Toxwology, 72 (1992) 99-105 Elsevier Scientific Pubhshers Ireland Ltd

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Comparison of several oximes against poisoning by soman, tabun and GF Paul M. Lundy, Arnold S. Hansen, Brian T. Hand and Camille A. Boulet Defence Research Estabhshrnent SuffieM, Ralston, Alberta (Canada) (Received October 4th, 1991, accepted December l lth, 1991)

Summary Three oxlmes currently being evaluated for adoption as replacement nerve agent therapy by various countries were compared for therapeutic efficacy against the toxic organophosphate mh~b~tors soman and tabun under a standard set of condmons These oxlmes together with PAM-CI and toxogonm, were also compared for efficacy against GF, an agent weaponlzed by Iraq The order of effectiveness against soman was HI-6 > HLo-7 > pynmldoxlme HLo-7 was very effectwe against tabun poisoning while HI-6 and pynmldox~me were of moderate value Against GF, HI-6 and HLo-7 were extremely effectwe, toxogonln was moderately effectwe, and PAM-CI and pynmldoxlme were the least effective HI-6 prowded a high level of protection against all of the agents tested as &d HLo-7 to a shghtly lesser degree The other oxlmes suffered from their lack of effects against one or more of the organophosphates

Key words Ox~mes, Acetylchohnesterase mhlbltors

Introduction The practical sigmficance of developmg safe effective oxtmes to combat the effects of the 'potent' nerve agents has never been more clear than it has been under threat of conflict such as occurred m the Middle East. Iraq has been reported to have an arsenal including nerve agents such as tabun (ethyl N,N-&methylphosphoramldocyamdate), sarin (tsopropyl methylphosphonofluorldate) and possibly soman (pinacolyl methylphosphonofluoridate). In ad&tlon, Iraq appears to have weaponlzed an agent known as G F (cyclohexyl methylphosphonofluorldate) (P.A Lockwood, pers. commun, l) which as a semi-persistent extremely potent 'nerve' agent [1] It appears from the literature that relatively little is known about treating G F poisoning. The importance of having effective therapies for a variety of agents is now abundantly clear. Thousands of casualties have been reported to have been produced by some of these agents m the Iran/Iraq conflict. These agents were and continue to be a very real threat to coalition forces as well as cwdians [2]. Correspondence to Paul M Lundy, Pharmacology and Therapeutics. Defence Research Estabhshment Suffield, Box 4000, Me&cme Hat, Alberta, T I A 8K6, Canada 1p A Lockwood was a member of the Umted Nations Special Commission Inspection Team which examined the chemical weapon production faclhtles in Iraq 0300-483X/92/$05 00 1992 Elsevier Scientific Pubhshers Ireland Ltd Printed and Pubhshed m Ireland

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The standard therapeutic regimens for treatment of chemical casualties resulting from the use of the agents hsted above consists of atropine together with an oxlme such as PAM-CI or toxogonin Each oxlme is known to be basically ineffective against one or more of the standard nerve agents. Possible beneficial effects of oximes against G F poisoning are not well documented Recently, HI-6 has been developed as an alternate oxame because, among other attributes, It has a wider spectrum of activity than toxogonm or PAM-C1. Unlike the other oximes, it is particularly effective against soman [3]. It has also been reported safe and effective against acute organophosphate poisoning an man [4,5] Recently, two other compounds, an imldazo-pyradinium compound, pyrlmldoxime,4-[(hydroxyamino)methyl]-1-[3-(3-methyl- 1H-imadazollum - 1-yl)propyl] pyrldmium dibromide (CAS no. 69445-02-9) [6,7] and HLo-7 1-[[[4-(aminocarbonyl)pyrldinio]methoxy]methyl]-2,4-bis[(hydroxyimlno) methyl]pyrldinlum diaodide (CAS no. 120103-35-7) [8] have been suggested to have promise as therapeutic adjuncts against organophosphate poisoning [9]. Although we know of no direct evidence to corroborate the effectiveness of these two compounds in vivo against soman, they have been reported to have interesting reactivating properties against various potent AChE inhibitors in vitro [6,10]. HL6-7 has been shown to be superior to HI-6 as a reactivator of both soman and tabun (ethyl N,N-dlmethylphosphoramldocyanidate) inhibited acetylcholinesterase in vitro [10] but its an vivo activity has not, to our knowledge, been documented Judging from its ability to reactivate the enzyme, HL6-7 might be a better therapeutic compound than HI-6, and, as such, might warrant further development for human use The present study was carried out for two reasons The first was to compare the effectiveness of the can&date oxames HI-6, HLo-7 and pyrlmldoxIme against threat agents, soman and tabun Soman cannot be effectively treated with PAM-CI or toxogonin while tabun poisoning is only marginally affected by PAM-CI The second aim of the study was to determine which oxlmes would be the most effective, or at least which would be acceptable treatments, against G F poisoning. The study was carried out under standard con&taons without the use of pretreatment compounds such as pyridostigmine The guinea pig was the ammal model chosen since it is generally regarded as the best non-primate model to assess the treatment of nerve agent poisoning and extrapolation to man. Methods

