Metabolic Profile of [¹⁴C]Bendamustine in Rat Urine and Bile: Preliminary Structural Identification of Metabolites

Abstract

ABSTRACT: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 [14 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 n 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 14 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 (␥-hydroxy-and 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 understan...

Metabolite Profiling of Bendamustine in Urine of Cancer Patients after Administration of [14C]Bendamustine

“…The identification was supported by the highresolution m/z value and by the major product ions 258.1232 (Ϫ18.0104 Da, loss of water), 230.1283 (Ϫ46.0053 Da, loss of carboxyl moiety), and 217.0972 (Ϫ59.0364, loss of aminoacetaldehyde), which are consistent with those reported for this compound in rat urine (Chovan et al, 2007).…”

“…The types of metabolites that were observed in this study were generally consistent with those reported previously for rat (Chovan et al, 2007). The most abundant metabolite in rat urine and bile was a mercapturic acid conjugate of bendamustine, corresponding with Met22 in this study.…”

“…On the basis of this list, however, three metabolites were initially thought to be sulfate conjugates. Sulfate conjugation was assumed in rat urine (Chovan et al, 2007), and, moreover, the normal-resolution MS 2 spectra of these metabolites showed intense product ions resulting from loss of 80 Da, which is characteristic for sulfate conjugates (Levsen et al, 2005). However, evaluation of the elemental composition of the proposed metabolite structures with high-resolution MS revealed that the mass deviations were substantially higher than deviations of the other metabolites (range, Ϫ1 to Ϫ3 ppm).…”

“…Clinical and preclinical studies showed that bendamustine is extensively metabolized in vivo (Teichert et al, 2005(Teichert et al, , 2009Chovan et al, 2007). Two phase 1 metabolites resulting from metabolic conversions at the benzimidazole/butyric acid moiety: ␥-hydroxybendamustine and N-desmethylbendamustine have been identified (Teichert et al, 2007).…”

“…Among the metabolites detected in rat urine and bile, three metabolites were identified as glutathione conjugates and six as mercapturic acid conjugates. In addition, products of N-dealkylation, oxidation, carboxylic acid formation, and sulfate conjugation were postulated (Chovan et al, 2007). Human urine has been investigated for the presence of cysteine S-conjugates (Teichert et al, 2005), but the presence of other metabolites and the excretion of bendamustine metabolites over time have not been described.…”