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, 42 (1), 162-71

Studies on the Role of Metabolic Activation in Tyrosine Kinase Inhibitor-Dependent Hepatotoxicity: Induction of CYP3A4 Enhances the Cytotoxicity of Lapatinib in HepaRG Cells

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Studies on the Role of Metabolic Activation in Tyrosine Kinase Inhibitor-Dependent Hepatotoxicity: Induction of CYP3A4 Enhances the Cytotoxicity of Lapatinib in HepaRG Cells

Klarissa D Hardy et al. Drug Metab Dispos.

Abstract

Idiosyncratic hepatotoxicity has been associated with the oral tyrosine kinase inhibitor lapatinib, which is used in metastatic breast cancer therapy. Lapatinib is extensively metabolized by cytochrome P450 3A4/5 to yield an O-debenzylated metabolite, which can undergo further oxidation to a reactive quinone imine. A recent clinical study reported that concomitant use of lapatinib with dexamethasone increased the incidence of hepatotoxicity in metastatic breast cancer patients treated with lapatinib, and so we hypothesized that induction of CYP3A enhances the bioactivation of lapatinib to reactive intermediates that contribute to hepatotoxicity. Therefore, we examined the effect of CYP3A4 induction on the cytotoxicity and metabolism of lapatinib in the HepaRG human hepatic cell line. Differentiated HepaRG cells were pretreated with dexamethasone (100 μM) or the prototypical CYP3A4 inducer rifampicin (4 μM) for 72 hours, followed by incubation with lapatinib (0-100 μM) for 24 hours. Cell viability was monitored using WST-1 assays, and metabolites were quantified by liquid chromatography coupled to tandem mass spectrometry. Induction of CYP3A4 by dexamethasone or rifampicin enhanced lapatinib-induced cytotoxicity, compared with treatment with lapatinib alone. A direct comparison of the cytotoxicity of lapatinib versus O-debenzylated lapatinib demonstrated that the O-debenzylated metabolite was significantly more cytotoxic than lapatinib itself. Furthermore, pretreatment with 25 μM l-buthionine sulfoximine to deplete intracellular glutathione markedly enhanced lapatinib cytotoxicity. Cytotoxicity was correlated with increased formation of O-debenzylated lapatinib and cysteine adducts of the putative quinone imine intermediate. Collectively, these data suggest that CYP3A4 induction potentiates lapatinib-induced hepatotoxicity via increased reactive metabolite formation.

