Pharmacological and toxicological in vitro and in vivo effect of higher doses of oxime reactivators.

The major function of compounds with an oxime moiety attached to a quarternary nitrogen pyridinium ring is to reactivate acetylcholinesterase inhibited by organophosphorus agent (OP). However, other oxime mechanisms (e.g. modulation of cholinergic or glutamatergic receptor) may be involved in the recovery. The main disadvantage of positively charged reactivators is their low ability to penetrate into the brain although crossing the blood brain barrier could be supported via increasing the dose of administered oxime. Thus, this study presents maximal tolerated doses (MTD) for marketed oximes (TMB-4, MMB-4, LüH-6, HI-6, 2-PAM) and the most promising K-oximes (K027, K048, K203) which can be used in OP therapy in the future. No signs of sarin intoxication were observed in mice treated with 100% MTD of HI-6 in contrast to those treated with atropine and only 5% LD50 of HI-6. 100% MTD of HI-6 resulted in levels of 500 µM and 12 µM in plasma and brain, respectively. This concentration is by a far margin safe with respect to direct effects on neuronal cell viability and, on the other hand, does not have any effects on central NMDA receptors or central nACh receptors. However, a weak antimuscarinic activity in case of LüH-6 and a weak peripheral antinicotinic action in case of TMB-4 and 2-PAM could be observed at their respective 100% MTD dose. These high doses, represented by MTD, are, however, irrelevant to clinical practice since they led to mild to moderate toxic side effects. Therefore, we conclude that clinically used doses of marketed oxime reactivators have no significant direct pharmacological effect on the tested receptors.

Toxicity and median effective doses of oxime therapies against percutaneous organophosphorus pesticide and nerve agent challenges in the Hartley guinea pig.

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, HLö-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 HLö-7 DMS, whereas 2-PAM Cl and obidoxime Cl2 were ineffective. Against VX, MMB4 DMS > HLö-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.

Efficacy of N-Acetylcysteine, Glutathione, and Ascorbic Acid in Acute Toxicity of Paraoxon to Wistar Rats: Survival Study.

There are a great number of reports with assertions that oxidative stress is produced by organophosphorus compound (OPC) poisoning and is a cofactor of mortality and morbidity in OPC toxicity. In addition, antioxidants have been suggested as adjuncts to standard therapy. However, there is no substantial evidence for the benefit of the use of antioxidants in survival after acute intoxication of OPCs. The present study was conducted to assess the effectiveness of three non-enzymatic antioxidants (NEAOs), N-acetylcysteine (NAC), glutathione (GSH), and ascorbic acid (AA), in acute intoxication of adult male Wister rats with paraoxon. The efficacy of the antioxidants was estimated as both a pretreatment and a concurrent application along with the standard oxime, pralidoxime (2-PAM). Relative risk of death after 48 hours of application was estimated by Cox regression analysis. The results revealed no benefit of either tested NEAO to the improvement in survival of experimental rats. The application of these antioxidants was found to be deleterious when administered along with pralidoxime compared to the treatment with pralidoxime alone. It has been concluded that the tested non-enzymatic antioxidants are not useful in acute toxicity for improving survival rates. However, the individual toxic dynamics of diversified OPCs should not be overlooked and further studies with different OPCs are suggested.

A trivalent approach for determining in vitro toxicology: Examination of oxime K027.

Unforeseen toxic effects contribute to compound attrition during preclinical evaluation and clinical trials. Consequently, there is a need to correlate in vitro toxicity to in vivo and clinical outcomes quickly and effectively. We propose an expedited evaluation of physiological parameters in vitro that will improve the ability to predict in vivo toxicity of potential therapeutics. By monitoring metabolism, mitochondrial physiology and cell viability, our approach provides insight to the extent of drug toxicity in vitro. To implement our approach, we used human hepatocellular carcinoma cells (HepG2) and neuroblastoma cells (SH-SY5Y) to monitor hepato- and neurotoxicity of the experimental oxime K027. We utilized a trivalent approach to measure metabolism, mitochondrial stress and induction of apoptosis in 96-well formats. Any change in these three areas may suggest drug-induced toxicity in vivo. K027 and pralidoxime, an oxime currently in clinical use, had no effect on glycolysis or oxygen consumption in HepG2 and SH-SY5Y cells. Similarly, these oximes did not induce oxidant generation nor alter mitochondrial membrane potential. Further, K027 and pralidoxime failed to activate effector caspases, and these oximes did not alter viability. The chemotherapeutic agent, docetaxel, negatively affected metabolism, mitochondrial physiology and viability. Our studies present a streamlined high-throughput trivalent approach for predicting toxicity in vitro, and this approach reveals that K027 has no measurable hepatotoxicity or neurotoxicity in vitro, which correlates with their in vivo data. This approach could eliminate toxic drugs from consideration for in vivo preclinical evaluation faster than existing toxicity prediction panels and ultimately prevent unnecessary experimentation.

