRs, resulting in inhibition ofnAChR function. Consistent with this, it has been shown that, at lowconcentrations, (partial) agonists can act as antagonists at a4b2

nAChRs [35,53,54].

Figure I. Time course of the current evoked by the agonist nicotine (black diamond) in HEK cells expressing ha4b2 nAChRs (patch clamp), with a schematicrepresentation of the corresponding transitions among the three functional modes of the nAChRs.

Opinion TRENDS in Pharmacological Sciences Vol.28 No.7 317

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dynamic array of responses probably contributes to both thereinforcing effects of nicotine and the craving symptomsupon smoking cessation, but it is not known exactly howchanges in transmitter release and receptor dynamics inter-act to cause craving and to suppress craving betweencigarettes and during smoking, respectively.

This article is based on the hypothesis that led to thedevelopment of partial agonists of a4b2 nAChRs as effec-tive smoking-cessation agents: that is, that smokingregulates the rapid increase and decrease in levels ofnicotine in the brain that result in a4b2-nAChR-mediatedneurobiological changes that lead to reinforcement andcraving.

Why a4b2 nAChR partial agonists for smokingcessation?Given the central role of a4b2 nAChRs in the reinforcementand maintenance of nicotine dependence, modulating theactivity of these receptors would be expected to havetherapeutic benefits. Specifically, partial agonists of a4b2

nAChRs that enhance the activity of these receptors suffi-ciently to blunt craving and withdrawal, but withoutassociated abuse potential, are attractive options. Further-more, high-affinity partial agonists of a4b2 nAChRs would

have the additional potential benefit of preventing nicotinefrom activating a4b2 nAChRs, thereby blocking its reinfor-cing effects. These considerations prompted the search forligands that act as partial agonists at the a4b2 subtype ofnAChR as novel treatments for smoking cessation.

By definition, partial agonists act as agonists with asmallermaximal effect at full receptor occupancy than doesthe full agonist (i.e. they have lower intrinsic functionalactivity). Additionally, partial agonists with high bindingaffinity act as antagonists of co-administered agonists andsuppress their effects. By mimicking some of the agonistrewarding effects of nicotine, partial agonists of the a4b2

nAChR will, theoretically, relieve craving and withdrawalwhen quitting. Furthermore, during smoking, the presenceof a partial agonist will reduce reinforcement by diminish-ing the repetitive nicotine-induced phasic DA increasesthat are mediated by a4b2 nAChRs (Figure 2).

In theory, the combination of these effects wouldreduce smoking in the short term and could be expected,over a period of months, to attenuate the salient cuesassociated with nicotine-based positive reinforcement,thus facilitating the extinction of behaviors associatedwith smoking and, ultimately, the achievement of long-term abstinence.

Figure II. Hypothetical effects of full agonist and partial agonist binding on the equilibrium between the closed and open modes of nAChRs. Full agonists are denoted byblack diamonds; partial agonists are denoted by white triangles. Traces on the right correspond to data [35] regarding the effects of nicotine and varenicline on evokedcurrents in vitro (patch clamp in HEK cells) and on DA release (microdialysis in rat nucleus accumbens).

318 Opinion TRENDS in Pharmacological Sciences Vol.28 No.7


Agonist and antagonist combinations: a proof ofconcept for partial agonistsRational approaches to pharmacotherapy for nicotineaddiction would include direct activation of a4b2 nAChRswith an agonist to reduce craving and withdrawal symp-toms when quitting, or blockade of a4b2 nAChRs with anantagonist to reduce reinforcement and reward from smok-ing. The first approach, represented by nicotine replace-ment therapy (NRT), uses nicotine as an agonist in safedelivery forms by eliminating the smoke that causestobacco-related illnesses. NRT is effective [3] at reducingcraving and withdrawal associated with quitting; however,

users are still theoretically able to obtain additionalreinforcement during NRT because of a rapid and steeprise in nicotine peak levels when smoking during treat-ment [14] [Figure 1b(ii)], coupled with the sensory cuesthat further maintain tobacco dependence [8].

The second approach has been exemplified by treatmentwith the nonselective nAChR antagonist mecamylamine,the use of which was one of the earliest suggestions forsmoking cessation pharmacotherapy [25]. Mecamylaminereduced subjective nicotine experiences in a dose-depend-ent manner [26] but produced inconsistent effects, increas-ing cigarette consumption in some studies, and its side

Figure 1. Role of a4b2 nAChRs in nicotine dependence. (a) After inhalation, nicotine is absorbed from the lungs into the arteries and is quickly delivered to the brain, whereit binds to nAChRs located on DA and GABA neurons in the VTA. Differential activation and desensitization of a4b2-containing, and possibly a6- and/or a7-containing,nAChRs results in the stimulation of DA release in the mesolimbic reward system, which initiates and maintains nicotine dependence. Abbreviation: GLU, glutamate.(b) A theoretical schematic representation of the pulsatile characteristics of the response (e.g. DA release) to fluctuating brain levels of nicotine during smoking and betweencigarettes, and hypothetical pharmacological effects of smoking cessation treatments. (i) Cigarette smoking (arrows) produces a rapid increase (smoking) and decrease (notsmoking) in nicotine levels, triggering responses (e.g. changes in DA release) that are thought to maintain the cycle of reward and craving. (ii) NRT provides a stableexposure to nicotine (broken line) that reduces craving (gray area), but cigarette smoking still causes steep increases in nicotine levels and in DA release, maintaining itsreinforcing effect. (iii) The use of partial agonists of a4b2 nAChRs would provide a stable exposure to a compound with a mild nicotinic effect (broken line) – reducingcraving when not smoking (gray area) and preventing the reinforcing effects of smoking by preventing full activation of a4b2 nAChRs by nicotine, thus ‘cutting off’ the peakresponses. This results in comparable effects of a partial agonist in the presence and the absence of nicotine (‘stabilization’ of mesolimbic DA release).



effects compromised compliance [27]. Rose and Levin [28]proposed a combination of these two approaches using theco-administration of nicotine andmecamylamine, hypothe-sizing that an agonist–antagonist mixture, when interact-ing in an appropriate ratio at a4b2 nAChRs, would not onlyprevent withdrawal symptoms but also attenuate thereinforcing effects of nicotine. In initial small clinical trialsusing transdermal nicotine and oral mecamylamine, thiscombination achieved higher abstinence rates than didtransdermal nicotine alone [29], providing conceptual vali-dation of the mixed agonist–antagonist approach. How-ever, the major practical challenge of maintaining anarrow agonist:antagonist ratio while administering twoseparate compounds with different pharmacokinetic andmetabolic profiles remained. Nevertheless, this exper-iment defined the clear therapeutic objective of delivering

dual agonist–antagonist a4b2-selective action for smokingcessation. Could this be better achieved by combining theseproperties into a single molecule (i.e. an a4b2-nAChRpartial agonist) [Figure 1b(iii)]? Such an approach hasthe potential to provide an optimal ratio of agonist andantagonist, avoid the pitfalls associatedwithmanaging theexposure of two separate, nonselective compounds andachieve an optimized balance of partial agonist activityand duration of action.

Design of a4b2 nAChR partial agonists: clues fromnatureCytisine (Figure 3), which is a plant alkaloid that has beenused for >40 years in Eastern Europe as a smoking cessa-tion agent (see later), provided early support for the partialagonist theory. In 1994, evidence revealed cytisine to be a

Figure 2. Theoretical representation of the differences in functional efficacy of a full agonist (nicotine) and a weak or potent partial agonist, alone and combined withnicotine. (a) Partial agonists have a smaller maximal effect than does the agonist nicotine (i.e. increasing their concentration or exposure does not further increase theireffect). When not smoking, the partial agonist has a mild nicotine-like effect and can relieve craving and withdrawal, dependent on the relative functional efficacy versusnicotine, the receptor-binding affinity and free levels in the brain. (b) To exert an antagonist effect (i.e. to block the reinforcing effect of nicotine when smoking), a partialagonist should have high binding affinity and sufficiently free levels in the brain (Ceff) to inhibit the effect of nicotine completely, resulting in the same maximal effect as thepartial agonist alone. Partial agonists with poor binding affinity and/or low brain penetration have insufficient Ceff to be efficacious as an antagonist in vivo.



partial agonist of nAChRs [30], providing a rationale for itsreported efficacy. Direct chemical modifications of cytisinedid not lead to viable drug candidates (Figure 3). Althoughentirely synthetic variations of the cytisine [3.3.1]-bicyclicframework generated a series of compounds with improvedbrain penetration and longer half-life, these compoundsexhibited mostly antagonist behavior [31] (Figure 3). Asearch for novel nicotinic structural motifs with partialagonist activity uncovered the related [3.2.1]-bicyclic ben-zazapine (Figure 3). Although it was reported in the 1970sto lack opioid activity [32], [3.2.1]-bicyclic benzazapine wasfound to be an a4b2 nAChR antagonist [33]. Further stu-dies showed that, by merging structural elements of othernicotinic agents, partial agonist profiles could bemodified –ultimately leading to varenicline (Figure 3), the first par-tial nicotinic agonist approved as a therapeutic aid tosmoking cessation [34,35].

Another nicotinic partial agonist, dianicline(Sanofi-Aventis: SSR591813) (Figure 3), shares severalpharmacological properties with varenicline but has loweraffinity at the a4b2 nAChR [36]. Only limited clinical dataare available for dianicline, which is in Phase III develop-ment (see later).

The discovery and neuropharmacology of a4b2

nAChR partial agonistsAlthough the pharmacological characteristics of partialagonists are easily formulated, the actual identification ofa partial agonist with the desired profile presents a formid-able challenge. The characterization of nAChR partial ago-nists became more practical only in the 1990s, with theapplication of functional in vitro assays – most notablypatch clamp and fluorescence imaging plate reader (FLIPR)methodology – to ion channels expressed in oocytes ormammalian cell lines. This enabled the evaluation of thewhole spectrum of functional efficacies of partial agonists.Screening strategies to identify compoundswith the desired

properties of a4b2 nAChR partial agonists have beendescribed [33–36]. Figure 4 summarizes a typical screeningstrategy – including receptor binding, electrophysiology,and in vitro and in vivo DA-release assays – before thecompound is tested in animal models that can predict ef-ficacy for smoking cessation (Box 2). Inmost of these assays,exemplified by data for varenicline (Figure 4), the effectsof the partial agonist alone and in combination withnicotine are compared with the effects of nicotine alone.This provides a measurement of the partial agonist activityof a compound per se and of its antagonist activity inthe presence of nicotine because potent partial agonistshave comparable effects in the absence and the presenceof nicotine.

The ideal partial agonist has a high binding affinity andachieves high free levels in the brain because theseparameters determine whether sufficient levels of DAare released to relieve craving and whether the partialagonist can act as an antagonist of nicotine. With peakbrain levels of !300 nM [15] and a Ki for a4b2 nAChRs of!2 nM [35], nicotine can readily displace compounds witheither poor binding affinity or low brain concentrations.The free levels of varenicline in the brain (equivalent to itsfree plasma levels of !30 nM) [37] and its high a4b2-nAChR-binding affinity Ki of 0.1 nM [35] are sufficient toprevent nicotine levels in smokers from fully activatinga4b2 nAChRs. However, high-affinity compoundswith poorbrain penetration, such as cytisine, or low-affinity com-pounds such as dianicline [36] might be less effective atcompeting with nicotine for a4b2 nAChRs and could beexpected to display only agonist activity (i.e. efficacy com-parable to NRT), without sufficiently potent antagonistactivity. Finally, no clinical data are available regardingthe optimal agonist:antagonist ratio for smoking cessationtreatment but, in our search for partial agonists, weassumed that 30–70% efficacy relative to nicotine wouldprovide an optimal range. We reasoned that compounds

Figure 3. Pathway from cytisine (R=H) and morphine substructures to varenicline. Chemical modifications of cytisine did not lead to viable drug candidates (i.e. R6"H), butcombining knowledge from morphine substructural studies with structural elements of other nicotinic agents led to partial agonist profiles and, ultimately, to varenicline[31–34]. Dianicline is a structurally related a4b2 nAChR partial agonist in Phase III [36].



with higher intrinsic activity could produce dependenceliability comparable to that of nicotine – although theirbrain penetration ratesmight be amore critical factor thanrelative efficacy – and that compounds with low intrinsicactivity would act as antagonists and could precipitatewithdrawal without relieving craving.

Clinical efficacy: do partial agonists of a4b2 nAChRswork?As noted, the potential benefits of dual agonist andantagonist action, as proposed by Rose and Levin [28],were originally examined by simultaneous nAChR agonist(NRT) and antagonist (mecamylamine) administration

Figure 4. Screening strategy for partial agonists of nAChRs. Corresponding varenicline data [34,35] are shown as an example.



[38,39]. Two small studies showed that mecamylamineaugmented the efficacy of NRT; however, there are nopublished data from larger studies confirming these results,and the combination has not yet become an established

treatment for smoking cessation [29]. Interestingly, theantidepressant bupropion, which is an accepted first-linepharmacotherapy for smoking cessation [40], has alsorecently been shown to have antagonist activity at thea4b2 nAChR [41,42].

