Effect of some acetylcholinesterase reactivators on human platelet aggregation in vitro

Acetylcholinesterase (AChE) reactivators are employed for the prophylaxis and treatment of intoxications with organophosphorus AChE inhibitors, including nerve agents and pesticides. For the recovery of inhibited enzyme, derivatives from the group of pyridinium or bispyridinium aldoximes (called oximes) are used. Adverse effects of these substances are not well elucidated, because of their narrow and one-shot usage. Owing to this fact, the study evaluated the influence of some currently applied oximes on human platelet aggregation in vitro. The antiplatelet activity of pralidoxime, obidoxime, HI-6, methoxime and HL? 7 was assayed in human platelet rich plasma (2.5 x 10(8) platelets.ml(-1)) at a concentration of 1.35 mM. Arachidonic acid (AA), adenosine diphosphate (ADP), collagen (COL) and thrombin (TR) were used as agonists of platelet aggregation. All tested substances, except pralidoxime and methoxime, caused a significant inhibition of the aggregation process induced by AA, ADP and COL. Of the oximes assayed, none was found to influence TR triggered aggregation. Since reduced platelet aggregation can play an important role as an adverse effect in reactivator administration, further evaluation is needed for the estimation of the real impact of active oximes to the aggregation process in humans.     

A comparison of reactivating and therapeutic efficacy of bispyridinium acetylcholinesterase reactivator KR-22934 with the oxime K203 and commonly used oximes (obidoxime,trimedoxime, HI-6) in tabun-poisoned rats and mice

The potency of bispyridinium acetylcholinesterase reactivator KR-22934 in reactivating tabun-inhibited acetylcholinesterase and reducing tabun-induced lethal toxic effects was compared with the oxime K203 and commonly used oximes. Studies determining percentage of reactivation of tabun-inhibited blood and tissue acetylcholinesterase in rats showed that the reactivating efficacy of KR-22934 was slightly higher than the reactivating efficacy of K203 and roughly corresponded to the reactivating efficacy of obidoxime and trimedoxime in blood and diaphragm. On the other hand, the oxime KR-22934 was not able to reactivate tabun-inhibited acetylcholinesterase in the brain. The therapeutic efficacy of all oximes studied approximately corresponded to their reactivating efficacy. Based on the results, one can conclude that the oxime KR-22934 is not suitable for the replacement of commonly used oximes for the antidotal treatment of tabun poisoning in spite of its potency to reactivate tabun-inhibited acetylcholinesterase in the peripheral compartment (blood, diaphragm).     

The forgotten or underestimated relevance of biopharmaceutical-based assessments for the oral absorption studies of oxime reactivators

Introduction: The absorption, distribution, metabolism, excretion and toxicity (ADME(T)) of oxime reactivators have been assessed with respect to their polarity, a fundamental requirement for their specific mechanism of action in the intoxication with organophosphorous compounds. The limitations of the therapeutic outcome have been associated not only with the severity of intoxication and to particularities of the toxicants, but also to the reduced lipophilicity and consequent restricted permeability across biological barriers.

Areas covered: This article inventories the plethora of mnemotic rules developed throughout the years for defining chemical spaces where drugs share one or more structural and ADME(T) characteristics. Their applicability to oxime is analyzed, especially in relation to intestinal absorption and brain distribution. Other aspects of oximes for antidotal outcome are also reviewed.

Expert opinion: The drugability rules are not applicable to oxime reactivators, because the increase in lipophicity and consequent improved permeability across biological barrier comes together with amplified (neuro)toxicity and reduced reactivating capacity. The available data suggest a high solubility and reduced metabolism, assigning the quaternary oximes to the fourth class of Biopharmaceutical Classification Systems. Reliance upon oral absorption data for designing safe centrally acting oximes can be of potential value, with adequate characterization of uptake-influx transporters interplay.     

In vitro reactivation potency of some acetylcholinesterase reactivators against sarin- and cyclosarin-induced inhibitions

In our study, we have tested six acetylcholinesterase (AChE) reactivators (pralidoxime, obidoxime, HI-6, trimedoxime, BI-6 and Hl?-7) for reactivation of sarin- and cyclosarin-inhibited AChE using an in vitro reactivation test. We have used rat brain homogenate as the suitable source of enzyme. All oximes are able to reactivate sarin-inhibited AChE. On the other hand, only HI-6 is able to reactivate satisfactorily cyclosarin-inhibited AChE.     

