Treatment of poisoning by soman

FUNDAMENTAL

AND

APPLIED

TOXICOLOGY

5,

S225-S23 1 (1985)

Treatment of Poisoning by Soman L.LEADBEATER,'

R.H.INNS,AND

J.M. RYLANDS

Chemical Defence Establishment, PortonDown,Salisbury,Wiltshire,Sp4OJQ,

Treatment of Poisoning by Soman.

LEADBEATER,

L., INNS,

R. H. AND

RYLANDS,

Great

Britain

J. M. (1985).

Fundam. Appl. Toxicol.5, S225-S23 1. The efficaciesof a number of drug treatment combinations in protecting guinea pigs against the lethal and incapacitating effects of soman (and satin) have been determined. Incapacitation was studied using a swimming test which is a measure of gross motor perfotmance. The drug combinations employed had no effect on the swimming performance of unpoisoned animals. Pyridostigmine pretreatment supported by postpoisoning therapy with atropine, pralidoxime mesylate (P2S), and diazepam protected guinea pigs against the lethal actions of soman and satin, but the treatment was less effective in protecting against the agent-induced decrements in swimming performance. Replacing pyridostigmine (a quatemary carbamate) by physostigmine (which readily enters the CNS) and introducing aprophen (an anti-cholinergic drug with a range of pharmacological actions) improved the protection achieved against both lethality and incapacitation. When the postpoisoning therapy was omitted, pretreatment with physostigmine and aprophen (or some other anti-choline&c drug) gave significant levels of protection against both soman- and satin-induced lethality and incapacitation. It is concluded that a number of different pharmacological actions are required to antagonize nerve agent-induced incapacitation and that they, and their relative importance, remain to be identified. o 1985Academic mess, IN.

In a number of species a most effective treatment for soman (1,2,2-trimethylpropyl methylphosphonofluoridate) poisoning has been shown to be pretreatment with a carbamate supported by therapy with an anticholinergic drug (Berry and Davies, 1970; Dimhuber et al., 1979; Gordon et al., 1978; Hey1 et al., 1980; Inns and Leadbeater, 1983; Karlsson et al., 1984). The United Kingdom has recently introduced for its Military Services a treatment regime against nerve agent poisoning consisting of pyridostigmine pretreatment supported by postpoisoning therapy with atropine, pralidoxime mesylate, (P2S) and diazepam (Gall, 198 1). Although this combination is effective in protecting animals against poisoning by a number of nerve agents (Inns and Leadbeater, 1983), the animals take many hours to recover completely. If servicemen exposed to nerve agents are to be able to continue to function effectively ’ To whom correspondence should be addressed.

rather than to be casualties requiring medical support, it is essential that the drug treatment available should prevent, or at least markedly reduce, nerve agent-induced decrement in military performance. Modifications to the drug treatment have been devised on the premise that to prevent incapacitation, the drugs must be given in anticipation of poisoning rather than in response to the signs and symptoms of poisoning (i.e., attention has been concentrated on modifications to the pretreatment rather than to therapy). This paper describes studies designed to improve the drug treatment for soman poisoning. Work with sarin (isopropyl methylphosphonofluoridate) has been included for comparative purposes. Pyridostigmine is a quatemary compound which does not readily penetrate the CNS (Birtley et al., 1966; Karlsson et al., 1984). The inability of pyridostigmine to enter the CNS may be the basis of its failure to protect against nerve agents’ incapacitating effects. Physostig-

S225

0272-0590185 $3.00 Copyright 0 1985 by the Society of Toxicology. All rights of reproduction in any form reserved.