A recently published systematic review of clinical datafor partial agonists of nicotinic receptors in smoking cessa-tion [43] included cytisine and varenicline. A third a4b2

nAChR partial agonist, dianicline [36], is – like varenicline– the product of rational drug design for smoking cessation.Preliminary Phase II data indicated significantly higherend-of-treatment quit rates than those seen with placebo:19% and 16% for 80 mg and 40 mg, respectively, comparedwith 8% for placebo [44]. Phase III clinical trials are inprogress.

Cytisine is available in Eastern Europe in 1.5-mgtablets, under the trade name Tabex1 (Sopharma, Sofia,Bulgaria). Recommended dosing begins with six tabletsdaily, taken every 2 h, tapering to 1–2 tablets daily over 25days (http://www.tabex.net/41814_packageinsert.phtml).Although most of the clinical trials conducted between1967 and 1987 do not conform to current standards ofstudy design and conduct for smoking cessation trials,the data collectively indicate that cytisine is effective forsmoking cessation [45]. In pooled data from three random-ized placebo-controlled studies (one with long-term follow-up), self-reported responder rates are approximately twicethose occurring with placebo (Table 1), which is similar tothe treatment effect achieved with NRT [3]. It is possiblethat the efficacy of cytisine is limited by poor brain penetra-tion, as discussed earlier.

Varenicline (Pfizer: CP526555) was taken into fullclinical development because it combined high bindingaffinity at a4b2 nAChRs and 40–60% partial agonistactivity with good oral bioavailability and predictablepharmacokinetics (highly absorbed, low plasma proteinbinding, almost completely renally excreted as unchangedvarenicline, with an elimination half-life of !24 h [37,46]).Varenicline, as CHANTIXTM, was approved by regulatoryagencies as an aid to smoking cessation treatment in theUSA in May 2006 and, subsequently, as CHAMPIXTM inthe EU; it has now been approved for marketing in >40countries. Efficacy at smoking cessation has been demon-strated in four clinical trials that followed post-treatment

Box 2. Predicting smoking cessation efficacy with animal

models

Animalmodels used in nicotine research [55] can identify compoundsthat: (i) animals perceive as being nicotine like; (ii) decrease nicotineself-administration; (iii) are significantly less reinforcing than nicotine;and (iv) do not produce withdrawal symptoms upon discontinuation.(i) Drug discrimination assesses the ability of an animal to detect

the subjective effects of centrally acting drugs (i.e. whether atest compound feels more like saline or more like a referenceagent, in this case nicotine). Because this procedure does notallow ‘in-between’ responses, partial agonists from variousdrug classes can fully substitute for the reference compound, ashas been shown for varenicline [35].

(ii) Nicotine self-administration procedures are good translationalmodels for the reinforcing effects of tobacco [56–58]. Intake thatis essentially unrestricted, similar to that of a smoker, can beobserved when nicotine is obtained after a fixed number oflever presses, enabling animals to establish their own patternsof intake. Acute pretreatment with nicotine agonists (nicotine),antagonists (mecamylamine and erysodine) and partial ago-nists (varenicline and dianicline) decreases nicotine self-admin-istration by !50% – similar to the effect of substituting saline fornicotine [35,36,59].

(iii) The reinforcing properties of a compound are assessed byrequiring an increasing number of lever presses (‘raised costs’)to obtain each subsequent nicotine injection; in this paradigm,the reinforcement strength is reflected by the effects ofsubstituting a drug for nicotine. For instance, the substitutionof increasing doses of varenicline significantly increasedresponding at only one dose, compared with saline substitu-tion, in contrast to three doses of nicotine [35]. It is important tonote that acute saline substitution for nicotine causes only a50% reduction in responding, which indicates that some self-administration is maintained by secondary reinforcementsassociated with the self-administration session, such as signal-ing lights and pump noise (perhaps analogous to the feel of acigarette in the hand and other contextual cues associated withsmoking). It takes several sessions of saline substitution foranimals trained to self-administer nicotine to cease lever-pressing behavior completely, illustrating the complex natureof the primary and secondary reinforcing effects of nicotine [60].

(iv) Withdrawal symptoms can be assessed by the sudden dis-continuation of chronic drug administration and by notingwithdrawal signs, either through observations or throughmonitoring disruptions in operant behavior.

Table 1. Smoking cessation efficacy of a4b2 nAChR partial agonists in randomized controlled clinical trialsa

a4b2 nAChR partial agonist(number of trialsb)

Odds ratio(95% CI)vs placebo

Criterion Refs

End-of-treatment smoking cessationVarenicline (2) 3.69 (2.88, 4.72) CO-confirmed continuous abstinence

during the last four weeks of the 12-weektreatment period

c

Cytisine (3) 1.93 (1.21, 3.06) Point prevalence of self-reportedabstinence at 3–8 weeks

[45]

Long-term abstinenceVarenicline (4) 3.22 (2.43, 4.27) CO-confirmed continuous abstinence in

weeks 9–52[43]

Cytisine (1) 1.77 (1.30, 2.40) Point prevalence of self-reportedabstinence at two years

[43]

aAbbreviations: CI, confidence interval; CO, carbon monoxide.bNumber of clinical trials contributing data for meta-analysis.cGonzales, D. et al. (2007) A pooled-analysis of varenicline, an a4b2 nicotinic receptor partial agonist, vs bupropion, and placebo, for smoking cessation. Oral presentation atthe 12th Annual Meeting of the Society for Research on Nicotine and Tobacco, Orlando, February 2006.



http://www.tabex.net/41814_packageinsert.phtml

smoking status to the end of one year [43]. Two Phase IIItrials compared 12-week treatment with varenicline (1 mgtwice daily), 150 mg of sustained-release bupropion twicedaily or placebo [47,48]. For varenicline, rates of end-of-treatment smoking cessation [measured as carbon monox-ide (CO)-confirmed continuous abstinence during the lastfour weeks of the treatment period] and long-term absti-nence (measured as continued abstinence from the last fourweeks of treatment to the end of one year) were approxi-mately three times those occurring with placebo (Table 1)and 1.5–1.9 times those caused by bupropion [47,48]. Com-pared with placebo, varenicline significantly reduced crav-ing (urge to smoke) and negative effects associated withnicotine withdrawal. Varenicline also reduced smokingsatisfaction, psychological reward and enjoyment of respir-atory-tract sensations, as reported by individuals whosmoked during treatment [47,48].

Concluding remarksTreatment of tobacco addiction with partial agonists ofa4b2

nAChRs offers a novel and well-validated pharmacothera-peutic approach. Mechanistically, these agents target thereceptors that are believed tomediate the reinforcing effectsof nicotine and, to some extent, mimic the agonist effects ofnicotine sufficiently to reduce craving when quitting.Furthermore, partial agonists with sufficiently high recep-tor-binding affinity and free concentration in the brain willact asantagonists in thepresenceofnicotine,attenuating itsreinforcing effects when smoking. Treatment of tobaccoaddiction with partial agonists builds on early experimentswith agonist–antagonist combinations and on historicalanecdotal data regarding the moderate effectiveness ofthe alkaloid cytisine. This approach has conferred robustclinical efficacy in the case of varenicline, which is nowmarketed as an aid to smoking cessation treatment. Thisnovel treatment option is a welcome addition to availablepharmacotherapies of nicotine dependence, providing im-provement in abstinence rates that should provide a sub-stantial public and individual health benefit.

Disclosure statementVarenicline is a product of Pfizer and is marketed as CHANTIXTM (USA)and CHAMPIXTM (Europe and elsewhere). H.R., J.W.C., L.K.C., R.S.H.and K.E.W. are employees of Pfizer and own Pfizer stock. S.M.S. hasreceived grants from and is a consultant for several pharmaceuticalcompanies, including Pfizer, for which he is on the speakers’ bureau.

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47 Gonzales, D. et al. (2006) Varenicline, an a4b2 nicotinic acetylcholinereceptor partial agonist, vs sustained-release bupropion and placebo

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48 Jorenby, D.E. et al. (2006) Efficacy of varenicline, an a4b2 nicotinicacetylcholine receptor partial agonist, vs placebo or sustained-releasebupropion for smoking cessation. A randomized controlled trial. J. Am.Med. Assoc. 296, 56–63

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59 Mansbach, R.S. et al. (2000) Effects of the competitive nicotinicantagonist erysodine on behavior occasioned or maintained bynicotine: comparison with mecamylamine. Psychopharmacology(Berl.) 148, 234–242

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Pharmacokineticsofcytisine,anα4β2nicotinicreceptorpartialagonist,inhealthysmokersfollowingasingledose

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Pharmacokinetics of cytisine, an α4β2 nicotinicreceptor partial agonist, in healthy smokersfollowing a single doseSoo Hee Jeong,a* David Newcombe,b Janie Sheridanc and Malcolm Tinglea

Cytisine, an α4β2 nicotinic receptor partial agonist, is a plant alkaloid that is commercially extracted for use as a smoking cessationmedication. Despite its long history of use, there is very little understanding of the pharmacokinetics of cytisine. To date, no pre-vious studies have reported cytisine concentrations in humans following its use as a smoking cessation agent. A high performanceliquid chromatography-ultraviolet (HPLC-UV) method was developed and validated for analysis of Tabex® and nicotine-free oralstrips, two commercial products containing cytisine. A sensitive liquid chromatography-mass spectrometry (LC-MS) method wasdeveloped and validated for the quantification of cytisine in human plasma and for the detection of cytisine in urine. Single-dosepharmacokinetics of cytisine was studied in healthy smokers. Subjects received a single 3mg oral dose administration of cytisine.Cytisine was detected in all plasma samples collected after administration, including 15min post-dose and at 24h. Cytisine wasrenally excreted and detected as an unchanged drug. No metabolites were detected in plasma or urine collected in the study.No adverse reactions were reported. Copyright © 2014 John Wiley & Sons, Ltd.

Keywords: cytisine; α4β2 nicotinic partial agonist; pharmacokinetics; LC-MS; human plasma

Introduction

Smoking is a leading cause of many diseases including cancer, car-diovascular disease, and respiratory disease.[1,2] Smoking cessationimproves health risks and survival,[3] but only 3 to 5% of smokerswho try to quit smoking without the use of medication succeed,as determined by abstinence at six months.[4]

Cytisine, an alkaloid found in plants such as Golden Rain (Cytisuslaburnum), is an α4β2 nicotinic receptor partial agonist that has beenused for smoking cessation since the 1960s.[5,6] Cytisine has beenwidely used in many countries in Central and Eastern Europe andCentral Asia,[7] but unlike its pharmacologically related analoguevarenicline, cytisine has not been approved for use as a smokingcessation medication in many countries including the USA, theUK, Australia and New Zealand. A systematic review and meta-analysis of clinical studies with cytisine suggests that cytisine iseffective for smoking cessation.[8] There is growing interest ingetting cytisine approved as a smoking cessation agent because ofits potential to be a much more affordable medication for smokingcessation than any other current pharmacotherapy available.[8–11]

In an economic evaluation that compared cytisine to varenicline forsmoking cessation, cytisine was estimated to be more cost-effectivethan varenicline.[12] Despite its long history of use, significant gapsremain in our knowledge of cytisine. Animal pharmacokinetic dataexist[13,14] but we have found no publicly available informationabout the pharmacokinetics/metabolism of cytisine in humans.

Cytisine is commercially available as an oral tablet marketed bySopharma, a Bulgarian pharmaceutical company, under its tradename Tabex®. The standard dosing schedule of Tabex® is complexand the rationale is not well understood. One Tabex® tablet con-tains 1.5mg of cytisine and it is recommended that a person takes1 tablet every 2h (maximum of 6 tablets per day) as a starting dose

for the first 3 days of the treatment. The dosing frequency and inter-val are changed throughout the course of the treatment. The doseis reduced to 5 tablets per day on days 4–12 (1 tablet every 2.5 h),4 tablets per day on days 13–16 (1 tablet every 3 h) and 3 tabletsper day on days 17–20 (1 tablet every 5 h). During the last 5 daysof treatment (days 21–25) the recommended dose is 1 or 2 tabletsevery 6 h (maximum of 2 tablets per day). Recently, another com-mercial product of cytisine became available in the form of an oralstrip. This has been marketed in Australia and each strip is reportedto contain 1mg of cytisine (instead of 1.5mg as in tablets). Thisproduct has a similar dosing schedule to Tabex®.

Several methods for the determination of cytisine have been re-ported in the literature, including methods that have not been vali-dated in human tissues[14–16] and methods that have been shownto be applicable for herbal intoxication or drug abuse cases[17–19];however, human PK analysis has not been conducted. No methodshave yet studied the commercial forms of cytisine and no methodhas quantified cytisine in clinical samples following administrationof these products.

* Correspondence to: Soo Hee Jeong, University of Auckland, Private Bag 92019Auckland 1142 New Zealand.E-mail: [email protected]

a University of Auckland, Pharmacology & Clinical Pharmacology, Auckland,New Zealand

b University of Auckland, School of Population Health, Auckland, New Zealand

c University of Auckland, School of Pharmacy, Auckland, New Zealand

Drug Test. Analysis (2014) Copyright © 2014 John Wiley & Sons, Ltd.

Research articleDrug Testing

and Analysis

Received: 24 June 2014 Revised: 29 July 2014 Accepted: 30 July 2014 Published online in Wiley Online Library

(www.drugtestinganalysis.com) DOI 10.1002/dta.1707

This paper has two overarching aims:

A. To describe the development and validation of an analytical assayfor cytisine and its use in the analysis of two commercially availableproducts Tabex® tablets and Quit4Good Nicotine Free Oral Strips.

