Mechanism of inhibition in vitro of mammalian acetylcholinesterase and cholinesterase in solutions of O, O-dimethyl 2,2,2-trichloro-1-hydroxyethyl phosphonate (trichlorphon)

Abstract

The rate of decomposition of trichlorphon into DDVP was measured polarographically at pH 7.4. The first order rate constants of decomposition at 25° are 7.27 × 10 −4 and 6.05 × 10 −4 min −1 for trichlorphon concentrations of 0.150 and 15.0 mM respectively; at 37° the corresponding rate constants are 53.1 × 10 −4 and 37.1 × 10 −4 min −1. The rate of decomposition of trichlorphon was also calculated from the kinetics of inhibition of acetylcholinesterase (EC 3.1.1.7) and cholinesterase (EC 3.1.1.8) in trichlorphon solutions at 25° and 37° (pH 7.4). The following enzyme sources were used: bovine erythrocytes and rat brain acetylcholinesterase, and human, horse and rat plasma cholinesterase. The rate of decomposition of trichlorphon was calculated by assuming that only DDVP formed from trichlorphon is the enzyme inhibitor, while trichlorphon itself does not act as an inhibitor. The calculated rate constants for the decomposition of trichlorphon are lower or just within the range of the rate constants obtained by the polarographic method. This agreement was taken as kinetic evidence that trichlorphon is not an inhibitor of mammalian cholinesterases. The effect of pH on enzyme inhibition supports this conclusion. The rate of inhibition of bovine erythrocyte acetylcholinesterase by DDVP is the same at pH 7.4 and pH 6.0 (37°). However, the rate of enzyme inhibition in trichlorphon solutions is 30 times faster at pH 7.4 than at pH 6.0, and this agrees with the greater stability of trichlorphon at the lower pH value. The rate of spontaneous reactivation of the enzyme was measured (37°, pH 7.4) after inhibition in trichlorphon solutions of acetylcholinesterase (human and bovine erythrocytes) and cholinesterase (human plasma). For all three enzyme preparations, the rate of spontaneous reactivation was the same as that obtained after inhibition by DDVP. All results point to the conclusion that trichlorphon in vitro is not an inhibitor of mammalian cholinesterases.