Enantioselective Metabolism of Ifosfamide by the Kidney KATARINA ALEKSA, SHINYA ITO, AND GIDEON KOREN * Division of Clinical Pharmacology and Toxicology, Hospital for Sick Children, Toronto, Canada and Department of Pharmacology, Faculty of Medicine, University of Toronto ABSTRACT Ifosfamide (IF), a potent chemotherapeutic agent for solid tumors, is known to cause high rates of nephrotoxicity, which is most likely due to the renal pro- duction of the metabolite chloroacetaldehyde. Enantioselective oxidation of IF has been shown in the liver but has never been reported in the kidney. Using porcine and human kidney samples, as well as the renal porcine cell line LLCPK-1, we document enantioselective metabolism of IF with prevalent production of the N-dechloroethylifos- famide (DCEIF) metabolites from the (S)-IF enantiomer compared to the amount of N-DCEIF metabolites produced from the (R)-IF enantiomers. Since IF enantiomers appear to be equally effective in chemotherapy, these results suggest that replacing the clinically standard racemic mixture of IF with (R)-IF may decrease renal metabo- lism of the drug and hence may decrease nephrotoxicity. Chirality 18:398–405, 2006. V V C 2006 Wiley-Liss, Inc. KEY WORDS: kidney; CYP3A; CYP2B6; ifosfamide; nephrotoxicity; enantioselective metabolism Ifosfamide (IF) is a pro-drug which requires in vivo acti- vation to produce pharmacologically active, cytotoxic spe- cies. 1–3 Since the molecule contains an asymmetrically sub- stituted phosphorous atom, it exists in two enantiomeric forms, (R)-ifosfamide [(R)-IF] and (S)-ifosfamide [(S)-IF]. 4,5 The medicinal preparation is a 50:50 [(R/S):50/50-ifosfa- mide] mixture of the two enantiomers. 5 Both enantiomers are metabolized by the CYP P450 mixed-function oxidase system in the liver, 2,6,7 specifically, CYP3A4, 3A5, and 2B6, to exert their chemotherapeutic effect (Fig. 1). 4,5,8 IF is metabolized by two main pathways. In the first path- way it undergoes ring hydroxylation, resulting in the pro- duction of the chemically unstable intermediate 4-hydroxy- ifosfamide (4-OHIF). 9 This metabolite rapidly converts to the active alkylating agent isophosphoramide mustard, which is responsible for the DNA-alkylating and cross-link- ing activities that ultimately cause cell death in the prolifer- ating target cells. 6,10,11 In the second pathway, the oxazophosphorine ring of IF may undergo side-chain dealkylation (up to 48% of the dose), 1,11,12 resulting in the production of N-2-dechlroethyl- ifosfamide (2-DCEIF) and N-3-dechloroethylifosfamide (3- DCEIF), which are also chiral and exist in both R and S forms (Fig. 1). 5 The metabolism of IF via dechloroethylation is both regioselectives and enantioselective. 5 The (R)-IF enantiomer is metabolized to (R)-2-DCEIF by CYP3A4 and (S)-3-DCEIF by CYP2B6, while the (S)-IF enantiomer is metabolized to (R)-3-DCEIF by CYP3A4 and (S)-2-DCEIF by CYP2B6. 4,5 The production of the dechloroethyl metabolites results in the production of equimolar amounts of chloro- acetaldehyde, 4,6,13 the agent largely believed to be responsi- ble for the nephrotoxic 9,14,15 and neurotoxic 16 adverse effects commonly associated with the administration of rac-IF. The metabolism of IF by the kidney has been shown by several groups. 17–24 The renal formation of chloroace- taldehyde has been hypothesized to be the mechanism of nephrotoxicity by rac-IF. However, unlike the liver, the enantioselective metabolism of rac-IF by the kidney has not been investigated. The objective of the present study is to investigate the enantioselective metabolism of IF by human and porcine kidney and by a renal cell line. MATERIALS AND METHODS Chemicals. Racemic ifosfamide (rac-IF) [(R/S):50/50- ifosfamide], racemic 2-dechloroethylifosfamide (2-DCEIF) [(R/S):50/50À2-DCEIF], racemic 3-dechloroethylifosfamide (3-DECIF) [R/S):50/50À3-DCEIF] and trophosphamide were kindly provided by Dr. Ulf Neimyer of Asta Medica This work was supported by a grant from the Canadian Institutes for Health Research (CIHR); K. Aleksa was supported by a studentship from the CIHR. G. Koren holds the Ivey Chair in Molecular Toxicology, The University of Western Ontario. *Correspondence to: Gideon Koren, MD, FRCPC, Division of Clinical Pharmacology and Toxicology, The Hospital for Sick Children, 555 Uni- versity Avenue, Toronto, Ontario, Canada, M5G 1X8. E-mail: gkoren@sickkids.ca Received for publication 29 October 2005; Accepted 20 January 2006 DOI: 10.1002/chir.20268 Published online 30 March 2006 in Wiley InterScience (www.interscience.wiley.com). Nonstandard abbreviations: IF (ifosfamide); 2-DCEIF (2-dechloroethylifosfa- mide); 3-DCEIF (3-dechloroethylifosfamide); N-DCEIF (2-dechloroethylifos- famide and 3-dechloroethylifosfamide); 4-OHIF (4-hydroxyifosfamide); race- mic ifosfamide [(þ)-(R)-ifosfamide þ (À)-(S)-ifosfamide) (50:50)]. CHIRALITY 18:398–405 (2006) V V C 2006 Wiley-Liss, Inc.