The Effects of Anticholinesterase Agents on the Blood Cholinesterases Levels of Normal and Glaucoma Subjects*

Home , Demecarium bromide, Echothiophate, Neostigmine, Physostigmine

THE EFFECTS OF ANTICHOLINESTERASE AGENTS ON THE BLOOD CHOLINESTERASES LEVELS OF NORMAL AND GLAUCOMA SUBJECTS*

BY Irving H. Leopold, M.D. AND (BY INVITATION) JVarendra Krishna, B.SC., M.B.B.S.,** AND Robert A. Lehman, PH.D.***

ANTICHOLINESTERASE AGENTS (cholinesterase inhibitors) are used ex- tensively in the-management of the glaucomas, in the form of local application to the eyes. In primary glaucomas, these drugs are used to lower intraocular pressure because of their effects on the iris, the ciliary body, and the vascular system of the eye. In closed angle or angle closure glaucoma, they act by freeing the angle of the obstructive effect of the iris, by producing miosis. In open angle or wide angle glaucoma, it is not known whether they exert their beneficial effect primarily by their action on the ciliary body musculature or by their action on the blood vessels, but it is known that they improve the facility of aqueous outflow. Inhibition of cholinesterases (ChE) results in the %preservation of acetylcholine from hydrolysis so that it can exert its muscarinic action on the iris, the ciliary body, and the blood vessels. These tissues, like the erythrocytes of the normal human being, possess Group I cholinesterases or acetylcholinesterases (AChE) (other equivalent terms pro- posed: specific ChE, true ChE, e-ChE, aceto-ChE), which are concerned in the neurohumeral function of acetylcholine. The blood plasma contains Group II cholinesterases (various terms designated: non-specific ChE, unspecific ChE, pseudo-ChE, s-ChE, butyro or butyryl-ChE, propiono-ChE, X-ChE) which are not concerned with neurohumeral transmission (1, 2, 3, 4). No reliable reports are avail- able on the cholinesterase content of the iris and ciliary body of glaucomatous eyes because of the obvious difficulty of obtaining the *From the Department of Ophthalmology, Graduate School of Medicine of the University of Pennsylvania, and the Research and Clinical Departments of the Wills Eye Hospital, Philadelphia, Pa. **"Fight for Sight" Research Fellow of the National Council to Combat Blind- ness, Inc., New York, N.Y. ***Director of Research, Campbell Pharmaceuticals, Inc., New York, N.Y. TR. AM. OPHTH. Soc., vol. 57, 1959 64 Leopold, Krishna and Lehman material in the early stages of the disease. The cholinesterases levels in the whole blood of glaucoma patients have been shown to lie within the normal range (5). In clinical use, anticholinesterase drugs are instilled repeatedly and' for an indefinite period into glaucomatous eyest It is reasonable to expect that some absorption may take place from the conjunctival sac, the nasal mucosa, and even the gastrointestinal tract into the general blood stream. That this is indeed so has been shown for DFP and Humorsol, with which the blood cholinesterases levels were depressed a few hours following a single instillation into the eyes (6, 7, 8). The short- acting cholinesterase inhibitors, physostigmine and prostigmine, ap- proximately equally inhibit Group I ChE or AChE and Group II ChE; whereas among the long-acting cholinesterase inhibitors DFP inhibits mainly group II ChE, while Phospholine and Humorsol inhibit chiefly AChE (9, 10, 11, 12). The present investigation has been undertaken to determine the effects of these anticholinesterase agents on the red blood cells and plasma cholinesterases levels of normal human subjects and glaucoma patients when administered locally into the eyes over periods from a few hours to two months.

METHODS Cholinesterases determinations were carried out by the method of continuous titration at constant pH. To a reaction vessel bearing calomel and glass electrodes is added 100 c.c. of a solution having the following composition: acetylcholine bromide, recrystallized from alcohol (0.012M), 0.27 percent; NaCl, 0.9 percent; KC1, 0.03 percent; CaC12 * 2H20, 0.034 percent; q.s. with glass distilled water, 100 percent. The vessel is inserted into a water bath at 37.5° + 0.010 C. and con- nection made to apparatus for automatic titration with 0.02N sodium hydroxide solution with simultaneous plotting of volume of base added against time. After the reaction vessel has reached the bath temperature, sodium hydroxide is run in until the pH is 7.4. Then, 1 to 2 c.c. of sample is added and the acetic acid, liberated by hydrolysis of the substrate, is titrated continuously at a pH of 7.4 + 0.05. After 20 min. the reaction' is stopped. The slope of the line is estimated graphically from the chart and the rate is expressed in milliequivalents of acetylcholine hydrolyzed per hour per cubic centimeter of sample. The titration equipment has been described previously (13). This study was carried out on out-patients at Wills Eye Hospital, Philadelphia. "Normal subjects" were patients with no ocular pathology Anticholinestierase Agents 65 other than refractive error. Venous blood samples were used in all cases and clotting was prevented by adding 1 mg. of heparin per 10 c.c. All samples were refrigerated until analyses were completed. Hematocrits were determined within 24 hours and whole blood and plasma were analysed for cholinesterases within five days. Packed red cell values were obtained by calculation. Drugs used in this study were physostigmine salicylate (eserine salicylate); Prostigmine methylbromide (neostigmine methylbromide); DFP (Floropryl, Isoflurophate) Phospholine iodide (echothiophate iodide, 217-MI); Humorsol (demecarium bromide, BC-48).

