Author Report for: Novotny L

Purpose: The aim of our study was to determine and compare the activity of acetylcholinesterase (AChE) in different parts of dog brain after the exposure to nerve agent sarin with or without HI-6 oxime treatment. Material and methods: Before intoxication, beagle dogs were intravenously anaesthetized and premedicated with atropine sulphate (0.01 mg/kg). Three experimental groups were established - control, sarin (0.03 mg/kg, intramuscularly, 5 min after anaesthesia onset), and sarin + HI-6 dichloride (11.4 mg/kg, intramuscularly, 30 min after sarin poisoning). Brain (amygdaloid body, head of caudate nucleus, somatosensory cortex, Amon's horn of hippocampus, hypothalamus, brain stem ventral respiratory group, and medial nuclei of thalamus) samples were taken 4 h after sarin administration. AChE activity was detected by histochemistry using the Karnovsky-Roots method and computer image analysis. Results: Sarin poisoning decreased AChE activity in all selected brain areas. HI-6 did not affect this outcome. Conclusion: HI-6 does not reactivate brain AChE in dogs when administered 30 min after sarin poisoning.

The purpose of this in vivo study was to assess a new, putatively optimised method for mass casualty decontamination ("ORCHIDS protocol") for effectiveness in removing the chemical warfare agent VX from the skin of anaesthetised, domestic white pigs. ORCHIDS protocol consists of a 1.5-minute shower with a mild detergent (Argos) supplemented by physical removal. A standard method of wet decontamination was used for comparison. Experimental animals were divided into four groups (A-D). Two groups were exposed to a supra-lethal percutaneous dose (5 x LD(50); 300 mug kg(-1)) of VX for 1 h prior to decontamination with either the ORCHIDS (C) or standard protocol (D). A third (B, positive control) group was exposed but not subject to decontamination. Blank controls (A) received anaesthesia and the corresponding dose of normal saline instead of VX. Observations of the clinical signs of intoxication were supplemented by measurements of whole blood cholinesterase (ChE) performed on samples of arterial blood acquired at 30-minute intervals for the duration of the study (up to 6 h). Untreated (B) animals displayed typical cholinergic signs consistent with VX intoxication (local fasciculation, mastication, salivation, pilo-erection and motor convulsions) and died 165-240 min post exposure. All animals in both decontamination treatment groups (C, D) survived the duration of the study and exhibited less severe signs of cholinergic poisoning. Thus, both the standard and ORCHIDS protocol were demonstrably effective against exposure to the potent nerve agent VX, even after a delay of 1 h. A critical advantage of the ORCHIDS protocol is the relatively short shower duration (1(1/2) min compared to 3 min). In practice, this could substantially improve the rate at which individuals could be decontaminated by emergency responders following exposure to toxic materials such as chemical warfare agents.

The penetration of acetylcholinesterase reactivators (oximes) into the central nervous system is typically restricted by the blood-brain barrier. Although oximes are highly hydrophilic compounds, some contradictory results confirming permeation into the brain exist. The aim of this study is to verify the penetration of oximes through the blood-brain barrier and to detect their levels achieved in different brain regions 60 min after the administration. It was confirmed that oximes are able to penetrate into the brain after injection of therapeutic doses corresponding with 5% of LD(50). The level in whole brain was 0.58% for trimedoxime and 0.85% for the experimental drug oxime K074 as the percentage of their plasma concentration. The highest concentration was found in frontal cortex (trimedoxime 2.27%; oxime K074 0.95%) and lowest in basal ganglia (trimedoxime 0.86%; oxime K074 0.42%). Entry of oximes into the brain is minimal, but some low reactivation effect should be expected. The reactivation potency of oximes might be higher or lower, depending on the real oxime concentration in a given area.

Metrifonate (trichlorfon) is an inhibitor of acetylcholinesterase (AChE). It was used as an Alzheimer's disease (AD) drug; however, the application was withdrawn due to adverse effects. Implication of metrifonate for the antioxidant status and regulation of apoptotic processes was evaluated in the present study. Wistar rats (six per group) were exposed subcutaneously to either 60 or 120 mg/kg of body weight of metrifonate and compared with the controls treated with saline only. Cerebral cortex and liver tissues were collected from animals 40 min after exposure. Activities of AChE, glutathione reductase, glutathione-S-transferase, caspase 3, total protein level, thiobarbituric acid reactive substances, reduced glutathione level and ferric reducing antioxidant power (FRAP) were assayed in the tissue samples. Metrifonate had only lower impact on oxidative stress in the liver. Cerebral cortex tissues had decreased AChE and increased caspase 3 activities as well as the FRAP level. Owing to the novel findings, suitability of metrifonate for AD therapy is discussed.

