Oxidative stress status assessment of rats' brains injury following subacute exposure to K-oximes.

Oxidative stress status and morphological injuries in the brain of Wistar rats induced by repeated application of selected acetylcholinesterase reactivators - asoxime, obidoxime, K027, K048, K074, and K075 were evaluated. Each oxime in a dose of 0.1 of LD50/kg im was given 2x/week for 4 weeks. Markers of lipid peroxidation (malondialdehyde, MDA), and protein oxidation (advanced oxidation protein products, AOPP), as well as the activity of antioxidant enzymes (catalase, CAT, superoxide dismutase, SOD, glutathione reductase, GR, and glutathione peroxidase, GPx), were estimated in the brain tissue homogenates on day 35 of the study. Brain alterations were carefully quantified by semiquantitative grading scales - brain damage score (BDS). Oxidative stress parameters, MDA and AOPP were significantly highest in the asoxime-, obidoxime- and K075-treated groups (p < 0.001). The activity of SOD and CAT was significantly elevated in the obidoxime-, K048-, and K075-treated groups (p < 0.001). Besides, GR was markedly decreased in the obidoxime- and K074-treated groups (p < 0.01), while treatment with K048, K074 and K075 induced extremely high elevation in GPx levels (p < 0.001). In the same groups of rats, brain alterations associated with polymorphonuclear cell infiltrate were significantly more severe than those observed in animals receiving only asoxime or K027 (p < 0.001). The presented results confirmed that treatment with different oximes significantly improved the oxidative status and attenuated signs of inflammation in rats' brains. Presented results, together with our previously published data can help to predict likely adverse systemic toxic effects, and target organ systems, which are crucial for establishing risk categories, as well as in dose selection of K-oximes as drug candidates.

Human HepaRG liver spheroids: cold storage protocol and study on pyridinium oxime-induced hepatotoxicity in vitro.

Oximes play an essential role in the therapy of organophosphorus compound (OP) poisoning by reactivating inhibited acetylcholinesterase. Impairment of liver function was observed in OP poisoning and associated with obidoxime treatment by some reports. In this study human three-dimensional HepaRG spheroids were used as complex in vitro model to investigate oxime-induced liver toxicity. In this context, cold storage of liver spheroids at 4 °C in standard culture medium and in optimized tissue preservation solutions of up to 72 h was assessed. Cold storage in standard culture medium resulted in a complete loss of viability whereas an optimized tissue preservation solution preserved viability. Separately from that liver spheroids were exposed to the four oximes pralidoxime, obidoxime, HI-6, MMB-4 and cytotoxicity (effective concentration, EC50) was determined with an ATP-based assay at several time points. The release of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin secretion was measured in supernatants. The same parameters were assessed with diclofenac as positive hepatotoxic control and with the OP pesticides malathion and malaoxon alone or in the presence of obidoxime. All individual tested oximes and OP showed a low cytotoxicity with effective concentrations mostly >2,000 µM. In contrast, the exposure to malaoxon in the presence of 1,000 µM obidoxime resulted in a marked decrease of viability and an increased release of AST indicating risk of liver injury only if oxime antidotes are strongly overdosed.

Pro: Oximes should be used routinely in organophosphate poisoning.

In poisoning with organophosphorus compounds (OP), patients can only profit from the regeneration of acetylcholinesterase, when the poison load has dropped below a toxic level. Every measure that allows an increase of synaptic acetylcholinesterase (AChE) activity at the earliest is essential for timely termination of the cholinergic crisis. Only drug-induced reactivation allows fast restoration of the inhibited AChE. Obidoxime and pralidoxime have proved to be able to reactivate inhibited cholinesterase thereby saving life of poisoned animals. A plasma level of obidoxime or pralidoxime allowing reactivation in humans poisoned by OP can be adjusted. There is no doubt that obidoxime and pralidoxime are able to reactivate OP-inhibited AChE activity in poisoned patients, thereby increasing AChE activity and contributing substantially to terminate cholinergic crisis. Hence, a benefit may be expected when substantial reactivation is achieved. A test system allowing determination of red blood cell AChE activity, reactivatability, inhibitory equivalents and butyrylcholinesterase activity is available for relatively low cost. If any reactivation is possible while inhibiting equivalents are present, oxime therapy should be maintained. In particular, when balancing the benefit risk assessment, obidoxime or palidoxime should be given as soon as possible and as long as a substantial reactivation may be expected.