BASE LINE CHOLINESTERASES LEVELS OF THE BLOOD OF NORMAL HUMAN SUBJECTS AND GLAUCOMA PATIENTS Twenty-two normal subjects and 19 patients with primary glaucoma were selected. An attempt was made in all cases to exclude any other disease which would influence the blood cholinesterases levels, such as liver diseases, blood dyscrasias, etc. (14, 15). Only those patients with glaucoma were included who had been on pilocarpine therapy at

WHOLE BLOOD PLASMA R.B.C. NORMAL GLAUCOMA NORMAL GLAUCOMA NORMAL GLAUCOMA

.6- *0.0 0 - E .5-. I.- 0 0 ,4-. 40 000

_J 0) * 0 .3- 0 0 . wC.) -~ *00 _J - ow' .2+ 0 *4t 0 .1- 00 0

FIGURE 1 Comuparison of base line blood, plasma, and red blood cells cholinesterases levels in normal and glaucoma patients, 66 Leopold, Krishna and Lehman

TABLE 1. BASE LINE BLOOD PLASMA AND RED BLOOD CELLS CHOLINESTERASES LEVELS OF NORMAL HUMAN SUBJECTS

Cholinesterase activity (mM acetylcholinc hydrolyzed,ljhr.1'c.c.) Patient Whole blood Plasma Red blood cells 1 .309 .182 .493 2 .334 .193 .521 3 .430 .220 .620 4 .269 .124 .500 5 .379 .255 .550 6 .303 .173 .505 7 .394 .309 .507 8 .295 .169 .448 9 .309 .136 .617 10 .240 .109 .483 11 .283 .164 .448 12 .415 .307 .571 13 .227 .098 .420 14 .326 .154 .518 15 .281 .136 .481 16 .352 .167 .577 17 .302 .164 .510 18 .378 .312 .476 19 .385 .260 .567 20 .19:3 .120 .314 21 .328 .169) .486 22 .224 .087 .438 the time of this study and in whom no cholinesterase inhibitor had been used for a period of at least two months prior to the collection of blood samples. One blood sample from each patient was subjected to analysis and the results are given in Tables 1 and 2. The mean and standard deviations for normals and glaucoma patients were computed from these data and compared with those of Sawitsky et al. (16), who employed a similar procedure in their determinations. These have been summarized in Table 3. Our normals are quite consistent with the normals reported by Sawitsky et al. However, significantly lower red blood cell cholinesterases levels were found in glaucoma patients. Rados (5) failed to find a change in whole blood cholinesterases in glaucoma which is not surprising since the unchanged plasma would obscure changes occurring in the erythrocytes. Studies to elucidate this point further are in progress and wvill be reported subsequently. It Anticholinesterase Agents 67

TABLE 2. BASE LINE BLOOD PLASMA.AND RED BLOOD CELLS CHOLINESTERASES LEVELS OF GLAUCOMA PATIENTS

Cholinesterase activity (mM acetylcholine hydrolyzed/hr./c.c.) Paticnt Whole blood Plasma Red blood cells 23 .307 .149 .510 24 .326 .211 .485 25 .285 .206 .403 26 .366 .252 .544 27 .212 .082 .352 28 .316 .181 .485 29 .316 .195 .453 30 .292 .223 .400 31 .292 .170 .468 32 .219 .052 .440 33 .269 .161 .400 34 .172 .072 .323 35 .323 .172 .542 36 .224 .146 .293 37 .248 .145 .430 38 .238 .114 .425 39 .354 .166 .549 40 .302 .184 .487 41 .251 .143 .421

TABLE 3. MEAN AND STANDARD DEVIATIONS OF BLOOD PLASMA AND RED BLOOD CELLS CHOLINESTERASES LEVELS OF NORMAL HUMAN SUBJECTS AND GLAUCOMA PATIENTS

Cholinesterase activity (mM acetylcholine hydrolyzed/hr./c.c.) No. of pts. Plasma Red blood cells Normals (Wills Eye Hospital) 22 0.182 ±0.068 0.502 A0.068 Normals ( Sawitsky et al.)* 15 0.199 ±0.060 0.525 ±0.053 Glaucoma patients (Wills Eye Hospital) 19 0.159 ±0.052 0.443 ±0.072 *Figures obtainied by multiplyinig the values by 0.06 for activity in mM/hr./c.c. of sample (16). 68 Leopold, Krishna and Lehman

