TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
117, 189- 193 ( 1992)
Use of Cholinesterases as Pretreatment Drugs for the Protection of Rhesus Monkeys against Soman Toxicity’ ALAN
D. WOLW,*
*Division
W. BLICK,t MICHAEL R. MURPHY,$ STEPHANIE A. MILLER,+ STANLEY L. HARTGRAVES,$ AND BHUPENDRA P. DOCTOR*-*
DENNIS
MARY
of Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100; and TSystems Research and #United States Air Force Armstrong Laboratory, Brooks Air Force Base, Texas 78235-5000
K. GENTRY,* Laboratories,
Received April 6, 1992; accepted August 4, 1992
OP poisoning consist of pretreatment with a reversible ChE inhibitor, pyridostigmine bromide, and treatment with a combination of atropine citrate, to counteract the effect of accumulated acetylcholine, and pralidoxime chloride (2M. K., HARTGRAVES, S. L., AND DOCTOR, B. P. (1992). Tuxicol. PAM) to reactivate inhibited AChE (Dunn and Sidell, 1989). Appl. Pharmacol. 117, 189- 193. Although this drug regimen is highly effective in protecting Purified fetal bovine serum acetylcholinesterase (FBS AChE) experimental animals against death by OP poisoning, it is and horse serum butyrylcholinesterase (BChE) were successfully ineffective in protecting against convulsions, performance used as single pretreatment drugs for the prevention of pinacolyl deficits, or permanent brain damage (Dirnhuber et al., 1979; methylphosphonofluoridate (soman) toxicity in nonhuman pri- McLeod, 1985; Kluwe et al., 1987; Dunn and Sidell, 1989). mates. Eight rhesus monkeys, trained to perform Primate Equilibrium Platform (PEP) tasks, were pretreated with FBS AChE Recently, the anticonvulsant drug diazepam was included as a treatment to minimize convulsions, thereby minimizing or BChE and challenged with a cumulative level of five median the risk of permanent brain damage. lethal doses (LD& ofsoman. All ChE-pretreated monkeys surOne approach to prevent lethality and minimize side efvived the soman challenge and showed no symptoms of soman toxicity. A quantitative linear relation was observed between the fects or performance decrements is the use of enzymes such soman dose and the neutralization of blood ChE. None of the as cholinesterases as a single pretreatment drug to sequester four AChE-pretreated animals showed PEP task decrements, highly toxic OP anti-ChEs before they reach their physioeven though administration of soman irreversibly inhibited nearly logical targets (Wolfe et al., 1987; Raveh et al., 1989; Ashani all of the exogenously administered AChE. In two of four BChE- et al., 1991; Doctor et al., 1991; Maxwell et al., 1991; Wolfe pretreated animals, a small transient PEP performance decrement occurred when the cumulative soman dose exceeded 4 et al., 199 1; Maxwell et al., 1992). We have pursued this goal with exogenous acetylcholinesterase as a single pretreatLD-. Performance decrements observed under BChE protection were modest by the usual standards of organophosphorus com- ment drug. This approach turns the irreversible nature of pound toxicity. No residual or delayed performance decrements the OP-cholinesterase interaction from disadvantage to ador other untoward effects were observed during 6 weeks of post- vantage; instead of focusing on the OP as an anticholinesexposure testing with either ChE. o 1992 Academic press, IN. terase, we have focused on the cholinesterase as an anti-OP. Using this approach, we have shown that administration of fetal bovine serum (FBS) AChE or human serum butyrylThe toxicity of organophosphorus (OP) compounds is at- cholinesterase (BChE)-protected animals from multiple LDsO tributed to their irreversible inhibition of acetylcholinesterase of a variety of highly toxic OPs without any toxic effects or (AChE). The resultant accumulation of acetylcholine, both performance decrements (Wolfe et al., 1987; Raveh et al., centrally and peripherally, produces an acute cholinergic 1989; Ashani et al., 1991; Doctor et al., 1991; Maxwell et crisis characterized by convulsions, bronchoconstriction, al., 1991, 1992; Wolfe et al., 1991). tracheobronchial and salivary secretions, bradycardia, beWe report here the protection of nonhuman primates, havioral incapacitation, muscle weakness, and, ultimately, rhesus monkeys, from soman toxicity as high as 5 LD5c by death due to respiratory failure. Present drug regimens for pretreatment with FBS AChE (De La Hoz et al., 1986) or horse serum BChE (Ralston et al., 1983) without the occur’ Research was conducted in compliance with the Animal Welfare Act rence of performance deficits, as measured by the Primate and other federal statutes and regulations relating to animals and experiments Equilibrium Platform (PEP) task (Farrer et al., 1982). This involving animals and adhered to the principles stated in the Guide for the task, in which a seated monkey is trained to manipulate a Care of Use of Laboratory Animals, NIH publication 86-23, 1985 edition. * To whom correspondence should be addressed. joystick to maintain orientation in space, was originally deUse of Cholinesterases as Pretreatment Drugs for the Protection of Rhesus Monkeys against Soman Toxicity. WOLFE, A. D., BLICK, D. W., MURPHY, M. R., MILLER, S. A., GENTRY,
189
0041-008X/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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WOLFE ET AL.
veloped as an animal model of pilot performance for studies in aerospace medicine. PEP performance is a very sensitive measure of cholinergic toxicity, and quantities of OPs or drugs that do not produce overt signs of intoxication produce significant performance deficits on the PEP task (Blick et al., 1989, 1991).
