John G. Clement

Efficacy of HLö-7 and pyrimidoxime as antidotes of nerve agent poisoning in mice

The toxicity and efficacy of two oximes, HLö-7 and pyrimidoxime, were evaluated in mice and compared to those obtained with HI-6. HLö-7 and pyrimidoxime produced 24 h LD50 values of 356 and 291 mg/kg (i.p.), respectively. In combination with atropine (17.4 mg/kg, i.p.), HLö-7 was a very efficient therapy against poisoning by 3×LD50 dose of soman, sarin and GF and 2×LD50 dose of tabun with ED50 values of 12.4, 0.31, 0.32 and 25.2 mg/kg, respectively. In contrast, pyrimidoxime was a relatively poor therapy which resulted in ED50 values of >150, 5.88, 100 and 71 mg/kg against poisoning by soman, sarin, GF and tabun, respectively. HLö-7 produced significant (p <0.05) reactivation of phosphorylated acetylcholinesterase, in vivo, resulting in 47, 38, 27 and 10% reactivation of sarin, GF, soman and tabun inhibited mouse diaphragm acetylcholinesterase, respectively. HLö-7 also antagonized sarin-induced hypothermia in mice suggesting that it reactivated central acetylcholinesterase. The potential of HLö-7 as a replacement oxime for the treatment of nerve agent poisoning is discussed.

Central activity of acetylcholinesterase oxime reactivators

The ability of various oximes to antagonize the sarin-induced hypothermia and reactivate phosphorylated acetylcholinesterase was used as an indicator of the central activity of oximes. HI-6, but neither toxogonin nor PAM Cl, antagonized sarin-induced hypothermia and reactivated brain acetylcholinesterase, in particular hypothalamic acetylcholinesterase. The sarin-induced hypothermia appears to be a muscarinic cholinergic action since atropine was also an effective antagonist of sarin-induced hypothermia. Neither HI-6 nor toxogonin antagonized oxotremorine-induced hypothermia, indicating that these oximes do not possess central cholinolytic activity. The results demonstrated that HI-6 penetrated the blood-brain barrier in a sufficient concentration to produce a biochemical and physiological action against sarin poisoning.

Efficacy of a combination of acetylcholinesterase reactivators, HI-6 and obidoxime, against tabun and soman poisoning of mice

The bispyridinium oxime HI-6, 1-((((4-amino-carbonyl) pyridinio)methoxy) methyl)-2-(hydroxyimino)-methyl) pyridinium dichloride monohydrate, combined with atropine is an effective treatment for soman (pinacolyl methylphosphonofluoridate) poisoning but is relatively ineffective against tabun (ethyl N-dimethyl phosphoroamidocyanidate) poisoning in mice. This contrasts with those results obtained using the bispyridinium oxime obidoxime [1,1′-(oxy bis(methylene)) bis(4-(hydroxyimino)-methyl) pyridinium dibromide]. The purpose of this study was to investigate the efficacy of the combination of HI-6 and obidoxime plus atropine against poisoning by tabun and soman in mice. The combination of ineffective single doses of obidoxime (5 or 10 mg/kg) and HI-6 (25 or 50 mg/kg) improved the treatment of tabun poisoning over either oxime alone. Combinations employing higher concentrations of obidoxime (25 or 50 mg/kg) and HI-6 (100 or 200 mg/kg) resulted in significant toxicity in the absence of organophosphate poisoning. Against soman poisoning the addition of obidoxime to HI-6 did not attenuate the efficacy of HI-6. The half-life of elimination and peak serum concentrations of HI-6 and obidoxime were not altered following administration of the combined injection. Reactivation of tabun-inhibited acetylcholinesterase was found consistently in the diaphragm but not in the brain. Using response surface methods it was possible to estimate the optimal therapy against soman and tabun poisoning (74.5 mg/kg HI-6+31.9 mg obidoxime against 1052 μg/kg challenge of tabun and 129 mg/kg HI-6 +0 mg/kg obidoxime against 390 μg/kg challenge of soman). It is proposed that reactivation of tabun inhibited acetylcholinesterase at the diaphragm may be responsible for the increased efficacy of the combination of HI-6 and obidoxime against tabun poisoning in mice.

