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Oxime-based acetylcholinesterase reactivators (briefly oximes) regenerate organophosphate-inactivated acetylcholinesterase and restore its function. Poor blood-brain-barrier passage and fast elimination from blood limit their actual use in treatment of patients exposed to organophosphates. Previous in vitro results implicated further testing of cucurbit[7]uril as a delivery vehicle for bisquaternary oximes. The present paper focuses on cell toxicity, in vivo safety and influence of cucurbit[7]uril on oxime pharmacokinetics and pharmacodynamics. Neither the K027 nor the complex caused any cell toxicity, changes in blood biochemistry or hepato- or nephrotoxicity in tested concentrations. The encapsulation of K027 increased and accelerated the blood-brain-barrier penetration. The peripheral oxime exposure also increased, supporting the suggestion that cucurbit[7]uril protects the circulating oxime from rapid renal clearance. Contrary to the comparable in vitro reactivation power of K027 and the encapsulated K027, we failed to confirm this in vivo. In theory, this might result from the non-specific binding of molecules to the cucurbit[7]uril or the interaction of K027 with cucurbit[7]uril being too strong for acetylcholinesterase reactivation. Precise explanation requires additional in silico, in vitro and also in vivo experiments., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The antidotal property of oximes is attributed to their ability to reactivate acetylcholinesterase (AChE) inhibited by organophosphorus compounds (OP) such as pesticides and nerve warfare agents. Understanding their interactions within the active site of phosphylated AChE is of great significance for the search for more efficient reactivators, especially in the case of the most resistant OP to reactivation, tabun. Therefore, herein we studied the interactions and reactivation of tabun-inhibited AChE by site-directed mutagenesis and a series of bispyridinium oximes. Our results indicated that the replacement of aromatic residues with aliphatic ones at the acyl pocket and choline binding site mostly interfered with the stabilisation of the oxime's pyridinium ring(s) within the active site gorge needed to obtain the proper orientation of the oxime group toward the phosphorylated active site serine. However, in the case of W286A, the mutation in the peripheral binding site by preventing a π-π interaction with one of the oxime's pyridinium rings allowed a more favourable position of the oxime for a nucleophilic attack on the phosphorylated catalytic serine. The mutation resulted in a 2-5 fold increase in the reactivation rates when compared to the AChE wild type. Therefore, it seems that aromatic amino acids at the peripheral binding site presented a limitation in bispyridinium oxime reactivation efficiency of tabun-phosphorylated AChE. Moreover, this is further corroborated by the reactivation by mono-pyridinium oxime 2-PAM, in which mutations at the peripheral site did not influence either the affinity or reactivation of tabun-inhibited AChE., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Highly toxic organophosphorus (OP) nerve agents are well characterized regarding chemical, biological and toxicological properties and the effectiveness of standard atropine and oxime therapy. Open literature data on the key nerve agent precursor methylphosphonic difluoride (DF) are scarce. To fill this gap the reactions of DF and its main degradation product methylphosphonofluoridic acid (MF) with human acetylcholinesterase (AChE) and the oximes obidoxime, HI-6 and 2-PAM were investigated in vitro. DF and MF were found to be weak inhibitors of human AChE being at least five orders less potent compared to the nerve agent sarin. Incubation of human AChE with millimolar DF and MF and subsequent addition of obidoxime and HI-6 resulted in a concentration-dependent decrease of AChE activity. This effect was not observed when incubating highly diluted AChE with oximes. The most likely explanation for this phenomenon is an inhibitory effect of phosphonyloximes formed by direct reaction of DF or MF with obidoxime and HI-6. These data indicate that high DF doses, resulting in millimolar blood and tissue DF/MF concentrations, are