Reactivation of organophosphate-inhibited serum butyrylcholinesterase by novel substituted phenoxyalkyl pyridinium oximes and traditional oximes

Abstract

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

Keywords: Acetylcholinesterase and butyrylcholinesterase inhibitors; Organophosphorus compounds; Oxime assisted catalysis; Oxime reactivators.

Fig. 1.

Structure of nerve agents, nerve agent surrogates, and OP insecticidal oxons.

Fig. 2.

Structure of novel phenoxyalkyl pyridinium oximes (US patent 9,277,937).

Fig. 3.

Serum BChE reactivation by oximes after inhibition by phthalimidyl isopropyl methylphosphonate (PIMP; sarin surrogate), nitrophenyl ethyl methylphosphonate (NEMP; VX surrogate), nitrophenyl isopropyl methylphosphonate (NIMP; sarin surrogate) and diisopropyl fluorophosphate (DFP). Rat serum, 3.A and 3. B; guinea pig serum, 3.C and 3.D; human serum, 3.E and 3.F. Data are expressed as mean ± SEM. N = 3 p*≤0.05 compared to 2-PAM.

Fig. 4.

Serum BChE reactivation by oximes after inhibition by nitrophenyl cyclohexyl methylphosphonate (NCMP; cyclosarin surrogate). Rat serum, 4.A; guinea pig serum, 4.B; human serum, 3.C. Data are expressed as mean ± SEM. N = 3 p*≤0.05 compared to 2-PAM.

Fig. 5.

Serum BChE reactivation by oximes after inhibition by phorate-oxon, phorate-oxon sulfoxide and paraoxon. Rat serum, 5.A and 5.B; guinea pig serum, 5.C and 5.D; human serum, 5.E and 5.F. Data are expressed as mean ± SEM. N = 3 p*≤0.05 compared to 2-PAM.