Time course of lapatinib M1-glucuronide formation. Lapatinib M1 (2 μM) was incubated with pooled human liver microsomes (0.1 mg protein/ml) for 0, 30, and 60 minutes in the presence and absence of UGT cofactor UDPGA. Formation of the M1-glucuronide conjugate was analyzed by LC-MS/MS using selected reaction monitoring (m/z 649 > 473). Relative metabolite levels were measured by the peak area ratio of M1-glucuronide to internal standard (d₄-M1, m/z 477 > 352). Results are presented as the mean ± S.D. from one experiment conducted in triplicate.

Lapatinib M1 glucuronidation by human recombinant UGT enzymes. Lapatinib M1 at (A) 0.2 μM and (B) 2 μM was incubated with individual recombinant UGT enzymes (Supersomes) or pooled human liver microsomes (0.2 mg protein/ml) for 30 minutes in the presence of UDPGA. Relative levels of M1-glucuronide conjugate were measured by LC-MS/MS analysis using selected reaction monitoring (m/z 649 > 473). Peak area ratios of M1-glucuronide to internal standard (d₄-M1, m/z 477 > 352) are shown. Results are presented as the mean ± S.D. from (A) two or (B) one experiment(s) conducted in triplicate each. HLM, human liver microsome.

Effect of UGT1A1 inhibition on lapatinib M1-glucuronide formation. Lapatinib M1 (2 μM) was incubated with pooled human liver microsomes (0.1 mg protein/ml) supplemented with UDPGA for 30 minutes in the presence and absence of UGT1A1 inhibitors atazanavir (1 and 10 μM) and erlotinib (3 and 10 μM). Relative levels of M1-glucuronide conjugate (m/z 649 > 473) were measured by LC-MS/MS analysis. The percentage (%) of M1-glucuronide conjugate formation was compared with vehicle control incubations without inhibitors (mean ± S.D. peak area ratio: 5.25 ± 0.80). Results are presented as the mean ± S.D. from one experiment conducted in triplicate.

Formation of lapatinib M1-glucuronide and quinoneimine-GSH conjugate in UGT1A1-genotyped human liver microsomes. Lapatinib M1 (2 μM) was incubated with pooled and individual genotyped human liver microsomes (0.2 mg protein/ml) for 30 minutes in the presence and absence of NADPH and UDPGA supplemented with GSH (5 mM). Relative levels of (A) M1-glucuronide conjugate (m/z 649 > 473) and (B) quinoneimine-GSH conjugate (m/z 778 > 655) were measured by LC-MS/MS analysis. Peak area ratios of analyte to internal standard (d₄-M1, m/z 477 > 352) are shown. Values are presented as the mean ± S.D. from two independent experiments conducted in triplicate each for six individual donors and pooled HLM. Donor lot numbers and UGT1A1 genotypes are shown: UGT1A1*1/*1 (n = 2), UGT1A1*1/*28 (n = 3), and UGT1A1*28/*28 (n = 1). Sample size was based on the number of commercially available UGT1A1-genotyped human liver microsomes. HLM, human liver microsome.

Correlation of lapatinib M1-glucuronide and quinoneimine-GSH conjugate formation with UGT1A1 and CYP3A activity, respectively, in human liver microsomes. (A) Correlation of lapatinib M1-glucuronide formation with UGT1A1 activity measured by 17β-estradiol 3-glucuronidation. (B) Correlation of quinoneimine-GSH conjugate formation with CYP3A activity measured by testosterone 6β-hydroxylation. Values are presented as the mean from two independent experiments conducted in triplicate for each of six individual donors and pooled HLM (n = 7 total). Donor genotypes were UGT1A1*1/*1 (n = 2), UGT1A1*1/*28 (n = 3), and UGT1A1*28/*28 (n = 1). Donor lot 710414 is highlighted as an outlier among UGT1A1*1/*28 donors with respect to high M1 glucuronidation (Grubbs’s outlier analysis). Sample size was based on the number of commercially available UGT1A1-genotyped human liver microsomes. UGT1A1 and CYP3A enzyme activity values are from XenoTech. GraphPad Prism 8 software was used for linear regression, Pearson r correlation, and Grubbs’s outlier analyses. HLM, human liver microsome.