S226

LEADBEATER,

INNS, AND RYLANDS

mine enters the CNS (Harris et al., 1978; Karlsson et al., 1984; Meyer, 1950) and is an effective pretreatment against nerve agent poisoning (Berry and Davies, 1970; Gordon et al., 1978). The efficacy of physostigmine in protecting against nerve agent incapacitation and lethality has been determined. The second approach was to incorporate an anti-cholinergic drug into the pretreatment, since in many studies of the treatment of nerve agent poisoning, atropine given before challenge with the agent has been found to be extremely effective (Brimblecombe et al., 1970; Clement, 198 1). Aprophen (adiethyl-aminoethyl aa-diphenylproprionate) possesses anti-muscarinic, anti-nicotinic, local anesthetic, and anti-spasmolytic activities (Unpublished results from CDE; Martindale, 1982; Mashkovsky and Liberman, 1957). It was therefore decided to evaluate the efficacy of this broad-spectrum anti-cholinergic drug in combination with carbamate pretreatment rather than a more specific anti-muscarinic drug such as atropine. Work at the Chemical Defence Establishment (CDE) has suggested that the guinea pig is a good model for predicting the efficacy of treatments for organophosphorus poisoning in primate species (Inns and Leadbeater, 1983). This species has therefore been employed in this study. Incapacitation has been measured in the swimming test developed by Rylands (1982), which gives a measure of gross motor behavior. Since the aim is to produce a drug treatment to be given in anticipation of nerve agent poisoning, the treatments were shown to be without any effect in the guinea pig swimming test. METHODS Animals. Male Dunkin-Hartley guinea pigs (200-300 g) were used. Materials. Sarin and soman (at least 95% pure), P2S, aprophen hydrochloride, and G3063 were prepared within the CDE. Atropine sulfate, adiphenine hydrochloride, and physostigmine salicylate were purchased from Sigma Ltd. (London) and hyoscine hydrobromide from MacFarlanSmith Ltd. Caramiphen hydrochloride, dicyclomine hy-

drochloride, and diazepam were donated by Geigy Ltd., Merrell Ltd., and by Berk Chemicals Ltd., respectively. Experimental methods. All drugs were dissolved in isotonic saline except for diazepam, which was dissolved in 50% polyethylene glycol 200 in saline. Atropine and P2S were dissolved in the same solution for postpoisoning therapy. Drugs were injected subcutaneously into the scruff of the neck at a dose volume of 1 ml/kg. Pretreatment drugs were given 30 min before the nerve agent and the therapeutic drugs immediately after poisoning. The guinea pig swim test (Rylands, 1982) consists of measuring the time taken by a naive animal to swim a distance of 2.65 m. The criterion for a significant decrement in performance is defined as a swim time greater than that of control animals (5.99 set) plus three standard deviations (i.e., 8.64 set). To determine the sign-free dose of the individual treatment drugs, serial doses (varying by a factor of 2) were administered to groups of six to eight animals: the signfree dose was taken to be half of the lowest dose which produced an increase in swimming time. Drug combinations were tested using the sign-free doses of the individual components and adjusting where necessary. The minimum effective dose of pyridostigmine was considerably higher than that required to give protection against soman poisoning (Gordon et al., 1978) and the latter dose (0.38 pmol/kg) was used in these experiments. Similarly the dose of physostigmine chosen (0.48 pmol/kg) was considerably less than its minimum effective dose but gave a similar level of carbamoylation of guinea pig erythrocyte acetylcholinesterase (70%) as the dose of pyridostigmine. P2S did not produce a decrement in swimming performance until near-lethal doses were used, therefore a dose was chosen (65 pmol/kg) which is effective against satin poisoning. The lowest effective doses of atropine and diazepam were 23 and 14 pmol/k& respectively.Higher doses caused an increase in the number of animals turning during the swim test. In establishing the sign-free therapeutic combination, the effects of atropine and diazepam were synergistic in increasing turning during the test. The dose of atropine was maintained as high as possible (11.5 pmol/ kg), and the dose of diazepam was reduced to 0.35 pmol/ kg when the total combination of atropine, P2S, and diazepam did not affect the guinea pig swimming performance. The sign-free dose of each pretreatment anti-cholinergic drug was determined in conjuction with pyridostigmine or physostigmine in guinea pigs given the standard therapy 30 min later. The sign-free doses were: adiphenine 46 pmollkg; aprophen 88 amollkg; atropine 5.7 rmol/kg; caramiphen 12 pmol/kg; dicyclomine 45 pmol/kg; G3063 2.4 pmol/kg; and hyoscine 0.26 rmol/kg. The ED50 of the organophosphonates were determined in untreated and treated guinea pigs using groups of six to eight animals at four or more doses by probit analysis (Finney, 1977). The efficacy of the drug treatments is expressed as a protection ratio, the ratio between the ED50 of the agent in treated animals to that in untreated animals. The 95% confidence. limits of the ratio are presented.

TREATMENT

OF POISONING

Incapacitation was determined I hr after poisoning by sarin and 4 hr after poisoning by soman (see Results) unless recorded otherwise. Lethality protection ratios were obtained similarly and were based on 24-hr mortalities.

