Dataset Information

Interindividual Variability in Imatinib Metabolism in Human Liver Microsomes and Primary Human Hepatocytes: Impact of CYP2C8 and CYP3A Phenotypes

ABSTRACT: Imatinib is a tyrosine kinase inhibitor (TKI) indicated for chronic myeloid leukemia (CML) and other cancers. It is metabolized by CYP2C8 and CYP3A into a major active metabolite N-desmethyl-imatinib. The purpose of the study was to determine in vitro the influence of CYP2C8 and CYP3A phenotypes, such as protein concentration and activity, and genotypes on imatinib clearance and N-desmethyl imatinib formation. Individual-donor and pooled human liver microsomes (HLM) and primary human hepatocytes (PHH) were incubated with imatinib to determine the rate of clearance of imatinib and formation of N-desmethyl-imatinib. HLMs were also investigated to determine their CYP2C8 and CYP3A activity and protein concentrations. Results were compared to evaluate the relationship between the CYP phenotypes and imatinib clearance and N-desmethyl-imatinib formation. For PHH samples, CYP2C8, CYP3A4, and CYP3A5 genotypes were also determined and imatinib metabolism was compared between the genotype groups. CYP3A5 metabolizer phenotype groups were also used to compare CYP phenotypes and imatinib metabolism in the PHH samples. The CYP targeted proteomics concentration measurements for the HLMs were undertaken on a nanoLC-QTRAP 5500 system (SCIEX mass spectrometer, Framingham, MA, USA) and the PHH measurements were determined on a microLC-Triple quadrupole 7500 system (also SCIEX mass spectrometer). Unlike the HLMs, membrane protein was extracted from the PHH samples before being trypsin digested. The data showed that, in HLM donors, CYP3A activity was the most significant predictor of imatinib clearance while CYP2C8 activity was the most significant predictor of N-desmethyl-imatinib formation. Additionally, in PHH, imatinib clearance was more significantly correlated with CYP2C8 phenotypes than CYP3A phenotypes. There was a significant difference in imatinib clearance between CYP2C8*1/*1 individuals and CYP2C8*3 carriers in PHH donors. Overall, the results support previous work that CYP2C8 plays a key role in imatinib metabolism and could provide insight into interindividual variability seen in patients taking the medication.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Liver

SUBMITTER: John Fallon

LAB HEAD: Klarissa Jackson

PROVIDER: PXD066482 | Pride | 2025-12-13

REPOSITORIES: Pride

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Source:

			Action	DRS
	HLM_QC_Digests_6th_April_2023__UGTs_CYP450s_.qsession	Other
	HLMs_6th_April_2023__UGTs.CYP450s_.wiff	Wiff
	HLMs_6th_April_2023__UGTs.CYP450s_.wiff.scan	Wiff
	HLMs_Digests_6th_April_2023__UGTs_CYP450s_.qsession	Other
	HLMs_QC_6th_April_2023.wiff	Wiff

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Project description:Metabolic dysfunction-associated steatotic liver disease (MASLD) is estimated to affect approximately 30% of adults worldwide. The progressive form of MASLD, namely metabolic-dysfunction steatohepatitis (MASH), is a leading cause of chronic liver disease. MASH is marked by hepatocellular fat accumulation (steatosis), ballooning, and inflammation. Although many in vitro and in vivo models replicate MASH pathophysiology, no in vitro hepatocyte MASH model has been evaluated for its ability to reflect clinically observed changes in drug metabolizing enzyme (DME) and transporter characteristics such as abundance and activity. This study addressed this knowledge gap by developing a model using sandwich-cultured human hepatocytes (SCHH) that mimics both MASH pathophysiology and alterations in DME and transporter concentrations and function. Lipid-cytokine treatments were first optimized using differentiated HuH-7 cells based on cellular toxicity profiles and their ability to induce a MASH-like phenotype. Three final treatments, all including TNF-α (1 ng/mL) and IL-6 (1.2 ng/mL), were selected for evaluation in SCHH: (1) oleic acid (OA):palmitic acid (PA) (1:2, 0.5 mM), (2) a lipid mix (lysophospholipids mixture + OA:PA), and (3) lipid mix+0.01 mM cholesterol. Treatments were incubated for 72 h with SCHH from three donors. Quantitative targeted absolute proteomics (QTAP), employing stable isotope labeled (SIL) peptide standards and microLC-MS/MS measurement, was used to quantify transporter and DME concentrations in the SCHH total membrane fraction. Concentrations of ten transporters, ten CYP450s, seven UGTs and five other DMEs could be measured above the lower limit of quantification of 0.1 pmol/mg membrane protein. B-CLEAR® technology was used, also, to evaluate transporter function with [3H]-taurocholate (TCA) and [3H]-estradiol-17β-glucuronide (E217G) employed as probe substrates of function. All three treatments significantly increased lipid droplet formation and peroxidation, characteristics of MASH, in SCHH with minimal toxicity. The treatments also altered DME and transporter concentrations in a similar manner to changes observed in liver tissue from patients with MASH. Across treatments, concentrations of bile salt export pump (BSEP), sodium taurocholate co-transporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, and multidrug resistance-associated protein (MRP) 2 were reduced by 0.66–0.57-fold, 0.71–0.52-fold, 0.74–0.63-fold, 0.82–0.80-fold, and 0.71–0.48-fold, respectively. Correspondingly, TCA apparent uptake clearance and biliary clearance were reduced by 0.70–0.26-fold and 0.61–0.27-fold, respectively. E217G apparent uptake clearance was reduced by 0.67–0.35-fold while biliary excretion index values for E217G were reduced to negligible levels. Overall, the findings demonstrate that lipid–cytokine treatments induce MASH-like changes in SCHH, including clinically relevant reductions in DME and transporter concentrations and transporter function. This model may serve as a valuable tool for predicting altered hepatobiliary drug disposition in MASH.

Dataset Information

Interindividual Variability in Imatinib Metabolism in Human Liver Microsomes and Primary Human Hepatocytes: Impact of CYP2C8 and CYP3A Phenotypes

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