David J. Sellers

In vitro studies on the reactivation by oximes of phosphylated acetylcholinesterase—I: On the reactions of P2S with various organophosphates and the properties of the resultant phosphylated oximes

The rates of formation and decomposition of a series of phosphylated oximes derived from P2S (2-hydroxy-iminomethyl-1-methylpyridinium methane-sulphonate) have been measured. The rates of inhibition of AChE by these phosphylated oximes and the parent (and related) organophosphates have also been measured. Possession of these rate data now permits a detailed analysis of the reactivation of phosphylated AChE by P2S to be made (see following paper).

Differences between some biological properties of enantiomers of alkyl S-alkyl methylphosphonothioates.

BASELT, R. C. & CASARETT, L. J. (1972). Biochem. Pharmac., 21, 2705-2712. BECKETT, A. H., MITCHARD, M. & SHIHAB, A . A. (1971). J. Pharm. Pharmac., 23, 347-353. BECKETT, A. H. , VAUGHAN, D. P. & ESSIEN, E. E. (1972). Ibid., 24, 244. DAVIS, C. M. & FENIMORE, D. C. (1975). J . Chromat., 104, 193-195. MISRA, A. L., MULE, S. J . , BLOCK, R. & VADLAMANI, . L. (1973). J. Pharmac. exp. Ther., 185, 287-299. MISRA, A. L., BLOCH, R., VADLAMANI, . L. & MULL, S. J. (1974). Ibid., 188, 3 4 4 4 . SULLIVAN, H. R . & DUE, S. L. (1973). J . medl Chem., 16, 909-913. SULLIVAN, H. R., DUE, S. L. & MCMAHON, R. E. (1973). J. Pharm. Pharmac., 25, 1009-1010. Clin. Pharmac. Ther., 17, 258-266.

The reactivation by oximes of phosphonylated acetylcholinesterase: The possible erroneous interpretation of reactivating potency

A comparative study of the reactivation by two oximes of acetylcholinesterase inhibited by several organophosphates has been made, with particular reference to the dependence of the degree of reactivation produced by an oxime (reactivating potency) upon the concentration of inhibited enzyme. In the case of one inhibitor it is demonstrated that the relative reactivating potency of the two oximes can be reversed by a change in experimental conditions. It is concluded that the measurement of the reactivation produced by two or more oximes, particularly when carried out under standardized conditions, is of little value in determining their relative reactivating potencies, and of negligible value in predicting their likely therapeutic effectiveness against organophosphate poisoning.

A comparison of methods for measuring acetyl cholinesterase activity in blood samples inhibited by carbamates.

Abstract An unambiguous method based on the measurement of the acetylcholinesterase activity resulting from complete decarbamoylation of the acetylcholinesterase in blood samples inhibited by a carbamate is described and compared with a spectrophotometric modification of Ellmans method.

In vitro studies on the reactivation by oximes of phosphylated acetylcholinesterase—II: On the formation of O, O-diethyl phosphorylated AChE and O-ethyl methylphosphonylated AChE and their reactivation by P2S

The in vitro reactivation profiles of O,O-diethyl phosphorylated AChE and O-ethyl methyl phosphonylated AChE by P2S (2-hydroxy iminomethyl-1-methyl-pyridinium methane sulphonate) have been determined. Whilst reinhibition of the reactivated AChE by phosphorylated oxime (POX) is not important in determining the reactivation profile of O,O-diethyl phosphorylated AChE, reinhibition of the reactivated AChE by phosphonylated oxime can, however, be important in determining the reactivation profile of O-ethyl methylphosphonylated AChE and the extent of this reinhibition is determined by the initial concentration of phosphonylated AChE. Kinetic analysis of the reactivation profiles demonstrated that the generally accepted scheme for this reactivation process is incorrect and that phosphylated AChE cannot be considered as a single species although an adequate description of the present data is afforded by a model using a 1:1 mixture of two species each with its own rate of reactivation. In the case of O,O-diethyl phosphorylated AChE the main kinetic difference between these two species is found not in the formation or stability of the phosphorylated AChE-P2S complex but in its subsequent reaction. From results with O-ethyl methylphosphonylated AChE prepared from two pairs of enantiomers as well as from the racemic fluoridate it was concluded that phosphonylation of AChE may not always occur via a mechanism involving inversion of configuration at phosphorus but can also occur with retention of configuration. Reactivation by P2S of O-ethyl methylphosphonylated AChE prepared from (S) organophosphates proceeds with inversion of configuration at phosphorus. Inversion also occurs in the reinhibition of AChE by the POX produced in the initial reactivation.

Interactions between acetylcholinesterase and tetra-N-alkylammonium ions

A study of the mechanism of interaction of acetylcholinesterase with some simple tetra-N-alkyammonium ions has been made. Kinetic schemes have been proposed which are consistent with the experimental results observed in the enzyme-tetra-N-alkylammonium system in the presence of substrate and in the presence of an organophosphorus inhibitor.

Stereodependent bioactivation of R(+)-ethyl S-propyl methylphosphonothioate: Is the S-oxide the active metabolite?

Abstract R (+)-Ethyl S -propyl methylphosphonothioate is bioactivated both in vivo and when perfused through isolated liver to give a product which is much more active as an inhibitor of acetylcholinesterase than the parent compound. The bioactivation does not occur in hepatectomised animals. Acetylcholinesterase inhibited by the active metabolite is not reactivated by pyridine-2-aldoxime methanesulphonate (P2S), whereas enzyme inhibited by the parent compound and its S (−) enantiomer is reactivatable. Attempts to identify the active metabolite were unsuccessful and experiments to explore its stability were inconclusive. Extensive in vitro studies of the inhibition of acetylcholinesterase by the enantiomers of ethyl S -propyl methylphosphonothioate and ethyl S -diisopropylaminoethyl methylphosphonothioates and subsequent reactivation of the enzyme by P2S showed that (a) there are large differences between the rates of inhibition of the R and S enantiomers of both compounds, (b) reactivation profiles are critically dependent on reaction conditions, and (c) the reactivation profiles of the R and S enantiomers of the former compound are indistinguishable under all conditions whereas differences are observed under some conditions for the latter pair of enantiomers. The results are discussed in terms of the possibility that the S -oxide of R (+)-ethyl S -propyl methylphosphonothioate is the active metabolite and it is concluded that this is unlikely.

The development of a method to measure the spatial and temporal heterogeneity of vapour clouds in the field

One of the main difficulties in assessing the downwind hazard from an atmospheric pollutant or an accidental release of a toxic chemical is a lack of information on the likely spatial and temporal heterogeneities in concentration which may occur. If field trials are to be conducted, an environmentally acceptable tracer coupled with a detector with both a quantitative and rapid response is required. Ethanol is suggested as a tracer and the development and characterization of a detector is discussed.