necessary to induce cholinergic signs and that under these conditions treatment with obidoxime and HI-6 may even worsen the poisoning., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The organophosphorus (OP) pesticide malathion is a highly neurotoxic compound and its toxicity is primarily caused by the inhibition of acetylcholinesterase (AChE), leading to cholinergic syndrome. Although oximes have been used as potential antidotal treatments in malathion poisoning because of their potential capability to reactivate the inhibited enzyme, the clinical experience with the clinically available oximes (e.g. pralidoxime) is disappointing and their routine use has been questioned. In the present study, we investigated the potency of pralidoxime and K074 in reactivating AChE after acute exposure to malathion, as well as in preventing malathion-induced changes in oxidative-stress related parameters in mice. Malathion (1.25 g/kg, s.c.) induced a significant decrease in cortico-cerebral, hippocampal and blood AChE activities at 24h after exposure. Oxime treatments (1/4 of LD(50), i.m., 6h after malathion poisoning) showed that pralidoxime significantly reversed malathion-induced blood AChE inhibition, although no significant effects were observed after K074 treatment. Interestingly, both oximes tested were unable to reactivate the cortico-cerebral and hippocampal enzymes after intramuscular or intracerebroventricular injection (1/4 of LD(50), 6h after malathion poisoning). Biochemical parameters related to oxidative stress (cerebro-cortical and hippocampal glutathione peroxidase, glutathione reductase and catalase activities, as well as lipid peroxidation) were not affected in animals treated with malathion, oximes or atropine alone. However, pralidoxime and K074, administered intramuscularly 6h after malathion poisoning, were able to increase the endogenous activities of these antioxidant enzymes in the prefrontal cortex and hippocampus. Taken together, the results presented herein showed that pralidoxime (the most common clinically used oxime) and the recently developed oxime K074, administered 6h after malathion poisoning, were unable to reactivate the inhibited AChE in mouse prefrontal cortex and hippocampus. However, only pralidoxime significantly reversed the blood AChE inhibition induced by malathion poisoning. This indicates that peripheral and central AChE activities are not necessarily correlated after the treatment of OP compounds and/or oximes, which should be taken into account in the diagnosis and management of OP-exposed humans. In addition, considering that the available treatments to malathion poisoning appear to be ineffective, the present study reinforce the need to search for potential new AChE reactivators able to efficiently reactivate the brain and blood AChEs after malathion poisoning., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The US Army utilizes pralidoxime (2-PAM) for the reactivation of OP-inhibited AChE. While 2-PAM effectively reactivates acetylcholinesterase (AChE) in the body, it does not cross the blood-brain barrier (BBB) at therapeutically relevant levels. To address this problem of central nervous system AChE reactivation, novel sugar-oxime conjugates were utilized. These 'sugar-oximes' would potentially be transported across the BBB because they contain a sugar moiety which would be recognized by the facilitative glucose transporters. Eight previously reported, but understudied sugar-oximes, as well as six novel sugar-oximes were synthesized, and their ability to reactivate both human red blood cell AChE and plasma butyrylcholinesterase poisoned with DFP, paraoxon, sarin and VX were tested. The results show that the novel sugar-oxime 13c was more active than the other compounds with a reactivation potential similar to 2-PAM. The sugar-oxime 8b had low toxicity with a LD(50) of 1,590 mg/kg from a single IM dose in the guinea pig and >2,000 mg/kg IP in the mouse. Histopathological analysis showed that there were no apparent differences in hippocampus, heart, liver, kidney sciatic nerve, or skeletal muscle between treated and untreated animals. These results show that sugar-oximes can be effective reactivators and suggest that high treatment doses may be possible., (Published by Elsevier Ireland Ltd.)