HI-6, an oxime which is an effective antidote of soman poisoning: A structure-activity study☆

Abstract In contrast to other organophosphates, soman poisoning is resistant to atropine (AT) + oxime therapy. However, new bispyridinium-type oximes + AT are effective antidotes of soman poisoning. In structure-activity studies, T 4925 (1,1′-[oxybis(methylene)]bispyridinium dichloride) had an LD 50 of 178 mg/kg and HS-14 (1-[[[pyridinio]methoxy]methyl]-2[(hydroxyiminio)methyl]pyridinium dichloride) had an LD 50 of 130 mg/kg. DL-10 (1-[[[3-(aminocarbonyl)pyridinio]methoxy]methyl]pyridinium dichloride) and DL-11 (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]pyridinium dichloride) had LD 50 s of 326 and 715 mg/kg, respectively, whereas HS-6 (Reg. No. 22625-23-6) and HI-6 (Reg. No. 34433-31-3) had LD 50 s of 316 and 514 mg/kg (ip), respectively. T 4925, HS-14, DL-10, and DL-11 at an LD 1 dose + AT (17.4 mg/kg) were relatively ineffective against soman (540 μg/kg; sc) compared to HI-6 and HS-6. Prophylactic ED 50 s for HI-6 and HS-6 vs soman were 17 and 110 mg/kg, producing safety ratios of 30 and 2.9, respectively. The therapeutic ED 50 for HI-6 was 20 mg/kg. Brain cholinesterase (ChE) in mice surviving soman (540 μg/kg; sc) + HI-6 (94 mg/kg) + AT was 0% of control activity at 1 and 2 hr, 5% at 24 hr, and 15% 48 hr later. Mice which died postsoman (540 μg/kg; sc) had 20% brain ChE activity. These results show that HI-6 was one of the least toxic and most efficacious compounds studied and suggest brain ChE inhibition was not the primary lesion in soman poisoning.

In vitro oxime-induced reactivation of various molecular forms of soman-inhibited acetylcholinesterase in striated muscle from rat, monkey and human

The purpose of this study was to compare the in vitro reactivation of the various molecular forms of soman-inhibited acetylcholinesterase by oximes such as HI-6, toxogonin and PAM, in striated muscle tissue from three species-rat, monkey and human. To simulate the various in vivo conditions the oxime was present either 5 min before and after (Pre-Post) or 5 min after (Post) exposure to the nerve agent soman. In the Pre-Post mode the oxime effects would result from a combination of not only shielding of acetylcholinesterase from soman inhibition but also from immediate reactivation of soman-inhibited acetylcholinesterase. In the Post experimental group the increase in soman-inhibited acetylcholinesterase activity was due to reactivation. HI-6 (Pre-Post) increased significantly the activity of soman-inhibited acetylcholinesterase in the rat, human and monkey muscle. HI-6 (Post) was a highly effective reactivator of soman-inhibited acetylcholinesterase in the rat muscle and moderately so in the human and monkey muscle. Toxogonin (Pre-Post) and toxogonin (Post) were effective in increasing soman-inhibited acetylcholinesterase activity in rat muscle but were relatively ineffective in the human and monkey muscle. PAM (Pre-Post) and PAM (Post) were ineffective in increasing soman-inhibited acetylcholinesterase activity in muscle from all species examined. Effectiveness of oxime-induced reactivation of soman-inhibited acetylcholinesterase could be estimated from the total acetylcholinesterase activity which appears to reflect the results found with the individual molecular forms of acetylcholinesterase. In addition, SAD-128, a non-oxime bispyridinium compound, appeared to enhance significantly the HI-6 induced reactivation of soman-inhibited acetylcholinesterase in human but not rat striated muscle.

Efficacy of various oximes against GF (cyclohexyl methylphosphonofluoridate) poisoning in mice

The efficacy of oxime (HI-6, toxogonin or PAM Cl) therapy against GF (cyclohexyl methylphosphonofluoridate) poisoning was assessed in mice. It was found that the combinations of atropine and either toxogonin or HI-6 were effective therapies against GF poisoning. PAM therapy was ineffective. HI-6 was the only oxime which reactivated GF inhibited acetylcholinesterase. This might explain the reason why the HI-6 treated mice appeared to recover more quickly from the incapacitating effects following GF poisoning.

Toxicity of the combined nerve agents GB/GF in mice : efficacy of atropine and various oximes as antidotes

The toxicity of a combination of isopropyl methylphosphonofluoridate (sarin; GB) and cyclohexyl methylphosphonofluoridate (GF) and the efficacy of various oxime reactivators in combination with atropine against the combined GB/GF challenge were evaluated in mice. The 24-h s.c. LD50 of the GB/GF combination was 1.15 μmol/kg (1.10–1.21; 95% confidence limits). Mice administered GB/GF displayed typical signs of nerve agent poisoning such as tremors and convulsions, with death most likely due to anoxia subsequent to respiratory arrest. The GB/GF LD50 value was comparable to the s.c. LD50 of 1.35 and 1.21 μmol/kg for GF and GB in mice, respectively. Combining the two nerve agents did not result in potentiation of the toxicity. In combination with atropine sulfate (17.4 mg/kg, i.p.), which alone did not reduce mortality, the oximes tested, 2-PAM, obidoxime and HI-6, were all effective when administered 5 min before 3×LD50 dose of GB/GF with 24-h ED50 values of 102.5, 18.22 and 1.96 μmol/kg, respectively. Use of the GB/GF combination does not appear to confer any unique toxicity profile and appears to be easily treated with the standard therapy of a cholinolytic and oxime.

