**3. Results**

### *3.1. Inhibition of UGT Enzyme Activities by Mertansine in Human Liver Microsomes*

Mertansine inhibited UGT1A1-catalyzed SN-38 glucuronidation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-β-glucuronidation, and UGT1A4-catalyzed trifluoperazine *N*-β-d-glucuronidation with IC<sup>50</sup> values of 16.2 µM, 6.4 µM, and 23.3 µM, respectively, but negligibly inhibited UGT1A6-catalyzed *N*-acetylserotonin β-d-glucuronidation, UGT1A9-catalyzed mycophenolic acid β-d-glucuronidation, and UGT2B7-catalyzed naloxone 3-β-d-glucuronidation in human liver microsomes at 50 µM (Figure 2, Table 1). β β acetylserotonin β mycophenolic acid β β

Mertansine noncompetitively inhibited UGT1A1-catalyzed SN-38 glucuronidation with a *K*<sup>i</sup> value of 13.5 µM, and competitively inhibited UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation and UGT1A4-catalyzed trifluoperazine *N*-β-D-glucuronidation, with *K*<sup>i</sup> values of 4.3 and 21.2 µM, respectively (Figure 3, Table 1). μ β μ

5′ **Figure 2.** Inhibitory effects of mertansine on six uridine 5 ′ -diphospho-glucuronosyltransferase (UGT) enzyme activities in ultrapooled human liver microsomes. The cocktail UGT substrate concentrations contained 0.5 µM SN-38 for UGT1A1, 2 µM chenodeoxycholic acid for UGT1A3, 0.5 µM trifluoperazine for UGT1A4, 1 µM *N*-acetylserotonin for UGT1A6, 0.2 µM mycophenolic acid for UGT1A9, and 1 µM naloxone for UGT2B7. Data are expressed as means ± SD (*n* = 3).

β

β

β

acetylserotonin β Mycophenolic acid β

**Omeprazole 50 μM**

**Omeprazole 50 μM**

– – – – – μ

– –

**Omeprazole 50 μM**

**Omeprazole 50 μM**

– –

– – – – – μ




β μ μ μ μ μ μ μ μ μ μ μ μ **Figure 3.** Dixon plots for the inhibitory effects of mertansine on (**A**) UGT1A1-catalyzed SN-38 glucuronidation, (**B**) UGT1A3-catalyzed chenodeoxycholic acid 24-acyl glucuronidation, and (**C**) UGT1A4-catalyzed trifluoperazine *N*-β-d-glucuronidation in ultrapooled human liver microsomes. Several substrate concentrations were evaluated: (**A**) SN-38; 0.2 µM ( β μM () μ μ μ μ μ μ μ μ μ μ μ ); 0.5 µM – 1 ((), ); 1 µM (H); 2 µM (▽); (**B**) chenodeoxycholic acid; 0.5 µM ( β μM () μ μ μ μ μ μ μ μ μ μ μ – ); 1 µM ( 1 (), ); 2 µM (H); 5 µM (▽); and (**C**) trifluoperazine; 0.2 µM ( β μM () μ μ μ μ μ μ μ – ); 0.5 µM (1 (), ); 1 µM (H); 2 µM (▽). Data are expressed as means ± SD (*n* = 3).

#### μ μ μ μ *3.2. E*ff*ects of Mertansine on CYP and UGT mRNA Levels in Human Hepatocytes*

– – – In the MTS colorimetric assay, mertansine did not cause toxicity in human hepatocytes (lot 319), as the viability of hepatocytes following 48 h mertansine treatment (1.25–6250 nM) was over 96.2%.

– – μ – – – – μ – – – – – – μ – – μ – – – – – – – – – μ – – μ – – – – μ – – – – μ – – μ The functionality of the hepatocyte was confirmed by the increase of mRNA levels and enzyme activities of CYPs following 48 h treatment with prototypical inducers using RT-PCR and LC-MS/MS, respectively, compared to the vehicle (Table 2). Fifty micromoles of omeprazole, a representative aromatic hydrocarbon receptor inducer (AHR), increased the CYP1A2 mRNA levels by enhancing the AHR binding to the promoter region of CYP1A2 [38] and CYP1A2-mediated phenacetin *O*-deethylase activity by 58.7–299.3 and 11.7–61.8 fold, respectively (Table 2). 10 µM rifampin, a potent pregnane X receptor (PXR) inducer, increased mRNA levels of CYP3A4 by enhancing the PXR binding to the promoter region of CYP3A4 [39] and CYP3A4-mediated midazolam 1 ′ -hydroxylase by 74.0–146.7 and 3.6–9.8 fold, respectively (Table 2). Additionally, 10 nM CITCO increased CYP2B6 mRNA levels and CYP2B6-mediated bupropion hydroxylase activity by 5.6–8.7 and 3.8–15.7 fold, respectively (Table 2), which was mediated by the transcriptional activation by the enhancement of constitutive androstane receptor binding to the promoter region of CYP2B6 [40]. 10 µM rifampin increased mRNA levels of CYP2C8, CYP2C9, CYP2C19, UGT1A1, UGT1A4, and UGT1A9 by 3.7–4.8, 2.9–5.3, 2.0–2.2, 2.5–3.0,

