Two cases of organophosphate poisoning with development of intermediate syndrome

Two cases of intermediate syndrome caused by organophosphorus poisoning are reported. Trichlorfon, propoxur (a carbamate pesticide) and fenthion were ingested in both attempts at suicide. After successful conventional therapy during the cholinergic phase, but before the time when the onset of delayed neuropathy might be expected, an intermediate syndrome developed. It affected the proximal limb muscles, neck flexors and respiratory muscles 2 d after pesticide ingestion. The two patients needed respiratory support. Recovery from the intermediate syndrome was complete in both patients, although one subsequently developed delayed neuropathy.     

Prophylactic administration of non‐organophosphate cholinesterase inhibitors before acute exposure to organophosphates: assessment using terbufos sulfone

Poisoning with organophosphorus compounds (OPCs) poses a serious threat worldwide. OPC‐induced mortality can be significantly reduced by prophylactic administration of reversible acetylcholinesterase (AChE) inhibitors. The only American Food and Drug Administration (FDA)‐approved substance for such pre‐treatment (to soman exposure) is presently pyridostigmine, although its efficacy is controversial. In search for more efficacious and broad‐spectrum alternatives, we have assessed in vivo the mortality‐reducing efficacy of a group of five compounds with known AChE inhibitory activity (pyridostigmine, physostigmine, ranitidine, tacrine and K‐27), when given in equitoxic dosage (25% of LD₀₁) 30 min before exposure to the OPC terbufos sulfone. Protection was quantified in rats by determining the relative risk of death (RR) using Cox analysis, with RR = 1 for animals given only terbufos sulfone, but no pre‐treatment. All tested AChE inhibitors reduced terbufos sulfone‐induced mortality significantly (p ≤ 0.05) as compared with the non‐treatment group (RR = 1: terbufos sulfone only). Best in vivo protection from terbufos sulfone‐induced mortality was achieved, when K‐27 was given before terbufos sulfone exposure (RR = 0.06), which was significantly (P ≤ 0.05) superior to the pre‐treatment with all other tested compounds, for example tacrine (RR = 0.21), pyridostigmine (RR = 0.28), physostigmine (RR = 0.29) and ranitidine (RR = 0.33). The differences in efficacy between tacrine, pyridostigmine, physostigmine and ranitidine were not statistically significant. Prophylactic administration of an oxime (such as K‐27) in case of imminent OPC exposure may be a viable option. Copyright © 2013 John Wiley & Sons, Ltd.     

Usefulness of administration of non‐organophosphate cholinesterase inhibitors before acute exposure to organophosphates: assessment using paraoxon

Reversible acetylcholinesterase (AChE) inhibitors can protect against the lethal effects of irreversible organophosphorus AChE inhibitors (OPCs), when administered before OPC exposure. We have assessed in vivo the mortality‐reducing efficacy of a group of known AChE inhibitors, when given in equitoxic dosage before exposure to the OPC paraoxon. Protection was quantified in rats by determining the relative risk (RR) of death. Best in vivo protection from paraoxon‐induced mortality was observed after prophylactic administration of physostigmine (RR = 0.30) or the oxime K‐27 (RR = 0.34); both treatments were significantly superior to the pre‐treatment with all other tested compounds, including the established substance pyridostigmine. Tacrine (RR = 0.67), ranitidine (RR = 0.72), pyridostigmine (RR = 0.76), tiapride (RR = 0.80) and 7‐MEOTA (RR = 0.86) also significantly reduced the relative risk of paraoxon‐induced death, but to a lesser degree. Methylene blue, amiloride and metoclopramide had an unfavorable effect (RR ≥ 1), significantly increasing mortality. When CNS penetration by prophylactic is undesirable K‐27 is a promising alternative to pyridostigmine. Copyright © 2012 John Wiley & Sons, Ltd.     