Bioscavengers are molecules able to neutralize neurotoxic organophosphorus compounds (OP) before they can reach their biological target. Human butyrylcholinesterase (hBChE) is a natural bioscavenger each molecule of enzyme neutralizing one molecule of OP. The amount of natural enzyme is insufficient to achieve good protection. Thus, different strategies have been envisioned. The most straightforward consists in injecting a large dose of highly purified natural hBChE to increase the amount of bioscavenger in the bloodstream. This proved to be successful for protection against lethal doses of soman and VX but remains expensive. An improved strategy is to regenerate prophylactic cholinesterases (ChE) by administration of reactivators after exposure. But broad-spectrum efficient reactivators are still lacking, especially for inhibited hBChE. Cholinesterase mutants capable of reactivating spontaneously are another option. The G117H hBChE mutant has been a prototype. We present here the Y124H/Y72D mutant of human acetylcholinesterase; its spontaneous reactivation rate after V-agent inhibition is increased up to 110 fold. Catalytic bioscavengers, enzymes capable of hydrolyzing OP, present the best alternative. Mesophilic bacterial phosphotriesterase (PTE) is a candidate with good catalytic efficiency. Its enantioselectivity has been enhanced against the most potent OP isomers by rational design. We show that PEGylation of this enzyme improves its mean residence time in the rat blood stream 24-fold and its bioavailability 120-fold. Immunogenic issues remain to be solved. Human paraoxonase 1 (hPON1) is another promising candidate. However, its main drawback is that its phosphotriesterase activity is highly dependent on its environment. Recent progress has been made using a mammalian chimera of PON1, but we provide here additional data showing that this chimera is biochemically different from hPON1. Besides, the chimera is expected to suffer from immunogenic issues. Thus, we stress that interest for hPON1 must not fade away, and in particular, the 3D structure of the hPON1 eventually in complex with OP has to be solved.

A simple and reliable HPLC method for determination of rat plasma levels of clinically used acetylcholinesterase (AChE) reactivators (HI-6 and obidoxime) is presented in our study. Separation was carried out by HPLC using an octadecyl silica stationary phase and a mobile phase consisting of 24% acetonitrile and containing 5 mM sodium octanesulfonate and 5 mM tetramethylammonium chloride (pH 2.3). Following intramuscular administration of equimolar doses of both oximes (22.23 mg/kg), the maximum of HI-6 concentration in rat plasma was reached in about 20 min giving 15.26 +/- 1.71 microg/mL. The distribution of obidoxime was fast; the single maximum 23.62 +/- 3.563 microg/mL was recorded at about 10 min. HPLC with UV detection presented in our study is a general method which could be applied for quick measurements of bisquaternary AChE reactivators in rat plasma.

Acetylcholinesterase (AChE) reactivators are crucial antidotes to organophosphate intoxication. A new series of 26 monooxime-monocarbamoyl xylene-linked bispyridinium compounds was prepared and tested in vitro, along with known reactivators (pralidoxime, HI-6, obidoxime, trimedoxime, methoxime, K107, K108 and K203), on a model of tabun- and paraoxon-, methylparaoxon- and DFP-inhibited human erythrocyte AChE. Although their ability to reactivate tabun-inhibited AChE did not exceed that of the previously known compounds, some newly prepared compounds showed promising reactivation of pesticide-inhibited AChE. The acute toxicity of the novel compounds was also determined. Docking studies using tabun-inhibited AChE were performed for three compounds of interest. The structure-activity relationship (SAR) study confirmed the apparent influence of the xylene linkage and carbamoyl moiety on the reactivation ability and toxicity of the agents.

Seven new oxime-based acetylcholinesterase reactivators were compared with three currently available ones (obidoxime, trimedoxime, HI-6) for their ability to lessen cholinesterase inhibition in blood and brain of cyclosarin-treated rats. Oximes were given at doses of 5% their LD(50) along with 21 mg/kg atropine five min before the LD(50) of cyclosarin (120 ug/kg) was administered. Blood and brain samples were collected 30 minutes later. The greatest difference between acetylcholinesterase inhibition in blood of cyclosarin-treated rats was found after administration of HI-6 (40%), compared to 22% for trimedoxime and 6% for obidoxime. Only two of the seven newly synthesized oximes had any effect (K203 at 7%, K156 at 5%). Effective oximes against cyclosarin-inhibited plasma butyrylcholinesterase were HI-6 (42%), trimedoxime (11%), and K156 (4%). The oximes were less effective in brain than in blood, with reactivation values for HI-6 30% against acetylcholinesterase and 10% against butyrylcholinesterase. Values for newly synthesized oximes were less than 10% for K206, K269 and K203.

Newly developed acetylcholinesterase reactivators K117 [1,5-bis(4-hydroxyiminomethylpyridinium)-3-oxapentane dichloride] and K127 [(1-(4-hydroxyiminomethylpyridinium)-5-(4-carbamoylpyridinium)-3-oxapentane dibromide)] were tested for their potency to reactivate tabun-inhibited human brain cholinesterases. Pralidoxime and trimedoxime were chosen as standard reference reactivators. Human tissue was used, as that was closer on the real treatment of human beings. As a result, oxime K127 was found as the best tested reactivator according to the constant k(r), characterizing the overall reactivation process. On the contrary, the maximal reactivation ability expressed as percentage of reactivation was the best for trimedoxime. This differences were caused as a result of using the enzyme from different species. Due to this, experiments on human tissue should be conducted after in vitro and in vivo tests on animals to eliminate such important failures of promising oximes.