Effect of adjuvant disease in rats on cyclophosphamide and isophosphamide metabolism

Abstract

After the injection of a variety of arthritogenic adjuvants into male Wistar rats, hepatic activation of cyclophosphamide and isophosphamide is rapidly and profoundly depressed. This selective injury is largely reversible with phenobarbital and principally restricted to the liver microsomal protein fraction, which demethylates aminopyrine and N, N-dimethylaniline and generates “alkylating metabolites” from cyclophosphamide in vitro. Evidence is presented, based upon both metabolite excretion studies and the duration of hexabarbital-induced hypnosis, that this phenomenon is not an artifact in vitro and must be seriously considered in evaluating both the efficacy of potential anti-arthritic drugs against the rat adjuvant arthritis and their toxicity in these arthritic animals. A quantitative separation of two pathological responses to the same adjuvant may be obtained: (1) in Buffalo rats, whose liver metabolism may be profoundly impaired while they suffer minimal (or no) arthritis after being inoculated with adjuvants which are truly arthritogenic in other rat strains; (2) with Mycobacterium tuberculosis dispersed in methyl oleate, which induces minimal arthritis in Wistar rats, but nevertheless impairs their liver metabolism over a prolonged period (14 days or more). Drug metabolism appeared to be normal in rats with two other immunologically mediated “inflammatory diseases” (graft vs host disease and allergic encephalomyelitis) and in other rodents examined after adjuvant inoculations. A novel bioassay for cyclophosphamide and isophosphamide metabolites is described which utilizes their ability to prevent grafted rat lymphocytes from initiating a graft vs host reaction in tolerant recipient rats. At least four alkylating metabolites of cyclophosphamide were found in rat bile and tentatively identified by thin-layer chromatography. The possible error in relying on changes in urinary excretion (rather than biliary excretion) of drug metabolites as a guide to changes in hepatic xenobiotic metabolism is discussed.