Dose selection of oximes The selection of the dose of oxlmes used was based on predetermined criteria. First, the use of doses of the oximes which were both maximally effective and of similar relative toxicity was desired in order to carry out comparative therapeutic effectiveness, based on standard fractions of their toxicity Second, it was our desire to use as few animals and as little drug as possible since both HI-6 and HLo-7 are relatively difficult and expensive compounds to synthesize. In order to satisfy the desires of obtaining an estimate of toxicity in a relatively large animal species like the guinea pig (as opposed to the mouse) and hence the requirement of considerable quantities of drugs, we used the 'up and down' method for LDs0 determination [11-13] and the LDs0 was calculated as outlined by Dixon [14]

101 This method involved the administration of an oxlme to an ammal at the estimated LDs0 dose. If the animal survived, the dose was increased, zf It did not, the dose was decreased, in each case by a predetermined factor (i e + 1.3 × original dose). Each animal was observed (24 h) and the next animal was given a larger or smaller dose depending on the outcome. The LDs0 estimate obtained in this manner compares favourably with conventional tests requiring many more ammals and much larger quantities of drugs [13] In the present experiments the LDs0 of each oxlme was determined using between 6 and 8 animals each Near maximally effective doses of HI-6, the compound we have had the most expermnce with, are about 1/5 of the LDs0 (LDs0 -- 550 mg/kg) Therefore, that fraction of the LDs0 was chosen for lmtlal studies with HLo-7 (LDs~ = 285 mg/kg) and pyrlmldoxlme (LDs0 = 120 mg/kg) In some cases the therapeutic doses of HLo-7 and pyrimidoxlme were raised to 1/4 and even 1/3 of the LDs0 to account for the possibility that better results could be obtained with a higher proportion of their respective LDs0.

LDso of organophosphate3 The determination of the subcutaneous LD~u of soman and tabun have been carried out several times over the past few years in this laboratory and details have been reported elsewhere [15] Multiples of the LDs0 values (soman 28 ~g/kg, tabun 94 ~¢g/kg) were then chosen against which the effects of the oxlmes were examined The LDs0 of GF, which had not previously been carried out in this laboratory, wa~ determined using 5 groups of 6 animals each and the LDs0 calculated by probJt analysis A value of 44 ~g/kg subcutaneously (s c ) was obtained Only whole multiples of the LDs0 of the organophosphates were employed when examining the therapeutic regimen since the aim of the experiments was to determine the relatJve effectiveness of the oxlmes, one against the other, and to determine ff the two new oxlmes examined were meaningfully superior to HI-6

Treatment sequence Male guinea pigs (250-350 g) (Hartley strain) were given atropine sulfate 17 mg/kg intramuscularly (l.m, right thigh), the organophosphate was rejected s c under the skin in the back of the neck 15 mm later, and the oxlme was administered i m 1 min following the organophosphate (left thigh) Mortality was assessed at various time intervals but is recorded here at 24 h following organophosphate

Drug preparatton The three oximes and atropine were made up in distilled water and injected in a volume of 1 ml/kg. HLo-7 was dissolved in water heated to 40°C just prior to injection. Tabun, soman and G F were synthesized in the chemistry section of Defence Research Establishment Suffield, (DRES), the details and purity have been published elsewhere [15] Briefly, organophosphates were stored refrigerated under nitrogen and repurlfied by Kugelrohr distillation before use HI-6 (DRES batch 32) and 'HLo-7 [8] were synthesized in the chemistry laboratories of DRES Pyrlmldoxlme was kindly supplied by M H Garrlgue (Centre d'Etudes du Bouchet, Paris).