Brain-targeted delivery of obidoxime, using aptamer-modified liposomes, for detoxification of organophosphorus compounds.

Effective intracerebral delivery acetylcholinesterase (AChE) reactivator is key for the acute organophosphorus (OPs) poison treatment. However, the blood-brain barrier (BBB) restricts the transport of these drugs from blood into the brain. Herein, we developed transferrin receptor (TfR) aptamer-functionalized liposomes (Apt-LP) that could deliver AChE reactivator (obidoxime) across the BBB to act against paraoxon (POX) poisoning. The aptamer had strong affinity for TfR and was modified with 3'-inverted deoxythymidine (dT) to improve serum stability. The uptake of Apt-LP by bEnd.3 cells was significantly higher than that of non-targeting liposomes. The ability of Apt-LP to penetrate intact BBB was confirmed in in vitro BBB mice model and in vivo biodistribution studies. Treatment of POX-poisoned mice with Apt-LP-LuH-6 reactivated 18% of the brain AChE activity and prevented brain damage to some extent. Taken together, these results showed that Apt-LP may be used as a promising brain-targeted drug delivery system against OPs toxicity.

Pharmacokinetics and efficacy of atropine sulfate/obidoxime chloride co-formulation against VX in a guinea pig model.

Nerve agent exposure is generally treated by an antidote formulation composed of a muscarinic antagonist, atropine sulfate (ATR), and a reactivator of acetylcholinesterase (AChE) such as pralidoxime, obidoxime (OBI), methoxime, trimedoxime or HI-6 and an anticonvulsant. Organophosphates (OPs) irreversibly inhibit AChE, the enzyme responsible for termination of acetylcholine signal transduction. Inhibition of AChE leads to overstimulation of the central and peripheral nervous system with convulsive seizures, respiratory distress and death as result. The present study evaluated the efficacy and pharmacokinetics (PK) of ATR/OBI following exposure to two different VX dose levels. The PK of ATR and OBI administered either as a single drug, combined treatment but separately injected, or administered as the ATR/OBI co-formulation, was determined in plasma of naïve guinea pigs and found to be similar for all formulations. Following subcutaneous VX exposure, ATR/OBI-treated animals showed significant improvement in survival rate and progression of clinical signs compared to untreated animals. Moreover, AChE activity after VX exposure in both blood and brain tissue was significantly higher in ATR/OBI-treated animals compared to vehicle-treated control. In conclusion, ATR/OBI has been proven to be efficacious against exposure to VX and there were no PK interactions between ATR and OBI when administered as a co-formulation.

Cholinergic syndrome: a case report of acute organophosphate and carbamate poisoning.

Cholinergic syndrome is a common topic at western medical universities yet rarely observed in clinical practice. The treatment involves muscarinic antagonists, acetylcholinesterase reactivation, seizure control, and supportive measures. Here we report a case of a 52-year old Caucasian male who attempted suicide by ingesting a purple crystal powder that turned out to be a mixture of carbofuran and chlormephos. At clinical examination, the patient presented with salivation, perspiration, diarrhoea, bradypnoea, loss of consciousness, and epileptic seizures. Laboratory tests showed low plasma cholinesterase, and we started obidoxime along with supportive intensive care treatment. He was later transferred to the psychiatry department for further diagnostics and treatment.

In Vitro Interaction of Organophosphono- and Organophosphorothioates with Human Acetylcholinesterase.

