ROUTES OF IFOSFAMIDE METABOLISM

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Ifosamide (IFOS) and its isomer, cyclophosphamide (CPA), are members of the oxazaphosphorine class of alkylating agents used in cancer chemotherapy. IFOS is a recently approved drug whereas CPA has been used extensively as an antitumor and immunosuppressant agent for the last 30 years. IFOS, like CPA, undergoes microsomal activation. Unlike CPA, IFOS has N-dealkylation metabolites which may comprise a major route of elimination for some patients and may comprise a major route of elimination for some patients and may account for its different toxicity profile. A limitation in further understanding the pharmacology of IFOS and potential drug interactions is a lack of method to measure its tumor active metabolites. The specific aims of this project include: a) developing techniques to quantitate the metabolism of IFOS b) to compare the metabolism of CPA to IFOS and identify factors significantly influencing IFOS' disposition c) to determine whether the uroprotective agent mesna, significantly alters the amount of cytotoxic IFOS formed by oxidative metabolism and (d to characterize IFOS' metabolism in patients. The major limitation in studying oxazaphosphorine pharmacology has been the lack of chromophore in the ring structure. Methods to measure IFOS an its metabolites will involve modifying a TLC and HPLC method used for CPA. We have previously used a thin-layer chromatography (TLC) assay to measure CPA's metabolism in animals and humans. We wish to modify this TLC assay for the detection of IFOS' major metabolic products. 3H-IFOS is to be used. The radiolabel on the chloroethyl side chain attached to the endocyclic nitrogen atom will allow for the measurement of all metabolites except for acrolein and dechloroethyl-cyclophosphamide. A recently described ion pair HPLC method for CPA will be adapted for IFOS and compared to results obtained using the TLC method. After the optimal methods are developed for quantitating IFOS and its metabolic products, human studies will follow. With the development of TLC or HPLC techniques to quantitate IFOS' metabolism, factors which affect CPA's metabolism can be evaluated for IFOS. These factors include the influence of genetic P-450 isoenzyme inheritance patterns and the influence of known inhibitors of various P- 450 isoenzymes. IFOS' metabolism in female Sprague-Dawley rats will be compared with metabolism in female Dark Agouti rats, which lack cytochrome IID6. The metabolism of IFOS by isolated perfused liver preparations will be measured in the presence of agents which inhibit cytochrome P-450 including cimetidine, SKF-525A, metyrapone and ketoconazole. The glutathione scavenging agent, mesna, which is used in combination with IFOS, protects patients from the urotoxic IFOS metabolite, acrolein. Whether mesna alters the amount of IFO antitumor metabolites formed will be another factor evaluated by these models. In vitro microsomal IFOS activation will be used to further identify mechanisms of inhibition. When IFOS' metabolic routes have been quantitated using the HPLC and TLC methodology in animal models, IFOS pharmacokinetics can then be extended to newly diagnosed cancer patients. Using these techniques, the potential interaction between IFOS and mesna can be studied in humans.