Irwin Koplovitz

A comparison of the efficacy of HI6 and 2-PAM against soman, tabun, sarin, and VX in the rabbit

This study compared the efficacy of HI6 and 2-PAM against nerve agent (soman, tabun, sarin, and VX)-induced lethality in the atropinesterase-free rabbits pretreated with vehicle (controls) or pyridostigmine. Treatment was administered at signs or 2 min after agent challenge and consisted of oxime (100 mumol/kg) + atropine (13 mg/kg) (alone or together with diazepam). Twenty-four-h LD50 values were calculated for soman- and tabun-intoxicated animals, whereas 24-h survival was noted in animals given 10 LD50s of sarin or VX. In pyridostigmine and control rabbits intoxicated with soman and treated with oxime + atropine (alone or together with diazepam), HI6 was 3-5 times more effective than 2-PAM. In contrast, HI6 was less effective than 2-PAM against tabun poisoning. In pyridostigmine-pretreated animals exposed to tabun, efficacy was increased more than 3-fold when compare to tabun-challenged animals treated with atropine + HI6 alone. Both oximes were highly effective against sarin and VX. These findings suggest that HI6 could replace 2-PAM as therapy for nerve agent poisoning, because it is superior to 2-PAM against soman, and when used in pyridostigmine-pretreated animals, it affords excellent protection against all four nerve agents when used in combination with atropine (alone or together with diazepam) therapy.

Prophylaxis with human serum butyrylcholinesterase protects guinea pigs exposed to multiple lethal doses of soman or VX

Human serum butyrylcholinesterase (Hu BChE) is currently under advanced development as a bioscavenger for the prophylaxis of organophosphorus (OP) nerve agent toxicity in humans. It is estimated that a dose of 200mg will be required to protect a human against 2×LD(50) of soman. To provide data for initiating an investigational new drug application for the use of this enzyme as a bioscavenger in humans, we purified enzyme from Cohn fraction IV-4 paste and initiated safety and efficacy evaluations in mice, guinea pigs, and non-human primates. In mice, we demonstrated that a single dose of enzyme that is 30 times the therapeutic dose circulated in blood for at least four days and did not cause any clinical pathology in these animals. In this study, we report the results of safety and efficacy evaluations conducted in guinea pigs. Various doses of Hu BChE delivered by i.m. injections peaked at ∼24h and had a mean residence time of 78-103h. Hu BChE did not exhibit any toxicity in guinea pigs as measured by general observation, serum chemistry, hematology, and gross and histological tissue changes. Efficacy evaluations showed that Hu BChE protected guinea pigs from an exposure of 5.5×LD(50) of soman or 8×LD(50) of VX. These results provide convincing data for the development of Hu BChE as a bioscavenger that can protect humans against all OP nerve agents.

Bioscavenger for protection from toxicity of organophosphorus compounds.

Current antidotal regimens for organophosphorus compound (OP) poisoning consist of a combination of pretreatment with a spontaneously reactivating AChE inhibitor such as pyridostigmine bromide, and postexposure therapy with anticholinergic drugs such as atropine sulfate and oximes such as 2-PAM chloride (Gray, 1984). Although these antidotal regimens are effective in preventing lethality of animals from OP poisoning, they do not prevent postexposure incapacitation, convulsions, performance deficits, or, in many cases, permanent brain damage (Dunn and Sidell, 1989). These problems stimulated the development of enzyme bioscavengers as a pretreatment to sequester highly toxic OPs before they reach their physiological targets. Several studies over the last two decades have demonstrated that exogenously administered human serum butyrylcholinesterase (Hu BChE) can be used successfully as a safe, efficacious, and single prophylactic treatment to counteract the toxicity of OPs. It also has potential use for first responders (civilians) reacting to terrorist nerve gas release, pesticide overexposure, or succinylcholine-induced apnea. A dose of 200 mg of Hu BChE in humans is envisioned as a prophylactic treatment that can protect from exposure of 2-5 x LD50 of nerve agents (Ashani, 2000).

A structure-activity analysis of the variation in oxime efficacy against nerve agents.