Formation of lapatinib M1-glucuronide and quinoneimine-GSH conjugate in liver microsomes from human, rat, and dog. Lapatinib M1 (2 μM) was incubated with pooled liver microsomes from human, rat, and dog (0.1 mg protein/ml) for 30 minutes with and without cofactors NADPH and UDPGA in the presence of GSH (5 mM). Relative levels of (A) M1-glucuronide conjugate (m/z 649 > 473) and (B) quinoneimine-GSH conjugate (m/z 778 > 655) were measured by LC-MS/MS analysis. Peak area ratios of analyte to internal standard (d₄-M1, m/z 477 > 352) are shown. Results are presented as the mean ± S.D. from two experiments conducted in triplicate each.

Time course for lapatinib M1 depletion and metabolite formation in human, rat, and dog liver S9 fractions. Lapatinib M1 (1 μM) was incubated with pooled human, rat, and dog liver S9 fractions (2.0 mg protein/ml) for 0, 5, 10, 15, and 20 minutes. Incubations were supplemented with MgCl₂ (2.5 mM), PAPS (0.1 mM), and UDPGA (2 mM). Control incubations were without PAPS and UDPGA. Relative levels of M1 and M1 metabolites were measured by LC-MS/MS analysis using selected reaction monitoring for (A) M1 (m/z 473 > 350), (B) M1-glucuronide conjugate (m/z 649 > 473), (C) M1-sulfate conjugate (m/z 553 > 473), and (D) M3 (m/z 489 > 366). The percentage (%) of remaining M1 substrate was calculated based on the M1 peak area ratio at time 0 minutes (A). Peak area ratios of metabolite to internal standard (d₄-M1, m/z 477 > 352) are shown (B–D). Results are presented as the mean ± S.D. from one experiment conducted in triplicate. Ln, natural log.

Phase II metabolism of lapatinib in cryopreserved human hepatocytes. Lapatinib (1 and 10 μM) was incubated with pooled human hepatocytes in suspension for 2 hours. Cryopreserved hepatocytes were from a pool of five donors (mixed sex). Control incubations were with no cells. Relative metabolite levels were measured by LC-MS/MS analysis using selected reaction monitoring: (A) lapatinib M1 (m/z 473 > 350), (B) M1-glucuronide (m/z 649 > 473), (C) M1-sulfate (m/z 553 > 473), and (D) quinoneimine-GSH conjugate (m/z 778 > 655). Metabolite peak areas are shown. Bars are the mean from one experiment conducted in duplicate.

Phase II metabolism of lapatinib M1 in CYP3A5-genotyped cryopreserved human hepatocytes. Lapatinib (1 and 10 μM) was incubated with pooled human hepatocytes in suspension for 2 hours. Cryopreserved human hepatocytes were from a pool of three donors for each CYP3A5 genotype: CYP3A5*3/*3, CYP3A5*1/*3, and CYP3A5*1/*1. Relative metabolite levels were measured by LC-MS/MS analysis for M1-glucuronide (m/z 649 > 473) and M1-sulfate (m/z 553 > 473). Formation of M1-glucuronide (A and B) and M1-sulfate (C and D) from hepatocyte incubations with 1 μM lapatinib (A and C) and 10 μM lapatinib (B and D) is shown as the peak area for each metabolite. Results are presented as the mean ± S.D. from one experiment conducted in four to five replicates per hepatocyte lot. The peak area value for one of four replicates from incubations with 1 μM lapatinib in CYP3A5*3/*3 hepatocytes was identified as an outlier based on Grubbs’ outlier analysis (GraphPad Prism 8 software); thus, this outlier was excluded.