Pre- and post-treatment effect of physostigmine on soman-inhibited human erythrocyte and muscle acetylcholinesterase in vitro.

Standard treatment of organophosphorus (OP) poisoning includes administration of an antimuscarinic (e.g., atropine) and of an oxime-based reactivator. However, successful oxime treatment in soman poisoning is limited due to rapid aging of phosphylated acetylcholinesterase (AChE). Hence, the inability of standard treatment procedures to counteract the effects of soman poisoning resulted in the search for alternative strategies. Recently, results of an in vivo guinea pig study indicated a therapeutic effect of physostigmine given after soman. The present study was performed to investigate a possible pre- and post-treatment effect of physostigmine on soman-inhibited human AChE given at different time intervals before or after perfusion with soman by using a well-established dynamically working in vitro model for real-time analysis of erythrocyte and muscle AChE. The major findings were that prophylactic physostigmine prevented complete inhibition of AChE by soman and resulted in partial spontaneous recovery of the enzyme by de-carbamylation. Physostigmine given as post-treatment resulted in a time-dependent reduction of the protection from soman inhibition and recovery of AChE. Hence, these date indicate that physostigmine given after soman does not protect AChE from irreversible inhibition by the OP and that the observed therapeutic effect of physostigmine in nerve agent poisoning in vivo is probably due to other factors.

In vitro kinetic interactions of pyridostigmine, physostigmine and soman with erythrocyte and muscle acetylcholinesterase from different species.

The low effectiveness of atropine and oxime treatment in soman poisoning may be enhanced by carbamates pre-treatment. For ethical reasons medical countermeasures can only be tested in animal models despite the fact of substantial species differences. With this kinetic in vitro study the interactions between pyridostigmine, physostigmine and soman with human, Rhesus monkey, swine and guinea pig erythrocyte AChE were investigated. In addition, the effect of the carbamates on the residual activity and enzyme recovery after soman inhibition was examined with erythrocyte and intercostal muscle AChE from these species with a dynamic in vitro model with real-time determination of AChE activity. Only small to moderate species differences of the inhibition and decarbamylation kinetics were recorded. It was possible to show that with erythrocyte and muscle AChE a similar level of protection by carbamates and reactivation after discontinuation of the carbamates and soman could be observed. Thus, these data indicate that carbamate pre-treatment is expected to protect a critical level of muscle AChE and confirm the presumption that erythrocyte AChE may serve as a surrogate for synaptic AChE. Hence, these and previous data fortify the notion that erythrocyte AChE is a proper tool for in vitro kinetic studies as well as for therapeutic monitoring in experimental and clinical studies.

Interaction of pentylsarin analogues with human acetylcholinesterase: a kinetic study.

Previous kinetic studies investigating the interactions between human acetylcholinesterase (AChE), structurally different organophosphorus compounds (OP) and oximes did not reveal a conclusive structure-activity relationship of the different reactions. The only exception was for a homologous series of methylphosphonofluoridates bearing C1-C4 O-n- or O-i-alkyl residues. Hence, it was tempting to investigate the kinetic interactions between different pentylsarin analogues, human AChE and two oximes, obidoxime and HI 6, in order to increase the understanding of structure-activity relationship between highly toxic OP and human AChE. The rate constants for the inhibition of human erythrocyte AChE by four pentylsarin compounds (k(i)), for the spontaneous dealkylation (aging, k(a)) and reactivation (k(s)) of inhibited AChE as well as for the oxime-induced reactivation of inhibited AChE by obidoxime and HI 6 reflected by the dissociation constant (K(D)) and the reactivity constant (k(r)) were determined. All pentylsarin analogues had a high inhibitory potency towards AChE. Inhibited AChE was subject to spontaneous reactivation which outweighed aging substantially. Pentylsarin-inhibited AChE could be reactivated by oximes, HI 6 being more potent than obidoxime. The determination of inhibition, reactivation and aging kinetics of pentylsarin analogues with human AChE extends the database on interactions between AChE and methylphosphonofluoridate homologues with C1-C4 n- and i-alkyl residues demonstrating a structure-activity relationship depending on the chain length with certain differences regarding inhibition and post-inhibitory reactions. Unfortunately, no structure-activity relationship could be observed for the oxime-induced reactivation of inhibited AChE. In view of previous results with numerous structurally different organophosphates, organophosphonates and phosphoramidates it has to be concluded that up to now kinetic studies did not provide decisive information for the development of more effective oxime-based reactivators.