Plasma and urine samples taken from a healthy subject who hadtaken Tabex® were analyzed to determine whether this methodwas sensitive enough to detect levels of cytisine following oraladministration. For simplification, a single dose was chosen overthe recommended split dosing. Initially, a high performance liquidchromatography-ultraviolet (HPLC-UV) assay was developed andvalidated for analysis of the two commercial products of cytisine.However, the sensitivity of this method was insufficient (not fit forpurpose) to detect cytisine in human plasma after the administereddose. Therefore, it was necessary to develop and validate a moresensitive method using high performance liquid chromatographycoupled with mass spectrometry (LC-MS) that could be used toinvestigate pharmacokinetics of cytisine in subsequent human studies.

B. To describe the pharmacokinetic characteristics of cytisine inhealthy smokers after a single dose, over a 24 h period.

The objectives of the pharmacokinetic study were:

1. To measure the concentrations of cytisine in plasma over a 24 hperiod, to determine clearance, volume of distribution andhalf-life of cytisine in humans, and to screen for the presenceof metabolite(s) in human plasma and urine in healthysmokers following a 3mg single oral dose administration.

2. To measure cytisine’s effect on heart rate, blood pressure andbreathing rate over 24h following a 3mg single doseadministration.

Methods – development of analytical assays

Chemicals and reagents

Cytisine (≥99% purity) and sulfanilamide (internal standard, IS)(p-aminobenzenesulfonamide, ≥99% purity) were purchased fromSigma Aldrich (Auckland, New Zealand). Methanol (>99%, HPLCgrade, Sigma Aldrich) was used in sample preparation. LC-gradewater (Millipore®, Milli-Q system) and methanol (>99%, HPLCgrade, Sigma Aldrich) were used for the mobile phase in theHPLC-UV method and LC-grade water and acetonitrile (ACN,>99%, HPLC-grade, Sigma Aldrich) were used for the mobile phasein the LC-MS method.

HPLC-UV

Standard solutions

Cytisine stock solutions for calibration standards were prepared indimethyl sulfoxide (DMSO). Stock solution was further diluted inDMSO to give appropriate working solutions. The IS solution wasprepared in DMSO at a concentration 400μM.Chromatographic separation was achieved on a Phenomenex

Gemini C18 HPLC column (4.6mm×150mm, 5μm) with a guardcolumn (C18, 4.6× 10mm, 5μm). Mobile phase consisted ofmethanol and 50mM ammonium acetate buffer adjusted topH6.5 (1 to 5% methanol gradient; flow rate 1.0mL/min, pressure120bar), with UV monitoring of the column effluent. Wavelengthsfrom 220 to 310nm were monitored and quantification wasperformed at absorbance of 310 nm (cytisine) and 280 nm(sulfanilamide). Signals areas were obtained from chromatograms

usingmanual integration. Cytisine/IS peak area ratio was calculatedas a quantitative measure to prepare calibration curves.

The assay was validated in accordance to the US FDA guidelinesfor bioanalytical methods validation over the range of 130 to4150ng on column for selectivity/specificity, precision and accu-racy and linearity.[20]

Tablet cytisine (external QC)

Ten cytisine tablets (Tabex® 1.5mg film-coated tablets, Sopharma)were obtained by Sopharma Pharmaceuticals, Sofia, Bulgaria.Tablets from the same batch (Batch number 10211) were crushed,weighed and four tubes containing an equivalent weight of onetablet were prepared. To each tube, 1mL of DMSO was added.The tubes were then vortex-mixed for 120 s, left to stand for20 min at room temperature then centrifuged at 22 000g for5min. The supernatant was transferred to a fresh tube, mixed withinternal standard and vortex-mixed for 120 s. An aliquot (10μL) wasthen injected onto the HPLC column to determine the amount ofcytisine on the column and to calculate the amount of cytisinepresent in one tablet. The value was then compared to the QualityCertificate (Sopharma) documentation (analytical certificate No.324/ 17.03.2011).

Oral strip cytisine (external QC)

Five cytisine oral strips were obtained from an Australianmarketer –Quit4Good (www.quit4good.com.au). Each strip was dissolved in1mL DMSO then processed as described above.

LC-MS

Standard solutions

Cytisine stock solutions for calibration standards were prepared inACN:formate buffer (20:80, v/v). Stock solution was further dilutedin ACN:formate buffer (20:80, v/v) to give appropriate working solu-tions. The stock solution of the IS was prepared inmethanol and theworking solution was prepared at a concentration 400μM in meth-anol. Standard samples for the calibration curve of cytisine wereprepared in human plasma. The final concentrations of cytisine instandard plasma samples were 1.5, 3, 6, 12, 24, 48, 95, 190, 380,760, and 1522ng/mL. Plasma samples used for calibration werestored in �80°C until analysis.

Chromatographic separation

Chromatography was performed using an Agilent 1100 liquid chro-matography (LC) system coupled with an Agilent MSD model Dsingle stage quadrupole mass spectrum (MS) detector. AgilentChemStation software (Version B.04.03-SP2) (Agilent Technologies,Goettingen, Germany) was used to access processed data andchromatograms. Chromatographic separation was achieved on aPhenomenex Gemini C18 HPLC column (4.6mm×150mm, 5μm)with a guard column (C18, 4.6 × 10mm, 5μm). A mobile phase of50mM ammonium formate buffer, pH4.5 (solvent A) and acetoni-trile (solvent B) with a phase gradient 1% (B) from 0 to 3min, 10%from 3 to 9min and 1% at 10min was used for separation. MS de-tection using electrospray ionization (ESI) was performed. Detectionby selective ion monitoring (SIM) (positive ion mode) for each massion was used: m/z 191.2 and 173.2 for cytisine and IS, respectively.Drying gas flow was 12.0 L/min and the nebulizer pressure was35 psig. The total run time was 10min with a flow rate of 0.5mL/minand sample injection size was 15 μL. Areas of signals wereobtained from chromatograms using manual integration.

S. H. Jeong et al.

Drug Testing

and Analysis

wileyonlinelibrary.com/journal/dta Copyright © 2014 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)

http://www.quit4good.com.au

Validation procedures

The assay was validated in human plasma in accordance with theUS FDA guidelines on bioanalytical method over the range of 2.97to 3043.84 pg on column for selectivity/specificity, precisionaccuracy and linearity.[20]

Selectivity/specificity was examined by using blank plasmasamples collected from seven different individuals to look for anyendogenous peaks that could interfere with peaks for cytisineand IS. Intra- and inter-day accuracy and precision was evaluatedby analyzing quality control (QC) samples at four concentrationlevels, low QC, two mid-QCs, and high QC (1.5, 24, 48,1522 ng/mL). Five replicates were evaluated per concentration. Ac-curacy was calculated by comparing the measured concentrationwith the true concentrations spiked in plasma. Precision, expressedas relative standard deviations (RSD, %), was calculated on threeseparate days. Calibration standards of 10 concentrations in therange 1.5 to 1522ng/mL were prepared in human plasma and an-alyzed (n=3) in three separate analytical runs. Calibration curves in-cluded a blank sample (no IS), a zero sample (plasma spikedwith IS)and 10 non-zero samples including the limit of quantification(LOQ). LOQ was evaluated based on signal to noise ratio of 5:1 withprecision and accuracy within 20% of the nominal value. Linearitywas assessed by preparing calibration curves plotting the peak arearatios of cytisine to IS against the concentrations of cytisine.

Absolute recovery was assessed by comparing the peak areas ofcytisine obtained from extracted spiked plasma standards withpeak areas from un-extracted standards in ACN:formate buffer(20:80%, v/v).

Short-term temperature stability of cytisine in plasma was exam-ined by using QC samples (n=3) and comparing freshly spikedplasma samples to the same samples left at room temperature for24 h. Freeze-thaw stability was also studied by comparing freshlyprepared spiked plasma samples to the same samples that werekept at of �80°C for 24 h and thawed at room temperature andsamples that underwent three freeze-thaw cycles.

For urine, selectivity/specificity was examined by using blankurine samples collected from six different individuals to look forany endogenous peaks that could interfere with peaks for cytisine.LOQ was evaluated with precision and accuracy within 20% of thenominal value.

Sample handling and preparation

Blood samples were allowed to stand at room temperature for30min prior to centrifugation at 3000g for 10min to separate theplasma and red blood cell fractions. Plasma samples were storedfrozen at �80°C until analysis. Aliquots (100μL) of plasma sampleswere thawed at room temperature and IS (10μL of 400μM) wasadded along with ice-cold methanol (2:1 v/v). Samples were vortexmixed for 120 s and left overnight at �20°C to precipitate protein.Samples were then centrifuged (15min at 22000g) and 200μL ofthe clear supernatant was removed and evaporated to dryness(SC210A SpeedVac® Plus, medium drying rate). The dry extractwas then reconstituted with 30μL ACN:formate buffer (20:80, v/v),centrifuged (5min at 22 000g) and 15μL of the final extract wasinjected onto column.

Urine samples were stored at �20°C until analysis. Prior toprocessing, the samples were thawed at room temperature.0.5 mL of urine sample was taken, IS was added and diluted(1:1, v/v) with MilliQ® water. Samples were vortex mixed andcentrifuged for 10min at 22000g. Solid-phase extraction (SPE)column (Alltech Prevail C18) was conditionedwith 0.5mLmethanol

then equilibrated with 0.5mL MilliQ® water under vacuum. Afterloading the sample, the column was washed with 0.5mL ofmethanol:water (5:95, v/v) and dried under full vacuum for 10mins.Methanol (0.5mL) was used to elute the compounds of interest andthe methanol eluate was collected and 15μL was injected.

Method – clinical application

Pilot testing for sensitivity

Initially, one healthy subject took a single 3mg oral dose (twoTabex®, film-coated 1.5mg oral tablets) to investigate whetherthe assay could be used to quantify drug concentrations inblood plasma. Whilst this involved a higher dose (double the rec-ommended dose taken at one time) it was selected to increasethe chance of detecting cytisine in human tissue using an analyt-ical method described in this paper. Blood samples (6mL) werecollected in heparinised tubes immediately prior to dosing(t=0), and then at 2 h post-dose. Urine was also collected up to390min after dosing. Samples were processed and analyzed asdescribed above. The developed LC-MS assay was then used tostudy single dose pharmacokinetics of cytisine in seven healthyparticipants who were smokers at the time, following a single3mg oral dose administration.

Single-dose pharmacokinetics study

Participants

Seven healthy subjects (aged between 20 and 39 years) took partin the study. Subjects were eligible if they were 18 years or olderand were current cigarette smokers at the time of the study(confirmed by saliva cotinine with NicAlert® – a commercialsemi-quantitative assay that measures cotinine). On average,subjects smoked 10.6 cigarettes per day. Two subjects hadpreviously tried to quit smoking. Subjects were excluded fromthe study if they self-reported being pregnant or breastfeeding,suffering from cardiovascular problems, having been diagnosedwith schizophrenia or were currently using nicotine replacementproducts or non-nicotine based medications to aid them quitsmoking. Tests were undertaken to exclude severe renal impair-ment. There was no restriction on diet or smoking during thestudy. Participant demographics were collected and an assess-ment of nicotine dependence using Fagerström Test for NicotineDependence (FTND)[21] was undertaken.

Study drug

Subjects received a single 3mg dose of cytisine given as an oraltablet form (Tabex®, Sopharma Pharmaceuticals, Sofia, Bulgaria).

Sample collection and analysis

Ten blood samples were collected in heparinised tubes at the fol-lowing times from each subject: 0 (just before dosing), 0.25, 0.5, 1,2, 3, 4, 6, 8 and 24h after cytisine administration. Blood sampleswere processed to obtain plasma and stored at�80°C until analysis.After providing a blood sample at 8 h, subjects went home andreturned the nextmorning 24h post-dosing. Subjects also providedspot urine samples. Plasma and urine samples were processed andanalyzed as described above.

Pharmacokinetics of cytisine in healthy smokers following a single dose

Drug Testing

and Analysis

Drug Test. Analysis (2014) Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta

Estimation of PK parameters

Non-linear mixed effects modelling (NONMEM) was used formodelling and estimating population pharmacokinetic parameters(CL, Vd). A single compartment model was fit to the data.

Other measurements

Blood pressure, heart rate and respiratory rate were measured at 0(just before dosing), 2, 4, 8, and 24h after drug administration.

Ethical approval and consent

All subjects provided a written consent before taking part in thestudy. Ethical approval to carry out this study was granted by theNorthern X Regional Ethics Committee of NZ (NTX/11/05/038).

Results

HPLC-UV method

Linearity was verified for this method by visual inspection and usingvalues for coefficients of determinations (R2) obtained fromnine lin-ear standard curves generated on three separate days. Correlationcoefficients (R2) of calibration curves were all above 0.99. Intra-dayand inter-day accuracy and precision values in the range 130 to4150ng on column were less than 15% of the actual values.

