TOXICOLOGY AND APPLIED PHARMACOLOGY 82, 14- 18 ( 1986)

Alteration of in Viva and in Vitro Effects of Heroin by Esterase Inhibition

GERALD GIANUTSOS,**' STEVEND.COHEN,* GUSTAF~ARLSON,* RICHARDHEYMAN,* PAULSALVA,=~GALEMORROW,* ANDGILBERTJ.HITE~

*Section of Pharmacology and ToxicoIogy; and tSection ofMedicinal Chemistry and Pharmacognosy, University of Connecticut, School of Pharmacy, Storrs, Connecticut 06268

Received September 5, 1984; accepted August 16, 1985

Alteration of in Vivo and in Vitro Effects of Heroin by Esterase Inhibition. GLWUTSOS, G., COHEN, S. D., CARLSON, G., HEYMAN, R., SALVA, P., MORROW, G., AND Hm, G. J. (1986).

Toxicol. Appl. Pharmacol. 82, 14-l 8. Selective inhibition of peripheral esterases by tri-orthc+tolyl phosphate in the mouse resulted in an increase in the analgetic activity of heroin, without affecting the activity of morphine. In vitro inhibition of esterases by paraoxon reduced the affinity of heroin for the opiate receptor, while that of morphine was una&cted. These results suggest that both central and peripheral esterases are involved in the metabolism of heroin and that interference with critical e&erases can alter its pharmacologic and toxicologic effects. Q 1986 AC&C& FWB, IIK.

It is generally accepted that heroin (diacetyl morphine) is metabolized by sequential deacetylation to 6-monoacetyl morphine (MAM) and then to morphine, with concom- itant changes in its pharmacological activity (Way et al., 1965). However, the enzymatic basis of its metabolism is not clearly estab lished. Wright ( 194 1) compared the ability of rabbit tissues to hydrolyze heroin and found that liver and kidney were more active than brain in contrast to their ability to hydrolyze acetylcholine. This led to the conclusion that cholinesterase (ChE) was not responsible for the metabolism. Nevertheless, it is known that in many species, including man, the half-life of heroin in blood is only a few minutes (Cohn et al., 1973; Lockridge et al., 1980). Ellis (1948) reported that hydrolysis of the phenolic acetate was closely associated with the activity of plasma tributyrinase, while Smith and Cole ( 1976) identified an arylesterase as the enzyme

’ To whom correspondence should be addrez& Section of Pharmacology 6’~ Toxicol~, University of Connecticut, Box U-92, Storrs, Conn. 06268.

responsible for metabolism. Lockridge and co- workers ( 1980) proposed that human serum ChE hydrolyzed heroin in vitro, while more recently, an RBC-associated esterase has been implicated (Owen and Nakatsu, 1983). Since. lipophilicity is in the order heroin > MAM > morphine, the greater toxicity of heroin on peripheral administration may be attributed to its enhanced ability to cross the blood-brain barrier. On the other hand, the greater molar toxicity of morphine upon intracerebral ad- ministration would suggest that heroin and MAM must be deacetylated for optimal phar- macological activity (Way et al., 1965). Similar LD50 values for heroin and MAM after pe- ripheral administration suggest that heroin is rapidly deacetylated to MAM, which then crosses the blood-brain barrier in order to ex- ert its action (Way and Adler, 1960; Way et al., 1965).

In this study, we have attempted to assess the importance of peripheral and central es- terases in mediating the action of heroin, Tri- ortho-tolyl phosphate (TOT?) was chosen for the in vivo studies because it has been shown

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ESTERASES AND HEROIN 15

to selectively inhibit peripheral ChE and car- boxylesterase (CE) activities without inhibiting brain ChE activity. Paraoxon was employed to inhibit brain ChE in vitro and to determine its effect on the binding of heroin and its me- tabolites to brain opiate receptors. If esterases play a role in the metabolism of heroin, it would be expected that its pharmacological activity would be altered in the presence of esterase inhibition.

METHODS

Analgesia. Analgetic activity was measured by the acetic acid-induced writhing method described by Taber and co- workers (1969). Male mice (CD-I; 25-35 g, obtained from Charles River Farms, Wilmington, Mass.) were housed eight per cage in a climate-controlled environment under a 12-hr period of light (lights on 6 AM). The mice were

allowed free access to feed (Purina Lab Chow) and water. During the analgesia tests, they were injected ip with 0.6% acetic acid in a volume of 10 ml/kg. Groups of two to three mice were immediately placed in an opaque plastic container and, after a delay of 5 min, the number of writhes were counted for the next 10 min. Writhing was defined as hind-limb extension with both rotation and an arching of the back. To assess analgetic activity, morphine sulfate, MAM HCI, or heroin HCI was injected sc 30, 20, or 10 min before the acid challenge, respectively. These pre- treatment intervals provided the most consistent and re- producible anafgetic activities. All drug solutions were freshly prepared in deionized, distilled water and admin- istered in a volume of 10 ml/kg, sc. Doses are expressed in terms of milligrams per kilogram of salt.

