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Volume 14 Reports 159 Number 2

pargyline, Pharm. Res. Commun. 1: 20-24, ethyl-N-pyridyl-4-methyltropamide), cyclopcnto- 1969. late [2-dimethylaminoethyl-ff-( 1-hydroxycyclopen- 8. Schumann, H. |., and Philippu, A.: Release of tyl )-«-phenyl-acetate] and phospholine iodide catechnlamines from isolated medullary gran- (echothiolate, S-ester of (2-mercaptoethyl)-tri- ules by sympathomimetic amines, Nature 193: methylammonium iodide with 0,0-diethylphos- 890, 1962. phorothioate) were ordinary ophthalmic solutions. 9. Mosnaim, A. D., Inwang, E. E., and Sabelli, • HBr and were obtained H. C: The inHuence of psychotropic on the levels of endogenous 2-phenylethylamine in from Aldrich and DFP (diisopropylphospho- rabbit brain, Biol. Psychiat. 8: 227-34, 1974. fluoridate) from Fluka; DMAEA (2-acetoxy-l- dimethylaminoethanol) and were syn- thesized by acylation of the appropriate amino with acetic anhydride in pyridine. A rapid method for measuring miotic A mouse is placed under a binocular microscope activity of drugs in the intact mouse (Nikon, :<40). The eye of the mouse is sharply focused and the lengths of the horizontal diameter eye. GIOKA THEISTEH, SAUL MAAYANI, AND of the whole eye and the pupil are accurately MORDECHAI SOKOLOVSKY. read by one observer, using a scale located be- A rapid and. precise method for evaluating the tween the ocular (x20) and the objective (x2). miotic activity of drugs has been de- The scale contains 100 divisions per centimeter. veloped based on Long's method, for measuring The light source was kept at a fixed distance of the rate of mydriasis. The rate of reversal of 10 cm. from the eye of the mouse. The illumina- mydriasis developed, previously in the intact mouse tion source (6 volts, 5 amperes) was obtained eye by a mild mydriatic () is used from Eliza (Tokyo). Illumination was kept con- stant by the use of a transformer (Eliza, Tokyo). to evaluate the miotic activity. The method -1 provides a useful tool for measuring and compar- A drop (ca. 50 A*l) of 10 - M phencyclidine in ing the miotic activity of acctylcholine 0.1 M phosphate buffer, pH 7.8, is then placed and cholinestcrasc inhibitors. carefully on the eye of the animal without touch- ing the cornea and wiped out gently 20 seconds Measuring the change in the diameter of the later. After an interval of more than three min- pupil in response to certain autonomic drugs is a utes, the diameter of the pupil is again measured well-known method whereby several parameters under the microscope. The 50 /xl drop covers the of the eflect can be evaluated, namely, its surface of the eye, and its excess was removed onset period, rate of action, magnitude, and dura- quantitatively at the appropriate intervals simply tion. by absorption with a piece of cotton without In order to measure accurately a miotic re- touching the cornea. This procedure which did sponse, however, one has to eliminate the disturb- not influence the diameter of the pupil was pre- ing light reflex. A simple way involves only the ferred over washing with, e.g., saline, since the use of a transparent ruler in a dimly lighted latter can cause a damage to the corneal epithe- room,1' 2 but this method is not ideal, mainly lium. because it is difficult to measure accurately in Those animals having a pre-experimental pupil the dim light needed to eliminate most of the diameter more than 25 per cent of the whole light reflex. eye diameter or mice which did not develop a Several types of pupillometers were developed mydriasis of at least 80 per cent of the whole during the last three decades,3- •* but none were eye, are rejected. These criteria eliminate about able to provide highly accurate measurements 20 per cent of the tested animals. The population totally without light reflex, save for the complex of rejected animals was not influenced by age or infra-red electronic pupillograph constructed by sex. Loewenstein and co-workers.57 A drop of the miotic agent is then applied to In the present communication, we present a the eye and the rate of the reversal of the simple, accurate method for measuring miotic and mydriasis is measured every 30 seconds. mydriatic pupillary responses in the mouse eye To study an effect of a mydriatic drug after which completely eliminates the light reflex. The the initial measurements of the diameters of the method is based on that previously described by whole eye and the pupil, a drop of the drug Armaly and Long.8 is applied to the eye and the development of Materials and methods. Phencyclidine-HCI [1(1- mydriasis is followed continuously, according to phenylcyclohexyl)] was prepared accord- Armaly and Long.8 The interval between read- ing to Kalir and co-workers." (sulphate, ings depends on the rate of onset and the dura- hydrate) and eserine () in the tion of the elTect. Pupillary change is expressed salycilate form were obtained from Sigma Chemi- as percentage of the pupil diameter of the whole cal Company. Mydriaticum (tropic amide, N- eye. Values presented throughout this report are

