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Oxime cholinesterase reactivators (oximes) are used to counteract organophosphate intoxication. Charged oximes are administered via intramuscular or intravenous injection when the majority of dose is unmetabolized and is excreted as urine. In this study, the effects of selected double charged oximes were determined in the HK-2 cell line as a model for renal toxicity screening. Some effects on dehydrogenase activity were found for obidoxime, asoxime (syn. HI-6), K027, and K203. The effects of K868 and K869 were found to be unreliable due to rapid degradation of both chlorinated oximes in the assay medium, resulting for K868 in an isoxazole-pyridinium product. Oximové reaktivátory cholinesterázy (oximy) se používají k potlačení intoxikace organofosfáty. Nabité oximy se podávají intramuskulární nebo intravenózní injekcí, dokud není většina z podané dávky nemetabolizována a je vylučována močí. V této studii byly účinky vybraných oximů s dvojitým nábojem stanoveny v buněčné linii HK-2 jako modelu pro screening renální toxicity. Některé účinky na aktivitu dehydrogenázy byly zjištěny u obidoximu, asoximu (syn. HI-6), K027 a K203. Bylo zjištěno, že účinky K868 a K869 jsou nespolehlivé v důsledku rychlé degradace obou chlorovaných oximů v testovacím médiu, což má za následek pro K868 isoxazol-pyridiniový produkt.

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.

Organophosphorus nerve agents (NA) inhibit acetylcholinesterase (AChE) which results in the over-stimulation of both the central and peripheral nervous systems, creating a toxic syndrome that can be lethal if left untreated (Cannard, 2006). It is standard practice to treat Sarin (GB) intoxication with an oxime, an antimuscarinic such as atropine and an anticonvulsant. Three common oximes are available: HI-6, Pralidoxime (2-PAM) and Obidoxime (Obi), all possess a nucleophile that can break the NA-AChE covalent bond. However, each oxime's efficacy profile against various agents is different (Thiermann and Worek, 2018). In an effort to broaden therapeutic efficacy against a range of possible NA's, consideration should be given to the use of two oximes in combination. Using a guinea pig model, the first arm of this study was to determine the pharmacokinetics (PK) of HI-6 DMS, 2-PAM chloride and Obi chloride (at autoinjector equivalent doses) following intramuscular (i.m.) co-administration along with atropine to replicate either a single isometrically scaled dose (referred to in this study as a single autoinjector equivalent) of 2-PAM (and equimolar doses of Obi and HI-6) or double doses (referred to in this study as two autoinjector equivalents). The second arm of the study evaluated the efficacy of Obi and 2-PAM individually at a single or double autoinjector dose and also in combination against GB exposure. Pharmacokinetic profiles of each oxime were evaluated for both arms of the study and no significant change in parameters were reported. Improved cholinesterase reactivation was observed in a dose dependent manner with combined therapy showing similar reactivation to individual oximes alone at a two autoinjector equivalent dose. Seizure activity was reduced when combined oxime therapy was administered. This improvement was also reflected in the Racine seizure index score assigned at the end of the experimental period. To the best of our knowledge, this study is the first to evaluate and compare the pharmacokinetics of three oximes and the combination of two oximes (2-PAM and Obi) administered in naïve animals or those exposed to GB. Combined oxime therapy (Obi and 2-PAM) resulted in improved seizure control, increased cholinesterase reactivation peripherally and centrally and improved behavioral signs (Racine score). This study provides evidence that combination of oximes is effective, does not result in adverse events and that the pharmacokinetics of each oxime are not affected when administered in combination.

A 63-year-old male was admitted to a district hospital after ingesting ethanol and pirimiphos-methyl (PM) with suicidal intentions. History included alcoholic cirrhosis with alcoholism, adiposity, diabetes with cerebral microangiopathy, chronic renal insufficiency, heparin-induced thrombocytopenia, and status post necrotizing fasciitis. Emergency medical service reported an alert patient without signs of cholinergic crisis; activated charcoal and atropine were administered. Upon hospital arrival, he received fluid resuscitation, activated charcoal, and atropine. He was transferred to a toxicology unit the next day. On admission, he had no cholinergic signs (dry mucous membranes, warm skin, and mydriatic pupils) requiring small atropine doses (0.5 mg per hour). Four hours after admission, he developed bradycardia and respiratory distress, necessitating intubation. He received atropine by continuous infusion for 7 days (248 mg total) and obidoxime (bolus and continuous infusion). PM, pirimiphos-methyl-oxon (PMO), and phosphorylated tyrosine (Tyr) adducts derived from human serum albumin were analyzed in vivo. Cholinesterase status (acetylcholinesterase (AChE), butyrylcholinesterase (BChE), inhibitory activity of patient plasma and reactivatability, and phosphorylated BChE-derived nonapeptides) was measured in vivo. Obidoxime and atropine were monitored. PM and PMO were detectable, PM with maximum concentration ∼24 h post admission (p.a.) and PMO at ∼18 h p.a. Tyr adducts were detectable. AChE in vivo was suppressed on admission, increased continuously after starting obidoxime, and reached maximum activity after ∼30 h. AChE in vivo and reactivatability remained at the same level until the end of monitoring. BChE was already suppressed on admission; termination of the antidote treatment was possible after BChE had recovered to 1/5th of its normal value and extubation was possible after BChE had recovered to 2/5th. While a substantial part of BChE was already aged on admission, aging continued peaking at ∼24 h p.a. After initiating obidoxime treatment, plasma levels increased until obidoxime plasma levels reached a steady state. On admission, plasma atropine level was low; it increased with the start of the continuous infusion. Afterward, the level dropped to a steady state. The clinical course was characterized by bouts of pneumonia, necessitating re-intubation and prolonged ventilation, sepsis, delirium, and a peripheral neuropathy. After psychiatric evaluation, the patient was discharged to a neurological rehabilitation facility after 77 days of hospital care.

Organophosphorus poisoning has significant clinical importance, especially in the developing countries, due to its major health consequences. The study evaluated role of obidoxime in reactivation of cholinesterase (ACHE, BuCHE) enzymes in acute organophosphorus (OP) intoxicated patients. It was conducted on group I: 50 patients received obidoxime therapy after acute OP intoxication, and group II: 50 patients did not receive obidoxime. Patients were subjected to neurological evaluation, determination of ACHE and BuCHE levels. Results revealed that there were statistically significant differences between both groups regarding clinical outcomes, with variability of significance regarding initial and final levels of cholinesterase enzymes. It was concluded that BuCHE enzymes' levels can be considered sensitive biomarker for OP exposure and severity, while, ACHE activity reflected the recovery.

