Epilepsy Res., 7 (1990) 105-116
105
Elsevier EPIRES 00357
Anticonvulsant effects of diazepam and MK-801 in soman poisoning*
Tsung-Ming Shih Biochemical Pharmacology Branch, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010.5425 (U. S.A.) (Received 9 January 1990; revision received 25 May 1990; accepted 1 June 1990)
Key words: Soman; Diazepam; MK-801; Atropine sulfate; Convulsions; Anticonvulsants; Secretions; Drug interaction
An animal model was developed to evaluate the anticonvulsant effects of diazepam and MK-801 in soman poisoning and to examine the possible mechanism of soman-induced convulsions. The oxime HI-6 (125 mg/kg, i.p.) was given to male rats, to increase survival, 30 min prior to 180/~g/kg, s.c. (.equivalent to 1.6 x LDs0) of soman, which produced 100% occurrence of convulsions. Initially, diazepam was studied with or without the concomitant adminis~xation of various doses of atropine sulfate 30 nfin prior to soman challenge. Diazepam (1.25-10.0 mg/kg, i.m.) alone did not prevent soman-induced convulsions. In the presence ef 2, 4, 8, and 16 mg/kg of atropine, the anticonvulsant EDs0 doses of diazepam were 0.490, 0.257, 0.132 and 0.136 mg/kg, respectively. Atropine sulfate at a dose of 16 mg/kg prevented the soman-induced hypersecretion, showed some anticonvulsant activity and provided a good motor recovery. MK-801 by itself, at or above 1 mg/kg, prevented convulsions, but markedly potentiated the lethal effects produced by soman. With atropine (16 mg/kg), the anticonvulsant EDs0 for MK-801 was 0.037 mg/kg, which indicated that MK-801 was about 4 times as potent as diazepam, and the lethal interactions between MK-801 and soman were suppressed. The findings indicate that, in soman poisoning, diazepam and MK-801 are effective anticonvulsants in the presence of the anticholinergic atropine sulfate. The possible sequence of events and neuropharmacological mechanism of soman-induced convulsions are discussed.
INTRODUCTION
Organophosphorus (OP) cholinesterase (ChE) * The experiments reported here were conducted according to the 'Guide for Care and Use of Laboratory Animals' (1985), as prepared by the Committee on Care and Use of Laboratory Animals, National Research Council, NIH Publication No. 85-23. Portions of this work were presented in abstract form54. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting views of the Department of the Army or the Department of Defense.
Correspondence to: Dr. T.-M. Shih, U.S. Army Medical Research Institute of Chemical Defense, SGRD-UV-PB, Aberdeen Proving Ground, MD 21010-5425, U.S.A.
inhibitors such as soman can cause a progression of signs, including hypersecretion, convulsions and death 58. The toxicity of soman is generally believed to be due to its inhibition of acetylcholinesterase (ACHE) and subsequent increase of acetylcholine (ACh) at central and peripheral neuronal synapses 5s. Pretreatment with carbamate antiChE significantly increases the 24-h survival rate in animals exposed to multiple LDso doses of soman 4'1s' 2A,33.However, this pretreatment does not ameliorate soman-induced convulsions ls'24'a2. Convulsive activity in soman intoxication creates a problem for medical management of exposed subjects and has been linked to irreversible brain damage 34' 43,47. Therefore, the most effective employment of
106 carbamate pretreatment appears to require concomitant administration of an adjunct compound selected for its anticonvulsant activityt9. Diazepam, a modulator of the receptor activity of the inhibitory amino acid neurotransmitter yaminobutyric acid (GABA) 14'~, has been shown to prevent the onset of soman-induced brain seizure activity and convulsions 5'21'30'35'36'40-42'52. It has been suggested as the drug of choice in the symptomatic treatment of poisoning by OP compounds 5°'59, including soman s'as'36. However, it possesses undesirable side effects3'17'27. On the other hand, MK-801, an excitatory amino acid antagonist that blocks the N-methyI-D-aspartate (NMDA) receptor, has been reported to be a very potent anticonvulsant against maximal electroshock (MES)- and drug-induced seizures n'6°. Given the recent evidence for its ability to prevent either seizure-mediated 1~ or anoxic-ischemic22 neuronal degeneration in vivo, MK-801 may be an alternative neuroprotective and anticonvulsant agent in soman poisoning. Due to the extremely lethal nature of soman intoxication, a treatment adjunct is needed to prolong the survival time sufficiently to permit observation of anticonvulsant activity. During the past several years, we have been investigating the mechanisms of antidotal action of the bis-pyridinium oxime HI-6 in rats, It was demonstrated that HI-6 by itself is effective in delaying the time-todeath without altering the time-course of toxic motor signs (convulsions) in rats exposed to soman and provides a protective ratio of 2.531,ss. The protective effect of HI-6 is attributed to its ability to reactivate soman-inhibited ChE activity in the periphery ss'56. Therefore, it was felt that rats treated with HI-6 may be an excellent animal model to evaluate compounds that would antagonize soman-induced convulsive activity. In this study, rats pretreated with HI-6 and subsequently given soman were used as the animal model to evaluate the anticonvulsant effects of 2 amino acid receptor modulators, diazepam and MK-801, in the absence and presence of the cholinergic, muscarinic receptor blocker atropine sulfate and to examine the possible mechanism of soman-induced convulsions.
MATERIALS AND METHODS
Animals Male Sprague-Dawley rats (crl:CD[SD]BR), weighing 200-300 g, were obtained from Charles River Labs. (Wilmington, MA), housed 3/plastic cage in temperature- and humidity-controlled quarters and maintained on a 12-h light/dark cycle with lights provided between 06.00 and 18.00 h. Laboratory rat chow and water were freely available. At least 7 days were allowed for the rats to become acclimated to the animal quarters prior to experimental use. In all experiments rats were randomly assigned to treatment groups. All drug injections were given between 09.00 and 12.00 h.
Materials Saline (0.9% NaCI) injection, USP, was purchased from Cutter Labs. Inc. (Berkeley, CA). Injectable Valium® (diazepam) solution was obtained from Hoffmann-LaRoche Inc. (Nutley, NJ), atropine sulfate, USP, from C.H. Boehringer-Sohn (Ingelheim, F.R.G.) and MK-801 ([+]-5methyl-10,11-dihydro-SH-dibenzo[a,d]cyclohep. ten-5,10-imine) from Merck, Sharp and Dohme Research Labs. (Essex, U.K.). HI-6 ([[[(4-aminocarbonyl)pyridino]methoxy]methyl]-2-[(hydroxy. imino)methyl]pyridinium dichloride) was a gift from the Defence Research Establishment Suffield (Ralston, Alberta, Canacla). Soman (pinacolyl methylphosphonofluoridate), obtained from the
Chemical Research, Development and Engineering Center (Aberdeen Proving Ground, MD), was diluted in ice-cold saline prior to injection. Atropine sulfate and HI-6 were prepared in saline solution. MK-801 and diazepam were prepared and diluted in a vehicle containing 40% propylene glycol, 10% ethanol, 1.5% benzyl alcohol and 48.5% distilled water. All drug solutions were prepared and injected separately. Soman was administered subcutaneously (s.c.), HI-6 intraperitoneally (i.p.), and atropine sulfate, diazepam and MK-801 intramuscularly (i.m.).