RESULTS
PEP tests after soman challenge were conducted with one monkey at a time (Farrer et al., 1982; Blick et al., 1989, 199 1). After a 30-min baseline test, (Figs 1 and 2, panel 1, period between start of test and E), monkeys were given FBS AChE or BChE intravenously in an amount calculated to be sufficient to neutralize in vitro 32 pg/kg (~5 LD50) of METHODS soman (Benschop et al., 1984; Raveh et al., 1989; Ashani et al., 199 1; Doctor er al., 199 1). As serum proteins, both these Healthy, adult, male rhesus monkeys (Mucuca mulutta), ranging in weight enzymes have long half-lives, 30-40 hr, (Raveh et al., 1989; from 5 to 8 kg, trained to perform the PEP task at least weeWy for a minimum of 6 months were used in these studies (Blick et al., 1991). None of the Ashani et al., 1991; Wolfe et al., 1991; Doctor et al., 1991) monkeys had been previously exposed to soman, AChE, or BChE. The LD,, and in the absence of soman challenge remained at a constant of soman in rhesus monkeys, is estimated to be 6.4 &kg (Hamilton and level in the blood for over 8 hr, well beyond the duration of Lundy, 1989). Routine care of the animals was provided by the Veterinary the PEP test (3 hr). A total of eight monkeys were tested, Sciences Division, Occupation and Environmental Health Directorate, Armstrong Laboratory. The monkeys were individually housed in stainless four receiving FBS AChE and four receiving BChE. No other treatment or prophylactic agent was given at any time. After steel cages in animal rooms that were maintained at 20-25°C and relative humidity of 50 + 10% on a 12-hr 1ight:dark cycle with no twilight. Cages ChE injection, monkeys were tested for 30 min on the PEP were washed twice daily, and a complete cage change was made every 2 to determine whether either enzyme affected performance. weeks. The monkeys were fed certified Primate Chow (Purina No. 5048. Next, the level of enzyme in whole blood was measured, Purina Mills, Inc., Richmond, IN), and permitted tap water ad libitum. followed by the first soman challenge ( 16 pg/kg, ~2.5 LDsO, Both enzymes were purified by procainamide affinity gel chromatography im) and 30 min of PEP performance. Blood ChE levels were (Ralston et al., 1983; De La Hoz et a/., 1986) and titrated in vitro with soman as previously described (Raveh et al., 1989; Ashani et a/., 199 1: again determined, and the second soman challenge dose (9.6 Doctor et al., 1991). One microgram of soman completely inhibited 1050 /Nkg, N 1.5 LD=J was administered. After another 30 min units of FBS AChE and approximately 165 units of BChE. Two of the four of PEP testing, blood ChE levels were measured for the third stereoisomers of racemic soman (Benschop et al., 1984) react with AChE. time. The result of this measurement was then used to caland three of the four stereoisomers react with BChE (for details see Ashani culate a third soman challenge dose (up to 6.4 pg/kg, N 1.0 et al., 1991). The specific activity of FBS AChE was 5600 U/mg (Raveh et al., 1989); 1 mg AChE = 1.428 X 10M8 M. The specific activity of horse LD& based on residual circulating enzyme activity. serum BChE was 750 U/mg. Both ChEs were assayedcolorimetricahy (EllAlthough blood ChE levels were elevated more than lOOman et a/., 1961) using acetylthiocholine (ATC) as a substrate for AChE fold, ChE injections alone caused no apparent physiological and butyrylthiocholine (BTC) as a substrate for BChE. Soman was supplied or neurological effect or deficit, as measured by the PEP task by the Chemical Research, Development, and Engineering Center, Aberdeen performance (Figs. 1 and 2, left, period between E and Sl). Proving Ground, MD. Soman was 98.6% pure when analyzed by 3’P-nuclear magnetic resonance. Soman was supplied in dilute solutions of 2 mg/ml in None of the eight monkeys showed any OP toxicity after propylene glycol. soman challenges; protection was so complete that there were The PEP (Fairer et al., 1982; Blick et al.. 1989, 1991) is a continuous no fasciculations even at the site of soman injections. Folcompensatory tracking device that measures the ability of a monkey to comlowing the first and second soman injections (totaling 25.6 pensate for unpredictable perturbations in pitch induced by a filtered random a/kg. -4 LD5& the PEP performance of all eight monkeys noise signal. The monkey was seated in a chair that rotated about the pitch axis in response to a random external signal providing unpredictable per- was completely normal. The four monkeys pretreated with turbations. The animal could control the pitch by manipulating a joystick FBS AChE and two pretreated with BChE continued this control mounted on the platform. The animal’s task was to use the joystick level of PEP performance even after the third soman chalto compensate for the external signal and thereby keep the platform as level lenge (Figs. 1 and 2, left). However, the two remaining monas possible. The platform position (angle in degrees) was measured by computer 10 times per second, and the standard deviation (a) of all the mea- keys that had been pretreated with BChE exhibited significant but minor PEP deficits after the third soman injection (Fig. surements for each 5-min epoch, i.e., 3000 points, was the metric for quality 2, left No. 604 and No. 564, S3 to end); these transient PEP of PEP performance. Whenever the platform deviated from level by more than 15”, the animal received mild tail shocks (lOO-ms duration, I-Hz rep- performance deficits were similar to those observed after lowetition rate, 5 mA maximum current) until the platform was returned to dose soman (~2.8 pg/kg) in unprotected monkeys (Blick et ..I. me .I 13 .-n*. .. ror - eacn* suqecr, . . tail-snack .. . ’ Iintensity was adjusted to wnnin - urnits. al., 1989, 199 1). Based upon this comparison, we believe the minimum level required to maintain motivated pelloiiila ---‘---rice in baseline tests. Well-trained subjects typically reduced the v+lbiti+v .I lY”.ll LJ3f c platform po- that the protective ratio afforded by our ChE pretreatment sition from a 5 of approximately 12”, which wo utd be producedby the can be estimated to be between 10 and 15. external input alone, to about 3-4” and thus received very few tail shocks Blood ChE levels measured before and after each soman (~1 shock/hour on average). Subjects performed the PEP task for 2.5 hr on challenge (Figs. 1 and 2, middle) showed a linear decline each soman challenge testing day. and results are presented for each 5-min with increasing soman dose. For AChE, this pattern continblock of testing time (Figs. I and 2, left). During the 6 weeks of long-term follow-up, PEP tests were conducted for 2 hr; 0 was computed for each 5- ued until nearly all the FBS AChE in the blood was inhibited; however, for BChE, 20% of the initial blood levels remained min block of time, and the mean of the 24 resulting data points was calculated to yield one performance score for the entire 2 hr. after administration of the same amount of soman. The
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FIG. 1. (Left) Effects of iv administered purified FBS AChE on PEP task performance before and after challenge with approximately 2.5, 1.5, and 1.O LD,, of soman. Four male rhesus monkeys (5-8 kg), trained to perform the primate equilibrium platform (PEP) task, each received approximately 0.4-0.5 rmol of AChE iv (greater than 1:l stoichiometry with soman). The sequence of behavioral testing and soman challenges was (a) 30-min PEP task (baseline); (b) AChE injection, E (15 min): (c) 30-min PEP task to determine the effect of administration of AChE alone, followed by a 15-min pause for obtaining blood samples, AChE assay, and soman injection, S I (16.0 pg/kg, ~2.5 LDSO, im): (d) 30-min PEP testing, followed by a 15-min pause for obtaining blood samples, AChE assay, and soman injection, S2 (9.6 rg/kg, = 1.5 LDSo, im); (e) 30-min PEP testing, followed by a 15-min pause for obtaining blood samples, AChE assay,and the final im soman injection, S3 (6.4 &kg, = 1.O LDSo, was planned but would be reduced if residual AChE activity was judged insufficient); (f) final 30 min of PEP testing. For each 30 min of PEP testing, the data (filled circles) from six sequential 5-min blocks of time are presented (15). (Middle) In vivo titrations of blood AChE in four rhesus monkeys pretreated by iv injection with FBS AChE. Details are as described above. The cumulative dose of soman which reduced ChE activity to the indicated final levels exceeded the amount of AChE administered, suggesting involvement of endogenous esterase. (Right) Long-term effects on PEP task performance of iv administered FBS AChE and challenge with a total of approximately 5 LDSo of soman and residual blood AChE levels. PEP performance and blood AChE levels of four monkeys were tested weekly for 6 weeks; filled circles, PEP performance: open circles, enzyme level. PEP performance scores are the mean of data from 24 separate 5-min blocks that compose the 2-hr test.