Reactivation of organophosphate-inhibited human AChE by combinations of obidoxime and HI 6 in vitro

Highly toxic organophosphorus-type (OP) chemical warfare agents (nerve agents) and OP pesticides may be used by terrorists and during military conflicts emphasizing the necessity for the development of effective medical countermeasures. The standard treatment with atropine and acetylcholinesterase (AChE) reactivators (oximes) is considered to be ineffective with certain nerve agents due to low oxime efficacy. Despite research over decades none of the oximes has turned out to be a broad spectrum reactivator to cover the whole range of potential threat agents. The prospective oxime HI 6 is a weak reactivator of tabun- and pesticide-inhibited AChE, while the established oxime obidoxime mainly lacks efficacy with cyclosarin-inhibited enzyme. In order to investigate the feasibility of combining obidoxime and HI 6, human AChE inhibited by sarin, cyclosarin, VX, tabun and paraoxon was reactivated by these oximes either alone or in combination. Two major findings of this study were that a combination of HI 6 and obidoxime did not impair reactivation, compared with HI 6 or obidoxime alone, but broadened the spectrum compared with the individual oximes. By using different oxime concentrations a combination of oxime doses may be suggested which could be an alternative to individual obidoxime or HI 6 autoinjectors.     

The evaluation of the reactivating and therapeutic efficacy of two novel oximes (K361 and K378) in comparison with the oxime K203 and trimedoxime in tabun-poisoned rats and mice

The potency of two newly developed oximes (K361 and K378) to reactivate tabun-inhibited cholinesterase and to reduce acute toxicity of tabun was compared with the oxime K203 and trimedoxime using in vivo methods. The study determining percentage of reactivation of tabun-inhibited diaphragm cholinesterase in poisoned rats showed that the reactivating efficacy of the oxime K378 is slightly lower than the reactivating potency of the oxime K203 and trimedoxime while the ability of the oxime K361 to reactivate tabun-inhibited cholinesterase is markedly lower compared with the oxime K203 and trimedoxime. In the brain, the potency of both newly developed oximes to reactivate tabun-inhibited cholinesterase was negligible. The therapeutic efficacy of both newly developed oximes roughly corresponds to their weak reactivating efficacy. Their potency to reduce acute toxicity of tabun was significantly lower compared with the oxime K203 as well as trimedoxime. In conclusion, the reactivating and therapeutic potency of both newly developed oximes does not prevail the effectiveness of the oxime K203 and trimedoxime and, therefore, they are not suitable for their replacement of commonly used oximes for the treatment of acute tabun poisoning.     

Bisquaternary oximes as reactivators of tabun-inhibited human brain cholinesterases: an in vitro study

Intoxications caused by tabun nerve agent are generally very hard to treat by convential acetylcholinesterase (AChE) reactivators. Due to this, new AChE reactivators are still developed. In this study, we have tested three new promising bisquaternary AChE reactivators: K027, K033 and K048. These reactivators were previously tested on rat brain homogenate. To mimic reality, we studied the potency of these new oximes to reactivate tabun-inhibited human brain cholinesterases. As is evident from the results, reactivator K048 (reactivation 40%) surpassed all reactivators tested in this study [including the most promising ones, namely trimedoxime (37%) and obidoxime (33%)]. Moreover, if compared to our previous results from rat brain studies, species differences were demonstrated.     

Effect of metoclopramide and ranitidine on the inhibition of human AChE by VX in vitro

The repeated misuse of highly toxic organophosphorus-type (OP) chemical warfare agents ('nerve agents') emphasizes the necessity for the development of effective medical countermeasures. The standard treatment with atropine and acetylcholinesterase (AChE) reactivators ('oximes') is considered to be ineffective with certain nerve agents due to low oxime efficacy. Therefore, pretreatment with carbamate-type compounds, e.g. pyridostigmine, was recommended to improve antidotal efficacy. Recently, the clinically used reversible AChE inhibitors metoclopramide (MCP) and ranitidine (RAN) were shown to exhibit some protective effect against the OP pesticide paraoxon in vitro and in vivo. The present study was undertaken to investigate a potential protective effect of MCP and RAN against inhibition of human AChE by the nerve agent VX (O-ethyl S-[2-(diisopropylamino)ethyl)methylphosphonothioate). Hemoglobin-free human erythrocyte membranes were incubated with various, human relevant MCP (0.5-2 microm) and RAN (0.5-5 microm) concentrations starting 1 min before addition of VX (1-40 nm). Both compounds failed to increase VX IC(50) values. In addition, human AChE was incubated with higher than human relevant therapeutic concentrations of MCP (1 microm-1 mm) and RAN (1 microm-2.0 mm) and inhibited by 40 nm VX. At concentrations higher than 100 microm MCP and RAN caused a concentration dependent increase of residual AChE activity 15 min after addition of VX. These data indicate that MCP and RAN may be ineffective in protecting human AChE against inhibition by the nerve agent VX at human relevant doses.     

