Search

Organophosphorus (OP) pesticides and nerve agents still pose a threat to the population. Treatment of OP poisoning is an ongoing challenge and burden for medical services. Standard drug treatment consists of atropine and an oxime as reactivator of OP-inhibited acetylcholinesterase and is virtually unchanged since more than six decades. Established oximes, i.e. pralidoxime, obidoxime, TMB-4, HI-6 and MMB-4, are of insufficient effectiveness in some poisonings and often cover only a limited spectrum of the different nerve agents and pesticides. Moreover, the value of oximes in human OP pesticide poisoning is still disputed. Long-lasting research efforts resulted in the preparation of countless experimental oximes, and more recently non-oxime reactivators, intended to replace or supplement the established and licensed oximes. The progress of this development is slow and none of the novel compounds appears to be suitable for transfer into advanced development or into clinical use. This situation calls for a critical analysis of the value of oximes as mainstay of treatment as well as the potential and limitations of established and novel reactivators. Requirements for a straightforward identification of superior reactivators and their development to licensed drugs need to be addressed as well as options for interim solutions as a chance to improve the therapy of OP poisoning in a foreseeable time frame.

Organophosphorus (OP) compounds inhibit central and peripheral acetylcholinesterase (AChE) activity, overstimulating cholinergic receptors and causing autonomic dysfunction (e.g., bronchoconstriction, excess secretions), respiratory impairment, seizure and death at high doses. Current treatment for OP poisoning in the United States includes reactivation of OP-inhibited AChE by the pyridinium oxime 2-pyridine aldoxime (2-PAM). However, 2-PAM has a narrow therapeutic index and its efficacy is confined to a limited number of OP agents. The bis-pyridinium oxime MMB4, which is a more potent reactivator than 2-PAM with improved pharmaceutical properties and therapeutic range, is under consideration as a potential replacement for 2-PAM. Similar to other pyridinium oximes, high doses of MMB4 lead to off-target effects culminating in respiratory depression and death. To understand the toxic mechanisms contributing to respiratory depression, we evaluated the effects of MMB4 (0.25-16 mM) on functional and neurophysiological parameters of diaphragm and limb muscle function in rabbits and rats. In both species, MMB4 depressed nerve-elicited muscle contraction by blocking muscle endplate nicotinic receptor currents while simultaneously prolonging endplate potentials by inhibiting AChE. MMB4 increased quantal content, endplate potential rundown and tetanic fade during high frequency stimulation in rat but not rabbit muscles, suggesting species-specific effects on feedback mechanisms involved in sustaining neurotransmission. These data reveal multifactorial effects of MMB4 on cholinergic neurotransmission, with the primary toxic modality being reduced muscle nicotinic endplate currents. Evidence of species-specific effects on neuromuscular function illustrates the importance of comparative toxicology when studying pyridinium oximes and, by inference, other quaternary ammonium compounds.

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.

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.

Irreversible inhibition of acetylcholinesterase (AChE) by organophosphates leads to many failures in living organism and ultimately in death. Organophosphorus compounds developed as nerve agents such as tabun, sarin, soman, VX and others belong to the most toxic chemical warfare agents and are one of the biggest threats to the modern civilization. Moreover, misuse of nerve agents together with organophosphorus pesticides (e.g. malathion, paraoxon, chlorpyrifos, etc.) which are annually implicated in millions of intoxications and hundreds of thousand deaths reminds us of insufficient protection against these compounds. Basic treatments for these intoxications are based on immediate administration of atropine and acetylcholinesterase reactivators which are currently represented by mono- or bis-pyridinium aldoximes. However, these antidotes are not sufficient to ensure 100 % treatment efficacy even they are administered immediately after intoxication, and in general, they possess several drawbacks. Herein, we have reviewed new efforts leading to the development of novel reactivators and proposition of new promising strategies to design novel and effective antidotes. Structure-activity relationships and biological activities of recently proposed acetylcholinesterase reactivators are discussed and summarized. Among further modifications of known oximes, the main attention has been paid to dual binding site ligands of AChE as the current mainstream strategy. We have also discussed new chemical entities as potential replacement of oxime functional group.

Acute organophosphorus (OP) intoxication is a worldwide clinical and public health problem. In addition to cholinergic crisis, neurodegeneration and brain damage are hallmarks of the severe form of this toxidrome. Recently, we generated a chemical model of severe acute OP intoxication in zebrafish that is characterized by altered head morphology and brain degeneration. The pathophysiological pathways resulting in brain toxicity in this model are similar to those described in humans. The aim of this study was to assess the predictive power of this zebrafish model by testing the effect of a panel of drugs that provide protection in mammalian models. The selected drugs included “standard therapy” drugs (atropine and pralidoxime), reversible acetylcholinesterase inhibitors (huperzine A, galantamine, physostigmine and pyridostigmine), N-methyl-d-aspartate (NMDA) receptor antagonists (MK-801 and memantine), dual-function NMDA receptor and acetylcholine receptor antagonists (caramiphen and benactyzine) and anti-inflammatory drugs (dexamethasone and ibuprofen). The effects of these drugs on zebrafish survival and the prevalence of abnormal head morphology in the larvae exposed to 4 µM chlorpyrifos oxon [1 × median lethal concentration (LC50)] were determined. Moreover, the neuroprotective effects of pralidoxime, memantine, caramiphen and dexamethasone at the gross morphological level were confirmed by histopathological and transcriptional analyses. Our results demonstrated that the zebrafish model for severe acute OP intoxication has a high predictive value and can be used to identify new compounds that provide neuroprotection against severe acute OP intoxication. © 2016, The Author(s)., This work was supported in part by the US Army ERDC-IRO (W912HZ-13-BAA-01; D.R.), the NATO SfP project MD.SFPP 984777 (D.R.), the Portuguese Foundation for Science and Technology Grant SFRH/BPD/78342/2011 (Programme POPH—QREN through the Portuguese Ministry of Education and Science and the European Social Fund; M.F.) and the Spanish Government (CTM2014-51985-R; D.R., E.P, M.F.). We also are grateful to Dr. Martí Pumarola from the Departament de Medicina i Cirurgia Animals of the UAB for their valuable comments on neuropathology.