strong linear relationship between OP dose and residual ChE in vivo agreed with previous observations made with mice (Raveh et al., 1989; Ashani et al., 1991; Doctor et al., 1991) and nonhuman primates (Wolfe et al., 199 1; Maxwell et al., 1992) and confirms the hypothesis that stoichiometric reaction is responsible for the observed protection. Soman was sequestered by exogenous and endogenous ChEs in the blood before it could reach physiologically critical target AChE. Thus, the extent of protection afforded by exogenous ChE
is dependent on the concentration of ChE in the blood and the OP dose; to double the protection, twice the amount of ChE will be required. In addition, the rate of circulation and the clearance of exogenous enzyme and OP will influence the extent of protection. During the period after soman challenge, blood AChE levels quickly returned to pre-FBS AChE levels (Fig. 1, right). BChE, however, appeared to decrease below its original (preexogenous BChE administration) level and then returned
WOLFE ET AL.
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(min) FIG. 2. (Left) Effects of iv administered purified horse serum BChE on PEP test performance before and after challenge with approximately 2.5, 1.5, and I .O LD,, of soman. See legend to Fig. 1. for a detailed explanation. (Middle) In viva titrations of blood BChE in four rhesus monkeys pretreated by iv injection with horse serum BChE. See legend to Fig. 1 for details. (Right) Long-term effects on PEP task performance of iv administered horse serum BChE and challenge with a total of approximately 5 LDSo of soman and residual blood BChE levels. For details, see legend to Fig. 1.
to normal levels (Fig. 2, right). Perhaps the exogenous BChE triggered a feedback mechanism responsible for maintaining endogenous BChE levels. During 6 weeks of postsoman testing, no monkey, including the two monkeys that received BChE and had shown PEP deficits on the challenge day, showed any signs of delayed toxicity, convulsions, or other OP symptoms, or any abnormality on PEP performance (Figs. 1 and 2, right). DISCUSSION The results presented here are in sharp contrast to the findings of previous studies that tested the effectiveness of the multiple-drug regimen currently used against OP (soman) toxicity. In those studies, pyridostigmine-pretreated rhesus monkeys receiving soman and the appropriate regimen of 2-PAM chloride, atropine, and diazepam were partially pro-
tected against lethality and extensive convulsions but still displayed serious symptoms of OP poisoning, including convulsions, a period of unconsciousness (Blick et al., 1989; Castro et al., 199 1; Murphy et al., 1992) and in one study (Murphy et al., 1992) exhibited a period of delayed and extended performance debilitation. We have shown that ChE pretreatment protected against OP-induced lethality, convulsions, and other overt symptoms and performance deficits, and there were no delayed effects. Based on the results presented here with nonhuman primates, the 1: 1 stoichiometry between ChE and the OP dose, and the amount of endogenous scavenger (ChE, carboxylesterases) normally present (Boskovic, 1979; Sterri et al., 198 1; Clement, 1984; Maxwell et al., 1987) the LDso of soman in humans may be extrapolated to be approximately 300 pg/ 70 kg. To achieve a protective ratio of 2, a pretreatment dose of lOO- 150 mg enzyme might be required. This pretreatment
CHOLINESTERASES
AS PRETREATMENT
should be effective for 70-90 hr. Adverse immunological reactions are among the possible drawbacks to the present approach, and current research is addressing this problem. We are also evaluating the possibility that the addition of one or more currently used drugs may multiply the effectiveness of exogenous ChE. The extensive use of OPs by the pesticide industry, the continued threat of their use in warfare or by terrorists, and the need for the destruction of the extensive existing stocks of these chemicals makes pharmacological and environmental protection against OP toxicity an important issue. We have shown that pretreatment with a ChE is the most effective approach found to date for protection against OP toxicity. The approach demonstrated here should provide a foundation for future prophylaxis and therapy for organophosphate toxicity. ACKNOWLEDGMENTS This work was partially conducted by Systems Research Laboratories, Inc., under contract to the United States Air Force. We wish to thank Frank R. Weathersby, Laurie De La Pana, Denise De La Hoz, German Caranto, Hugh Hively, and Roberta Larrison for expert technical assistance and Drs. Yacov Ashani, G. Carroll Brown, and John W. Fanton for stimulating discussion and advice during the course of this investigation.