An injection was deemed to be analgetic if the number of writhes exhibited by a mouse differed from (was less than) the average obtained in controls on the same day by a factor of at least twice the standard error of the control mean. Percentage of protection (i.e., number showing an- algesia over total number of mice tested at a specific dose) was then calculated for each narcotic dose. The analgesic ED50,95% confidence intervals, and potency ratios were obtained by the method of Litchtield and Wilcoxon ( 1949).

Drug and treatment schedules. TOTP (Eastman; tech- nical grade) was dissolved in corn oil to provide a dose of 125 m&kg in an injection volume of 5 ml/kg (Cohen and Ehrich, 1976). TOTP or a corn oil control was injected I8 hr prior to treatment with narcotic drug. To minimize circadian effects, pretreatments were given in late after- noon, and all analgesia tests were performed the following morning. Control or TOTP-treated mice used in the an- algesia test were terminated immediately aBetwards for ChE and CE determinations.

For in vitro inhibition of brain ChE, paraoxon (1 PM) was preincubated with the opiate receptor preparation for

30 min prior to the start of the binding assay. Paraoxon (Chem Service Co.; ~-95% pure) was stored in the freezer as a lo-* M stock solution in 100% ethyl alcohol. Dilutions were prepared with appropriate buffer immediately before -Y.

Esteruse assays. Tissue ChE activity was measured spectrophotometrically with acetylthiocholine iodide serving as the substrate for hydrolysis by the method of Ellman et al. (1961) as modified by Ehrich and Cohen (1977).

Tissue hydrolysis of alpha naphthyl acetate was mea- sured as an indicator of CE activity (Ecobichon, 1970). Tissue was incubated with substrate for 30 min at 37°C inO. Mphosphatebuffer(pH7.4).Thereactionwasstopped with TCA, and the resulting a-naphthol was measured at 332 nm.

Receptor binding. Drug affinity for opiate binding sites was determined by the displacement of [3H]etorphine as described by Simon et al. (1973). Brains were removed from male rats (CD, Charles River) and the tissue minus cerebellum was homogenized in 6 vol of chilled 0.32 M sucrose. The homogenate was centrifuged first at IOOOg for 5 min to remove nuclear and tissue debris, and the resulting supemate was centrifuged at 47,000g for 20 min to obtain the crude membrane pellet. This pellet was washed twice in ice-cold, 50 mM Tris buffer (pH 7.4) and frozen for the assays. On the day of assay, the pellet was again washed in 45 vol of the Tris buffer, centrifuged, and resuspended. A 1.95-ml ahquot of the suspension was in- cubated at 37’C for 5 min with various concentrations of unlabeled drug (or no drug) in the presence or absence of 1 pM paraoxon. [3H]Etorphine (sp act 46 Ci/mmol, Amersham) was added to a final concentration of 0.3 nM, and the incubation proceeded for 15 min. The samples were then rapidly filtered under reduced pressure through Whatman GF/B filters and the trapped tissue was washed three times with 3 ml of ice-cold Tris buffer with a Brandell Cell Harvester. Filters were placed in scintillation vials with 0.5 ml of Protosol (NEN). AAer addition of ScintiVerse E (Fisher), the samples were quantified by liq- uid scintillation spectrometry. AU samples were performed in triplicate. Specific binding was defined as label displac- able by 1 PM levorphanol. Affinity for the binding she was expressed as ICSO values, i.e., the concentration of drug needed to displace 50% of labeled etorphine from the binding site as calculated by linear regression. Values in the tables am derived from data pooled from three separate experiments performed at different times with tissue from different sets of animals (except for MAM which was run once).