RESULTS Organophosphonts

Poisoning

Both sarin and soman increased the swim time in a dose- and time-dependent manner. The effect was maximal OS-2 hr after the injection of sarin and l-4 hr after soman. The minimum ED50 values for both compounds were not significantly different from their 24hr LD5Os (Table 1). Carbamate Pretreatment Pretreating animals with pyridostigmine gave no protection against incapacitation or lethality induced by satin or soman (Table 2). Physostigmine pretreatment gave significant protection against both satin and soman lethality, and protected against incapacitation induced by soman, but not that by sarin. Therapy with a combination of atropine, P2S, and diazepam markedly enhanced the protection afforded by either carbamate against organophosphonate-induced incapacitation or lethality (Table 2). Physostigmine was a more effective pretreatment than pyridostigmine. For both pretreatment drugs, protection was maximal 1 and 4 hr after poisoning with satin and soman, respectively, i.e., TABLE 1 THE

TOXICITY OF SARIN AND SOMAN IN THE GUINEA

PIG

kin

Soman

ED50

39.0 (36.9-41.5)

28.2 (24.1-30.1)

LDSO

41.7 (38.6-44.7)

29.0 (25.8-32.6)

Note. The ED50 and LD50 values are in &kg confidence limits).

(95%

BY SOMAN

s221

at the times when the two agents were maximally incapacitating in unpoisoned animals. The drug treatment moved the swimming time-dose curve to the right, demonstrating that it afforded complete protection against a dose of organophosphonate which caused an increased swimming time in untreated guinea Pigs. In these experiments the doses of carbamate used gave a peak carbamoylation of the erythrocyte acetylcholinesterase (AChE) of about 70%. Reducing the dose so that the inhibition of AChE was about 40% had no significant effect on the protection afforded by pyridos&nine, but the efficacy of physostigmine was reduced against soman-induced incapacitation and against lethality induced by either organophosphonate. In all the subsequent experiments the higher doses of the carbamates were used. Anti-cholinergic

and Carbamate Pretreatment

Inclusion of aprophen with carbamate pretreatment in guinea pigs receiving the standard therapy increased the efficacy of the treatment (Table 3). In combination with pyridostigmine pretreatment, aprophen increased the protection against organophosphonate-induced incapacitation and lethality, but with physostigmine it only raised the protection against incapacitation. The increased protection achieved by aprophen pretreatment was so high that the aprophen-carbamate pretreatments were evaluated without any supporting postpoisoning therapy (Table 3). The omission of the therapy reduced the levels of protection provided against organophosphonate poisoning but nevertheless, sign&+ protection was achieved. The combination of aprophen and physostigmine was the more effective, giving protection ratios greater than 5 for both incapacitation and lethality produced by satin or soman. Because of the efficacy of the aprophenphysostigmine pretreatment, a number of other anti-cholinergic drugs were evaluated (Table 4). Atropine and hyoscine are more

S228

LEADBEATER,

INNS, AND RYLANDS TABLE 2

THE

PROTECYTION

A!=F~RDED

BY CARBAMATE THERAPY WITH

PRETREATMENT ALONE OR COMBINED ATROPINE, P2.3, AND DIAZEPAM

Protective ratio (95%

WITH

POSTP~ISONING

confidence

limits)

Peak

Carbamate

inhibition of erythrocyte AChE (%)

Postpoisoning therapy

0.048

40

Yes

2.1 (1.6-4.3)

0.38

68

Yes

Dose (mWkg)

Pyridostigmine

Physostigmine

Satin

Soman

Incapacitation

Lethality

Incapacitation

Lethality

15.6 (8.0-30)

1.7 (1.6-1.9)

2.6 (2.3-3.2)

2.2 (1.9-2.5)

(7.OZ.O)

(LZ.4)

3.6 (2.5-5.2)

0.38

68

No

(o.;%)

(LO!;f3)

(0.91.fLO)

(Z3)

0.03

39

Yes

8.5 (7.4-9.7)

8.9 (5-l-15.2)

2.1 (2.1-3.5)

(2.3ti.O) 14.4 (10.9-19.0)

0.48

14

Yes

8.6 (5.3-14.0)

(17%)

6.0 (5.1-7.1)

0.48

74

No

(0.:::.6)

(1.9I;tz)

(Z.3)

2.5 (2.2-2.8)

Note. The doses and timings of drug administration are given under Methods.