Cytisine tablets and oral strips

From the calibration curve generated, cytisine concentration wascalculated to be 1.43mg/mL, corresponding to 1.43mg per tablet.The deviation from the value reported on the Quality Certificate(1.41mg) was 1.27% which was within acceptable limits.The amount of cytisine in the each of the oral strips was also

calculated. All strips had on average 1.00 to 1.07mg of cytisine.The average amount of cytisine in the five oral strips was calculatedto be 1.03mg. Although no QC documentation is available, this iswithin 3% of the stated 1mg/strip.

LC-MS

Method validation

Spiked plasma samples showed a symmetrical peak for cytisine andIS. The retention times for cytisine and IS were 7.6 and 9.0min,respectively (Figures 1 and 2). Selectivity/specificity was exam-ined by comparing chromatograms of blank plasma sampleswith spiked plasma samples. No peaks were observed at the

retention time of cytisine and sulfanilamide in blank plasmasamples collected from seven different individuals. The RSD(%) of instrument response of the different sources of blankplasma was within 15% in the seven independent plasmasamples tested.

Accuracy and precision was assessed using QC samples andvalues are reported in Tables 1 and 2. Variation for intra-dayand inter-day accuracy and precision was less than 15% forQC samples. Linearity was verified for this method by usingvalues for coefficients of determinations (R2) obtained fromnine linear standard curves generated on three separate days.Correlation coefficients (R2) of calibration curves were allabove 0.99. The LOQ for cytisine for this method is 2.97 pgon column.

The absolute recovery of cytisine was consistent and, onaverage, 75% for the QC samples. Stability of cytisine inspiked plasma samples after storage in room temperaturefor 24 h and after 3 freeze-thaw cycles was examined and ispresented in Table 3.

No endogenous peaks were observed at the retention time ofcytisine in blank urine samples collected from six different individ-uals. The LOQ in urine was 152pg on column.

Figure 1. Mean plasma concentrations (ng/mL) of cytisine over 24hoursfollowing a single 3mg oral dose. Values are shown as mean±SEM (n=7).

Figure 2. Log[Cytisine] vs. time showing a single linear elimination phaseafter maximum concentration is reached (2hours). Values shown aremean±SEM (n=7).

Table 1. Intra-day variation between spiked plasma samples (n=5)

Spiked concentration(ng/mL)

Mean concentration(ng/mL)

Accuracy(%)

Precision(RSD, %)

1.5 1.42 95.34 7.56

24 23.59 99.21 11.58

48 50.35 105.87 1.29

1522 1518.61 99.78 2.22

Table 2. Inter-day variation between spiked plasma samples (n=5) an-alyzed on 3 separate days


Mean concentration(ng/mL)

Accuracy(%)

Precision(RSD, %)

1.5 1.97 90.17 9.37

24 23.93 92.35 9.44

48 51.08 104.16 3.40

1522 1529.81 100.51 2.32

S. H. Jeong et al.

Drug Testing

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Clinical application

Pilot testing

Plasma samples collected from a healthy subject immediately priorto cytisine administration (t=0) did not show any endogenous inter-ference peaks for cytisine or IS. This assay was able to detect andquantify cytisine in the plasma sample collected from the subjectat 2 h post-dose following a single 3mg administration. The plasmaconcentration of cytisine was determined to be 23.38 ng/mL. Thisassay was also able to detect cytisine in urine samples collectedup to 390min post dose. Urine samples were collected just beforedosing, 0–90min, 90–180min, 180–300min and 300–390min.Cytisine was detected in all of the urine samples collected.

Single dose study

Study participants

Participant characteristics are described in Table 4. Subjects were allmales, with a mean age of 26.3 years. On average, subjects hadbeen smoking for 9.5 years and only 2 subjects had previouslymade an attempt to quit smoking. Six of the seven subjects in thestudy had FTND scores of 4 or below (range 1–7) and the meanFTND score was 3 (low to moderate dependence).

Pharmacokinetics

The current study is the first to describe the pharmacokinetics ofcytisine in humans. After a single 3mg oral administration, cytisinewas absorbed into the bloodstream, with cytisine detectable inplasma as early as 15min after dosing. Peak plasma concentrationswere typically observed at 2h after administration. In two subjects,the peak plasma concentration was observed at 1 h post-dose,suggesting that the peak plasma concentration may actually havebeen achieved between 1 and 2h for all subjects. The peak concen-trations (Cmax) of cytisinemeasured in these subjects were between23.37 and 32.04 ng/mL. Themean Cmax was 27.76ng/mL. Followingthe peak plasma concentration, cytisine concentrations declined ina monophasic manner after a single oral dose. Cytisine was still de-tectable in the urine collected at 24h for all subjects (mean 24hconcentration was 428.15 ng/mL).

Cytisine was detected in urine as an unchanged drug and nometabolites were detected in plasma or in urine in any of thesubjects in the study.

Data collected in this study weremodelled using NONMEM to es-timate PK parameters of volume of distribution (VD) and clearance(CL). The values for VD and CL were estimated to be 115 L and16.7 L/h, respectively with standard error values 0.003. Half-life ofcytisine was calculated to be 4.8 h.

Safety

There were no reports of adverse events in the study. Blood pres-sure, heart rate and respiratory rate did not appear to be adverselyaffected after 3mg single dose administration of cytisine.

Discussion

There is currently limited data on the pharmacokinetics of cytisinein animal studies and none to date reported for humans. Theanimal data that exist describe pharmacokinetic parameters inrabbits and mice but the doses studied are not clinically relevantin humans.[13,14]

Although both HPLC-UV and LC-MS methods are commonlyused in the detection and quantification of drugs in biologicalfluids, an HPLC-UV method was developed and validated first as ithad the advantages of being a relatively simple and low costprocedure.

The HPLC-UV assay developed had acceptable intra- and inter-assay accuracy and precision and was accurate in determining theamount of cytisine in two commercial forms of cytisine includingTabex® tablets and oral strips. This method was determined to beexternally valid. The main objective of method validation, however,is to show that the method can be used for its intended purposewith acceptable reliability and reproducibility. Unfortunately, thetrue usefulness (fit for purpose) of the assay (for pharmacokineticanalysis in humans) was only able to be determined by obtaininga ‘real’ sample, that is, a plasma sample collected from an individualafter a therapeutically-relevant dose of cytisine. Only after analyzingthe plasma sample collected following a 3mg single dose adminis-tration was it revealed that the HPLC-UV method was not suitablefor the quantification of cytisine with dosages used for smokingcessation. Therefore, a more sensitive method was required.

The developed LC-MS analytical method for the determination ofcytisine in human plasma is more sensitive compared to both theHPLC-UV method described in this paper and the previously pub-lished analytical method.[14] This newmethodwas found to complywith limits set by US FDA guidelines including accuracy, precision,specificity and linearity.[20] This method has been successfully usedto quantitatively determine the concentration of cytisine in a hu-man subject following a single 3mg dose of cytisine at 2 h post-dose. This method, therefore, has the sensitivity required to studythe pharmacokinetics of cytisine in human smokers at clinicallyrelevant doses.

Plasma cytisine concentrations declinedwith an average elimina-tion half-life of 4.8h. The half-life of cytisine has been previouslyreported in two animal species including mice and rabbits andcytisine has been described as a drug with a short half-life. In rab-bits, the half-life of cytisine following an oral administration hasbeen reported to be less than 1h.[14]

The logarithmic plot of mean cytisine concentration versus timeafter Cmax (2 h) showed a single elimination phase (i.e., no distinc-tion could bemade between the distribution and elimination phase

Table 3. Recovery of cytisine from spiked plasma samples after storagein room temperature for 24h and after 3 freeze-thaw cycles


Recovery after 24h in roomtemperature (%)

3 Freeze-thawcycles (%)

1.5 91.91 89.56

24 93.00 90.94

48 99.15 92.99

1522 90.29 86.95

Table 4. Participant characteristics

Participant characteristics N=7

Male, % 100

Age, mean±SD, yr 26.3±6.58

Smoking history, mean±SD, yr 9.5±6.98

Previously tried to quit, % 28.6

FTND score, mean± SD 3±2.08


Drug Testing

and Analysis


of the drug). This indicates that single dose pharmacokinetics ofcytisine may be described using a one-compartment model. Thismay propose two possible explanations for how cytisine is distri-buted in the body. Cytisine may either stay in the blood compart-ment or it may distribute to other compartments very rapidly. Theapparent volume of distribution for cytisine estimated for thepopulation in the study (115 L) is more than three times that ofthe blood compartment (35 L), which suggests that the latter expla-nation is more likely.These results show that pharmacokinetics of cytisine are differ-

ent from that of its synthetic analogue, varenicline. Cytisine not onlyhas a much shorter half-life than varenicline (approximately 5h vs24 h),[22] but is also different in the way it is distributed in the body.Varenicline concentrations in plasma decrease in a biphasicmannerand is best described as a two-compartment model.[23] The volumeof distribution of varenicline is approximately three-fold greaterthan cytisine[23] which suggests that varenicline is distributed totissues more extensively. The effect of these pharmacologicalagents is expected to be dependent upon the concentrationsachieved in the brain (centrally acting drugs) and potencies at thetarget receptor (α4β2 nAChR) and, therefore, it is desirable to lookat the extent of brain penetration of these drugs. Unlike varenicline,which is known to readily cross the blood brain barrier,[24] cytisinehas been shown to have poor entry into the brain in animalmodels.[24–27]

In rats, average brain concentration of 145 ng/mL has beenshown 15min after a subcutaneous (s.c.) injection of 1mg/kg ofcytisine, which was less than 30% of the plasma concentration.[27]

Interestingly, the acid dissociation constant (pKa) of cytisine is 7.8(cf. 9.3 for varenicline) which indicates that cytisine exists in itsionised form in lower levels than varenicline at physiological pH(7.4). Therefore, ionisation alone does not explain cytisine’s limitedbrain entry and suggests that cytisinemay be removed or excludedfrom the brain via active effluxmechanisms. However, cytisine doesnot appear to be a substrate for P-gp and its susceptibility to breastcancer resistance protein (BCRP) transporters has not been found todiffer significantly from varenicline.[24] More work is needed toexplore whether cytisine is a substrate for other active effluxtransporters. Cytisine, however, is potent and binds to α4β2 nAChRsat nanomolar concentrations (Ki values ranging from 0.45 to2.4 nM)[24,28–30] which may suggest that even with limited bloodbrain barrier penetration, the exposure of cytisine at α4β2nAChRs in the brain may be sufficient to result in activation ofthese receptors.Animal studies have shown that cytisine is renally eliminated.[13]

Consistent with this, this study demonstrates that cytisine is renallyeliminated in humans and detectable in urine. Nometabolites weredetected in any plasma or urine sample obtained in this study. Themetabolism of cytisine has not been studied extensively, butpreclinical studies have found that cytisine undergoes minimalmetabolism with 90–95% of the administered dose excretedunchanged in the urine[31] and animal studies in rabbits did notreport the presence of metabolic products.[13] Consistent withanimal studies, this study found that unchanged cytisine is renallyeliminated in humans. For varenicline, two minor metabolites havebeen identified in human urine (hydroxyquinoxaline andN-carbamoylglucuronide metabolites), but more than 90% of theadministered dose is in the blood and urine as unchangedvarenicline.[32] As with varenicline, the metabolism appears not tobe a primary route of elimination for cytisine. Therefore, unlikebupropion or nortriptyline (other drugs used for smoking cessation)which are extensively metabolised by hepatic enzymes (primarily

by CYP2B6 and CYP2D6, respectively,[33,34] it is unlikely that cytisinewill have drug-drug interactions (DDIs) due to competition forhepatic enzymes. Furthermore, even if cytisine is metabolised, itsmetabolites will be present in very low concentrations comparedto cytisine and so it is unlikely that the metabolites will be pharma-cologically active. Hepatic insufficiency is, therefore, unlikely to leadto changes in the pharmacokinetics of cytisine.

On the other hand, as cytisine is eliminated primarily through re-nal clearance, renal insufficiency would need to be explored to seewhether renal impairment leads to increased systemic exposure tocytisine and a prolonged half-life in plasma and whether this leadsto increased adverse effects. For varenicline, severe renal impair-ment leads to 2.1-fold increase in area under the curve (AUC) andreduced dosing is recommended for these subjects.[35] In addition,if cytisine clearance involves active renal secretion (transporters),there is potential for DDIs with other renally secreted drugs thatare substrates for the same transporters. Varenicline is excretedpartially via active renal secretion and has been shown to be asubstrate for human organic cation transporter 2 (hOCT2), butnot for other major renal transporters such as the human organicanion transporters (hOAT1 and hOAT3) and human organiccation/carnitine transporters (hOCTN1 and hOCTN2).[36] As cytisinewould partly exist as cations at physiological pH, it is expected thatthe drug would also be a substrate for active renal transport involv-ing organic cation transporters.

Cytisine administration did not appear to adversely affect bloodpressure, heart rate and respiratory rate in this study despite thedose under study being double the normal amount that isrecommended to be ingested at one time.[31] No side effectswere reported and 3mg of cytisine was well-tolerated in all sub-jects. The most common feature of cytisine toxicity reported inthe literature (both animal studies and clinical studies) includesdistresses in the gastrointestinal (GI) tract such as nausea[37–40]

although a meta-analysis found no significant differencebetween cytisine and placebo.[8] Nausea is a commonly reporteddose-related adverse effect with the use of varenicline.[28,35,41] Astudy found that varenicline is less well-tolerated under fastingconditions and nausea and vomiting may be reduced whenvarenicline is taken with food.[42] It would be interesting toexplore whether this is the same for cytisine as this study wasdone with non-fasting subjects.