Experimental design and procedures The animal model was designed to meet the following objectives: (1) a selected dose of soman should cause all of the animals to convulse, but the
107 majority, if not all, of the HI-6-treated rats should survive for a period of time without any additional form of therapy, and (2) the determination of the ability of candidate compounds to prevent somaninduced convulsions should be rapid and reproducible. Acceptable candidate anticonvulsant compounds should not enhance the lethality of soman, but should prevent the onset of convulsions with minimal side effects (e.g., brief loss of coordination or consciousness). (1) Development of the rat model. We previously determined that 0.9 x LDs0 of soman did not induce convulsive activity in all the animals tested 29'51. More recently we demonstrated that when rats were treated with HI-6 immediately after 1.8 x LDs0 of soman, all treated animals convulsed, but only 34% died in 24 h 53. We do not have data available to determine whether HI-6 given prior to soman challenge would provide better protection. Consequently, in this preliminary experiment, HI-6 (125 mg/kg, i.p.) was given to the animals either 30 min prior to ('HI-6 pretreatment') or within 10 sec following ('HI-6 therapy') soman challenge. This was done to select an optimal HI-6 treatment mode that would insure survival long enough for appropriate measurement of any anticonvulsant activity of the tested compounds. This dose of HI-6 has been routinely used with this species in our studies with reproducible results 38'53' 55.56. Ten HI-6-treated rats (n - 10) were injected at each of 4 doses (1.2, 1.4, 1.6 and 1.8 x LD50, s.c.) of soman. The optimal treatment mode of HI-6 and an appropriate dosage of soman were obtained from this preliminary experiment and then used in the following experiments to determine anticonvulsant activity of the test compounds in this animal model. (2) Studies of diazepam and MK-801. Since diazepam has consistently been shown to prevent soman-induced brain seizure activity and convulsions 21'3°'35'4°'41'52,it was used initially to evaluate this rat model and to serve as a standard for comparison. In the 1st study, utilizing the HI-6 (125 mg/kg, i.p.) pretreatment mode from the experiment described above, 5 doses of diazepam (1.25, 2.5, 5.0,
7.5 and 10 mg/kg, i.m.) each administered with HI-6 to rats, 30 min prior to soman challenge, were used to generate a dose-efficacy profile. In the 2nd study, atropine sulfate was given concurrently with diazepam and HI-6. Rats were pretreated with HI-6, 1 of 4 doses (2, 4, 8, and 16 mg/kg, i.m.) of atropine sulfate and one of several doses (from 0.039 to 10 mg/kg, i.m.) of diazepam, 30 min prior to soman challenge. Atropine sulfate is currently one of the antidotes for OP poisoning5s. In this study we attempted to determine the combined effects of diazepam and atropine sulfate and to determine an optimal dose of atropine sulfate in combination with diazepam as an anticonvulsant in soman-intoxicated rats. Subsequently, MK-801 was investigated in the absence and presence of atropine. In this 3rd study, various doses (0.25-10.0 mg/kg, i.m.) of MK-801 were injected with HI-6, 30 min prior to soman challenge, while in the 4th study, rats were pretreated with HI-6, atropine sulfate (16 mg/kg, i.m.) and one of several doses (from 0.0125 to 0.1 mg/kg, i.m.) of MK-801, 30 min prior to soman administration. (3) Method of observation. Each rat was observed for gross behavioral changes and signs of intoxication. Dampness of the lips and nose was taken to indicate the presence of hypersecretion. Particular attention was paid to motor activity and consciousness because some of the anticonvulsants, such as diazepam, also possess muGcle relaxant and sedative properties and may induce ataxia and unconsciousness 3'27. Unconsciousness was determined by loss of both righting and corneal reflexes. Rats were observed for convulsions and other visible signs of intoxication continuously for the first 15 rain and then at 0.5, 1, 2, 3, 4, and 24 h after soman injection. Our experience with soman and diazepam indicated that convulsions occurred within 10 min after soman and muscle relaxation occurred within 15 min after diazepam 52. Therefore, monitoring during the first 4 h covered the time-course of protection by the test compound against soman-induced convulsions, as well as the development and progress of signs of intoxication. The mortality was recorded at 24 h after soman injection. The respective EDs0 values for protection from soman-induced hypersecretion and convul-
108 sions were calculated using the probit method 2°. RESULTS
Development of the rat model Two HI-6 treatment modes, pretreatment and therapy, and various doses of soman were used to establish a suitable test model. Tables IA and IB show the results obtained for HI-6 pretreatment and therapy, respectively. Soman produced a dose-related effect on the time-to-onset of convulsions. The higher the soman dose, the quicker was the onset of convulsions. Doses of soman at or above 1.6 x LD50 were required to produce convulsions in all rats, no matter whether HI-6 was administered prior to (Table IA) or immediately after (Table IB) soman. Increasing the dose of soman from 1.6 to 1.8 x LDs0 decreased the 24-h survival rate. At 1.6 x LDs0 of soman, HI-6 pretreatment yielded 40% mortality, whereas HI-6 therapy yielded 70% mortality. HI6 pretreatment produced less variance in the timeto-onset of convulsions. Therefore, the rat given HI-6 (125 mg/kg, i.p.) 30 min prior to intoxication with 1.6 x LDs0 (180/zg/kg, ~.c.) of soman was chosen as the test model.
Effect of diazepam Diazcpam treatment alone produced dose-de-
pendent decrements of muscle tone and motor activity, and sedation at higher doses. The dose-related effects of diazepam on the time-to-onset of convulsions and the time-to-death after 1.6 x LD50 of soman in the presence of HI-6 are presented in Table II. Surprisingly, all animals pretreated with diazepam developed convulsions, even at 10 mg/kg of diazepam. Rats developed severe convulsions in 4-7 rain and only at the highest dose (10 mg/kg) was the time-to-onset of convulsions doubled. During convulsions, the limbs became rigid and were fully extended. An anticonvulsant EDs0 of diazepam could not be obtained. As the dose of diazepam was increased, more of the treated rats showed severe muscle fasciculations and the survival rate decreased.
Effects of diazepam with atropine sulfate The dose-related effects of diazepam, acting jointly with 4 different doses (2, 4, 8 and 16 mg/kg) of atropine sulfate, on the number of rats that convulsed and the calculated EDs0 after 1.6 × LDs0 of soman in the test model are presented in Table III. In contrast to diazepam alone, diazepam combined with atropine sulfate showed potent anticonvulsant activity. The time-to-onset of convulsions (5-6 rain) was approximately the same as that of the vehicle controls or rats pretreated with diazepam but not atro-
TABLE I
Effects of HI.6 on soman.induced toxicity in rats Dose of soman
laglkg,s.c. (,4) HI.6 pretreatmenta 132.4 154.0 175.6 197.1
Convulsions
No. of LDsos
Mortality
Time-to.onset (n) (rain + S.E.M,)
Response
Time.to.death (n)
Response
fraction
(h +_S.E.M.)
fraction
1.2 1.4 1.6 1.8
13.4 4- 5.2 8.2 4- 3.3 6,1 _ 1.2 4,6 + 0.6
(8) (9) (10) (10)
8/10 9/10 10/10
5,0 4- 2,7 5.8 4- 4,1 1.8 --.0,4
(7) (5) (4)
7/10 5/10 4/10
10/10
2.0 __0.5
(9)
9/10
1.2 1.4 1.6 1,8
16.0 ± 5.0 17.3 + 6.2 9.9 ± 2.5 5.8 4- 1.0
(9) (8) (10) (10)
9/10 8/10
8.3 4- 3.2 7.8 __ 3.6
(8) (7)
8/10 7/10
10/10 10/10
7.6 __3.9 2.4 4- 0.6
(7) (9)
7/10 9/10
(B) HI-6 therapy b 132.4 154.0 175.6 197.1
a HI-6 (125 mg/kg, i.p.) was given 30 min prior to soman, b HI°6 (125 mg/kg, i.p.) was given immediately after soman.
109 TABLE II
Dose-related effects of diazepam on soman-induced toxicity in rats Dose of diazepam (mglkg, i.m.)
Convulsions
Mortality
Time-to-onset (min 4. S.E.M.)
(n)
Response fraction
Time-to-death (h 4- S.E.M.)
(n)
Response fraction
ControP 1.25 2.5 5.0 7.5 10.0
5.1 3.7 5.5 4.5 4.6 8.9
(6) (6) (6) (6) (6) (6)
6/6 6/6 6/6 6/6 6/6 6/6
11.25 0.2 20.0 0.56 0.71 5.54
(2) (2) (1) (2) (3) (4)
2/6 2/6 1/6 2/6 3/6 4/6
+_ 1.3 4- 0.6 +_ 0.7 4. 0.6 + 0.9 4. 2.1
4- 8.75 4- 0.1 4. 0.02 4. 0.32 4. 4.82
a Control injection includes HI-6 (125 mg/kg, i.p.) and vehicle containing 40% propylene glycol, 10% ethanol, 1.5% benzyl alcohol and 48.5% water; volume of injection was 0.5 ml/kg, i.m. All antidotes were given separately 30 rain prior to soman (1.6 x LDs0 , s.c.) challenge.
pine sulfate. At the onset of convulsions, the animals' bodies were fully extended, forelimbs upward and hind limbs downward. Both convulsing and non-convulsing animals fell on their sides and remained unconscious for 15-20 sec. Afterwards, limb and trunk movements ceased, and the animals were prostrate. Later, they gradually resumed locomotion with moderate ataxia (primarily due to hind limb weakness). The convulsive episodes recurred periodically for several hours. As shown in Table III, the calculated doses (with 95% fiducial limits) of diazepam required, in the
presence of different doses of atropine sulfate, to protect 50% of rats against soman-induced convulsions decreased as the doses of atropine were increased. The EDs0 values of diazepam for both 8 and 16 mg/kg of atropine sulfate are significantly (P < 0.05) different from the EDs0 value of diazepam for 2 mg/kg of atropine sulfate. One or two rats in each dosage group died within 24 h. Animals that exhibited severe, whole body muscle fssciculations usually died. Survivors at 24 h were in much better physical condition than those pretreated with diazepam without atropine
TABLE III
Dose.related effects of diazepam and atropine sulfate on soman.induced convulsions in rats
Dose of diazepam (mglkg, i.m.) Controla 0.039 0.078 0.156 0.313 0.625 1.25 2.5 EDso (95% fiducial limits) of diazepam (mg/kg)
Atropine sulfate (mg/kg, i.m.)
2 8/8 8/8 5/8 3/8 1/8 0/1 0.490 (0.315-0.782)
4
8
8/8 7/8 5/8 4/8 1/8 1/8 -
5/8 6/8 5/8 4/8 3/8 1/8 0/6 -
0.257 (0.132-0.455)
0.132' (0.054-0.247)
16 11/14 9/10 7/10 3/10 2/10 1/6 1/6 0/6 0.136" (0.071-0.234)
a Control injection includes HI-6 (125 mg/kg, i.p.), atropine and vehicle containing 40% propylene glycol, 10% ethanol, 1.5% benzyl alcohol and 48.5% water; volume of injection was 0.5 ml/kg, i.m. All antidotes were given separately 30 min prior to soman (1.6 x LDs0, s.c.) challenge. *P < 0.05 when compared to the ED50 for diazepam with 2 mg/kg atropine sulfate.