REFERENCES Ashani. Y., Shapira, S., Levy, D., Wolfe, A. D., Doctor, B. P., and Raveh, L. ( 199 I ). Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice. Biochem. Pharrnacol. 41, 37-41. Benschop, H. P., Konings, C. G. A., Genderen, J. G., and DeLong, L. P. A. (1984). Isolation, anticholinesterase properties, and acute toxicity in mice of the four stereoisomers of the nerve agent soman. Toxicol. Appl. Pharmacol. 72, 6 1-74. Blick, D. W., Murphy, M. R., Fanton, J. W., Kerenyi, S. Z., Miller, S. A., and Hartgraves, S. L. (1989). Incapacitation and performance recovery after high-dose soman: Effects of diazepam. Proceedings of the Medical Chemical Defense Bioscience Review, pp. 2 19-222. Columbia, MD. Blick, D. W., Kerenyi, S. Z., Miller, S. A., Murphy, M. R., Brown, G. C., and Hartgraves, S. L. (199 1). Behavioral toxicity of anticholinesterases in primates: Chronic pyridostigmine and soman interactions. Pharmacol Biochem. Behav. 38,527-532. Boskovic, B. (1979). The influence of 2-(O-cresyl)-4 H-1:3:2:-benzodioxaphosphorin-2-oxide (CBDP) on organophosphate poisoning and its therapy. Arch. Toxicol. 42, 207-2 16. Castro, C. A., Larsen, T., Finger, A. V., Solana, R. P., and McMaster, S. B. ( 199 1). Behavioral efficacy of diazepam against nerve agent exposure in rhesus monkeys. Pharmacol. Biochem. Behav. 41, 159-164. Clement, J. G. (1984). Importance of ahesterase as a detoxification mechanism for soman (pinacolyl methylphosphonofluoridate) in mice. Biochem. Pharmacol. 33,3807-38 11. De La Hoz, D. M., Doctor, B. P., Ralston, J. S., Rush, R. S., and Wolfe, A. D. (1986). A simplified procedure for the purification of large quantities of mammalian acetylcholinesterase. Life Sci. 39, 195-199.
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Dirnhuber, P., French, M. C., Green, D. M., Leadbeater, L., and Stratton, J. A. (1979). The protection of primates against soman poisoning by pretreatment with pyridostigmine. J. Pharm. Pharmacol. 31,295-299. Doctor, B. P., Brecht, K., Castro, C., De La Hoz, D., Finger, A., Gentry, M. K., Gold, G., Hively, H., Larrison, R., Maxwell, D., McMaster, S., Solana, R.. Wolfe, A. D., and Woodward, C. (1991). Protection against soman toxicity and prevention of performance decrement in rhesus monkeys by pretreatment with acetylcholinesterase. Proceedings of the 1991 Medical Defense Bioscience Review. pp. 407-4 13. Edgewood, MD. Doctor, B. P., Raveh, L., Wolfe, A. D., Maxwell, D. M., and Ashani, Y. (199 1). Enzymes as pretreatment drugs for organophosphate toxicity. Neurosci. Biobehav. Rev. 15, 123-128. Dunn, M. A., and Sidell, F. R. (1989). Progress in medical defense against nerve agents. JAMA 262, 649-652. Ellman, G. L., Courtney, D., Andres, V., Jr., and Featherstone, R. M. (1961). A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. Farrer, D. N., Yochmowitz, M. G., Mattson, J. L., Lof, N. E., and Bennett, C. T. (1982). Effects of benactyzine on an equilibrium and multiple response task in rhesus monkeys. Pharmacol. Biochem. Behav. 16, 605609. Hamilton, M. G., and Lundy, P. M. (1989). HI-6 therapy of soman and tabun poisoning in primates and rodents. Arch. Toxicol. 63, 144-149. Kluwe, W. M., Chinn, J. L., Feder, P., Olson, C., and Joiner, R. (1987). Efficacy of pyridostigmine pretreatment against acute soman intoxication in a primate model. Proceedings of the Sixth Medical Chemical Defense Bioscience Review, pp. 227-234. Edgewood, MD. Maxwell, D. M., Brecht, K. M.. and O’Neill, B. L. (1987). The effect of carboxylesterase inhibition on interspecies differences in soman toxicity. Toxicol. Len. 39, 35-42. Maxwell, D. M., Wolfe, A. D., Ashani, Y., and Doctor, B. P. (1991). In Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology (J. Massoulie et al., Eds.), pp. 206-209. American Chemical Society, Washington, D.C. Maxwell, D. M., Castro, C. A., De La Hoz, D. M., Gentry, M. K.. Gold, M. B., Solana, R. P., Wolfe, A. D., and Doctor, B. P. (1992). Protection of rhesus monkeys against soman and prevention of performance decrement by pretreatment with acetylcholinesterase. Toxicol. Appl Pharmacol. 115. 44-49. McLeod, C. G., Jr. (I 985). Pathology of nerve agents: perspectives on medical managements. Fundam. .4ppl. Toxicol. 5, SlO-Sl5. Murphy, M. R.. Blick, D. W., Dunn, M., Fanton, J. W., and Hartgraves, S. L. (1992). Diazepam as a treatment for nerve agent poisoning in primates. Aviation, Space, Environ. Med., in press. Ralston, J. S., Main, A. R., Kilpatrick, J. L., and Chasson, A. L. (1983). Use of procainamide gels in the purification of human and horse serum butyrylcholinesterase. Biochem. J. 211,243-25 1. Raveh, L., Ashani. Y., Levy, D., De La Hoz, D., Wolfe, A. D., and Doctor, B. P. (1989). Acetylcholinesterase prophylaxis against organophosphate poisoning: Quantitative correlation between protection and blood enzyme level in mice. Biochem. Pharmacol. 38, 529-534. Sterri, S. H., Lyngaas, S., and Fonnum, F. (198 1). Toxicity of soman after repetitive injection of sublethal doses in guinea pig and mouse. Acta Pharmacol. Toxicol. 49, 8- 13. Wolfe, A. D., Rush, R. S., Doctor, B. P., Koplovitz, I., and Jones. D. (1987). Acetylcholinesterase prophylaxis against organophosphate toxicity. Fundam. Appl. Toxicol. 9,266-270. Wolfe, A. D.. Maxwell, D. M., Raveh, L., Ashani, Y., and Doctor, B. P. (199 1). In vivo detoxification of organophosphate in marmosets by acetylcholinesterase. Proceedings of the 1991 Medical Defense Bioscience Review, pp. 547-550. Edgewood, MD.