A structure-activity analysis was used to evaluate the variation in oxime efficacy of 2-PAM, obidoxime, HI-6 and ICD585 against nerve agents. In vivo oxime protection and in vitro oxime reactivation were used as indicators of oxime efficacy against VX, sarin, VR and cyclosarin. Analysis of in vivo oxime protection was conducted with oxime protective ratios (PR) from guinea pigs receiving oxime and atropine therapy after sc administration of nerve agent. Analysis of in vitro reactivation was conducted with second-order rate contants (k(r2)) for oxime reactivation of agent-inhibited acetylcholinesterase (AChE) from guinea pig erythrocytes. In vivo oxime PR and in vitro k(r2) decreased as the volume of the alkylmethylphosphonate moiety of nerve agents increased from VX to cyclosarin. This effect was greater with 2-PAM and obidoxime (>14-fold decrease in PR) than with HI-6 and ICD585 (<3.7-fold decrease in PR). The decrease in oxime PR and k(r2) as the volume of the agent moiety conjugated to AChE increased was consistent with a steric hindrance mechanism. Linear regression of log (PR-1) against log (k(r2)[oxime dose]) produced two offset parallel regression lines that delineated a significant difference between the coupling of oxime reactivation and oxime protection for HI-6 and ICD585 compared to 2-PAM and obidoxime. HI-6 and ICD585 appeared to be 6.8-fold more effective than 2-PAM and obidoxime at coupling oxime reactivation to oxime protection, which suggested that the isonicotinamide group that is common to both of these oximes, but absent from 2-PAM and obidoxime, is important for oxime efficacy.

Reactivation of brain acetylcholinesterase by monoisonitrosoacetone increases the therapeutic efficacy against nerve agents in guinea pigs.

Current oxime therapies do not readily cross the blood-brain barrier to reactivate organophosphorus nerve agent-inhibited cholinesterase (ChE) within the CNS. We investigated the ability of monoisonitrosoacetone (MINA), a tertiary oxime, to reactivate ChE inhibited by the nerve agent sarin (GB), cyclosarin (GF), or VX, in peripheral tissues and brain of guinea pigs and determined whether reactivation in the CNS will enhance protection against the lethal effects of these three agents. In the reactivation experiment, animals were pretreated with atropine methylnitrate (1.0mg/kg, i.m.) 15 min prior to subcutaneous (s.c.) challenge with 1.0 x LD(50) of GB, GF, or VX. Fifteen minutes later animals were treated intramuscularly (i.m.) with MINA (ranging from 22.1 to 139.3mg/kg) or 2-PAM (25.0mg/kg). At 60 min after nerve agent, CNS (brainstem, cerebellum, cortex, hippocampus, midbrain, spinal cord, and striatum) and peripheral (blood, diaphragm, heart, and skeletal muscle) tissues were collected for ChE analysis. MINA reactivated nerve agent-inhibited ChE in the CNS and peripheral tissues in a dose-dependent manner in the following order of potency: GB>GF>VX. In a survival experiment, animals were injected i.m. with atropine sulfate (0.5mg/kg), 2-PAM (25.0mg/kg), or MINA (35.0, 60.0, or 100.0mg/kg) alone or in combination 1 min after challenge with varying s.c. doses of GB, GF, or VX to determine the level of protection. The rank order of MINAs efficacy in guinea pigs against nerve agent lethality was the same as for reactivation of inhibited ChE in the CNS. These data show that MINA is capable of reactivating nerve agent-inhibited ChE and that the extent of ChE reactivation within the CNS strongly relates to its therapeutic efficacy.

Evaluation of the toxicity, pathology, and treatment of cyclohexylmethylphosphonofluoridate (CMPF) poisoning in rhesus monkeys

Cyclohexylmethylphosphonofluoridate (CMPF) is an organophosphate cholinesterase inhibitor with military significance. The purpose of these studies was 1) to determine the acute toxicity of CMPF in the male rhesus monkey, 2) to evaluate the efficacy of pyridostigmine (PYR) pretreatment plus atropine and oxime (2-PAM or HI6) treatment, and 3) to evaluate the pathological consequences of acute poisoning. An i. m. LD50 of CMPF was estimated using an up-and-down dose selection procedure and 12 animals. The 48-h and 7-day LD50 was 46.6 μg/kg, i.m. In the protection experiments, pyridostigmine (0.3–0.7 mg/kg/24 h) was administered by surgically implanted osmotic minipumps for 3–12 days resulting in 21–65% inhibition of erythrocyte acetylcholinesterase activity. Animals were challenged with 5 × LD50 CMPF (233 μg/kg) and treated with atropine (0.4 mg/kg) and either 2-PAM (25.7 mg/kg) or HI6 (37.8 mg/kg) at the onset of signs or 1 min after challenge. Osmotic pumps were removed within 30 min after agent challenge. Pyridostigmine, atropine, and either 2-PAM or HI6 were completely effective against CMPF, saving ten of ten animals in each group. In comparison, three of five animals challenged with 5 × LD50 of soman and treated with atropine and 2-PAM survived 7 days. The primary histologic lesions in the acute toxicity group were neuronal degeneration/necrosis and spinal cord hemorrhage. The CMPF treated groups (total of 20 animals) had minimal nervous system changes with no significant lesion difference resulting from the different oxime therapies. The primary non-neural lesions were degenerative cardiomyopathy and skeletal muscle degeneration which occasionally progressed to necrosis and mineralization. The results indicate that PYR pretreatment in conjunction with atropine and oxime treatment is an effective regimen against battlefield relevant levels (5 × LD50) of CMPF.