0.5 % ESERINE (0.1cc 02) 5.0% PROSTIGMINE (Olccc02) WHOLE PWLHSMA ROLEB.C | D L O C. BLOOD PLSA RBC BLOOD PLSA RBC .6- .5~~2 4 V ~ ~ ~ ~~~~V`

,,i'~~\,,A.N \/':. 3 : *..2 'NAN~~~~~~~~~~~D I.~ ~~H

BEFORE 35 1 3 5 135 1 3 5 1 3 5 1 3 5 INSTILL.4) - HOURS A F T E R I NSTILLATION -

FIGURE 2 Hourly influence of eserine and prostigmine on blood, plasma, and red blood cells cholinesterases levels. seems pertinent to mention here that the cholinesterases activity may vary widely from individual to individual, but tends to remain fairly constant in one individual. Age, sex, diet, physical exertion, heart rate, and blood pressure are without influence on it (16, 17).

CHOLINESTERASES LEVELS OF THE BLOOD OF NORMAL HUMAN SUBJECTS BEFORE AND AFTER ANTECHOLINESTERASE THERAPY A control blood sample was drawn and 0.1 c.c. of the anticholinesterase drug was put in each eye immediately afterwards, care being taken to keep the eyes closed for at least 2 min. following the instillation. The maximum recommended therapeutic concentrations of the drugs were generally employed. Following instillation into the eyes, blood samples were collected from each patient after intervals of one, three, and five hours. Tables 4, 5, 6, 7, 8, and 9 show the results with 0.5 percent physostigmine, 5.0 percent Prostigmine, 0.1 percent DFP, 0.25 percent Phospho- line, 0.25 percent and 0.5 percent Humorsol instillations respectively. Since we did not know the diurnal fluctuation of the blood cholin- Anticholinesterase Agents 69

PHOSPHOLINE 0.1 % D FP (0.1cCC02) 0.25% (O I CC.02) BLOOD WHOLECPLASMA R.B.C.

,.E 5 _--'_

'. L N.D.

0 I~

BEFORE( 135 1 3 5 35 1 3 13 135 INSTILL. OHOURS AFTER I NSTILLLATI N-N FIGURE 3 Hotirly influence of DFP and phospholine iodide on blood, plasma, and red blood cells cholinesterases levels. esterases levels of these normal individuals, it was decided to regard only those values as significant which were less than the normal mean minus two standard deviations. According to this criterion only one subject (no. 1, Table 9) showed significant depression of the red blood cells cholinesterases levels after a single instillation of 0.5 percent Humorsol. This depression was noticeable one hour after instillation and persisted after an interval of five hours. The other drugs studied produced no change.

CHOLINESTERASES LEVELS OF THE BLOOD OF GLAUCOMA PATIENTS BEFORE AND AFTER ANTICHOLINESTERASE THERAPY

Several patients with long-standing primary glaucoma, well con- trolled on pilocarpine therapy alone, and in whom no anticholinesterase agent had been employed for a period of at least two months prior to this study, were selected and control determinations were made. Each of these subjects was then put on minimum local anticholinesterase therapy of one form or another which would effectively control the intraocular pressure. Blood samples were collected, one, two, and 70 Leopold, Krishna and Lehman

TABLE 4. BLOOD PLASMA AND RED BLOOD CELLS CHOLINESTERASES LEVELS OF NORMAL HUMAN SUBJECTS* BEFORE AND AFTER INSTILLATION OF 0.1 C.C. OF 0.5 PERCENT PHYSOSTIGMINE SALICYLATE (ESERINE SALICYLATE) INTO EACH EYE

Patient 1 Patient 2 Cholinesterase activity (mM acetylcholine hydrolyzed /hr.! Whole Red blood Whole Red blood c.c.) blood Plasma cells blood Plasma cells Before instillation 0.328 0.169 0.486 .224 .087 .438 1 hr. after instillation 0.296 0.142 0.485 .266 .109 .395 3 hrs. after instillation 0.327 0.170 0.484 .239 .077 .473 5 hrs. after instillation 0.292 0.147 0.455 .223 .110 .416 *Patients 1 and 2 are patients 21 and 22 in Table 1.

0.25% HUMORSOL (0.1 cc. 02) 0.5% HUMORSOL (O.1ct 02) WHOLE WHOLE BLOODB8D PLASMA R.B.C. BLOODOOD PLSAPLASMA RSCR.B.C.

. .