可通过血脑屏障的乙酰胆碱酯酶重活化剂的研究进展

神经性毒剂和有机磷农药是乙酰胆碱酯酶的不可逆抑制剂,对人类生命安全构成重大威胁。目前可用于临床预防和治疗的药物大都为季铵型乙酰胆碱酯酶重活化剂,对外周组织和血液中乙酰胆碱酯酶中毒具有较好的活化作用,但季铵盐结构限制了血脑屏障通过率,使这类重活化剂在中枢神经系统发挥作用有限。因此,开发可通过血脑屏障的重活化剂是当前主要发展趋势。本文对可通过血脑屏障的重活化剂的研究进展进行了综述。     

1,3-Bispyridinium-dimethylether mono- and dioximes: synthesis,reactivating potency and therapeutic effect in experimental poisoning by organophosphorus compounds

Nine bispyridinium oximes containing two pyridinium rings linked by dimethylether were synthesised. Each compound had on one of the pyridinium rings a hydroxyiminomethyl group in position 2 or 4, while the other ring was unsubstituted or had a methyl or a hydroxyiminomethyl group in position 2 or 4. The reactivating potency and therapeutic effect of the oximes were tested on two organophosphates: O,O-dimethyl-2,2-dichlorovinylphosphate (DDVP) and O-ethyl-S-(2-diisopropylaminoethyl)-methylphosphonothioate (VX). The reactivation was measured on human erythrocyte acetylcholinesterase and the therapeutic effect was evaluated on male albino rats. The oximes with a hydroxyiminomethyl group in position 4 in the pyridinium ring were good reactivators of both phosphorylated and phosphonylated acetylcholinesterase. They were also very effective given together with atropine against VX and DDVP poisoning. The compounds are almost as effective as PAM-2, but PAM-2 is less toxic.     

Reactivation of immobilized acetylcholinesterasetabun complex by pralidoxime,its isomers,and homologs

Reactivation efficacy of three homologous and three isomeric series of pralidoxime-type reactivators with aldoxime group in position 2, 3 and 4 of the heterocycle was tested in reactivation of tabun-inhibited AChE. The experiments were performed with immobilized and stabilized porcine brain AChE. The enzyme acticity was measured by Ellman method. Reactivation efficacy was determined by measurement of indicator fabric coloration intensity as a measure of AChE activity. Of the studied group of nine reactivators, isomers with the functional group in position 2 were the most effective. The highest value (30 %) for reactivation of inhibited AChE was found for 2PAE after treatment for 15?min at concentration 0.5?mg/cm³. The efficacy of the isomers decreased in the order ortho > para > meta. No marked effect on the efficacy of the reactivators was observed on prolongation of the reactivation time. The reactivators efficacy decreased with decreasing concentration of their solutions.     

Cytotoxicity of acetylcholinesterase reactivators evaluated in vitro and its relation to their structure

The development of acetylcholinesterase reactivators, i.e., antidotes against organophosphorus poisoning, is an important goal of defense research. The aim of this study was to compare cytotoxicity and chemical structure of five currently available oximes (pralidoxime, trimedoxime, obidoxime, methoxime, and asoxime) together with four perspective oximes from K-series (K027, K074, K075, and K203). The cytotoxicity of tested substances was measured using two methods – colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and impedance based real-time cytotoxicity assay – in three different cell lines (HepG2, ACHN, and NHLF). Toxicity was subsequently expressed as toxicological index IC₅₀. The tested compounds showed different cytotoxicity ranging from 0.92 to 40.06?mM. In HepG2 cells, K027 was the least and asoxime was the most toxic reactivator. In ACHN and NHLF cell lines, trimedoxime was the compound with the lowest adverse effects, whereas the highest toxicity was found in methoxime-treated cells. The results show that at least five structural features affect the reactivators’ toxicity such as the number of oxime groups in the molecule, their position on pyridinium ring, the length of carbon linker, and the oxygen substitution or insertion of the double bond into the connection chain. Newly synthetized oximes with IC₅₀?≥?1?mM evaluated in this three cell lines model might appear suitable for further testing.     