The implementation of the Chemical Weapons Convention (CWC) in 1997 was a milestone in the prohibition of chemical warfare agents (CWA). Yet, the repeated use of CWA underlines the ongoing threat to the population. Organophosphorus (OP) nerve agents still represent the most toxic CWA subgroup. Defensive research on nerve agents is mainly focused on the "classical five", namely tabun, sarin, soman, cyclosarin and VX, although Schedule 1 of the CWC covers an unforeseeable number of homologues. Likewise, an uncounted number of OP pesticides have been produced in previous decades. Our aim was to determine the in vitro inhibition kinetics of selected organophosphono- and organophosphorothioates with human AChE, as well as hydrolysis of the agents in human plasma and reactivation of inhibited AChE, in order to derive potential structure-activity relationships. The investigation of the interactions of selected OP compounds belonging to schedule 1 (V-agents) and schedule 2 (amiton) of the CWC with human AChE revealed distinct structural effects of the P-alkyl, P-O-alkyl and N,N-dialkyl residues on the inhibitory potency of the agents. Irrespective of structural modifications, all tested V-agents presented as highly potent AChE inhibitors. The high stability of the tested agents in human plasma will most likely result in long-lasting poisoning in vivo, having relevant consequences for the treatment regimen. In conclusion, the results of this study emphasize the need to investigate the biological effects of nerve agent analogues in order to assess the efficacy of available medical countermeasures.

A case report of cholinesterase inhibitor poisoning: cholinesterase activities and analytical methods for diagnosis and clinical decision making.

Suicidal ingestion of organophosphorus (OP) or carbamate (CM) compounds challenges health care systems worldwide, particularly in Southeast Asia. The diagnosis and treatment of OP or CM poisoning is traditionally based on the clinical appearance of the typical cholinergic toxidrome, e.g. miosis, salivation and bradycardia. Yet, clinical signs might be inconclusive or even misleading. A current case report highlights the importance of enzymatic assays to provide rapid information and support clinicians in diagnosis and rational clinical decision making. Furthermore, the differentiation between OP and CM poisoning seems important, as an oxime therapy will most probably not provide benefit in CM poisoning, but-as every pharmaceutical product-it might result in adverse effects. The early identification of the causing agent and the amount taken up in the body are helpful in planning of the therapeutic regimen including experimental strategies, e.g. the use of human blood products to facilitate scavenging of the toxic agent. Furthermore, the analysis of biotransformation products and antidote levels provides additional insights into the pathophysiology of OP or CM poisoning. In conclusion, cholinesterase activities and modern analytical methods help to provide a more effective treatment and a thorough understanding of individual cases of OP or CM poisoning.

A novel high-performance liquid chromatography with diode array detector method for the simultaneous quantification of the enzyme-reactivating oximes obidoxime, pralidoxime, and HI-6 in human plasma.

Oximes such as pralidoxime (2-PAM), obidoxime (Obi), and HI-6 are the only currently available therapeutic agents to reactivate inhibited acetylcholinesterase (AChE) in case of intoxications with organophosphorus (OP) compounds. However, each oxime has characteristic agent-dependent reactivating efficacy, and therefore the combined administration of complementary oximes might be a promising approach to improve therapy. Accordingly, a new high-performance liquid chromatography method with diode-array detection (HPLC-DAD) was developed and validated allowing for simultaneous or single quantification of 2-PAM, Obi, and HI-6 in human plasma. Plasma was precipitated using 5% w/v aqueous zinc sulfate solution and subsequently acetonitrile yielding high recoveries of 94.2%-101.0%. An Atlantis T3 column (150 × 2.1mm I.D., 3 µm) was used for chromatographic separation with a total run time of 15 min. Quantification was possible without interferences within a linear range from 0.12 to 120 µg/mL for all oximes. Excellent intra-day (accuracy 91.7%-98.6%, precision 0.5%-4.4%) and inter-day characteristics (accuracy 89.4%-97.4%, precision 0.4%-2.2%) as well as good ruggedness were found. Oximes in processed samples were stable for at least 12 h in the autosampler at 15°C as well as in human plasma for at least four freeze-thaw cycles. Finally, the method was applied to plasma samples of a clinical case of pesticide poisoning.

Oximes as pretreatment before acute exposure to paraoxon.