A potential safety concern with cytisine, as for any other smokingcessation drug, is that prolonged usemay indirectly affect the phar-macokinetics of concomitantly administered drugs. This is becausechemical constituents (e.g. polycyclic aromatic hydrocarbons) incigarette smoke can interact with drug metabolising enzymesand the use of cytisine may decrease (or stop) cigarette smokingwhich in turn could affect the pharmacokinetics and toxicity ofdrugs that are metabolically cleared by such enzymes. The mostwell-known example is CYP1A2 induction in smokers.[43,44] Giventhat several antipsychotic drugs are metabolised primarily byCYP1A2,[45,46] this would be particularly important for patients withschizophrenia, a population with a high incidence of smoking.[47]

An interesting feature in this study is the little variability observedbetween the drug concentrations measured between subjects.However, the main limitation of this study is the small number ofsubjects and thus the little diversity in the study population. Thesubjects in this study were relatively homogenous in terms of sexand build (all relatively fit and no one was overweight); most ofthem were in their 20s and their relatively young age was reflectedin the number of years they had smoked (on average less than10years). All were screened for adequate renal function.

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Another limitation of this study was that no data were collectedon the effect of food or drink on the pharmacokinetics of cytisine.Effects of foods on absorption of cytisine would be an area toexplore, whether food affects the oral bioavailability of cytisine.Given its water solubility and renal elimination, the use of diureticssuch as caffeine or alcohol may have a greater effect on the phar-macokinetics of cytisine.

More recently, a buccal strip containing cytisine has beenmarketed in Australia. Interestingly, cytisine is not a registeredmedication in Australia and no quality assurance documentationof these products is available. It would be interesting to studythe bioavailability of these two different dosage forms of cytisineto determine the differences in the absorption profile of the twoformulations and more importantly whether this has an impacton craving for cigarettes. However, as there is no formal docu-mentation for these oral strips, it is uncertain whether theseproducts contain only cytisine.

Future studies will need to look at cytisine pharmacokinetics inrenally impaired patients and other special population groups todetermine whether dose adjustments should be made to promotesafe use of cytisine. The data generated in the present study will aidthe pharmacometric modelling for future multi-dose pharmacoki-netic studies in humans.

Conclusion

This paper reports two methods for the detection and quantifica-tion of cytisine, a nicotinic partial agonist that has been used asan aid to smoking cessation. AnHPLC-UV assay was able to quantifycytisine in the two commercial forms of cytisine with acceptableaccuracy and precision, but was not sensitive enough to quantifycytisine in human plasma after clinically relevant doses. TheLC-MS bioanalytical assay was therefore developed to supportpharmacological studies of cytisine in humans. This method hasbeen validated and results are within the acceptable range as setby the US FDA guidelines. This method was successfully used todetect and quantify cytisine in human plasma after a single oraladministration of Tabex®.

Cytisine was well tolerated after a 3-mg single dose in smokersand no safety concerns were identified at this dose. Cytisineshowed a simple pharmacokinetic profile with maximum concen-trations reached typically at 2 h post-dose. Cytisine was detectedas an unchanged drug in plasma and urine and no metaboliteswere detected.

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S. H. Jeong et al.

Drug Testing

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RESEARCH PAPER

Pre-clinical properties of the a4b2 nicotinicacetylcholine receptor partial agonists varenicline,cytisine and dianicline translate to clinical efficacyfor nicotine dependencebph_682 334..345

H Rollema1, A Shrikhande1, KM Ward1, FD Tingley III1, JW Coe2, BT O’Neill2, E Tseng3,EQ Wang3, RJ Mather1, RS Hurst1, KE Williams4, M de Vries5, T Cremers5, S Bertrand6 andD Bertrand6,7

1Neuroscience Biology, Groton, CT, USA, 2Neuroscience Chemistry, Groton, CT, USA, 3Pharmacokinetics, Dynamics andMetabolism and 4Clinical Sciences, Pfizer Global Research and Development, Groton, CT, USA, 5Brains On-line and Universityof Groningen, Antonius Deusinglaan 1, Groningen, The Netherlands, 6HiQScreen Sàrl, Conches, Geneva, Switzerland and7Neuroscience, Medical Faculty, Geneva, Switzerland

Background and purpose: Smoking cessation trials with three high-affinity partial agonists of a4b2 neuronal nicotinicacetylcholine receptors (nAChRs) have demonstrated differences in their clinical efficacy. This work examines the origin of thedifferences by taking into account brain exposure and pharmacological effects at human a4b2 nAChRs.Experimental approach: Rat plasma and brain pharmacokinetics were characterized and used to predict human steady-stateplasma and brain concentrations following recommended doses of each of the three compounds. The pharmacologicalcharacterization included in vitro affinities at different nAChR subtypes, functional efficacies and potencies at the human a4b2nAChR, as well as in vivo effects on rat mesolimbic dopamine turn-over.Key results: A comparison of predicted human brain concentrations following therapeutic doses demonstrated that vareni-cline and nicotine, but not dianicline and cytisine, can extensively desensitize and, to a lesser extent, activate a4b2 nAChRs.The limited clinical efficacy of dianicline may be accounted for by a combination of weak functional potency at a4b2 nAChRsand moderate brain penetration, while recommended doses of cytisine, despite its high in vitro potency, are predicted to resultin brain concentrations that are insufficient to affect a4b2 nAChRs.Conclusions and implications: The data provide a plausible explanation for the higher abstinence rate in smoking cessationtrials following treatment with varenicline than with the two other a4b2 nAChR partial agonists. In addition, this retrospectiveanalysis demonstrates the usefulness of combining in vitro and in vivo parameters with estimated therapeutic human brainconcentrations for translation to clinical efficacy.British Journal of Pharmacology (2010) 160, 334–345; doi:10.1111/j.1476-5381.2010.00682.x; published online 22March 2010

Keywords: cytisine; dianicline; dopamine; nAChR partial agonists; nicotine; rat pharmacokinetics; unbound brain concentra-tions; varenicline; voltage clamp

Abbreviations: aCSF, artificial CSF; AUC, area under the curve; BCRP, breast cancer resistance protein; B/P, brain-to-plasmaratio; Bu, unbound brain concentrations; CL, plasma clearance; Css,avg, steady-state plasma concentration;DOPAC, 3,4-dihydroxyphenylacetic acid; ECF, extracellular fluid; FLIPR, fluorimetric imaging plate reader; fu,unbound fraction; HPLC, high-performance liquid chromatography; HVA, homovanillic acid; Ka, acid disso-ciation constant; Ki, receptor affinity; MDR1, multidrug resistance protein; P-gp, P-glycoprotein; Tmax, time tomaximum plasma concentration; Vd, volume of distribution.

Introduction

Nicotine dependence is a chronic relapsing condition oftenrequiring multiple quit attempts with only few smokersachieving long-term abstinence. Successful quitting isextremely difficult because of the distressing nicotine

Correspondence: Hans Rollema, Neuroscience Biology, Pfizer Global Researchand Development, MS 8220-4358, Groton, CT 06340, USA. E-mail:[email protected] 1 October 2009; revised 14 December 2009; accepted 4 January2010

British Journal of Pharmacology (2010), 160, 334–345© 2010 Pfizer IncJournal compilation © 2010 The British Pharmacological Society All rights reserved 0007-1188/10www.brjpharmacol.org

withdrawal symptoms that accompany smoking cessation,and are intensified by social and environmental sensory cues.Smoking tobacco gives almost immediate relief of these with-drawal symptoms as inhaled nicotine rapidly reaches peakconcentrations in the brain to produce the pleasurable expe-rience that reinforces smoking behaviour (Fiore et al., 2008;Benowitz, 2009).

Pharmacotherapies that provide relief of nicotine with-drawal symptoms during a quit attempt have been shown tobe effective, but treatments that would also reduce the plea-surable reward-producing effects of nicotine from smokingshould further improve efficacy. To be maximally effective,such a treatment should not only have high affinity for thetarget receptor to compete with nicotine and prevent itsbinding to the receptor, but also possess adequate pharmaco-kinetic properties that provide sufficient and long-lastingbrain exposure to cover the period of repeated high nicotineexposures during a relapse to smoking.

A recent approach to achieve such dual activity is the use ofpartial agonists of the central high affinity a4b2-containingnicotinic acetylcholine receptors (nAChRs; nomenclaturefollows Alexander et al., 2009), believed to play a key role inthe addictive effects of nicotine (Picciotto et al., 1998; 2008;Mansvelder and McGehee, 2002; Marubio et al., 2003; Lavio-lette and Van der Kooy, 2004; Hogg and Bertrand, 2005).High-affinity a4b2 nAChR partial agonists should relievecraving and withdrawal symptoms during a quit attempt byactivating a4b2 nAChRs, and will reduce the reinforcingeffects of nicotine upon smoking by competing with nicotinefor the binding site (Cohen et al., 2003; Coe et al., 2005; Hoggand Bertrand, 2007; Rollema et al., 2007a). Compounds withreduced agonist efficacy at a4b2 nAChRs with a slower onsetand much longer half-life than inhaled nicotine shouldexhibit a low potential for abuse liability.

Three a4b2 nAChR partial agonists varenicline (Coe et al.,2005), cytisine (Papke and Heinemann, 1994; Tutka andZatonski, 2005) and dianicline (Cohen et al., 2003; structuresin Figure 1) have been studied in clinical trials, and found toexhibit differing efficacies as smoking cessation aids. Achievedabstinence rates and odds ratios (ORs) versus placebo arehigher for varenicline (Cahill et al., 2008; Nides et al., 2008)than for either cytisine (Etter, 2006; Cahill et al., 2008) ordianicline (Fagerström and Balfour, 2006). The purpose of this

study was to examine whether the reported differences inabstinence rates can be attributed to key pharmacokinetic andpharmacodynamic parameters that determine clinical efficacyof the agent, that is, its unbound brain concentration andfunctional interaction with the target receptor at therapeuticdoses.

To correlate pharmacologically active concentrations tohuman brain exposure after recommended doses of thepartial agonists, rat plasma and CNS pharmacokinetics weremeasured in order to predict human unbound steady-stateplasma and brain exposures. Disparities between central expo-sures of each partial agonist prompted us to study key prop-erties that determine their penetration into the brain.

For a quantitative in vitro analysis of the receptor interac-tions, we determined binding affinities at different nAChRsubtypes, their functional efficacies and concentration depen-dence for both activation and desensitization at the humana4b2 nAChRs. Finally, dose-dependent effects on mesolimbicdopamine turn-over in rat brain were determined as a rel-evant measure of in vivo potencies of the partial agonists.

Our results show that differences in predicted unboundhuman brain concentrations and in in vitro potencies todesensitize and activate central a4b2 nAChRs, provide a plau-sible explanation for the lower abstinence rates with diani-cline and cytisine than observed with varenicline, and can beused as predictors of clinical outcome.

Methods

AnimalsAll animal care and experimental procedures complied withand were approved by, as appropriate, the InstitutionalAnimal Care and Use Committees of Pfizer Global Researchand Development (Groton, CT, USA) and the University ofGroningen (The Netherlands), and the animal rights rulesfrom Geneva, Switzerland. Male Sprague-Dawley rats (n = 75,250–320 g, Charles River Laboratories, Wilmington, MA, USAand Harlan, The Netherlands), male FVB/N (n = 12) andmdr1a/1b (–/–,–/–) (n = 12) mice (Taconic Laboratories, Ger-mantown, NY, USA) were used. The animals were housed ona 12 h light schedule (lights on at 7:00 AM) in temperature-controlled (20°C) colony rooms with free access to standardchow and water. Xenopus laevis ovaries were harvested fromfemales (n = 5) maintained at the Medical Faculty of Geneva.

Pharmacokinetic studies

In vitro protein bindingTo determine unbound concentrations and to calculate brain-to-plasma ratios (B/P), concentration-time profiles were deter-mined in rat plasma, brain, CSF and extracellular fluid (ECF),and total concentrations were corrected with the unboundfractions. Protein binding was determined with 1 mM testcompound in pooled human plasma, pooled rat plasma or20% rat brain homogenate by equilibrium dialysis in Dulbec-co’s phosphate-buffered saline (pH 7.4) as described previ-ously (Reed-Hagen et al., 1999). The unbound fraction (fu) ofthe test compound in undiluted brain tissue was calculated

N

N

H

N

O

N

H

N

O

NHH

H

A B

C D

Nicotine Cystisine

Dianicline Varenicline

Figure 1 Chemical structures of nicotine and the a4b2 nAChRpartial agonists tested clinically as aids to smoking cessation: (A)nicotine; (B) Cytisine (Tabex); (C) Dianicline (SSR5918113); and (D)varenicline (Chantix, Champix).

a4b2 Partial agonists: PK–PD efficacy predictionsH Rollema et al 335

British Journal of Pharmacology (2010) 160 334–345

from the fu in the homogenate and the homogenate dilutionfactor according to Kalvass and Maurer (2002).