The implementation of the Chemical Weapon Convention (CWC), prohibiting the development, production, storage and use of chemical weapons by 192 nations and the ban of highly toxic OP pesticides, especially class I pesticides according to the WHO classification, by many countries constitutes a great success of the international community. However, the increased interest of terrorist groups in toxic chemicals and chemical warfare agents presents new challenges to our societies. Almost seven decades of research on organophosphorus compound (OP) toxicology was mainly focused on a small number of OP nerve agents despite the fact that a huge number of OP analogues, many of these agents having comparable toxicity to classical nerve agents, were synthesized and published. Only limited physicochemical, toxicological and medical information on nerve agent analogues is available in the open literature. This implies potential gaps of our capabilities to detect, to decontaminate and to treat patients if nerve agent analogues are disseminated and may result in inadequate effectiveness of newly developed countermeasures. In summary, our societies may face new, up to now disregarded, threats by toxic OP which calls for increased awareness and appropriate preparedness of military and civilian CBRN defense, a broader approach for new physical and medical countermeasures and an integrated system of effective detection, decontamination, physical protection and treatment.

Current treatment of organophosphate poisoning is insufficient, and survivors may suffer from long-lasting adverse effects, such as cognitive deficits and sleep-wake disturbances. In the present study, we aimed at developing a guinea pig model to investigate the benefits of immediate and delayed stand-alone therapy on the development of clinical signs, EEG, heart rate, respiration and AChE activity in blood and brain after soman poisoning. The model allowed the determination of the therapeutic effects at the short-term of obidoxime, atropine and physostigmine. Obidoxime exerted the highest therapeutic efficacy at administration of the lowest dose (3.1 mg/kg i.m.), whereas two higher doses (9 and 18 mg/kg) were less effective on most parameters. Addition of atropine at 0.03 and 3 mg/kg (i.m.) to the treatment did not improve the therapeutic effects of obidoxime alone. Physostigmine (0.8 mg/kg im) at 1 min after poisoning increased mortality. Two lower doses (0.1 and 0.3 mg/kg i.m.) showed improvements on all parameters but respiration. The middle dose was most effective in preventing seizure development and therefore assessed as the most efficacious dose. Combined treatment of obidoxime and physostigmine shortened the duration of seizures, if present, from up to 80 min to ∼10-15 min. In practice, treatment will be employed when toxic signs appear, with the presence of high levels of AChE inhibition in both blood and brain. Administration of physostigmine at that moment showed to be redundant or even harmful. Therefore, treatment of OP poisoning with a carbamate, such as physostigmine, should be carefully re-evaluated. © 2010 The Author(s).

Organophosphorus nerve agents are still in use today in warfare and as terrorism compounds. Classical emergency treatment of organophosphate poisoning includes the combined administration of a cholinesterase reactivator (an oxime), a muscarinic cholinergic receptor antagonist (atropine) and a benzodiazepine anticonvulsant (diazepam). However, recent experiments with primates have demonstrated that such treatment, even when administered immediately after organophosphate exposure, does not rapidly restore normal electroencephalographic (EEG) activity and fails to totally prevent neuronal brain damage. The objective of this study was to evaluate, in a realistic setting, the therapeutic benefit of administration of GK-11 (gacyclidine), an antiglutamatergic compound, as a complement to the available emergency therapy against organophosphate poisoning. GK-11 was injected at a dose of 0.1 mg/kg (i.v) after a 45-min latency period to heavily intoxicated (8 LD50) primates. Just after intoxication, man-equivalent doses of one autoinjector containing atropine/pralidoxime/diazepam were administered. The effects of GK-11 were examined on survival, EEG activity, signs of toxicity, recovery after challenge and central nervous system histology. The present data demonstrate that treatment with GK-11 prevents the mortality observed after early administration of classical emergency medication alone. EEG recordings and clinical observations also revealed that GK-11 prevented soman-induced seizures and motor convulsions. EEG analysis within the classical frequency bands (beta, theta, alpha, delta) demonstrated that central activity was totally restored to normal after GK-11 treatment, but remained profoundly altered in animals receiving atropine/pralidoxime/diazepam alone. GK-11 also markedly accelerated clinical recovery of soman-challenged primates. Lastly, this drug totally prevented the neuropathology observed 3 weeks after soman exposure in animals treated with classical emergency treatment alone. GK-11 represents a promising adjuvant therapy to the currently available emergency polymedication to ensure optimal management of organophosphate poisoning in man. This drug is presently being evaluated in a human clinical trial for a different neuroprotective indication.

