Jana Zdarova Karasova

Colorimetric dipstick for assay of organophosphate pesticides and nerve agents represented by paraoxon, sarin and VX.

A dipstick for fast assay of nerve agents and organophosphate pesticides was developed. Indicator pH papers were used as detectors. The principle of the assay is based on enzymatic hydrolysis of acetylcholine into acetic acid and choline by acetylcholinesterase. Acidification of the reaction medium due to accumulation of acetic acid was visible. The colour changed from dark red to yellow as the pH indicator recognized pH shift. Presence of an organophosphate pesticide or a nerve agent results in irreversible inhibition of acetylcholinesterase intercepted on the dipstick. The inhibition stops the enzymatic reaction. The inhibition appears as no change of the medium pH. Three compounds were assayed: paraoxon-ethyl as representative organophosphate pesticides and nerve agents sarin and VX. The achieved limit of detection was 5 x 10(-8)M for paraoxon-ethyl and 5 x 10(-9)M for sarin and VX. Dipsticks were found stable for at least one month. Suitability of these dipsticks for routine assay is discussed.

Passive diffusion of acetylcholinesterase oxime reactivators through the blood–brain barrier: Influence of molecular structure

In this in vitro study, high-performance liquid chromatography (HPLC) was used to determinate the penetration of 30 acetylcholinesterase (AChE) reactivators through the blood-brain barrier (BBB). According to our method, monoquaternary AChE reactivators were found to be able to penetrate the BBB. In addition to molecular structure, molecular weight appears to be an important factor for passive transport of oximes through the BBB. For bisquaternary reactivators, the connecting linker plays a key role in the ability to penetrate into the central nervous system (CNS): simple, short linkers tend to facilitate permeation. The location of groups on the pyridine ring also influences passive transport into the brain; the optimum position of the oxime group was found to be position four (para) and substitution of the oxime group on the pyridine ring by carbamoyl or amidoxime group markedly decreased penetration of AChE reactivators into the CNS.

The Development of New Oximes and the Evaluation of their Reactivating, Therapeutic and Neuroprotective Efficacy Against Tabun

Tabun (O-ethyl-N,N-dimethyl phosphoramidocyanidate) belongs to highly toxic organophosphorus compounds misused as chemical warfare agents for military as well as terroristic purposes. The antidotal treatment of tabun acute poisonings still represents a serious problem and the development of new, more effective AChE reactivators to achieve the satisfactorily effective antidotal treatment of acute poisonings with tabun still represents very important goal. Since 2003, we have prepared around 200 new AChE reactivators. Their potency to reactivate tabun-inhibited acetylcholinesterase has been subsequently evaluated using our in vitro screening test. Afterwards, promising compounds were selected and kinetic parameters and reactivation constants were determined. Then, the best reactivators were subjected to the in vivo studies (toxicity test, the evaluation of therapeutic, reactivating and neuroprotective efficacy) and their potency to counteract the acute toxicity of tabun is compared to the therapeutic, reactivating and neuroprotective efficacy of commonly used oximes - obidoxime and the oxime HI-6. According to the results obtained, the newly synthesized oxime K075 showed the highest potency to reduce tabun-induced acute lethal toxicity while the therapeutic potency of obidoxime and the oxime HI-6 was significantly lower. The therapeutic efficacy of oximes studied corresponds to their reactivating efficacy in vivo as well as in vitro. The potency of all newly synthesized oximes to reactivate tabun-inhibited AChE is comparable with obidoxime with the exception of K074 that is significantly more efficacious in the brain. In addition, all newly synthesized oximes combined with atropine seem to be effective antidotes for a decrease in tabun-induced acute neurotoxicity. While the neuroprotective efficacy of obidoxime in combination with atropine is similar to the potency of newly synthesized oximes, the ability of the oxime HI-6 combined with atropine to counteract tabun-induced acute neurotoxicity is significantly lower. Due to their therapeutic, reactivating and neuroprotective efficacy, all newly synthesized oximes appear to be suitable oximes for the antidotal treatment of acute tabun poisonings.