The global use of organophosphorus compounds (OP) for pest control and nerve agents being used in military conflicts and for assassinations renders intoxications by these agents a public health concern. OP-poisoned patients often suffer from dysrhythmias which may ultimately result in death. In this study, human-induced pluripotent stem cells derived cardiomyocytes were exposed to OP compounds in a microelectrode array system (MEA). The MEA system is widely accepted to assess the proarrhythmic properties of (candidate) drugs. The directly acting cholinergic compounds acetylcholine and carbachol and the irreversible acetylcholinesterase inhibitor cyclosarin - a highly toxic nerve agent - were assessed. All three compounds induced a dose-dependent (up to 600 nmol/L) corrected field potential duration (FPDc) prolongation of 9.7 ± 0.6% for carbachol, for 9.7 ± 1.2% acetylcholine and 9.4 ± 0.5% for cyclosarin. Additionally, the electrophysiological alterations of the clinically approved oxime reactivators obidoxime, pralidoxime and the oximes in development HI-6 and MMB-4 were investigated in the absence of OP. Neither of these oximes (up to a concentration of 300 μmol/L) caused dysrhythmia nor beat arrest. The competitive muscarinic receptor antagonist atropine as a cornerstone in the treatment of OP poisoning was also analyzed. Interestingly, atropine caused a drop in the beat rate which might result from a non-receptor action of this substance in the absence of OP. Atropine in combination with the OP nerve agent cyclosarin and the direct cholinergics acetylcholine or carabachol completely reversed the induced FPDc prolongation. However, the oxime HI-6 as potent reactivator of cyclosarin-inhibited AChE was not able to prevent the FPDc prolongation in this model. In conclusion, the current model allows the assessment of FPDc prolongation by the nerve agent cyclosarin, the cholinergic compounds carbachol, acetylcholine and the block of this effect by atropine.

In previous experiments, human and rat small bowel samples have been successfully used to study the spasmolytic effect of (potential) therapeutics in carbamate-constricted bowel specimens. Additionally, transferability from rat to human data was shown in the previous study. In the present study, the effects of atropine, scopolamine, MB327, HI-6 as well as obidoxime were examined in organophosphorus-poisoned human small bowel specimens. All substances were tested with at least seven concentrations in samples previously exposed to the nerve agent sarin. Furthermore, the cholinesterase reactivation potential of all substances was investigated. The test substances displayed a spasmolytic effect allowing the calculation of dose-response curves and EC50s. The parasympatholytic compound scopolamine had the strongest relaxing effect (EC50 = 0.05 μM) followed by atropine (EC50 = 0.07 μM). HI-6 and obidoxime were capable to reactivate the sarin-inhibited cholinesterase activity in small bowel samples. Both substances restored AChE activity in a dose-dependent way, with HI-6 being more potent (HI-6 EC50 = 3.8 μM vs obidoxime EC50 = 197.8 μM). Summarizing, our isolated human small bowel setup is a suitable tool to investigate the smooth muscle relaxing effect of (candidate) therapeutics for organophosphorus compound poisoning i.e. sarin exposure in a complex 3D tissue model.

Background: One of the most toxic effects of organophosphorus poisoning (OP) is the paralysis of skeletal muscles. The oximes are a group of available antidotes. This study investigated the effects of different concentrations of paraoxon on the function of skeletal muscle and reversal or prevention of these effects by three different oximes (i.e., pralidoxime, obidoxime, and HI-6). Materials and Methods: This study was conducted based on the chicken biventer cervices (CBC) nerve-muscle preparation and the use of twitch tension recording technique. The twitches of the CBC were evoked by stimulating the motor nerve at 0.1 Hz with pulses of 0.2 msec duration and a voltage greater than that required to produce the maximum response. Moreover, twitches and contractures were recorded isotonically using Grass Biosystems. Results: Paraoxon at 0.1 µM induced a significant increase (more than 100%) in the twitch amplitude, while higher concentrations (0.3 and 1µM) induced partial or total contracture. Therefore, paraoxon at a concentration of 0.1 µ M was used to examine the capability of oximes to prevent or reverse its effects. Pralidoxime, obidoxime, and HI-6 dose-dependently prevented (when it was used as pre-treatment, 20 min before or at the same time of administration of the toxin) and reversed (when it was used as post-treatment, 20 min after the administration of the toxin) the effect of paraoxon. Conclusion: In conclusion, these results revealed that oximes were very useful in the prevention and reversal of the OP toxic effects on the skeletal muscle. Moreover, it was suggested that oximes were more effective when used as pre-treatment. Pralidoxime was more potent than obidoxime and HI-6. The HI-6, which is a newer oxime, was unexpectedly less effective than the other two.

Organophosphorus compounds (OPs) include nerve agents and insecticides that potently inhibit acetylcholinesterase (AChE), an essential enzyme found throughout the nervous system. High exposure levels to OPs lead to seizures, cardiac arrest, and death if left untreated. Oximes are a critical piece to the therapeutic regimen which remove the OP from the inhibited AChE and restore normal cholinergic function. The current oximes 2-PAM, MMB-4, TMB-4, HI-6, and obidoxime (OBD) have two drawbacks: lack of broad spectrum protection against multiple OP structures and poor brain penetration to protect against OP central neurotoxicity. An alternative strategy to enhance therapy is reactivation of serum butyrylcholinesterase (BChE). BChE is stoichiometrically inhibited by OPs with no apparent toxic result. Inhibition of BChE in the serum followed by reactivation could create a pseudo-catalytic scavenger allowing numerous regenerations of BChE to detoxify circulating OP molecules before they can reach target AChE. BChE in serum from rats, guinea pigs or humans was screened for the reactivation potential of our novel substituted phenoxyalkyl pyridinium oximes, plus 2-PAM, MMB-4, TMB-4, HI-6, and OBD (100μM) in vitro after inhibition by highly relevant surrogates of sarin, VX, and cyclosarin, and also DFP, and the insecticidal active metabolites paraoxon, phorate-oxon, and phorate-oxon sulfoxide. Novel oxime 15 demonstrated significant broad spectrum reactivation of OP-inhibited rat serum BChE while novel oxime 20 demonstrated significant broad spectrum reactivation of OP-inhibited human serum BChE. All tested oximes were poor reactivators of OP-inhibited guinea pig serum BChE. The bis-pyridinium oximes were poor BChE reactivators overall. BChE reactivation may be an additional mechanism to attenuate OP toxicity and contribute to therapeutic efficacy.