Oxime-type acetylcholinesterase reactivators (oxime-AChER) are used as an adjunct in the treatment for organophosphorus anticholinesterase poisoning. Because of the widespread usage and exposure of organophosphorus compounds (OPCs), its poisoning and fatalities is obvious in pregnant women, embryos and fetuses. OPCs irreversibly inhibit acetylcholinesterase (AChE) at nerve synapses. Furthermore, the role of AChE other than neurotransmission termination has been defined in the literature. The growing evidences show that cholinergic mechanisms are involved during growth and development of other organ systems. In contrary to the fact, the data on the use of oxime-AChER in OPC poisoning in pregnancy are scanty. The present review aimed to comprehend the status of oximes in pregnancy in lieu of the published literature. A thorough literature search was performed in January 2013, using ten popular search engines including Medline/PubMed, Google scholar, etc., using nine standard keywords. The search period was set from 1966 to present. The search did not reveal substantial data. No considerable studies were retrieved which could really demonstrate either the beneficial, harmful or even null effect of oxime-AChER usage in pregnancy. Only eighteen relevant articles were obtained for a period of about 47 years. In the literature, there is no report available to demonstrate the risk of using oxime-AChER in pregnancy for the treatment of OPC poisoning. The study reveals that the use of oxime-AChER in pregnancy is largely un-addressed, inconclusive and based on speculation albeit the incidences of OPC poisoning are quite prevalent. Well-designed studies are warranted for a tangible conclusion.

Organophosphorus nerve agents are the most toxic chemical warfare agents that are known to have been produced, stockpiled and weaponised. Their development, production, stockpiling and use are prohibited under the terms of the Chemical Weapons Convention and, together with their precursors, are subject to strict controls and verification procedures. The detection and identification of biological markers of exposure to nerve agents are required for three main purposes: confirmation of exposure for forensic purposes in cases of alleged use; diagnosis to guide appropriate medical countermeasures in the event of an exposure; and occupational health monitoring of workers in defence laboratories and demilitarisation facilities. Biomarkers of nerve agents fall into two main groups, free metabolites and adducts to proteins. These are reviewed together with analytical methods for their identification. Examples are provided of applications in cases of human exposure.

Organophosphorus inhibitors (OP) of acetylcholinesterase (AChE) represent a group of highly toxic compounds. The treatment of OP intoxication is, however, insufficiently ensured. Currently, two main categories of drugs—anticholinergics and oxime reactivators— are employed as antidotes. Oximes have been reported to act at several levels of the cholinergic transmission, and among the non-reactivation effects, the interaction with cholinergic receptors stands out. This review addresses issues correlated with non-reactivating effects of oxime reactivators with a special focus on the muscarinic and nicotinic receptors, but involvement of other cholinergic structures such as AChE and choline uptake carriers are discussed too. It can be concluded that the oxime reactivators show a variation in their antagonistic effect on the muscarinic and nicotinic receptors, which is likely to be of significance in the treatment of OP poisoning. In vitro data reported oximes to exert higher efficacy on the muscarinic M2 subtype than on the AChE. However, this effect seemed to be subtype specific since the antagonistic M3 effect was lower. Also, and importantly, the antimuscarinic effect was larger than that on nicotinic receptors. Even though atropine showed a much higher muscarinic antagonism, it is supposed that non-reactivation properties of oxime reactivators play a significant role in the treatment of OP poisoning.

We measured in nine patients, poisoned by organophosphorus agents (ethyl parathion, ethyl and methyl parathion, dimethoate, or bromophos), erythrocyte and serum cholinesterase activities, and plasma concentrations of the organophosphorus agent. These patients were treated with pralidoxime methylsulphate (Contrathion), administered as a bolus injection of 4.42 mg.kg-1 followed by a continuous infusion of 2.14 mg.kg-1/h, a dose regimen calculated to obtain the presumed "therapeutic" plasma level of 4 mg.l-1, or by a multiple of this infusion rate. Oxime plasma concentrations were also measured. The organophosphorus agent was still detectable in some patients after several days or weeks. In the patients with ethyl and methyl several days or weeks. In the patients with ethyl and methyl parathion poisoning, enzyme reactivation could be obtained in some at oxime concentrations as low as 2.88 mg.l-1; in others, however, oxime concentrations as high as 14.6 mg.l-1 remained without effect. The therapeutic effect of the oxime seemed to depend on the plasma concentrations of ethyl and methyl parathion, enzyme reactivation being absent as long as these concentrations remained above 30 micrograms.l-1. The bromophos poisoning was rather mild, cholinesterases were moderately inhibited and increased under oxime therapy. The omethoate inhibited enzyme could not be reactivated.

