Glutathione-dependent metabolism of cis-3-(9H-purin-6-ylthio)acrylic acid to yield the chemotherapeutic drug 6-mercaptopurine: evidence for two distinct mechanisms in rats.

cis-3-(9H-Purin-6-ylthio)acrylic acid (PTA) is a structural analog of azathioprine, a prodrug of the antitumor and immunosuppressive drug 6-mercaptopurine (6-MP). In this study, we examined the in vitro and in vivo metabolism of PTA in rats. Two metabolites of PTA, 6-MP and the major metabolite, S-(9H-purin-6-yl)glutathione (PG), were formed in a time- and GSH-dependent manner in vitro. Formation of 6-MP and PG occurred nonenzymatically, but 6-MP formation was enhanced 2- and 7-fold by the addition of liver and kidney homogenates, respectively. Purified rat liver glutathione S-transferases enhanced 6-MP formation from PTA by 1.8-fold, whereas human recombinant alpha, mu, and pi isozymes enhanced 6-MP formation by 1.7-, 1.3-, and 1.3-fold, respectively. In kidney homogenate incubations, PG accumulation was only observed during the first 15 min because of further metabolism by gamma-glutamyltranspeptidase, dipeptidase, and beta-lyase to yield 6-MP, as indicated by the use of the inhibitors acivicin and aminooxyacetic acid. Based on these results and other lines of evidence, two different GSH-dependent pathways are proposed for 6-MP formation: an indirect pathway involving PG formation and further metabolism to 6-MP, and a direct pathway in which PTA acts as a Michael acceptor. HPLC analyses of urine of rats treated i.p. with PTA (100 mg/kg) showed that 6-MP was formed in vivo and excreted in urine without apparent liver or kidney toxicity. Collectively, these studies show that PTA is metabolized to 6-MP both in vitro and in vivo and may therefore be a useful prodrug of 6-MP.