A comparison of reactivating efficacy of newly developed oximes (K074, K075) and currently available oximes (obidoxime, HI-6) in soman, cyclosarin and tabun-poisoned rats

The potency of newly developed oximes (K074, K075) and commonly used oximes (obidoxime, HI-6) to reactivate nerve agent-inhibited acetylcholinesterase was evaluated in rats poisoned with soman, tabun or cyclosarin at a lethal dose corresponding to their LD(50) value. In vivo determined percentage of reactivation of soman-inhibited blood and brain acetylcholinesterase in poisoned rats showed that only the oxime HI-6 was able to reactivate soman-inhibited acetylcholinesterase in the peripheral (blood) as well as central (brain) compartment. In vivo determined percentage of reactivation of tabun-inhibited blood and brain acetylcholinesterase in poisoned rats showed that obidoxime is the most efficacious reactivator of tabun-inhibited acetylcholinesterase among studied oximes in the peripheral compartment (blood) while K074 seems to be the most efficacious reactivator of tabun-inhibited acetylcholinesterase among studied oximes in the central compartment (brain). In vivo determined percentage of reactivation of cyclosarin-inhibited blood and brain acetylcholinesterase in poisoned rats showed that HI-6 is the most efficacious reactivator of cyclosarin-inhibited acetylcholinesterase among studied oximes. Due to their reactivating effects, both newly developed K oximes can be considered to be promising oximes for the antidotal treatment of acute tabun poisonings while the oxime HI-6 is still the most promising oxime for the treatment of acute soman and cyclosarin poisonings.

Partition of bispyridinium oximes (trimedoxime and K074) administered in therapeutic doses into different parts of the rat brain

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.

Ascorbic Acid: An Old Player with a Broad Impact on Body Physiology Including Oxidative Stress Suppression and Immunomodulation: A Review

Ascorbic acid is a low molecular weight antioxidant well known as anti-scorbut acting vitamin C in humans, primates and guinea pigs. This review summarizes basic data about ascorbic acid in its physiological action point of view. It is divided into biochemistry of ascorbic acid synthesis, mechanism of antioxidant action and participation in anabolism, pharmacokinetics and excretion, exogenous ascorbic acid immunomodulatory effect and participation in infectious diseases, impact on irradiation and intoxication pathogenesis, and supplementary demands. The primary intention was to consider ascorbic acid not only as an antioxidant but also as a chemical compound affecting multiple pathways with a potential beneficial impact in many diseases and processes in human body.

Effect of five acetylcholinesterase reactivators on tabun-intoxicated rats: induction of oxidative stress versus reactivation efficacy.

Oxime reactivators HI‐6, obidoxime, trimedoxime, K347 and K628 were investigated as drugs designed for treatment of tabun intoxication. The experiments were performed on rats in order to simulate real conditions. Rats were intoxicated with one LD50 of tabun and treated with atropine and mentioned reactivators. Activities of erythrocyte acetylcholinesterase (AChE), plasma butyrylcholinesterase (BChE) and brain AChE were measured as markers of reactivation efficacy. An estimation of low molecular weight antioxidant levels using cyclic voltammetry was the second examination parameter. The evaluation of cholinesterases activity showed good reactivation potency of blood AChE and plasma BChE by commercially available obidoxime and newly synthesized K347. The potency of oximes to reactivate brain AChE was lower due to the poor blood–brain barrier penetration of used compounds. Commercially available reactivator HI‐6 and newly synthesized K628 caused oxidative stress measured by cyclic voltammetry as antioxidant level. The oxidative stress provoked by HI‐6 and K628 was found to be significant on probability level P = 0.05. The others reactivators did not affect antioxidant levels. Copyright

A Comparison of neuroprotective efficacy of newly developed oximes (K203, K206) and commonly used oximes (obidoxime, HI-6) in tabun-poisoned rats