Reactions of methylphosphonic difluoride with human acetylcholinesterase and oximes--Possible therapeutic implications.

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

Effects of K074 and pralidoxime on antioxidant and acetylcholinesterase response in malathion-poisoned mice.

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

Blood-brain barrier penetration of novel pyridinealdoxime methiodide (PAM)-type oximes examined by brain microdialysis with LC-MS/MS.

To develop a new reactivator of inhibited acetylcholinesterase (AChE) that can easily penetrate the blood-brain barrier (BBB), BBB penetration of 6 known and novel pyridinealdoxime methiodide (PAM)-type oximes (alkylPAMs) with relatively high reactivation activities was examined by in vivo rat brain microdialysis with liquid chromatography-mass spectrometry (LC-MS/MS). The 50% lethal dose (LD(50)) of alkylPAMs was intravenously determined for Wistar rats, then the limit of detection, quantification range and linearity of the calibration curve of the alkylPAMs in dialysate and blood were determined by LC-MS/MS. Following 10% LD(50) intravenous administration of the alkylPAMs, 4-[(hydroxyimino) methyl]-1-(2-phenylethyl) pyridinium bromide (4-PAPE) and 4-[(hydroxyimino) methyl]-1-octylpyridinium bromide (4-PAO) appeared in the dialysate. Striatal extracellular fluid/blood concentration ratios were 0.039+/-0.018 and 0.301+/-0.183 (mean+/-SEM), respectively, 1 h after treatment. This is the first report of BBB penetration of 4-PAPE, and the concentration ratio was smaller than that of 2-PAM. The mean BBB penetration of 4-PAO was approximately 30%, indicating that intravenous administration of 4-PAO may be effective for the reactivation of blocked cholinesterase in the brain. However, the toxicity of 4-PAO (LD(50); 8.89 mg/kg) was greater than that of 2-PAM. Further investigation is required to determine the effects of these alkylPAMs in organophosphate poisoning.

The "intermediate syndrome" as critical sequelae of organophosphate poisoning: the first report of two cases in Thailand.

The authors report 2 cases of organophosphate poisoning which developed intermediate syndrome. The first case was a man who took an organophosphate insecticide, monocrotophos, and developed severe organophosphate poisoning. Respiratory support was needed. He was treated with atropine and 2-PAM. Weakness of neck muscles, proximal limb and respiratory muscle developed in the 3rd day after ingestion. By supportive treatment and careful monitoring, however, he recovered after 11 days of the poisoning. The second case was a lady who took dicrotophos. She developed severe organophosphate poisoning for which respiratory support was also needed High dose of atropine, but without 2-PAM, was administered. She developed bulbar palsy, proximal muscle and respiratory weakness 3 day after the ingestion. Ventilation support was needed for 13 days before weaning was successful. This report did not support an efficacy of pralidoxime (2-PAM) in alleviation of the intermediate syndrome, but aims to alert physicians to recognize the intermediate syndrome for which adequate respiratory care is the crucial key for its management.

Sulfur derivatives of 2-oxopropanal oxime as reactivators of organophosphate-inhibited acetylcholinesterase in vitro: synthesis and structure-reactivity relationships.

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

Potential acetylcholinesterase reactivators: pyridine and alpha-oxooxime derivatives.

The synthesis and pharmacological screening in vitro and in vivo of pyridine-2-carbaldoxime derivatives I and alpha-oxooximes II are described. Four compounds elicited reactivating effect on phosphorylated acetylcholinesterase comparable with that of pralidoxime used as reference substance. Among the compounds tested, interesting structures are those of oximes bearing a thioether substituent [RA 49 (Table 1) and RA 59 (Table 2)] chloro derivative of MINA [RA 55 (Table 2)] and dipyridyl glyoxime methiodide RA 56 (Table 1).

The neurobehavioral effects of pralidoxime mesylate in the rat.