The toxicity and efficacy of two oximes, HLö-7 and pyrimidoxime, were evaluated in mice and compared to those obtained with HI-6. HLö-7 and pyrimidoxime produced 24 h LD50 values of 356 and 291 mg/kg (i.p.), respectively. In combination with atropine (17.4 mg/kg, i.p.), HLö-7 was a very efficient therapy against poisoning by 3 x LD50 dose of soman, sarin and GF and 2 x LD50 dose of tabun with ED50 values of 12.4, 0.31, 0.32 and 25.2 mg/kg, respectively. In contrast, pyrimidoxime was a relatively poor therapy which resulted in ED50 values of greater than 150, 5.88, 100 and 71 mg/kg against poisoning by soman, sarin, GF and tabun, respectively. HLö-7 produced significant (p less than 0.05) reactivation of phosphorylated acetylcholinesterase, in vivo, resulting in 47, 38, 27 and 10% reactivation of sarin, GF, soman and tabun inhibited mouse diaphragm acetylcholinesterase, respectively. HLö-7 also antagonized sarin-induced hypothermia in mice suggesting that it reactivated central acetylcholinesterase. The potential of HLö-7 as a replacement oxime for the treatment of nerve agent poisoning is discussed.

The hydrolysis of acetylthiocholine iodide (ATCh) by pralidoxime chloride (2-PAM Cl), trimedoxime (TMB(4)) and obidoxime chlpride (LUH(6)) was studied at pH 5.8-8.0 and incubation temperature from 5 to 40 degrees C in vitro. Significant ATCh hydrolysis by 2-PAM Cl, TMB(4) and LUH(6) was found, with the exceptions of those at pH 7.0, 6.2 and 5.8 at 5 degrees C and those at pH 6.2 and 5.8 at 15 degrees C. The hydrolysis by TMB(4) and LUH(6) was significantly stronger than that by 2-PAM Cl. The hydrolysis increased with increasing pH, incubation temperature and three oxime or ATCh concentration. Significant hydrolysis of ATCh by the three oximes could be found when the terminal concentration of oxime was higher than 0.01 mM at pH 7.0 and 7.4 at 30 and 37 degrees C. However, no hydrolysis of natural substrate (acetylcholine iodide) by the three oximes was found when very high terminal concentrations of oximes were used. In addition, the three oximes displayed an extraordinary efficiency in the reactivation of phoxim-inhibited acetylcholinesterase (AChE) from fish (Carassius auratus) or rabbit (Oryctolagus cuniculus domestic) brain in vitro. Parallel to the level of ATCh hydrolysis by the oximes, TMB(4) and LUH(6) displayed significantly higher reactivation efficiency than 2-PAM Cl to phoxim-inhibited AChE. And, the extent of reactivation by 2-PAM Cl was also lower than the other two. Plausible antidotal actions of the oximes against organophosphate poisoning AChE and erroneously high estimation of AChE activity by the Ellman method were discussed.

The extrapolation from animal data to therapeutic effects in humans, a basic pharmacological issue, is especially critical in studies aimed to estimate the protective efficacy of drugs against nerve agent poisoning. Such efficacy can only be predicted by extrapolation of data from animal studies to humans. In pretreatment therapy against nerve agents, careful dose determination is even more crucial than in antidotal therapy, since excessive doses may lead to adverse effects or performance decrements. The common method of comparing dose per body weight, still used in some studies, may lead to erroneous extrapolation. A different approach is based on the comparison of plasma concentrations at steady state required to obtain a given pharmacodynamic endpoint. In the present study, this approach was applied to predict the prophylactic efficacy of the anticholinergic drug caramiphen in combination with pyridostigmine in man based on animal data. In two species of large animals, dogs and monkeys, similar plasma concentrations of caramiphen (in the range of 60-100 ng/ml) conferred adequate protection against exposure to a lethal-dose of sarin (1.6-1.8 LD(50)). Pharmacokinetic studies at steady state were required to achieve the correlation between caramiphen plasma concentrations and therapeutic effects. Evaluation of total plasma clearance values was instrumental in establishing desirable plasma concentrations and minimizing the number of animals used in the study. Previous data in the literature for plasma levels of caramiphen that do not lead to overt side effects in humans (70-100 ng/ml) enabled extrapolation to expected human protection. The method can be applied to other drugs and other clinical situations, in which human studies are impossible due to ethical considerations. When similar dose response curves are obtained in at least two animal models, the extrapolation to expected therapeutic effects in humans might be considered more reliable.

Organophosphorous poisoning is a common method of deliberate self-harm in countries where the pesticides are readily available and can result in type I, II and/or III paralysis. The in-hospital morbidity and mortality of the poisoning are mostly associated with type II paralysis (intermediate syndrome). The aim of this study was to determine the role of oxidative stress in relation to the severity of poisoning and development of type II paralysis in patients suffering from acute organophosphate poisoning. This prospective study was carried out at the Christian Medical College Hospital. Thirty-two patients with acute organophosphorous poisoning, admitted in one medical unit over 17 months, were included in the study. They were clinically assessed for severity of poisoning and paralysis during the first 10 days of their hospitalisation. Temporal profiles of butyrylcholinesterase (BuChE) and oxidative stress parameters, for 4, 7 and 10 days of hospitalisation, were established in 25 of these patients. Type I and II paralysis were associated with severe poisoning. The majority of patients with type II paralysis had prior evidence of type I paralysis. The pattern of muscles that were paralysed in type I paralysis occurring alone and in type I paralysis proceeding to type II paralysis were similar. BuChE was significantly inhibited in all patients. Oxidative stress occurred in acute organophosphate poisoned patients and was greater in severe poisoning. The results suggest that type I paralysis may progress to type II paralysis in severely poisoned patients. They demonstrate early occurrence of oxidative stress in severe acute organophosphate poisoning. However, the development of type II paralysis is not associated with the level of oxidative stress. They suggest that mechanisms other than acetylcholine induced oxidative stress may be involved in the progression of type I to type II paralysis.