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Results Against s o m a n p o i s o n i n g (4 LDs0), H1-6 was superior to HLo-7 when both were administered as c o n s t a n t fractions of their LD50 (Table I). Both c o m p o u n d s were superior to pyrlmldoxame. If the HLo-7 dose was raised to 1/3 of its LDs0, some m i n o r additional therapeutic benefit was observed, but HLo-7 appeared to be inferior to HI-6 even when the latter c o m p o u n d was given at only 1/5 of its LDs0 Pyrlmldoxlme had no lasting effect on the outcome of p o i s o n i n g by 4 LD50 s o m a n and, in fact, appeared to not even p r o l o n g the time until death, although measurements of this p a r a m e t e r were not made. HLo-7 was more effective against t a b u n challenge than either HI-6 or pyrlmldoxime (Table II) All a n i m a l s treated with HLo-7 survived the challenge of 8 LDs0 of t a b u n Within a few minutes, t a b u n poisoned, HLb-7 treated animals entered a severe crisis stage a n d suffered convulswe activity and respiratory difficulty. However within a b o u t 30 rain, they began to recover a n d by 2 h it was a p p a r e n t that they would survive and they became more mobile and alert By 24 h, they appeared normal except that they were r e m a r k a b l y responsive to any external stimuli Against t a b u n challenge, HI-6 a n d p y r l m l d o x l m e appeared to be similarly effective, however, the animals treated with HI-6 appeared to be in better c o n d i t i o n for longer periods of time even if they failed to survive. Against G F poisoning, there were m a r k e d differences in protection a m o n g the five oxlmes tested Neither P A M - C I n o r p y r l m l d o x l m e offered protection against 4-times

TABLE I THERAPEUTIC EFFICACY OF VARIOUS OXIMES AGAINST SOMAN POISONING (4 LDs0) Ammals received atropine (17 mg/kg a p ) 15 mm prior to soman challenge, followed 1 mm later by thc oxlme (i m ) Oxlme

HI-6 (1/5 LDs0) 110 mg/kg HI-6 (1/4 LDs0) 137 mg/kg HLo-7 (1/5 LDs0) 57 mg/kg HLo-7 (1/4 LDs0) 71 mg/kg HLo-7 (1/3 LDso) 95 mg/kg Pyrlmldoxlme (1/5 LDso) 24 mg/kg Pynmldoxlrne (1/4 LDs0) 30 mg/kg Pyrlmldoxlme (1/3 LDs0) 40 mg/kg

Survivors (No ahve/no treated) 2h

24h

6/6

3/6

6/6

4/6

0/6

0/6

0/6

0/6

2/6

I/6

0/6

0/6

0/6

0/6

0/6

0/6

103 TABLE II THERAPEUTIC E F F I C A C Y OF VARIOUS OXIMES A G A I N S T TABUN POISONING Animals were treated with atropine sulphate 17 mg/kg 0 m ) 15 mm prior to agent (s c ) which was gwen 1 mm prior to oxlme 0 m ) HI-6, HLo-7 and pynmldox~me were given as constant fractions of their LDs0 The LDs0 of tabun was 94 t~g/kg s c The mortality was examined 24 h following challenge Oxxme

H1-6 (1/5 LDs0) 110 mg/kg HLo-7 (1/5 LDso) 57 mg/kg Pynmldoxlme (1/5 LDs0) 24 mg/kg

Survivors (No alive/no treated) 4 × LDs0

6 × LDs0

8 × LDs0

6/6

4/6

0/6

6/6

6/6

6/6

3/6

3/6

0/6

the LDs0 of GF. Neither oxime protected animals, even for as httle as 2 h, against this dose. HI-6 and HL6-7, on the other hand, were effective against the highest doses of G F which were used (8 LDs0). Those animals given 8-times the LDs0 followed by HL6-7 or HI-6 recovered quickly and appeared quite normal by 24 h (Table III). Discussion