Drug synthesis. An analytically pure sample of heroin was synthesized in 97% yield by a modification of the pro- cedure of Garrett and Gurkan (1979). Morphine base (0.93 g) was dissolved in 50 ml of acetic anhydride and 2 drops of trietbylamine was added. The reaction vessel was stop pered, heated to 60°C for 4 hr, and then stirred at room temperature for 2 days. Excess acetic anhydride was vol- atilized at room temperature under a stream of nitrogen

16 GIANUTSOS ET AL.

and the resulting yellow oil was dissolved in 30 ml of cold 5% sodium carbonate. This mixture was extracted five times with cold chloroform. The chloroform solution was dried over sodium sulfate, diluted with ether, decolorized with No&-A, filtered, and evaporated to yield 1.17 g of heroin base. The heroin was recrystallized twice from ethyl acetate to yield white plates melting at 172-l 73°C (lit. 170- 17 1 “C, Garrett and Gurkan, 1979). The crystals were dissolved in a large volume of anhydrous ether and titrated with dilute, dry etheral-HCl to yield the HCl salt as a white precipitate melting at 228°C (lit. 227-229”C, Garrett and Gurkan, 1979).

An analytically pure sample of MAM was prepared in 87% yield according to the method of Wright (1941). Morphine base (500 mg) was dissolved in 40 ml of acetic anhydride and the solution was heated to 90°C for 24 hr. After removal of the acetic anhydride under a nitrogen stream, 20 ml of water and 360 mg of hydroxylamine HCI were added. The mixture was heated to 90°C for 30 min and was left to stand at room temperature. A crystalline solid formed which was collected and washed with ether, dissolved in water, and decolorized with No&A. The aqueous solution was reduced in volume and crystals formed which sintered at 130°C and melted with decom- position at 3 10°C. The material was dried under reduced pressure at 55°C. The resulting solid melted with decom- position at 285-290°C (lit. 295°C; Wright, 1941).

RESULTS

In preliminary studies, acetic acid-induced writhing was compared in mice pretreated with corn oil or TOTP (125 mg/kg). Table 1 indicates that under the conditions of this test, TOTP did not exhibit analgesic activity. Also shown in Table 1 are the results of tissue es-

terase analyses from control and TOTP-pre- treated mice. TOTP significantly inhibited mouse plasma ChE and CE activity by 80 to 90% without any significant effect on ChE ac- tivity in the brain. Together, these findings in- dicate that TOTP would be a useful tool to evaluate the importance of peripheral esterases to narcotic analgesic ester action.

To determine the effect of peripheral ester- ase inhibition on the analgesic action of nar- cotic esters, groups of mice were pretreated with corn oil or TOTP (125 mg/kg) and were challenged 18 hr later with morphine, MAM, or heroin for the assessment of analgesia. The results are presented in Table 2. In control mice, MAM and heroin were of similar po- tency, being two to three times more potent than morphine. TOTP pretreatment caused a six-fold increase (p < 0.05) in the analgesic potency of heroin without significantly altering the potency of MAM or morphine.

The above results suggest that peripheral esterases play a significant role in the analgesic action of heroin. To evaluate the role of central esterases on the action of heroin, as deter- mined by the affinity of heroin for the opiate receptor, we added paraoxon to a receptor- enriched membrane fraction from control rat brain. This pretreatment resulted in complete inhibition of ChE activity in the receptor preparation. Paraoxon alone did not alter the binding of the radioligand, [3H]etorphine,

TABLE 1

EFFECT OF I8-hr PRETREATMENT WITH TOTP ON MOUSE TISSUE ESTERASE AC?-IVITIES

AND ACETIC ACIDINDUCED WRITHING * b

Corn oil TOTP (5 ml/kg) (125 mg/5 ml/kg)

Brain cholinesterase 12.4 + 4.5 (10) 11.4 f 1.6 (20) Plasma cholinesterase 2.9 f 0.6 (10) 0.6 f 0.2 (20)’ Plasma carboxylesterase 23.0 f 1.8 (10) 2.1 + 0.4 (2O)C

Writhina 30 + 7 (17) 25 f 6 (8)

’ Esterase activities are expressed as micromoles of substrate hydrolyzed per minute per gram (wet weight) or milliliter of tissue, X f SE (n).

b Writhing data are expressed as the total number of writhes detected per mouse during a IO-min observation period, 2 t SE (n).

cSignificantly different from corn oil control (p < 0.05) by Student’s t test.

ESTERASES AND HEROIN 17

TABLE 2

E!=FECX OF TOTP PRETREATMENT ON NARCOTIC- INDUCED ANALGESIA

Analgesia EDSO’

Narcotic Control TOTPb Ratio’

Morphine 0.62 (0.33-l. 17) 0.48 (0.23-0.82) 1.3 MAM 0.28 (0.18-0.43) 0.14 (0.09-0.22) 2.0 Heroin 0.24 (0.20-0.28) 0.04 (0.02-0.07) 6.0d

’ A minimum of six doses and five mice per dose was used to estimate the dose which would cause analgesia in 50% of the animals (ED50).

