Jacob W. Skovira

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.

In Vivo Reactivation by Oximes of Inhibited Blood, Brain and Peripheral Tissue Cholinesterase Activity Following Exposure to Nerve Agents in Guinea Pigs

This study compared the ability of nine oximes (HI-6, HLö7, MMB-4, TMB-4, carboxime, ICD585, ICD692, ICD3805, and 2-PAM) to reactivate in vivo cholinesterase (ChE) in blood, brain, and peripheral tissues in guinea pigs intoxicated by one of four organophosphorus nerve agents. Two bis-pyridinium compounds without an oxime group, SAD128 and ICD4157, served as non-oxime controls. Animals were injected subcutaneously with 1.0 x LD(50) of the nerve agents sarin, cyclosarin, VR or VX and treated intramuscularly 5 min later with one of these oximes. Toxic signs and lethality were monitored; tissue ChE activities were determined at 60 min after nerve agent. Some animals exposed to sarin or cyclosarin, with or without non-oxime treatment, died within 60 min; however, no animal treated with an oxime died. For VR or VX, all animals survived the 60 min after exposure, with or without non-oxime or oxime therapy. The four nerve agents caused differential degrees of inhibition in blood, brain regions and peripheral tissues. The tested oximes exhibited differential potency in reactivating nerve agent-inhibited ChE in various peripheral tissues, but did not affect ChE activity in the brain regions. There was no direct relation between blood and peripheral tissues in the reactivating efficacy of oxime treatments. ChE inhibited by sarin was the most susceptible to oxime reactivation while cyclosarin the least susceptible. There was no difference in the ChE reactivating potency between the dimethanesulfonate and dichloride salts of HI-6. MMB-4 significantly reactivated the ChE inhibited by these four nerve agents in blood and all three peripheral tissues of the guinea pig, and among all the oximes tested it was the most effective in vivo ChE reactivator against all four nerve agents.

Evaluation of nine oximes on in vivo reactivation of blood, brain, and tissue cholinesterase activity inhibited by organophosphorus nerve agents at lethal dose

The capability of several oximes (HI-6, HLö7, MMB-4, TMB-4, carboxime, ICD 585, ICD 692, ICD 3805, and 2-PAM) to reactivate in vivo AChE inhibited by the nerve agents sarin, cyclosarin, VX, or VR in blood, brain regions, and peripheral tissues in guinea pigs was examined and compared. Animals were injected subcutaneously with 1.0 LD50 of sarin, cyclosarin, VR, or VX, and treated intramuscularly 5 min later with one of these compounds. Toxic signs and lethality were monitored, and tissue AChE activities were determined at 60 min after nerve agent. The animals exposed to sarin or cyclosarin, alone or with non-oxime treatment, some died within 60 min; however, when treated with an oxime, no animal died. For VR or VX, all animals survived for 60 min after exposure, with or without non-oxime or oxime therapy. These nerve agents caused differential degrees of inhibition: in whole blood sarin = cyclosarin > VR = VX; in brain regions sarin > cyclosarin > VX > VR; and in peripheral tissues sarin > VX > cyclosarin > VR. These oximes exhibited differential potency in reactivating nerve agent-inhibited AChE in various peripheral tissues, but not AChE activity in the brain regions. There was no difference in the AChE reactivating potency between the dichloride and dimethanesulfonate salts of HI-6. AChE inhibited by sarin was the most and cyclosarin the least susceptible to oxime reactivation. Overall, MMB-4 appeared to be, among all oximes tested, the most effective in vivo AChE reactivator against the broadest spectrum of nerve agents.

In vivo oxime administration does not influence Ellman acetylcholinesterase assay results

Organophosphorus compounds (OPs) are potent inhibitors of acetylcholinesterase (AChE). Treatment for OP poisoning is by administration of atropine sulfate, an oxime, and diazepam. Oximes such as 2-PAM are used to reactivate OP-inhibited AChE so as to restore normal enzymatic function and serve as a true antidote. There are reports of non-enzymatic hydrolysis by oximes of acetylthiocholine in in vitro preparations in the widely used Ellman assay for AChE activity, which may confound the interpretation of AChE activity by producing elevated results. The purpose of this experiment was to determine if there is appreciable interference by therapeutic levels of oximes on the results of the Ellman assay in assessing AChE reactivation by oxime compounds in vivo. When therapeutic doses of oximes (2-PAM, HI-6, MMB-4, or MINA) were administered intramuscularly to guinea pigs and samples collected 60 min later, there was no statistical difference between oxime and saline control groups in measured AChE activity in various tissue samples, including blood. With appropriate dilution of samples prior to spectrophotometric assay, the Ellman assay is an acceptable method to measure in vivo oxime reactivation of inhibited AChE. Inclusion of an oxime control group to insure that this particular type of interference is not causing false readings in the assay is a prudent step.

Anticonvulsant efficacy of antihistamine cyproheptadine in rats exposed to the chemical warfare nerve agent soman.

HighlightsCyproheptadine at1 min after soman reduced seizures and increased survival rate.Cyproheptadine at seizure onset stopped seizures at doses above 10 mg/kg.Cyproheptadine at 5 min after seizure onset stopped ≥75% of seizures.Cyproheptadine controls seizures and improves survival following soman exposure. Abstract Organophosphate compounds, such as soman and sarin, are highly toxic chemical warfare nerve agents that cause a build‐up of acetylcholine in synapses and neuromuscular junctions. Current therapies aim to prevent seizures and protect against brain injury following exposure. The present study was designed to evaluate the effectiveness of the antihistamine cyproheptadine in improving survival and controlling seizures in rats exposed to soman. Rats were pretreated with the oxime reactivator HI‐6 (125 mg/kg, ip) 30 min prior to soman exposure (225 &mgr;g/kg, sc) and then treated with atropine methylnitrate (AMN, 2.0 mg/kg, im) 1 min after soman. Cyproheptadine (10, 13, 16 or 20 mg/kg, ip) was given at one of three time points: 1 min after soman intoxication, at the onset of soman‐induced seizures or 5 min after seizure onset. Control animals were exposed to soman and given an equivalent volume of sterile water instead of cyproheptadine. The incidence of seizures, mortality, neuron counts, neuropathology and apoptosis in specific regions of the brain were evaluated. In animals given HI‐6 and AMN the incidence of soman‐induced seizure and mortality rate within the first 24 h were 100%. When cyproheptadine was given at a dose of 13 or 20 mg/kg 1 min after soman exposure, the incidence of seizures was reduced from 100% to 13% and 30%, respectively. In addition, cyproheptadine given at 1 min after soman exposure increased the survival rate to 100% regardless of dose. When cyproheptadine was administered at seizure onset, seizures were terminated in 100% of the animals at doses above 10 mg/kg. The survival rate with cyproheptadine treatment at the onset of seizure was ≥83%. Seizures terminated in ≥75% of the animals that received cyproheptadine 5 min after soman‐induced seizure onset. When given at 5 min after seizure onset the survival rate was 100% at all tested doses of cyproheptadine. The neuropathology scores and the number of TUNEL positive cells in the brain regions examined decreased at all time points and cyproheptadine doses tested. These observations indicate that cyproheptadine treatment can effectively control seizures, improve survival, reduce seizure duration and reduce the number of dying cells in the brain following soman exposure.