有机磷与拟除虫菊酯农药联合作用的毒性评价

目的 探讨农药混配后农药之间发生的相互作用,为合理混配农药提供参考依据。方法 用等毒剂量法评价有机磷类与拟除虫菊酯类农药混剂对大鼠急性经口毒性的联合作用。结果 辛酸磷＋溴氰菊酯、辛硫磷＋高效氯氰菊酯、甲基对硫磷＋高效氯氰菊酯和水胺硫磷＋甲氰菊酯毒性增加非常明显呈协高作用。辛硫磷＋氰戊菊酯虽属相加作用,但毒性是相加作用的１．５倍,唯独敌敌畏＋溴氰菊酯虽属相加作用,但毒性略有降低。结论 有机磷与拟除虫     

Electrophysiologic changes associated with chronic administration of organophosphates

Changes in the electrophysiologic activity of the rat sciatic nerve were examined after repeated dosing with either of two organophosphate insecticides, parathion, and trichlorfon. Although the parathion treated animals showed overt signs of systemic toxicity, there were no significant changes in any of the measured parameters of sciatic nerve excitability. Trichlorfon, on the other hand, produced dose-dependent changes in the duration, rise time, relative area, and refractory period of the sciatic nerve compound action potential. The observed changes indicated an increased excitability of the nerve. During the early development of these electrophysiologic changes there were no accompanying histologic changes in the nerve. This suggests that changes in nerve excitability may be a sensitive indicator of neurotoxicity, and that continued trichlorfon exposure may lead to a cumulative alteration in nerve function.     

On the development of behavioral tolerance to organophosphates. I: Behavioral and biochemical aspects

The development of tolerance to organophosphates (OPs) was investigated by SC injections of saline and sublethal doses of DFP or soman three times per week or every other day for at least 4 weeks. Shuttlebox performance was tested 1 hr and 24 hr after the injections. Notwithstanding a progressive inhibition of AChE to very low values in various organs, shuttlebox performance was virtually normal 24 hr after the OP injections. However, whereas the performance decrements measured 1 h after the injection of DFP practically disappeared in the course of weeks, the decrements 1 hr after soman remained approximately the same. These differences between the effects of DFP and soman cannot be explained: 1) by differences in inhibition or de novo synthesis of AChE in various regions of the CNS, the striated muscle or blood, 2) by differences in the reductions of the muscarinic receptors in various regions of the CNS, 3) by differences in the number of nicotinic receptors in the diaphragm muscle, or 4) by differences in phosphorylphosphatase (DFP-ase or somanase) activity in blood plasma or liver.     

A pregnane X receptor agonist with unique species-dependent stereoselectivity and its implications in drug development

Pregnane X receptor (PXR) is an orphan nuclear receptor that regulates the expression of genes encoding drug-metabolizing enzymes and transporters. In addition to affecting drug metabolism, potent and selective PXR agonists may also have therapeutic potential by removing endogenous and exogenous toxins. In this article, we report the synthesis and identification of novel PXR agonists from a library of peptide isosteres. Compound S20, a C-cyclopropylalkylamide, was found to be a PXR agonist with both enantiomer- and species-specific selectivity. S20 has three chiral carbons and was resolved into its two enantiomers. The individual S20 enantiomers exhibited striking mouse/human-specific PXR activation, whereby enantiomer (+)-S20 preferentially activated hPXR, and enantiomer (-)-S20 was a better activator for mPXR. As a human PXR (hPXR) agonist, (+)-S20 was more potent and efficacious than rifampicin. Mutagenesis studies revealed that the ligand binding domain residue Phe305 is critical for the preference for the (-)-S20 enantiomer by the rodent PXR. Treatment of S20 induced the expression of drug-metabolizing enzymes and transporters in reporter gene assays, in primary human hepatocytes, and in "humanized" hPXR transgenic mice. To our knowledge, S20 represents the first compound whose enantiomers have opposite species preference in activating a xenobiotic receptor. The stereoselectivity may be used to guide the development of safer drugs to avoid drug-drug interactions or to achieve human-specific therapeutic effects when a xenobiotic receptor is being used as a drug target.     