Organophosphates, useful agents as pesticides, also represent a serious danger due to their high acute toxicity. There is indication that oximes, when administered before organophosphate exposure, can protect from these toxic effects. We have tested at equitoxic dosage (25% of LD01 ) the prophylactic efficacy of five experimental (K-48, K-53, K-74, K-75, K-203) and two established oximes (pralidoxime and obidoxime) to protect from mortality induced by the organophosphate paraoxon. Mortalities were quantified by Cox analysis and compared with those observed after pretreatment with a strong acetylcholinesterase inhibitor (10-methylacridine) and after the FDA-approved pretreatment compound pyridostigmine. All nine tested substances statistically significantly reduced paraoxon-induced mortality. Best protection was conferred by the experimental oxime K-48, reducing the relative risk of death (RR) to 0.10, which was statistically significantly superior to pyridostigmine (RR = 0.31). The other oximes reduced the RR to 0.13 (obidoxime), 0.20 (K-203), 0.21 (K-74), 0.24 (K-75) and 0.26 (pralidoxime), which were significantly more efficacious than 10-methylacridine (RR = 0.65). These data support the hypothesis that protective efficacy is not primarily due to cholinesterase inhibition and indicate that the tested experimental oximes may be considered promising alternatives to the established pretreatment compound pyridostigmine.

Interspecies and intergender differences in acute toxicity of K-oximes drug candidates.

K-oximes were developed as modern drug candidates acting as AChE reactivators. In this study, it has been investigated which interspecies and intergender differences changes could be observed in Wistar rats and Swiss mice, both genders, after the treatment with increasing doses of selected acetylcholinesterase reactivators - asoxime, obidoxime, K027, K048, and K075. After the 24 h, a number of died animals was counted and the median lethal dose (LD50) for each oxime was calculated. By using the intramuscular route of administration, asoxime and K027 had the least toxicity in female rats (640.21 mg/kg and 686.08 mg/kg), and in female mice (565.75 mg/kg and 565.74 mg/kg), respectively. Moreover, asoxime and K027 showed 3, 4 or 8 times less acute toxicity in comparison to K048, obidoxime and K075, respectively. Beyond, K075 had the greatest toxicity in male rats (81.53 mg/kg), and in male mice (57.34 mg/kg), respectively. Our results can help to predict likely adverse toxic effects, target organ systems and possible outcome in the event of massive human overexposure, and in establishing risk categories or in dose selection for the initial repeated dose toxicity tests to be conducted for each oxime.

Molecular modeling studies on the interactions of 7-methoxytacrine-4-pyridinealdoxime, 4-PA, 2-PAM, and obidoxime with VX-inhibited human acetylcholinesterase: a near attack conformation approach.

7-methoxytacrine-4-pyridinealdoxime (7-MEOTA-4-PA, named hybrid 5C) is a compound formerly synthesized and evaluated in vitro, together with 4-pyridine aldoxime (4-PA) and commercial reactivators of acetylcholinesterase (AChE). This compound was designed with the purpose of being a prophylactic reactivator, capable of interacting with different subdomains of the active site of AChE. To investigate these interactions, theoretical results from docking were first compared with experimental data of hybrid 5C, 4-PA, and two commercial oximes, on the reactivation of human AChE (HssAChE) inhibited by VX. Then, further docking studies, molecular dynamics simulations, and molecular mechanics Poisson-Boltzmann surface area calculations, were carried out to investigate reactivation performances, considering the near attack conformation (NAC) approach, prior to the nucleophilic substitution mechanism. Our results helped to elucidate the interactions of such molecules with the different subdomains of the active site of HssAChE. Additionally, NAC poses of each oxime were suggested for further theoretical studies on the reactivation reaction.

Repetitive antidotal treatment is crucial in eliminating eye pathology, respiratory toxicity and death following whole-body VX vapor exposure in freely moving rats.