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20

120 240 360 720 Time (sec.)

20

80 120 Time (min.) Fig. 1. Onset (a) and duration (b) of niyclriatic activity of clinically used niyclriatic drugs: A—A ahopine sulphate (101 M), O—O mydriaticum (fi x 1Q-' M), :•: — :•: eyclopentolate (3 >'- 10"' M), and •—• phencyclidine (10"' M). Each point represents the mean results of experiments on six animals. Horizontal bars denote standard error of the mean. 1. Time of drug application.

the mean of at least six experiments, carried out the pupil diameter of the whole eye was found by one observer, on six dilierent animals. to be 90 ± 2 per cent scale units (S.E.M.). It Reproduction of results was checked by taking should be noted that similar results were obtained six successive pupillary diameter readings of the when several observers participate in the measure- resting pupil under the conditions of strong and ments. constant illumination described above, and six Results and discussion. The method of compar- successive dilierent readings of the diameter of a ing the miotic activity of drugs is based on com- maximum niyclriatic pupil. Measurements were petition between the miotic drugs tested and the carried out at 15-second intervals by one observer. mild niyclriatic effect of phencyclidine1" in the Diameter of the resting pupil expressed as per- cholinergic system of the sphincter of the intact centage of the pupil diameter of the whole eye, nuirine eye. The rate of reversal of the previously was found to be 20 i 2 per cent scale units developed mydriasis is measured under standard (S.E.M.). Diameter of the pupil at the maximum defined conditions. obtainable mydriasis expressed as percentage of The optimal niyclriatic agent should induce a

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ARECOLINE

100

80 CYCLO.

g ® •o

20 PHEN.

0 30 120 210 300 390 480 Time (sec.) Fig. 2. Rate of reversal of previously developed mydriasis by 10 ^ M arecoline. One drop of the drug placed on the mouse eye: full mydriasis had been developed previously by (3 x 10-' M), mydriaticum (6 x 10-' M), or phencyclidine ( 10 ;1 M). Each point represents the mean results of experiments on six animals. Horizontal bars denote standard error of the mean.

full and rapid mydriasis while having a sufficiently action, the long duration of its effect, and its high low affinity to the cholinergic receptor to permit affinity to the receptor—evidenced by the low efficient competition by the miotic drugs. In the concentration needed to induce full mydriasis. search for optimal performance, the mydriatic The mydriasis developed by phencyclidine (Fig. activities of atropine, cyclopentolate, and mydriat- 2) was antagonized by arecoline faster than that icum at the minimal concentrations needed to developed by either of the other two anti- induce full mydriasis in the mouse eye were com- cholinergic drugs. pared to that of phencyclidine. From the data presented in Figs. 1 and 2, it A measure of the relative affinity of the drugs seems that among the four drugs to the sphincter cholinergic receptors may be tested, phencyclidine has the lowest affinity to obtained by determining both the minimal con- the cholinergic: receptors in the sphincter muscle; centrations at which maximal mydriasis may be both a relatively high concentration is needed to induced and the duration of mydriatic activity. develop full mydriasis (3 x 10"'1 M), and its As shown in Fig. 1, phencyclidine activity had mydriatic activity is of short duration ( 15 min- the shortest duration (the plateau, 15 minutes), utes). Indeed, the affinity constant of phen- while atropine activity persisted for more than cyclidine to a peripheral muscarinic receptor was two hours. The time of onset of complete mydria- found to be lower by three orders of magnitude sis for mydriaticum and cyclopentolate is similar than that of atropine.1" to that for phencyclidine, around 3.4 minutes— The sensitivity of the mouse eye to phencycli- while the time of onset of atropine activity is dine is not influenced by age (among the age about 15 minutes. The relative anticholinergic group of 3 to 6 months) or sex of the animals potencies of the drugs may also be compared by (see also methods). competing them with a cholinergic drug on the The effect of the contact time (see methods) same receptor. One drop of 10~'-' M arecoline (in of phencyclidine solution with the mouse eye on 0.1 M phosphate buffer, pH = 7.4) was applied the time -of onset of mydriasis was measured locally to the same eye in which mydriasis had (Figs. 3 and 4). The rate of the developing been induced previously by one of the mydriatic mydriasis after contact times of five and twenty drugs. The rate of the reversal of the mydriasis seconds is shown in Fig. 3 and data are presented caused by the miotic agent was measured using in Fig. 4 for the five different contact times the technique described above (Fig. 2). Atropine measured. Increasing the contact time from 5 to was omitted in this experiment due to its slow 40 seconds shortened the time needed for the