Therapeutic application of newly developed oximes is limited due to their adverse effects on different tissues. Within this article, it has been investigated which morphological changes could be observed in Wistar rats after the treatment with increasing doses of selected acetyl cholinesterase reactivators - asoxime, obidoxime, K027, K048, and K075. Subsequently, heart, diaphragm and musculus popliteus were obtained for pathohistological and semiquantitative analysis 24 hrs and 7 days after im administration of a single dose of 0.1 LD50, 0.5 LD50, and 1.0 LD50 of each oxime. Different muscle damage score was based on an estimation scale from 0 (no damage) to 5 (strong damage). In rats treated with 0.1 LD50 of each oxime, muscle fibres did not show any change. The intensive degeneration was found in all muscles after treatment with 0.5 LD50 of asoxime and obidoxime, respectively. Acute toxic muscle injury was developed within 7 days following treatment with 0.5 LD50 and 1.0 LD50 of each oxime, with the highest values in K048 and K075 group (P

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 (pKa), 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).

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. © 2018 Elsevier B.V.

The primary toxic mechanism of organophosphorus compounds, i.e. nerve agents or pesticides, is based on the irreversible inhibition of acetylcholinesterase. In consequence of the impaired hydrolysis, the neurotransmitter acetylcholine accumulates in cholinergic synapses and disturbs functional activity of nicotinic and muscarinic acetylcholine receptors by overstimulation and subsequent desensitization. The resulting cholinergic syndrome will become acute life-threatening, if not treated adequately. The current standard treatment, consisting of administration of a competitive mAChR antagonist (e.g. atropine) and an oxime (e.g. obidoxime, pralidoxime), is not sufficient in the case of soman or tabun intoxications. Consequently, alternative therapeutic options are necessary. An innovative approach comprises the use of compounds selectively targeting nAChRs, especially positive allosteric modulators, which increase the population of the conducting receptor state. MB327 (1,1′-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)) is able to restore soman-blocked muscle-force in preparations of various species including human and was recently identified as “resensitizer”. In contrast to the well-studied MB327, the pharmacological efficacy of the 2- and 3-tert-butylpyridinium propane regioisomers is unknown. As a first step, MB327 and its 3-regioisomer (PTM0001) and 2-regioisomer (PTM0002) were pharmacologically characterized using [3H]epibatidine binding assays, functional studies by solid supported membranes based electrophysiology, and in vitro muscle-force investigations of soman-poisoned rat hemidiaphragm preparations by indirect field stimulation technique. The results obtained from targets of different complexity (receptor, muscle tissue) showed that the pharmacological profiles of the 2- and 3-regioisomers were relatively similar to those of MB327. Furthermore, high concentrations showed inhibitory effects, which might critically influence the application as an antidote. Thus, more effective drugs have to be developed. Nevertheless, the combination of the methods presented is an effective tool for clarifying structure-activity relationships.

Organophosphorus compounds (OPCs), inhibitors of acetylcholinesterase (AChE), are useful agents as pesticides, but also represent a serious health hazard. Standard therapy with atropine and established oxime-type enzyme reactivators (pralidoxime, obidoxime) is unsatisfactory. Better therapeutic results are obtained, when reversible AChE inhibitors are administered before OPC exposure. This review summarizes the history of such a pretreatment approach and sums up a set of experiments undertaken in search of compounds that are efficacious when given before a broad range of OPCs. The prophylactic efficacy of 10 known AChE inhibitors, either already used clinically for different indications (physostigmine, pyridostigmine, ranitidine, tiapride, tacrine, amiloride, metoclopramide, methylene blue) or developed for possible therapeutic use in the future (7-methoxytacrine, K-27) was compared, when administered before exposure to six chemically diverse OPCs in the same experimental setting: ethyl-paraoxon, methyl-paraoxon, diisopropylfluorophosphate, terbufos sulfone, azinphos-methyl and dicrotophos. The experimental oxime K-27 was the most efficacious compound, affording best protection, when administered before terbufos sulfone, azinphos-methyl and dicrotophos, second best before ethyl- and methyl-paraoxon exposure and third best before diisopropylfluorophosphate administration. This ranking was similar to that of physostigmine, which was superior to the Food and Drug Administration-approved pretreatment for soman with pyridostigmine. Tiapride, amiloride, metoclopramide, methylene blue and 7-methoxytacrine did not achieve protection. No correlation was observed between the IC50 of the reversible AChE inhibitors and their protective efficacy. These studies indicate that K-27 can be considered a very promising broad-spectrum prophylactic agent in case of imminent organophosphate exposure, which may be related to its AChE reactivating activity rather than its AChE inhibition.

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.

Background Based on in vitro and in vivo rat experiments, the newly developed acetylcholinesterase (AChE) reactivator, K203, appears to be much more effective in the treatment of tabun poisonings than currently fielded oximes. Methods To determine if this reactivating efficacy would extend to humans, studies were conducted in vitro using human brain homogenate as the source of AChE. The efficacy of K203 was compared with commercially available oximes; pralidoxime, obidoxime and asoxime (HI-6). Results Reactivation studies showed that K203 was the most effective reactivator with a second order kinetic constant (k r) of 2142 min− 1. M− 1, which was 51 times higher than that obtained for obidoxime (k r = 42 min− 1. M− 1). Both pralidoxime and asoxime (HI-6) failed to significantly reactivate tabun-inhibited human AChE. Discussion According to these results and previous studies, using K203, it appears that oxime K203 is the most effective reactivator of tabun-inhibited cholinesterase in several species including humans and should be considered as a possible medical countermeasure to tabun exposure.

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 Cl 2 , MMB4-DMS, and HLo7 DMS. The inhibition rate constants ( k i ) for PHO were calculated for AChE derived from each species and found to be low (i.e., 4.8 × 10 3 to 1.4 × 10 4 M − 1 min − 1 ) compared to many other OPs. Obidoxime Cl 2 was the most effective reactivator tested. The aging rate of PHO-inhibited AChE was very slow (limited aging was observed out to 48 h) for all three species. Conclusions: (1) Obidoxime Cl 2 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.

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 (O-2 (center dot-)) 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.

Organophosphate (OP) toxic compounds remain a great threat for humans because they are used as pesticides or misused as chemical warfare nerve agents. Their mechanism of toxicity involves the irreversible inhibition of the acetylcholinesterase (AChE) enzyme important in the control of cholinergic neurotransmission at the periphery and in the brain. An available pharmacological treatment are reactivators of OP-inhibited AChE that are not equally effective for every possible OP and they cross the blood-brain barrier (BBB) poorly. Novel oximes are being designed and synthesized at a high rate and scale and their pharmacological efficiency is being addressed mostly with the in vitro reactivation assay. Nevertheless, none of the newly synthesized oximes have shown better pharmacological properties than the ones developed more than 65 years ago since their potential to act as efficient antidotes in vivo depends on their pharmacokinetic and neuropharmacokinetics profiles. This paper provides an overview of all the important aspects that should be accounted for in the search for a centrally active oxime. Furthermore, it lists the most important BBB oxime delivery strategies employed until now, and the available pharmacokinetic data on old and new oximes. It should, therefore, serve as a guideline for the future development and evaluation of novel antidotes for OP poisoning.