Serum carboxylesterase activity in various strains of rats: sensitivity to inhibition by CBDP (2-/o-cresyl/4H:1:3:2-benzodioxaphosphorin-2-oxide).

Rodents are relatively insensitive to the neurotoxic effects of various organophosphorus compounds. The purpose of this investigation was to determine if differences in inactivation of CBDP could explain the strain differences in the sensitivity to neurotoxicity following administration of TOCP (tri-o-cresyl phosphate) observed by Carrington and Abou-Donia (1988). Serum carboxylesterase but not cholinesterase is an important detoxification route for organophosphates. Serum carboxylesterase and cholinesterase activity were significantly different (p<0.05) among the various strains of rats (Table 1). The rank order of carboxylesterase activity was Sprague Dawley (6158 nmole/ml serum/min)>Long Evans (5589)>Fischer 344 (5010) whereas the rank order for cholinesterase activity was Fischer 344 >Sprague Dawley >Long Evans. TOCP is metabolized to the active neurotoxicant CBDP (2-/o-cresyl/4H ∶ 1∶3∶2-benzodioxaphosphorin-2-oxide). The ED50 for CBDP inhibition of serum carboxylesterase activity was found to vary considerably for the various strains of rats. The rank order of CBDP ED50 concentration in the various strains was Fischer 344 (437 μM) >Long Evans (339 μM) >Sprague Dawley (78 μM), indicating that there was a difference between the carboxylesterase of the various strains with regard to interaction with CBDP. It is suggested that the differences in the quantity of serum carboxylesterase combined with the differences in the interaction of the inhibitor with the enzyme(s) may be responsible for the strain differences observed by Carrington and Abou-Donia (1988).

Soman and sarin induce a long-lasting naloxone-reversible analgesia in mice

Soman (50 micrograms/kg) and sarin (120 micrograms/kg), potent organophosphate anticholinesterase agents, produced an analgesic response in the mouse hotplate latency test. Naloxone antagonized but did not completely reverse the soman- and sarin-induced analgesia, whereas atropine antagonized completely the soman-and sarin-induced analgesia. Soman poisoning did not potentiate morphine-induced analgesia. It was simply an additive response. In survivors of soman (287 micrograms/kg) poisoning, the analgesia was more pronounced and was still apparent 96 hr after administration. This analgesia was completely antagonized by naloxone. Similar results were found in survivors of sarin (510 micrograms/kg) poisoning. The organophosphate-induced analgesia was not due to physical incapacitation as evidenced by performance on the accelerating rotorod. It is suggested that the organophosphate-induced analgesia is due to a combination of an increased concentration of acetylcholine due to inhibition of acetylcholinesterase combined with a reduced destruction of endogenous opioid-like substances due to organophosphate inhibition of proteases.

Stereoisomers of soman (pinacolyl methylphosphonofluoridate): Inhibition of serum carboxylic ester hydrolase and potentiation of their toxicity by CBDP (2-(2-methylphenoxy)-4H-1,3,2-benzodioxaphosphorin-2-oxide) in mice

The interaction of C(+/-)P(+/-)-soman (pinacolyl methylphosphonofluoridate) and its individual stereoisomers with serum carboxylic-ester hydrolase and potentiation of their toxicity by a carboxylic-ester hydrolase inhibitor CBDP (2-(2-methylphenoxy)-4H-1,3,2-benzodioxaphosphorin-2-oxide) was investigated. C(+/-)P(+/-)-Soman and the individual stereoisomers all inhibited purified mouse serum carboxylic-ester hydrolase to different degrees. C(+/-)P(+/-)-Soman and the C(-)P(-)- and C(+)P(-)-isomers had Ki values of 30.6, 18.7, and 35.7 nM, respectively, and C(-)P(+)- and C(+)P(+)-isomers had Ki values of 1412 and 2523 nM, respectively. In toxicity experiments CBDP (0.5 mg/kg; iv 1 hr prior to soman) pretreatment potentiated the toxicity of C(+/-)P(+/-)-, C(+)P(-)-, and C(-)P(-)-soman to a similar degree. Thus, it appears that the toxic stereoisomers of soman have a similar affinity for mouse serum carboxylic-ester hydrolase, and CBDP pretreatment does not enhance selectively the toxicity of one stereoisomer over the other.