Exposure to the chemical warfare nerve agent VX is extremely toxic, causing severe cholinergic symptoms. If not appropriately treated, death ultimately ensues. Based on our previously described whole-body vapor exposure system, we characterized in detail the clinical outcome, including respiratory dynamics, typical of whole-body exposure to lethal doses of VX vapor in freely moving rats. We further evaluated the efficacy of two different antidotal regimens, one comprising a single and the other repeated administration of antidotes, in countering the toxic effects of the exposure. We show that a 15 min exposure to air VX concentrations of 2.34-2.42 mg/m3 induced a late (15-30 min) onset of obvious cholinergic signs, which exacerbated over time, albeit without convulsions. Marked eye pathology was observed, characterized by pupil constriction to pinpoint, excessive lacrimation with red tears (chromodacryorrhea) and corneal damage. Respiratory distress was also evident, characterized by a three-fourfold increase in Penh values, an estimate of lung resistance, and by lung and diaphragm histological damage. A single administration of TAB (the oxime TMB-4, atropine and the anticholinergic and antiglutamatergic benactyzine) at the onset of clinical signs afforded only limited protection (66% survival), with clinical deterioration including weight loss, chromodacryorrhea, corneal damage, increased airway resistance and late death. In contrast, a combined therapy of TAB at the onset of clinical signs and repeated administration of atropine and toxogonin (ATOX) every 3-5 h, a maximum of five i.m. injections, led to 100% survival and a prompt recovery, accompanied by neither the above-described signs of eye pathology, nor by bronchoconstriction and respiratory distress. The necessity of recurrent treatments for successful elimination of VX vapor toxicity strongly supports continuous penetration of VX following termination of VX vapor exposure, most likely from a VX reservoir formed in the skin due to the exposure. This, combined with the above-described eye and respiratory pathology and absence of convulsions, are unique features of whole-body VX vapor exposure as compared to whole-body vapor exposure to other nerve agents, and should accordingly be considered when devising optimal countermeasures and medical protocols for treatment of VX vapor exposure.

Oxime K203: a drug candidate for the treatment of tabun intoxication.

For over 60 years, researchers across the world have sought to deal with poisoning by nerve agents, the most toxic and lethal chemical weapons. To date, there is no efficient causal antidote with sufficient effect. Every trialed compound fails to fulfil one or more criteria (e.g. reactivation potency, broad reactivation profile). In this recent contribution, we focused our attention to one of the promising compounds, namely the bis-pyridinium reactivator K203. The oxime K203 is very often cited as the best reactivator against tabun poisoning. Herein, we provide all the available literature data in comprehensive and critical review to address whether K203 could be considered as a new drug candidate against organophosphorus poisoning with the stress on tabun. We describe its development from the historical point of view and review all available in vitro as well as in vivo data to date. K203 is easily accessible by a relatively simple two-step synthesis. It is well accommodated in the enzyme active gorge of acetylcholinesterase providing suitable interactions for reactivation, as shown by molecular docking simulations. According to a literature survey, in vitro data for tabun-inhibited AChE are extraordinary. However, in vivo efficiency remains unconvincing. The K203 toxicity profile did not show any perturbations compared to clinically used standards; on the other hand versatility of K203 does not exceed currently available oximes. In summary, K203 does not seem to address current issues associated with the organophosphorus poisoning, especially the broad profile against all nerve agents. However, its reviewed efficacy entitles K203 to be considered as a backup or tentative replacement for obidoxime and trimedoxime, currently only available anti-tabun drugs.

Efficacy of atropine sulfate/obidoxime chloride co-formulation against sarin exposure in guinea pigs.

The efficacy and pharmacokinetics of the aqueous co-formulation contents of the Trobigard™ (atropine sulfate, obidoxime chloride) auto-injector were evaluated in a sarin exposed guinea pig model. Two subcutaneous (sc) sarin challenge doses were evaluated in guinea pigs instrumented with brain and heart electrodes for electroencephalogram (EEG) and electrocardiogram (ECG). Sarin challenge doses were chosen to reflect exposure subclasses with sublethal (moderate to severe clinical signs) and lethal consequences. The level of protection of intramuscular human equivalent doses of the co-formulation was defined by (1) the mitigation of signs and symptoms at a sublethal level and (2) the increase of survival time at the supralethal sarin dose levels. Pharmacokinetics of both atropine sulfate and obidoxime were proportional at 1 and 3 human equivalent doses, and only a small increase in heart rate was observed briefly as a side effect. At both sarin challenge doses, 54 µg/kg and 84 µg/kg, the co-formulation treatment was effective against sarin-induced effects. Survival rates were improved at both sarin challenge levels, whereas clinical signs and changes in EEG activity could not in all cases be effectively mitigated, in particular at the supralethal sarin challenge dose level. Reactivation of sarin inhibited cholinesterase was observed in blood, and higher brain cholinesterase activity levels were associated with a better clinical condition of the co-formulation treated animals. Although the results cannot be directly extrapolated to the human situation, pharmacokinetics and the effects over time related to plasma levels of therapeutics in a freely moving guinea pig could aid translational models and possibly improve prediction of efficacy in humans.