In vivo brain disposition studies in rats and in P-glycoprotein(P-gp)-deficient micePlasma and brain exposures of cytisine and dianicline weremeasured in wild-type (WT) mice and in mice lacking theP-gp transporter to examine whether these compounds weresubstrates for this transporter. Male rats received a single1 mg kg–1 p.o. dose of partial agonist or 1 mg kg–1 s.c. nicotine.The rats were killed by CO2 inhalation at 0.5, 1.5, 3 and 6 hpost-p.o. dosing, and at 0.167, 0.5, 1 and 2 h post-s.c. dosing.Whole blood was collected by cardiac puncture, and centri-fuged to obtain plasma. CSF was collected via puncture of thecisterna magna, and samples were immediately frozen on dryice upon collection. Whole brains were collected, rinsed inphosphate-buffered saline, weighed and immediately frozenon dry ice upon collection.

Male FVB/N (WT) and mdr1a/1b (–/–, –/–) mice (30 g, n = 3per genotype/time point/compound) were given a single3 mg kg–1 p.o. dose of cytisine or dianicline in phosphate-buffered saline, and killed in a CO2 chamber at 0.5 and 2 hpost-dose. Whole blood and brains were collected as describedabove, and all samples were stored at –20°C until analysis.

In vivo rat brain (ECF) disposition studyFree brain concentrations were measured directly in the extra-cellular space by ultra slow-flow microdialysis. Unbound con-centrations in rat brain ECF of each test compound after1 mg kg–1 p.o. (nicotine s.c.) were determined by ultra-slow-flow microdialysis in rat cortex, as described by Cremers et al.(2009). A 4 mm MetaQuant probe (BrainLink, Groningen,The Netherlands) was stereotaxically implanted into the pre-frontal cortex (coordinates from bregma: AP 3.4 mm and ML0.8 mm; from dura: DV –5.8 mm) under 2% isoflurane ana-esthesia, and experiments were performed 48 h later. Theprobe was perfused with artificial CSF (aCSF) at a flow rate of0.15 mL min–1, while ultra-purified water was deliveredthrough the dilution inlet of the probe to create a carrier flowof 0.80 mL min–1. After equilibrating for 1 h, the test com-pound was administered at t = 0 min, and nine microdialysissamples were collected for 4.5 h at 30 min intervals in taredvials in an automated fraction collector (CMA 142). For invitro recoveries, a MetaQuant probe was placed in a stirredaCSF solution containing 10-8 M of test compound, and per-fused with aCSF and water at the same flow rates as in the invivo experiment. The exact slow-flow rate was verified byweighing each vial during the experiment, and two additionalvials at the end of the experiment, 15 min after stopping theslow flow. All samples were stored at –80°C until analysis.

Rat i.v. and p.o. studies for plasma pharmacokinetic and humanpharmacokinetic predictionFor the estimation of human plasma exposure of dianiclineand cytisine by allometric scaling, pharmacokinetic param-eters were obtained from a rat i.v. and p.o. pharmacokineticstudy. Male rats received a single 1 mg kg–1 i.v. or p.o. dose of

dianicline or cytisine in saline. Blood samples were collectedvia a jugular vein catheter at 0.083, 0.167, 0.5, 1, 2, 4 and 7 hpost-dose, and centrifuged to obtain plasma samples thatwere stored at –20°C until analysis. Unbound human clear-ance for dianicline and cytisine was predicted based onobserved rat pharmacokinetic data using single-species allo-metric scaling as described by Hosea et al. (2009).

Steady-state human brain exposures following therapeuticdoses can be predicted from the rat B/P and the humanaverage steady-state plasma concentration (Css,avg), assumingthat rat and human have the same B/P ratio for each com-pound. The predicted human Css,avg for each compound wascalculated according to the equation:

CFCL

ss avgDose

, = ×× τ

(1)

where F is the oral availability, CL is the clearance and t is thedosing interval.

Assays and pharmacokinetic analysisAll samples from the protein binding, brain disposition andpharmacokinetic studies were analysed by HPLC–mass spec-trometry (detailed assay conditions for each compounds aregiven in the Supporting Information Appendix S1).

The i.v. and p.o. pharmacokinetic analyses were conductedusing individual animal concentration data. Data from thebrain penetration study were analysed using mean concentra-tions at each time-point. All pharmacokinetic analyses wereperformed using WinNonlin Enterprise version 5.2 (PharsightCorporation, Mountainview, CA, USA).

Pharmacodynamic studies

In vitro binding affinity to nAChRsBinding affinities of the test compounds to nAChR subtypeswere determined as previously described (Rollema et al.,2007b) using [3H]-epibatidine to label a4b2, a3b4 and a6/4b4nAChRs expressed in HEK293 cells, [125I]-a-bungarotoxin tolabel a7 nAChRs expressed in IMR32 cells and a1bgd nAChRsin Torpedo electroplax membrane. Ki values (Table 1) werecalculated according to Ki = IC50/(1 + [3H-ligand]/Kd), andexpressed as mean � SEM (n = 5).

In vitro functional activity at a4b2 nAChRs expressedin oocytesExperiments were carried out on human a4b2 nAChRsexpressed in Xenopus laevis oocytes using complementaryDNA expression. Currents evoked by ACh and nicotinicligands were recorded using a standard two electrode voltageclamp configuration as described by Hoda et al. (2008).Ovaries were harvested from Xenopus females, and a smallpiece of ovary was isolated for immediate preparation, whilethe remaining part was placed at 4°C in a sterile Barth solu-tion containing (in mM) NaCl, 88; KCl, 1; NaHCO3, 2.4;HEPES, 10; MgSO4.7H2O, 0.82; Ca(NO3)2.4H2O, 0.33;CaCl2.6H2O, 0.41 at pH 7.4, supplemented with 20 mg mL–1 of

a4b2 Partial agonists: PK–PD efficacy predictions336 H Rollema et al


kanamycin, 100 unit mL–1 penicillin and 100 mg mL–1 strepto-mycin. All recordings were performed at 18°C, and cells weresuperfused with OR2 medium containing (in mM): NaCl,82.5; KCl, 2.5; HEPES, 5; CaCl2.2H2O, 2.5; MgCl2.6H2O, 1 atpH 7.4, and 0.5 mM atropine to prevent possible activation ofendogenous muscarinic receptors. Cells were held at –80 mV,and data were captured and analysed using data acquisitionand analysis software running under Matlab (Mathworks Inc.,Natick, MA, USA). Concentration–activation curves werefitted using either a single or dual empirical Hill equation(Buisson and Bertrand, 2001):

Y a x a xn n= + ( )⎡⎣ ⎤⎦ + −( ) + ( )⎡⎣ ⎤⎦1 1 150 50EC ECH LHH HL (2)

where Y is the fraction of evoked current, a is the fraction ofhigh-affinity component, EC50H is the concentration for 50%activation of the high affinity, nHH is the apparent cooperat-ivity for the high affinity, EC50L is the concentration for 50%activation of the low affinity, nHL is the apparent cooperativityfor the low affinity and x is the agonist concentration. For thesingle Hill curve fitting, the second term was not used, and ais the unity.

Concentration–inhibition curves were fitted with a compa-rable equation:

Y a x a xn n= + ( )⎡⎣ ⎤⎦ + −( ) + ( )⎡⎣ ⎤⎦1 1 150 50IC ICH LHH HL (3)

where a, nHH and nHL are the same as above; Y is the fraction ofremaining current, IC50H is the concentration for 50% inhibi-tion of the high-affinity component; IC50L is the concentra-tion for 50% inhibition of the low-affinity component; and xis the antagonist concentration.

Whenever needed, a dual Hill equation, similar to that usedfor the concentration–activation curves, was used. For statis-tical analysis, the unpaired, two-tailed Student’s t-test wascomputed either with Excel (Microsoft) or Matlab (MathworksInc.).

Dopamine turn-over in rat nucleus accumbensThe dose-dependent effects of the partial agonists on dopam-ine turn-over in rat nucleus accumbens were determined byoral administration of test compound 2 h prior to rapidremoval of the nucleus accumbens, and compared with the

response to a maximally effective dose of 1 mg kg–1 s.c. nico-tine (Rollema et al., 2007b). Concentrations of dopamine andits metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) andhomovanillic acid (HVA) were measured in the tissue samplesby HPLC and electrochemical detection (details in SupportingInformation Appendix S1). Dopamine turn-over was calcu-lated as the ratio ([DOPAC] + [HVA])/[dopamine], andexpressed as percentage of controls � SEM (n = 4–6). Statisticalsignificance was analysed by one-way ANOVA using Dunnett’spost hoc test.

MaterialsVarenicline (6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine) was synthesized according to Coe et al.(2005), and dianicline (SSR591813; 5aS,8S,10aR)-5a,6,9,10-tetrahydro,7H,11H-8,10a-methanopyrido[2′,3′:5,6]pyrano[2,3-d]azepine) according to Galli et al. (2000). Cytisine (1R,5S)-1,2,3,4,5,6-hexahydro-1,5-methano-8H-pyrido[1,2a][1,5]diazocin-8-one) was purchased from Austin Chemical Co.(Buffalo Grove, IL, USA), and nicotine bitartrate from Sigma-Aldrich (St Louis, MO, USA). All compounds were dissolved insaline, administered as 1 mL kg–1 solutions, and doses areexpressed as the active compound (base). Radioligands werepurchased from PerkinElmer Life and Analytical Sciences Inc.(Boston, MA, USA); all other analytical grade chemicals werefrom Sigma-Aldrich.

Results

Pharmacokinetic dataFour types of pharmacokinetic studies were performed topredict human unbound brain concentrations and to accountfor differences observed in rat in brain penetration of thecompounds.

Unbound exposure and brain penetrationTime-courses of drug concentrations measured in rat plasma,brain, CSF and ECF following p.o. administration of 1 mg kg–1

of each compound (nicotine s.c.) are shown in Figure 2, andcorresponding calculated areas under the concentration time

Table 1 Protein binding, unbound exposure and exposure ratios in rat plasma, brain, CSF and ECF after 1 mg kg–1 p.o. of the partial agonists,and 1 mg kg–1 s.c. of nicotine

Nicotine Varenicline Cytisine Dianicline

Protein binding (fraction unbound, fu)fu Plasma 0.81 0.55 1.00 0.95fu Brain 1.00 0.67 1.00 0.44

Unbound exposure (AUC0–6h, h·ng·mL-1)Plasmaunbound 512 397 702 448Brainunbound 1070 1570 80 176CSF 639 495 190 150ECF 495 380 36 77

Brainunbound AUC/Plasmaunbound AUC 2.10 3.90 0.11 0.39CSF AUC /Plasmaunbound AUC 1.25 1.25 0.27 0.33ECF AUC /Plasmaunbound AUC 0.97 0.96 0.05 0.17

fu, protein binding expressed as fraction unbound in plasma and brain; AUC0–6h, unbound exposure expressed as area under the curve over 0–6 h in h·ng·mL-1.



curve over 0–6 h (AUC0–6h) are shown in Table 1. Measure-ments of unbound fractions (fu) of each of the four com-pounds show that nicotine and cytisine are essentially non-protein bound in plasma and brain (fu > 0.8), that dianiclineis unbound in plasma and 60% bound in brain, while vareni-cline is moderately protein bound (fu > 0.5) in plasma andbrain (Table 1).

Oral administration to rats of 1 mg kg–1 of each compoundyielded comparable unbound plasma concentrations for thefour compounds, with AUC0–6h values of 512, 397, 702 and448 h ng–1 mL–1 for nicotine, varenicline, cytisine and diani-cline respectively. However, CNS exposures were significantlyhigher for nicotine and varenicline than for dianicline andcytisine (Figure 2, Table 1). CSF exposures of nicotine andvarenicline are two- to threefold lower than their corre-sponding unbound brain exposures, suggesting either over-estimation of unbound brain exposures or active transport ofboth compounds into the brain or underestimation of CSFconcentrations.

In vivo rat brain ECF disposition studyThe direct measurement of free drug exposures in the extracel-lular space by ultra-slow-flow microdialysis sampling revealedthat ECF exposures of the test compounds are approximatelytwo- to fivefold lower than their unbound brain concentra-tions. ECF concentrations of nicotine and varenicline are thuspractically identical to their unbound plasma and CSF expo-sures, indicating that entry of varenicline and nicotine into thebrain is likely to be mediated primarily via passive diffusion. Incontrast, unbound brain exposures, as well as CSF and ECFexposures of dianicline and cytisine, are several fold lower thantheir unbound plasma exposures, while in addition, centralexposures were 4- to 19-fold lower for dianicline and cytisinethan for nicotine and varenicline.

In vivo brain disposition studies in P-gp-deficient miceBecause these data could indicate either reduced brain entryor active brain efflux of cytisine and dianicline, we examinedwhether the two compounds are substrates for the P-gp trans-

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Figure 2 Time-courses of unbound concentrations of nicotine and partial agonists in rat plasma, brain, CSF and ECF after 1 mg kg–1 of eachcompound. Data are expressed as ng·mL–1 or ng·g–1 (brain) � SD (n = 3–6). (A) Nicotine 1 mg kg–1 s.c.; (B) varenicline 1 mg kg–1 p.o.; (C)cytisine 1 mg kg–1 p.o.; and (D) dianicline 1 mg kg–1 p.o.



porter in WT and P-gp knock-out (KO) mice. The data (Sup-porting Information Table S2) show that CNS penetration ofcytisine, with a KO-to-WT total brain-to-total plasma (KOB/P/WTB/P) ratio of 0.9, is not influenced by mdr1 P-gp, and thatfor dianicline, with a KOB/P/WTB/P ratio of 2.2, P-gp does notimpair CNS penetration.