Racemic and optically active trans 2,6-dimethyl analogues of the reversed ester of pethidine

The preparation and stereochemical characterization of (+/-)-1,trans 2,6-trimethyl-4-phenyl-4-propionoxypiperidine hydrochloride and its (+)- and (-)-antipodes are described. The absolute configurations of the antipodes were established by X-ray analysis of the corresponding (-)-4-piperidinol hydrobromide. In antinociceptive tests on mice and rats, the (+)-2S,6S ester proved the more potent antipode by factors of at least 10 (rats) and 20 (mice), a result consistent with earlier proposals made about the probable uptake conformation of pethidine reversed esters at opioid receptors.     

The unusual gradient elution for reversed phase HPLC of a strong chelator as an active drug substance

The new approach to drug development, especially for cardiovascular and brain diseases, brought to synthesis of new lipophillic derivatives of strong calcium chelator BAPTA — DP-b99 and DP-109. Due to their chelating ability, these compounds require metal-free stationary phases, and their high hydrophobicity resulted in unusually steep gradient elution. Novel HPLC methods for analysis of these two compounds were developed. Purospher® RP-C18, 5 μm, 125×3.0 mm and XTerra™ RP18, 3.5 μm, 100×4.6 mm columns with a steep gradient from: 1% acetic acid to acetonitrile were used for DP-b99, and Hypersil HyPurity™ C4, 5 μm, 100×4.6 mm column with a steep gradient from 1% Acetic acid to 5% THF in methanol — for DP-109. Versatile detection techniques could be used with these LC procedures. The methods appeared to be sensitive, selective, reproducible and stability indicating. They could be easily upgraded to bioanalytical methods with LC-MS technique.     

Effects of organophosphates on bioelectrical activity of the brain

Effects on electroencephalogram of acute and chronic exposure of two main groups of organophosphates: classic anticholinesterases (OP) and new bicyclic organophosphates (PTBO) are described. The role of muscarinic receptors of the midbrain reticular formation in the mechanism of action of OP and the possible mechanism of action of PTBO as blockers of a chloride ionophore of the GABA receptor complex are presented. The mechanism of convulsive activity of both groups are also discussed.     

Tomoxetine and the stereoselectivity of drug action

The phenoxypropylamine derivative tomoxetine has been shown to inhibit noradrenaline (NA) uptake into synaptosomes from rat hypothalamus (Wong et al 1982). Differences in the potencies of the resolved optical isomers, (-)-tomoxetine (I) and (+)-tomoxetine were also noted, the (-)-isomer being more potent than the racemate or the (+)-isomer in-vitro and in-vivo.     

Probing the active sites of butyrylcholinesterase and cholesterol esterase with isomalathion: conserved stereoselective inactivation of serine hydrolases structurally related to acetylcholinesterase.

Previous work has shown that acetylcholinesterase (AChE), a member of the alpha/beta-hydrolase superfamily, is stereoselectively inhibited by the four stereoisomers of isomalathion. Recent kinetic and mass spectral data demonstrated that a difference in mechanism of inactivation exists for AChE treated with (1R)- versus (1S,3S)-stereoisomers. This study sought to determine whether other alpha/beta-hydrolases are stereoselectively inhibited by isomalathion and if the difference in mechanism of AChE inactivation between (1R)- and (1S,3S)-isomers is conserved for other alpha/beta-hydrolases. Bimolecular rate constants of inhibition (k(i)) were measured for human and equine butyrylcholinesterase (HBChE and EBChE, respectively) and bovine cholesterol esterase (BCholE) with all four isomers. Isomalathion isomers inhibited these enzymes with the following order of potency: (1R,3R) > (1R,3S) > (1S,3R) > or = (1S,3S). Ratios of k(i) values for the most potent to the least potent isomer were 10.5 (HBChE), 11.9 (EBChE), and 68.6 (BCholE). Rate constants of reactivation (k(3)) were measured for enzyme inhibited by isomalathion isomers. HBChE, EBChE, and BCholE inactivated by the (1R)-isomers readily reactivated. However, enzymes modified by (1S)-isomalathions were refractory toward reactivation, and k(3) values were not significantly different from zero for HBChE and BCholE treated with the (1S,3S)-isomer. Computer-based docking experiments were performed for BCholE with (1R,3R)- and (1S,3S)-enantiomers. Calculated structures predicted a difference in primary leaving group: diethyl thiosuccinate for (1R,3R)-isomalathion and thiomethyl for the (1S,3S)-isomer. The data demonstrate that the alpha/beta-hydrolases used in this study are stereoselectively inhibited by isomalathion. Furthermore, the results suggest that the mechanistic shift demonstrated to occur for inhibition of AChE by (1R)- versus (1S,3S)-isomers is conserved for butyrylcholinesterase and cholesterol esterase.     