The toxicity of a combination of isopropyl methylphosphonofluoridate (sarin; GB) and cyclohexyl methylphosphonofluoridate (GF) and the efficacy of various oxime reactivators in combination with atropine against the combined GB/GF challenge were evaluated in mice. The 24-h s.c. LD50 of the GB/GF combination was 1.15 mumol/kg (1.10-1.21; 95% confidence limits). Mice administered GB/GF displayed typical signs of nerve agent poisoning such as tremors and convulsions, with death most likely due to anoxia subsequent to respiratory arrest. The GB/GF LD50 value was comparable to the s.c. LD50 of 1.35 and 1.21 mumol/kg for GF and GB in mice, respectively. Combining the two nerve agents did not result in potentiation of the toxicity. In combination with atropine sulfate (17.4 mg/kg, i.p.), which alone did not reduce mortality, the oximes tested, 2-PAM, obidoxime and HI-6, were all effective when administered 5 min before 3 x LD50 dose of GB/GF with 24-h ED50 values of 102.5, 18.22 and 1.96 mumol/kg, respectively. Use of the GB/GF combination does not appear to confer any unique toxicity profile and appears to be easily treated with the standard therapy of a cholinolytic and oxime.

The objective of the present study was to investigate whether reduction of central acetylcholine (ACh) accumulation by adenosine receptor agonists could serve as a generic treatment against organophosphate (OP) poisoning. The OPs studied were tabun ( O-ethyl- N-dimethylphosphoramidocyanidate), sarin (isopropylmethylphosphonofluoridate), VX ( O-ethyl- S-2-diisopropylaminoethylmethylphosphonothiolate) and parathion ( O, O-diethyl- O-(4-nitrophenyl)phosphorothioate). The efficacy of the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) against an OP intoxication was examined on the basis of the occurrence of clinical symptoms that are directly associated with such intoxication. CPA (1-2 mg/kg) effectively attenuated the cholinergic symptoms and prevented mortality in lethally tabun- or sarin-intoxicated rats. In contrast, CPA (2 mg/kg) proved to be ineffective against VX or parathion intoxication. Intracerebral microdialysis studies revealed that survival of sarin-poisoned and CPA-treated animals coincided with a minor elevation of extracellular ACh concentrations in the brain relative to the baseline value, whereas an 11-fold increase in transmitter levels was observed in animals not treated with CPA. In VX-intoxicated rats, however, the ACh amounts increased 18-fold, irrespective of treatment with CPA. The striatal acetylcholinesterase (AChE) activity following a lethal sarin intoxication was completely abolished in the vehicle-treated animals, whereas 10% and 60% AChE activity remained in animals treated with 2 mg/kg CPA 1 min after or 2 min prior to the poisoning, respectively. In VX-intoxicated animals the AChE activity in the brain was strongly reduced (striatum 10%, hippocampus 1%) regardless of the CPA treatment. These results demonstrate that CPA is highly effective against tabun or sarin poisoning, but fails to protect against VX or parathion. Survival and attenuation of clinical signs in tabun- or sarin-poisoned animals are associated with a reduction of ACh accumulation and with protection of AChE activity in the brain.

The increased international concern about the threat of military and terroristic use of nerve agents, prompted us to critically consider the expected value of the currently available oxime treatment of nerve agent poisoning. Although oximes have been designed to reactivate the inhibited acetylcholinesterase (AChE), clinical experience has indicated that they are not always very effective as reactivators and at this very moment none of them can be regarded as a broad-spectrum antidote. In spite of this drawback, oximes are worth further investigating, since recent data derived from soman or tabun lethally intoxicated non-human primates suggest that the oxime HI-6 may exert a pharmacological effect that is not related to reactivation of inhibited AChE, but still leads to survival. This pharmacological effect causes recovery of neuronal transmission in the respiratory centres of the brain and recovery of neuromuscular transmission in the diaphragm. These findings have stimulated research to reveal the pharmacological basis of these effects in order to find drugs which could be more effective and less toxic than the available oximes. Since cholinergic drugs were able to exert this effect, a new concept for further treatment is suggested: maintenance of neuronal transmission in spite of continued AChE-inhibition by pharmacological manipulation of the cholinergic receptor. This should renew interest in the diverse pharmacological effects of oximes to reach a more effective treatment in the future.

The therapeutic effectiveness of a new binary autoinjector containing 500 mg HI-6 and 2 mg atropine sulphate was tested in anesthetized pigs poisoned by a lethal dose of soman i.v. (9 micrograms/kg per 20 min). Pharmacokinetics and pharmacodynamics of HI-6 were studied concomitantly on administration of HI-6 alone, together with atropine sulphate, or together with atropine sulphate during soman intoxication. Cardiopulmonary parameters were monitored and serum concentrations of oxime and acetylcholinesterase (AChE) were measured in blood samples taken at intervals over a 6-h period postinjection. Five minutes after the start of soman infusion, mean AChE activity was decreased to 27 +/- 4.3% of baseline and signs of poisoning appeared. The antidotes, HI-6 and atropine sulphate, were then administered i.m. One minute after this injection there was a transient significant increase in AChE activity of 76 +/- 8.2% of baseline (p < 0.01). It then again decreased and remained suppressed throughout the experiment. Mean respiratory rate was significantly decreased (p < 0.01) to 20 +/- 3.2% of baseline after 20 min of soman infusion and remained low during the rest of the experiment. The poisoning signs were counteracted 15-20 min after antidote therapy and all pigs survived soman intoxication without ventilatory assistance. Administration of either atropine or atropine and soman had no significant effect on the pharmacokinetics of HI-6 in anesthetized pigs.

