Short Communication Structural Characterization of Novel Adenine Dinucleotide Phosphate Conjugates of Imatinib in Incubations with Rat and Human Liver Microsomes

Imatinib, a potent and selective protein tyrosine kinase inhibitor, has been approved for the treatment of chronic myelogenous leukemia and metastatic and unresectable malignant gastrointestinal stromal tumors. In vitro metabolism of imatinib was investigated in rat and human liver microsomes. Besides several oxidative metabolites and an N-desmethyl metabolite, as previous reported, a novel metabolite with a mass addition of 621 atomic mass units to the parent was detected as the major metabolite in the incubations with rat liver microsomes, using NADPH as a cofactor. The analysis of MS and MS data revealed that this metabolite corresponded to adenine dinucleotide phosphate (ADP ) conjugate of imatinib. The ADP adduct was scaled up from rat liver microsomal incubations and isolated for NMR analysis. NMR data confirmed and conclusively showed the conjugation had occurred between the pyridine nitrogen of imatinib to the ribose ring of ADP moiety. The formation of this adduct was enzymatic and required NADP as a reactant. In addition, ADP adducts of imatinib N-oxide and desmethyl imatinib were also detected as minor metabolites in the incubations with rat liver microsomes. In contrast, only trace levels of ADP adducts of imatinib and desmethyl imatinib were detected in the incubations with human liver microsomes. Imatinib-ADP adducts have been observed only in in vitro studies to date. The physiological role of these adducts is not clear, nor is their in vivo relevance. Imatinib (Gleevec) is an anticancer drug that selectively inhibits the BCR-ABL tyrosine kinase, and -platelet-derived growth factor receptors, as well as c-Kit receptor tyrosine kinase (Capdeville et al., 2002; Savage and Antman, 2002). The metabolism and disposition of imatinib was investigated in healthy volunteers after a single oral dose of 239 mg of C-labeled imatinib mesylate (Gschwind et al., 2005). Approximately 65 and 9% of the total systemic exposure corresponded to imatinib and its active N-desmethyl metabolite, respectively. In the excreta, imatinib and its N-desmethyl metabolites accounted for 28 and 13% of administrated dose, respectively. The remaining proportion represented mainly oxidative metabolites and Nor O-glucuronides. Thus, imatinib was predominantly cleared through oxidative metabolism in humans. Imatinib was metabolized in vitro mainly by CYP3A4 and to a lesser extent by CYP1A2, CYP2C9, CYP2C19, and CYP2D6 (Peng et al., 2005). In the present study, we report the detection and structural characterization of novel adenine dinucleotide phosphate (ADP ) adducts of imatinib after incubations with rat and human liver microsomes (HLM). Materials and Methods Materials. Imatinib mesylate was purchased from AK Scientific Inc. (Mountain View, CA). Rat liver microsomes (RLM; lot no. 33152) and HLM (lot no. 36170) were obtained from BD Gentest (Woburn, MA). Formic acid was purchased from Pierce (Rockford, IL). Methanol, acetonitrile, and water were obtained from Thermo Fisher Scientific (Waltham, MA). NADPH, NADP , and NAD glycohydrolase from porcine brain were purchased from Sigma-Aldrich (St. Louis, MO). In Vitro Incubations. Imatinib (10 M) was incubated with 1 mg/ml rat or HLM in 100 mM phosphate buffer, pH 7.4, containing 1 mM NADPH or NADP at 37°C for 1 h. Incubations of imatinib with boiled RLM in the presence of NADPH or NADP were also performed as negative controls. Imatinib (10 M) was also incubated with NAD glycohydrolase (NADase; 5 mg/ml suspension in 100 mM phosphate buffer) containing 1 mM NADP at 37°C for 1 h. The total incubation mixture was 500 l. The incubations were quenched with 250 l of acetonitrile. The samples were then mixed on a vortex mixer and centrifuged at 14,000 rpm for 10 min. An aliquot of 20 l was injected into the liquid chromatography-mass spectrometry (LC-MS) system for analysis. Analytical Methods. The LC-MS system consisted of an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA) and a LTQ mass spectrometer (Thermo Fisher Scientific). Separation was achieved on an YMC basic column (2.0 150 mm, 5 m; Waters, Milford, MA) at a flow rate of 0.2 ml/min. Mobile phases consisting of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B) were used under the following gradient conditions: 0 to 3 min, 10% B; 3 to 45 min, 10 to 50% B; 45 to 46 min, 50 to 95% B; 46 to 50 min, 95% B; 50 to 51 min, 95 to 10% B; and 51 to 56 min, 10% B. The UV detector was set at a wavelength of 270 nm. The LTQ mass spectrometer was operated in the positive ion mode with an electrospray ionization source. The ion source conditions were as follows: spray voltage, 5 kV; sheath and auxiliary gas (N2) flow, 60 and 10 (arbitrary units), respectively; capillary voltage, 48 V; tube lens, 136 V; capillary temperature, 250°C. Tandem mass spectrometry (MS/MS), MS, and MS experiments were performed under collision energy of 20 to 30% with an isolation mass window of 2 Da. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.023085. ABBREVIATIONS: ADP , adenine dinucleotide phosphate; HLM, human liver microsomes; RLM, rat liver microsomes; NADase, NAD glycohydrolase; LC-MS, liquid chromatography-mass spectrometry; MS/MS, tandem mass spectrometry. 0090-9556/08/3612-2414–2418$20.00 DRUG METABOLISM AND DISPOSITION Vol. 36, No. 12 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 23085/3408712 DMD 36:2414–2418, 2008 Printed in U.S.A. 2414 at A PE T Jornals on A ril 0, 2017 dm d.aspurnals.org D ow nladed from