Nerve agents (NA) are chemical warfare munitions and their exposure causes a progressive inhibition of acetylcholinesterase (AChE). This inhibition causes NA-induced brain damage in central nervous system (CNS). Oximes reactivate AChE in both the peripheral nervous system and the CNS. Transport of the oxime across the blood–brain barrier (BBB) in the existed therapeutic concentrations at the brain parenchyma determines the effectiveness of antidote therapy on respiratory depression and NA-induced brain damage. However, oximes could not cross the BBB in therapeutic concentrations. The aim of this study was to load AChE reactivator obidoxime chloride to PLGA and PEG-b-PLGA nanoparticles and to improve the BBB transport of the molecule. Brain microvascular endothelial cells were used as the BBB model. 79.3 ± 4.2% of obidoxime was released from PLGA nanoparticles and 88.2 ± 4.4% of obidoxime was released from PEG-b-PLGA nanoparticles within 24 h. It was found that PEG-b-PLGA nanoparticles were ideal drug carrier because of its low tissue toxicity, few side effects, and controllable drug release profile. Transport efficiency of obidoxime across the BBB is a major challenge in the prevention of the CNS, the effectiveness of NA poisoning and new strategies like using obidoxime-loaded PEG-b-PLGA nanoparticles could overcome this challenge for the management of NA-induced brain damage.

Prophylactic antidotes are needed primarily in the case of soman poisoning, since its complex with acetylcholinesterase (AChE) ages very quickly and becomes resistant to the posttherapeutic administration of AChE reactivators. Among the many compounds tried as prophylactic antidotes, reversible AChE inhibitors (physostigmine, pyridostigmine, galantamine and huperzine A), noncompetitive antagonists of NMDA/AMPA receptors for excitatory amino acid glutamine (memantine, dizocilpine, procyclidine, ketamine), transdermal patches with a combination of physostigmine and procyclidine or patches with asoxime and stoichiometric and catalytic scavengers of nerve agents gave best results. Postexposure treatment of nerve agent intoxication consists of a triple regimen: anticholinergics, AChE reactivators, and anticonvulsants. Antimuscarinics, mainly atropine, but also more liposoluble scopolamine, oximes pralidoxime (2-PAM, P2S), trimedoxime (TMB-4), obidoxime LuH-6, asoxime (HI-6), HLo-7 and, most recently, K-oximes and benzodiazepines, diazepam, midazolam, and clonazepam, are discussed. Special attention is paid to glutamate receptor antagonists, memantine, ketamine, and procyclidine, as anticonvulsants that prevent the perpetuation of seizures following poisoning with nerve agents.

Poisoning with organophosphorus compounds (OPCs) is a major problem worldwide. Standard therapy with atropine and established oxime-type enzyme reactivators (pralidoxime, obidoxime) is unsatisfactory. In search of more efficacious broad-spectrum oximes, new bispyridinium (K-) oximes have been synthesized, with K027 being among the most promising. This review summarizes pharmacokinetic characteristics of K027, its toxicity and in vivo efficacy to protect from OPC toxicity and compares this oxime with another experimental bisquaternary asymmetric pyridinium aldoxime (K048) and two established oximes (pralidoxime, obidoxime). After intramuscular (i.m.) injection, K027 reaches maximum plasma concentration within ∼30 min; only ∼2% enter the brain. Its intrinsic cholinesterase inhibitory activity is low, making it relatively non-toxic. In vitro reactivation potency is high for ethyl-paraoxon-, methyl-paraoxon-, dichlorvos-, diisopropylfluorophosphate (DFP)- and tabun-inhibited cholinesterase. When administered in vivo after exposure to the same OPCs, K027 is comparable or more efficacious than pralidoxime and obidoxime. When given as a pretreatment before exposure to ethyl-paraoxon, methyl-paraoxon, DFP, or azinphos-methyl, it is superior to the Food and Drug Administration-approved compound pyridostigmine and comparable to physostigmine, which because of its entry into the brain may cause unwanted behavioral effects. Because of its low toxicity, K027 can be given in high dosages, making it a very efficacious oxime not only for postexposure treatment but also for prophylactic administration, especially when brain penetration is undesirable.

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.

The available antidotal therapy against acute poisoning by organophosphates involves the use of atropine alone or in combination with one of the oximes, e.g. 2-PAM, Obidoxime, TMB-4 or HI-6. Each of these oximes has some limitation, raising the question of the universal antidotal efficacy against poisoning by all OPs/nerve agents. In the present study, newly synthesized bis quaternary 2-(hydroxyimino)-N-(pyridin-3yl) acetamide derivatives (HNK-series) oximes were evaluated for their antidotal efficacy against DDVP intoxicated Swiss mice, in terms of the Protection Index (PI) and AChE reactivation in brain and serum. The inhibition concentration (IC50) was determined in brain and serum after optimizing the time point for maximum inhibition (60 min post DDVP exposure). AChE reactivation efficacy of the HNK series was evaluated at IC50 and compared with 2-PAM. HNK-102 showed a ~2 times better Protection Index (PI) as compared to 2-PAM against DDVP toxicity. IC50 at 60 min DDVP post exposure was found to be approximately one fifth and one half of the LD50 dose for brain and serum AChE, respectively. Out of three HNK oximes, HNK-102 & 106 at 0.20 LD50 dose significantly reactivated DDVP intoxicated brain AChE (p50 dose of DDVP. In light of double PI and higher AChE reactivation, HNK 102 was found to be a better oxime than 2-PAM in the treatment of acute poisoning by DDVP.

As oxime-based structures are the only causal antidotes to organophosphate (OP)-inhibited acetylcholinesterase (AChE), the majority of studies on these have been directed towards their synthesis and testing. In this study, experimental bispyridinium oximes K027 and K203, which have shown promising results in the last decade of research, were examined in vivo for their therapeutic and reactivating ability in acute poisoning by the direct AChE-inhibitor dichlorvos (DDVP), used as a dimethyl OP structural model. Additionally, the efficacy of oximes K027 and K203 was compared with the efficacy of four oximes (pralidoxime, trimedoxime, obidoxime and HI-6), already used in efficacy experiments and human medicine. To evaluate therapeutic efficacy, groups of Wistar rats were treated with equitoxic doses of oximes (5% LD50, i.m.) and/or atropine (10 mg/kg, i.m.) immediately after s.c. DDVP challenge (4-6 doses). Using the same antidotal protocol, AChE activity was measured in erythrocytes, diaphragm and brain 60 min after s.c. DDVP exposure (75% LD50). The oxime K027 was the most efficacious in reducing the DDVP induced lethal effect in rats, while the oxime K203 was more efficacious than trimedoxime, pralidoxime and HI-6. Significant reactivation of DDVP inhibited AChE was achieved only with oxime K027 or its combination with atropine in erythocytes and the diaphragm. Moreover, the acute i.m. toxicity of oxime K027 in rats was lower than all other tested oximes. The results of this study support previous studies considering the oxime K027 as a promising experimental oxime structure for further testing against structurally-different OP compounds.