Naphthalene dicarboxaldehyde as an electrophilic fluorogenic moiety for affinity labeling: application to opioid receptor affinity labels with greatly improved fluorogenic properties

To develop ligands with fluorogenic properties amenable for following the kinetics of cross-linking to receptors, a naphthalene dicarboxaldehyde moiety has been attached to an opiate pharmacophore 2 and evaluated in mu-opioid receptors. The fluorescence of the benzo[f]isoindole formed upon cross-linking of mu-opioid receptors by 2 permitted the time-course of covalent bonding to be followed. This demonstrated proof-of-concept suggests the usefulness of naphthalene dicarboxaldehyde-containing affinity labels as kinetic probes.     

Verapamil interaction with the muscarinic receptor: stereoselectivity at two sites

Verapamil, in addition to blocking calcium channels, exhibits such "non-specific" effects on myocardium as inhibition of sodium and potassium conductances and modifications of muscarinic receptor-ligand interactions. To characterize further the effects of verapamil on the cardiac muscarinic receptor, we examined the abilities of the enantiomers of verapamil to modify the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB) to purified canine sarcolemmal vesicles. Membranes were incubated with [3H]QNB and various concentrations of racemic, (+)-, or (-)- verapamil (25 or 37 degrees, pH 7.4), and reactions were terminated by rapid filtration. (-)-Verapamil (Ki of 5.3 +/- 0.2 microM) was twice as potent an inhibitor of equilibrium binding as (+)-verapamil (Ki of 11.4 +/- 0.6 microM), and this effect resulted from the ability of each enantiomer to slow [3H]QNB-receptor association. This degree of stereoselectivity, albeit at nanomolar concentrations, was similar to that observed for each enantiomer to compete for the specific phenylalkylamine site in this preparation. Verapamil also inhibited [3H]QNB-receptor dissociation, but this effect required high concentrations and demonstrated stereoselectivity opposite to that observed for association. These findings support the view that verapamil interacts with two distinct sites, possibly within membrane lipid, each with a different affinity and preference for (+)- and (-)-verapamil, to modify the muscarinic receptor.     

Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential

The assay of neurotoxic esterase (NTE) in brains taken from dosed hens enables potential neurotoxicity of organophosphate pesticides, plasticers, etc. to be assessed. The original assay [Johnson, M. K. Biochem. J. 114, 711–717 (1969)] has been simplified to eliminate centrifugation and transfer steps and both the selectivity and the sensitivity have been increased. The procedures necessary to obtain stable reagent stocks are described.

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Zusammenfassung Durch Bestimmung der neurotoxischen Esterase (NTE) ist es möglich, im Gehirn von mit phosphororganischen Pflanzenschutzmitteln, Weichmachern und anderen Stoffen behandelten Hühnern die potentielle Neurotoxizität dieser Stoffe zu erfassen. Die ursprüngliche Methode [Johnson, M. K. Biochem. J. 114, 711–717 (1969)] wurde vereinfacht, so daß Zentrifugieren und Transferschritte nicht mehr erforderlich sind. Die Selektivität und Empfindlichkeit der Methode wurde verbessert. Die Herstellung stabiler Reagentienstammlösungen wird beschrieben.

