SUBSENSITIVITY TO PILOCARPINE O F THE AQUEOUS OUTFLOW SYSTEM IN

MONKEY EYES AFTER TOPICAL ANTICHOLINESTERASE TREATMENT

P A U L L. KAUFMAN, M.D.

Madison, Wisconsin

AND

E R N S T H . B Ä R A N Y , M . D .

Uppsala, Sweden

Topical anticholinesterase treatment induced subsensitivity to direct-acting cholinomimetics in the irides of sev­eral mammalin species, including the rhesus monkey.1 - 4 We have shown that chronic topical treatment with echothio-phate caused subsensitivity to pilocar-pine in the accommodative mechanism of vervet monkeys5 and cynomolgus mon­keys (unpublished data). A similar phe­nomenon affects the aqueous outflow apparatus of vervet and cynomolgus monkeys.

M A T E R I A L AND METHODS

Six young cynomolgus (Macaca fas-cicularis) and two adult vervet (Cerco-pithecus ethiops) monkeys were studied. All monkeys were treated in one eye with topical echothiophate iodide and in the other with a control solution by essential­ly the technique we previously de­scribed.5 However, general anesthesia was used less frequently, the cynomolgus

From the Department of Medical Pharmacology, University of Uppsala, Uppsala, Sweden (Drs. Kauf­man and Bârâny), and the Department of Ophthal­mology, University of Wisconsin Medical School, Madison, Wisconsin (Dr. Kaufman). This study was supported by National Institutes of Health grants EY 00231-10-11, 5 SOI RR-05435-14, and special fellowship 1 F03 EY 55678-01-02; The Seeing Eye, Inc., Morristown, New Jersey; and the ALZA Cor­poration, Palo Alto, California.

Reprint requests to Paul L. Kaufman, M.D., De­partment of Ophthalmology, University of Wiscon­sin Hospitals, 1300 University Ave., Madison, WI 53706.

monkeys were handled without a net, and the topical eyedrop volume was 2.5 μΐ. The effects of echothiophate on accom­modation and on the lens were also being studied and, therefore, some eyes had undergone total removal of the iris6 and pharmacological treatment before the present experiments were started. The Table gives the history of each eye and the treatment protocol of the present ex­periments. Because of the monkey's low body weight, systemic toxicity from topi­cally applied echothiophate can occur. The use of multiple small eyedrops ap­plied to the central cornea, with blinking prevented between drops, increases the proportion of the dose entering the eye and decreases the proportion absorbed systemically. However, preliminary ex­periments showed that challenging mon­keys with these echothiophate doses, even given in this careful manner, caused some of them to die or become ill within a few days. Therefore, we started treatment with lower doses and the dose was gradu­ally increased over several weeks to the level indicated in the Table.

The echothiophate we used (Phospho-line Iodide) was diluted to the desired concentration with the commercial dilu­ent. The control solution was identical except for the absence of echothiophate iodide. Pilocarpine hydrochloride for sensitivity testing was of pharmaceutical quality. Hexamethonium bromide was 99% pure. All dosages refer to the salts.

883

884 AMERICAN JOURNAL OF OPHTHALMOLOGY DECEMBER, 1976

TABLE SUMMARY OF EXPERIMENTAL PROTOCOLS*

Species, Monkey No.

Cynomolgus, 108

109

208

227

276

277

Vervet, 34

38

Eye

R h R L R L R L É L R L R L R L

Iris Present

Yes Yes Yes Yes No Yes Yes Yes Yesf No Yesf No No No No No

Drug Treatment

PI 75 μ8 Diluent Diluent PI 75 μ8 PI 88 »g Diluent PI 250 μg Diluent PI 88 μg Diluent Diluent PI 88 μg Diluentf PI 63 μ8§ PI 63 μ8§ DiluentJ

Duration, Days

158

158

72

56

172

172

110

146

Perfused After Treatment Began, days

156,194,229

156,196,239

73

57,95,138,193

73,105,137,228,267 302

73,102,137,228,268 303,

111

146,181,217

*R indicates right eye; L, left eye; PI, echothiophate iodide; diluent, control solution identical to PI solution except for absence of echothiophate iodide (not commercial diluent).

f Iris totally removed four days after topical treatment stopped. The other aniridic eyes had undergone total iris removal two months (276, left eye; 277, left eye) or 18 months (34 and 38, both eyes) before starting the current experiments.