110 sulfate. Toxic signs usually associated with the recovery phase in soman-intoxicated rats, such as 'bloody' tears (chromodacryorrhea) around the eyelids, abnormality in walking or hunched back, diminished as the doses of atropine were increased. Full recovery was observed in rats treated with 16 mg/kg atropine, while no recovery within 24 h was observed with 2 mg/kg atropine. None of the 72 (0%), 17 of the 46 (37%), 30 of the 40 (75%) and 20 of the 33 (88%) rats showed hypersecretion in the presence of 16, 8, 4 and 2 mg/kg of atropine sulfate, respectively. The calculated EDs0 of atropine sulfate for prevention of soman-induced hypersecretion in the presence of various doses of diazepam in rats was 6.2 (5.07.7) mg/kg, i.m.
Effects of atropine sulfate The dose-related effects of atropine sulfate on the numbers of animals that salivated, convulsed and died after 1.6 x LD50of soman in the presence of HI-6 are summarized in Table IV. Atropine, at or below 8 mg/kg, did not provide adequate protection against soman-induced hypersecretion. The calculated anti-secretory EDs0 of atropine sulfate for blocking 1.6 × LDs0 of soman effect was 7.3 mg/kg. At doses of 8 and 16 mg/kg, atropine sulfate exerted some anticonvulsant activity against soman. The soman-induced convulsive postures of these rats wore modified considerably by the presence of atropine sulfate. At the onset of convui-
TABLE IV
Dose.related antidotal effects of atropine sulfate on soman-induced toxicity in rats Dose of atropine (mg/kg, i,m,) ControP 2.0 4.0 8.0 16.0
Response fiactions Secretions
Convulsions
Mortality
6/6 6/6 6/6 2/8 0/14
6/6 6/6 6/6 6/8 11/14
2/6 0/6 2/6 1/8 6/14
a Control injection includes HI-6 (125 mg/kg, i.p.) and saline (0.5 ml/kg, i.m.). All antidotes were given separately 30 min prior to soman (1.6 × LD50, s.c.) challenge.
sions, animals' tails shook and were partially erect. The limbs of the animals were not splayed; instead, the whole body was fully extended with forelimbs upward and hind limbs downward, and spasms occurred in all 4 limbs. The convulsive episode lasted for 60-90 sec and then subsided; convulsions recurred periodically for several hours. Head bobbing and gasping-for-air behavior was seen only in those rats that showed copious salivary and mucus secretions. With 2 and 4 mg/kg of atropine, all treated rats developed convulsions within 5 min, and the signs of intoxication were similar to those of soman controls. Sub-convulsive jerking with head bobbing occurred throughout the 4-h observation period. High incidences of severe muscle fasciculations and mortality were observed with 16 mg/kg of atropine. At 24 h, survivors from the 2, 4, 8 and 16 mg/kg groups showed, respectively, the typical behavioral abnormality, muscular weakness, slight weakness, and none of these signs associated with soman exposure. An atropine dose of 16 mg/kg adequately suppressed soman-induced hypersecretion, and in combination with diazepam, it also provided excellent recovery from abnormal motor behavior induced by soman poisoning. Therefore, this dose of atropine sulfate was used for the rats in subsequent studies.
Effects of MK.801 The dose-related effects of MK-801 alone on gross motor behaviors were as follows: at 0.25 mg/kg, i.m., ataxia, side-to-side head movements and increased motor activity; at 0.5 mg/kg, i.m., ataxia, side-to-side head movements and increased motor activity with aimless movements; at 1.0 mg/kg, i.m., in addition to the above, loss of muscle tone; and at 5.0 mg/kg, i.m., and above, no head movements, but prostration. Severe adverse interactions between MK-801 (0.25-10.0 mg/kg) and soman (180/~g/kg) were observed: (1) excessive urination; (2) copious salivary and bronchial secretions; (3) severe exophthalmos (eyeball protrusion) and chromodacryorrhea; (4) modified convulsive postures with flexion of all 4 limbs; (5) moribund with loss of consciousness; and (6) severe suppression of respira-
111 TABLE V
Dose-related effects of MK-801 on soman-induced toxicity in rats
Dose of MK-801 (mg/kg, i.m.)
Convulsions Time-to-onset (min + S.E.M.)
(n)
Response fraction
Time-to-death (h + S.E.M.)
(n)
Response fraction
Control a 0.25 0.5 1.0 5.0 10.0
5.8 + 1.3 2.8 + 0.4 3.8 + 1.0 N.C. c N.C. N.C.
(6) (6) (5) b
6/6 6/6 5/6 0/6 0/3 0/2
11.25 + 0.74 + 0.32 + 1.24 + 0.07 + 0.08 +
(2) (3) (2) (5) (3) (2)
2/6 3/6 2/6 5/6 3/3 2/2
Mortality
8.75 0.42 0.26 0.56 0.01 0.00
a Control injection includes HI-6 (125 mg/kg, i.p.) and vehicle containing 40% propylene glycol, 10% ethanol, 1.5% benzyl alcohol and 48.5% water; volume of injection was 0.5 ml/kg, i.m. All antidotes were given separately 30 min prior to soman (1.6 x LDs0, s.c.) challenge. b Transient effect; animals convulsed for less than 30 sec. c N.C., none convulsed.
tion and death apparently by immediate respiratory arrest. The dose-related effects of MK-801 in the presence of HI-6 on time-to-onset of soman-induced convulsions and time-to-death are presented in Table V. At 0.25 mg/kg, all rats developed intermittent convulsions for 15 min and then a moribund state with very shallow respiration. At 0.5 mg/kg, rats exhibited convulsions, but these activities usually subsided within 30 sec; then immobilization and severe respiratory distress were observed. At a dose of 1.0 mg/kg, none of the animals exhibited convulsions, but 3 of 5 died within 5 min and the others were prostrate and gasping for
air. At 5.0 and 10.0 mg/kg, severe lethal interactions occurred. At the first sign of soman-induced abnormal muscle movement, usually about 4-5 min after soman, each animars body became fully extended and asphyxia developed; all the animals died immediately without convulsing. MK-801 potentiated the respiratory depression by soman.
Effects of MK-801 with atropine sulfate In the presence of atropine sulfate, MK-801 at or below 0.05 mg/kg had no apparent behavioral effect, but at 0.1 mg/kg it produced ataxia initially, followed by crawling and side-to-side head movements.
TABLE VI
Dose-related effects of MK-801 acting jointly with a fixed dosage of atropine sulfate on soman-induced toxicity in rats
Dose of MK.801 (mg/kg, i.m.)
Convulsions Time.to-onset (rain + S.E.M.)
(n)
Response fraction
Time.to-death (h + S.E.M.)
O0
Response fraction
ControP 0.013 0.025 0.050 0.100
5.2 + 4.7 + 5.2 + 5.8 + 4.8
(11) (9) (7) (3) (1)
11/12 9/10 7/10 3/10 1/6
0.21 +_.0.03
(6)
6/12 0/10 5/10 1/10 1/6
0.8 0.6 1.6 2.5
Mortality
N.D. b
0.21 + 0.05 0.08 0.33
(5) (1) (1)
a Control injection includes HI-6 (125 mg/kg, i.p.), atropine sulfate (16 mg/kg, i.m.) and vehicle containing 40% propylene glycol, 10% ethanol, 1.5% benzyl alcohol and 48.5% water; volume of injection was 0.5 ml/kg, i.m. All antidotes were given separately 30 min prior to soman (1.6 x LDs0, s.c.) challenge. b N.D., none died.
