Jiri Bajgar

Currently Used Cholinesterase Reactivators Against Nerve Agent Intoxication: Comparison of Their Effectivity in Vitro

In vitro comparison of reactivation efficacy of five currently used oximes—pralidoxime, obidoxime, trimedoxime, methoxime, and HI-6 (at two concentrations: 10−5 and 10−3 M)—against acetylcholinesterase (AChE; E.C. 3.1.1.7) inhibited by six different nerve agents (VX, Russian VX, sarin, cyclosarin, tabun, soman) and organophosphorus insecticide chlorpyrifos was the aim of this study. As a source of AChE in the experiments, rat brain homogenate was used. According to the results obtained, no AChE reactivator was able to reach sufficient potency for AChE inhibited by all nerve agents used. Moreover, oxime HI-6 (the most effective one) was not able to reactivate tabun- and soman-inhibited AChE. Due to this fact, it could be designated as a partially broad-spectrum reactivator.

Potency of new structurally different oximes to reactivate cyclosarin-inhibited human brain acetylcholinesterases

Antidotes currently used for organophosphorus pesticide and nerve agent intoxications consist of anticholinergics (atropine mainly) and acetylcholinesterase (AChE, EC 3.1.1.7) reactivators called oximes. Owing to the wide-spread of these toxic compounds worldwide, development of antidotes in the case of first aid is needed. To select the most promising AChE reactivators is a very time consuming process, which is necessary before approval of these compounds to be used as human antidotes. Because of ethical reasons, many developing experiments have been conducted on laboratory animals. However, these results often could not be transferred directly to human. Here, we have tested five newly developed AChE reactivators – K027, K033, K048, K074 and K075, which showed promising reactivation activity on rodents, as reactivators of inhibited human brain cholinesterases. For this purpose, cyclosarin was used as member of the nerve agent family. Oxime HI-6 and pralidoxime were used as AChE reactivator standards. Two AChE reactivators, K027 and K033, achieved comparable reactivation potency as HI-6. Moreover, oxime K033 reached its maximal reactivation potency at the lowest concentration which could be attained in humans.

An attempt to assess functionally minimal acetylcholinesterase activity necessary for survival of rats intoxicated with nerve agents.

Acetylcholinesterase (AChE, EC 3.1.1.7) is an important enzyme for cholinergic nerve transmission. The action of toxic organophosphates such as nerve agents is based on AChE inhibition. The death following acute nerve agent poisoning is due to central or peripheral respiratory/cardiac failure. Therefore, the changes in AChE activity following nerve agents acting predominantly on the central (sarin, soman) or peripheral (VX) level were studied. It is known that AChE activity in different structures exists in relative excess. Female Wistar rats intoxicated with sarin, soman, and VX in different doses (0.5-2.0 x LD(50)) were divided into groups of survived and died animals. AChE activities in diaphragm, brain parts (pontomedullar area, frontal cortex, basal ganglia, in some cases other parts of the brain) were determined and the rest of activity (in %) was correlated with survival/death of animals. More precise elucidation of action of nerve agents and the assessment of minimal AChE activity in different organs compatible with the survival of organism poisoned with nerve agents were the aims of this study.

Asoxime (HI-6) impact on dogs after one and tenfold therapeutic doses: Assessment of adverse effects, distribution, and oxidative stress

Asoxime (HI-6) is a well known oxime reactivator used for counteracting intoxication by nerve agents. It is able to reactivate acetylcholinesterase (AChE) inhibited even by sarin or soman. The present experiment was aimed to determine markers of oxidative stress represented by thiobarbituric acid reactive substances and antioxidants represented by ferric reducing antioxidant power, reduced and oxidized glutathione in a Beagle dog model. Two groups of dogs were intramuscularly exposed to single (11.4 mg/kg.b.wt.) or tenfold (114 mg/kg.b.wt.) human therapeutically doses of HI-6. HI-6 affinity for AChE in vitro was evaluated in a separate experiment. Complete serum biochemistry and pharmacokinetics were also performed with significant alteration in blood urea nitrogen, creatine phosphokinase, glucose and triglycerides. Blood samples were collected before HI-6 application and after 30, 60, and 120 min. The overall HI-6 impact on organism is discussed.

Tabun-inhibited rat tissue and blood cholinesterases and their reactivation with the combination of trimedoxime and HI-6 in vivo.

Up to now, intensive attempts to synthesize a universal reactivator able to reactivate cholinesterases inhibited by all types of nerve agents/organophosphates were not successful. Therefore, another approach using a combination of two reactivators differently reactivating enzyme was used: in rats poisoned with tabun and treated with combination of atropine (fixed dose) and different doses of trimedoxime and HI-6, changes of acetylcholinesterase activities (blood, diaphragm and different parts of the brain) were studied. An increase of AChE activity was observed following trimedoxime treatment depending on its dose; HI-6 had very low effect. Combination of both oximes showed potentiation of their reactivation efficacy; this potentiation was expressed for peripheral AChE (blood, diaphragm) and some parts of the brain (pontomedullar area, frontal cortex); AChE in the basal ganglia was relatively resistant. These observations suggest that the action of combination of oximes in vivo is different from that observed in vitro.