{Treated topically with 40 μg of PI twice daily for eight weeks, ending 12 months before the start of the current experiments.

^Treated topically with 63 μg of PI and 750 μg of atropine sulfate twice daily for 121 days ending the day before the start of the current experiments.

Gross outflow facility was measured by two-level constant pressure perfusion using mock aqueous humor7 and a one-needle technique. All values were correct­ed for the internal resistance of the perfu­sion circuit. Outflow facility was deter­mined for about 30 minutes before and after each pilocarpine injection. Pilocar-pine dissolved in mock aqueous humor (pH adjusted to ~ 6.5) was injected via a micrometer syringe into the inflow tubing 3 μΐ from the eye. After allowing five minutes for the drug to wash into the eye, the anterior chamber contents were mixed by blowing cold air on the cornea for three minutes. The first eight minutes after the start of drug injection were not used in the calculation of outflow facility. Intramuscular hexamethoniüm bromide, 10 to 20 mg/kg, was given either 40 min­

utes before the start of the outflow facility determinations or after baseline outflow facility had been measured.

Slit-lamp examination was performed periodically on all animals, and immedi­ately before most perfusions. Successive perfusions in individual animals were always separated by at least four weeks and usually more. This was sufficient time for the mild postperfusion anterior segment inflammatory reaction to sub­side. Anesthesia for perfusions was by intramuscular methohexital sodium (Brie-tal), 15 mg/kg, followed by intramuscular peritobarbital sodium 35 mg/kg. For slit-lamp examinations, 5 to 10 mg/kg of a 1:1 (weightrweight) mixture of tiletamine hydrochloride and zolazepam hydro-chloride was given intramuscularly.8 For topical treatments under anesthesia, me-

VOL. 82, NO. 6 SUBSENSITIVITY OF OUTFLOW FACILITY 885

thohexital was used for the vervets and tiletamine-zolazepam mixture for the cy-nomolgi.

RESULTS

Monkey 227—One day after the cessa­tion of 56 days of echothiophate treat­ment (250 μg twice daily), the outflow facility response of the echothiophate-treated eye to graded pilocarpine doses was less than the response of the control eye (Fig. 1, A). Thirty-nine days after echothiophate treatment ended, the re­

sponses had increased considerably (Fig. 1, B), but even 82 days after echothio­phate therapy ended, the responses were still less than those in the control eye (Fig. 1, C); By 137 days after treatment, the responses in the two eyes were essen­tially the same (Fig. 1, D).

Monkey 109—The treatment period was 156 days and an echothiophate dose of 75 μg was given twice daily. Outflow facility was measured approximately five hours after the last dose of echothiophate. There was marked subsensitivity of the

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Fig. 1 (Kaufman and Bârâny). Cynomolgus monkey No. 227. A, Subsensitivity to intracameral pilocarpine after intense topical echothiophate treatment. B-D, Recovery. Abscissa indicates time; ordinates, outflow facility; C6, hexamethonium bromide; PILO, pilocarpine hydrochloride; PI, echothiophate iodide; i.m., intramuscular injection; and a.c, intracameral injection. Time interval between individual outflow facility points is four minutes. Intervals between groups of points are drawn approximately to scale, but interval between hexamethonium injection and start of initial outflow facility determination (~ 40 minutes) is foreshortened.

886 AMERICAN JOURNAL OF OPHTHALMOLOGY DECEMBER, 1976

treated eye (Fig. 2, A). Thirty-eight days after treatment ended, considerable re­covery of sensitivity had occurred (Fig. 2, B), but 81 days after treatment ended, the eyes still responded differently (Fig. 2 ,C) .