'.3

R -M

c.~~~ ~ ~ ~~~~~.A

BEFORE I 3 5 13 5 13 135 35 135 INSTILL.O) I- HOURS A FTER INSTILLATI-N 4

FIGURE 4 Hourly influence of 0.25 percent and 0.5 percent Humorsol on blood, plasma, and red blood cells cholinesterases levels. z _ cr 0 0- t~- eq 100C -t t- eq co C

z eq 0 rt- 3-C -eq- - u- CO CO CO eq 0 eq _O100 o o 0 00koLOeq eqCO ueq 3-, - -- 0- Z [. .' 10 U- z 00 O C 00) 0 m)Z ¢ P4 0o UZ 00 - CO 0) 10 vU)0 3-o t. . 10 10 .4 . CO ¢> ¢ e0 0)0

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- )U 4 . Cj 000 1% 0 0 0*O- H 0* 4) _ 0 _~ ' -t .e 4 00 4- 4U)i t - 0 .0 0 (n-:-= X (L) CU CZ) s _~ _ c c 0 7 .u_ 0 o .0; m 0010 z 0 00 Co Cl

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00 C C) N OCO 10 ?,:S ._ Cu " ,s." % .t> -4- C C.) CuC *2 74 Leopold, Krishna and Lehman

TABLE 10. BLOOD PLASMA AND RED BLOOD CELLS CHOLINESTERASES LEVELS OF GLAUCOMA PATIENTS BEFORE AND AFTER PHYSOSTIGMINE (ESERINE) THERAPY

Cholinesterase activity (mM Whole Red blood acetylcholine hydrolyzed/hr./c.c.) blood Plasma cells Patient 1 (0.25% O.U. o.d.) Before therapy .248 .145 .430 1 week after therapy .238 .149 .403 2 weeks after therapy .239 .157 .386 3 weeks after therapy .244 .160 .394 Patient 2 (0.25% O.U. o.d.) Before therapy .224 .146 .293 1 week after therapy .238 .161 .328 2 weeks after therapy .262 .178 .374 3 weeks after therapy .244 .164 .369

TABLE 1 1. BLOOD PLASMA AND RED BLOOD CELLS CHOLINESTERASES LEVELS OF GLAUCOMA PATIENTS BEFORE AND AFTER DFP (FLOROPRYL) THERAPY

Cholinesterase activity (mM Whole Red blood acetylcholine hydrolyzed/hr./c.c.) blood Plasma cells Patient 1 (.01% O.U. o.d.) Before therapy .292 .170 .468 1 week after therapy .272 .173 .410 2 weeks after therapy .281 .163 .460 3 weeks after therapy .275 .145 .467 Patient 2 (.01% O.D. o.d.) Before therapy .307 .149 .510 1 week after therapy .279 .212 .372 2 weeks after therapy .278 .133 .464 3 weeks after therapy .278 .138 .472 Patient 3 (.01% O.U. o.d.) Before therapy .219 .052 .440 1 week after therapy .224 .058 .444 2 weeks after therapy .208 .043 .436 3 weeks after therapy .211 .051 .423 Anticholinesterase Agents 75

TABLE 12. BLOOD PLASMA AND RED BLOOD CELLS CHOLINESTERASES LEYELS OF GLAUCOMA PATIENTS BEFORE AND AFTER PHOSPHOLINE IODIDE (ECHOTHIOPHATE IODIDE) THERAPY

Cholinesterase activity (mM Whole Red blood acetylcholine hydrolyzed/hr./c.c.) blood Plasma cells Patient 1 (.1% O.U. o.d.) Before therapy .292 .223 .400 1 week after therapy .264 185 .387 2 weeks after therapy .224 149 .347 3 weeks after therapy .223 167 .310 Patient 2 (.1% O.D. o.d.) Before therapy 172 .072 .323 1 week after therapy .242 .117 .438 2 weeks after therapy .209 .086 .393 3 weeks after therapy

TABLE 13. BLOOD PLASMA AND RED BLOOD CELLS CHOLINESTERASES LEVELS OF GLAUCOMA PATIENTS BEFORE AND AFTER HUMORSOL (DEME- CARIUM BROMIDE) THERAPY

Cholinesterase activity (mM Whole Red blood acetylcholine hydrolyzed/hr./c.c.) blood Plasma cells Patient 1 (.1% O.U. o.d.) Before therapy .238 .114 .425 1 week after therapy .209 .107 .369 2 weeks after therapy .201 .095 .422 3 weeks after therapy .202 .104 .370 Patient 2 (.1% O.U. o.d.) Before therapy .354 .166 .549 1 week after therapy .332 .155 .540 2 weeks after therapy .347 .169 .540 3 weeks after therapy .317 .143 .529 Patient 3 (.1 % O.D. o.d.) Before therapy .302 .184 .487 1 week after therapy .262 .184 .387 2 weeks after therapy .257 .181 .425 3 weeks after therapy .260 .178 .389 Patient 4 (.1% O.U. o.d.) Before therapy .251 .143 .421 1 week after therapy .220 .107 .416 2 weeks after therapy .210 .149 .302 3 weeks after therapy .201 .169 .025 76 Leopold, Krishna and_ Lehman