112 The dose-related joint effects of MK-801 with 16 mg/kg of atropine sulfate on the time-to-onset of convulsions and the time-to-death after 1.6 x LDs0 of soman in the presence of HI-6 are presented in Table VI. Whether the animals convulsed or not, at the onset of extreme motor activities (tremors or muscle twitching), they would suddenly turn and drop on their sides and remain unconscious for several seconds. They were prostrate, but there was no marked respiratory distress. Afterwards, they recovered gradually and resumed walking with moderate ataxia. Animals that exhibited convulsions usually exhibited weakness and decreased respiratory rate. The EDs0 of MK-801 for protection against soman-induced convulsions was 0.037 mg/kg, i.m., in the presence of 16 mg/kg of atropine sulfate. In the presence of atropine, the toxic interactions between MK-801 and soman were no longer apparent and survivors observed at 24 h appeared to be normal and free of the usual signs associated with the recovery phase in soman-intoxicated rats. DISCUSSION In the present study, the use of HI-6 to protect against soman-induced lethality was based on its beneficial effects against soman reported earlier3~'53's5'56.Lundy and Shaw37 also reported that HI-6 did not alter soman-induced convulsions at any time before or after injection of various doses of soman. The protection by HI-6 is limited to the periphery and not necessarily to any action on the brain 53'55'56.This is consistent with its limited entry to the central nervous system (CNS) due to the bisquaternary structure of the compound. In unprotected rats, doses of soman lower than one LD50 did not produce convulsions in all animals challenged29,5~, whereas doses greater than one LDs0 caused death too rapidly3~,53for observation of biochemical changes and anticonvulsant potential of the test compounds. Throughout this study, HI-6 pretreatment alone resulted in a reproducible onset time for convulsions (mean = 5,2 rain) and yielded 70% survival at 24 h. Those pretreated animals that died did so hours after soman administration as compared to minutes in rats challenged with an equivalent dose of soman
alone 53'55. HI-6 has also been utilized by other investigators in studies of biochemical correlates of soman-induced convulsive activity37. Thus, treatment with HI-6 is essential in this rat model to prolong the survival time and permit observation of the anticonvu!sant activity of test compounds. The test model yields results quite rapidly; within the first 15 rain after soman injection a judgment can be made as to whether the animal will develop convulsions. With an effective combination of treatments, no convulsions were observed after 15 min, even though the animal may have had fasciculations during the later observation periods. Although HI-6 was proven to be useful in this and other experiments37, it should be noted that a relatively high dose (0.25 x LDs0) of HI-6 was used. Even though the role played by HI-6 in the CNS may be limited, the peripheral mechanisms affected by HI-6, at the neuromuscular junction and elsewhere, may have altered the effects of soman or other test compounds. The pharmacological properties of HI-6 include ganglionic blockade 39 and hemicholinium-like and antimuscarinic activity~°'sS. Therefore, further studies are needed to clarify the role played by HI-6 in our tests of the effectiveness of anti~onvulsants in soman poisons mg. In our test model, pretreatment with a GABA pathway agonist diazepam alone, even at a close as high as 10,0 mg/kg, did not prevent convulsions induced by 1.6 x LDso of soman, but only delayed the onset of convulsions. This finding is contrary to prior reports of successful use of diazepam in preventing soman-induced convulsions5'2L3°,35,36, 40-42,52. Several differences between this study and other earlier reports can be identified. First of all, the differences could be due to a dose-effect relationship between diazepam and soman. While the majority of earlier experiments 21,4°-42, including o u r o w n 52, involved a dose of soman at or below one LD50, in the present study we used 1.6 x LD50 of soman. In addition to its activity on the GABAergic system ~4'26,diazepam has been shown to possess some anticholinergic activity13,52. It is possible that diazepam suppressed the cholinergic activity induced by certain levels of soman, above which the activity of diazepam was overwhelmed by excessive cholinergic stimulation. Secondly, differ-
1_13 ences in the routes of administration of diazepam and the species of experimental animals used may account for the discrepant results. Lipp 35'36 demonstrated the effectiveness of intravenous diazepare in suppressing soman-induced brain seizure activity and convulsions in rhesus monkeys, while in the present study an intramuscular route was applied in rats. Differences in the absorption rate and plasma profile of diazepam have been observed after oral, intravenous and intramuscular administrations of diazepam 1'2. Thirdly, inclusion of an anticholinergic compound with diazepam may also explain the different results. For example, Boskovic5, Johnson and Lowndes 3°, Lipp 35 and McDonough et al. 42 reported the synergistic effects of concomitant administration of diazepam and atropine sulfate in soman intoxication. In our initial study with diazepam, atropine was not included. In a subsequent study, however, we confirmed their findings and showed that diazepam was extremely effective in preventing soman-induced convulsions when atropine sulfate was included in the pretreatment regimen. There appears to be a threshold dose of atropine sulfate below which the interactive effect with diazepam, the acute motor debilitation and the later recovery phase were severely affected in somanintoxicated rats. This is paralleled by the dose-related block by atropine sulfate of soman-induced hypersecretion, i.e., with increase in dosage, the suppression of hypersecretion by atropine and the anticonvulsant activity of diazepam vary in the same direction (Table III). Thus, it would seem that modifications in cholinergic function do affect OABA/benzodiazepine function. However, diazepam did not potentiate the block by atropine of soman-induced hypersecretion in the periphery, because the anti-secretory EDs0 doses for atropine were practically the same whether diazepam was included or not (6.2 vs. 7.3 mg/kg; P > 0.05). Thus, our present data do not support other reports 3°'42that diazepam can potentiate the anti-secretory activity of atropine, although a trend toward .~ lower anti-secretory ED50 was observed with diazepam plus atropine. Moreover, it was found that atropine sulfate modified the convulsive postures produced by somati and the gaspingfor-air and head bobbing behavior were seen only
in rats that showed hypersecretion. These results confirm the notion that a titration of more atropine (atropinization) against the end points of good control of secretions and adequate respiratory airway exchange is important and that suppression of hypersecretion is a reliable clinical indicator of adequate atropinization of OP nerve agent c:~sualties 19. An atropine dose of 16 mg/kg prevented hypersecretion induced by 1.6 × LD50of soman, and in combination with diazepam, it also provided excellent recovery from perturbed motor behavior in soman poisoning. Therefore, this dose of atropine sulfate is recommended for rats in future studies. We also found that blockade of NMDA receptors with MK-801 in the presence of atropine sulfate (16 mg/kg, i.m.) and HI-6 (125 mg/kg, i.p.) would prevent convulsions induced by soman in rats. The anticonvulsant ED50 of MK-801 in the presence of the atropine is 0.037 mg/kg, which is significantly lower (P < 0.05) than that of diazepam (0.136 mg/kg) plus atropine. With atropine sulfate, 0.1 mg/kg of MK-801 prevented convulsions in 5 of 6 animals (Table VI), whereas a dosage of MK-801 approximately 10 times higher was required to prevent convulsions in the absence of atropine sulfate (Table V). Thus, with atropine sulfate, MK-801 is about 10 times as potent an anticonvulsant as it is without atropine, and it is 3 times more potent than diazepam plus atropine in preventing soman-induced convulsions. In this rat model, although MK-801 per se is a potent anticonvulsant against soman-induced convulsions, it not only produces abnormal behavioral effects, but also strongly potentiates soman-induced toxicity, especially respiratory depression Is. Exposed animals died very rapidly. These untoward toxic effects can be totally suppressed by the co-administration of atropine sulfate. Therefore, MK-801 is clearly not recommended to prevent brain damage in soman poisoning without concomitant use of an anticholinergic compound. Atropine sulfate, the most common antidote in OP poisoning, protects the muscarinic cholinergic receptors from ACh overstimulation 5s. In the present study, we found that atropine is also an important adjunct to the GABA agonist diazepam and the NMDA antagonist MK-801 in the prevention of soman-induced convulsions. Atropine sul-
114 fate pretreatment significantly potentiates the anticonvulsant effectiveness of diazepam and MK801 and markedly improves the physical condition in the recovery phase. This provides some clues to the possible sequence of events and neuropharmacological mechanisms in soman-induced convulsive activity. Soman acts indirectly on the cholinergic system via irreversible inhibition of AChE and subsequent elevation of ACh levels 58. It is also a potent convulsive agent that acts presumably through the elevated levels of ACh 5~ and subsequent activation of other excitatory neuronal systems in the CNS~'45'48. This sequence of events leading to convulsions in soman intoxication is clearly implicated in view of our findings that the muscarinic receptor blocker atropine sulfate potentiated the anticonvulsant actions of 2 amino acid neurotransmission modulators, diazepam and MK-801. Thus, it appears that in soman poisoning the excitatory amino acids (aspartate/glutamate) 45 and the inhibitory amino acid ( G A B A ) 16'23'57 are being invoked secondarily rather than primarily as in the genesis of other types of convulsions. Cholinergic hyperactivity plays the major and initial role in soman-induced convulsions. With hyperstimulation of cholinergic receptors by excessive ACh blocked by atropine sulfate, an inhibitory amino acid receptor (GABA) agonist, diazepam, or an excitatory amino acid receptor (NMDA) antagonist, MK-801, dampens the subsequent stimulation of excitatory neuronal activity. The convulsive activity of soman is thus readily suppressed. In fact, the sequence of events is very similar to that proposed for the lithium-pilocarpine model of status epilepticus in rats ~. Another important finding in the present study was the clear anticonvulsant activity of atropine sulfate when given 30 min prior to soman challenge, although a high dosage of atropine was required. McDonough et al. 42 also reported that only a high dose of atropine was able to suppress the motor signs of soman intoxication. It is conceivable that at such large doses atropine might exert some effect through nicotinic 7or NMDA 9 receptors. Further experiments are needed to explore the nicotinic and NMDA mechanism of soman-induced convulsions. Nevertheless, following this lead, we have recently demonstrated the anticonvulsant activity of a variety of tertiary anticholinergic compounds in soman poisoning, with atro-
pine sulfate having the least activity s. This has opened up a new field of research in seeking or designing a class of compounds with dual antidotal properties 25'49. When such compounds are available, the concerns about the unwanted psychopharmacologic effects of diazepam and MK-8013' n,27, the abuse potential of diazepam 17 and the prostration and unconsciousness in animals treated with a combination of atropine and diazepam or MK-801 in soman poisoning, observed in the present study and in others 6'2s, may be dismissed and a substantial improvement in protecting both the lives and functional capacity of persons at risk for OP nerve agent exposure may be achieved 19. In conclusion, a rat model for evaluation of potential anticonvulsant compounds in soman poisoning was developed in which HI-6 was used to prolong the survival time and increase the number of survivors. In our test model diazepam by itself is not effective as an anticonvulsant. MK-801 by itself is a ?, tent anticonvulsant, but it has severe toxic interactions with soman, especially that of respiratory suppression. In high dosage (16 mg/kg, i.m.) atropine sulfate treatment alone has some anticonvulsam activity. In the presence of atropine sulfate, diazepam becomes an effective anticonvulsant, and MK-801 demonstrates a 10-fold increase in anticonvulsant potency in the test model. Furthermore, MK-801 is about 3 times as potent as diazepam. With both diazepam and MK801, brief periods of unconsciousness ensue in soman-poisoned rats. Blockade of muscarinic receptors by atropine has some anticonvulsant effect, and in the presence of such blockade, 2 anticonvul~ant compounds with different mechanisms of action each demonstrate increased anticonvulsant potency. Our results appear to suggest that excessive cholinergic stimulation plays the major and initial role in soman-induced convulsions and that excitatory amino acid neurotransmission may also be involved subsequent to cholinergic hyperstimulation. ACKNOWLEDGEMENTS The author wishes to thank Dr. Paul M. Lundy for his generous supplies of HI-6 and Mr. Thomas A. Koviak for the technical assistance.
