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Objective: This study focuses on the pharmacokinetics of trofosfamide (TRO) and metabolites after oral administration of TRO. Methods: Twelve patients with solid tumors and non-Hodgkin lymphomas were treated with 450 mg TRO orally for 7 days. TRO and the stable metabolites ifosfamide (IFO), cyclophosphamide (CYC), 2-and 3-dechloroethylifosfamide (2-DCE, 3-DCE) were determined by GC and the sum of the 4-OH-metabolites was measured by HPLC. Results: A fast metabolism of TRO with a half-life of about I h was observed. IFO was the main stable metabolite, whereas CYC was only detected in minor quantities. The peak levels and the AUC of the 4-OH-metabolites were 9.5 and 4.3 times higher than observed after an equimolar IFO dose. Only 6% of the administered dose was recovered in urine within 24 hours as stable metabolites. TRO was under limit of detection. Conclusions: Our results confirm that dechloroethylation of TRO to IFO is a major metabolic pathway. Additionally, we found considerable 4-hydroxylation not shown previously. With respect to the low levels of IFO and CYC observed, the sum of 4-OH-metabolites cannot be explained by hydroxylation of these metabolites only. Hence, we assume a direct 4-hydroxylation of TRO occurring to a high extent. Bioavailability of TRO could not be calculated directly, because TRO is only available as an oral formulation. The bioavailability of oral IFO, however, is reported to be almost 100%. Therefore, after normalization of the dose, a bioavailability of 32% for IFO after oral TRO could be calculated. Thus, in contrast to previous reports, direct 4-hydroxylation of TRO seems to be the main metabolic pathway.

Metabolic activation of ifosfamide (IFO) leads to the active 4-hydroxy-metabolite and to a substantial liberation of chloroacetaldehyde (CAA). CAA has been presumed responsible for side effects of IFO. We recently have shown cytotoxic effects of CAA against human tumor cells in vitro. The aim of this study was to demonstrate antitumor effects of CAA in vivo, and to compare its potency to 4-OH-IFO. Pharmacokinetics of IFO and metabolites were evaluated after infusion of 250 mg/kg IFO in mice. The area under the curve (AUC) for 4-hydroxyifosfamide (4-OH-IFO) and CAA were 138. 5 and 102.4 micromol. h/liter, respectively. To compare pharmacokinetics and antitumor effects, the mice received isolated infusion of 4-OH-IFO or CAA in equimolar doses to IFO. Administration of 4-OH-IFO yielded AUC values comparable with those obtained after administration of the parent drug. In contrast, infusion of isolated CAA via tail vein gave a low AUC value of 51.5 micromol. h/liter due to slow flow in the tail vein and rapid degradation. Administration of the parent drug gave highly cytotoxic intratumoral peak concentrations of 25 and 12 micromol/kg tumor weight for 4-OH-IFO and CAA in MX1 xenotransplanted nude mice. Both IFO and isolated 4-OH-IFO led to complete remissions. Administration of isolated CAA (75 mg/kg) delayed tumor growth significantly. The equitoxic dose of isolated 4-OH-IFO was 40 mg/kg. On a molar basis CAA was seven times less potent as 4-OH-IFO. However, on the basis of achieved AUC values, CAA seems to exhibit a similar antitumor activity to 4-OH-IFO.

The initial metabolism of ifosfamide (IFO) consists of two different pathways, which lead to the alkylating metabolite 4-hydroxy-IFO and to chloroacetaldehyde (CAA). CAA has been reported to cause side effects, such as neuro- and nephrotoxicity, whereas no direct antitumor effect has been described thus far. Therefore, two human tumor cell lines (MXI and S117) and a renal tubular cell line (Landa Leiden) were exposed to 4-hydroxy-IFO, CAA, and a combination of both. The concentrations used were in the same range as measured in the blood of 10 patients treated with 5 g/m2 IFO. The cell survival was measured using the MTT assay. Similar dose-response curves were found for both metabolites. For the MX1 tumor, the IC50s of 4-hydroxy-IFO and CAA were 10.8 and 8.6 microM, respectively. For the reduction of S117 cell survival, higher concentrations of the metabolites were needed (25.0 microM 4-hydroxy-IFO and 15.3 microM CAA). Combination treatment of the cells resulted in an approximately additive effect. Both metabolites exhibited similar toxicity against Landa Leiden cells. Our results indicate that CAA has its own cytotoxic profile against tumor cells. Hence, we conclude that the molecular mechanism of action of IFO seems to be only in part an alkylating effect and that CAA may play an important role in the therapeutic efficacy of IFO.