| 0.25% ESERINE (02) [ 0.1 e7 DFP (02) WHOLEBLOODPLASMA R.B.C. WBHLOEBLOD PLASMPLASMA j1R.B.C.R.B.C .6j

I~~~~~~~~~~~~~~

Lo 2

I-. ~ ~ ~ ~ ~ ~ ~ ~ ~ M....A.A L. .2 l ,, .l

BEFORE (4) 2 3 2 3 1 2 3 2 3 2 3 2 3 THERAPY *1t| W+_--~~~~~~~~~~~~~~~~~.E E K S A F T E RM|+j...... T HE R ..A..APY 1 FIGURE 5 Weekly influence of eserine and DFP on blood, plasma, and red blood cells cholinesterases levels. three weeks after institution of anticholinesterase therapy and the cholinesterases activity determined. Care was taken that there was a time interval of at least 12 hours between the last instillation of the cholinesterase inhibitor and the collection of the blood sample. The difficulties inherent in a study of this nature are quite obvious. Several patients who were originally included in this series had to be abandoned in course of time for one reason or the other. In some individuals intraocular pressure could not be maintained on the new therapeutic regimen. Others complained of local side effects and refused to carry on with the new medication. Still others did not return for a follow-up. In the ultimate analysis only 11 patients were included. Tables 10, 11, 12, and 13 show the results in this series. If the glaucoma mean minus two standard deviations is taken as the criterion for a significant depression of the blood cholinesterase levels, only one patient (no. 4 in Table 13), who was on Humorsol therapy 0.1 percent O.U. once a day, showed a significant depression of the erythrocyte cholinesterase activity after two weeks of medication, and which persisted after a three-week interval. No change occurred with the other drugs tested. Anticholinesterase Agents 77 CHOLINESTERASES LEVELS OF THE BLOOD OF GLAUCOMA PATIENTS ON PROLONGED ANTICHOLINESTERASE THERAPY Primary glaucoma patients who had been on anticholinesterase therapy for the control of intraocular pressure for a period of at least two months were selected and blood samples were taken from each. Any value lower than the glaucoma mean minus two standard deviations was regarded as showing significant depression of the cholinesterases activity. Tables 14, 15, 16, and 17 show the results. Two

TABLE 14. BLOOD PLASMA AND RED BLOOD CELLS CHOLINESTERASES LEVELS OF GLAU- COMA PATIENTS ON PROLONGED PHYSOSTIGMINE (ESERINE) THERAPY

Cholinesterase activity (mM acetylcholine hydrolyzed/hr./cc.) Patient Dosage Whole blood Plasma Red blood cells 1 Physostigmine 14% O.U. q.i.d. .284 .168 .411 2 Physostigmine 1% O.S. t.i.d. .316 .170 .487 3 Physostigmine 1% O.U. q.i.d. .227 .132 .495 4 Physostigmine 1% O.D. q.i.d. .252 .087 .481 5 Physostigmine 2% O.D. q.i.d. .358 .306 .427 6 Physostigmine 2% O.S. t.i.d. .331 .190 .504 7 Physostigmine 1% O.U. q.i.d. .316 .215 .452 8 Physostigmine 4% O.U. q.i.d. .296 .160 .159* 9 Physostigmine 2% O.S. q.i.d. .271 .138 .138* *Signiificant depression.

TABLE 15. BLOOD PLASMA AND RED BLOOD CELLS CHOLINESTERASES LEVELS OF GLAUCOMA PATIENTS ON PROLONGED DFP (FLOROPRYL) THERAPY

Cholinesterase activity (mM acetylcholine hydrolized/hr./c.c.) Patient Dosage Whole blood Plasma Red blood cells 1 DFP 0.05% O.U. o.d. .262 .165 .391 2 DFP 0.025% O.D. o.d. .291 .146 .453 3 DFP 0.025% O.D. o.d. .190 .122 .328 4 DFP 0.05% O.S. b.i.d. .350 .200 .513 5 DFP 0.05% O.S. o.d. .310 .164 .520 6 DFP 0.05% O.U. o.d. .269 .127 .483 7 DFP 0.025% O.S. q.i.d. .296 .100 .526 8 DFP 0.05% O.U. b.i.d. .335 .163 .615 9 DFP 0.01% O.S. b.i.d. .412 .060 .987 *Significant depression. 78 Lcopold, Krishna and Lehman

TABLIE 16. BLOOD PLASMA ANI) RED BLOOD CELLS CHOLINfESTERASES LEVELS IN GLAUCOMA PATIENTS ON PROLONGED PHOS PHOLI NE IODIDE (ECHOTHIOPHATE) THERAPY

