Jiri Kassa

Tacrine–Trolox Hybrids: A Novel Class of Centrally Active, Nonhepatotoxic Multi-Target-Directed Ligands Exerting Anticholinesterase and Antioxidant Activities with Low In Vivo Toxicity

Coupling of two distinct pharmacophores, tacrine and trolox, endowed with different biological properties, afforded 21 hybrid compounds as novel multifunctional candidates against Alzheimers disease. Several of them showed improved inhibitory properties toward acetylcholinesterase (AChE) in relation to tacrine. These hybrids also scavenged free radicals. Molecular modeling studies in tandem with kinetic analysis exhibited that these hybrids target both catalytic active site as well as peripheral anionic site of AChE. In addition, incorporation of the moiety bearing antioxidant abilities displayed negligible toxicity on human hepatic cells. This striking effect was explained by formation of nontoxic metabolites after 1 h incubation in human liver microsomes system. Finally, tacrine-trolox hybrids exhibited low in vivo toxicity after im administration in rats and potential to penetrate across blood-brain barrier. All of these outstanding in vitro results in combination with promising in vivo outcomes highlighted derivative 7u as the lead structure worthy of further investigation.

A Comparison of the Efficacy of New Asymmetric Bispyridinium Oximes (K027, K048) with Currently Available Oximes Against Tabun by In Vivo Methods

The potency of newly developed asymmetric bispyridinium oximes (K027, K048) in reactivating tabun-inhibited acetylcholinesterase (AChE) and in eliminating tabun-induced acute toxic effects was compared with commonly used oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies determined the percent of reactivation of tabun-inhibited blood and tissue AChE in poisoned rats and showed that the reactivating efficacy of both newly developed oximes is comparable with obidoxime and trimedoxime, the most efficacious known reactivators of tabun-inhibited AChE. These were also found to be sufficiently efficacious in the elimination of acute lethal toxic effects in tabun-poisoned rats. The oxime HI-6, relatively efficacious against soman, did not seem to be an adequately effective oxime in reactivation of tabun-inhibited AChE and in counteracting acute lethal effects of tabun. In addition, our results confirm that the efficacy of oximes in reactivating tabun-inhibited AChE in blood, diaphragm, and brain correlates with the potency of oximes in protecting rats poisoned with supralethal doses of tabun.

Potency of several oximes to reactivate human acetylcholinesterase and butyrylcholinesterase inhibited by paraoxon in vitro.

Organophosphorus pesticides (e.g. chlorpyrifos, malathion, and parathion) and nerve agents (sarin, tabun, and VX) are highly toxic organophosphorus compounds with strong inhibition potency against two key enzymes in the human body-acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8). Subsequent accumulation of acetylcholine at synaptic clefts can result in cholinergic crisis and possible death of intoxicated organism. For the recovery of inhibited AChE, derivatives from the group of pyridinium or bispyridinium aldoximes (called oximes) are used. Their efficacy depends on their chemical structure and also type of organophosphorus inhibitor. In this study, we have tested potency of selected cholinesterase reactivators (pralidoxime, obidoxime, trimedoxime, methoxime and H-oxime HI-6) to reactivate human erythrocyte AChE and human plasma BuChE inhibited by pesticide paraoxon. For this purpose, modified Ellmans method was used and two different concentrations of oximes (10 and 100 microM), attainable in the plasma within antidotal treatment of pesticide intoxication were tested. Results demonstrated that obidoxime (96.8%) and trimedoxime (86%) only reached sufficient reactivation efficacy in case of paraoxon-inhibited AChE. Other oximes evaluated did not surpassed more than 25% of reactivation. In the case of BuChE reactivation, none of tested oximes surpassed 12.5% of reactivation. The highest reactivation efficacy was achieved for trimedoxime (12.4%) at the concentration 100 microM. From the data obtained, it is clear that only two from currently available oximes (obidoxime and trimedoxime) are good reactivators of paraoxon-inhibited AChE. In the case of BuChE, none of these reactivators could be used for its reactivation.