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105
Elsevier EPIRES 00357
Anticonvulsant effects of diazepam and MK-801 in soman poisoning*
Tsung-Ming Shih Biochemical Pharmacology Branch, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010.5425 (U. S.A.) (Received 9 January 1990; revision received 25 May 1990; accepted 1 June 1990)
Key words: Soman; Diazepam; MK-801; Atropine sulfate; Convulsions; Anticonvulsants; Secretions; Drug interaction
An animal model was developed to evaluate the anticonvulsant effects of diazepam and MK-801 in soman poisoning and to examine the possible mechanism of soman-induced convulsions. The oxime HI-6 (125 mg/kg, i.p.) was given to male rats, to increase survival, 30 min prior to 180/~g/kg, s.c. (.equivalent to 1.6 x LDs0) of soman, which produced 100% occurrence of convulsions. Initially, diazepam was studied with or without the concomitant adminis~xation of various doses of atropine sulfate 30 nfin prior to soman challenge. Diazepam (1.25-10.0 mg/kg, i.m.) alone did not prevent soman-induced convulsions. In the presence ef 2, 4, 8, and 16 mg/kg of atropine, the anticonvulsant EDs0 doses of diazepam were 0.490, 0.257, 0.132 and 0.136 mg/kg, respectively. Atropine sulfate at a dose of 16 mg/kg prevented the soman-induced hypersecretion, showed some anticonvulsant activity and provided a good motor recovery. MK-801 by itself, at or above 1 mg/kg, prevented convulsions, but markedly potentiated the lethal effects produced by soman. With atropine (16 mg/kg), the anticonvulsant EDs0 for MK-801 was 0.037 mg/kg, which indicated that MK-801 was about 4 times as potent as diazepam, and the lethal interactions between MK-801 and soman were suppressed. The findings indicate that, in soman poisoning, diazepam and MK-801 are effective anticonvulsants in the presence of the anticholinergic atropine sulfate. The possible sequence of events and neuropharmacological mechanism of soman-induced convulsions are discussed.
INTRODUCTION
Organophosphorus (OP) cholinesterase (ChE) * The experiments reported here were conducted according to the 'Guide for Care and Use of Laboratory Animals' (1985), as prepared by the Committee on Care and Use of Laboratory Animals, National Research Council, NIH Publication No. 85-23. Portions of this work were presented in abstract form54. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting views of the Department of the Army or the Department of Defense.
Correspondence to: Dr. T.-M. Shih, U.S. Army Medical Research Institute of Chemical Defense, SGRD-UV-PB, Aberdeen Proving Ground, MD 21010-5425, U.S.A.
inhibitors such as soman can cause a progression of signs, including hypersecretion, convulsions and death 58. The toxicity of soman is generally believed to be due to its inhibition of acetylcholinesterase (ACHE) and subsequent increase of acetylcholine (ACh) at central and peripheral neuronal synapses 5s. Pretreatment with carbamate antiChE significantly increases the 24-h survival rate in animals exposed to multiple LDso doses of soman 4'1s' 2A,33.However, this pretreatment does not ameliorate soman-induced convulsions ls'24'a2. Convulsive activity in soman intoxication creates a problem for medical management of exposed subjects and has been linked to irreversible brain damage 34' 43,47. Therefore, the most effective employment of
106 carbamate pretreatment appears to require concomitant administration of an adjunct compound selected for its anticonvulsant activityt9. Diazepam, a modulator of the receptor activity of the inhibitory amino acid neurotransmitter yaminobutyric acid (GABA) 14'~, has been shown to prevent the onset of soman-induced brain seizure activity and convulsions 5'21'30'35'36'40-42'52. It has been suggested as the drug of choice in the symptomatic treatment of poisoning by OP compounds 5°'59, including soman s'as'36. However, it possesses undesirable side effects3'17'27. On the other hand, MK-801, an excitatory amino acid antagonist that blocks the N-methyI-D-aspartate (NMDA) receptor, has been reported to be a very potent anticonvulsant against maximal electroshock (MES)- and drug-induced seizures n'6°. Given the recent evidence for its ability to prevent either seizure-mediated 1~ or anoxic-ischemic22 neuronal degeneration in vivo, MK-801 may be an alternative neuroprotective and anticonvulsant agent in soman poisoning. Due to the extremely lethal nature of soman intoxication, a treatment adjunct is needed to prolong the survival time sufficiently to permit observation of anticonvulsant activity. During the past several years, we have been investigating the mechanisms of antidotal action of the bis-pyridinium oxime HI-6 in rats, It was demonstrated that HI-6 by itself is effective in delaying the time-todeath without altering the time-course of toxic motor signs (convulsions) in rats exposed to soman and provides a protective ratio of 2.531,ss. The protective effect of HI-6 is attributed to its ability to reactivate soman-inhibited ChE activity in the periphery ss'56. Therefore, it was felt that rats treated with HI-6 may be an excellent animal model to evaluate compounds that would antagonize soman-induced convulsive activity. In this study, rats pretreated with HI-6 and subsequently given soman were used as the animal model to evaluate the anticonvulsant effects of 2 amino acid receptor modulators, diazepam and MK-801, in the absence and presence of the cholinergic, muscarinic receptor blocker atropine sulfate and to examine the possible mechanism of soman-induced convulsions.
MATERIALS AND METHODS
Animals Male Sprague-Dawley rats (crl:CD[SD]BR), weighing 200-300 g, were obtained from Charles River Labs. (Wilmington, MA), housed 3/plastic cage in temperature- and humidity-controlled quarters and maintained on a 12-h light/dark cycle with lights provided between 06.00 and 18.00 h. Laboratory rat chow and water were freely available. At least 7 days were allowed for the rats to become acclimated to the animal quarters prior to experimental use. In all experiments rats were randomly assigned to treatment groups. All drug injections were given between 09.00 and 12.00 h.
Materials Saline (0.9% NaCI) injection, USP, was purchased from Cutter Labs. Inc. (Berkeley, CA). Injectable Valium® (diazepam) solution was obtained from Hoffmann-LaRoche Inc. (Nutley, NJ), atropine sulfate, USP, from C.H. Boehringer-Sohn (Ingelheim, F.R.G.) and MK-801 ([+]-5methyl-10,11-dihydro-SH-dibenzo[a,d]cyclohep. ten-5,10-imine) from Merck, Sharp and Dohme Research Labs. (Essex, U.K.). HI-6 ([[[(4-aminocarbonyl)pyridino]methoxy]methyl]-2-[(hydroxy. imino)methyl]pyridinium dichloride) was a gift from the Defence Research Establishment Suffield (Ralston, Alberta, Canacla). Soman (pinacolyl methylphosphonofluoridate), obtained from the
Chemical Research, Development and Engineering Center (Aberdeen Proving Ground, MD), was diluted in ice-cold saline prior to injection. Atropine sulfate and HI-6 were prepared in saline solution. MK-801 and diazepam were prepared and diluted in a vehicle containing 40% propylene glycol, 10% ethanol, 1.5% benzyl alcohol and 48.5% distilled water. All drug solutions were prepared and injected separately. Soman was administered subcutaneously (s.c.), HI-6 intraperitoneally (i.p.), and atropine sulfate, diazepam and MK-801 intramuscularly (i.m.).
