Interindividual Variability in Cytochrome P450 3A and 1A Activity Influences Sunitinib Metabolism and Bioactivation

Abstract

Sunitinib is an orally administered tyrosine kinase inhibitor associated with idiosyncratic hepatotoxicity; however, the mechanisms of this toxicity remain unclear. We have previously shown that cytochromes P450 1A2 and 3A4 catalyze sunitinib metabolic activation via oxidative defluorination leading to a chemically reactive, potentially toxic quinoneimine, trapped as a glutathione (GSH) conjugate (M5). The goals of this study were to determine the impact of interindividual variability in P450 1A and 3A activity on sunitinib bioactivation to the reactive quinoneimine and sunitinib N-dealkylation to the primary active metabolite N-desethylsunitinib (M1). Experiments were conducted in vitro using single-donor human liver microsomes and human hepatocytes. Relative sunitinib metabolite levels were measured by liquid chromatography-tandem mass spectrometry. In human liver microsomes, the P450 3A inhibitor ketoconazole significantly reduced M1 formation compared to the control. The P450 1A2 inhibitor furafylline significantly reduced defluorosunitinib (M3) and M5 formation compared to the control but had minimal effect on M1. In CYP3A5-genotyped human liver microsomes from 12 individual donors, M1 formation was highly correlated with P450 3A activity measured by midazolam 1'-hydroxylation, and M3 and M5 formation was correlated with P450 1A2 activity estimated by phenacetin O-deethylation. M3 and M5 formation was also associated with P450 3A5-selective activity. In sandwich-cultured human hepatocytes, the P450 3A inducer rifampicin significantly increased M1 levels. P450 1A induction by omeprazole markedly increased M3 formation and the generation of a quinoneimine-cysteine conjugate (M6) identified as a downstream metabolite of M5. The nonselective P450 inhibitor 1-aminobenzotriazole reduced each of these metabolites (M1, M3, and M6). Collectively, these findings indicate that P450 3A activity is a key determinant of sunitinib N-dealkylation to the active metabolite M1, and P450 1A (and potentially 3A5) activity influences sunitinib bioactivation to the reactive quinoneimine metabolite. Accordingly, modulation of P450 activity due to genetic and/or nongenetic factors may impact the risk of sunitinib-associated toxicities.

Scheme 1.

Proposed Metabolic Pathway of Sunitinib^{a a}Adapted from Xie et al. and Amaya et al. Abbreviations: M1 (N-desethylsunitinib), M2 (monooxygenated metabolites of sunitinib), M3 (defluorosunitinib), M4 (sunitinib−glucuronide conjugate), and M5 (quinoneimine−glutathione conjugate); GSH (reduced glutathione); P450 (cytochrome P450); and UGT (uridine diphospho-glucuronosyltransferase).

Figure 1.

Correlation of sunitinib metabolite formation with P450 3A and 1A2 activity in single-donor human liver microsomes. Sunitinib (10 μM) was incubated with human liver microsomes (0.1 mg/mL) from 12 individual donors (n = 12) for 10 min in the presence of an NADPH-regenerating system and GSH (5 mM). Sunitinib metabolite formation was measured by LC−MS/MS analysis. Relative metabolite levels were determined by the peak area ratio of analyte to internal standard (sunitinib-d₄). (A−C) Correlation of sunitinib metabolite formation to P450 3A activity, as measured by midazolam 1′-hydroxylation. (D−F) Correlation of sunitinib metabolite formation to P450 1A2 activity, as measured by phenacetin O-deethylation. Rates of phenacetin O-deethylation were provided by Corning Life Sciences and Xenotech. Data were analyzed by linear regression and Pearson r correlation using GraphPad Prism software to determine R², r, and P values. The results shown for sunitinib metabolite formation are the mean values from three independent experiments conducted in triplicate each.

Figure 2.

Correlation of quinoneimine−GSH conjugate (M5) formation with defluorosunitinib (M3) formation in single-donor human liver microsomes from 12 individual donors (n = 12). Data were analyzed by linear regression and Pearson r correlation using GraphPad Prism software to determine R², r, and P values. The results shown are the mean values from three independent experiments conducted in triplicate each.

Figure 3.

Formation of N-desethylsunitinib (M1) and defluorosunitinib (M3) in single-donor CYP3A5-genotyped human hepatocytes and comparison with P450 3A and 1A2 activity. Sunitinib (10 μM) was incubated with human hepatocytes (0.5 × 10⁶ cells/mL) from individual donors (n = 12) in suspension for 2.2 h. The formation of sunitinib metabolites (M1 and M3) was measured by LC−MS/MS analysis. Relative metabolite levels were determined by the peak area ratio of analyte to internal standard (sunitinib-d₄). (A) Correlation of M1 formation with individual P450 3A activity, as measured by midazolam 1′-hydroxylation. (B) Correlation of M3 formation with P450 1A2 activity, as measured by phenacetin O-deethylation. Rates of phenacetin O-deethylation for single-donor hepatocytes were provided by BioIVT. (C, D) Comparison of sunitinib metabolite formation by donor sex (8 males, 4 females). (E, F) Comparison of P450 3A and 1A2 activity by donor sex. Statistical analysis was performed using GraphPad Prism software. Data were analyzed by linear regression and Pearson r correlation using GraphPad Prism software to determine R², r, and P values (parts A and B). Comparisons by males and females were analyzed by an unpaired t-test (parts C−F).

Figure 4.

Representative LC−MS/MS chromatogram from analysis of sunitinib metabolites in sandwich-cultured human hepatocytes. Sunitinib metabolites were analyzed by LC−MS/MS analysis with System 2 using selected reaction monitoring in positive ion mode [M + H]⁺: M1 (m/z 371 > 283), M3 (m/z 397 > 281), sunitinib (m/z 399 > 283), sunitinib-d₄ (internal standard, m/z 403 > 283), M6 (m/z 516 > 400), and M4 (m/z 575 > 459). Data are shown using Thermo Xcalibur Qual Browser software. The peak detection threshold was set to 10% of relative abundance. Labels for analytes are added for clarity.

Figure 5.

Effect of P450 1A and 3A induction on sunitinib metabolism in sandwich-cultured human hepatocytes. Hepatocytes from a single donor (HC3–38, female) were incubated with P450 1A inducer omeprazole (Omep, 50 μM), P450 3A inducer rifampicin (Rif, 25 μM), or vehicle control (0.1% DMSO) for 72 h. After the induction period, cells were pretreated with P450 nonselective inhibitor 1-ABT (1 mM) for 1 h, followed by incubation with sunitinib (Sun, 10 μM) for 24 h. Sunitinib and sunitinib metabolites were analyzed by LC−MS/MS analysis: sunitinib (A), M1 (B), M3 (C), M4 (D), and M6 (E). Relative metabolite levels were determined by the peak area ratio of analyte to internal standard (sunitinib-d₄). The results are the mean ± SD from a single experiment conducted in triplicate.