Changes of Cholinesterase Activities in the Rat Blood and Brain After Sarin Intoxication Pretreated with Butyrylcholinesterase

After sarin inhalation exposure of rats pretreated with equine serum butyrylcholinesterase (EqBuChE), cholinesterase activities of the whole blood, acetylcholinesterase (AChE) in erythrocytes, pontomedullar area, frontal cortex, and striatum of the brain, and plasma butyrylcholinesterase (BuChE) were determined. Using different doses of EqBuChE as a pretreatment (intraperitoneal injection), dose-dependent increases in plasma BuChE activity and no changes in the erythrocyte and brain AChE activities were demonstrated. Decreases in plasma BuChE activity and red blood cells (RBC) and brain AChE activities were observed in control rats after sarin inhalation exposure without EqBuChE pretreatment. In rats pretreated with EqBuChE, this inhibition was lower compared with control animals not only in the blood but also in the brain structures studied. These results demonstrate protective effects of EqBuChE pretreatment in rats intoxicated with sublethal concentrations of sarin by inhalation.

Different inhibition of acetylcholinesterase in selected parts of the rat brain following intoxication with VX and Russian VX

Differences between acetylcholinesterase (AChE) inhibition in the brain structures following VX and RVX exposure are not known as well as information on the possible correlation of biochemical and histochemical methods detecting AChE activity. Therefore, inhibition of AChE in different brain parts detected by histochemical and biochemical techniques was compared in rats intoxicated with VX and RVX. AChE activities in defined brain regions 30 min after treating rats with VX and Russian VX intramuscularly (1.0 × LD50) were determined by using biochemical and histochemical methods. AChE inhibition was less expressed for RVX, in comparison with VX. Frontal cortex and pontomedullar areas containing ncl. reticularis has been found as the most sensitive areas for the action of VX. For RVX, these structures were determined to be frontal cortex, dorsal septum, and hippocampus, respectively. Histochemical and biochemical results were in good correlation (Rxy = 0.8337). Determination of AChE activity in defined brain structures was a more sensitive parameter for VX or RVX exposure than the determination of AChE activity in the whole-brain homogenate. This activity represents a “mean” of the activities in different structures. Thus, AChE activity is the main parameter investigated in studies searching for target sites following nerve-agent poisoning contributing to better understanding of toxicodynamics of nerve agents.

Changes of cholinesterase activities in the plasma and some tissues following administration of l-carnitine and galanthamine to rats

Changes of acetylcholinesterase (AChE) activities in the hypophysis and brain (frontal cortex, hippocampus, medial septum and basal ganglia), and butyrylcholinesterase in plasma and liver following galanthamine (GAL) administration were studied in rats pretreated with L-carnitine (CAR). Following only GAL administration (10 mg/kg, i.m.), both cholinesterases (without clinical symptoms of GAL overdosage) were significantly inhibited. Pretreatment with CAR (3 consecutive days, 250 mg/kg, p.o.) followed by GAL administration showed higher AChE inhibition in comparison with single GAL administration. However, a statistically significant difference was observed for AChE in the hippocampus only. The activity of peripheral cholinesterases was not influenced by CAR pretreatment. Thus, pretreatment with CAR enhanced AChE inhibition in some brain parts of the rat following GAL administration.

CHAPTER 66 – Novel Oximes

Publisher Summary This chapter describes the synthesis of the new acetylcholinesterase (AchE) reactivators that have been developed within the last few years. AChE plays a key role in cholinergic transmission in the peripheral and central nervous system and, consequently, its inhibition is life-endangering. The acute toxicity of nerve agents is usually attributed to the excessive cholinergic stimulation caused by excess of ACh, followed by subsequent overstimulation of the cholinergic pathways and desensitization of the cholinergic peripheral and central receptor sites. When an individual is exposed to low amounts of a nerve agent, the initial symptoms include runny nose, contraction of the pupils, miosis, deterioration of visual accommodation, headache, slurred speech, nausea, hallucination, pronounced chest pain, and an increase in the production of saliva. At higher doses of a nerve agent the aforementioned symptoms are more pronounced. Coughing and breathing problems also occur. Antidotes developed for treatment of nerve agent intoxication could be divided into two parts: prophylaxis as preexposure administration of antidotes and postexposure treatment consisting of anticholinergic drugs, AChE reactivators, and anticonvulsants.

Continual monitoring of the reactivation effect of oximes on blood acetylcholinesterase in the rats poisoned with organophosphates

Acetylcholinesterase activity in rat blood was continuously monitored following O-ethyl-S-(2-dimethylaminoethyl)-methylphosphonothioate intoxication (p.o.) alone and in combination with atropine and the reactivators trimedoxime, obidoxime and methoxime. Decrease of acetylcholinesterase activity was not influenced by atropine alone but following treatment with a combination of atropine with the reactivators mentioned, an increase (reactivation) of the blood enzyme was demonstrated. This increase was highest for the combination atropine-trimedoxime and the lowest for the combination atropine-obidoxime.