Abstract

The reaction kinetics of the hydrolysis, phosphatolysis, glutathionyl conjugation, and alpha-glutathione-S-transferase (GST)-catalyzed glutathione conjugation of [3H-ring]melphalan were investigated at pH 6.5 and 7.4. The distribution of products relative to the initial parent compound radioactivity over time was measured by HPLC and analyzed by nonlinear regression techniques using a system of rate and distribution equations that describe the complete precursor-product pathways applicable to each reaction condition. The kinetic parameters calculated in the analysis were the first- and second-order rate constants of formation and the product ratios of the aziridinium intermediates. The second-order rate constants were normalized to those obtained for the hydroxylation reactions to yield relative rate constants. The first-order rate constants of aziridinium ion formation from melphalan and from all the monosubstituted melphalan species, except chloro, hydroxyl melphalan, were similar under all reaction conditions. The relative second-order rate constant for nonenzymatic glutathionylation of the aziridinium intermediate was 7 times larger at pH 7.4 than at pH 6.5. GST was found to react only with the aziridinium intermediate formed from melphalan and to dissociate slowly from the resultant GST-product complex (dissociation half-life, 1 hr at pH 7.4 and 3.5 hr at pH 6.5). The kinetic parameter estimates found in this study can be used to make preliminary calculations of the impact that cellular phosphate, glutathione, and GST concentrations will have on the intracellular detoxication of melphalan.