AND
APPLIED
PHARMACOLOGY
117, 189- 193 ( 1992)
Use of Cholinesterases as Pretreatment Drugs for the Protection of Rhesus Monkeys against Soman Toxicity’ ALAN
D. WOLW,*
*Division
W. BLICK,t MICHAEL R. MURPHY,$ STEPHANIE A. MILLER,+ STANLEY L. HARTGRAVES,$ AND BHUPENDRA P. DOCTOR*-*
DENNIS
MARY
of Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100; and TSystems Research and #United States Air Force Armstrong Laboratory, Brooks Air Force Base, Texas 78235-5000
K. GENTRY,* Laboratories,
Received April 6, 1992; accepted August 4, 1992
OP poisoning consist of pretreatment with a reversible ChE inhibitor, pyridostigmine bromide, and treatment with a combination of atropine citrate, to counteract the effect of accumulated acetylcholine, and pralidoxime chloride (2M. K., HARTGRAVES, S. L., AND DOCTOR, B. P. (1992). Tuxicol. PAM) to reactivate inhibited AChE (Dunn and Sidell, 1989). Appl. Pharmacol. 117, 189- 193. Although this drug regimen is highly effective in protecting Purified fetal bovine serum acetylcholinesterase (FBS AChE) experimental animals against death by OP poisoning, it is and horse serum butyrylcholinesterase (BChE) were successfully ineffective in protecting against convulsions, performance used as single pretreatment drugs for the prevention of pinacolyl deficits, or permanent brain damage (Dirnhuber et al., 1979; methylphosphonofluoridate (soman) toxicity in nonhuman pri- McLeod, 1985; Kluwe et al., 1987; Dunn and Sidell, 1989). mates. Eight rhesus monkeys, trained to perform Primate Equilibrium Platform (PEP) tasks, were pretreated with FBS AChE Recently, the anticonvulsant drug diazepam was included as a treatment to minimize convulsions, thereby minimizing or BChE and challenged with a cumulative level of five median the risk of permanent brain damage. lethal doses (LD& ofsoman. All ChE-pretreated monkeys surOne approach to prevent lethality and minimize side efvived the soman challenge and showed no symptoms of soman toxicity. A quantitative linear relation was observed between the fects or performance decrements is the use of enzymes such soman dose and the neutralization of blood ChE. None of the as cholinesterases as a single pretreatment drug to sequester four AChE-pretreated animals showed PEP task decrements, highly toxic OP anti-ChEs before they reach their physioeven though administration of soman irreversibly inhibited nearly logical targets (Wolfe et al., 1987; Raveh et al., 1989; Ashani all of the exogenously administered AChE. In two of four BChE- et al., 1991; Doctor et al., 1991; Maxwell et al., 1991; Wolfe pretreated animals, a small transient PEP performance decrement occurred when the cumulative soman dose exceeded 4 et al., 199 1; Maxwell et al., 1992). We have pursued this goal with exogenous acetylcholinesterase as a single pretreatLD-. Performance decrements observed under BChE protection were modest by the usual standards of organophosphorus com- ment drug. This approach turns the irreversible nature of pound toxicity. No residual or delayed performance decrements the OP-cholinesterase interaction from disadvantage to ador other untoward effects were observed during 6 weeks of post- vantage; instead of focusing on the OP as an anticholinesexposure testing with either ChE. o 1992 Academic press, IN. terase, we have focused on the cholinesterase as an anti-OP. Using this approach, we have shown that administration of fetal bovine serum (FBS) AChE or human serum butyrylThe toxicity of organophosphorus (OP) compounds is at- cholinesterase (BChE)-protected animals from multiple LDsO tributed to their irreversible inhibition of acetylcholinesterase of a variety of highly toxic OPs without any toxic effects or (AChE). The resultant accumulation of acetylcholine, both performance decrements (Wolfe et al., 1987; Raveh et al., centrally and peripherally, produces an acute cholinergic 1989; Ashani et al., 1991; Doctor et al., 1991; Maxwell et crisis characterized by convulsions, bronchoconstriction, al., 1991, 1992; Wolfe et al., 1991). tracheobronchial and salivary secretions, bradycardia, beWe report here the protection of nonhuman primates, havioral incapacitation, muscle weakness, and, ultimately, rhesus monkeys, from soman toxicity as high as 5 LD5c by death due to respiratory failure. Present drug regimens for pretreatment with FBS AChE (De La Hoz et al., 1986) or horse serum BChE (Ralston et al., 1983) without the occur’ Research was conducted in compliance with the Animal Welfare Act rence of performance deficits, as measured by the Primate and other federal statutes and regulations relating to animals and experiments Equilibrium Platform (PEP) task (Farrer et al., 1982). This involving animals and adhered to the principles stated in the Guide for the task, in which a seated monkey is trained to manipulate a Care of Use of Laboratory Animals, NIH publication 86-23, 1985 edition. * To whom correspondence should be addressed. joystick to maintain orientation in space, was originally deUse of Cholinesterases as Pretreatment Drugs for the Protection of Rhesus Monkeys against Soman Toxicity. WOLFE, A. D., BLICK, D. W., MURPHY, M. R., MILLER, S. A., GENTRY,
189
0041-008X/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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veloped as an animal model of pilot performance for studies in aerospace medicine. PEP performance is a very sensitive measure of cholinergic toxicity, and quantities of OPs or drugs that do not produce overt signs of intoxication produce significant performance deficits on the PEP task (Blick et al., 1989, 1991).