The oxime HI 6 (in combination with atropine) is considered to be an effective antidote in soman intoxication but was shown to be less effective in tabun poisoning. In contrast to HI 6, first in vitro studies with HLo 7 demonstrated a reasonable reactivating potency at acetylcholinesterase (AChE) inhibited by soman and tabun. Therefore, the therapeutic efficacy of HLo 7, HI 6 and obidoxime (with and without atropine) was compared in tabun poisoned guinea-pigs. In addition, the therapeutic effect of atropine in guinea-pigs poisoned by various doses of tabun was investigated. Female Pirbright-white guinea-pigs were anaesthetized with urethane (1.8 g/kg) and the carotid artery, jugular vein and trachea were cannulated. After baseline measurements the animals received tabun, 60, 180 or 300 μg/kg, and 2 min later the antidotes (all i.v.): obidoxime, HLo 7, or HI 6 (30 or 100 μmol/kg, each) or atropine 10 mg/kg or a combination of atropine and one of the oximes. Respiratory and circulatory parameters were recorded for 60 min or until the death of the animal. Erythrocyte, brain and diaphragm AChE activity was determined in every animal after the experiment. Poisoning by tabun resulted in a rapid deterioration of respiratory function and respiratory arrest within 5 min. Atropine treatment was very effective in improving the respiratory function after tabun 60 μg/kg but was ineffective after tabun 300 μg/kg. However, circulatory parameters were restored almost completely in all atropine therapy groups. Therapy of tabun 300 μg/kg poisoned animals with atropine plus oxime (30 μmol/kg) improved respiration to a variable extent and restored circulation. The efficacy decreased in the order obidoxime>HLo 7> >HI 6. Use of oximes 100 μmol/kg did not further increase the therapeutic effect. Oximes alone were completely ineffective. The considerable therapeutic efficacy of atropine and oximes was not accompanied by a reactivation of diaphragm or brain AChE. Erythrocyte AChE was partially reactivated by obidoxime. Tabun primarily impaired central respiratory control but peripheral neuromuscular block developed already at low tabun doses. Atropine was very effective in restoring circulation but respiration was improved only after low doses of tabun. The results of this investigation demonstrate a considerable effect of atropine plus obidoxime or HLo 7 in improving respiration and circulation after high dose tabun. This effect was not accompanied by a noticeable AChE reactivation, indicating the involvement of some other “direct” mechanisms. HLo 7 has to be considered as a broad-spectrum antidote, being superior to obidoxime or HI 6.

Cyclohexylmethylphosphonofluoridate (CMPF) is an organophosphate cholinesterase inhibitor with military significance. The purpose of these studies was 1) to determine the acute toxicity of CMPF in the male rhesus monkey, 2) to evaluate the efficacy of pyridostigmine (PYR) pretreatment plus atropine and oxime (2-PAM or H16) treatment, and 3) to evaluate the pathological consequences of acute poisoning. An i.m. LD50 of CMPF was estimated using an up-and-down dose selection procedure and 12 animals. The 48-h and 7-day LD50 was 46.6 micrograms/kg, i.m. In the protection experiments, pyridostigmine (0.3-0.7 mg/kg/24 h) was administered by surgically implanted osmotic minipumps for 3-12 days resulting in 21-65% inhibition of erythrocyte acetylcholinesterase activity. Animals were challenged with 5 x L50 CMPF (233 micrograms/kg) and treated with atropine (0.4 mg/kg) and either 2-PAM (25.7 mg/kg) or HI6 (37.8 mg/kg) at the onset of signs or 1 min after challenge. Osmotic pumps were removed within 30 min after agent challenge. Pyridostigmine, atropine, and either 2-PAM or H16 were completely effective against CMPF, saving ten of ten animals in each group. In comparison, three of five animals challenged with 5 x LD50 of soman and treated with atropine and 2-PAM survived 7 days. The primary histologic lesions in the acute toxicity group were neuronal degeneration/necrosis and spinal cord hemorrhage. The CMPF treated groups (total of 20 animals) had minimal nervous system changes with no significant lesion difference resulting from the different oxime therapies. The primary non-neural lesions were degenerative cardiomyopathy and skeletal muscle degeneration which occasionally progressed to necrosis and mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)

The efficacy of oxime (HI-6, toxogonin or PAM Cl) therapy against GF (cyclohexyl methylphosphonofluoridate) poisoning was assessed in mice. It was found that the combinations of atropine and either toxogonin or HI-6 were effective therapies against GF poisoning. PAM therapy was ineffective. HI-6 was the only oxime which reactivated GF inhibited acetylcholinesterase. This might explain the reason why the HI-6 treated mice appeared to recover more quickly from the incapacitating effects following GF poisoning.