These data are consistent with in vitro data that predict alack of susceptibility to human P-gp of the three compounds(Supporting Information Table S3). Other molecular proper-ties that can affect brain penetration, such as ELogD7.4, polarsurface area, membrane permeability, multidrug resistanceprotein (MDR1) and breast cancer resistance protein (BCRP)transporter susceptibility, do not significantly differ betweenthe three partial agonists (Supporting Information Table S3).

Pharmacokinetics of dianicline and cytisine in ratsFor the prediction of unbound brain levels of cytisine anddianicline, their human plasma levels were estimated by allo-metric scaling from rat pharmacokinetic parameters obtainedin an i.v. and p.o. pharmacokinetic study of dianicline andcytisine in rats following a 1 mg kg–1 dose (Table 2).

Cytisine showed a moderate plasma clearance (CL~35 mL min–1 kg–1), whereas the CL of dianicline approxi-mates rat hepatic blood flow (CL ~66 mL min–1 kg–1). Bothcompounds showed moderate volumes of distribution (Vd)with values ranging from 2.7 to 3.8 L kg–1, and short half-livesof 1.5 and 0.95 h respectively. Following oral administration,both compounds were rapidly and fully absorbed into thesystemic circulation as indicated by the short time tomaximum plasma concentration (Tmax) (0.63 and 0.5 h) andcomplete oral bioavailability.

Prediction of human steady-state brain concentrations afterrecommended dosesThe predicted human free Css,avg of cytisine after 1.5 mg fourtimes a day (QID) (Etter, 2006) and of dianicline after 40 mg

twice a day (BID) (http://clinicaltrials.gov/ct2/results?term=dianicline) was calculated from estimated human pharmaco-kinetic parameters (Table 2) and measured human plasmaprotein binding (Table 1), to be 36 and 216 nM, respectively(Table 3). The human free Css,avg of varenicline following oraladministration of the recommended 1 mg BID dose has beenreported to be approximately 33 nM (Burstein et al., 2006;Faessel et al., 2006a,b). Subsequently, an upper and lowerrange of predicted unbound exposure in human brain wascalculated for each compound by multiplying the human freeplasma Css,avg with the highest and lowest unbound B/P deter-mined in rats with the methods described above and shownin Table 1, that is, unbound brain-to-unbound plasma ratio(Bu/Pu), CSF-to-unbound plasma ratio (CSF/Pu) and ECF-to-unbound plasma ratio (ECF/Pu). In this way, the unboundCss,avg in human brain after therapeutic doses was predicted torange from 32 to 131 nM for varenicline, from 2 to 10 nM forcytisine and from 37 to 84 nM for dianicline (Table 3).

Pharmacodynamic dataIn vitro binding affinities of the compounds were measured atseveral nAChR subtypes to assess their binding potency andselectivity. Functional studies determined concentration-dependent activation and desensitization profiles at thehuman a4b2 nAChR to provide functional potencies andfunctional efficacies. These in vitro experiments were carriedout with a mixed population of a4b2 nAChRs not intention-ally enriched for any particular stoichiometry of a and bsubunits. Effects on dopamine turn-over in the rat mesolimbicsystem were determined to assess the functional activities andpotencies of the compounds in vivo.

Receptor binding affinitiesIn vitro receptor binding data (Table 4) show that the testcompounds have some selectivity for a4b2 nAChRs, and that

Table 2 Rat pharmacokinetic parameters (mean � SD) for cytisine and dianicline following a 1 mg kg–1 i.v. and p.o. dose

Route(1 mg·kg–1) CL(mL·min–1·kg–1) Vd(L·kg-1) t1⁄2(h) Cmax (ng·mL–1) Tmax(h) AUC0–• (h·ng·mL-1) F

Cytisine i.v. 35.0 � 7.5 2.7 � 1.2 1.5 � 0.1 517 � 125p.o. 38.3 � 8.3 4.9 � 1.4 146 � 26 0.63 � 0.25 483 � 94 0.94

Dianicline i.v. 66.5 � 14.2 3.8 � 1.7 0.95 � 0.1 233 � 27p.o. 66.1 � 27 7.2 � 1.1 126 � 16 0.5 � 0 303 � 88 1.30

Vd, volume of distribution; t½, half-life; Cmax, maximum observed concentration; Tmax, time when maximum concentration is observed; AUC0–•, area under the curvefrom t = 0 to end of dosing interval; F, oral bioavailability.

Table 3 Measured human pharmacokinetic data for varenicline and predicted human pharmacokinetic data for cytisine and dianicline, afterrecommended therapeutic doses of each compound

Compounda Dose(mg) CL(mL·min–1·kg–1) Vd(L·kg–1) t1⁄2(h) fu,PL F Css,avg(nM) [Bu]predicted(nM)

Varenicline 2¥ 1 2.3b nd 31.5 0.80 nd 33 32–131Cytisine 4¥1.5 5.2 1.6 3.6 0.64 1 36 2–10Dianicline 2¥ 40 14.1 3.3 2.7 0.82 1 214 37–84

aVarenicline data from Faessel et al. (2006b); cytisine and dianicline data were predicted by allometric scaling of rat pharmacokinetic data (Supporting InformationTable S3).bCalculated from dose/AUC0–t (area under the curve from t = 0 to end of dosing interval) for 70 kg body weight.nd, not determined; Vd, volume of distribution; t½, half-life; fu,PL, unbound plasma fraction; F, oral bioavailability; Css,avg, average unbound concentration in humanplasma at steady state; [Bu], unbound brain concentration.



varenicline binds to the a4b2 subtype with higher receptoraffinity (Ki = 0.4 nM) than the other compounds. It has15-fold higher affinity than nicotine (Ki = 6.1 nM), a require-ment for effectively competing with nicotine for the a4b2nAChR. Although varenicline has significantly lower affinityfor a7 (Ki = 125 nM) than for a4b2 nAChRs, it binds to thisnAChR subtype with at least 20-fold higher affinity than theother compounds (Ki values > 2.1 mM). Cytisine is the onlycompound showing moderate affinity for the musclea1-containing nAChRs (Ki = 430 nM) to which nicotine bindswith low affinity (Ki = 2 mM), while varenicline and dianiclinedo not bind to this subtype (Ki values > 8 mM).

Functional efficacies and potencies at a4b2 nAChRsTo correlate the predicted human free brain concentrationsafter recommended doses to drug concentrations required todesensitize and activate a4b2 nAChRs, we performed voltageclamp analyses of the functional interaction of each com-pound at the human a4b2 nAChR expressed in Xenopusoocytes (Table 5, Figure 3). Brief applications of nicotine orpartial agonists cause activation of the receptors, whereassustained exposures desensitize the receptor. Examples ofconcentration-dependent responses evoked by the partialagonists and of concentration-dependent desensitization bythe compounds are illustrated in Figure 4. Consistent withdata from earlier studies (Briggs and McKenna, 1998; Hoggand Bertrand, 2005; Picciotto et al., 2008), receptor desensiti-zation was observed at lower concentrations than thoseneeded for activation. The EC50 values for high-affinity acti-vation of a4b2 nAChRs ranged from 0.06 to 18 mM, while the

IC50 values for high-affinity desensitization were 0.05–2.8 nM(Table 5, Figure 3).

The concentration–activation curves show that agonist effi-cacies relative to the effect of 100 mM ACh are 22 � 2.5% forvarenicline, 6.5 � 0.2% for cytisine and 8.0 � 0.3% for diani-cline. Parallel studies in HEK293 cells expressing human a4b2nAChRs, using calcium flux measurements with a fluorimetricimaging plate reader (FLIPR; data not shown), yielded com-parable results with higher functional efficacies relative to theresponse evoked by 100 mM nicotine of 48 � 1.8% for vareni-cline, 46 � 5% for cytisine and 34 � 5.6% for dianicline.Table 5 shows desensitization–activation potencies and effica-cies, as well as Hill parameters obtained from voltage clampdata in Figure 3, using the sum of two isotherms for the curvefitting (Buisson and Bertrand, 2001). Overall, the in vitrobinding and functional data are in good agreement withbinding potencies and functional efficacies relative to eithernicotine or ACh that have been reported for these compoundspreviously (Cohen et al., 2003; Coe et al., 2005; Mihalak et al.,2006; Rollema et al., 2007b; Smith et al., 2007).

The overlap of the concentration-dependent activation anddesensitization curves defines a concentration range for eachcompound thought to represent a pharmacologically activerange where the receptor can be partially activated withoutcomplete desensitization (Hogg and Bertrand, 2005). Thisconcentration range between onset of activation and com-plete desensitization is approximately 10–100 mM for diani-cline, and 0.01–10 mM for the other three compounds(Figure 3). The predicted unbound human brain concentra-tions (Table 3) are compared with this pharmacological rangefor each compound (grey bars in Figure 3).

Table 4 In vitro receptor binding affinities of test compounds and radioligands used for binding assays at different nAChR subtypes

Ki (means � SEM, nM)

nAChR Subtype radioligand Nicotine Varenicline Cytisine Dianiclinea4b2 ([3H]-epibatidine) 16.1 � 4.9 0.4 � 0.1 2.0 � 0.2 105 � 14a7 ([3H]-a bungarotoxin) 2110 � 852 125 � 18 5890 � 1250 >12 500a3b4 ([3H[-epibatidine) 520 � 120 86 � 16 480 � 63 5130 � 180a6/a4b4 ([3H[-epibatidine) 270 � 75 110 � 13 329 � 33 2320 � 875a1b1gd ([3H]-a bungarotoxin) 2090 � 620 8200 � 1530 492 � 11 >12 500

Data represent mean Ki values � SEM in nM.

Table 5 Agonist efficacies and functional potencies of nicotine, varenicline, cytisine and dianicline at human a4b2 nAChRs expressed inoocytes

Compound High affinity Low affinity Efficacy versus ACh

Activation a EC50 mM nH EC50 mM nH %Nicotine 0.25 0.06 � 0 1 � 0.0 4.6 � 1 1.4 � 0.03 100 � 1Varenicline 1.0 1.4 � 0.1 1.3 � 0.01 22 � 2.5Cytisine 1.0 2.0 � 0.1 1.2 � 0.02 6.5 � 0.2Dianicline 1.0 18.4 � 1.2 1.24 � 0.04 8.0 � 0.3

Desensitization a IC50 nM nH IC50 nM nHNicotine 0.25 � 0.01 2.8 � 1.1 1.5 � 0.45 167 � 63 1.3 � 0.05Varenicline 0.48 �.0.04 0.07 � 0.01 1.3 � 0.01 50 � 9 1Cytisine 0.53 � 0.07 0.05 � 0.01 1.3 � 0.10 28 � 6 1Dianicline 0.33 � 0.05 2.0 � 0.1 1.3 � 0.08 3000 � 860 1

Parameters are obtained from best fits of the data using a dual empirical Hill equation (solid lines in Figure 3).a, fraction of high-affinity component (Hill equation); %, relative agonist efficacy versus ACh or versus nicotine; EC50, concentration in mM for 50% activation; IC50,concentration in nM for 50% inhibition of ACh- or nicotine-evoked currents; nH, Hill coefficient.



Effect on dopamine turn-over in rat nucleus accumbensIn vivo activities of the three compounds were assessed bymeasuring effects on dopamine turn-over, compared with themaximal effect of nicotine. Each compound, in a dose-dependent manner, increased dopamine turn-over in ratnucleus accumbens. The highest test dose of each partialagonist produced increases to less than 140% of controls,compared with the maximal nicotine effect of 180% of con-trols after 1 mg kg–1 s.c. nicotine (Figure 5). Higher doses ofthe partial agonists were either poorly tolerated and notfurther assessed, or caused lower turn-over increases thanafter the previous test dose, resulting in an inverted U-shapeddose–response curve, consistent with previous findings(Rollema et al., 2007b). Relative potencies of the three partialagonists were estimated by comparing oral doses required toproduce a 20% increase in dopamine turn-over above controllevels (‘ED20’). These data revealed that varenicline (ED20

~0.12 mg kg–1) is about 20-fold more potent in increasingdopamine turn-over than cytisine (ED20 ~2.5 mg kg–1), and atleast 100-fold more potent than dianicline (ED20 ~20 mg kg–1).From the dose–response curves, we also estimated theunbound rat brain peak concentrations associated with a dose

that significantly increased dopamine turn-over. These dosesand unbound concentrations were for nicotine 1 mg kg–1 s.c.and ~4 mM, for varenicline 1 mg kg–1 p.o. and ~0.4 mM, forcytisine 10 mg kg–1 p.o. and ~0.7 mM and for dianicline20 mg kg–1 p.o. and ~3.2 mM.

