Metabolic Profile of [C]Bendamustine in Rat Urine and Bile: Preliminary Structural Identification of Metabolites

Bendamustine, a bifunctional alkylating agent, is currently in clinical trials for the treatment of hematological and other malignancies. Although it has been used in the former East Germany for more than 30 years, very limited information is available on its biotransformation. The objective of this investigation was to obtain information on the structures of metabolites excreted into rat urine and bile to understand the metabolic fate of bendamustine in vivo. Metabolites of [C]bendamustine hydrochloride in rat urine and bile were determined using liquid chromatography-mass spectrometry (MS) in parallel with on-line radioactivity detection in samples obtained after i.v. dosing of 3 mg/kg. A total of 17 radioactive peaks were identified in rat urine and 10 in rat bile (2 were unique to bile). Four of these metabolites had been previously reported, whereas 15 are novel. Proposed structures of all metabolites detected are based on MS spectra generated from a linear ion trap mass spectrometer. These results suggest that the major metabolic pathways in rat are oxidative and/or hydrolytic dehalogenation, oxidation, carboxylic acid formation, N-dealkylation, sulfation, and glutathione and cysteine (probably via glutathione) conjugation. The cysteine-conjugated compounds are observed in their N-acetylated cysteine (mercapturic acid) forms. Bendamustine, a bifunctional alkylating agent (Fig. 1), is currently in late-stage clinical trials for the treatment of hematological and other malignancies (Pönisch et al., 2006). Although it has been used in the former East Germany for more than 30 years (Teichert et al., 2005; Gandhi, 2002), limited information is available on its biotransformation. Pharmacokinetic studies in mice indicated that bendamustine concentrations in plasma rapidly decreased after i.v. dosing (Weber et al., 1991). Bendamustine metabolites excreted into rat bile and urine were investigated with C-labeled compound using thin-layer chromatography analysis, and conjugated and hydroxylated metabolites were observed (Bezek et al., 1991). Hydroxylated, N-demethylated, and cysteine-conjugated metabolites of bendamustine were identified in human bile, urine, and plasma (Teichert et al., 2005), and two phase I metabolites ( -hydroxyand N-desmethyl-bendamustine) were further characterized by LC-MS and nuclear magnetic resonance spectroscopy after isolation from cytochrome P450 incubation mixtures (Teichert et al., 2007). Although seven metabolites were tentatively identified from the above investigations, four of which were also observed in the studies reported herein and designated M6, M16, M20, and M21 (see below), the complete metabolic fate of bendamustine in vivo remains to be fully elucidated. The other three metabolites previously observed were mono (with or without hydrolysis of the opposite chlorine)or di-cysteine conjugates of the Nchloroethyl groups. The objective of this investigation was to obtain information on the structures of metabolites excreted into rat urine and bile to understand the routes of metabolic clearance of bendamustine in vivo. The rat is the principle rodent species used for safety evaluation of bendamustine. In a mass balance study in rats, approximately 90% of the dose was recovered in excreta during 7 days after a single 3-mg/kg i.v. dose of [C]bendamustine HCl, with most ( 77%) of the radioactivity being recovered during the first 24 h. Urine fractions (0–4, 4–8, and 8–24 h) were obtained on dry ice during this mass balance study. After analysis for total radioactivity, the samples were refrozen at approximately 80°C for later LC-MS analysis. Because substantial radioactivity ( 49%) had been recovered in the feces during this study, biliary excretion of compound-derived radioactivity was presumed. Therefore, the bile duct of a male Sprague-Dawley rat was cannulated, a 3-mg/kg i.v. dose of [C]bendamustine HCl was administered, and bile was collected in fractions on wet ice through 2 h after dosing and analyzed immediately by LC-MS. The preliminary results for the identification of the radioactive components observed in rat urine and bile are reported herein. Besides the previously characterized metabolites (Teichert et al., 2005), 15 novel metabolites were identified in this study. Materials and Methods Materials. Reference standards were obtained from AMCIS (Bubendorf, Switzerland) for bendamustine hydrochloride, 5-[bis(2-chloroethyl)amino]-1methyl-1H-benzimidazole-2-butanoic acid monohydrochloride (purity 97.0– 102.0%); a hydroxylated metabolite (M20 in this report), 4{5-[bis-(2-chloroethyl)amino]-1-methyl-1H-benzoimidazol-2-yl}-4-hydroxybutyric acid (purity 89.4%); a N-desmethyl metabolite (M21), 4{5-[bis-(2-chloroethyl)-amino]1H-benzoimidazol-2-yl}-butyric acid (purity 71.1%); the monohydrolysis metabolite (M16; purity 82.5%); and the dihydrolysis metabolite (M6; purity 92.0%). Naming of metabolites in this report is different from naming in previous publications (e.g., Teichert et al., 2005). [C]Bendamustine HCl (purity 98%; specific activity 26.1 Ci/mg) was obtained from BioDynamics Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.107.015958. ABBREVIATIONS: LC, liquid chromatography; MS, mass spectrometry. 0090-9556/07/3510-1744–1753$20.00 DRUG METABOLISM AND DISPOSITION Vol. 35, No. 10 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 15958/3250812 DMD 35:1744–1753, 2007 Printed in U.S.A. 1744 at A PE T Jornals on A uust 4, 2017 dm d.aspurnals.org D ow nladed from (Rushden, UK). Acetonitrile, methanol, and formic acid were high-performance liquid chromatography grade and were obtained from VWR (West Chester, PA) or J.T. Baker (Phillipsburg, NJ). Water was obtained from a Milli-Q Ultrapurification System (Millipore Corporation; Milford, CT). Ultima-Flo liquid scintillation cocktail was purchased from PerkinElmer Life and Analytical Sciences (Shelton, CT). Other chemicals used were generally of reagent grade or better. Animals and Sampling Procedures. A male Sprague-Dawley rat was commercially obtained from Charles River Laboratories (Raleigh, NC) for the bile studies. After receipt at Cephalon (Cephalon, Inc., West Chester, PA), the animal was housed under standard conditions with food and water provided ad