Organophosphates (OPs) and carbamates exhibit their toxicity by inhibiting acethylcholinesterase (AChE, EC 3.1.1.7) due to phosphorylation, or carbamylation in case of carbamate poisoning. OP poisoning is treated by atropine (muscarinic cholinergic receptor antagonist) and cholinesterase reactivators - oximes (PAM-2Cl, TMB-4, and HI-6). In contrast to the irreversible inhibition of AChE by OPs, carbamylated enzyme reactivates rapidly and antidotal treatment is limited to atropine. The objective of this study was to evaluate the efficiency of thienyl phencyclidine (tenocyclidine, TCP) and its new synthesized adamantyl derivatives containing piperidine (TAPIP), pyrolidine (TAPIR) and morpholine (TAMORF) group in mice poisoned with OPs and carbamates. These new compounds were characterized by m.p. determination, elemental analysis, and spectroscopic methods (IR, 1H-NMR, 13C-NMR, MS). They showed low acute toxicity (LD50), and their LD50 values varied from 106.00 mg kg -1 b.m. (TCP) to >504.00 mg kg -1 b.m. (TAMORF). TCP and its adamantyl derivatives were administered intraperitoneally (2.5 mg kg-1 b.m.) together with atropine (10.0 mg kg-1 b.m.) and with or without 1/4 LD50 of HI-6. Each compound administered with atropine had a therapeutic effect in the poisoning with carbamates propoxur, aldicarb, and Ro 02-0683 (therapeutic dose was from 3.18 LD50 of aldicarb to 16.00 LD50 for propoxur). However, the efficiency of those compounds in combination with atropine was lower in poisoning with OP insecticide dichlorvos (DDVP) and warfare agents soman and tabun. In soman-poisoned mice the best therapeutic effects were obtained with the combination of HI-6 plus atropine and tested compounds, which ensured the survival of all treated animals with 4.00-6.40 LD50 of soman. The results suggest that TCP and adamantyl tenocyclidines could be used in combination with atropine as antidotes in carbamate poisoning and as adjuvant therapy to HI-6 and atropine in soman poisoning.

Whether or not extracorporeal hemodialysis or hemoperfusion with coated activated charcoal might be used in eliminating organophosphates following poisoning with nitrostigmine, demeton-S-methyl sulfoxide, or dimethoate was here examined. Nitrostigmine could not be hemodialysed. The other two organophosphates, on the other hand could be well eliminated from the blood by hemodialysis. The clearance rates for demeton-S-methyl sulfoxide and dimethoate were 52.98 ml/min and 59.07 ml/min respectively, at a blood flow rate of 100 ml/min. The clearance values for hemoperfusion with coated activated charcoal were higher under the same trial conditions, the values being 83.70 ml/min for demeton-S-methyl sulfoxide and 87.84 ml/min for dimethoate. Nitrostigmine, too, could be eliminated from the blood by hemoperfusion, its clearance being 59.20 ml/min.

Investigations into the therapeutic properties of various combinations of the bispyridinium salts HS-3 and HS-6 and the cholinolytics atropine and benactyzine against soman poisoning in unanesthetized male beagles were performed. In our investigations we observed that: 1. The most effective protection against soman poisoning was attained if both oximes were applied early i.m. 6 min after intoxication together with the cholinolytics. 2. On the basis of clinical symptoms HS-6 proved to have a more intensive therapeutic effect than HS-3 upon early application. 3. If HS-3 was applied early after s.c. intoxication with low concentrations of soman (up to 3 LD50), a significant protection or reactivation effect on serum cholinesterase was measured. 4. When HS-3 was applied at the beginning of convulsions — generally 28 min after s.c. intoxication — it also raised the rate of surviving animals. 5. The maximal blood levels for HS-3 and HS-6 were measured 20–30 min after i.m. injection; the half-life values of HS-3 and HS-6 in plasma were 45–60 min.

The therapeutic effects of pipethanate (sycotrol) and two newly synthetized cholinolytics, DPX-8 and ANC-51, were compared with atropine in mice poisoned by DDVP, fluostigmine, phospholine, and paraoxon. The antagonistic activotagonistic activity of tested drugs against acetylcholine-induced contraction of rat ileum and oxotremorine-induced salivation and tremor in the mouse was also studied. The anticholinergic activity of pipethanate, DPX-8, and ANC-51 was weaker than that of atropine. However,the therapeutic effect of pipethanate was higher than that of atropine in mice poisoned by the organophosphates. DPX-8 and ANC-51 afforded a better antidotal effect than atropine only in DDVP-poisoned mice.

Twenty-four male volunteers were given obidoxime tablets in quantities ranging from 1.84-3.58 g in a single dose, or 7.36 g divided into 4 equal doses. With the lowest dose, average peak plasma level of the drug was 1.9 mug/ml and after the highest single dose it was 5.6 mug/ml, both attained 1.5 h after administration. In the multiple-dosed individuals, plasma levels of the oxime increased gradually following each additional dose, reaching a peak of 3.5 mug/ml after the last dose. Thirteen individuals complained of one or more of the following side effects: pallor, nausea, pyrosis, headache, generalized weakness, sore throat, and paresthesia of the face muscles. Activities of blood cholinesterase, glutamic oxalacetic transaminase, glutamic pyruvic transaminase, as well as hematocrit values, heart rate, and blood pressure were not affected. It is postulated that due to the undesirable side effects, the general use of obidoxime tablets should not be recommended. However, prophylactic oral treatment with obidoxime could be considered for persons at high risk of organophosphate poisoning or when parenteral administration might not be feasible.