Discussion and conclusions

Because of their inherent activation and desensitization prop-erties, a4b2 nAChR partial agonists are believed to relievecraving during abstinence and to reduce nicotine reinforce-ment during smoking lapses, resulting in improved efficacy asaids to smoking cessation compared with other treatments.This hypothesis was borne out from the observation of higherabstinence rates in smokers treated with varenicline, a partialagonist approved as a smoking cessation aid (Chantix,Champix), than with any other treatment (Cahill et al., 2008;Nides et al., 2008). Although no head-to-head comparisonshave been made, the available data from smoking cessationtrials with two other a4b2 nAChR partial agonists, cytisine(Tabex) and dianicline (SSR591813), show lower abstinence

Figure 3 Plots of concentration activation and concentration inhibition curves obtained for nicotine, varenicline, cytisine and dianicline at thehuman a4b2 nAChR expressed in Xenopus oocytes measured by voltage clamp. The activation responses are normalized to the maximalresponse to 100 mM ACh (=1); inhibition responses are normalized to the half maximal response (=0.5) obtained with 30 mM ACh. Continuouslines through the data points are the best fits obtained for each compound (parameters in Table 5). The dotted vertical lines indicate themidpoint concentration of the area defined by the overlap of activation and concentration inhibition curves. The grey bars represent thepredicted unbound brain concentration of nicotine after smoking and of each partial agonist after recommended therapeutic doses fromTable 3. (A) Nicotine, (B) varenicline, (C) cytisine and (D) dianicline.



rates (Etter, 2006; Fagerström and Balfour, 2006; Cahill et al.,2008) than for varenicline, raising questions about optimalrequirements for efficacy. As with other centrally acting drugs,the clinical outcome of treatment with partial agonists isdetermined both by their unbound human brain concentra-tions and their functional potencies at the target a4b2nAChR, and our aim was to evaluate whether key pharmaco-kinetic and pharmacodynamic properties of the three partialagonists would have predicted differences in their clinicalefficacies.

In vivo pharmacokinetic studies in rat demonstrated thatoral administration of varenicline, cytisine or dianiclineresulted in comparable unbound plasma exposures, but thereare important differences between the unbound brain con-centrations of the agents. Varenicline is the only compound

for which human plasma levels after recommended therapeu-tic doses are available (Faessel et al., 2006a,b). For the predic-tion of unbound brain levels of cytisine and dianicline, it wastherefore necessary to estimate their human plasma levels byallometric scaling. Rat single species allometry predicted thathuman unbound Css,avg after 1.5 mg QID cytisine was in thesame range as reported values for unbound Css,avg after 1 mgBID varenicline, while 40 mg BID dianicline was estimated toresult in a proportionally higher unbound Css,avg (Table 2).However, in brain, human steady-state unbound concentra-tions (Bu) after recommended doses of varenicline are pre-dicted to be much higher than those of cytisine due to thereduced brain entry of cytisine (Table 2). The poor brain pen-etration of cytisine has previously been attributed to its highhydrophilicity, that is, a lower logP value than nicotine and

Figure 4 Activation and desensitization experiments at human a4b2 nAChRs expressed in Xenopus oocytes. Typical concentration-dependentresponses evoked by cytisine, varenicline and dianicline are illustrated in the left panels. Cells were exposed once every 2 min for 10 s toincreasing concentrations of partial agonist (0.03, 0.1, 0.3, 1, 3, 10, 30, 100 and 300 mM) followed by a pulse of ACh (30 mM) to determinethe maximal response. Typical concentration-dependent desensitization data obtained for cytisine, varenicline and dianicline are illustrated inthe right panels. To evaluate the desensitization effects caused by sustained agonist exposure, a protocol of repetitive challenges with a fixedACh pulse (30 mM, 10 s, applied every 2 min) was used. To assess stability of the recordings, cells were first challenged with ACh (30 mM, 10 s)four times in the control medium and then with the same ACh test pulse in the presence of a fixed concentration of agonist (100 nM).



the other agonists (Reavill et al., 1990; Tutka and Zatonski,2005; Supporting Information Table S3). It is, however,unlikely that hydrophilicity is the sole or major reason for thelow brain exposure of cytisine. At physiological pH, thepartial agonists have approximately the same distributioncoefficient (ElogD7.4), because at pH 7.4 much less cytisine,which has an acid dissociation constant (pKa) of 7.8, is in theprotonated form than varenicline and dianicline (pKa 9.3–9.8; Supporting Information Table S3). Moreover, there arevery small differences in other molecular properties of thepartial agonists that impact permeability and transport, andnone of the compounds was found to be a substrate for P-gpor BCRP efflux transporters (Supporting Information Tables S2and S3). We speculate that the low brain concentrations ofcytisine could be due to an active, non-P-gp, non-BCRP brainefflux mechanism. Dianicline has comparable permeabilityand hydrophilicity as varenicline, and is not a substrate forP-gp or BCRP efflux transporters (Supporting InformationTables S2 and S3), but has limited brain penetration. Recom-mended doses of 40 mg BID dianicline are predicted to resultin relatively low unbound human brain concentrations, com-parable to those of varenicline after 1 mg BID (Table 3).

To correlate the predicted human Bu of the partial agonistsand nicotine with their in vitro properties, binding and func-tional studies were conducted at human nAChRs. Results ofthese experiments confirmed that each compound bindsselectively to a4b2 nAChRs, and that varenicline, cytisine anddianicline act as partial agonists (Figure 3; Tables 4 and 5).However, there are substantial quantitative differences inbinding affinities and functional potencies of dianiclineversus the other two partial agonists, and much higher diani-cline concentrations are required for an interaction with a4b2nAChRs. This pharmacologically relevant range between theactivation and desensitization curves is 10–100 mM for diani-

cline, compared to 0.01–10 mM for nicotine, varenicline andcytisine (Figure 3). Plots of predicted Bu values of eachcompound over their desensitization and activationconcentration–response curves at the human a4b2 nAChRreveal whether drug concentrations are sufficient for func-tional receptor interactions. These data (grey bars in Figure 3)show that unbound nicotine concentrations from tobaccosmoking (50–300 nM; Rose et al., 1999) and varenicline con-centrations after therapeutic doses (30–130 nM) will mark-edly, but not fully, desensitize the receptors. Thus, steady-state concentrations of varenicline are expected to result inlow, but sustained activation of the a4b2 receptor. In contrast,the low Bu for 1.5 mg QID cytisine (2–10 nM) is predicted tocause significant desensitization, but not a4b2 nAChR activa-tion. Finally, treatment with 40 mg BID dianicline is predictedto result in unbound brain concentrations of 40–80 nM,which will cause only minimal desensitization of a4b2nAChRs. This is likely insufficient to significantly reducecraving when quitting, and will only marginally reducenicotine-evoked reinforcing effects.

The translation of these data to the clinic is based on thehypothesis that differences in functional interactionsbetween the partial agonist and the a4b2 nAChR will result invarying degrees of relief of craving and attenuation of nico-tine reinforcement, and hence in clinical efficacy. Fluctuatingnicotine concentrations in smokers, with a fast rising phase inseconds and a slow decline over hours, are expected toproduce receptor activation followed by desensitization thatlikely contributes to the wide range of effects of nicotine(Picciotto et al., 2008). In contrast, drug concentrations with aslow onset and long half-life will mainly cause receptor desen-sitization and limited activation of a4b2 nAChRs, providedsteady-state drug concentrations are near the area of overlapbetween the desensitization and activation profiles. Thepresent data could therefore explain the higher abstinencerates observed with varenicline in smoking cessation trialsthan with cytisine and dianicline. Compared with placebo,the OR for varenicline for end-of-treatment continuous absti-nence during weeks 9–12 of a 12 week treatment period is 3.7[95% confidence interval (CI): 2.86–4.68; Nides et al., 2008].Cytisine was reported to have an OR versus placebo of 1.9(95% CI: 1.2–3.1), based on a meta-analysis of point preva-lence data of self-reported abstinence at weeks 3–8 of an8 week treatment (Etter, 2006). At present, only preliminaryphase 2 data are available for dianicline with end-of-treatment quit rates for 40 mg BID dianicline of 16 versus 8%for placebo (Fagerström and Balfour, 2006). Because in addi-tion, further development of dianicline was halted in 2008(Sanofi-Aventis press release, 2008), it is reasonable to assumethat the OR of dianicline is comparable to or lower than thatof cytisine. The abstinence rates of varenicline are also higherthan those observed with various nicotine replacement thera-pies, where nicotine acts as a full agonist at the nAChR. Acomparative meta-analysis of abstinence rates versus placeboat 6 months post-quit for available smoking cessation medi-cations showed an estimated OR (95% CI) of 3.1 (2.5–3.8) forvarenicline, while those for individual nicotine replacementtherapies were 2.3 (1.7–3.0) or lower (Fiore et al., 2008).

The notion that the capacity to interact with a4b2 nAChRssignificantly contributes to differences in pharmacological

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Figure 5 Effects of test compounds on dopamine (DA) turn-over inrat nucleus accumbens. Dose–response curves for the effects of orallyadministered varenicline, cytisine and dianicline, compared with theeffect of 1 mg kg–1 s.c. nicotine and controls. Data are expressed as %of controls � SEM. *P < 0.05, **P < 0.01 compound versus controls.



activity is supported by the relative in vivo potencies toincrease rat mesolimbic dopamine turn-over. After oraladministration, varenicline was found to be 20- and 100-foldmore potent than cytisine and dianicline, respectively, instimulating dopamine turn-over in rat nucleus accumbens(Figure 5). Interestingly, each compound produced a signifi-cantly increased dopamine turn-over in nucleus accumbensafter doses at which unbound brain concentrations fall at thecentre between their a4b2 nAChR desensitization and activa-tion curves. This is consistent with nAChR-mediated dopam-ine release being predominantly mediated via activation anddesensitization of a4b2-containing nAChRs located ondopaminergic and GABAergic neurons (Picciotto et al., 1998;2008; Mansvelder and McGehee, 2002; Mansvelder et al.,2002; Laviolette and Van der Kooy, 2004; Maskos et al., 2005).

These data illustrate that correlating unbound brain con-centrations with in vitro functional potencies can translate toclinical efficacy of partial agonists based on studies with a4b2nAChRs. In addition to b2-containing subtypes, the a7nAChR has been implicated in the effects of nicotine (Mans-velder and McGehee, 2002; Mansvelder et al., 2002). Asvarenicline has moderate affinity for a7 nAChRs (Ki = 125 nM;Table 4), we are also investigating the interactions of vareni-cline with this receptor subtype. Functional data confirm thatvarenicline acts as a full a7 nAChR agonist (107% v ACh)(Mihalak et al., 2006), but at concentrations that are signifi-cantly higher than estimated human brain concentrations. Attherapeutic doses, varenicline is predicted to minimallydesensitize a7 nAChRs and not causing detectable activationof this receptor. This suggests that a7 nAChRs plays a minorrole in the clinical effects of varenicline as a smoking cessa-tion aid. Additional nAChR subtypes, including a3*, a5* anda6* nAChRs, have been proposed to contribute to mesolimbicactivity and nicotine dependence (Drenan et al., 2008; Exleyet al., 2008; Pons et al., 2008; Grady et al., 2009; Livingstoneet al., 2009; Ramiro et al., 2009). Further work is thereforeneeded for a more thorough evaluation of the effects ofvarenicline, but the good correlation between the presentpre-clinical and clinical data argues in favour of a dominantrole of a4b2-containing nAChRs in mediating effects ofvarenicline.

Taken together, translation of pre-clinical pharmacokineticand pharmacodynamic data to the clinic suggests that annAChR partial agonist will be most efficacious as an aid forsmoking cessation if four criteria are simultaneously met. Thecompound should exhibit potent binding affinity to the a4b2nAChR, should reach sufficiently high unbound brain con-centrations to allow desensitization and some activation ofa4b2 nAChRs and should prevent inhaled nicotine frombinding at nAChRs to block its reinforcing effect. Workingtogether, these properties result in a significant increase inefficacy over other treatments that do not meet these criteria,either because of insufficient CNS drug exposure at the targetsite or because of inadequate functional potency.

In conclusion, this retrospective analysis for the first timeidentified predictors of clinical efficacy of three a4b2 nAChRpartial agonists as smoking cessation aids, providing a basisfor further studies aimed at designing more efficacious com-pounds for the treatment of nicotine dependence. The studyalso illustrates that in vitro properties of centrally acting drugs

have limited predictive value for clinical outcome withouttaking into account pharmacokinetic data to estimate humanbrain exposures.

Acknowledgements

Electrophysiology studies by S.B. and D.B. were supported byfunding from Pfizer Inc. and by the Swiss National ScienceFoundation to D.B. We greatly appreciate the contributionsand helpful discussions with Marina Shalaeva, Gus Campos,Christopher Shaffer, Hélène Faessel, Bo Feng and CharlesPotter (Pfizer Global Research and Development).

Conflicts of interests

All the authors, with the exception of the last four authorsM.dV, T.C., S.B. and D.B., are employed by Pfizer Inc., themanufacturer of varenicline (Chantix). Editorial supportfor the development of this paper was provided by PennyGorringe of UBC Scientific Solutions, and was funded byPfizer, Inc.

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Supporting Information

Additional Supporting Information may be found in theonline version of this article:

Table S1 Summary of assay conditions.Table S2 Total plasma and total brain concentrations(mean � SD) at 0.5 and 2 h after 3 mg kg–1 p.o. cytisine ordianicline in FVB/N (WT) and mdr1a/1b (–/–) (P-gp-deficient)mice.Table S3 Molecular properties of nicotine and a4b2 nAChRpartial agonists.Appendix S1 Supplementary materials and methods.

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting materials suppliedby the authors. Any queries (other than missing material)should be directed to the corresponding author for the article.



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