Cholinzesterase activity (m .11 acetylcholine hydrolyzed /hr. 'cc.) Patient Dosage Whole blood Plasma Red blood cells 1 0.25% O.tT. 1).i.cl. 0.054 ).(030* 0. 126* 2 0.25%0.D). .nd. 0.089 0.070 0. 114* 3 0.25% 0.t'. o.d. 0(. 156 0. 133 0. 203* 4 0(.1% 0.t. bi.d. 0.141 0.062 0.248* a )0.1% 0.1). b.i.d. (.366 0.21:3 0.724 6 0.1% 0.1). 0.25% O.S. b).i.d. 0 091 0.098 0.082* 0.1% 0.1t. od. 0. 25X) 0.157 0.528* 8 0.1% OP.).od.d. 0.1() .086 0.127* 9 0. 1% 0.1). (.i.d. 0. 1% 0.S. o.d. 0.002 0. 0* 0.005* 10 0.1% o.n. o.cl. 0. 1% O.S. b.i.d. 0.117 0. 109 0.129* 1 0.1%l 0. b.i.d. 0.052 0.082 0.008* 12 0. 25%0.I b.i.d. 0.132 0.11( 0. 157* *sigfjiifcailt depressioni. patients on physostigmine therapy (no. 8 and no. 9 in Table 14), all patients except one (no. 5 in Table 16) on Phospholine therapy, and five patients on Humorsol therapy (nos. 1, 2, 3, 4, and 8 in Table 17) showed significant depression of red blood cells cholinesterases activity. Two patients (no. 1 and no. 9 in Table 16) on phospholine therapy in addition showed significant inhibition of blood plasma cholinesterases activity. No inhibition of blood plasma or red blood cells cholinesterases activity could be demonstrated in patients on DFP therapy.

COMMENTS AND CONCLUSIONS This studyv has revealed certain interesting points and raised some new problems for which an answer must be sought. Further studies are needed to corroborate or substantiate our findings of lower erythrocyte cholinesterases activity in glaucoma patients as compared to normal individuals. An ideal situation would be to take the blood sample for analysis as soon as the patient is found to have glaucoma and before any antiglaucomatous therapy is instituted. Any condition Anticholinesterase Agents 79 which may affect the blood cholinesterases levels must be excluded (jaundice, cirrhosis of liver, hepatitis, anemias, subacute bacterial endocarditis, rheumatic fever, arthritis, hyperthyroidism, carcinoma- tosis, cachexia, sepsis, ulcerative colitis, pneumonia, hyperpyrexia) and care taken that such an individual is not taking any drugs (physostig-

TABLE 17. BLOOD PLASMA AND RED BLOOD CELLS CHOLINESTERASES LEVELS IN GLAUCOMA PATIENTS ON PROLONGED HUMORSOL (DEMECARIUM) THERAPY

Cholinesterase activity (mM acetylcholine hydrolyzedl'hr. Ic.c.) Patient Dosage Whole blood Plasma Red blood cells 1 BC-48 0.25% O.U. b.i.d. .218 .189 .251* 2 BC-48 0.25% O.U. b.i.d. .141 .130 .152* 3 BC-48 0.25% O.U. b.i.d. .175 .173 .177* 4 BC-48 0.25% O.U. t.i.d. .197 .161 .268* 5 BC-48 0.1 % O.U. o.d. .265 .172 .422 6 BC-48 0.1 % O.U. o.d. .278 .158 .420 7 BC-48 0.1 % O.U. o.d. .295 .224 .411 8 BC-48 0.25% O.S. o.d. .091 .105 .104* *Sigiiificaiit depression.

.17% PHOSPHOLINE IODIDE (02) .1 S. HUMQRSOL (OL) WHOLE PLASMA R.S.C WHOLE PLASMA R. .C. BLOOD BLOOD _ .6

Z~~~~~ t;

~ -r------A-C- BEFOREt 1 2 3 1 2 3 1 2 3 1 2 3 123 123 THERAPYv ) I- -WEEKS AFTER THERAPY I

FIGURE 6 Weekly influence of phospholine iodide and Humorsol on blood, plasma, and red blood cells cholinesterases levels. 80 Leopold, Krishna and Lehman DOSAGE: 0-.01., On=.025%, O=.05% WHOLE BLOOD PLASMA R. 8 C. NORM. GLAUC. DFP NORM. GLAUC. DFP NORM. GLAUC. DFP

00~~~0 .6 .5 ~~~~~~~~~~~~~~00 0FIUR .00% .4 O0 0 0 0~~~~~~~~~~~~~~ 000~o .00 000 QI- b3 0 0 000 00 No~~~~~t0 SlN0CDPESO