In vitro reactivation of sarin-inhibited brain acetylcholinesterase from different species by various oximes

In vitro as well as in vivo evaluation of the reactivating efficacy of various oximes against nerve agent-inhibited acetylcholinesterase has been usually done with the help of animal experiments. Nevertheless, previously published data indicate that the reactivation potency of oximes may be different in human and animal species, which may hamper the extrapolation of animal data to human data. Therefore, to better evaluate the efficacy of various oximes (pralidoxime, obidoxime, HI-6, K033) to reactivate brain acetylcholinesterase inhibited by sarin by in vitro methods, human, rat and pig brain acetylcholinesterase were used to calculate kinetic parameters for the reactivation. Our results show differences among the species, depending on the type of oxime, and indicate that data from animal experiments needs to be carefully evaluated before extrapolation to humans.

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.

Fluorinated pyridinium oximes as potential reactivators for acetylcholinesterases inhibited by paraoxon organophosphorus agent

A series of fluorinated oxime compounds was designed and synthesized in order to probe the effect of fluorine substitution on reactivation of inhibited acetylcholinesterase (AChE) by organophosphorus agents. Permeability measurements, using the Parallel Artificial Membrane Permeation Assays (PAMPA) method, were employed to experimentally demonstrate that membrane permeabilities of the series of oximes increase in proportional to the increase in the number of fluorine atoms. Among the compounds explored in this study, the mono-fluorinated carbamoyl aldoxime 4b was the most potent reactivator for paraoxon-inhibited red blood cell (RBC) AChE.

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.

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.

The reactivating and therapeutic efficacy of oximes to counteract Russian VX poisonings.

Russian VX (O-isobutyl-S-(2-diethylaminoethyl)methylphosphonothioate) is the structural analogue of VX agent. It differs from VX agent (O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothioate) by two alkyl groups. The potency of currently available oximes (pralidoxime, obidoxime, HI-6) to reactivate Russian VX–inhibited acetylcholinesterase and to eliminate Russian VX–induced acute toxic effects was evaluated using in vivo methods. In vivo determined percentage of reactivation of Russian VX–inhibited blood and brain acetylcholinesterase in poisoned rats shows that HI-6 seems to be the most efficacious reactivator of Russian VX–inhibited acetylcholinesterase among currently used oximes in the peripheral compartment, whereas no difference between reactivating efficacy of all tested oximes was observed in the central compartment. The oxime HI-6 was also found to be the most efficacious oxime in the elimination of acute lethal toxic effects in Russian VX–poisoned mice among all studied oximes. Thus, the oxime HI-6 seems to be the most suitable oxime for the antidotal treatment of acute poisonings with Russian VX as in the case of VX, sarin, cyclosarin, and soman poisonings.

New K-Oximes (K-27 and K-48) in Comparison with Obidoxime (LuH-6), HI-6, Trimedoxime (TMB-4), and Pralidoxime (2-PAM): Survival in Rats Exposed IP to the Organophosphate Paraoxon

ABSTRACT Oximes are cholinesterase reactivators used in organophosphorus compound poisoning. The purpose of the study was to compare the protective effect of the K-oximes (K-27 and K-48) in male rats with that of obidoxime (LuH-6), trimedoxime (TMB-4), and HI-6, using paraoxon (POX) as a cholinesterase inhibitor. Pralidoxime (2-PAM) was also retested. Seven groups of six rats each were used. Group 1 (G1) received 1 μmol/rat POX (≈ LD75), the other groups (G2–7) received 1 μmol/rat POX + one of the six reactivators. The animals were monitored for 48 h and time of mortality was recorded. The procedure was repeated seven times. Subsequently, experiments as described were repeated using 10 and 15 μmol/rat POX. Mortality data were compared and hazards ratios (relative risks) ranked with the Cox proportional hazards model using the POX dose and group (reactivator) as time-independent covariables. K-27 followed by K-48 were the most potent reactivators. K-27 was statistically significantly superior to all other reactivators except K-48. The relative risk of death estimated by Cox analysis in K-27– and K-48–treated animals when compared with untreated animals, adjusted for the POX dose, was 0.22 (95% confidence interval [CI], 0.15 to 0.31) and 0.26 (95% CI, 0.18 to 0.37), respectively. We concluded that in the animal model used K-27 and K-48 are superior to older oximes in their ability to protect from paraoxon effects. They should be tested further using methyl- and propyl-organophosphates as toxic agents.