DOSE-RESPONSE EFFECTS OF ATROPINE AND HI-6 TREATMENT OF ORGANOPHOSPHORUS POISONING IN GUINEA PIGS'

HI-6 (1-2-hydroxyiminomethyl-1-pyridino-3-(4-carbamoyl-1-pyridino -2- oxapropane dichloride) has been evaluated as an oxime alternative to pralidoxime, and toxogonin in the treatment of organophosphorus (OP) poisoning. The dose response effects of atropine (ATR) and HI-6 were investigated to more fully explore the interaction of these compounds in the treatment of OP poisoning. ATR, HI-6 and various combinations of the two drugs were evaluated against lethal poisoning by soman (GD) and tabun (GA) in guinea pigs. The effect of adjunctive diazepam treatment on the efficacy of atropine and HI-6 against soman was also investigated. Animals of either sex were challenged s.c. with OP and treated i.m. 1 min later with ATR and/or HI-6. When used, diazepam was injected immediately after ATR+HI6. LD50s of each treatment were calculated from probit models based on 24-hour survival against 5 levels of nerve agent and 6 animals per challenge level. A protective index (PI) was calculated by dividing the nerve agent LD50 in the presence of treatment by the LD50 in the absence of treatment. Treatment with HI6 alone had little effect on the toxicity of either OP. Treatment with ATR alone was more effective than HI-6 alone and was significantly more effective against soman than against tabun. When used in combination atropine and HI-6 had a strong synergistic effect against both agents. The dose of atropine used with HI-6 was critical in determining the efficacy of HI-6 against either agent. The slopes of the dose-lethality curves were minimally affected by the dose of ATR or HI-6. Adjunctive treatment with diazepam enhanced the efficacy of HI-6 and atropine against soman. It is concluded that 1) ATR has a large effect on the efficacy of HI-6 against OP poisoning, 2) the dose of ATR must be carefully selected in studies investigating the efficacy of HI-6 against OP poisoning, 3) the effective dose of ATR in the guinea pig is approximately 16 mg/kg, and 4) diazepam is a useful adjunct to atropine and HI-6.

