Cytochrome P450 3A4 and CYP3A5-Catalyzed Bioactivation of Lapatinib

Abstract

Metabolic activation of the dual-tyrosine kinase inhibitor lapatinib by cytochromes CYP3A4 and CYP3A5 has been implicated in lapatinibinduced idiosyncratic hepatotoxicity; however, the relative enzyme contributions have not been established. The objective of this study was to examine the roles of CYP3A4 and CYP3A5 in lapatinib bioactivation leading to a reactive, potentially toxic quinoneimine. Reaction phenotyping experiments were performed using individual human recombinant P450 enzymes and P450-selective chemical inhibitors. Lapatinib metabolites and quinoneimine-glutathione (GSH) adducts were analyzed using liquid chromatography-tandem mass spectrometry. A screen of cDNA-expressed P450s confirmed that CYP3A4 and CYP3A5 are the primary enzymes responsible for quinoneimine-GSH adduct formation using lapatinib or O-dealkylated lapatinib as the substrate. The mean kinetic parameters (K m and k cat ) of lapatinib O-dealkylation revealed that CYP3A4 was 5.2-fold more efficient than CYP3A5 at lapatinib O-dealkylation (CYP3A4 k cat / K m = 6.8 mM 21 min 21 versus CYP3A5 k cat /K m = 1.3 mM 21 min 21). Kinetic analysis of GSH adduct formation indicated that CYP3A4 was also 4-fold more efficient at quinoneimine-GSH adduct formation as measured by k cat (maximum relative GSH adduct levels)/K m (CYP3A4 = 0.0082 vs. CYP3A5 = 0.0021). In human liver microsomal (HLM) incubations, CYP3A4-selective inhibitors SR-9186 and CYP3cide reduced formation of GSH adducts by 78% and 72%, respectively, compared with >90% inhibition by the pan-CYP3A inhibitor ketoconazole. The 16%-22% difference between CYP3A-and CYP3A4-selective inhibition indicates the involvement of remaining CYP3A5 activity in generating reactive metabolites from lapatinib in pooled HLMs. Collectively, these findings support the conclusion that both CYP3A4 and CYP3A5 are quantitatively important contributors to lapatinib bioactivation.

Identifying the reactive metabolites of tyrosine kinase inhibitors in a comprehensive approach: Implications for drug‐drug interactions and hepatotoxicity

“…Therefore, depending on the enzyme (CYP3A4 vs CYP3A5) and its predominantly catalyzed biotransformation pathways ( O ‐dealkylation vs N ‐oxidation) leading to different RM (nitroso vs p ‐quinone‐imine metabolite), lapatinib can cause quasi‐irreversible or irreversible CYP inactivation . CYP3A4 preferentially catalyzes lapatinib N ‐oxidation, while CYP3A5 preferentially catalyzes lapatinib O ‐dealkylation, which is consistent with the results of a docking study of lapatinib into CYP3A4 and CYP3A5 . A phenylalanine residue in CYP3A4 is involved in a π‐stacking interaction with the chlorophenyl ring, orienting lapatinib side chain toward the heme.…”

“…GSH and methoxylamine adducts have been evidenced, involving RM in CYP inhibition . CYP3A inactivation mechanisms have been extensively studied . For Teng et al, the formation of a p ‐quinone‐imine 32 that covalently modifies the CYP3A4 apoprotein and/or heme is considered to be the most likely mechanism to explain CYP3A4 MBI (Figure ).…”

Cytochromes P450 1A2 and 3A4 Catalyze the Metabolic Activation of Sunitinib

“…Recombinantly expressed P450 enzymes and human liver microsomal preparations used in this study were similar to those described by Towles et al 18 Briefly, Supersomes (baculovirus-infected insect cell microsomes) containing cDNA-expressed human P450 1A1, 1A2, 1B1, 2B6, 2C8, 2C9*1, 2C19, 2D6*1, 2E1, 2J2, 3A4, and 3A5 coexpressed with P450 reductase and cytochrome b 5 , except P450 1A2 and 2D6*1 (expressed without cytochrome b 5 ), were purchased from Corning Discovery Labware (Woburn, MA). Pooled (150-Donor Ultra Pooled, mixed gender) and single-donor human liver microsomes (Lots HH741 and HH581) were purchased from Corning Discovery Labware.…”

“…System A consisted of a Thermo Accela ultrahigh performance liquid chromatography (UHPLC) system equipped with a Thermo PAL autoinjector and a column oven coupled to a Thermo TSQ Quantum Triple Quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA), as described previously. 18 Analyte separation was achieved using a Kinetex C18 or EVO C18 octadecylsilane column (2.6 μ m, 50 mm × 2.1 mm, 100 Å) (Phenomenex, Torrance, CA) with a flow rate of 0.3 mL/min and column oven temperature 30 °C. Mobile phases were (A) 0.1% formic acid in LC/MS-grade water and (B) 0.1% formic acid in LC/MS-grade acetonitrile (all v/v).…”

Characterization of kinetics of human cytochrome P450s involved in bioactivation of flucloxacillin: inhibition of CYP3A‐catalysed hydroxylation by sulfaphenazole

“…These results suggest that flucloxacillin has a unique binding mode to the active site to CYP3A4, explaining the relatively weak inhibition by 1 μM ketaconazole and the unexpectedly strong inhibition by sulfaphenazole. A similar situation has been described recently in a study where sulfaphenazole significantly inhibited bioactivation of hydroxylapatinib by HLM, although recombinant CYP2C9 showed only very low activity (Towles et al ., ). Therefore, the CYP3A family in rare occurrences can be inhibited by sulfaphenazole in a substrate‐dependent manner.…”