RESULTS
PEP tests after soman challenge were conducted with one monkey at a time (Farrer et al., 1982; Blick et al., 1989, 199 1). After a 30-min baseline test, (Figs 1 and 2, panel 1, period between start of test and E), monkeys were given FBS AChE or BChE intravenously in an amount calculated to be sufficient to neutralize in vitro 32 pg/kg (~5 LD50) of METHODS soman (Benschop et al., 1984; Raveh et al., 1989; Ashani et al., 199 1; Doctor er al., 199 1). As serum proteins, both these Healthy, adult, male rhesus monkeys (Mucuca mulutta), ranging in weight enzymes have long half-lives, 30-40 hr, (Raveh et al., 1989; from 5 to 8 kg, trained to perform the PEP task at least weeWy for a minimum of 6 months were used in these studies (Blick et al., 1991). None of the Ashani et al., 1991; Wolfe et al., 1991; Doctor et al., 1991) monkeys had been previously exposed to soman, AChE, or BChE. The LD,, and in the absence of soman challenge remained at a constant of soman in rhesus monkeys, is estimated to be 6.4 &kg (Hamilton and level in the blood for over 8 hr, well beyond the duration of Lundy, 1989). Routine care of the animals was provided by the Veterinary the PEP test (3 hr). A total of eight monkeys were tested, Sciences Division, Occupation and Environmental Health Directorate, Armstrong Laboratory. The monkeys were individually housed in stainless four receiving FBS AChE and four receiving BChE. No other treatment or prophylactic agent was given at any time. After steel cages in animal rooms that were maintained at 20-25°C and relative humidity of 50 + 10% on a 12-hr 1ight:dark cycle with no twilight. Cages ChE injection, monkeys were tested for 30 min on the PEP were washed twice daily, and a complete cage change was made every 2 to determine whether either enzyme affected performance. weeks. The monkeys were fed certified Primate Chow (Purina No. 5048. Next, the level of enzyme in whole blood was measured, Purina Mills, Inc., Richmond, IN), and permitted tap water ad libitum. followed by the first soman challenge ( 16 pg/kg, ~2.5 LDsO, Both enzymes were purified by procainamide affinity gel chromatography im) and 30 min of PEP performance. Blood ChE levels were (Ralston et al., 1983; De La Hoz et a/., 1986) and titrated in vitro with soman as previously described (Raveh et al., 1989; Ashani et a/., 199 1: again determined, and the second soman challenge dose (9.6 Doctor et al., 1991). One microgram of soman completely inhibited 1050 /Nkg, N 1.5 LD=J was administered. After another 30 min units of FBS AChE and approximately 165 units of BChE. Two of the four of PEP testing, blood ChE levels were measured for the third stereoisomers of racemic soman (Benschop et al., 1984) react with AChE. time. The result of this measurement was then used to caland three of the four stereoisomers react with BChE (for details see Ashani culate a third soman challenge dose (up to 6.4 pg/kg, N 1.0 et al., 1991). The specific activity of FBS AChE was 5600 U/mg (Raveh et al., 1989); 1 mg AChE = 1.428 X 10M8 M. The specific activity of horse LD& based on residual circulating enzyme activity. serum BChE was 750 U/mg. Both ChEs were assayedcolorimetricahy (EllAlthough blood ChE levels were elevated more than lOOman et a/., 1961) using acetylthiocholine (ATC) as a substrate for AChE fold, ChE injections alone caused no apparent physiological and butyrylthiocholine (BTC) as a substrate for BChE. Soman was supplied or neurological effect or deficit, as measured by the PEP task by the Chemical Research, Development, and Engineering Center, Aberdeen performance (Figs. 1 and 2, left, period between E and Sl). Proving Ground, MD. Soman was 98.6% pure when analyzed by 3’P-nuclear magnetic resonance. Soman was supplied in dilute solutions of 2 mg/ml in None of the eight monkeys showed any OP toxicity after propylene glycol. soman challenges; protection was so complete that there were The PEP (Fairer et al., 1982; Blick et al.. 1989, 1991) is a continuous no fasciculations even at the site of soman injections. Folcompensatory tracking device that measures the ability of a monkey to comlowing the first and second soman injections (totaling 25.6 pensate for unpredictable perturbations in pitch induced by a filtered random a/kg. -4 LD5& the PEP performance of all eight monkeys noise signal. The monkey was seated in a chair that rotated about the pitch axis in response to a random external signal providing unpredictable per- was completely normal. The four monkeys pretreated with turbations. The animal could control the pitch by manipulating a joystick FBS AChE and two pretreated with BChE continued this control mounted on the platform. The animal’s task was to use the joystick level of PEP performance even after the third soman chalto compensate for the external signal and thereby keep the platform as level lenge (Figs. 1 and 2, left). However, the two remaining monas possible. The platform position (angle in degrees) was measured by computer 10 times per second, and the standard deviation (a) of all the mea- keys that had been pretreated with BChE exhibited significant but minor PEP deficits after the third soman injection (Fig. surements for each 5-min epoch, i.e., 3000 points, was the metric for quality 2, left No. 604 and No. 564, S3 to end); these transient PEP of PEP performance. Whenever the platform deviated from level by more than 15”, the animal received mild tail shocks (lOO-ms duration, I-Hz rep- performance deficits were similar to those observed after lowetition rate, 5 mA maximum current) until the platform was returned to dose soman (~2.8 pg/kg) in unprotected monkeys (Blick et ..I. me .I 13 .-n*. .. ror - eacn* suqecr, . . tail-snack .. . ’ Iintensity was adjusted to wnnin - urnits. al., 1989, 199 1). Based upon this comparison, we believe the minimum level required to maintain motivated pelloiiila ---‘---rice in baseline tests. Well-trained subjects typically reduced the v+lbiti+v .I lY”.ll LJ3f c platform po- that the protective ratio afforded by our ChE pretreatment sition from a 5 of approximately 12”, which wo utd be producedby the can be estimated to be between 10 and 15. external input alone, to about 3-4” and thus received very few tail shocks Blood ChE levels measured before and after each soman (~1 shock/hour on average). Subjects performed the PEP task for 2.5 hr on challenge (Figs. 1 and 2, middle) showed a linear decline each soman challenge testing day. and results are presented for each 5-min with increasing soman dose. For AChE, this pattern continblock of testing time (Figs. I and 2, left). During the 6 weeks of long-term follow-up, PEP tests were conducted for 2 hr; 0 was computed for each 5- ued until nearly all the FBS AChE in the blood was inhibited; however, for BChE, 20% of the initial blood levels remained min block of time, and the mean of the 24 resulting data points was calculated to yield one performance score for the entire 2 hr. after administration of the same amount of soman. The
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FIG. 1. (Left) Effects of iv administered purified FBS AChE on PEP task performance before and after challenge with approximately 2.5, 1.5, and 1.O LD,, of soman. Four male rhesus monkeys (5-8 kg), trained to perform the primate equilibrium platform (PEP) task, each received approximately 0.4-0.5 rmol of AChE iv (greater than 1:l stoichiometry with soman). The sequence of behavioral testing and soman challenges was (a) 30-min PEP task (baseline); (b) AChE injection, E (15 min): (c) 30-min PEP task to determine the effect of administration of AChE alone, followed by a 15-min pause for obtaining blood samples, AChE assay, and soman injection, S I (16.0 pg/kg, ~2.5 LDSO, im): (d) 30-min PEP testing, followed by a 15-min pause for obtaining blood samples, AChE assay, and soman injection, S2 (9.6 rg/kg, = 1.5 LDSo, im); (e) 30-min PEP testing, followed by a 15-min pause for obtaining blood samples, AChE assay,and the final im soman injection, S3 (6.4 &kg, = 1.O LDSo, was planned but would be reduced if residual AChE activity was judged insufficient); (f) final 30 min of PEP testing. For each 30 min of PEP testing, the data (filled circles) from six sequential 5-min blocks of time are presented (15). (Middle) In vivo titrations of blood AChE in four rhesus monkeys pretreated by iv injection with FBS AChE. Details are as described above. The cumulative dose of soman which reduced ChE activity to the indicated final levels exceeded the amount of AChE administered, suggesting involvement of endogenous esterase. (Right) Long-term effects on PEP task performance of iv administered FBS AChE and challenge with a total of approximately 5 LDSo of soman and residual blood AChE levels. PEP performance and blood AChE levels of four monkeys were tested weekly for 6 weeks; filled circles, PEP performance: open circles, enzyme level. PEP performance scores are the mean of data from 24 separate 5-min blocks that compose the 2-hr test.