Glycosylated-imidazole aldoximes as reactivators of pesticides inhibited AChE: Synthesis and in-vitro reactivation study

The present armamentarium of commercially available antidotes provides limited protection against the neurological effects of organophosphate exposure. Hence, there is an urgent need to design and develop molecules that can protect and reactivate inhibited-AChE in the central nervous system. Some natural compounds like glucose and certain amino acids (glutamate, the anion of glutamic acid) can easily cross the blood brain barrier although they are highly polar. Glucose is mainly transported by systems like glucose transporter protein type 1 (GLUT1). For this reason, a series of non-quaternary and quaternary glycosylated imidazolium oximes with different alkane linkers have been designed and synthesized. These compounds were evaluated for their in-vitro reactivation ability against pesticide (paraoxon-ethyl and paraoxon-methyl) inhibited-AChE and compared with standards antidote AChE reactivators pralidoxime and obidoxime. Several physicochemical properties including acid dissociation constant (pK_a), logP, logD, HBD and HBA, have also been assessed for reported compounds. Out of the synthesized compounds, three have exhibited comparable potency with a standard antidote (pralidoxime).     

Reversible and irreversible inhibition of rat brain muscarinic receptors is related to different substitutions on bisquaternary pyridinium oximes

The role of the functional substituents on the pyridinium ring of bisquaternary pyridinium compounds, mostly oximes, in exerting reversible and irreversible inhibition of binding of [³H]-N-methyl-4-piperidyl benzilate ([³H]-4NMPB) to rat brain stem muscarinic receptors was studied. The drugs tested, i.e. HGG-42, HGG-12, HGG-52, HI-6, obidoxim, SAD-128 and TMB-4, could reversibly inhibit binding of [³H]-4NMPB, with the highest potency (K_I=1.7–6 M) exhibited by analogs possessing hydrophobic substituents at position 3 or 4 of the pyridinium ring. Bisquaternary drugs possessing an oxime moiety at position 2, but not at position 4 of the pyridinium ring, could also induce about 30% reduction of maximal binding capacity (B_max) (loss of muscarinic receptors) in addition to their reversible effect. Thus the structural correlates of the reversible and the irreversible effects of these drugs are different.     

Effect of Toxogonin and P2S on the Toxicity of Carbamates and Organophosphorus Compounds

Abstract Toxogonin (80 mg/kg intraperitoneally) given 15 min. prior to the administration of organophosphorus insecticides dimethoate, malathion, parathion and azinphos-methyl, organophosphorus warfare agents soman and tabun, or carbamates physostigmine, pyridostigmine and aldicarb, reduced the toxicity in mice of these agents by increasing their LD50 dose 1.5-3 fold. The toxicity of the carbamate insecticide carbaryl, however, was significantly increased by toxogonin. Similar results were obtained for P2S (150 mg/kg intraperitoneally) with respect to the toxicity of dimethoate, soman and pyridostigmine, whereas no effect could be detected on the toxicity of tabun. Only a slight reduction in the toxicity of physostigmine was observed. The acetylcholinesterase activity in erythrocytes, cerebrum and diaphragm of surviving mice 20 hours after organophosphate intoxication was similar both in toxogonin and P2S treated animals and untreated animals.     

A patent review of butyrylcholinesterase inhibitors and reactivators 2010–2017

Introduction: Butyrylcholinesterase (BuChE) has obtained a renewed interest as therapeutic target in Alzheimer’s disease (AD), when changes in BuChE activity and expression along disease progression were highlighted as well as correlation between BuChE levels and cognitive function.

Areas covered: During the last eight years, fourteen patents on BuChE inhibitors (BuChEI) have been submitted. Only three of them relate to BuChE selective inhibitors, while four of them focus on multitarget inhibitors which address different key pathological factors other than BuChE. Two patents report on non-selective acetylcholinesterase (AChE)/BuChE inhibitors, while four patents deal with natural compounds and their derivatives. One patent relates to antitoxic agents to treat exposure to ChEI pesticides and nerve agents.

Expert opinion: Increasing evidence supports BuChE as a more beneficial target in moderate-to-severe forms of AD in comparison to the well-known AChE. However, hitting a single pathological target is likely not sufficient to halt the disease progression. Therefore, patented BuChE inhibitors with a multifunctional profile may open new therapeutic avenues, since the additional activities could reinforce the therapeutic effects. Unfortunately, in vivo studies are limited and key parameters, such as ADMET data, are missing. This lack of information makes difficult to forecast the development of patented BuChEIs into effective drug candidates.     