Figures

Fig. 1.
Fig. 1.
Proposed bioactivation pathway of lapatinib. MI, metabolic-intermediate.
Fig. 2.
Fig. 2.
Cytotoxicity of lapatinib in primary human hepatocytes and HepaRG cells. Cryopreserved plated human hepatocytes from two donors (Hu4246 and Hu1389) and HepaRG cells were treated with lapatinib (100 μM) for 24 hours. Cell viability was monitored using WST-1 assays, and viability is expressed as the percent viability compared with vehicle treatment. Data represent the mean ± S.D. of triplicate values for Hu4246 and Hu1389, and the mean ± S.D. of triplicate values from three independent experiments for HepaRG cells. *P < 0.05; **P < 0.01; ****P < 0.0001 compared with vehicle (unpaired t test, two-tailed P values).
Fig. 3.
Fig. 3.
Induction of CYP3A4 activity by dexamethasone and rifampicin, and the effect of CYP3A4 induction on the cytotoxicity of lapatinib in HepaRG cells. HepaRG cells were pretreated with varying concentrations of dexamethasone (A) or rifampicin (B) or vehicle for 72 hours, followed by incubation with midazolam (3 μM) for 1 hour. CYP3A4 activity was assessed by measurement of 1′-hydroxymidazolam using LC-MS/MS. Fold change in 1′-hydroxymidazolam was calculated by comparison with pretreatment with vehicle control (DMSO). Data represent mean ± S.E.M. of three to four values. For cytotoxicity studies, HepaRG cells were pretreated with 100 μM dexamethasone (C) or 4 μM rifampicin (D) or vehicle for 72 hours, followed by incubation with LAP (10, 50, 100 μM) or vehicle control for 24 hours. Cell viability was monitored using WST-1 assays. For (C), LAP + Dex was compared with incubation with LAP alone at each concentration. For (D), LAP + Rif was compared with incubation with LAP alone at each concentration. Data represent the mean ± S.D. of triplicate values. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired t test, two-tailed P values). Dex, dexamethasone; Rif, rifampicin.
Fig. 4.
Fig. 4.
Cytotoxicity of lapatinib versus O-debenzylated lapatinib in HepaRG cells and the effect of glutathione depletion by BSO on lapatinib-induced cytotoxicity. (A) HepaRG cells were incubated with LAP or LAP-OH (10, 50, 100 μM) or vehicle control for 24 hours. Cell viability was monitored using WST-1 assays. (B) HepaRG cells were pretreated with 25 μM BSO for 24 hours, prior to incubation with LAP (10, 50, 100 μM) or vehicle control for 24 hours. For (A), LAP-OH was compared with incubation with LAP at each concentration. Data represent means ± S.E.M. of triplicate values from three independent experiments. For (B), LAP + BSO was compared with incubation with LAP alone at each concentration. Data represent the mean ± S.D. of triplicate values. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired t test, two-tailed P values).
Fig. 5.
Fig. 5.
Effect of CYP3A4 induction and inhibition on the formation of O-debenzylated lapatinib in HepaRG cells. HepaRG cells were incubated with 100 μM dexamethasone (A) or 4 μM rifampicin (B) for 72 hours, followed by incubation with LAP (100 μM) for 24 hours. For CYP3A4 inhibition, HepaRG cells were coincubated with ketoconazole (4 μM) + LAP (100 μM). Formation of LAP-OH was quantified by LC-MS/MS utilizing MRM. Data represent the mean ± S.D. of triplicate values. ***P < 0.001 and ****P < 0.0001 for comparison of LAP versus LAP + Dex and LAP versus LAP + Rif; ƚƚƚP < 0.001 and ƚƚƚƚP < 0.0001 for comparison of LAP + Dex versus LAP + Dex + Keto and LAP + Rif versus LAP + Rif + Keto (unpaired t test, two-tailed P values). Dex, dexamethasone; Keto, ketoconazole; Rif, rifampicin.
Fig. 6.
Fig. 6.
Identification of reactive metabolite GSH and cysteine adducts. (A) Enhanced product ion scans of the putative quinone imine reactive metabolite-GSH adduct (RM-SG) (m/z 778) formed in pooled human liver microsomes incubated with O-debenzylated lapatinib (LAP-OH) (100 μM) for 1 hour and supplemented with NADPH and GSH (50 mM). (B) MRM chromatogram of RM-SG from incubation of LAP-OH (100 μM) in HepaRG cells for 24 hours. (C) Enhanced product ion scans of the putative quinone imine RM-Cys adduct (m/z 592). (D) MRM chromatogram of RM-Cys from incubation of LAP-OH (100 μM) in HepaRG cells for 24 hours.
Fig. 7.
Fig. 7.
Effect of CYP3A4 induction and inhibition on formation of RM-Cys adducts in HepaRG cells. HepaRG cells were incubated with 100 μM dexamethasone (A) or 4 μM rifampicin (B) for 72 hours, followed by incubation with lapatinib (100 μM) with or without coincubation with ketoconazole (4 μM) for 24 hours. Relative levels of RM-Cys adducts were quantified by LC-MS/MS MRM. Data represent the mean ± S.D. of triplicate values. **P < 0.01 for comparison of LAP versus LAP + Dex and LAP versus LAP + Rif; ƚƚP < 0.01 for comparison of LAP + Dex versus LAP + Dex + Keto and LAP + Rif versus LAP + Rif + Keto (unpaired t test, two-tailed P values). Dex, dexamethasone; Keto, ketoconazole; Rif, rifampicin.

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