Monkey 111—A surgically aniridic eye treated twice daily with 88 μg of echo­thiophate for 73 days did not react to pilocarpine doses up to 100 μg (Fig. 3, A), and after an additional 29 days of treat­ment, doses of 1 and 5 mg barely caused a reaction (Fig. 3, B) (nonechothiophate-treated normal and surgically aniridic cynomolgus monkey eyes showed a large outflow facility increase after these large doses). Fifty-six days after cessation of 172 days of echothiophate treatment, per­fusion revealed some recovery of sensitiv­ity. Ninety-six days after echothiophate treatment ended (92 days after total iris

removal in the control eye), perfusion showed a further recovery of sensitivity (Fig. 3, C), and 131 days after echothio­phate therapy ended, perfusion yielded the results shown in Figure 3, D.

Monkey 276—The experiments were similar to those in monkey 277 except that echothiophate was given to the eye that was originally not iridectomized. The findings were essentially identical. Eyes of monkeys 276 and 277 were perfused on three occasions while receiving echothio­phate treatment; the initial perfusions were done 73 days after echothiophate was started (Table). In each animal, the ganglion-blocked outflow facility was reasonably similar on all three occasions (Fig. 4, E and F).

Monkey 38—In this vervet monkey, there was marked subsensitivity to pilo­carpine after 146 days of echothiophate

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Fig. 2 (Kaufman and Bârâny). Cynomolgus mon­key No. 109. Subsensitivity (A) and partial recovery (B and C) after moderately intense but prolonged echothiophate treatment. C6 indicates hexametho-nium bromide; PILO, pilocarpine hydrochloride; PI, echothiophate iodide; i.m., intramuscular injec­tion; and a .c , intracameral injection. Axes and time intervals same as for Figure 1.

VOL. 82, NO. 6 SUBSENSITIVITY OF OUTFLOW FACILITY 887

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Fig. 3 (Kaufman and Bârâny). Cynomolgus monkey No. 277. A and B, Subsensitivity during moderately intense echothiophate treatment. C and D, Recovery. Echothiophate-treated eye had the iris totally removed eight weeks before start of treatment; control eye underwent total iris removal four days after treatment was stopped. C6 indicates hexamethonium bromide; PILO, pilocarpine hydrochloride; PI, echothiophate iodide; i.m., intramuscular injection; a.c, intracameral injection. Axes and time intervals same as for Figure 1.

treatment (Fig. 5, A) and recovery within 35 days after echothiophate treatment ended (Fig. 5, B and C). Both eyes of this animal had been treated before (Table). The present control eye reacted well de­spite previous echothiophate treatment 18 months earlier. However, we cannot be sure that this represents full recovery. The findings in the remaining monkeys were essentially the same.

Pilocarpine dose-outflow facility re­sponse curves for the six echothiophate-treated eyes perfused on multiple occa­sions during and after treatment showed marked subsensitivity during echothio­phate treatment and recovery after treat­ment (Fig. 4).

Several of the echothiophate-treated

cynomolgus monkey eyes, including one aniridic one, demonstrated mild anterior chamber cells and flare during the first two to three weeks of treatment. We did not see this reaction in the echo­thiophate-treated vervet monkey eyes, or in any of the control eyes. We saw no cysts in any irides. We did observe poster­ior or anterior subcapsular lens changes, or both, in all the echothiophate-treated eyes."

DISCUSSION

Strong continuous cholinergic stimula­tion was provided by echothiophate that protects endogenous acetylcholine from destruction by cholinesterases. Direct-acting agents were not used since they

888 AMERICAN JOURNAL OF OPHTHALMOLOGY DECEMBER, 1976

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Fig. 4 (Kaufman and Bârâny). Pilocarpine dose-outflow facility response relationship during and after topical echothi-ophate treatment for six monkey eyes. Each panel shows all the curves for one eye. Each curve was obtained at one per­fusion experiment, and numbered according to days after echothiophate treatment was started. Each point represents the average of the three high­est outflow facility values obtained after that pilo­carpine dose. Outflow fa­cility at 0 μg of pilocar­pine represents average outflow facility after in­tramuscular hexametho-nium bromide injection (10 to 20 mg/kg) but be­fore pilocarpine injection. For the 228-day perfu­sions in two eyes (E and F), we believe that for technical reasons the pilo­carpine doses (5 and 20 μg) were actually lower, and that the outflow facil­ity after the correct doses might have been higher (the responses in the con­trol eyes of both animals were abnormally low and both doses in both eyes of both animals came from the same solution). The irides of two eyes (C and F) were totally removed before starting topical treatment; one eye (E) un­derwent total iris removal on day 176; and the irides in three eyes (A, B, and D) were not removed. Cyno indicates cynomolgus; PI, echothiophate iodide; and a .c , intracameral injec­tion.

