The objective of the current study was to investigate the metabolism of cytochrome P450 (CYP) probe substrates in male Sprague Dawley rat liver microsomes and to determine their substrate specificities. Time and microsomal protein concentrations were varied to determine the linear conditions for each reaction. Appropriate substrate concentrations were chosen to determine the apparent K(m) and V(max) for 17 different reactions under initial rate conditions of protein and reaction time. All reactions appeared to follow Michaelis-Menten kinetics. Subsequently, each substrate was incubated at one to two times K(m) with each of 14 baculovirus cDNA-expressed rat CYP enzymes to determine the specificity of the reaction monitored. Of the 14 enzymes tested, seven were seen as the major rat CYP enzymes responsible for the majority of the substrate metabolism tested. Testosterone 2alpha- and 16alpha-hydroxylation reactions were conducted primarily by CYP2C11, and midazolam 4-hydroxylation and triazolam 1'-hydroxylation were preferentially catalyzed by CYP3A1/2, but specificity was otherwise generally poor. The results presented herein clearly indicate that care must be taken in interpretation of metabolism results obtained in rats using standard probe substrates, especially in extrapolation of those results to humans.
Chemical structures of seven cyanide adducts of imatinib have been identified or proposed based on high-resolution MS/MS data. Mechanisms for the formation of the conjugates were also proposed. The findings may help to understand the mechanism of hepatotoxicity of imatinib in humans.
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...
Medicinal chemists try to avoid certain organic functional groups, summarized in an ever-growing list, in order to avoid the potential bioactivation to reactive metabolites. To add to that alert list, we report herein that boronic acid-containing compound structures, such as those found in proteasome inhibitors bortezomib and ixazomib, can become bioactivated to chemically reactive imine amide metabolites. Test compounds, ixazomib and bortezomib, were incubated in vitro using human liver fractions containing cytosol and microsomes (S9) under conventional conditions in the presence of GSH. Metabolites were then analyzed using LC-MS(n) with or without online hydrogen-deuterium exchange (HDX) liquid chromatography coupled with an LTQ-Orbitrap. The exact mass measurements of both the precursor and product ions were acquired through data dependent acquisition and compared with theoretical values of proposed fragment ions. Upon deboronation catalyzed by cytochrome P450 enzymes, both test compounds formed imine amide metabolites that were identified by high resolution exact mass measurements in both normal aqueous and HDX HPLC-MS analysis. GSH conjugates were also identified and were postulated as nucleophilic addition of GSH to the imine amide metabolites. All mass spectrometric and HDX measurements of these GSH conjugates proved that the GSH unit was added to the carbon atom of the imine amide partial structure, hence demonstrating the electrophilic property of these imine amide metabolites. The awareness of the formation of electrophilic imine amide metabolites from boronic acid-containing compounds, where the boron atom is bonded to a carbon atom adjacent to an amide nitrogen, should help in drug candidate design and optimization with regard to avoiding potential bioactivation.
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