Elsevier

Biochemical Pharmacology

Volume 55, Issue 2, 15 January 1998, Pages 159-168
Biochemical Pharmacology

Research Papers
Combined Effect of Organophosphorus Hydrolase and Oxime on the Reactivation Rate of Diethylphosphoryl-Acetylcholinesterase Conjugates

https://doi.org/10.1016/S0006-2952(97)00430-9Get rights and content

Abstract

Reactivation of inhibited acetylcholinesterase (AChE) is essential for rapid recovery after organophosphate (OP) poisoning. However, following administration of an oxime reactivator, such as pralidoxime mesylate (P2S), in patients poisoned with certain diethylphosphorothioate pesticides, no reactivation is observed, presumably due to reinhibition by circulating anti-cholinesterase OPs. Pretreatment alone with organophosphorus hydrolases (OPH) that are capable of rapidly hydrolyzing OPs was demonstrated, in animals, to confer significant protection against OP toxicity. One strategy to augment the potentially therapeutic scope of OPHs is a combined post-exposure treatment consisting of a drug(s) commonly used against OP toxicity and a suitable hydrolase. In this study, we examined the in vitro ability of OPH from Pseudomonas sp. (OPHps) to prevent reinhibition of P2S-reactivated AChE by excess OPs. The kinetic parameters of the reactivation of a series of diethylphosphoryl-AChE (DEPAChE) conjugates, obtained by the use of various diethylphosphates, were determined and compared with the rates of reactivation in the presence of OPHps, with and without the OP inhibitors in the reactivation medium. Extrapolation of the in vitro results to in vivo conditions suggests that an OPHps concentration as low as 1 μg/mL blood would result in a 100-fold decrease in the concentration of circulating anti-AChE pesticides within less than one blood-circulation time, thereby minimizing reinhibition of the reactivated enzyme. Thus, for DEP-based pesticides, the combination of P2S–OPH treatment can significantly improve clinical recovery after OP intoxication. In addition, it is shown here for the first time that an OPH can effectively hydrolyze quaternary ammonium-containing OPs. This indicates that hydrolysis of phosphorylated oximes, toxic side products of oxime treatment, may also be accelerated by OPHs.

Section snippets

Materials

MEPQ [20], DEPQ [21], and TEPP [22] were prepared as described elsewhere 20, 21, 22. DEFP was prepared by fluorination of DECP as described by Saunders and Stacey [23]. DECP was obtained from the Aldrich Chemical Co. and distilled before use. O,O-Diethyl p-nitrophenyl phosphate (paraoxon) was purchased from the Sigma Chemical Co. and used as obtained. The structure and the purity (>96%) of all OPs were confirmed by 1H NMR and 31P NMR spectroscopy and gas chromatography. TEPP contained less than

Reactivation of Diethylphosphoryl-AChE Conjugates

The main goal of this study was to define the capacity of OPHps to prevent reinhibition of P2S-reactivated enzyme by a variety of (C2H5O)2P(O)X (Fig. 1; Table 1). In several cases, the inhibited enzyme was spiked with a different diethylphosphate than the OP that was used to obtain the DEPAChE conjugate. Thus, it was important first to ascertain that all DEPAChEs display the same reactivation characteristics. The data of the time–course of the reactivation were best fitted by a single

Discussion

In this work we aimed to demonstrate the in vitro efficacy of an oxime-OPH combination against the toxicity of the diethylphosphates because a large number of widely used pesticides (e.g. parathion, chlorpyrifos, diazinon, coumaphos) are metabolized to potent AChE inhibitors that eventually produce DEPAChEs. The results clearly suggest that a combination of a standard oxime reactivator, such as P2S, and a low dose of an OPH can significantly improve clinical recovery after poisoning with

Acknowledgements

We thank Mrs. Bracha Manisterski for excellent technical assistance, and Prof. P. Taylor and Dr. Z. Radic for donation of purified rMoAChE.

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