Experimental design and procedures The animal model was designed to meet the following objectives: (1) a selected dose of soman should cause all of the animals to convulse, but the
107 majority, if not all, of the HI-6-treated rats should survive for a period of time without any additional form of therapy, and (2) the determination of the ability of candidate compounds to prevent somaninduced convulsions should be rapid and reproducible. Acceptable candidate anticonvulsant compounds should not enhance the lethality of soman, but should prevent the onset of convulsions with minimal side effects (e.g., brief loss of coordination or consciousness). (1) Development of the rat model. We previously determined that 0.9 x LDs0 of soman did not induce convulsive activity in all the animals tested 29'51. More recently we demonstrated that when rats were treated with HI-6 immediately after 1.8 x LDs0 of soman, all treated animals convulsed, but only 34% died in 24 h 53. We do not have data available to determine whether HI-6 given prior to soman challenge would provide better protection. Consequently, in this preliminary experiment, HI-6 (125 mg/kg, i.p.) was given to the animals either 30 min prior to ('HI-6 pretreatment') or within 10 sec following ('HI-6 therapy') soman challenge. This was done to select an optimal HI-6 treatment mode that would insure survival long enough for appropriate measurement of any anticonvulsant activity of the tested compounds. This dose of HI-6 has been routinely used with this species in our studies with reproducible results 38'53' 55.56. Ten HI-6-treated rats (n - 10) were injected at each of 4 doses (1.2, 1.4, 1.6 and 1.8 x LD50, s.c.) of soman. The optimal treatment mode of HI-6 and an appropriate dosage of soman were obtained from this preliminary experiment and then used in the following experiments to determine anticonvulsant activity of the test compounds in this animal model. (2) Studies of diazepam and MK-801. Since diazepam has consistently been shown to prevent soman-induced brain seizure activity and convulsions 21'3°'35'4°'41'52,it was used initially to evaluate this rat model and to serve as a standard for comparison. In the 1st study, utilizing the HI-6 (125 mg/kg, i.p.) pretreatment mode from the experiment described above, 5 doses of diazepam (1.25, 2.5, 5.0,
7.5 and 10 mg/kg, i.m.) each administered with HI-6 to rats, 30 min prior to soman challenge, were used to generate a dose-efficacy profile. In the 2nd study, atropine sulfate was given concurrently with diazepam and HI-6. Rats were pretreated with HI-6, 1 of 4 doses (2, 4, 8, and 16 mg/kg, i.m.) of atropine sulfate and one of several doses (from 0.039 to 10 mg/kg, i.m.) of diazepam, 30 min prior to soman challenge. Atropine sulfate is currently one of the antidotes for OP poisoning5s. In this study we attempted to determine the combined effects of diazepam and atropine sulfate and to determine an optimal dose of atropine sulfate in combination with diazepam as an anticonvulsant in soman-intoxicated rats. Subsequently, MK-801 was investigated in the absence and presence of atropine. In this 3rd study, various doses (0.25-10.0 mg/kg, i.m.) of MK-801 were injected with HI-6, 30 min prior to soman challenge, while in the 4th study, rats were pretreated with HI-6, atropine sulfate (16 mg/kg, i.m.) and one of several doses (from 0.0125 to 0.1 mg/kg, i.m.) of MK-801, 30 min prior to soman administration. (3) Method of observation. Each rat was observed for gross behavioral changes and signs of intoxication. Dampness of the lips and nose was taken to indicate the presence of hypersecretion. Particular attention was paid to motor activity and consciousness because some of the anticonvulsants, such as diazepam, also possess muGcle relaxant and sedative properties and may induce ataxia and unconsciousness 3'27. Unconsciousness was determined by loss of both righting and corneal reflexes. Rats were observed for convulsions and other visible signs of intoxication continuously for the first 15 rain and then at 0.5, 1, 2, 3, 4, and 24 h after soman injection. Our experience with soman and diazepam indicated that convulsions occurred within 10 min after soman and muscle relaxation occurred within 15 min after diazepam 52. Therefore, monitoring during the first 4 h covered the time-course of protection by the test compound against soman-induced convulsions, as well as the development and progress of signs of intoxication. The mortality was recorded at 24 h after soman injection. The respective EDs0 values for protection from soman-induced hypersecretion and convul-
108 sions were calculated using the probit method 2°. RESULTS
Development of the rat model Two HI-6 treatment modes, pretreatment and therapy, and various doses of soman were used to establish a suitable test model. Tables IA and IB show the results obtained for HI-6 pretreatment and therapy, respectively. Soman produced a dose-related effect on the time-to-onset of convulsions. The higher the soman dose, the quicker was the onset of convulsions. Doses of soman at or above 1.6 x LD50 were required to produce convulsions in all rats, no matter whether HI-6 was administered prior to (Table IA) or immediately after (Table IB) soman. Increasing the dose of soman from 1.6 to 1.8 x LDs0 decreased the 24-h survival rate. At 1.6 x LDs0 of soman, HI-6 pretreatment yielded 40% mortality, whereas HI-6 therapy yielded 70% mortality. HI6 pretreatment produced less variance in the timeto-onset of convulsions. Therefore, the rat given HI-6 (125 mg/kg, i.p.) 30 min prior to intoxication with 1.6 x LDs0 (180/zg/kg, ~.c.) of soman was chosen as the test model.
Effect of diazepam Diazcpam treatment alone produced dose-de-
pendent decrements of muscle tone and motor activity, and sedation at higher doses. The dose-related effects of diazepam on the time-to-onset of convulsions and the time-to-death after 1.6 x LD50 of soman in the presence of HI-6 are presented in Table II. Surprisingly, all animals pretreated with diazepam developed convulsions, even at 10 mg/kg of diazepam. Rats developed severe convulsions in 4-7 rain and only at the highest dose (10 mg/kg) was the time-to-onset of convulsions doubled. During convulsions, the limbs became rigid and were fully extended. An anticonvulsant EDs0 of diazepam could not be obtained. As the dose of diazepam was increased, more of the treated rats showed severe muscle fasciculations and the survival rate decreased.
Effects of diazepam with atropine sulfate The dose-related effects of diazepam, acting jointly with 4 different doses (2, 4, 8 and 16 mg/kg) of atropine sulfate, on the number of rats that convulsed and the calculated EDs0 after 1.6 × LDs0 of soman in the test model are presented in Table III. In contrast to diazepam alone, diazepam combined with atropine sulfate showed potent anticonvulsant activity. The time-to-onset of convulsions (5-6 rain) was approximately the same as that of the vehicle controls or rats pretreated with diazepam but not atro-
TABLE I
Effects of HI.6 on soman.induced toxicity in rats Dose of soman
laglkg,s.c. (,4) HI.6 pretreatmenta 132.4 154.0 175.6 197.1
Convulsions
No. of LDsos
Mortality
Time-to.onset (n) (rain + S.E.M,)
Response
Time.to.death (n)
Response
fraction
(h +_S.E.M.)
fraction
1.2 1.4 1.6 1.8
13.4 4- 5.2 8.2 4- 3.3 6,1 _ 1.2 4,6 + 0.6
(8) (9) (10) (10)
8/10 9/10 10/10
5,0 4- 2,7 5.8 4- 4,1 1.8 --.0,4
(7) (5) (4)
7/10 5/10 4/10
10/10
2.0 __0.5
(9)
9/10
1.2 1.4 1.6 1,8
16.0 ± 5.0 17.3 + 6.2 9.9 ± 2.5 5.8 4- 1.0
(9) (8) (10) (10)
9/10 8/10
8.3 4- 3.2 7.8 __ 3.6
(8) (7)
8/10 7/10
10/10 10/10
7.6 __3.9 2.4 4- 0.6
(7) (9)
7/10 9/10
(B) HI-6 therapy b 132.4 154.0 175.6 197.1
a HI-6 (125 mg/kg, i.p.) was given 30 min prior to soman, b HI°6 (125 mg/kg, i.p.) was given immediately after soman.
109 TABLE II
Dose-related effects of diazepam on soman-induced toxicity in rats Dose of diazepam (mglkg, i.m.)
Convulsions
Mortality
Time-to-onset (min 4. S.E.M.)
(n)
Response fraction
Time-to-death (h 4- S.E.M.)
(n)
Response fraction
ControP 1.25 2.5 5.0 7.5 10.0
5.1 3.7 5.5 4.5 4.6 8.9
(6) (6) (6) (6) (6) (6)
6/6 6/6 6/6 6/6 6/6 6/6
11.25 0.2 20.0 0.56 0.71 5.54
(2) (2) (1) (2) (3) (4)
2/6 2/6 1/6 2/6 3/6 4/6
+_ 1.3 4- 0.6 +_ 0.7 4. 0.6 + 0.9 4. 2.1
4- 8.75 4- 0.1 4. 0.02 4. 0.32 4. 4.82
a Control injection includes HI-6 (125 mg/kg, i.p.) and vehicle containing 40% propylene glycol, 10% ethanol, 1.5% benzyl alcohol and 48.5% water; volume of injection was 0.5 ml/kg, i.m. All antidotes were given separately 30 rain prior to soman (1.6 x LDs0 , s.c.) challenge.
pine sulfate. At the onset of convulsions, the animals' bodies were fully extended, forelimbs upward and hind limbs downward. Both convulsing and non-convulsing animals fell on their sides and remained unconscious for 15-20 sec. Afterwards, limb and trunk movements ceased, and the animals were prostrate. Later, they gradually resumed locomotion with moderate ataxia (primarily due to hind limb weakness). The convulsive episodes recurred periodically for several hours. As shown in Table III, the calculated doses (with 95% fiducial limits) of diazepam required, in the
presence of different doses of atropine sulfate, to protect 50% of rats against soman-induced convulsions decreased as the doses of atropine were increased. The EDs0 values of diazepam for both 8 and 16 mg/kg of atropine sulfate are significantly (P < 0.05) different from the EDs0 value of diazepam for 2 mg/kg of atropine sulfate. One or two rats in each dosage group died within 24 h. Animals that exhibited severe, whole body muscle fssciculations usually died. Survivors at 24 h were in much better physical condition than those pretreated with diazepam without atropine
TABLE III
Dose.related effects of diazepam and atropine sulfate on soman.induced convulsions in rats
Dose of diazepam (mglkg, i.m.) Controla 0.039 0.078 0.156 0.313 0.625 1.25 2.5 EDso (95% fiducial limits) of diazepam (mg/kg)
Atropine sulfate (mg/kg, i.m.)
2 8/8 8/8 5/8 3/8 1/8 0/1 0.490 (0.315-0.782)
4
8
8/8 7/8 5/8 4/8 1/8 1/8 -
5/8 6/8 5/8 4/8 3/8 1/8 0/6 -
0.257 (0.132-0.455)
0.132' (0.054-0.247)
16 11/14 9/10 7/10 3/10 2/10 1/6 1/6 0/6 0.136" (0.071-0.234)
a Control injection includes HI-6 (125 mg/kg, i.p.), atropine and vehicle containing 40% propylene glycol, 10% ethanol, 1.5% benzyl alcohol and 48.5% water; volume of injection was 0.5 ml/kg, i.m. All antidotes were given separately 30 min prior to soman (1.6 x LDs0, s.c.) challenge. *P < 0.05 when compared to the ED50 for diazepam with 2 mg/kg atropine sulfate.
110 sulfate. Toxic signs usually associated with the recovery phase in soman-intoxicated rats, such as 'bloody' tears (chromodacryorrhea) around the eyelids, abnormality in walking or hunched back, diminished as the doses of atropine were increased. Full recovery was observed in rats treated with 16 mg/kg atropine, while no recovery within 24 h was observed with 2 mg/kg atropine. None of the 72 (0%), 17 of the 46 (37%), 30 of the 40 (75%) and 20 of the 33 (88%) rats showed hypersecretion in the presence of 16, 8, 4 and 2 mg/kg of atropine sulfate, respectively. The calculated EDs0 of atropine sulfate for prevention of soman-induced hypersecretion in the presence of various doses of diazepam in rats was 6.2 (5.07.7) mg/kg, i.m.