’ TOTP ( 125 mg/kg) was injected 18 hr before analgesia testing. Values are ED50 (mg/kg) for narcotic drug; 95% confidence intervals in parentheses.

’ ED50 control/ED50 TOTP. d Significantly different from control by method of

Litchfield and Wilcoxon (1949).

suggesting that it did not directly affect recep- tor binding. Furthermore, data in Table 3 in- dicate that paraoxon did not alter the binding of morphine. On the other hand, paraoxon pretreatment decreased the receptor-binding affinity of heroin approximately threefold and, to a lesser extent, that of MAM.

DISCUSSION

The present study was undertaken to de- termine the effect of selective inhibition of tis- sue esterases on the action of narcotic ester analgesics. Inhibition of peripheral esterases by TOTP markedly enhanced the analgetic activity of heroin without significantly affect- ing that of MAM or morphine. On the other hand, inhibition of brain esterases in vitro re- duced the affinity of heroin for opiate binding sites (at least of the mu type labeled by etor- phine), without affecting the affinity of mor- phine. Appropriate controls were included to demonstrate that TOTP pretreatment was not analgetic on its own, and that paraoxon did not alter the binding of radioligand to the opi- ate receptor.

The inhibition of plasma esterases by TOTP and the accompanying potentiation of the an- algetic potency of heroin are consistent with the idea that esterases, e.g., ChE and CE of plasma and other tissues, may play a role in

the metabolism of heroin. The enhanced po- tency could result from decreased hydrolysis in the periphery and enhanced CNS penetra- tion by the more lipophilic parent molecule. This is in agreement with Lockridge and co- workers ( 1980) who suggested that serum cho- linesterase may be the principal enzyme for heroin hydrolysis. The lack of potentiation of morphine analgesia by similar TOTP treat- ment is also consistent with the above expla- nation, since morphine contains no ester link- age and would not be expected to be a substrate for tissue esterases.

The in vitro findings are consistent with the idea that once it reaches the CNS, heroin, and possibly MAM, must be hydrolyzed for opti- mum receptor binding. Thus, in vitro addition of paraoxon at a concentration which inhibited esterase activity reduced the ability of heroin to interact with the opiate receptor. This would be expected if hydrolytic removal of the phe- nolic ester of heroin was normally mediated by central esterases. In the presence of para- oxon, the heroin would be expected to have remained intact and the phenolic ester group impeded optimal receptor interaction. These results, taken together, would be in agreement with the conclusion of Way et al. (1965) that the greater potency of injected heroin com- pared with morphine is due to pharmacoki- netic factors rather than an enhanced intrinsic pharmacological activity, since heroin (in the presence of esterase inhibition) was approxi- mately l/30 as potent as morphine in its af- finity for the opiate receptor.

TABLE 3

EFFECT OF PARAOXON ON AFFINITY OF NARCOTICS

FOR OPIATE BINDING IN VITRO

Affinity for etorphine displacement”

Dw Control Paraoxon Ratio b

Morphine 22 (14-35) 23 (15-35) 1.0 MAM 90 155 1.7 Heroin 200(130-300) 610(330-1100) 3.0

’ Affinity expressed as IC50 (nM); 95% confidence in- tervals in parentheses.

b IC50 paraoxon/IC50 control.

18 GIANUTSOS ET AL.

These results also illustrate the potential importance of peripheral and central esterases in modulating the activity and toxicity of nar- cotic analgesic esters as well as the many other drugs which may require ester hydrolysis for activation, inactivation, or both (Heyman, 1982). Toxic interactions might result when one takes such drugs after exposure to esterase- inhibiting doses of other substances. For ex- ample, exposure to organophosphate insecti- cides, at doses which produce no outward signs of toxicity, may greatly inhibit nonvital tissue esterases (Cohen, 1984). Under such condi- tions, there may be potential added risk in the use of narcotic esters like heroin.

In another light, the present study suggests the need for caution in the design and inter- pretation of in vitro structure-activity rela- tionship (SAR) studies which use receptor binding as an endpoint. For example, the binding performed in the absence of paraoxon would have led to a false estimate of the po- tency of heroin and would have underesti- mated the effect that the acetylation of mor- phine would have had on binding affinity.

In summary, this study has demonstrated that central and peripheral esterases play dif- ferent roles in the action of heroin. The former may activate heroin near its site of action while peripheral esterases may participate in its de- toxification.

ACKNOWLEDGMENTS

This work was supported by NIDA Grant DA 01612 (to G.J.H.). We thank Hoffmann-LaRoche (Nutley, N.J.) for providing levorphanol, and Ellen Ambelas for her con- tributions in measuring brain esterase activity.

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