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100

•2 60

20

120 240 Time (sec.) Fig. 3. Effect of contact time of phencyclidine (HCl) solution ( 10"- M in 0.1 M phosphate buffer, pH = 7.4) on the rate of the developing mydriasis. See text for experimental details. •—• 5 seconds, O—O 20 seconds. Each point represents the mean results of experiments on six animals. Horizontal bars denote standard error of the mean.

300

»° 100

10 30 180 Time contact (sec.)

Fig. 4. Effect of increasing the contact time of phencyclidine (HCl) solution with the mouse

eye on the time needed for development of full niydriasis (Tn,.,s). Each point represents the mean results of experiments on six animals. Vertical bars denote standard error of the mean.

development of full niydriasis from five to two The relative miotic potencies of some well- minutes, the minimal onset time obtainable (Fig. known cholinergic drugs as determined by this 4). A 20-second contact time was chosen as method are summarized in Table I. Three minutes optimal for all further experiments. after phencyclidine treatment, one drop of the On the basis of these experiments, the follow- drug to be .tested, in bullered solution, was ap- ing standard conditions were chosen for develop- plied to the treated eye and the pupil diameter ing niydriasis in order to evaluate the miotic was measured. Comparative values of miotic activity of choLinergic drugs: (1) the duration activity are obtained by determining the time of a single test is 10 minutes, which is the dura- needed for reversal of an arbitrarily selected per- tion of full niydriasis induced by 10"- M phen- centage of the previously developed niydriasis, cyclidine in 0.1 M phosphate bnlfer, pH 7.8; and e.g., 50 percent (T V2 ) (Table I). (2) the contact time of the phencyclidine solution There are several biological parameters which with the mouse eye is 20 seconds. might influence the overall biological response of