对硫磷与对氧磷抑制的人脑乙酰胆碱酯酶的老化及重活化

目的　了解对硫磷与对氧磷在试管内对人脑乙酰胆碱酯酶 (AChE)的抑制 ,膦酰化酶的老化速率及肟类药物的重活化效能有何差别。方法　微量羟胺比色法测定AChE活性。结果　对硫磷与对氧磷抑制人脑AChE 5 0 %活性的摩尔浓度的负对数值(pI50 )分别为 4 .10及 7.5 1,抑制人脑AChE 90 %活性的摩尔浓度的负对数值 (pI90 )值分别为 2 .5 5及6 .5 7。氯磷定、双复磷、双磷定和酰胺磷定 4种重活化剂对对硫磷与对氧磷抑制的人脑AChE的重活化作用有较大差异 ,等摩尔浓度条件下 ,其对对氧磷的重活化作用普遍好于对硫磷。且双肟类重活化剂作用普遍强于单肟类重活化剂 ;对硫磷与对氧磷的最佳重活化剂均为双复磷。对硫磷与对氧磷人脑AChE磷酰化酶的半老化时间 (t0 .5)分别为 14及 12h ,老化达到 99%的时间 (t0 .99)分别为 95及 81h。结论　对硫磷或对氧磷中毒时应尽早使用双复磷或双磷定。且在急性中毒症状控制后 ,仍须连续使用重活化剂 4d。     

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 © 2009 John Wiley & Sons, Ltd.     

In vitro P-glycoprotein activity does not completely explain in vivo efficacy of novel centrally effective oxime acetylcholinesterase reactivators

Novel-substituted phenoxyalkyl pyridinium oxime acetylcholinesterase (AChE) reactivators (US patent 9,227,937) that showed convincing evidence of penetration into the brains of intact rats were developed by our laboratories. The oximes separated into three groups based on their levels of brain AChE reactivation following exposure of rats to the sarin surrogate nitrophenyl isopropyl methylphosphonate (NIMP). P-glycoprotein (P-gp) is a major blood–brain barrier (BBB) transporter and requires ATP for efflux. To determine if P-gp affinity screening could be used to reduce animal use, we measured in vitro oxime-stimulated ATPase activity to see if the in vivo reactivation efficacies related to the oximes’ functions as P-gp substrates. High efficacy oximes were expected to be poor P-gp substrates, thus remaining in the brain longer. The high efficacy oximes (24–35% brain AChE reactivation) were worse P-gp substrates than the low efficacy oximes (0–7% brain AChE reactivation). However, the oxime group with medium in vivo reactivation of 10–17% were even worse P-gp substrates than the high efficacy group so their reactivation ability was not reflected by P-gp export. The results suggest that in vitro P-gp ATPase activity can remove the low efficacy oximes from in vivo testing, but is not sufficient to differentiate between the top two tiers.     

Emerging therapeutic potentials of dual‐acting MAO and AChE inhibitors in Alzheimer's and Parkinson's diseases

No drug has been approved to prevent neuronal cell loss in patients suffering from Parkinson's disease (PD) or Alzheimer's disease (AD); despite increased comprehension of the underlying molecular causes, therapies target cognitive functional improvement and motor fluctuation control. Drug design strategies that adopt the “one protein, one target” philosophy fail to address the multifactorial aetiologies of neurodegenerative disorders such as AD and PD optimally. On the contrary, restoring neurotransmitter levels by combined combinatorial inhibition of cholinesterases, monoamine oxidases, and adenosine A_2AA receptors, in conjunction with strategies to counter oxidative stress and beta‐amyloid plaque accumulation, would constitute a therapeutically robust, multitarget approach. This extensive review delineates the therapeutic advantages of combining dual‐acting molecules that inhibit monoamine oxidases and cholinesterases and/or adenosine A_2AA receptors, and describes the structure–activity relationships of compound classes that include, but are not limited to, alkaloids, coumarins, chalcones, donepezil–propargylamine conjugates, homoisoflavonoids, resveratrol analogs, hydrazones, and pyrazolines. In the wake of recent advances in network biology, in silico approaches, and omics, this review emphasizes the need to consider conceptually informed research strategies for drug discovery, in the context of the mounting burden posed by chronic neurodegenerative diseases with complex aetiologies and pathophysiologies involving multiple signalling pathways and numerous drug targets.