Taxane’s Substituents at C3 Affect Its Regioselective Metabolism: Different in Vitro Metabolism of Cephalomannine and Paclitaxel Jiang-Wei Zhang, Guang-Bo Ge, Yong Liu, Li-Ming Wang, Xing-Bao Liu, Yan-Yan Zhang, Wei Li, Yu-Qi He, Zheng-Tao Wang, Jie Sun, Hong-Bin Xiao, and Ling Yang Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (J.-W.Z., G.-B.G., Y.L., X.-B.L., Y.-Y.Z., W.L., L.Y.); Laboratory of Medical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (H.-B.X.); The Second Affiliated Hospital of Dalian Medical University, Dalian, China (L.-M.W., J.S.); Shanghai University of Traditional Chinese Medicine, Shanghai, China (Y.-Q.H., Z.-T.W.); and Graduate School of Chinese Academy of Sciences, Beijing, China (J.-W.Z., G.-B.G., Y.-Y.Z., W.L.) Received August 16, 2007; accepted November 21, 2007 ABSTRACT: To investigate how taxane’s substituents at C3 affect its metab- olism, we compared the metabolism of cephalomannine and pac- litaxel, a pair of analogs that differ slightly at the C3 position. After cephalomannine was incubated with human liver microsomes in an NADPH-generating system, two monohydroxylated metabolites (M1 and M2) were detected by liquid chromatography/tandem mass spectrometry. C4 (M1) and C6 (M2) were proposed as the possible hydroxylation sites, and the structure of M1 was con- firmed by 1 H NMR. Chemical inhibition studies and assays with recombinant human cytochromes P450 (P450s) indicated that 4- hydroxycephalomannine was generated predominantly by CYP3A4 and 6-hydroxycephalomannine by CYP2C8. The overall biotrans- formation rate between paclitaxel and cephalomannine differed slightly (184 vs. 145 pmol/min/mg), but the average ratio of metab- olites hydroxylated at the C13 side chain to C6 for paclitaxel and cephalomannine varied significantly (15:85 vs. 64:36) in five human liver samples. Compared with paclitaxel, the major hydroxylation site transferred from C6 to C4, and the main metabolizing P450 changed from CYP2C8 to CYP3A4 for cephalomannine. In the in- cubation system with rat or minipig liver microsomes, only 4-hy- droxycephalomannine was detected, and its formation was inhibited by CYP3A inhibitors. Molecular docking by AutoDock suggested that cephalomannine adopted an orientation in favor of 4-hydroxylation, whereas paclitaxel adopted an orientation favoring 3-p-hydroxyla- tion. Kinetic studies showed that CYP3A4 catalyzed cephalomannine more efficiently than paclitaxel due to an increased V m . Our results demonstrate that relatively minor modification of taxane at C3 has major consequence on the metabolism. Paclitaxel is one of the best antineoplastic drugs derived from the yew trees, and it has been found to be effective against a broad spectrum of cancers (Spratlin and Sawyer, 2007). The superior anti- tumor activity of paclitaxel, however, is undermined by limitations such as multidrug resistance, poor aqueous solubility, poor oral bio- availability, and toxicities (Frapolli et al., 2006). It is generally rec- ognized that poor oral bioavailability of paclitaxel results from its poor solubility (Singla et al., 2002) and P-glycoprotein efflux (Spar- reboom et al., 1997). To overcome these shortcomings, numerous novel taxanes superior to paclitaxel were synthesized (Ojima et al., 1996; Mastalerz et al., 2003; Barboni et al., 2005; Lockhart et al., 2007), and some of them such as BAY59-8862 (Sano et al., 2006), BMS-275183 (Broker et al., 2007), and MAC-321 (Lockhart et al., 2007) entered clinical trials. Many of these novel taxanes had modi- fied substituents at C3' of the C13 side chain, where two phenyls were replaced by other functional groups (Sano et al., 2006; Broker et al., 2007; Lockhart et al., 2007). These structural modifications made the novel taxanes effective against paclitaxel-resistant tumor cells, more soluble, and/or more potent. It is still not clear how these structural changes at C3' affect taxane’s metabolism property, which is crucial to efficacy, toxicity, route of administration, pharmacokinetics, and pharmacodynamics (Taniguchi et al., 2005; Spratlin and Sawyer, 2007). P450-mediated oxidative metabolism is the major elimination routine for taxanes (Anderson et al., 1995; Cresteil et al., 2002), which differ strikingly from one another in metabolism despite their structural similarity. For example, when benzamide at C3' in This work was supported by the 973 Program (2007CB707802) of the Ministry of Science and Technology of China, the National Natural Science Foundation of China (30640066), and the Dalian Institute of Chemical Physics Innovation and Ph.D. Exploration Fund of the Chinese Academy of Sciences. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.107.018242. ABBREVIATIONS: P450, cytochrome P450; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; HPLC, high-performance liquid chromatrography; RLM, rat liver microsome; HLM, human liver microsome; PLM, minipig liver microsome; PDB, protein data bank; RT, retention time; DH, bond dissociation energy; BAY59-8862, 13-(N-tert-butoxycarbonyl--isobutylisoserinyl)-14-hydroxy- baccatin-1,14-carbonate; BMS-275183, 3' -dephenyl-3' -tert-butyl-4-deacetyl-4-O-methoxycarbonyl-10-acetyldocetaxel; MAC-321, 3'-dephenyl-3'-furanyl-7-propionyl-docetaxel; SB-T-1102, 10-acetyl-3'-dephenyl-3'-(2-methylpropyl)docetaxel; SB-T-1214, 10-(cyclopropylcarbonyl)- 3'-dephenyl-3'-(2-methylpropenyl)docetaxel; SB-T-1216, 3'-dephenyl-10-(N,N-dimethylcarbamoyl)-3'-(2-methylpropenyl)docetaxel; IDN5390, 13-(N- Boc-3-i-butylisoserinoyl)-C-7,8-seco-10-deacetylbaccatin III. 0090-9556/08/3602-418–426$20.00 DRUG METABOLISM AND DISPOSITION Vol. 36, No. 2 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 18242/3302369 DMD 36:418–426, 2008 Printed in U.S.A. 418 at Univ of Chicago Sci Lib on August 7, 2008 dmd.aspetjournals.org Downloaded from