The neurobehavioral effects of pralidoxime mesylate (P2S) were characterized in the rat by comparing standard measures such as conditioned taste aversion, operant behavior and spontaneous locomotor activity. Male albino Sprague-Dawley rats were injected IP with either saline, 25, 50, or 100 mg/kg of P2S. None of these doses produced any overt toxic effects. P2S produced a conditioned taste aversion at the highest dose (100 mg/kg) using a single conditioning trial with sucrose in a one-bottle test. Acute administration of 100 mg/kg did not disrupt fixed ratio responding on a water reinforced schedule. All three doses, 25, 50, and 100 mg/kg, decreased spontaneous locomotor activity in a dose-dependent manner. The results of these studies support the findings that a single injection of P2S at the therapeutic levels would not disrupt the normal working performance in man.

Atropine sulfate and 2-pyridine aldoxime methylchloride elicit stress-induced convulsions and lethality in mice and guinea pigs.

The present study demonstrates that dose combinations of atropine sulfate and 2-pyridine aldoxime methylchloride (2-PAM), which do not produce any overt toxic effects on the behavior of mice or guinea pigs in a stable environment, elicit clonic-tonic convulsions and death when the animals are physically stressed by cold water swimming. Phenoxybenzamine (1-6 mg/kg), diazepam (0.625 and 1.25 mg/kg) and pilocarpine (2.5 and 5 mg/kg) significantly decreased (or abolished) the occurrence of atropine and 2-PAM stressed-induced convulsions and/or lethality. In contrast, propranolol (20 mg/kg), was ineffective in preventing either convulsions or lethality. Changes in plasma glucose levels and internal body temperature did not appear to explain the precipitation of convulsions or ensuing death. These results suggest that during acute physical stress, relatively low doses of atropine and 2-PAM produce toxic and lethal effects due to the activation of alpha-adrenergic mechanisms along with a concomitant inactivation of cholinergic mechanisms.

Adverse behavioral effects of the anticholinesterase poisoning protector pralidoxime methanesulfonate.

Pralidoxime methanesulfonate (P2S) has anticholinesterase protective properties, but it also has an array of gastrointestinal (GI) symptoms. Because such a symptom would be disadvantageous to occupational workers who handled and used organophosphorus anticholinesterase continuously, and to soldiers who have had oral pretreatment in a situation where anticholinesterase agent poisoning is a possibility, this question was investigated in rats using three behavioral paradigms to evaluate the feasibility of the oral prophylactic regimen. These are: (1) conditioned taste aversion (CTA), (2) operant behavior and (3) spontaneous locomotor activity (SMA); these three behavioral parameters are analogous to toxicant-induced gastrointestinal (GI) disturbances, performance of learned tasks and behavioral arousal, respectively. Dose-response studies of P2S in dose levels of 0.2, 0.4, 0.8 and 1.6 gm/kg (P.O.) were evaluated. The results consistently demonstrated that only the highest dose significantly produced marked decreases in consumption of flavored solution associated with its ingestion, suppressed keypress response maintained under a 20-response fixed-ratio schedule of water presentation, and inhibited SMA. By inference, if CTA, operant behavior and SMA are appropriate paradigms, P2S, on an acute single oral high dose level, would cause GI disturbances, impair task performance and induce sedation in man.

Behavioral toxicological assessment of oral pralidoxime methanesulfonate in the rat.

The behavioral toxicity of pralidoxime methanesulfonate (P2S) was examined in the rat by comparing standard measures such as conditioned taste aversion (CTA), drinking behavior and acute oral toxicity. P2S produced a weak CTA at doses of 0.4 and 0.8 g/kg (PO) and a profound CTA at the highest dose (1.6 g/kg) using a single sucrose-flavored conditioning trial with a one bottle test. The CTA produced by the highest dose of P2S was blocked by a specific, and exclusively peripheral, histamine-H2blocker, cimetidine (30 mg/kg, IP), which also has a cytoprotecting effect on gastric mucosal lesions. These data suggest that the H2 receptors may be involved in inducing the aversive effects of P2S through the inherent local irritating property of P2S on the rat gastric mucosa. There was no disruption of water drinking in thirsty rats with P2S at doses ranging from 0.2 to 1.6 g/kg. The survival time after an acute oral lethal dose of P2S (8-15 g/kg) was prolonged in non-fasted rats (16.5-38.5 min) compared to fasted ones (3.5-14.5 min), however the LD50's were identical (8.7 +/- 1.0 and 7.5 +/- 0.5 g/kg; respectively); indicating that P2S taken with food delays the lethal effects, but does not affect its lethal potency.

[Toxicity of oximes and their effect on acetylcholinesterase activity in the blood and myoneural synapses of animals]. / Vurkhu toksichnostta na niakoi oksimi i vliianieto im vurkhu aktivnostta na atsetilkholinesterazata v kruvta i mionevralnite sinapsi nazhivotnite.

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