– –

–

– –

μ

–

3.9–4.5, and 2.0–2.2 fold, respectively, and 50 µM omeprazole increased the mRNA levels of UGT1A1 and UGT1A4 by 3.9–7.0 and 3.3–4.1 fold, respectively, in three human hepatocytes (Table 2).

**Table 2.** Effects of prototypical inducers such as omeprazole, 6-(4-chlorophenyl)imidazo[2,1-b] (1,3)thiazole-5-carbaldehyde-*O*-(3,4-dichlorobenzyl)oxime (CITCO), and rifampicin on the mRNA expression of cytochrome p450s (CYPs) and UGTs and the enzyme activities of CYP1A2, CYP2B6, and CYP3A4 after 48 h treatment in three human hepatocytes (lots 319, 321, and 361). Data are expressed as means ± SD (*n* = 3).



Mertansine led to the dose-dependent suppression of mRNA expression of CYP1A2 (from 1.2 to 0.22 fold), CYP2B6 (from 1.2 to 0.18 fold), and CYP3A4 (from 1.1 to 0.29 fold) in three human hepatocytes (Figure 4A). Mertansine decreased the activities of CYP1A2-mediated phenacetin *O*-deethylase by 27.8–79.0%, CYP2B6-mediated bupropion hydroxylase by 23.9–93.1%, and CYP3A4-mediated midazolam 1′ -hydroxylase by 30.8–62.7%, compared to the enzyme activities treated with the vehicle in three human hepatocytes (Figure 4B).

Mertansine dose-dependently suppressed the mRNA levels of CYP2C8 (from 1.2 to 0.09 fold), CYP2C9 (from 1.2 to 0.32 fold), CYP2C19 (from 1.3 to 0.23 fold), UGT1A1 (from 1.1 to 0.37 fold), UGT1A4 (from 1.1 to 0.45 fold), and UGT1A9 (from 1.2 to 0.09 fold), in three human hepatocytes (Figure 5). Table 3 lists the IC<sup>50</sup> values for mertansine on the suppression of mRNA expression of CYPs and UGTs in three human hepatocytes.

– **Figure 4.** Effects of mertansine on (**A**) the mRNA levels of CYP1A2, CYP2B6, and CYP3A4; and (**B**) the activities of CYP1A2-catalyzed phenacetin *O*-deethylase, CYP2B6-catalyzed bupropion hydroxylase, and CYP3A4-catalyzed midazolam hydroxylase compared to the vehicle (0.1% DMSO) after 48 h mertansine treatment (1.25–2500 nM) in three human hepatocytes: lots 319 ( β μM () μ μ μ μ μ μ μ – ), 321 ( 1 (), ), and 361 (H). Data are expressed as means ± SD (*n* = 3).

μ μ μ μ

– – – – – μ

– –

**Omeprazole 50 μM**

– – μ

– –

–

– –

μ

–

 **Figure 5.** Effects of mertansine on mRNA levels of CYP2C8, CYP2C9, CYP2C19, UGT1A1, UGT1A4, and UGT1A9 after 48 h treatment in three human hepatocytes: lots 319 ( β μM () μ μ μ μ μ μ μ – ), 321 ( 1 (), ), and 361 (H). Data are expressed as means ± SD (*n* = 3).

–

– –

μ

–

μ μ μ μ

– – – – – μ

– –

**Omeprazole 50 μM**

– – μ

– –


**Table 3.** IC<sup>50</sup> values for mertansine on the suppression of mRNA expression of CYPs and UGTs after 48 h mertansine treatment (1.25–2500 nM) in three human hepatocytes (lots 319, 321, and 361).

#### **4. Discussion**

In this study, the effects of mertansine on the inhibition of UGT activities in human liver microsomes and its effects on mRNA expression of CYPs and UGTs in human hepatocytes were evaluated to assess the potential for mertansine-induced drug interactions.