The neuroprotective effects of newly developed oximes (K203, K206) and commonly used oximes (obidoxime, HI-6) in combination with atropine in rats poisoned with tabun at a sublethal dose (180 μg/kg i.m.; 80% LD50) were studied. The tabun-induced neurotoxicity was monitored by using a functional observational battery and an automatic measurement of motor activity. The neurotoxicity of tabun was monitored at 24 hours and 7 days following tabun challenge. The results indicate that K203 and obidoxime in combination with atropine allow all tabun-poisoned rats to survive within 7 days following tabun challenge, while 2 nontreated tabun-poisoned rats and 1 tabun-poisoned rat treated with K206 or HI-6 in combination with atropine died within 7 days. Only one of the newly developed oximes (K203) combined with atropine seems to be effective for a decrease in tabun-induced neurotoxicity within 24 hours after tabun sublethal poisoning, although it is not able to eliminate tabun-induced neurotoxicity completely. On the other hand, the neuroprotective efficacy of commonly used oximes (obidoxime and HI-6), as well as one of the new synthesized oximes (K206), is significantly lower in comparison with K203, according to the number of eliminated tabun-induced neurotoxic signs at 24 hours after tabun challenge. Due to its neuroprotective effects, K203 appears to be a suitable oxime for the antidotal treatment of acute tabun poisonings.

The influence of combinations of oximes on the reactivating and therapeutic efficacy of antidotal treatment of tabun poisoning in rats and mice

The influence of the combination of oximes on the reactivating and therapeutic efficacy of antidotal treament of acute tabun poisoning was evaluated. The ability of two combinations of oximes (HI‐6 + obidoxime and HI‐6 + K203) to reactivate tabun‐inhibited acetylcholinesterase and reduce acute toxicity of tabun was compared with the reactivating and therapeutic efficacy of antidotal treatment involving single oxime (HI‐6, obidoxime, K203) using in vivo methods. Studies determining percentage of reactivation of tabun‐inhibited blood and tissue acetylcholinesterase in poisoned rats showed that the reactivating efficacy of both combinations of oximes is higher than the reactivating efficacy of the most effective individual oxime in blood and diaphragm and comparable with the reactivating effects of the most effective individual oxime in brain. Moreover, both combinations of oximes were found to be slightly more efficacious in the reduction of acute lethal toxic effects in tabun‐poisoned mice than the antidotal treatment involving individual oxime. A comparison of reactivating and therapeutic efficacy of individual oximes showed that the newly developed oxime K203 is slightly more effective than commonly used obidoxime and both of them are markedly more effective than the oxime HI‐6. Based on the obtained data, we can conclude that the antidotal treatment involving chosen combinations of oximes brings beneficial effects for the potency of antidotal treatment to reactivate tabun‐inhibited acetylcholinesterase in rats and to reduce acute toxicity of tabun in mice. Copyright

Pharmacokinetic study of two acetylcholinesterase reactivators, trimedoxime and newly synthesized oxime K027, in rat plasma.

K027 [1‐(4‐hydroxyiminomethylpyridinium)‐3‐(4‐carbamoylpyridinium)–propane dibromide] is a promising new reactivator of organophosphate‐ or organophosphonate‐inhibited acetylcholinesterase (AChE) with low acute toxicity and broad spectrum efficacy. The aim of the present study was to compare the pharmacokinetics of both compounds. Male Wistar rats (body weight = 320 ± 10 g) were administered a single intramuscular dose of K027 (22.07 mg kg−1) and an equimolar dose of trimedoxime. Blood was collected at various time intervals until 180 min. Plasma samples were analyzed by reversed‐phase HPLC with ultraviolet (UV) detection. The recovery of both oximes from the plasma was approximately 90% and a linear relationship (R2 > 0.998) was observed between the peak areas and concentrations of calibrated standards in the range 1–100 µg ml−1. Near‐identical plasma profiles were obtained for both compounds. No differences were found in the mean ± SD values of Cmax (18.6 ± 2.5 vs 20.0 ± 6.3 µg ml−1, P = 0.72) and AUC0–180min (2290 ± 304 vs 2269 ± 197 min µg ml−1, P = 0.84). However, the percentage coefficient of variation of the first‐order rate constant of absorption (ka) was 3‐fold higher (P < 0.01) providing evidence for more erratic absorption of intramuscular trimedoxime as compared with K027. In conclusion, oxime K027 might have superior pK properties that may be translated in its faster absorption and subsequent tissue distribution. Copyright