TABLE 3 THE

EFFETE

OF APROPHEN ON THE EFFICACY OF CARBAMATE PRETREATMENT ALONE WITH POSTP~ISONING THERAPY WITH ATROPINE, PZS, AND DIAZEPAM

OR COMBINED

Protective ratio (95% confidence limits) Grin Pretreatment Pyridostigmine

Postpoisoning therapy

Incapacitation

Soman Lethality

incapacitation

Lethality

(L9?5)

(%,‘I:,,

(2.0%)

(2.5Ti2.2)

(8.k6)

$69)

(4.Z.3)

(4.Z)

(L.6::5)

(4.3%)

1.9 (1.6-2.3)

(2.6?4)

(5.3YP4)

$51)

(5.1%)

14 (1 I-19)

$28)

(2:2_48)

(9.:16)

8.8 (7.2-l 1)

(5.8Ti.6)

8.7 (6.7-l 1)

(4.3Tt.3)

1.3 (6.2-8.5)

YeS

Pyridostigmine plus aprophen

Yes

Pyridostigmine plus aprophen

No

Physostigmine

Yes

Physostigmine plus aprophen

Yes

Physostigmine plus aprophen

No

Note. The doses and timings of drug administration are given under Methods.

TREATMENT

OF POISONING

S229

BY SOMAN

TABLE 4 THE PROTECTION

AFFORDED

BY PRETREATMENT WITH COMBINATIONS AND ANTI-CHOLINERGIC DRUGS

OF FWS~STIGMINE

Protection ratio (95% confidence limits) Anti-cholinergic dw Satin Incapacitation Lethality Soman Incapacitation Lethality

Adiphenine 3.2 (2.6-3.9) <5

2.5 (2.2-2.9)

Aprophen

Atropine

(6.&)

(2.ii.9)

12.1 (7.9-19)

(2.Z.2)

(3.6!:9)

(L!z.O)

(4.OZO)

(2. :1:.5)

Caramiphen

Dicyclomine

(3.94576)

(2.4%)

(4.::.4)

<5

15

<5

<4

G3063

Hyoscine

(4.?6.3) (4?7,4)

(2.8%)

(2.3%)

(3.??6)

(3.tTi.5)

<4

<4

<4

4.0* (3.2-5.0)

Note. The doses and timings of drug administration are given under Methods. * Nof significantly different from the protection ratio obtained with aprophen.

potent and more specific anti-muscarinic drugs than aprophen, dicyclomine, adiphenine, and caramiphen. G3063 (N-methyl 4-pipyridinyl phenylcyclopentanecarboxylate) is the most effective anti-cholinergic drug reported to date in protecting against sarin lethality (Green et al., 1977). Aprophen was the most effective of all the drugs tested, although against soman poisoning hyoscine was not significantly less effective. In all the experiments reported above, the protection ratios were determined 1 hr after sarin poisoning and 4 hr after soman, i.e., at times when the incapacitation was maximal in untreated animals and in those given pyridostigmine and therapy. The incapacitation protection-time profiles were determined for aprophen, hyoscine, and atropine (Table 5). For all three drugs the maximum protection was achieved after 1 hr against sarin poisoning and at 4 hr after soman. DISCUSSION A sign-free combination of pyridostigmine pretreatment and postpoisoning therapy with atropine, P2S, and diazepam protected guinea pigs against the lethal actions of soman but was markedly less effective in protecting against agent-induced decrements in swim-

ming performance. Replacing pyridostigmine by physostigmine and introducing aprophen into the pretreatment improved the protection achieved against both lethality and incapacitation. Even when postpoisoning therapy was omitted, pretreatment with physostigmine and aprophen, or other anti-cholinergic drugs, gave very significant levels of protection. All the drug treatments were markedly more effective against sarin poisoning. Since sarin poisoning responds well to oxime therapy this result was not unexpected when postpoisoning therapy with P2S was used. However, even in the absence of oxime, the combinations of carbamate and anti-cholinergic drugs were more effective against satin than soman poisoning. The guinea pig swim test which has been used to measure nerve agent-induced incapacitation in these experiments is a measure of gross motor performance. Clearly, therefore, these results must be confirmed in a range of behavioral tests. If the efficacy of the combination could be confhmed and a suitable formulation were developed for use in man, it could be of considerable benefit against poisoning with a nerve agent. An effective pretreatment has been identified, but it is difficult to identify the precise mechanisms involved in the protection.