Poisoning with organophosphorus compounds (OPCs) represents an ongoing threat to civilians and rescue personal. We have previously shown that oximes, when administered prophylactically before exposure to the OPC paraoxon, are able to protect from its toxic effects. In the present study, we have assessed to what degree experimental (K-27, K-48, K-53, K-74, K-75) or established oximes (pralidoxime, obidoxime), when given as pretreatment at an equitoxic dosage of 25% of LD01, are able to reduce mortality induced by the OPC azinphos-methyl. Their efficacy was compared with that of pyridostigmine, the only FDA-approved substance for such prophylaxis. Efficacy was quantified in rats by Cox analysis, calculating the relative risk of death (RR), with RR=1 for the reference group given only azinphos-methyl, but no prophylaxis. All tested compounds significantly (p ≤ 0.05) reduced azinphos-methyl-induced mortality. In addition, the efficacy of all tested experimental and established oximes except K-53 was significantly superior to the FDA-approved compound pyridostigmine. Best protection was observed for the oximes K-48 (RR = 0.20), K-27 (RR = 0.23), and obidoxime (RR = 0.21), which were significantly more efficacious than pralidoxime and pyridostigmine. The second-best group of prophylactic compounds consisted of K-74 (RR = 0.26), K-75 (RR = 0.35) and pralidoxime (RR = 0.37), which were significantly more efficacious than pyridostigmine. Pretreatment with K-53 (RR = 0.37) and pyridostigmine (RR = 0.52) was the least efficacious. Our present data, together with previous results on other OPCs, indicate that the experimental oximes K-27 and K-48 are very promising pretreatment compounds. When penetration into the brain is undesirable, obidoxime is the most efficacious prophylactic agent already approved for clinical use.

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.

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.

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.

Isolated organs proofed to be a robust tool to study effects of (potential) therapeutics in organophosphate poisoning. Small bowel samples have been successfully used to reveal smooth muscle relaxing effects. In the present study, the effects of obidoxime, TMB-4, HI-6 and MB 327 were investigated on human small bowel tissue and compared with rat data. Hereby, the substances were tested in at least seven different concentrations in the jejunum or ileum both pre-contracted with carbamoylcholine. Additionally, the cholinesterase activity of native tissue was determined. Human small intestine specimens showed classical dose response-curves, similar to rat tissue, with MB 327 exerting the most potent smooth muscle relaxant effect in both species (human EC50=0.7×10-5M and rat EC50=0.7×10-5M). The AChE activity for human and rat samples did not differ significantly (rat jejunum=1351±166 mU/mg wet weight; rat ileum=1078±123 mU/mg wet weight; human jejunum=1030±258 mU/mg wet weight; human ileum=1293±243 mU/mg wet weight). Summarizing, our isolated small bowel setup seems to be a solid tool to investigate the effects of (potential) therapeutics on pre-contracted smooth muscle, with data being transferable between rat and humans.

The currently fielded pre-hospital therapeutic regimen for the treatment of organophosphorus (OP) poisoning in the United States (U.S.) is the administration of atropine in combination with an oxime antidote (2-PAM Cl) to reactivate inhibited acetylcholinesterase (AChE). Depending on clinical symptoms, an anticonvulsant, e.g., diazepam, may also be administered. Unfortunately, 2-PAM Cl does not offer sufficient protection across the range of OP threat agents, and there is some question as to whether it is the most effective oxime compound available. The objective of the present study is to identify an oxime antidote, under standardized and comparable conditions, that offers protection at the FDA approved human equivalent dose (HED) of 2-PAM Cl against tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), and VX, and the pesticides paraoxon, chlorpyrifos oxon, and phorate oxon. Male Hartley guinea pigs were subcutaneously challenged with a lethal level of OP and treated at approximately 1min post challenge with atropine followed by equimolar oxime therapy (2-PAM Cl, HI-6 DMS, obidoxime Cl2, TMB-4, MMB4-DMS, HLö-7 DMS, MINA, and RS194B) or therapeutic-index (TI) level therapy (HI-6 DMS, MMB4-DMS, MINA, and RS194B). Clinical signs of toxicity were observed for 24h post challenge and blood cholinesterase [AChE and butyrylcholinesterase (BChE)] activity was analyzed utilizing a modified Ellman's method. When the oxime is standardized against the HED of 2-PAM Cl for guinea pigs, the evidence from clinical observations, lethality, quality of life (QOL) scores, and cholinesterase reactivation rates across all OPs indicated that MMB4 DMS and HLö-7 DMS were the two most consistently efficacious oximes.

The ability of the acetylcholinesterase reactivator obidoxime (H2L2+) to bind palladium(II) cations was evaluated spectrophotometrically at different reaction conditions (pH, reaction time, metal-to-ligand molar ratio). The results showed that immediately after mixing the reagents, pH 7.4, complex species of composition [PdHL]3+ existed predominantly with a value of conditional stability constant lgβ'=6.52. The reaction was completed within 24 hours affording the formation of species [Pd2L]4+ with significantly increased stability (lgβ'=9.34). The spectral data suggest that obidoxime coordinates metal(II) ions through the oximate functional groups. The in vitro reactivation assay of paraoxon-inhibited rat brain acetylcholinesterase revealed that the new complex species were much less active than the non-coordinated obidoxime. The lack of reactivation ability could be explained by the considerable stability of complexes in solution as well as by the deprotonation of oxime groups essential for recovery of the enzymatic activity.

The ability of two novel oximes K378 and K348 and currently available oximes (HI-6, obidoxime) to reactivate sarin-inhibited acetylcholinesterase and to reduce acute toxicity of sarin was evaluated. Both new potential oxime reactivators were chosen for this study based on the data obtained during the extensive work on oximes development, from structure–activity relationship studies and in vitro evaluation of their ability to reactivate acetylcholinesterase inhibited by organophosphorus compounds. In vivo determined percentage of reactivation of sarin-inhibited rat blood and brain acetylcholinesterase showed that the potency of both novel oximes K378 and K458 to reactivate sarin-inhibited acetylcholinesterase roughly corresponds to low reactivating efficacy of obidoxime. On the other hand, the oxime HI-6 was found to be efficient reactivator of sarin-inhibited acetylcholinesterase in the peripheral compartment. While the oxime HI-6 was able to reduce the acute toxicity of sarin more than three times, both novel oximes decreased the acute toxicity of sarin less than two times. Based on the results, we can conclude that reactivating and therapeutic efficacy of both novel oximes K378 and K458 are significantly lower compared to the oxime HI-6 and, therefore, it is not suitable to replace the oxime HI-6 for the antidotal treatment of acute sarin poisoning.