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0~~~~~~~~~~ 0 ~~~~~~NoSIGNIFICANT DEPRESSION FIGURE 7 Comparison of base line blood, plasma, and red blood cells cholinesterases levels in normal and glaucoma patients and those treated with DFP for prolonged periods. mine, Prostigmine, DFP, TEPP, Mintacol, Phospholine, Humorsol, Carbachol) that may alter the cholinesterase activity. Granted that glaucoma patients have lower erythrocyte cholinesterases levels, the next problem is whether this is of any significance in the pathogenesis of the entity of primary glaucoma or whether it is merely a random finding. In general, it has been felt that blood cholinesterases have no apparent function and that their concentrations do not correlate well with cholinesterases levels at other sites. This study shows that sufficient absorption of certain cholinesterase inhibitors takes place after ocular instillation to affect the blood cholinesterases levels. The vehicle employed may play an important part in the facility and rate of absorption. This may explain the reason that DFP in peanut oil failed to produce significant changes in the blood levels even after more than two months of therapy (Table 15). Also this effect may be due to loss of potency of DFP in gastric and intestinal juices. Physostigmine and Prostigmine are short-acting cholinesterase inhibitors which inhibit cholinesterase reversibly (18). However, it was Anticholinesterase Agents 81 DOSAGE: *= I %;°= 1X%; *=%'O-02 qi-d.

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0 SIGNFI0CAN DEPRESSION FIGURE 8 Comparison of base line blood, plasma, and red blood cells cholinesterases levels in normal and glaucoma patients and those treated with eserine for prolonged periods. interesting to find significant depression in the erythrocyte cholinesterases levels of two patients who were on physostigmine therapy for more than two months (Table 18). Data on Prostigmine could not be collected because of the paucity of material, as this agent is rarely used in long-term therapy, its principal use being to abort an attack of acute glaucoma. Phospholine iodide depressed the erythrocyte cholinesterase levels in all patients who were on this medication for more than two months (Table 18). In addition, in two patients there was significant depression of the plasma cholinesterase levels as well (Table 16). With Humorsol, there was a significant lowering of erythrocyte cholinesterase levels not only in patients treated for more than two months, but also in one glaucoma patient treated for two weeks, and in one normal human subject following a single instillation (Table 18). Thus, Humorsol must be either an ultra-long-acting cholinesterase inhibitor or more readily absorbed when applied to the eye. It should be noted that none of the patients who showed changes in tQ 0F z 1-

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40 4- o 4*J 0 IO..; Z Anticholinesterase Agents 803 DOSAGE: *=0.1%; 0=0.25% WHOLE BLOOD PLASMA R.B.C. ECHO- ECHO- ECHO- GLAU- THIO- GLAU- THIO- GLAU- THIO- NORMAL COMA PHATE NORMAL COMA PHATE NORMAL COMA PHATE 0 .7

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.002~ ~ ~ .0*.005 .006 e SIGNIFICANT DEPRESSION) FIGURE 9 Comparison of base line blood, plasma, and red blood cells cholinesterases levels in normial and glauicoma patients and those treated with echothiophate for prolonged periods. the erythrocyte or plasma cholinesterases levels exhibited signs or symptoms which could be attributed to the systemic action of the drugs used (7, 10, 19). Nevertheless, the results show a definite spill over of drug into the blood stream and systemic side effects are to be expected occasionally.

SUMMARY Erythrocyte cholinesterases levels of 19 glaucoma patients have been shown to be significantly lower than those of normal human subjects. A plea is made for determining the blood cholinesterases levels of newly diagnosed glaucoma patients before any therapy is instituted, so that these findings may be confirmed or refuted. Among various cholinesterase inhibitors, the long-acting drugs Phospholine and Humorsol significantly depressed the erythrocyte 84 Leopold, Krishna and Lehman DOSAGE: *-0.1% 0=.25%

WHOLE BLOOD PLASMA R. B. C. GLAU- DEME- GLAU- DEME- GLAU- DEME- NORMAL COMA CARIUM NORMAL COMA CARIUM NORMAL COMA CARIUM

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*SIGNIFICANT DfPRESSION FIGURE 10 Comparison of base line blood, plasma, and red blood cells cholinesterases levels in normal and glaucoma patients and those treated with demecarium for prolonged periods. cholinesterases levels in glaucoma patients after more than two months therapy. Humorsol, apparently an ulta-long-acting cholinesterase inhibitor, brought about significant blood cholinesterase inhibition after a very short period of time, in one subject following a single instillation. DFP, another long-acting cholinesterase inhibitor, failed to inhibit the blood cholinesterase level, probably because of its oily vehicle. Physostigmine, though generally considered a short-acting cholinesterase inhibitor, depressed the erythrocyte cholinesterases level in two glaucoma patients after two months of treatment.