Mertansine was a noncompetitive inhibitor of UGT1A1-catalyzed SN-38 glucuronidation with a *K<sup>i</sup>* value of 13.5 µM, and a competitive inhibitor of UGT1A3-catalyzed 24-acyl-β-glucuronidation and UGT1A4-catalyzed trifluoperazine *N*-β-d-glucuronidation with *K<sup>i</sup>* values of 4.3 and 21.2 µM, respectively, in human liver microsomes (Figure 3). These findings suggest the potential for DDIs between mertansine and UGT1A1, UGT1A3, or UGT1A4 substrates when used concomitantly. However, the maximum plasma concentrations of mertansine were 7.2 ± 2.7 nM, with the highest level of 30 nM after the intravenous infusion of 3.6 mg/kg T-DM1 every 3 weeks in HER2-positve breast cancer patients [16–19]. Therefore, the ratio of maximal unbound plasma concentrations of mertansine to *K<sup>i</sup>* values (0.00004–0.0002) was much lower than the ratio indicating the likelihood of drug interaction (0.1), suggesting that mertansine-induced drug interactions via the inhibition of UGT activity are unlikely during T-DM1 therapies.

In addition, although mertansine is a competitive inhibitor of CYP2C8 and CYP2D6 activities with *K<sup>i</sup>* values of 11 and 14 µM, respectively, and it also irreversibly inhibits CYP3A4 activity with *K<sup>i</sup>* of 3.4 µM and *<sup>k</sup>*inact of 0.058 min−<sup>1</sup> , mertansine would not cause serious CYP-mediated DDI during the T-DM1 therapies considering the plasma concentrations [32].

The mRNA levels and enzyme activities of CYP1A2, CYP2B6, and CYP3A4 were induced to levels comparable to typical inducers, such as omeprazole, CITCO, and rifampin, following 48 h treatment in three human hepatocytes (Table 2), indicating that the induction system used herein was reliable. The induction effects of mertansine on CYPs and UGTs were assessed using therapeutic to clinically non-achievable high concentrations (1.25–2500 nM) in three human hepatocytes. Mertansine dose-dependently suppressed the mRNA expression of CYP1A2, CYP2B6, and CYP3A4 with IC<sup>50</sup> values of 93.7 ± 109.1, 36.8 ± 18.3, and 160.6 ± 167.4 nM, respectively, in thee human hepatocytes (Figure 4A, Table 3). Additionally, mertansine decreased the activities of CYP1A2-mediated phenacetin *O*-deethylase, CYP2B6-mediated bupropion hydroxylase, and CYP3A4-mediated midazolam 1 ′ -hydroxylase by mean values of 48.3%, 63.5%, and 39.7%, respectively, at the highest concentration (2500 nM) in three human hepatocytes (Figure 4B). Mertansine dose-dependently suppressed the mRNA expression of CYP2C8, CYP2C9, CYP2C19, UGT1A1, and UGT1A9 mRNA levels with IC<sup>50</sup> values of 32.1 ± 14.9, 578.4 ± 452.0, 539.5 ± 233.4, 856.7 ± 781.9, and 54.1 ± 29.1 nM, respectively, with a little suppression of UGT1A4 mRNA levels (IC<sup>50</sup> value > 2500 nM) (Figure 5, Table 3). These suppressions were not likely to be due to cytotoxic effects because the viability of hepatocytes was not affected by mertansine treatment (1.25–6250 nM). The suppression of CYP mRNA by mertansine was similar to those of other tubulin inhibitors, such as MMAE and colchicine [33–35]. In previous studies, 100 and 1000 nM MMAE treatment suppressed CYP1A2, CYP2B6, and CYP3A4 mRNA expression by 61–90% and 95–97%, respectively, and decreased CYP activities by 40–71% and 45–81%, respectively, in three human hepatocytes [32]. These findings support the idea that the suppression of CYP and UGT mRNA levels by mertansine may result from the disruption of cytoskeletal structures formed by microtubule networks, which are important for the functioning of the nuclear receptor signaling cascade [33–35,41]. These in vitro results suggest the clinical evaluation of the DDI potential of mertansine with CYP1A2, CYP2B6, CYP3A4, CYP2C8/9/19, UGT1A1, and UGT1A9 substrates.

Several ADCs with mertansine as a payload have been under clinical trials since the approval of T-DM1 [7,10–14]. Liver is the major organ for the distribution and metabolism of antibody maytansinoid conjugates and its catabolites, and the hepatic concentrations of mertansine or ravtansine therefore depend on the catabolism of ADC within the liver [7,18,29,42]. The extensive tissue distribution of mertansine after the administration of mertansine itself in rats led to higher hepatic levels of mertansine compared to plasma levels [28]. Although the maximal plasma concentration of the catabolite mertansine is low (≤7.2 ± 2.7 nM) in T-DM1 treated cancer patients [16–19], a clinical evaluation of DDIs regarding the reduced mRNA levels by repeated treatment of T-DM1 and the CYP1A2, CYP2B6, CYP2C8/9/19, CYP3A4, UGT1A1, and UGT1A9 substrates may be necessary on the basis of these in vitro findings.