Structure-Based Optimization of Nonquaternary Reactivators of Acetylcholinesterase Inhibited by Organophosphorus Nerve Agents.

Acetylcholinesterase (AChE), a key enzyme in the central and peripheral nervous systems, is the principal target of organophosphorus nerve agents. Quaternary oximes can regenerate AChE activity by displacing the phosphyl group of the nerve agent from the active site, but they are poorly distributed in the central nervous system. A promising reactivator based on tetrahydroacridine linked to a nonquaternary oxime is also an undesired submicromolar reversible inhibitor of AChE. X-ray structures and molecular docking indicate that structural modification of the tetrahydroacridine might decrease inhibition without affecting reactivation. The chlorinated derivative was synthesized and, in line with the prediction, displayed a 10-fold decrease in inhibition but no significant decrease in reactivation efficiency. X-ray structures with the derivative rationalize this outcome. We thus show that rational design based on structural studies permits the refinement of new-generation pyridine aldoxime reactivators that may be more effective in the treatment of nerve agent intoxication.

Some benefit from non-oximes MB408, MB442 and MB444 in combination with the oximes HI-6 or obidoxime and atropine in antidoting sarin or cyclosarin poisoned mice.

The effect of three newly developed bispyridinium non-oxime compounds (MB408, MB442, and MB444) on the therapeutic efficacy of a standard antidotal treatment (atropine in combination with the oxime HI-6 or obidoxime) of acute poisoning by two nerve agents (sarin and cyclosarin) in mice was studied. The therapeutic efficacy of atropine in combination with an oxime with or without one of the bispyridinium non-oximes was evaluated by determination of the 24 h LD50 values of the nerve agents studied and by measurement of the survival time after supralethal poisoning. Addition of all tested non-oximes increased the therapeutic efficacy of atropine in combination with an oxime against sarin poisoning; however, the differences were not significant. The non-oximes also positively influenced the number of surviving mice 6 h after supralethal poisoning with sarin. In the case of cyclosarin, they were also slightly beneficial in the treatment of acute poisoning. The higher dose of MB444 was able to significantly increase the therapeutic efficacy of standard antidotal treatment of poisoning with cyclosarin. The benefit of each bispyridinium non-oxime compound itself was obviously dose-dependent. In summary, the addition of MB compounds to the standard antidotal treatment of acute nerve agent poisoning was beneficial for the antidotal treatment of sarin or cyclosarin poisoning, although their benefit at 24 h after poisoning was not significant, with the exception of the higher dose of MB444 against cyclosarin.

Synthesis, Biological Evaluation, and Docking Studies of Novel Bisquaternary Aldoxime Reactivators on Acetylcholinesterase and Butyrylcholinesterase Inhibited by Paraoxon.

Nerve agents and oxon forms of organophosphorus pesticides act as strong irreversible inhibitors of two cholinesterases in the human body: acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8), and are therefore highly toxic compounds. For the recovery of inhibited AChE, antidotes from the group of pyridinium or bispyridinium aldoxime reactivators (pralidoxime, obidoxime, HI-6) are used in combination with anticholinergics and anticonvulsives. Therapeutic efficacy of reactivators (called "oximes") depends on their chemical structure and also the type of organophosphorus inhibitor. Three novel oximes (K131, K142, K153) with an oxime group in position four of the pyridinium ring were designed and then tested for their potency to reactivate human (Homo sapiens sapiens) AChE (HssACHE) and BChE (HssBChE) inhibited by the pesticide paraoxon (diethyl 4-nitrophenyl phosphate). According to the obtained results, none of the prepared oximes were able to satisfactorily reactivate paraoxon-inhibited cholinesterases. On the contrary, extraordinary activity of obidoxime in the case of paraoxon-inhibited HssAChE reactivation was confirmed. Additional docking studies pointed to possible explanations for these results.