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VOL. 82, NO. 6 SUBSENSITIVITY OF OUTFLOW FACILITY 889

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Fig. 5 (Kaufman and Bârâny). Marked subsensiti-vity (A) and recovery (B and C) after moderately intense but prolonged echothiophate treatment in vervet monkey No. 38. Both irides had been totally removed 18 months before the start of the present experiments and the control eye had received topical echothiophate for eight weeks ending 12 months before the start of the present experiments. C6 indicates hexamethonium bromide; PILO, pilocar­pine hydrochloride; PI, echothiophate iodide; i.m., intramuscular injection; and a.c, intracameral injec­tion. Axes and time intervals same as for Figure 1.

could not be eliminated when measuring outflow facility. Spontaneous release of acetylcholine during outflow facility measurement was prevented by ganglion-ic blockade with hexamethonium; the hexamethonium dosages we employed completely reversed echothiophate-in-duced accommodation for several hours in these animals. Agents such as acetyl­choline and methacholine that are sus­ceptible to cholinesterases were not used to test sensitivity of outflow facility. They would be quickly destroyed in the control eye, but protected in the echothiophate-treated eye, the degree of protection de­creasing progressively after echothio­phate treatment had ceased. This would preclude comparisons between echothio-

phate-treated and control eyes, and be­tween the echothiophate-treated eyes dur­ing and after treatment. Such agents could be protected in nonechothiophate-treated eyes for the few hours a perfusion experiment requires by a short-acting cholinesterase inhibitor such as physo-stigmine, but one cannot assume that pro­tection would be as complete as in the chronically echothiophate-treated eye. Since equivalence of dose is essential when comparing responses, only direct-acting cholinergics that are not destroyed by cholinesterases, such as pilocarpine or carbachol, could be used to test sensitivi­ty of outflow facility.

The data show that chronic topical echothiophate treatment causes a loss of

890 AMERICAN JOURNAL OF OPHTHALMOLOGY DECEMBER, 1976

the outflow facility response to intra-cameral pilocarpine doses of up to 5 mg, which are at least 50 times supramaximal in normal vervet and cynomolgus mon­key eyes. Since subsensitivity of the ac­commodative mechanism to pilocarpine is caused by chronic echothiophate treat­ment,5 at least part of the loss of the facility response is probably due to sub-sensitivity of the ciliary muscle. Howev­er, we do not know whether there are additional factors involved. For instance, there could be an effect on the outflow channels increasing their resistance. Out­flow resistance after hexamethonium in­jection tended to be slightly higher in the echothiophate-treated eyes, but the num­ber of animals was too small to prove this point. We did not study the time course of the development of subsensitivity. The earliest perfusion was done eight weeks after starting echothiophate treatment (cynomolgus monkey 227, Fig. 1, A), by which time subsensitivity was already marked. Profound subsensitivity persist­ed for the duration of the echothiophate treatment in all eyes.

We noted a significant recovery of the outflow facility response within five to six weeks after stopping echothiophate treat­ment. A partial recovery of sensitivity of the accommodative mechanism also oc­curs within this time (unpublished data). We do not know whether the recovery of the outflow response is partial or com­plete. If the initial outflow resistance of the two eyes is different, the conclusion may depend on whether one looks at outflow facility or resistance. Thus, re­covery may appear incomplete on the outflow facility scale (Fig. 3, D), but if the data are plotted on a resistance scale, the two eyes react similarly. Furthermore,, comparison with the control eye may not be ideal. For instance, if maximal con­traction of the ciliary muscle causes changes in the trabecular meshwork that are slowly reversible, the control eye

would be more affected by the previous pilocarpine infusion than the less sensi­tive echothiophate-treated eye. We ob­served such decreased responsiveness of a control eye (Fig. 1).