strong linear relationship between OP dose and residual ChE in vivo agreed with previous observations made with mice (Raveh et al., 1989; Ashani et al., 1991; Doctor et al., 1991) and nonhuman primates (Wolfe et al., 199 1; Maxwell et al., 1992) and confirms the hypothesis that stoichiometric reaction is responsible for the observed protection. Soman was sequestered by exogenous and endogenous ChEs in the blood before it could reach physiologically critical target AChE. Thus, the extent of protection afforded by exogenous ChE
is dependent on the concentration of ChE in the blood and the OP dose; to double the protection, twice the amount of ChE will be required. In addition, the rate of circulation and the clearance of exogenous enzyme and OP will influence the extent of protection. During the period after soman challenge, blood AChE levels quickly returned to pre-FBS AChE levels (Fig. 1, right). BChE, however, appeared to decrease below its original (preexogenous BChE administration) level and then returned
WOLFE ET AL.
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(min) FIG. 2. (Left) Effects of iv administered purified horse serum BChE on PEP test performance before and after challenge with approximately 2.5, 1.5, and I .O LD,, of soman. See legend to Fig. 1. for a detailed explanation. (Middle) In viva titrations of blood BChE in four rhesus monkeys pretreated by iv injection with horse serum BChE. See legend to Fig. 1 for details. (Right) Long-term effects on PEP task performance of iv administered horse serum BChE and challenge with a total of approximately 5 LDSo of soman and residual blood BChE levels. For details, see legend to Fig. 1.
to normal levels (Fig. 2, right). Perhaps the exogenous BChE triggered a feedback mechanism responsible for maintaining endogenous BChE levels. During 6 weeks of postsoman testing, no monkey, including the two monkeys that received BChE and had shown PEP deficits on the challenge day, showed any signs of delayed toxicity, convulsions, or other OP symptoms, or any abnormality on PEP performance (Figs. 1 and 2, right). DISCUSSION The results presented here are in sharp contrast to the findings of previous studies that tested the effectiveness of the multiple-drug regimen currently used against OP (soman) toxicity. In those studies, pyridostigmine-pretreated rhesus monkeys receiving soman and the appropriate regimen of 2-PAM chloride, atropine, and diazepam were partially pro-
tected against lethality and extensive convulsions but still displayed serious symptoms of OP poisoning, including convulsions, a period of unconsciousness (Blick et al., 1989; Castro et al., 199 1; Murphy et al., 1992) and in one study (Murphy et al., 1992) exhibited a period of delayed and extended performance debilitation. We have shown that ChE pretreatment protected against OP-induced lethality, convulsions, and other overt symptoms and performance deficits, and there were no delayed effects. Based on the results presented here with nonhuman primates, the 1: 1 stoichiometry between ChE and the OP dose, and the amount of endogenous scavenger (ChE, carboxylesterases) normally present (Boskovic, 1979; Sterri et al., 198 1; Clement, 1984; Maxwell et al., 1987) the LDso of soman in humans may be extrapolated to be approximately 300 pg/ 70 kg. To achieve a protective ratio of 2, a pretreatment dose of lOO- 150 mg enzyme might be required. This pretreatment
CHOLINESTERASES
AS PRETREATMENT
should be effective for 70-90 hr. Adverse immunological reactions are among the possible drawbacks to the present approach, and current research is addressing this problem. We are also evaluating the possibility that the addition of one or more currently used drugs may multiply the effectiveness of exogenous ChE. The extensive use of OPs by the pesticide industry, the continued threat of their use in warfare or by terrorists, and the need for the destruction of the extensive existing stocks of these chemicals makes pharmacological and environmental protection against OP toxicity an important issue. We have shown that pretreatment with a ChE is the most effective approach found to date for protection against OP toxicity. The approach demonstrated here should provide a foundation for future prophylaxis and therapy for organophosphate toxicity. ACKNOWLEDGMENTS This work was partially conducted by Systems Research Laboratories, Inc., under contract to the United States Air Force. We wish to thank Frank R. Weathersby, Laurie De La Pana, Denise De La Hoz, German Caranto, Hugh Hively, and Roberta Larrison for expert technical assistance and Drs. Yacov Ashani, G. Carroll Brown, and John W. Fanton for stimulating discussion and advice during the course of this investigation.