Effects of atropine sulfate The dose-related effects of atropine sulfate on the numbers of animals that salivated, convulsed and died after 1.6 x LD50of soman in the presence of HI-6 are summarized in Table IV. Atropine, at or below 8 mg/kg, did not provide adequate protection against soman-induced hypersecretion. The calculated anti-secretory EDs0 of atropine sulfate for blocking 1.6 × LDs0 of soman effect was 7.3 mg/kg. At doses of 8 and 16 mg/kg, atropine sulfate exerted some anticonvulsant activity against soman. The soman-induced convulsive postures of these rats wore modified considerably by the presence of atropine sulfate. At the onset of convui-
TABLE IV
Dose.related antidotal effects of atropine sulfate on soman-induced toxicity in rats Dose of atropine (mg/kg, i,m,) ControP 2.0 4.0 8.0 16.0
Response fiactions Secretions
Convulsions
Mortality
6/6 6/6 6/6 2/8 0/14
6/6 6/6 6/6 6/8 11/14
2/6 0/6 2/6 1/8 6/14
a Control injection includes HI-6 (125 mg/kg, i.p.) and saline (0.5 ml/kg, i.m.). All antidotes were given separately 30 min prior to soman (1.6 × LD50, s.c.) challenge.
sions, animals' tails shook and were partially erect. The limbs of the animals were not splayed; instead, the whole body was fully extended with forelimbs upward and hind limbs downward, and spasms occurred in all 4 limbs. The convulsive episode lasted for 60-90 sec and then subsided; convulsions recurred periodically for several hours. Head bobbing and gasping-for-air behavior was seen only in those rats that showed copious salivary and mucus secretions. With 2 and 4 mg/kg of atropine, all treated rats developed convulsions within 5 min, and the signs of intoxication were similar to those of soman controls. Sub-convulsive jerking with head bobbing occurred throughout the 4-h observation period. High incidences of severe muscle fasciculations and mortality were observed with 16 mg/kg of atropine. At 24 h, survivors from the 2, 4, 8 and 16 mg/kg groups showed, respectively, the typical behavioral abnormality, muscular weakness, slight weakness, and none of these signs associated with soman exposure. An atropine dose of 16 mg/kg adequately suppressed soman-induced hypersecretion, and in combination with diazepam, it also provided excellent recovery from abnormal motor behavior induced by soman poisoning. Therefore, this dose of atropine sulfate was used for the rats in subsequent studies.
Effects of MK.801 The dose-related effects of MK-801 alone on gross motor behaviors were as follows: at 0.25 mg/kg, i.m., ataxia, side-to-side head movements and increased motor activity; at 0.5 mg/kg, i.m., ataxia, side-to-side head movements and increased motor activity with aimless movements; at 1.0 mg/kg, i.m., in addition to the above, loss of muscle tone; and at 5.0 mg/kg, i.m., and above, no head movements, but prostration. Severe adverse interactions between MK-801 (0.25-10.0 mg/kg) and soman (180/~g/kg) were observed: (1) excessive urination; (2) copious salivary and bronchial secretions; (3) severe exophthalmos (eyeball protrusion) and chromodacryorrhea; (4) modified convulsive postures with flexion of all 4 limbs; (5) moribund with loss of consciousness; and (6) severe suppression of respira-
111 TABLE V
Dose-related effects of MK-801 on soman-induced toxicity in rats
Dose of MK-801 (mg/kg, i.m.)
Convulsions Time-to-onset (min + S.E.M.)
(n)
Response fraction
Time-to-death (h + S.E.M.)
(n)
Response fraction
Control a 0.25 0.5 1.0 5.0 10.0
5.8 + 1.3 2.8 + 0.4 3.8 + 1.0 N.C. c N.C. N.C.
(6) (6) (5) b
6/6 6/6 5/6 0/6 0/3 0/2
11.25 + 0.74 + 0.32 + 1.24 + 0.07 + 0.08 +
(2) (3) (2) (5) (3) (2)
2/6 3/6 2/6 5/6 3/3 2/2
Mortality
8.75 0.42 0.26 0.56 0.01 0.00
a Control injection includes HI-6 (125 mg/kg, i.p.) and vehicle containing 40% propylene glycol, 10% ethanol, 1.5% benzyl alcohol and 48.5% water; volume of injection was 0.5 ml/kg, i.m. All antidotes were given separately 30 min prior to soman (1.6 x LDs0, s.c.) challenge. b Transient effect; animals convulsed for less than 30 sec. c N.C., none convulsed.
tion and death apparently by immediate respiratory arrest. The dose-related effects of MK-801 in the presence of HI-6 on time-to-onset of soman-induced convulsions and time-to-death are presented in Table V. At 0.25 mg/kg, all rats developed intermittent convulsions for 15 min and then a moribund state with very shallow respiration. At 0.5 mg/kg, rats exhibited convulsions, but these activities usually subsided within 30 sec; then immobilization and severe respiratory distress were observed. At a dose of 1.0 mg/kg, none of the animals exhibited convulsions, but 3 of 5 died within 5 min and the others were prostrate and gasping for
air. At 5.0 and 10.0 mg/kg, severe lethal interactions occurred. At the first sign of soman-induced abnormal muscle movement, usually about 4-5 min after soman, each animars body became fully extended and asphyxia developed; all the animals died immediately without convulsing. MK-801 potentiated the respiratory depression by soman.
Effects of MK-801 with atropine sulfate In the presence of atropine sulfate, MK-801 at or below 0.05 mg/kg had no apparent behavioral effect, but at 0.1 mg/kg it produced ataxia initially, followed by crawling and side-to-side head movements.
TABLE VI
Dose-related effects of MK-801 acting jointly with a fixed dosage of atropine sulfate on soman-induced toxicity in rats
Dose of MK.801 (mg/kg, i.m.)
Convulsions Time.to-onset (rain + S.E.M.)
(n)
Response fraction
Time.to-death (h + S.E.M.)
O0
Response fraction
ControP 0.013 0.025 0.050 0.100
5.2 + 4.7 + 5.2 + 5.8 + 4.8
(11) (9) (7) (3) (1)
11/12 9/10 7/10 3/10 1/6
0.21 +_.0.03
(6)
6/12 0/10 5/10 1/10 1/6
0.8 0.6 1.6 2.5
Mortality
N.D. b
0.21 + 0.05 0.08 0.33
(5) (1) (1)
a Control injection includes HI-6 (125 mg/kg, i.p.), atropine sulfate (16 mg/kg, i.m.) and vehicle containing 40% propylene glycol, 10% ethanol, 1.5% benzyl alcohol and 48.5% water; volume of injection was 0.5 ml/kg, i.m. All antidotes were given separately 30 min prior to soman (1.6 x LDs0, s.c.) challenge. b N.D., none died.