We did not directly test for subsensi­tivity to acetylcholine by intracameral in­jection. However, since acetylcholine and pilocarpine presumably act on the same muscarinic cholinergic receptor, continu­ous exposure of tissue to high levels of either probably induces subsensitivity to both. In the few experiments in which outflow facility was determined before ganglionic blockade, outflow facility in the echothiophate-treated eye was no higher than in the nonechothiophate-treated opposite eye (Fig. 3, B). Presum­ably, excess acetylcholine was negated by subsensitivity to acetylcholine. However, we cannot say that echothiophate always becomes ineffective with prolonged use. Our monkeys were deeply anesthetized, which may have diminished spontaneous acetylcholine release. Moreover, the de­gree of response will depend on the bal­ance between subsensitivity to acetylcho­line on the one hand and potentiation of acetylcholine on the other. Thus, a full response may or may not be obtained in the presence of profound subsensitivity.5

If our findings are applicable to the glaucomatous human eye, three clinical consequences are possible: (1) cholines-terase inhibitors, and perhaps even direct-acting cholinomimetics, may lose their pressure-lowering effect with time not only because of progress of the dis­ease but also because of induced cho­linergic subsensitivity; (2) the com­mon practice of starting pilocarpine treatment after discontinuing cholinester-ase inhibitor treatment several weeks be­fore glaucoma or cataract surgery10 may be a fruitless exercise; and (3) if for some reason one wishes to institute long-term therapy with direct-acting cholinomim­etics after a period of treatment with

VOL. 82, NO. 6 SUBSENSITIVITY O F OUTFLOW FACILITY 891

cholinesterase inhibitors, one may have to wait several weeks to months to assess adequately the effectiveness of the di­rect-acting agent.

SUMMARY

Cynomolgus and vervet monkeys were treated unilaterally with topical echothio­phate iodide twice daily for eight to 25 weeks. The effect of intracameral pilocar-pine on outflow facility was determined in the ganglionic-blocked animal during and after echothiophate treatment. The echothiophate-treated eyes demonstrated marked subsensitivity to pilocarpine, and required several weeks to months to re­cover normal pilocarpine sensitivity.

REFERENCES 1. Bito, L. Z., Hyslop, K., and Hyndman, J.:

Antiparasympathomimetic effects of cholinesterase inhibitor treatment. J. Pharmcol. Exp. Ther. 157: 159, 1967.

2. Bito, L. Z.: The absence of sympathetic role in anti-ChE induced changes in cholinergic transmis­sion. J. Pharmacol. Exp. Ther. 161:302, 1968.

3. Bito, L. Z., and Dawson, M. J.: The site and mechanism of the control of cholinergic sensitivity. J. Pharmacol. Exp. Ther. 175:673, 1970.

4. Bito, L. Z., and Banks, N.: Effects of chronic cholinesterase inhibitor treatment. 1. The pharma­cological and physiological behavior of the anti-ChE-treated monkey (Macaca mulatto) iris. Arch. Ophthalmol. 82:681, 1969.

5. Kaufman, P. L., and Bârâny, E. H.: Subsensi­tivity to pilocarpine in primate ciliary muscle fol­lowing topical anticholinesterase treatment. Invest. Ophthalmol. 14:302, 1975.

6. Kaufman, P. L., and Lütjen-Drecoll, E.: Total iridectomy in the primate in vivo: surgical tech­nique and postoperative anatomy. Invest. Oph­thalmol. 14:766, 1975.

7. Bârâny, E. H.: Simultaneous measurement of changing intraocular pressure and outflow facility in the vervet monkey by constant pressure infusion. Invest. Ophthalmol. 3:135, 1964.

8. Kaufman, P. L., and Hahnenberger, R.: CI-744 anesthesia for ophthalmological examination and surgery in monkeys. Invest. Ophthalmol. 14:788, 1975.

9. Kaufman, P. L., Axelsson, U., and Bârâny, E. H.: Induction of subcapsular cataracts in cynomol­gus monkeys by echothiophate. Arch. Ophthalmol. In press.

10. Kolker, A. E., and Hetherington, J., Jr.: Becker-Shaffer's Diagnosis and Therapy of the Glaucomas, 3rd ed. St. Louis, C. V. Mosby, 1970, p. 308.