REFERENCES Ashani. Y., Shapira, S., Levy, D., Wolfe, A. D., Doctor, B. P., and Raveh, L. ( 199 I ). Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice. Biochem. Pharrnacol. 41, 37-41. Benschop, H. P., Konings, C. G. A., Genderen, J. G., and DeLong, L. P. A. (1984). Isolation, anticholinesterase properties, and acute toxicity in mice of the four stereoisomers of the nerve agent soman. Toxicol. Appl. Pharmacol. 72, 6 1-74. Blick, D. W., Murphy, M. R., Fanton, J. W., Kerenyi, S. Z., Miller, S. A., and Hartgraves, S. L. (1989). Incapacitation and performance recovery after high-dose soman: Effects of diazepam. Proceedings of the Medical Chemical Defense Bioscience Review, pp. 2 19-222. Columbia, MD. Blick, D. W., Kerenyi, S. Z., Miller, S. A., Murphy, M. R., Brown, G. C., and Hartgraves, S. L. (199 1). Behavioral toxicity of anticholinesterases in primates: Chronic pyridostigmine and soman interactions. Pharmacol Biochem. Behav. 38,527-532. Boskovic, B. (1979). The influence of 2-(O-cresyl)-4 H-1:3:2:-benzodioxaphosphorin-2-oxide (CBDP) on organophosphate poisoning and its therapy. Arch. Toxicol. 42, 207-2 16. Castro, C. A., Larsen, T., Finger, A. V., Solana, R. P., and McMaster, S. B. ( 199 1). Behavioral efficacy of diazepam against nerve agent exposure in rhesus monkeys. Pharmacol. Biochem. Behav. 41, 159-164. Clement, J. G. (1984). Importance of ahesterase as a detoxification mechanism for soman (pinacolyl methylphosphonofluoridate) in mice. Biochem. Pharmacol. 33,3807-38 11. De La Hoz, D. M., Doctor, B. P., Ralston, J. S., Rush, R. S., and Wolfe, A. D. (1986). A simplified procedure for the purification of large quantities of mammalian acetylcholinesterase. Life Sci. 39, 195-199.
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Dirnhuber, P., French, M. C., Green, D. M., Leadbeater, L., and Stratton, J. A. (1979). The protection of primates against soman poisoning by pretreatment with pyridostigmine. J. Pharm. Pharmacol. 31,295-299. Doctor, B. P., Brecht, K., Castro, C., De La Hoz, D., Finger, A., Gentry, M. K., Gold, G., Hively, H., Larrison, R., Maxwell, D., McMaster, S., Solana, R.. Wolfe, A. D., and Woodward, C. (1991). Protection against soman toxicity and prevention of performance decrement in rhesus monkeys by pretreatment with acetylcholinesterase. Proceedings of the 1991 Medical Defense Bioscience Review. pp. 407-4 13. Edgewood, MD. Doctor, B. P., Raveh, L., Wolfe, A. D., Maxwell, D. M., and Ashani, Y. (199 1). Enzymes as pretreatment drugs for organophosphate toxicity. Neurosci. Biobehav. Rev. 15, 123-128. Dunn, M. A., and Sidell, F. R. (1989). Progress in medical defense against nerve agents. JAMA 262, 649-652. Ellman, G. L., Courtney, D., Andres, V., Jr., and Featherstone, R. M. (1961). A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. Farrer, D. N., Yochmowitz, M. G., Mattson, J. L., Lof, N. E., and Bennett, C. T. (1982). Effects of benactyzine on an equilibrium and multiple response task in rhesus monkeys. Pharmacol. Biochem. Behav. 16, 605609. Hamilton, M. G., and Lundy, P. M. (1989). HI-6 therapy of soman and tabun poisoning in primates and rodents. Arch. Toxicol. 63, 144-149. Kluwe, W. M., Chinn, J. L., Feder, P., Olson, C., and Joiner, R. (1987). Efficacy of pyridostigmine pretreatment against acute soman intoxication in a primate model. Proceedings of the Sixth Medical Chemical Defense Bioscience Review, pp. 227-234. Edgewood, MD. Maxwell, D. M., Brecht, K. M.. and O’Neill, B. L. (1987). The effect of carboxylesterase inhibition on interspecies differences in soman toxicity. Toxicol. Len. 39, 35-42. Maxwell, D. M., Wolfe, A. D., Ashani, Y., and Doctor, B. P. (1991). In Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology (J. Massoulie et al., Eds.), pp. 206-209. American Chemical Society, Washington, D.C. Maxwell, D. M., Castro, C. A., De La Hoz, D. M., Gentry, M. K.. Gold, M. B., Solana, R. P., Wolfe, A. D., and Doctor, B. P. (1992). Protection of rhesus monkeys against soman and prevention of performance decrement by pretreatment with acetylcholinesterase. Toxicol. Appl Pharmacol. 115. 44-49. McLeod, C. G., Jr. (I 985). Pathology of nerve agents: perspectives on medical managements. Fundam. .4ppl. Toxicol. 5, SlO-Sl5. Murphy, M. R.. Blick, D. W., Dunn, M., Fanton, J. W., and Hartgraves, S. L. (1992). Diazepam as a treatment for nerve agent poisoning in primates. Aviation, Space, Environ. Med., in press. Ralston, J. S., Main, A. R., Kilpatrick, J. L., and Chasson, A. L. (1983). Use of procainamide gels in the purification of human and horse serum butyrylcholinesterase. Biochem. J. 211,243-25 1. Raveh, L., Ashani. Y., Levy, D., De La Hoz, D., Wolfe, A. D., and Doctor, B. P. (1989). Acetylcholinesterase prophylaxis against organophosphate poisoning: Quantitative correlation between protection and blood enzyme level in mice. Biochem. Pharmacol. 38, 529-534. Sterri, S. H., Lyngaas, S., and Fonnum, F. (198 1). Toxicity of soman after repetitive injection of sublethal doses in guinea pig and mouse. Acta Pharmacol. Toxicol. 49, 8- 13. Wolfe, A. D., Rush, R. S., Doctor, B. P., Koplovitz, I., and Jones. D. (1987). Acetylcholinesterase prophylaxis against organophosphate toxicity. Fundam. Appl. Toxicol. 9,266-270. Wolfe, A. D.. Maxwell, D. M., Raveh, L., Ashani, Y., and Doctor, B. P. (199 1). In vivo detoxification of organophosphate in marmosets by acetylcholinesterase. Proceedings of the 1991 Medical Defense Bioscience Review, pp. 547-550. Edgewood, MD.
