STABILITY OF CHEMICALLY AND METABOLICALLY GENERATED REACTIVE INTERMEDIATES

THOMAS HARRY KEENAN, Purdue University

Abstract

A method was presented that characterized the stability (half-life) of chemically and metabolically generated reactive intermediates and that determined the reaction order (S(,N)1 vs. S(,N)2) of the ultimate toxic species without requiring the isolation or synthesis of the intermediates. The methodology employed flow kinetics and trapping agents at physiological conditions. Intermediates were introduced into an ion exchange column via either an injection system (chemically activated) or an enzyme chamber (metabolically activated). A pH 7.4 buffer containing reactive intermediate was passed through the system at a known flow rate. While passing through the column the intermediate decomposed to an ultimate alkylating species which reacted with a thiol moeity on the ion exchange resin bound trapping agent (cysteamine). The amount of trapped ultimate toxic species was quantitated by either gas chromatography mass spectrometry or liquid scintillation. Half-lifes of the intermediates were determined by plotting the amount of trapped ultimate toxic alkylating species versus time. Investigations involving the chemically activated substrates N-methyl-N-nitrosourea and N-methyl-N-nitroso-N'-nitroquanidine provided half-life values of 14 and 0.2 minutes, respectively. This data is comparable to that reported in the literature. S(,N)1 and S(,N)2 reaction mechanisms were distinguished by varying the column nucleophilic concentration. Methyliodide (S(,N)2) and N-methyl-N-nitrosourea (S(,N)1) were employed in these experiments. Statistical analysis of the experimental data indicated constant slope values for the S(,N)1 species and different slope values for the S(,N)2 species at column nucleophilic concentrations of 1.5 and 2.0 meq/ml. Also, data was obtained for a series of nitrosamines (metabolically activated) which supported the postulated short half-lifes of their (alpha)-carbon hydroxylated metabolites (i.e. acetoxymethylmethylnitrosamine--30 seconds, diethylnitrosamine--40 seconds, and nitrosopyrrolidine--55 seconds). Attempts to characterize the half-lifes of dimethylnitrosamine and bromobenzene failed, possibly because the intermediates were extremely reactive and chemically decomposed before reaching the ion exchange column. Thus, the experimental data demonstrated that half-lifes of a few seconds to many minutes can be determined and that these values are obtained without isolating or synthesizing the intermediate. This information can have importance to investigations of mutagenic activity and organ specificity in tumorgenesis and drug design.

Degree

Ph.D.

Subject Area

Pharmaceuticals

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