A comparison of the chemical purity, toxicology and potency of HI-6 (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2- [(hydroxyimino)methyl]-pyridinium dichloride) obtained from various sources (Canada, Israel, Yugoslavia, The Netherlands, United Kingdom) was performed. There were no significant differences between HI-6 obtained from Israel, Yugoslavia, The Netherlands and Canada regarding their potency, when combined with atropine, as an antidote of organophosphate poisoning. HI-6 obtained from the United Kingdom was significantly more toxic and less potent than any of the other HI-6 samples. In addition, the results of this study showed that there was no significant difference between HI-6 prepared as a laboratory batch and HI-6 prepared commercially with regards to chemical purity, toxicology or potency.

The bis-pyridinium oxime HI-6, in conjunction with atropine, was found to offer significant protection against multiple LD50 challenges with the organophosphorus compounds soman and tabun. In adult rhesus macaques, the therapeutic administration of HI-6 with atropine and diazepam protected three of four animals from the lethal effects of 5 x LD50 of soman and three of three animals from 5 x LD50 of tabun. However, when toxogonin was substituted for HI-6 in the therapeutic mixture, all three animals poisoned with 5 x LD50 of soman died. In rats, the 24 h protective ratios against tabun and soman with HI-6 were 2 and 3.5, respectively, whereas in guinea pigs these values were between 4 and 6 for both agents. No evidence was obtained for acetylcholinesterase (AChE) reactivation by HI-6 in tissue from tabun-poisoned rodents or following soman or tabun in primate plasma. The results underscore the significant therapeutic benefit of HI-6 in primates, a species specific efficacy against tabun, and argue for some mechanism of action of HI-6 at least partly unrelated to AChE reactivation.

The aim of the experiments was to obtain more information on the toxicity of organophosphates and protection against them. Pretreatment of mice with CBDP increased the s.c. toxicity of soman 19.1-fold, and its i.p. toxicity 17.8-fold. The protective effect of atropine and the oximes HS-3, HS-6 and HI-6 in soman poisoning was much greater in CBDP pretreated than in control animals. Atropine + HI-6 raised the s.c. LD50 of soman in the CBDP pretreated animals from 6.8 micrograms/kg to 166 micrograms/kg (PI = 24.3), but in control animals the i.p. LD50 was only raised from 370 micrograms/kg to 608 micrograms/kg (PI = 0.6). CBDP inhibited blood and brain AChE activity, but had no effect on aliesterase (AE) activity in plasma, liver and brain of mice in vivo. CBDP increased s.d. toxicities of sarin 11-fold, of tabun 5-fold and of VX 0.24-fold. The protective index of atropine + HS-3 in sarin poisoning, as in the case of soman poisoning, was much higher in CBDP pretreated than in control animals (20.1 : 13.6), only slightly higher in tabun poisoning (4.3 : 3.4) and in the case of VX poisoning lower in CBDP pretreated than in control animals (32 : 47). The results indicate that CBDP potentiates soman, sarin and tabun toxicities mainly by blocking their binding to non-specific sites in the body.

The bispyridinium oxime HI-6, 1-((((4-amino-carbonyl) pyridinio)methoxy) methyl)-2-(hydroxyimino)-methyl) pyridinium dichloride monohydrate, combined with atropine is an effective treatment for soman (pinacolyl methylphosphonofluoridate) poisoning but is relatively ineffective against tabun (ethyl N-dimethyl phosphoroamidocyanidate) poisoning in mice. This contrasts with those results obtained using the bispyridinium oxime obidoxime [1,1′-(oxy bis(methylene)) bis(4-(hydroxyimino)-methyl) pyridinium dibromide]. The purpose of this study was to investigate the efficacy of the combination of HI-6 and obidoxime plus atropine against poisoning by tabun and soman in mice. The combination of ineffective single doses of obidoxime (5 or 10 mg/kg) and HI-6 (25 or 50 mg/kg) improved the treatment of tabun poisoning over either oxime alone. Combinations employing higher concentrations of obidoxime (25 or 50 mg/kg) and HI-6 (100 or 200 mg/kg) resulted in significant toxicity in the absence of organophosphate poisoning. Against soman poisoning the addition of obidoxime to HI-6 did not attenuate the efficacy of HI-6. The half-life of elimination and peak serum concentrations of HI-6 and obidoxime were not altered following administration of the combined injection. Reactivation of tabun-inhibited acetylcholinesterase was found consistently in the diaphragm but not in the brain. Using response surface methods it was possible to estimate the optimal therapy against soman and tabun poisoning (74.5 mg/kg HI-6+31.9 mg obidoxime against 1052 μg/kg challenge of tabun and 129 mg/kg HI-6 +0 mg/kg obidoxime against 390 μg/kg challenge of soman). It is proposed that reactivation of tabun inhibited acetylcholinesterase at the diaphragm may be responsible for the increased efficacy of the combination of HI-6 and obidoxime against tabun poisoning in mice.