Background and Purpose Eye exposure to the organophosphorus (OP) irreversible cholinesterase inhibitor sarin results in long-term miosis and impaired visual function. We have previously shown that tropicamide is better at ameliorating this insult than topical atropine or cyclopentolate. However, to minimize side effects associated with repeated tropicamide applications and high treatment doses, we evaluated the effects of oximes (ChE re-activators) alone and combined with tropicamide at ameliorating OP-induced ocular impairments. Experimental approach Rats were topically exposed to sarin, followed by topical treatment with various oximes alone or in combination with tropicamide. Pupil width and light reflex were measured by an infrared-based digital photograph system, while visual performance was assessed by employing the cueing version of the Morris water maze (MWM). Key Results Oxime treatment following sarin ocular exposure induced a slow persistent pupil widening with efficacy in the order of HLo-7 > HI-6 > obidoxime = TMB-4 = MMB-4. In the light reflex test, the ability of the iris to contract following oxime treatment was mostly impaired at 1 h and was back to normal at 4 h following sarin exposure. All oxime treatments ameliorated the sarin-induced visual impairment as tested in the visual task (MWM). The combined topical treatment of tropicamide with an oxime induced a rapid improvement in pupil widening, light reflex and visual performance, and enabled a reduction in tropicamide dose. Conclusions and Implications The use of tropicamide combined with an oxime should be considered as the topical treatment of choice against the toxic effects of ocular OP exposure.

Summary The neuroprotective effects of a novel oxime KR-22934, the oxime K203 and obidoxime in combination with atropine in rats poisoned with tabun at a sublethal dose (200 μg/kg i.m.; 80% LD 50 ) were studied. The tabun-induced neurotoxicity was monitored at 24 h following tabun challenge using a functional observational battery and an automatic measurement of motor activity. The results indicate that all tabun-poisoned rats treated with oximes in combination with atropine were able to survive within 24 h following tabun poisoning. One tabun-poisoned rat without antidotal treatment died within 24 h. The oximes KR-22934 and K203 combined with atropine showed a similar potency to decrease tabun-induced neurotoxicity at 24 h after tabun administration while the neuroprotective efficacy of obidoxime was slightly higher. However, no oxime was able to eliminate tabun-induced neurotoxicity completely. When atropine was administered alone, negligible neuroprotective efficacy was observed. Based on the results, a novel oxime KR-22934 did not bring any improvement of the neuroprotective efficacy of antidotal treatment of acute tabun poisonings.

Acetylcholinesterase reactivators (oximes) are highly hydrophilic compounds. This factor is considered to limit their distribution through the human body, especially into the central nervous system. However, some results contradict this hypothesis and confirm low concentrations of oximes inside the CNS. This study aims to verify oxime penetration through the blood–brain barrier, and into different brain regions. It has been confirmed that oximes can penetrate the BBB after therapeutic dose administration (0.73%, 0.69%, and 0.52% corresponding to obidoxime, HI-6, and new oxime K027). Although the whole brain level of K027 seems to be lower compared with other oximes we have to keep in mind that these numbers represent only a percentage of the concentration found in plasma. So, for more adequate comparison it is essential to have a look at the actual concentration of oximes in the wet brain tissue: 106.6 ± 19.87 ng/g (K027), 49.88 ± 10.21 ng/g (obidoxime) and 86.51 ± 42.37 ng/g (HI-6), where concentration of K027 was one order of magnitude higher than obidoxime and HI-6. In spite of the fact that the distribution of oximes into the CNS is minimal and concentrations differ from section to section, reasonable reactivation should still occur.

Obidoxime and asoxime (HI-6) are considered to be the most important acetylcholinesterase (AChE) reactivators applicable for treatment of poisoning by nerve agents. Unfortunately, toxicology of the oximes is not well known. For this reason, we decided to investigate the pertinent adverse effects on guinea pigs which are close to humans in toxicological point of view. HI-6 and obidoxime were administered intramuscularly in 5% of the median lethal dose. The animals were sacrificed 15, 30, 60, 120, and 240 min after exposure, and the brain, liver, spleen and kidneys were collected. Ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive substances (TBARS), glutathione S-transferase (GST) and glutathione reductase (GR) were measured. Results indicated that obidoxime acted on oxidative stress than HI-6. We found evidence of low molecular weight antioxidants depletion after obidoxime administration. On the other hand, TBARS assay showed significant decrease in brain and little increase in spleen and liver. The effect of HI-6 was more striking than the effect of obidoxime. There was a sign of higher metabolism and production of antioxidants in liver because GR was significantly increased after HI-6 exposure, and it is another sign of ongoing oxidative stress. Owing to the achieved results, obidoxime can be considered as a less toxic drug in counteracting oxidative stress despite its higher toxicity. Key words: Oxidative stress, obidoxime, HI-6, acetylcholinesterase, butyrylcholinesterase.

Poisoning by organophosphorus compounds (OP) still is a major therapeutic problem. Intentional OP pesticide poisoning results in up to 300.000 deaths each year and highly toxic OP nerve agents pose a permanent threat for the civilian population and military forces. The therapeutic value of clinically used oximes, pralidoxime, obidoxime and TMB-4, in human OP pesticide poisoning is under debate. Moreover, these oximes lack efficacy in poisoning by various nerve agents. An innumerable number of novel oximes have been synthesized in the past five decades to provide more effective oximes and compounds with improved blood–brain-barrier penetration. Novel compounds were tested with largely different experimental protocols in vitro and in animals in vivo. The lack of comparable experimental conditions and the absence of human in vivo studies hamper a well-founded evaluation of the available data. At present, it appears that only a small number of (bispyridinium) oximes show superior potency and efficacy against individual OP. However, until now, no oxime with sufficient broad-spectrum activity against structurally different OP pesticides and nerve agents is available. An interim solution may be the combination of two oximes with overlapping reactivation spectrum. In conclusion, the unsatisfying situation calls for studies with standardized and comparable experimental conditions in order to allow a sound assessment of available and novel oximes.