Comparative effecttveness of pyrtmtdoxime, HI-6 and HLo-7 There was wide vanabihty in the effectweness among HI-6, HL6-7 and pyrtmldoxTABLE III T H E R A P E U T I C E F F I C A C Y OF VARIOUS OXIMES AGAINST GF POISONING Ammals were treated with atropine sulphate 17 mg/kg 0.m ) 15 mm prior to agent (s c ) which was given 1 mm prior to oxlme (1 m ) HI-6, HLo-7 and pyrlmldoxlme were given as fracUons of their LDs0 while 2-PAM-CI and toxogonm were given as doses historically providing near maximum levels of protection against sensltwe organophosphates The LDs0 of G F was 44 t~g/kg The mortality was examined 24 h foUowmg challenge Oxlme

2-PAM-C1 30 mg/kg Toxogonm 20 mg/kg HI-6 (1/5 LDs0) 110 mg/kg HLo-7 (1/5 LDs0 ) 57 mg/kg Pynmldoxlme (1/4 LD50) 30 mg/kg

Survivors (No ahve/no treated) 4 × LDso

6 × LDs0

8 × LD50

0/5

0/5

--

3/5

1/5

--

5/5

5/5

6/6

6/6

6/6

6/6

0/6

--

__

104

lme depending on the organophosphate against which they were tested. HI-6, the least toxic oxlme, was the most effective against soman, but HLo-7, at a sufficiently high fraction of its LDs0, had slight protective effects at the soman dose chosen for this study. Pyrimidoxlme, the most toxic oxlme, was ineffective against the standard challenge of soman at any dose tested and appeared not to significantly prolong the survival time nor to reduce the severity of the symptoms. Thus against soman poisoning, HI-6 had the best protective effects judging from its obviously higher therapeutic index All three oxlmes exhibited some therapeutic activity against tabun poisoning with HLo-7 being the most effective oxlme This is an interesting finding because HLo-7, unlike HI-6, has been reported to be able to reactivate tabuninhibited enzyme [10]. Most of the considerable activity of HI-6 against tabun poisoning has been ascribed to undefined 'direct' effects because of its lnablhty to reactwate tabun-lnhxblted AChE [15] Therefore, the additional therapeutic benefit resulting from HL6-7 administration beyond that resulting from HI-6, may be due to enzyme reactivation. Studies to resolve the questions concerning enzyme reactivation with HL6-7 on tabun- and soman -lnhlblted enzyme are ongoing. Effecttveness o f vartous oxlmes agamst G F poisoning As was the case with the studies on the other agents, a standard challenge dose of G F was chosen. This was done merely to arrive at a convenient way to compare oxime effectiveness rather than to define the absolute limits of effectiveness of the individual oximes. Both HLo-7 and HI-6 were effective agamst doses of G F of at least 8 LDs0 Toxogonln had some protective activity against 4 LDs0 while PAM-CI and pyrimidoxime had no protective effect against the concentrations of agent used here. The mechanism of action of the oximes is not yet known to be partially, totally or not at all, due to enzyme reactivation or some combination of direct effects and enzyme reactivation. In unpublished studies, we have noted however, that these oxlmes are effective against G F when used in conjunction with carbamate (pyridostlgmlne) pretreatment Studies are presently underway to determine the mechanism of action of HI-6 and HL6-7 against G F poisoning. In conclusion, it appears as if the choice of an oxime for life saving therapy for chemical casualty treatment, especially in the absence of a pretreatment drug like pyridostigmine, is likely to be extremely important. This is especially so because of the wide differences in effectiveness of the individual oximes against any particular 'nerve agent' The casualties inflicted on military and civilians alike reported in the Iran/Iraq war and the threats posed by existing chemical weapons throughout the world, emphasizes the necessity of developing safe, effective treatment of these toxic agents [2]. In this regard, the present study and a survey of the literature, suggests that HI-6 has the broadest spectrum of acceptable protective effects and that HLo-7 appears to have some promise as well. References

1 H J McGeorge, Iraq's chemical and biological arsenal Appl Sc~ Anal Newsl , 22(1) (1991) 1 2 A Saghafinla, Emergencytreatment ofchemmal weapons casualties - Field experiences Appl Scl Anal Newsl, 24(3) (1991) 1

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Comparison of several oximes against poisoning by soman, tabun and GF.

Three oximes currently being evaluated for adoption as replacement nerve agent therapy by various countries were compared for therapeutic efficacy aga...
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