Evaluation of anticonvulsant drugs for soman-induced seizure activity

This report describes three lines of organophosphorus (OP) nerve-agent anticonvulsant studies: (a) a basic research effort to understand how different pharmacological classes of compounds influence the expression of seizure produced by soman in rats, (b) a drug-screening effort to determine whether clinically useful antiepileptic drugs can modulate soman-induced seizures in rats, and (c) an advanced testing effort in which anticholinergic compounds are evaluated in comparison to the current anticonvulsant treatment (i.e., diazepam) in guinea pigs. Electroencephalographic (EEG) recordings were used in all studies. Basic studies were conducted in rats pretreated with HI-6 and challenged with 1.6 x the median lethal dose (LD 50 ) soman. Antimuscarinic compounds were extremely effective in blocking (pretreatment) or terminating soman seizures when given 5 min after seizure onset; however, significantly higher doses were required when treatment was delayed >10 min, and some antimuscarinic compounds lost anticonvulsant efficacy when treatment was delayed 40 min. Diazepam blocked seizure onset, yet seizures could recur after an initial period of anticonvulsant effect at doses ≤2.5 mg/kg. Diazepam could terminate ongoing seizures when given 5 min after seizure onset, but doses up to 20 mg/kg were ineffective when treatment was delayed for 40 min. The γ-aminobutyric acid (GABA) uptake inhibitor tiagabine was ineffective in blocking or terminating soman motor convulsions or seizures. The glutamate receptor antagonists NBQX, GYKI 52466, and memantine had weak or minimal antiseizure activity, even at doses that virtually eliminated signs of motor convulsions. The antinicotinic mecamylamine was ineffective in blocking or stopping seizure activity. Pretreatment with a narrow range of doses of the alpha-2-adrenergic agonist clonidine produced variable protection (40-60%) against seizure onset; treatment after seizure onset with clonidine was not effective. Screening studies in rats, using HI-6 pretreatment, showed that the clinically effective antiepileptic drugs pentobarbital and valproic acid were modestly effective in terminating seizures when given shortly (5 min) after seizure onset. In contrast, phenytoin, carbamazepine, felbamate, magnesium sulfate, ketamine, primidone, and lamotrigine were ineffective. Advanced studies of eight anticholinergic drugs compared with diazepam anticonvulsant therapy were performed in guinea pigs pretreated with pyridostigmine, challenged with soman (2 x LS 50 ), and immediately treated with 2-PAM Cl and atropine. The rank of potency for diazepam and eight antimuscarinic compounds, when administered 5 min after seizure onzet (scopolamine, , biperiden, benactyzine, trihexyphenidyl, azaprophen, procyclidine, aprophen, diazempam, atropine), closely resembles that obtained from the rat model.

Characterization and Treatment of the Toxicity of O-isobutyl S-[2-(diethylamino)ethyl]methylphosphonothioate, a Structural Isomer of VX, in Guinea Pigs

O-isobutyl S-[2-(diethylamino)ethyl]methylphosphonothioate (VR), a structural isomer of the chemical warfare agent O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate (VX) was characterized with respect to its toxicity, acetylcholinesterase (AChE) inhibition, oxime reactivation, and antidotal treatment. VR was highly toxic in guinea pigs, with an s.c. LD 50 of 11.3 μLg/kg. The bimolecular rate constant for inhibition of AChE by VR was 1.4 x 10 8 M -1 min -1 , which is similar to the AChE inhibition rate constants of VX and of other chemical warfare agents whose toxicity is attributed to their inhibition of AChE. Complete in vitro reactivation of VR-inhibited AChE was produced by oximes, but reactivation with the bispyridinium oximes 1-(((4-(aminocarbonyl)pyridino)methoxy methyl)-2-((hydroximino)-methyl)pyridinium dichloride (HI-6), 1-(((4-(aminocarbonyl)pyridino)methoxy)-methyl)-2,4-bis((hydroxyimino)methyl)pyridinium dimethanesulfonate (HLo7), and toxogonin dichloride (TOXO) occurred at a much higher rate than with the monopyridinium oxime 2-pyridine aldoxime methylchloride (2-PAM). The reactivation rates with HLo7, HI-6, and TOXO were, respectively, 21-, 5-, and 3-fold greater than with 2-PAM. The aging of VR-inhibited AChE to an oxime-resistant state occurred slowly with a half-life of 43 h. Treatment of guinea pigs with equimolar (145 μLmol/kg) amounts of HI-6 (55 mg/kg) or 2-PAM (25 mg/kg) in combination with atropine (16 mg/kg) i.m. 1 min after VR exposure produced protective ratios of 43.9 for HI-6 and 6.5 for 2-PAM. Carbamate pretreatment prior to these oxime/atropine treatments caused no increase in their protective ratios against VR. These observations suggest that VR is a highly toxic anticholinesterase agent that can be effectively treated with an appropriate oxime/atropine regimen.

Efficacy of oxime plus atropine treatment against soman poisoning in the atropinesterase-free rabbit

The oximes pralidoxime chloride (2-PAM), MMB4, and HI-6 were evaluated in combination with atropine as treatments against soman poisoning in atropinesterase-free rabbits. Animals were challenged i.m. with 2 x LD50 soman and treated at the onset of toxic signs with 50 mumols/kg of oxime and 5 or 13 mg/kg atropine. Survival and time to death were compared at 48 hours post-soman challenge. Survival rates in MMB4 and HI-6 treated animals were higher than in 2-PAM-treated animals. The increase in survival was significant at the 13 mg/kg dose of atropine. MMB4 and HI-6 also significantly delayed time to death after soman compared to 2-PAM. The results suggest that MMB4 and HI-6 have potential as useful oximes for treating soman poisoning.