The ability of two newly developed bispyridinium oximes (K456, K458) to reduce tabun-induced acute neurotoxic signs and symptoms was compared with oxime K203 and trimedoxime using the functional observational battery. The neuroprotective effects of the oximes studied combined with atropine on rats poisoned with tabun at a sublethal dose (200 μg/kg i.m.; 85% of LD50 value) were evaluated. Tabun-induced neurotoxicity was monitored by the functional observational battery and automatic measurement of motor activity at 2 hr after tabun challenge. The results indicate that all tested oximes combined with atropine enable tabun-poisoned rats to survive till the end of experiment. Both newly developed oximes (K456, K458) combined with atropine were able to decrease tabun-induced neurotoxicity in the case of sublethal poisonings although they did not eliminate all tabun-induced acute neurotoxic signs and symptoms. Their ability to decrease tabun-induced acute neurotoxicity was slightly higher than that of trimedoxime and oxime K203, but the difference in neuroprotective efficacy among all oximes studied is not large enough to make a decision about replacement of commonly used oximes (especially trimedoxime and obidoxime) in the treatment of acute tabun poisonings.

Nerve agents such as tabun, cyclosarin and Russian VX inhibit the essential enzyme acetylcholinesterase (AChE) by organophosphorylating the catalytic serine residue. Nucleophiles, such as oximes, are used as antidotes as they can reactivate and restore the function of the inhibited enzyme. The oxime HI-6 shows a notably low activity on tabun adducts but can effectively reactivate adducts of cyclosarin and Russian VX. To examine the structural basis for the pronounced substrate specificity of HI-6, we determined the binary crystal structures of Mus musculus AChE (mAChE) conjugated by cyclosarin and Russian VX and found a conformational mobility of the side chains of Phe338 and His447. The interaction between HI-6 and tabun-adducts of AChE were subsequently investigated using a combination of time resolved fluorescence spectroscopy and X-ray crystallography. Our findings show that HI-6 binds to tabun inhibited Homo sapiens AChE (hAChE) with an IC50 value of 300μM and suggest that the reactive nucleophilic moiety of HI-6 is excluded from the phosphorus atom of tabun. We propose that a conformational mobility of the side-chains of Phe338 and His447 is a common feature in nerve-agent adducts of AChE. We also suggest that the conformational mobility allow HI-6 to reactivate conjugates of cyclosarin and Russian VX while a reduced mobility in tabun conjugated AChE results in steric hindrance that prevents efficient reactivation.

The potency of a novel oxime K203 in reactivating soman-inhibited acetylcholinesterase and reducing acute toxicity of soman was compared with commonly used oximes (HI-6, obidoxime, trimedoxime, methoxime) using in vivo methods. The study determining percentage of reactivation of soman-inhibited blood and tissue acetylcholinesterase in rats showed that the potency of the oxime K203 to reactivate soman-inhibited acetycholinesterase in the peripheral compartment is slightly higher than obidoxime and trimedoxime, especially in the diaphragm, slightly lower than methoxime and markedly lower compared to the oxime HI-6. The reactivating efficacy of the oximes studied in the peripheral compartment roughly corresponds to their potency to reduce acute toxicity of soman in mice. Based on the obtained data, we can conclude that the oxime K203 is not suitable for the replacement of the oxime HI-6 for the antidotal treatment of acute soman poisoning due to its relatively low potency to counteract acute toxicity of soman.

Anticholinesterases, such as organophosphorus pesticides and warfare nerve agents, present a significant health threat. Onset of symptoms after exposure can be rapid, requiring quick-acting, efficacious therapy to mitigate the effects. The goal of the current study was to identify the safest antidote with the highest therapeutic index (TI = oxime 24-hr LD50/oxime ED50) from a panel of four oximes deemed most efficacious in a previous study. The oximes tested were pralidoxime chloride (2-PAM Cl), MMB4 DMS, HLo-7 DMS, and obidoxime Cl2. The 24-hr median lethal dose (LD50) for the four by intramuscular (IM) injection and the median effective dose (ED50) were determined. In the ED50 study, male guinea pigs clipped of hair received 2x LD50 topical challenges of undiluted Russian VX (VR), VX, or phorate oxon (PHO) and, at the onset of cholinergic signs, IM therapy of atropine (0.4 mg/kg) and varying levels of oxime. Survival was assessed at 3 hr after onset clinical signs. The 3-hr 90th percentile dose (ED90) for each oxime was compared to the guinea pig pre-hospital human-equivalent dose of 2-PAM Cl, 149 µmol/kg. The TI was calculated for each OP/oxime combination. Against VR, MMB4 DMS had a higher TI than HLo-7 DMS, whereas 2-PAM Cl and obidoxime Cl2 were ineffective. Against VX, MMB4 DMS > HLo-7 DMS > 2-PAM Cl > obidoxime Cl2. Against PHO, all performed better than 2-PAM Cl. MMB4 DMS was the most effective oxime as it was the only oxime with ED90 < 149 µmol/kg against all three topical OPs tested.

Despite six decades of extensive research in medical countermeasures against nerve agent poisoning, a broad spectrum acetylcholinesterase (AChE) reactivator is not yet available. One current approach is directed toward synthesizing oximes with high affinity and reactivatability toward butyrylcholinesterase (BChE) in plasma to generate an effective pseudocatalytic scavenger. An interim solution could be the administration of external AChE or BChE from blood products to augment pseudocatalytic scavenging with slower but clinically approved oximes to decrease nerve agent concentrations in the body. We here semiquantitatively investigate the ability of obidoxime and HI-6 to decrease the inhibitory activity of VX with human AChE and BChE from whole blood, erythrocyte membranes, erythrocytes, plasma, clinically available fresh frozen plasma and packed red blood cells. The main findings are that whole blood showed a VX concentration-dependent decrease in inhibitory activity with HI-6 being more potent than obidoxime. Using erythrocytes and erythrocyte membranes again, HI-6 was more potent compared to obidoxime. With freshly prepared plasma, obidoxime and HI-6 showed comparable results for the decrease in VX. The use of the clinically available blood products revealed that packed red blood cells showed similar kinetics as fresh erythrocytes. Fresh frozen plasma resulted in a slower and incomplete decrease in inhibitory plasma compared to freshly prepared plasma. In conclusion, the administration of blood products in combination with available oximes augments pseudocatalytic scavenging and might be useful to decrease the body load of persistent, highly toxic nerve agents.

