P450 3A-Catalyzed O-Dealkylation of Lapatinib Induces Mitochondrial Stress and Activates Nrf2

Abstract

Lapatinib (LAP), an oral tyrosine kinase inhibitor for the treatment of metastatic breast cancer, has been associated with idiosyncractic hepatotoxicity. Recent investigations have implicated the importance of P450 3A4/5 enzymes in the formation of an electrophilic quinone imine (LAPQI) metabolite generated through further oxidation of O-dealkylated lapatinib (OD-LAP). In the current study, hepatic stress was observed via mitochondrial impairment. OD-LAP caused a time- and concentration-dependent decrease in oxygen consumption in HepG2 cells, whereas LAP did not alter the oxygen consumption rate. Interestingly, however, HepG2 cells transfected with human P450 3A4 did exhibit mitochondrial dysfunction via P450 3A4-mediated metabolism of LAP to OD-LAP. OD-LAP-induced mitochondrial toxicity was enhanced upon depletion of intracellular GSH levels, demonstrating that cellular GSH levels are important in the protection of mitochondrial function against LAPQI. Given the nature of LAPQI and the importance of GSH levels in LAP-induced mitochondrial stress, the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) was evaluated, as this transcription factor induces the expression of NAD(P)H quinone oxidoreductase 1, glutathione S-transferase, UDP-glucuronosyltransferases, and glutathione synthetase, all of which might be expected to decrease the toxicity of LAP. Using a FRET-based target gene assay in HepG2 cells, OD-LAP was indeed found to activate Nrf2. Follow-up assays showed increased mRNA levels of Nrf2 target genes after a 4 h treatment with OD-LAP but not with LAP. LAP activation of Nrf2 was observed only when HepG2 cells were transduced with P450 3A4. The significance of Nrf2 protection was established in vivo in Nrf2-KO mice. Increased transaminase levels were found after a single LAP dose in both Nrf2-KO and control mice, indicating elevated hepatic necrosis, although transaminase levels reverted to baseline levels in the control mice upon repeat dosing. They continued to rise in Nrf2-KO mice, however, indicating the likelihood that Nrf-2 plays a significant role in combatting the hepatotoxicity triggered by LAP.

Cytochrome P450 3A4 and CYP3A5-Catalyzed Bioactivation of Lapatinib

“…Contribution of CYP3A4 and CYP3A5 to Lapatinib Bioactivation administration to Nrf2-knockout and control mice led to elevated liver transaminases, which levels returned to baseline after repeat dosing in control mice but continued to rise in Nrf2 knockout mice, indicating that Nrf2 may be involved in cellular defense against lapatinib-induced hepatotoxicity (Eno et al, 2016).…”

“…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). Lapatinib Fig.…”

“…An alternative hypothesis was recently presented to suggest that lapatinib itself may trigger immune activation by enhancing ligand binding to HLA-DRB1*07:01 (Hirasawa et al, 2015). Inhibition of the bile salt export pump by LAP-OH (Castellino et al, 2012) and induction of mitochondrial impairment (Eno et al, 2016) have also been suggested to contribute to liver injury. Additional studies are required to elucidate the actual mechanisms involved.…”

Role of Cytochrome P450 Enzymes in the Metabolic Activation of Tyrosine Kinase Inhibitors

“…Eno et al examined the cellular effects of lapatinib vs its O -dealkylated metabolite in HepG2 cells [62]. Mitochondrial respiration, as measured by oxygen consumption rate, was monitored using a Seahorse XF24 analyzer.…”

“…The results of this study demonstrated that O -dealkylated lapatinib caused mitochondrial dysfunction in a time- and concentration-dependent manner. Treatment of HepG2 cells with 30 μM O -dealkylated lapatinib for 24 h significantly reduced basal and maximal oxygen consumption rate compared to untreated cells [62]. The parent drug lapatinib did not significantly alter mitochondrial respiration in HepG2 cells at the concentration tested (10 μM lapatinib).…”