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

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 mM) or the prototypical CYP3A4 inducer rifampicin (4 mM) for 72 hours, followed by incubation with lapatinib (0-100 mM) 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 mM 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.

Cytochrome P450 3A4 and CYP3A5-Catalyzed Bioactivation of Lapatinib

“…In addition, Teo et al (2012) demonstrated that concurrent use of lapatinib with dexamethasone (CYP3A4 inducer) in metastatic breast cancer patients increased the risk of lapatinib-induced hepatotoxicity, suggesting the involvement of CYP3A-mediated bioactivation. We have previously shown that LAP-OH was more cytotoxic than lapatinib itself in the HepaRG hepatic cell model (Hardy et al, 2014). CYP3A4 induction by dexamethasone and rifampin potentiated lapatinib-induced cytotoxicity, which was correlated with increased formation of LAP-OH and reactive quinoneimine-cysteine conjugates (Hardy et al, 2014).…”

“…We have previously shown that LAP-OH was more cytotoxic than lapatinib itself in the HepaRG hepatic cell model (Hardy et al, 2014). CYP3A4 induction by dexamethasone and rifampin potentiated lapatinib-induced cytotoxicity, which was correlated with increased formation of LAP-OH and reactive quinoneimine-cysteine conjugates (Hardy et al, 2014). More recently, Cameron and colleagues showed that O-dealkylated lapatinib induced mitochondrial stress and activation of the oxidative stress responsive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in HepG2 cells (Eno et al, 2016).…”

“…The following precursors to product MRM transitions were used for the detection of lapatinib and lapatinib metabolites based on structurally specific fragmentation obtained from collision-induced dissociation with collision energy 18-30 V: m/z 581.6 → 365 for lapatinib, m/z 473 → 350 for O-dealkylated lapatinib (LAP-OH); m/z 475 → 366 for N-dealkyl lapatinib (N-dealkyl-LAP), m/z 597 → 458 for N-hydroxy lapatinib (N-OH-LAP), and m/z 778 → 655 for quinoneimine-GSH adducts, as previously described (Hardy et al, 2014). The MRM transitions for stable isotope-labeled internal standards were: d 4 -lapatinib m/z 585 → 365, and d 4 -LAP-OH m/z 477 → 352.…”

Practical approaches to resolving reactive metabolite liabilities in early discovery

“…This is probably attributed to an increase in bioactivation of the drug ( Figure 11A) due to induction of P450 3A4. The effect of induction on toxicity of lapatinib was recently demonstrated by Hardy et al (2014) who showed that induction of P450 3A4 by dexamethasone or rifampicin in HepaRG cells markedly enhanced the cytotoxicity of lapatinib. This correlated with increased formation of GSH adducts of the drug (Hardy et al, 2014).…”

“…The effect of induction on toxicity of lapatinib was recently demonstrated by Hardy et al (2014) who showed that induction of P450 3A4 by dexamethasone or rifampicin in HepaRG cells markedly enhanced the cytotoxicity of lapatinib. This correlated with increased formation of GSH adducts of the drug (Hardy et al, 2014). Similarly, induction of P450 3A, which is the predominant catalyst of the activation of troglitazone in humans, is probably associated with an increased bioactivation of the drug (Tettey et al, 2001).…”