During more than five decades, pyridinium oximes have been developed as therapeutic agents used in the medical treatment of poisoning with organophosphorus compounds. Their mechanism of action is reactivation of acetylcholinesterase (AChE) inhibited by organophosphorus agents. Organophosphorus compounds (OPC) are used as pesticides and developed as warfare nerve agents such as tabun, soman, sarin, VX and others. Exposure to even small amounts of an OPC can be fatal and death is usually caused by respiratory failure resulting from paralysis of the diaphragm and intercostal muscles, depression of the brain respiratory center, bronchospasm, and excessive bronchial secretions. The mechanism of OPC poisoning involves phosphorylation of the serine hydroxyl group at the active site of AChE leading to the inactivation of this essential enzyme, which has an important role in neurotransmission. AChE inhibition results in the accumulation of acetylcholine at cholinergic receptor sites, producing continuous stimulation of cholinergic fibers throughout the central and peripheral nervous systems. Presently, a combination of an antimuscarinic agent, e.g. atropine, AChE reactivator such as one of the standard pyridinium oximes (pralidoxime, trimedoxime, obidoxime, HI-6) and diazepam are used for the treatment of organophosphate poisoning in humans. Despite of enormous efforts devoted to synthesis and development of new pyridinium oximes as potential antidotes against poisoning with OPC, only four compounds have found their application in human medicine so far. However, they differ in their activity in poisoning with warfare nerve agents and pesticides and there is still no universal broad-spectrum oxime capable of protecting against all known OPC. In this article the latest data on structure-activity relationship of pyridinium oximes including their efficacy in treatment of poisoning with organophosphorus compounds are reviewed.

More effective countermeasures against nerve-agent poisoning are needed, because current ones do not protect sufficiently, particularly the central nervous system (CNS). The purpose of the present study was to make a comparison of the antidotal capabilities of atropine/obidoxime/diazepam (termed the obidoxime regimen), atropine/HI-6 (1-[([4-(aminocarbonyl)pyridinio]methoxy)methyl]-2-[(hydroxyimino)methyl]pyridinium)/avizafone (termed the HI-6 regimen), and scopolamine/HI-6/physostigmine (termed the physostigmine regimen) against various doses of soman (2, 3, 4 x LD50). The results showed that each regimen administered twice (1 min and 5 min after exposure) effectively prevented or terminated epileptiform activity within 10 min. However, the regimens differed markedly in life-saving properties with the physostigmine regimen ranking highest followed in descending order by the HI-6 and obidoxime regimens. Pretreatment with pyridostigmine increased the potency of the HI-6 regimen, but not the obidoxime regimen. The latter regimen administered thrice (1 min, 5 min, and 9 min after exposure) did not compensate for the insufficiency. In half of the rats that lived for 7 days, neuropathology was unexpectedly observed predominantly in the left hemisphere unrelated to whether they seized or not. Local glutamatergic excitotoxic activity may occur even if manifest toxic signs are absent. The physostigmine regimen has excellent antidotal capacity, but the very narrow therapeutic window (< 10 min) makes it unsuitable for use in the field. The HI-6 regimen appears to constitute an efficacious therapy against lower doses of soman (2 and 3 x LD50). Copyright © 2012 John Wiley & Sons, Ltd.

Organophosphorus poisoning manifests as a cholinergic syndrome due to an inhibition of acetylcholinesterase. It is treated symptomatically by anticholinergics and oxime reactivators are used as causal antidotes. Reactivators possess a complex mechanism of action and interact at various levels of the cholinergic transmission. The aim of this study was to investigate the effect of standard oxime reactivators (HI-6, obidoxime, trimedoxime, methoxime and pralidoxime) on the hemicholinium-3 sensitive carriers, which are involved in the high-affinity choline uptake (HACU) transport, a key regulatory step in the synthesis of acetylcholine. The activity of the carriers was estimated in vitro on hippocampal synaptosomes using the substrate (3H)-choline and the competitive inhibitor (3H)-hemicholinium-3. Furthermore, the effect of the reactivators on the fluidity of hippocampal membranes was assessed. All tested compounds, except methoxime, showed an acute inhibitory effect on the carriers, however, only at μM concentrations. Trimedoxime showed the highest potency to inhibit HACU among all tested compounds ( I max 62%, IC 50 = 3 μM). All compounds, except HI-6, influenced also a membrane fluidity in the region of the hydrophilic heads of phospholipid bilayer, nevertheless, only methoxime was able to penetrate more deeply into the hydrocarbon core. We suggest that the direct interaction of oxime reactivators with the carrier protein (HI-6 and trimedoxime) and/or the changes in carrier conformation mediated by alterations in membrane fluidity (trimedoxime, obidoxime and pralidoxime) could occur here. The influence of reactivators on the carriers could be unfavorable in the case of their prolonged administration in vivo. From this point of view, the application of methoxime appears to be the best.

Methamidophos is one of the most toxic organophosphorus (OP) compounds. It acts via phosphorylation of a serine residue in the active site of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), leading to enzyme inactivation. Dif- ferent oximes have been developed to reverse this inhibition. Thus, our work aimed to test the protective or reactivation capabil- ity of pralidoxime and obidoxime, as well as two new oximes synthesised in our laboratory, on human and rat cholinesterases inhibited by methamidophos. In addition, we performed molecular docking studies in non-aged methamidophos-inhibited AChE to understand the mechanisms involved. Our results suggested that pralidoxime protected and reactivated methamidophos-inhib- ited rat brain AChE. Regarding human erythrocyte AChE, all oximes tested protected and reactivated the enzyme, with the best reactivation index observed at the concentration of 50 lM. Concerning BChE, butane-2,3-dionethiosemicarbazone oxime (oxime 1) was able to protect and reactivate the methamidophos-inhibited BChE by 45% at 50 lM, whereas 2(3-(phenylhydrazono) butan-2-one oxime (oxime 2) reactivated 28% of BChE activity at 100 lM. The two classical oximes failed to reactivate BChE. The molecular docking study demonstrated that pralidoxime appears to be better positioned in the active site to attack the O-P moiety of the inhibited enzyme, being near the oxyanion hole, whereas our new oximes were stably positioned in the active site in a manner similar to that of obidoxime. In conclusion, our work demonstrated that the newly synthesised oximes were able to reactivate not only human erythrocyte AChE but also human plasma BChE, which could represent an advantage in the treatment of OP compounds poisoning.

Summary Organophosphorous compounds cause fatal intoxication based on inhibition of acetylcholinesterase, an essential enzyme of neurosynapses and neuromuscular junctions. There is an obvious need to develop appropriate treatment against them due to their application in agriculture and chemical industry or their misuse in terrorist or war attack. In the Czech army some medicaments have been established to be used against this poisoning; Obidoxime, Methoxime, Atropine and Benactyzine. In present in vitro study we focused on potential cytotoxic and genotoxic effect evaluation of these drugs by the clonogenic and comet assay, respectively. Obtained results show that oximes exhibit pronounced toxic effect, namely obidoxime in term of genotoxicity and methoxime in term of cytotoxicity.