Influence of hepatic oxidative metabolism on the immunosuppressive activities of 6-thiopurines.

It has been suggested that in vitro and in vivo hepatic metabolism of 6-thiopurine and its prodrug azathioprine generates immunosuppressive metabolites with far greater activity than the parent drugs. To examine this possibility, in vitro and in vivo drug-metabolizing systems were interfaced with in vitro immune response assays for the detection of active 6-thiopurine metabolites. In this way, it was demonstrated that preincubation of 6-thiopurine with hepatic microsomes from Balb/c mice does not enhance the immunosuppressive activity of the parent drug in in vitro lymphocyte proliferation assays performed with Balb/c splenocytes stimulated by T and B cell mitogenic lectins. In fact, prior microsomal metabolism generally decreased the inhibitory effects of 6-thiopurine on lymphocyte mitogenic responses, indicating that any metabolites formed under these conditions were relatively inactive. In contrast, the immunosuppressive effects of cyclophosphamide, which was used as a positive control in this system, were profoundly increased by microsomal metabolism. It was shown, moreover, that in vivo metabolism of 6-thiopurine and azathiopurine did not lead to the generation of metabolites with detectably greater immunosuppressive activity than the parent drugs. The circulating immunosuppressive activity present in the serum of drug-treated Balb/c mice, when quantitated in vitro with a sensitive murine mixed lymphocyte culture system, was found to correlate strongly with the serum levels of the unmetabolized thiopurines, which were measured specifically by high-pressure liquid chromatography assay. Taken together, these results fail to support earlier hypotheses that blood-borne active metabolites contribute significantly to the immunosuppressive actions of 6-thiopurines.