     

Boronate can be the fluorogenic switch for the detection of hydrogen peroxide

Hydrogen peroxide (H(2)O(2)) has entrapped the abundant concern of numerous researchers in the cutting edge of chemistry, biology and medicine since it is thought to be associated with various biological and pathological conditions. Fluorescent probes are the promising tools for the detection and understanding of the physiological roles of H(2)O(2), considering that they are able to provide spatial and temporal information about target biomolecules in living cells. Nevertheless, the existent fluorescent probes have low selectivity and sensitivity on discerning H(2)O(2). This review would like to demonstrate a comprehensive examination on the design, recognition, and performance of state-of-the-art boronate-based fluorogenic switch for the detection of H(2)O(2), a field in which remarkable improvements have been accomplished over the last decade.     

Concentration-dependent interactions of the organophosphates chlorpyrifos oxon and methyl paraoxon with human recombinant acetylcholinesterase

For many decades it has been thought that oxygen analogs (oxons) of organophosphorus insecticides phosphorylate the catalytic site of acetylcholinesterase by a mechanism that follows simple Michaelis-Menten kinetics. More recently, the interactions of at least some oxons have been shown to be far more complex and likely involve binding of oxons to a second site on acetylcholinesterase that modulates the inhibitory capacity of other oxon molecules at the catalytic site. The current study has investigated the interactions of chlorpyrifos oxon and methyl paraoxon with human recombinant acetylcholinesterase. Both chlorpyrifos oxon and methyl paraoxon were found to have k(i)'s that change as a function of oxon concentration. Furthermore, 10 nM chlorpyrifos oxon resulted in a transient increase in acetylthiocholine hydrolysis, followed by inhibition. Moreover, in the presence of 100 nM chlorpyrifos oxon, acetylthiocholine was found to influence both the K(d) (binding affinity) and k(2) (phosphorylation constant) of this oxon. Collectively, these results demonstrate that the interactions of chlorpyrifos oxon and methyl paraoxon with acetylcholinesterase cannot be described by simple Michaelis-Menten kinetics but instead support the hypothesis that these oxons bind to a secondary site on acetylcholinesterase, leading to activation/inhibition of the catalytic site, depending on the nature of the substrate and inhibitor. Additionally, these data raise questions regarding the adequacy of estimating risk of low levels of insecticide exposure from direct extrapolation of insecticide dose-response curves since the capacity of individual oxon molecules at low oxon levels could be greater than individual oxon molecules in vivo associated with the dose-response curve.     

The effect of organophosphates on heart ATPase in the rat

The effect of organophosphate intoxication with diisopropylfluorophosphate (DFP), paraoxon and parathion on the activity of some ATPases of rat hearts was examined. Biochemical and histochemical methods were employed to determine the activities of the transport ATPase, of the myofibres and mitochondrial ATPases using different doses and antidote application. The organophosphates studied here had an inhibitory effect on the activities of enzymes. Biochemical measurements showed that the ATPase activities are dependent upon the duration of poisoning, the dose of poison and the applied organophosphates. DFP caused the highest restriction in activity of the examined ATPases. The Ca2+-stimulated ATPase was more sensitive than the Na+/K+-ATPase to the application of organophosphates. The antidote therapy determined the grade of influence on activity. Combined treatments of organophosphate poisoning with atropine and obidoxime chloride in the high doses of 7.5 mg/kg and 12.5 mg/kg body weight i.m. proved to be the most effective against the organophosphate intoxication. Antidote combination of small doses and also single application of atropine or obidoxime chloride might cause a stimulation of activity of the Na+/K+-ATPase and a significant decline of the Ca2+-ATPase.