112 The dose-related joint effects of MK-801 with 16 mg/kg of atropine sulfate on the time-to-onset of convulsions and the time-to-death after 1.6 x LDs0 of soman in the presence of HI-6 are presented in Table VI. Whether the animals convulsed or not, at the onset of extreme motor activities (tremors or muscle twitching), they would suddenly turn and drop on their sides and remain unconscious for several seconds. They were prostrate, but there was no marked respiratory distress. Afterwards, they recovered gradually and resumed walking with moderate ataxia. Animals that exhibited convulsions usually exhibited weakness and decreased respiratory rate. The EDs0 of MK-801 for protection against soman-induced convulsions was 0.037 mg/kg, i.m., in the presence of 16 mg/kg of atropine sulfate. In the presence of atropine, the toxic interactions between MK-801 and soman were no longer apparent and survivors observed at 24 h appeared to be normal and free of the usual signs associated with the recovery phase in soman-intoxicated rats. DISCUSSION In the present study, the use of HI-6 to protect against soman-induced lethality was based on its beneficial effects against soman reported earlier3~'53's5'56.Lundy and Shaw37 also reported that HI-6 did not alter soman-induced convulsions at any time before or after injection of various doses of soman. The protection by HI-6 is limited to the periphery and not necessarily to any action on the brain 53'55'56.This is consistent with its limited entry to the central nervous system (CNS) due to the bisquaternary structure of the compound. In unprotected rats, doses of soman lower than one LD50 did not produce convulsions in all animals challenged29,5~, whereas doses greater than one LDs0 caused death too rapidly3~,53for observation of biochemical changes and anticonvulsant potential of the test compounds. Throughout this study, HI-6 pretreatment alone resulted in a reproducible onset time for convulsions (mean = 5,2 rain) and yielded 70% survival at 24 h. Those pretreated animals that died did so hours after soman administration as compared to minutes in rats challenged with an equivalent dose of soman
alone 53'55. HI-6 has also been utilized by other investigators in studies of biochemical correlates of soman-induced convulsive activity37. Thus, treatment with HI-6 is essential in this rat model to prolong the survival time and permit observation of the anticonvu!sant activity of test compounds. The test model yields results quite rapidly; within the first 15 rain after soman injection a judgment can be made as to whether the animal will develop convulsions. With an effective combination of treatments, no convulsions were observed after 15 min, even though the animal may have had fasciculations during the later observation periods. Although HI-6 was proven to be useful in this and other experiments37, it should be noted that a relatively high dose (0.25 x LDs0) of HI-6 was used. Even though the role played by HI-6 in the CNS may be limited, the peripheral mechanisms affected by HI-6, at the neuromuscular junction and elsewhere, may have altered the effects of soman or other test compounds. The pharmacological properties of HI-6 include ganglionic blockade 39 and hemicholinium-like and antimuscarinic activity~°'sS. Therefore, further studies are needed to clarify the role played by HI-6 in our tests of the effectiveness of anti~onvulsants in soman poisons mg. In our test model, pretreatment with a GABA pathway agonist diazepam alone, even at a close as high as 10,0 mg/kg, did not prevent convulsions induced by 1.6 x LDso of soman, but only delayed the onset of convulsions. This finding is contrary to prior reports of successful use of diazepam in preventing soman-induced convulsions5'2L3°,35,36, 40-42,52. Several differences between this study and other earlier reports can be identified. First of all, the differences could be due to a dose-effect relationship between diazepam and soman. While the majority of earlier experiments 21,4°-42, including o u r o w n 52, involved a dose of soman at or below one LD50, in the present study we used 1.6 x LD50 of soman. In addition to its activity on the GABAergic system ~4'26,diazepam has been shown to possess some anticholinergic activity13,52. It is possible that diazepam suppressed the cholinergic activity induced by certain levels of soman, above which the activity of diazepam was overwhelmed by excessive cholinergic stimulation. Secondly, differ-
1_13 ences in the routes of administration of diazepam and the species of experimental animals used may account for the discrepant results. Lipp 35'36 demonstrated the effectiveness of intravenous diazepare in suppressing soman-induced brain seizure activity and convulsions in rhesus monkeys, while in the present study an intramuscular route was applied in rats. Differences in the absorption rate and plasma profile of diazepam have been observed after oral, intravenous and intramuscular administrations of diazepam 1'2. Thirdly, inclusion of an anticholinergic compound with diazepam may also explain the different results. For example, Boskovic5, Johnson and Lowndes 3°, Lipp 35 and McDonough et al. 42 reported the synergistic effects of concomitant administration of diazepam and atropine sulfate in soman intoxication. In our initial study with diazepam, atropine was not included. In a subsequent study, however, we confirmed their findings and showed that diazepam was extremely effective in preventing soman-induced convulsions when atropine sulfate was included in the pretreatment regimen. There appears to be a threshold dose of atropine sulfate below which the interactive effect with diazepam, the acute motor debilitation and the later recovery phase were severely affected in somanintoxicated rats. This is paralleled by the dose-related block by atropine sulfate of soman-induced hypersecretion, i.e., with increase in dosage, the suppression of hypersecretion by atropine and the anticonvulsant activity of diazepam vary in the same direction (Table III). Thus, it would seem that modifications in cholinergic function do affect OABA/benzodiazepine function. However, diazepam did not potentiate the block by atropine of soman-induced hypersecretion in the periphery, because the anti-secretory EDs0 doses for atropine were practically the same whether diazepam was included or not (6.2 vs. 7.3 mg/kg; P > 0.05). Thus, our present data do not support other reports 3°'42that diazepam can potentiate the anti-secretory activity of atropine, although a trend toward .~ lower anti-secretory ED50 was observed with diazepam plus atropine. Moreover, it was found that atropine sulfate modified the convulsive postures produced by somati and the gaspingfor-air and head bobbing behavior were seen only
in rats that showed hypersecretion. These results confirm the notion that a titration of more atropine (atropinization) against the end points of good control of secretions and adequate respiratory airway exchange is important and that suppression of hypersecretion is a reliable clinical indicator of adequate atropinization of OP nerve agent c:~sualties 19. An atropine dose of 16 mg/kg prevented hypersecretion induced by 1.6 × LD50of soman, and in combination with diazepam, it also provided excellent recovery from perturbed motor behavior in soman poisoning. Therefore, this dose of atropine sulfate is recommended for rats in future studies. We also found that blockade of NMDA receptors with MK-801 in the presence of atropine sulfate (16 mg/kg, i.m.) and HI-6 (125 mg/kg, i.p.) would prevent convulsions induced by soman in rats. The anticonvulsant ED50 of MK-801 in the presence of the atropine is 0.037 mg/kg, which is significantly lower (P < 0.05) than that of diazepam (0.136 mg/kg) plus atropine. With atropine sulfate, 0.1 mg/kg of MK-801 prevented convulsions in 5 of 6 animals (Table VI), whereas a dosage of MK-801 approximately 10 times higher was required to prevent convulsions in the absence of atropine sulfate (Table V). Thus, with atropine sulfate, MK-801 is about 10 times as potent an anticonvulsant as it is without atropine, and it is 3 times more potent than diazepam plus atropine in preventing soman-induced convulsions. In this rat model, although MK-801 per se is a potent anticonvulsant against soman-induced convulsions, it not only produces abnormal behavioral effects, but also strongly potentiates soman-induced toxicity, especially respiratory depression Is. Exposed animals died very rapidly. These untoward toxic effects can be totally suppressed by the co-administration of atropine sulfate. Therefore, MK-801 is clearly not recommended to prevent brain damage in soman poisoning without concomitant use of an anticholinergic compound. Atropine sulfate, the most common antidote in OP poisoning, protects the muscarinic cholinergic receptors from ACh overstimulation 5s. In the present study, we found that atropine is also an important adjunct to the GABA agonist diazepam and the NMDA antagonist MK-801 in the prevention of soman-induced convulsions. Atropine sul-
114 fate pretreatment significantly potentiates the anticonvulsant effectiveness of diazepam and MK801 and markedly improves the physical condition in the recovery phase. This provides some clues to the possible sequence of events and neuropharmacological mechanisms in soman-induced convulsive activity. Soman acts indirectly on the cholinergic system via irreversible inhibition of AChE and subsequent elevation of ACh levels 58. It is also a potent convulsive agent that acts presumably through the elevated levels of ACh 5~ and subsequent activation of other excitatory neuronal systems in the CNS~'45'48. This sequence of events leading to convulsions in soman intoxication is clearly implicated in view of our findings that the muscarinic receptor blocker atropine sulfate potentiated the anticonvulsant actions of 2 amino acid neurotransmission modulators, diazepam and MK-801. Thus, it appears that in soman poisoning the excitatory amino acids (aspartate/glutamate) 45 and the inhibitory amino acid ( G A B A ) 16'23'57 are being invoked secondarily rather than primarily as in the genesis of other types of convulsions. Cholinergic hyperactivity plays the major and initial role in soman-induced convulsions. With hyperstimulation of cholinergic receptors by excessive ACh blocked by atropine sulfate, an inhibitory amino acid receptor (GABA) agonist, diazepam, or an excitatory amino acid receptor (NMDA) antagonist, MK-801, dampens the subsequent stimulation of excitatory neuronal activity. The convulsive activity of soman is thus readily suppressed. In fact, the sequence of events is very similar to that proposed for the lithium-pilocarpine model of status epilepticus in rats ~. Another important finding in the present study was the clear anticonvulsant activity of atropine sulfate when given 30 min prior to soman challenge, although a high dosage of atropine was required. McDonough et al. 42 also reported that only a high dose of atropine was able to suppress the motor signs of soman intoxication. It is conceivable that at such large doses atropine might exert some effect through nicotinic 7or NMDA 9 receptors. Further experiments are needed to explore the nicotinic and NMDA mechanism of soman-induced convulsions. Nevertheless, following this lead, we have recently demonstrated the anticonvulsant activity of a variety of tertiary anticholinergic compounds in soman poisoning, with atro-
pine sulfate having the least activity s. This has opened up a new field of research in seeking or designing a class of compounds with dual antidotal properties 25'49. When such compounds are available, the concerns about the unwanted psychopharmacologic effects of diazepam and MK-8013' n,27, the abuse potential of diazepam 17 and the prostration and unconsciousness in animals treated with a combination of atropine and diazepam or MK-801 in soman poisoning, observed in the present study and in others 6'2s, may be dismissed and a substantial improvement in protecting both the lives and functional capacity of persons at risk for OP nerve agent exposure may be achieved 19. In conclusion, a rat model for evaluation of potential anticonvulsant compounds in soman poisoning was developed in which HI-6 was used to prolong the survival time and increase the number of survivors. In our test model diazepam by itself is not effective as an anticonvulsant. MK-801 by itself is a ?, tent anticonvulsant, but it has severe toxic interactions with soman, especially that of respiratory suppression. In high dosage (16 mg/kg, i.m.) atropine sulfate treatment alone has some anticonvulsam activity. In the presence of atropine sulfate, diazepam becomes an effective anticonvulsant, and MK-801 demonstrates a 10-fold increase in anticonvulsant potency in the test model. Furthermore, MK-801 is about 3 times as potent as diazepam. With both diazepam and MK801, brief periods of unconsciousness ensue in soman-poisoned rats. Blockade of muscarinic receptors by atropine has some anticonvulsant effect, and in the presence of such blockade, 2 anticonvul~ant compounds with different mechanisms of action each demonstrate increased anticonvulsant potency. Our results appear to suggest that excessive cholinergic stimulation plays the major and initial role in soman-induced convulsions and that excitatory amino acid neurotransmission may also be involved subsequent to cholinergic hyperstimulation. ACKNOWLEDGEMENTS The author wishes to thank Dr. Paul M. Lundy for his generous supplies of HI-6 and Mr. Thomas A. Koviak for the technical assistance.
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