S230

LEADBEATER,

INNS, AND RYLANDS

TABLE 5 PROTECTION-TIME PROFILESFOR PHYSOSTIGMINE-ANTI-CHOLINERGIC DRUG COMBINATIONS Protection ratio (95% confidence limits) Time after nerve agent administration (min) Nerve agent Sarin

Anticholinergic dwz

7.5

Aprophen

30

60

240 -

(I .f2.4)

(2.K.3)

1.3 (5.7-9.4)

8.6 (6.8-10.8)

(I K.4)

2.8* (2.4-3.3)

(3.tT4.6)

(4.Z.3)

Hyoscine Atropine Soman

15

(l.K.4)

1.7 (1.6-1.8)

2.3 (2.1-2.6)

(1.::2.4)

(2.if3.3)

(3.~~5.0)

(1.Z.2)

(l.ifi.8)

0.9 (0.7-1.2)

(1.:.-41.8)

Aprophen

(2.6%9)

(3X.9)

4.4 (3.8-5.2) * (3.;:.2)

(3.iz.5)

(l.f;-61.8)

(2.ii7)

(Z3.0)

Hyoscine Atropine

-

(3F5.9)

Nofe. The doses and timings of drug administration are given under Methods. * Not significantly different from the protection ratio obtained with aprophen.

Pyridostigmine alone does not protect against poisoning by sarin or soman but neither does it sensitize animals to the nerve agents, even though at the moment of challenge 70% of the erythrocyte AChE was carbamoylated. Physostigmine protected against both sarin and soman lethality but was only effective against the incapacitation induced by soman. The superiority of physostigmine was confirmed when postpoisoning therapy with atropine, P2S, and diazepam was given. It has been proposed (Berry and Davies, 1970) that carbamates are effective by protecting part of the vital tissue AChE from irreversible phosphonylation by the nerve agents. Thus the greater efficacy of physostigmine may be related to its ability to penetrate the CNS and protect the central AChE (Harris et al., 1978; Karlsson et al., 1984; Meyer, 1950). However, Wolthuis and Vanwersch (1984) have shown that pyridostigmine and physostigmine cause a range of behavioral effects at 9 and 3.5% of their LDSOs, respectively, suggesting similar abilities to modify the CNS. Albuquerque et al., (1984) have shown that pyridostigmine is

a weak agonist at the acetylcholine nicotinic receptor-ionic channel complex whereas physo&mine is an open channel blocker. It is possible that the ion-channel blocking activity of physostigmine may contribute to its efficacy in antagonizing nerve agent incapacitation. Of the anti-choline&z drugs evaluated as pretreatments in conjunction with physostigmine, aprophen was the most effective, although hyoscine and G3063 gave good protection against soman. Aprophen is a nonspecZc anti-cholinergic drug having antimuscarinic, anti-nicotinic, local anesthetic, and anti-spasmolytic activities (Unpublished results from CDE, Martindale, 1982; Mashkovsky and Liberman, 1957). G3063 has both anti-muscarinic and anti-nicotinic activity (Green et al., 1977). Mecamylamine is an antinicotinic drug (Chance et al., 1978) and its inclusion into pretreatment with pyridostigmine and atropine markedly enhances the protection afforded against soman poisoning (Harris et al., 1980; Hey1 et al., 1980). Thus anti-nicotinic activity appears to be beneficial in the treatment of nerve agent poisoning.

TREATMENT

OF POISONING

Atropine, which does not have any marked anti-nicotinic actions, is less effective than aprophen or G3063. However, hyoscine, which is an effective pretreatment, also does not have anti-nicotinic activity. Hyoscine differs from atropine in that it is about 16 and 3 times more potent as an anti-muscarinic drug in the central and peripheral nervous systems, respectively, it antagonizes seizures elicited by electroshock in rats (Green et al., 1977); and in man, unlike an-opine, it slows the heart, depresses cerebral cortex activity (especially that of the motor areas), and is effective in the treatment of motion sickness (Martindale, 1982; Weiner, 1980). It is clear that a variety of pharmacological actions contribute to the reduction of nerve agent-induced incapacitation. These activities and their significance in the treatment of nerve agent poisoning remain to be identified. ACKNOWLEDGMENTS It is a pleasure to acknowledge the very significant contributions made by our colleagues in the Biology Division of the Chemical Defenoe Establishment.

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BY SOMAN

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