**1. Introduction**

Maytansine was first isolated in 1972 from the plant *Maytenus* ovatus [1] and showed potent cytotoxic effects in cell-based systems and efficacy in animal tumor models by binding to tubulin and blocking microtubule assembly [1–5]. However, maytansine failed as an anticancer drug in human clinical trials because of its unacceptable systemic toxicity [5–7]. Many maytansinoids, chemical derivatives of maytansine, showed higher cytotoxicity—by 100–1000 times—than other tubulin inhibitors, vincristine and vinblastine, in cancer cell lines in vitro [7,8]. The structure–antitumor activity relationship revealed that the ester side chain of maytansine plays an important role in the anti-tumor activity as well as tubulin binding [8]. Maytansinoids with potent cytotoxicity are clinically used and studied as the cytotoxic component of antibody–drug conjugates (ADCs) or aptamer-drug conjugates to reduce side effects and increase treatment effectiveness [7–15]. Mertansine

(Figure 1, called DM1), a thiol-containing maytansinoid, is attached to a monoclonal antibody through a reaction of the thiol group with a linker to create an ADC. Several ADCs containing mertansine have been developed, including bivatuzumab mertansine, cantuzumab mertansine, lorvotuzummab mertansine, and trastuzumab emtansine (T-DM1, Kadcyla ®) [6–15]. T–DM1 is an ADC drug approved in early 2013 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer that combines the biological activity of HER2 antibody (Herceptin or trastuzumab) with the targeted delivery of a potent antimicrotubule agent mertansine to HER2-expressing breast cancer cells [16–20]. A meta-analysis of a total of five randomized clinical trials involving 3720 patients with HER2-positive metastatic breast cancer revealed that T-DM1 significantly prolonged the progression-free survival and overall survival with tolerated toxicity compared to other anti-HER2 therapies [20]. However, patients who received T-DM1 treatment exhibited a significantly higher risk ratio of hepatotoxicity and thrombocytopenia [20]. – – –

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**Figure 1.** The chemical structure of mertansine.

5′ – – Cytochrome P450s (CYPs) and uridine-5 ′ -diphospho-glucuronosyltransferases (UGTs) are critical drug-metabolizing enzymes and are often involved in drug–drug interactions (DDIs) [21–27]. The in vitro inhibitory and induction potentials of drugs on CYPs and UGTs in human liver microsomes and hepatocytes have been evaluated to help identify clinical DDIs [26].

α 1′ −1 After an intravenous injection of [ <sup>3</sup>H]-mertansine at 0.2 mg/kg in rats, the radioactivity of mertansine was rapidly cleared from the blood and extensively distributed to highly perfused organs such as liver, kidney, spleen, lungs, heart, adrenal, and the gastrointestinal tract with high tissue-to-blood radioactivity ratios (ca. 1~11) for 24 h, declining to minimal levels by 120 h [28]. The majority of dosed mertansine radioactivity was recovered in feces over 120 h, with biliary excretion as the major route (~46% of dosed radioactivity over 72 h), but 5% of dosed radioactivity was recovered in urine over 120 h [28,29]. Mertansine was extensively metabolized to 11 metabolites via *S*-oxidation, hydrolysis, *S*-methylation, and glutathione conjugation [28,30,31]. It competitively inhibited CYP2C8-mediated paclitaxel 6α-hydroxylation and CYP2D6-mediated dextromethorphan *O*-demethylation with *K<sup>i</sup>* values of 11 and 14 µM, respectively, in human liver microsomes; mertansine also inactivated midazolam 1 ′ -hydroxylation in recombinant human CYP3A4 with a *K<sup>i</sup>* of 3.4 µM and a *k*inact of 0.058 min −1 , but it exhibited no induction potential up to 1 µM [31,32].

– Other tubulin inhibitors, such as colchicine and monomethyl auristatin E (MMAE), have been reported to downregulate CYP mRNA expression through the disruption of the microtubulin cellular skeletal structure that is necessary for the proper functioning of nuclear receptor signaling cascades [33–35]. However, to our knowledge, no studies have investigated the inhibitory potential of mertansine on UGTs, the second major group of enzymes responsible for drug metabolism [27], in human liver microsomes and the suppression potential of mertansine on mRNA expression or activities of major CYPs and UGTs in human hepatocytes.

The purpose of this study was to investigate the in vitro inhibitory potentials of mertansine on human UGT activities including UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 in ultrapooled human liver microsomes and to evaluate the effect of mertansine on the mRNA levels of human CYP1A2, CYP2B6, CYP3A4, CYP2C8, CYP2C9, CYP2C19, UGT1A1, UGT1A4, and UGT1A9 in human hepatocytes to assess the potential for mertansine-induced drug interactions.