REFERENCES 1. De Roetth, A. Jr., Cholinesterase activity in ocular tissues and fluids, Arch. Ophth., 43: 1004, 1950. 2. Dardenne, U., W. Leydhecker, and E. Helferich, Die Cholinesterase in der Iris von Mensch und Rind, Graefes Arch. f. ophth., 158: 434, 1957. 3. Augustinsson, K. B., Cholinesterases: A study in comparative enzymology, Acta physiol. Scandinav., 15 (Suppl. 15): 1, 1948. 4. Augustinsson, K. B., Assay methods for cholinesterases, in methods of biochemical analysis, vol. 5, pp. 1-63. New York and London: Interscience Publishers, 1957. Anticholinesterase Agents 85 5. Rados, A., Blood cholinesterase values of patients with glaucoma, Arch. Ophth., 30: 371, 1943. 6. Leopold, I. H., and J. H. Comroe, Effect of diisopropyl fluorophosphate ("DFP") on the normal eye, Arch. Ophth., 36: 17, 1946. 7. Leopold, I. H., and J. H. Comroe, Use of diisopropyl fluorophosphate ("DFP") in treatment of glaucoma, Arch. Ophth., 36: 1, 1946. 8. Gittler, R., and B. Pillat. Die Verwendung von Dekamethylen-bis- (N-methylcarbaminoyl-m-trimethylammoniumphenol) (BC-48) bei der Therapie des Glaukoms, Graefes Arch. Ophth., 157: 473, 1956. 9. Stumpf, C., Anticholinesterases, Subsidia medica, 6: 201, 1954. 10. Krishna, N., and I. H. Leopold, Phospholine iodide or echothiophate iodide (217-MI) in treatment of glaucoma: Further observations, A.M.A. Arch. Ophth., in press. 11. Kraupp, O., C. Stumpf, E. Herzfeld, and B. Pillat, Pharmakologische Eigenschaften einiger langwirksamer Cholinesterase-Hemmkorper aus der Reihe der Polymethylen-bis-( Carbaminoyl-m-trimethylammoniumphenole), Arch. internat. Pharmacodyn., 102: 281, 1955. 12. Kraupp, O., H. G. Schwarzacher, and C. Stumpf, Ueber die pharmakologischen Eigenschaften einiger Polymethylen-Biscarbaminoyl-m-trimethylammoniumphenole, Arch. exper. Path. u. Pharmakol., 225: 117, 1955. 13. Lehman, R. A., and L. Pollack, Evalutation of antacid liquid suspensions and tablets in relation to secretory dynamics, Gastroenterology, 29: 46, 1955. 14. Wescoe, W. C., C. C. Hunt, W. F. Riker, and I. C. Litt, Regeneration rates of serum cholinesterase in normal individuals and in patients with liver damage, Am. J. Physiol., 149: 549, 1947. 15. Sabine, J. C., Choline-esterase of blood cells and plasma in blood dyscrasias, with special reference to pernicious anemia, J. Clin. Investigation, 9: 833, 1940. 16. Sawitsky, A., H. M. Fitch, and L. M. Meyer, A study of cholinesterase activity in the blood of normal subjects, J. Lab. & Clin. Med., 33: 203, 1948. 17. Hall, G. E., and C. C. Lucas, Cholinesterase activity of normal and patholo- gical sera, J. Pharmacol. & Exper. Therap., 59: 34, 1937. 18. Goldstein, A., Inhibitors of purified human plasma cholinesterase, Fed. Proc., 7: 223, 1948. 19. Krishna, N., and I. H. Leopold, Use of BC-48 (demecarium bromide) in treatment of glaucoma, Am. J. Ophth., in press.

DISCUSSION DR. ALBERT C. SNELL. An awareness of the systemic effects of locally applied drugs is as old as pharmacology. We are not reminded of this in the everyday practice of ophthalmology as frequently now as we were when reactions to mydriatic drugs were more common. Yet the very process of searching for and achieving safer, better drugs, a process proceeding now with almost explosive vigor, demands a lasting vigilance for possible side- effects. This paper by Dr. Leopold and his associates is a splendid example of this kind of vigilance. The most stimulating part of this work is the prospect it furnishes that glaucoma patients differ from the rest of the population in a nonocular metabolic respect. If this be true, and I say "if" because it seems desirable that a larger sample of persons be tested to assure randomness, it is a sort of 86 Leopold, Krishna and Lehmnan break-through in a new direction. What this means or where it may lead is so far pure speculation. DR. LEOPOLI). I want to thank Dr. Snell for his suggestions and for stressing that we are not yet certain about the cholinesterase values in glaucoma. We are in the process of enlarging the series and using other analytic methods. One method might overlook some aspects which would alter the true picture. There are many factors that may alter the red cell and plasma cholinesterase value. This does not seem to vary much in anv one individual but, from individual to individual, there are variations. Age, sex, physical exertion, or diet are usually without effect, but liver disease, blood dyscrasias, elevated temperature, as well as other factors, can produce enough variation that no significance should be attributed to this one finding at present. It simply has contributed a stimulus for further study.