Oximes are enzyme reactivators used in treating poisoning with organophosphorus cholinesterase (AChE) inhibitors. The oxime dose which can be safely administered is limited by the intrinsic toxicity of the substances such as their own AChE-inhibiting tendency. Clinical experience with the available oximes is disappointing. To meet this need, new AChE reactivators of potential clinical utility have been developed. The purpose of the study was to estimate in vitro both the intrinsic toxicity and the extent of possible protection conferred by established (pralidoxime, obidoxime, HI-6, methoxime, trimedoxime) and experimental (K-type) oximes, using diisopropyl-fluoro-phosphate (DFP) as an AChE inhibitor. The IC50 of DFP against human red blood cell AChE was determined ( approximately 120 nm). Measurements were then repeated in the presence of increasing oxime concentrations, leading to an apparent increase in DFP IC50. Calculated IC50 values were plotted against oxime concentrations to obtain an IC50 shift curve. The slope of this shift curve (tan alpha) was used to quantify the magnitude of the protective effect (nm IC50 increase per microm oxime). We show that, in the case of a linear relationship between oxime concentration and IC50, the binding constant K, determined using the Schild equation, equals IC50/DFP/tan alpha. Based on the values of tan alpha and of the binding constant K, some of the new K-oxime reactivators are far superior to pralidoxime (tan alpha = 0.8), obidoxime (1.5), HI-6 (0.8), trimedoxime (2.9) and methoxime (5.9), with K-107 (17), K-108 (20), and K-113 (16) being the outstanding compounds., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

The effect was followed up of 2-PAM and Toxogonin (T), reactivators of choline esterase, at a single muscular application on the activity of acetylcholine esterase (ES 3.1.1.7) (ACE) of whole blood (WB) and the myoneural synapses (MS) of an interrib muscle. It was found that a species-specific effect was produced by T and 2-PAM on ACE of WB and MS in lambs and sheep. Optimal doses of 50 and 80 mg/kg led to a slight drop in the activity of ACE at the 2nd hour following injection, after which it rose gradually up to values that were higher than the initial ones for more than ten days. 100 and 200 mg/kg led to a more sensitive and long-term drop in the activity of ACE (up to 48h--72nd hour), after which it likewise rose above the initial value, and came back to normal on the 13th day. With lambs the reaction was analogous, though weaker. In rats and rabbits there was no change in the activity of ACE of WB and MS following the use of oximes. The optimal nontoxic and inductoenzyme rates of the two oximes for lambs and sheep proved to be about 50 mg/kg. It was established that both for rats and for sheep and lambs T was almost twice as toxic as 2-PAM. Intoxication with T was characterized by severe hemorrhagic and necrotic nephroso-nephritis, acute toxic dystrophy of the liver, and ulcerous, necrotic enteritis. 2-PAM was found to potentiate the toxic effect of T.

We have prepared four new oximes, 1b-e, which conform to the general structure RCH2COCH = NOH where R = CH3S, CH3SO, CH3SO2, and (CH3)2S+, respectively, and have the same E configuration as the parent 2-oxopropanal oxime 1a (R = H, MINA). The pKa values range from 6.54 (1e) to 8.16 (1b), as compared with 8.30 for 1a. Rates of reaction (kappa 1) with 4-nitrophenyl acetate indicate that the oximate anions have a much higher nucleophilicity than common oxyanions of similar basicities: the alpha effects measured for 1a-e are of the order of 200-250. The abilities of 1b-e to reactivate acetylcholinesterase (AChE) inhibited by organophosphates have been evaluated. In vitro experiments reveal a significant reactivation potency of 1b-e against VX-, sarin-, and paraoxon-inhibited immobilized eel AChE. The highly lipophilic methylthio oxime 1b (log P greater than 1) is intrinsically (kappa 2) 3 times more reactive than the more basic MINA (log P less than 1). The sulfonium oxime 1e is a potent reactivator against paraoxon. Interestingly, both 1b and 1e have a low toxicity and they exhibit a significant antidotal effect at a relative low dose against paraoxon in rats.

Out of 12 oximes and amidoximes (3 of which were new to the literature) the following showed a distinct antilethal effect in DFP poisoning: RA14 (1-methylbenzimidazole-2-aldoxime methiodide), RA14 (pyridine-4-aldoxime dodecyl bromide), and RA24 (pyridine-2-aldoxime dodecyl bromide). These compounds also reactivated acetylcholinesterase (AChE) in vitro, but had no effect on this enzyme in vivo. Moreover, addition of the dodecyl chain to pyridinealdoxtimes, or in a less degree replacement of the pyridine ring with benzimidazole in aldoximes, distinctly increased acute toxicity and lipophilicity when compared with the parent pyridine compounds, along with protective action in DFP poisoning.