Trofosfamide metabolism in different species--ifosfamide is the predominant metabolite

Trofosfamide (TRO) belongs to the group of oxazaphosphorines and is a congener of cyclophosphamide (CYC) and ifosfamide (IFO). The precondition for the cytotoxic effect of all oxazaphosphorines is their metabolic activation by "ring" oxidation at the hepatic mixed-function oxidase system. In addition, an inactivating metabolic pathway ("side chain" oxidation) is known for CYC and IFO. The metabolic pattern of the substances gains special interest in the discussion of a growing incidence of side effects. Therefore, the in vitro biotransformation of TRO was studied. Liver microsomes were prepared from different species, including the rat, rabbit, and mouse as well as from one human sample. Microsomal proteins were incubated for various periods and concentrations of TRO and its metabolites were analyzed by reversed-phase high-performance liquid chromatography (HPLC). In vitro metabolism resulted in the formation of activated metabolites by hydroxylation at position 4. In addition, side-chain oxidation resulted in the formation of IFO and CYC. IFO was the predominating metabolite of this pathway, with a 5- to 6-fold excess being noted as compared with CYC in rats and mice. The rabbit species showed similar CYC and IFO formation; in the single human sample, only IFO could be detected. In rats, the Michaelis constant (Km) for biotransformation to IFO was 398 microM, with the maximal volume (Vmax) being 70.8 nmol 120 min-1 mg protein-1, the corresponding values for biotransformation to CYC were 348 microM and 13.30 nmol 120 min-1 mg protein-1. On the basis of its structural similarity and the current knowledge of oxazaphosphorine metabolism, CYC was expected to be the main metabolite of TRO. The predominance of IFO was unexpected, but the observed metabolic profile promises numerous interesting aspects for the clinical use of TRO.