Kinetic analysis of oxime-assisted reactivation of human, Guinea pig, and rat acetylcholinesterase inhibited by the organophosphorus pesticide metabolite phorate oxon (PHO).

Phorate is a highly toxic agricultural pesticide currently in use throughout the world. Like many other organophosphorus (OP) pesticides, the primary mechanism of the acute toxicity of phorate is acetylcholinesterase (AChE) inhibition mediated by its bioactivated oxon metabolite. AChE reactivation is a critical aspect in the treatment of acute OP intoxication. Unfortunately, very little is currently known about the capacity of various oximes to rescue phorate oxon (PHO)-inhibited AChE. To help fill this knowledge gap, we evaluated the kinetics of inhibition, reactivation, and aging of PHO using recombinant AChE derived from three species (rat, guinea pig and human) commonly utilized to study the toxicity of OP compounds and five oximes that are currently fielded (or have been deemed extremely promising) as anti-OP therapies by various nations around the globe: 2-PAM Cl, HI-6 DMS, obidoxime Cl2, MMB4-DMS, and HLö7 DMS. The inhibition rate constants (ki) for PHO were calculated for AChE derived from each species and found to be low (i.e., 4.8×103 to 1.4×104M-1min-1) compared to many other OPs. Obidoxime Cl2 was the most effective reactivator tested. The aging rate of PHO-inhibited AChE was very slow (limited aging was observed out to 48h) for all three species. CONCLUSIONS: (1) Obidoxime Cl2 was the most effective reactivator tested. (2) 2-PAM Cl, showed limited effectiveness in reactivating PHO-inhibited AChE, suggesting that it may have limited usefulness in the clinical management of acute PHO intoxication. (3) The therapeutic window for oxime administration following exposure to phorate (or PHO) is not limited by aging.

Effect of six oximes on acutely anticholinesterase inhibitor-induced oxidative stress in rat plasma and brain.

Beside the key inhibition of acetylcholinesterase (AChE), involvement of oxidative stress in organophosphate (OP)-induced toxicity has been supported by experimental and human studies. On the other hand, according to our best knowledge, possible antioxidant properties of oximes, the only causal antidotes to OP-inhibited AChE, have been examined only by a few studies. Thus, we have determined the effect of four conventional (obidoxime, trimedoxime, pralidoxime, asoxime) and two promising experimental oximes (K027, K203) on dichlorvos (DDVP)-induced oxidative changes in vivo. Wistar rats (5/group) were treated with oxime (5% LD50 i.m) immediately after DDVP challenge (75% LD50 s.c). Oxidative stress biomarkers were determined in plasma and brain 60 min after the treatment: prooxidative-superoxide anion (O2·-) and total oxidative status (TOS); antioxidative-superoxide dismutase (SOD), total thiol (SH) groups, total antioxidant status (TAS) and paraoxonase (PON1); tissue oxidative stress burden-prooxidative-antioxidative balance (PAB) and oxidative stress index (OSI); oxidative tissue damage-malondialdehyde (MDA) and advanced oxidation protein products (AOPP). All oximes were able to attenuate DDVP-induced oxidative stress in rat plasma and brain. Changes of determined parameters in brain were not as prominent as it was seen in plasma. Based on OSI, better abilities of oxime K027, K203 and obidoxime to maintain DDVP-induced oxidative stress in rat brain were shown as compared to trimedoxime, pralidoxime and asoxime. Oximes can influence the complex in vivo redox processes that might contribute to their overall therapeutic efficacy. Further research is needed to understand the underlying molecular mechanisms involved in this phenomenon.