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Table I. Reversal of activity by cholinergic drugs 2. Mamo, J. C, and Leopold, I. H.: Evaluation acting on previously developed mydriasis in the and use of oximes in ophthalmology, Am. J. intact mouse eye Ophthalmol. 46: 724, 1958. 3. Wolf, A. U., and Hodge, H. E.: Effect of Concen- r 50% f atropine sulfate, nitrate tration" (sec) (metropine), and homatorpine hydrobromide Drug (M) ± S.E.M. on adult human eyes, Arch. Ophthalmol. 36: 293, 1946. I. ACh-likc: 4. Cogan, D. G. P.: A simplified entopic pupil- Arecoline 10-- 53 ± l.fi loineter, Am. J. Ophthalmol. 24: 1431, 1941. Oxotremorine 10-- 203 ± 5.4 5. Loewenstein, O., and Loewenfeld, I. E.: Aceclidine 354 t 14.3 DMAEAt 2 x 10 - 387 t 7.5 Electronic pupillography. A new instrument and some clinical applications, Arch. Ophthal- II. ChE inhibitors: mol. 59: 352, 1958. Eserine (salycilate) 10-- 94 t 4.6 6. Loewenfeld, I. E.: The iris as pharmacologic Phospholine'( iodide) 1.6 x 10- * 425 t 17.3 indicator. I. Effect of physostigmine and of DFP§ 10-- 303 + 7.6 on pupillary movements in normal man, Arch. Ophthalmol. 70: 42, 1963. One drop of the mydrintic drug was applied locally to the intact mouse eye which was kept under strong and 7. Ogle, K. N., Whisnant, R. H., and Hazelrig, constant illumination. The pupil diameter was measured J. B.: Quantitative study of pupil response by a binocular microscope (x40) every 30 seconds. to miotic drugs, INVEST. OPHTHALMOL. 5: °ln 0.1 M phosphate buffer, pH = 7.0. fTime needed for reversal of the usually 90 per cent 176, 1966. 90-20 8. Armaly, M. F., and Long, J. P.: Factors developed mydriasis (from 20 per cent) to ( + 20) influencing the activity of topically applied = 55 per cent mydriasis. mydriatic agents in mice, Arch. Int. Phar- I Dimethylaminoethylneetate, the tertiary analog of acetyl- macodyn. 161: 423, 1966. . $ln 0.1 VI phosphate buffer, pH = 6.0. 9. Kalir, A., Edery, H., Pelah, Z., et al.: 1- Phenylcyclohexylnmine derivatives. II. Syn- thesis and pharmacological activity, J. Med. the iris sphincter to a miotic drug. Different lipid Chem. 12: 473, 1969. solubilities and degradation of the drug by the 10. Maayani, S., Weinstein, H., Ben-Zvi, N., et present in the iris are but two al.: Psychotomimetics as anticholinergic examples. It was possible by the method described agents. I. 1-Cyclohexylpiperidine derivatives: to evaluate the overall miotic activities of series anticholinesterase activity and antagonistic of ACh agonists and inhibitors in activity to , Biochem. Pharmacol. the mouse eye. By comparing the dose-response 23: 1263, 1974. dependence of non-ester acetylcholine-agonists (e.g., oxotremorine) to that of aminoacetates (e.g., aceclidine) it should be possible to estimate Intact omentum for ocular vascularization. to what extent the cholinesterase activity in- HARRY S. GOLDSMITH, WEI-FAN CHEN, lluences, e.g., the required drug concentration or AND PETER V. PALENA. the duration of miotic activity. Preliminary results, to be published, were found to be in good agree- Twenty clogs had their intact omentum exten- ment with cholinergic activity data obtained from sively lengthened by a series of surgical maneu- isolated organs, e.g., isolated guinea pig ileum. vers. Transverse incisions were then made along The skillful technical assistance of Miss H. the chest, shoulder, neck, and scalp which were Calron is gratefully acknowledged. undermined and connected to form a subcutaneous tunnel through which the omentum teas brought From the Department of Biochemistry, The up to the orbit. The lateral rectus muscle of the Ceorge Wise Center for Life Sciences, Tel-Aviv eye was divided and a sclcral flap developed along University, Tel-Aviv, Israel, and the Department the lateral superior region of the eye which ex- of Ophthalmology, The Sheba Medical Center, Tel-Aviv University, Tel-Hashomer, Israel. Sub- posed the choroid upon which the omentum was mitted for publication Sept. 26, 1974. Reprint secured. Subsequent studies demonstrated vascular requests: M. Sokolovsky, Department of Bio- connections between intraocular vessels and. those chemistry, The Ceorge Wise Center for Life of the omentum. Proof of the existence of these Sciences, Tel-Aviv University, Tel-Aviv, Israel. vascular connections was based upon fluorescent funduscopic, gross, and histologic evidence. Key words: miotic activity, mouse eye, cholinergic drugs. The intact omentum has been used surgically REFERENCES for a variety of clinical problems.1-'1 It has been 1. Swan, K. C, and Cehrsitz, L.: Competitive recently shown in our laboratory that the intact action of miotics on the iris sphincter, Arch. omentum can be successfully transposed to the Ophthalmol. 46: 477, 1957. brain1 and spinal cord5 of trie dog. The purpose

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