Modeling of Hepatic Drug Metabolism and Responses in CYP2C19 Poor Metabolizer Using Genetically Manipulated Human iPS cells

Abstract

Cytochrome P450 family 2 subfamily C member 19 (CYP2C19), in liver, plays important roles in terms of drug metabolism. It is known that CYP2C19 poor metabolizers (PMs) lack CYP2C19 metabolic capacity. Thus, unexpected drug-induced liver injury or decrease of drug efficacy would be caused in CYP2C19 substrate-treated CYP2C19 PMs. However, it is difficult to evaluate the safety and effectiveness of drugs and candidate compounds for CYP2C19 PMs because there is currently no model for this phenotype. Here, using human induced pluripotent stem cells (human iPS cells) and our highly efficient genome-editing and hepatocyte differentiation technologies, we generated CYP2C19-knockout human iPS cell-derived hepatocyte-like cells (CYP2C19-KO HLCs) as a novel CYP2C19 PM model for drug development and research. The gene expression levels of hepatocyte markers were similar between wild-type iPS cell-derived hepatocyte-like cells (WT HLCs) and CYP2C19-KO HLCs, suggesting that CYP2C19 deficiency did not affect the hepatic differentiation potency. We also examined CYP2C19 metabolic activity by measuring S-mephenytoin metabolites using ultraperformance liquid chromatography-tandem mass spectrometry. The CYP2C19 metabolic activity was almost eliminated by CYP2C19 knockout. Additionally, we evaluated whether clopidogrel (CYP2C19 substrate)-induced liver toxicity could be predicted using our model. Unexpectedly, there was no significant difference in cell viability between clopidogrel-treated WT HLCs and CYP2C19-KO HLCs. However, the cell viability in clopidogreland ketoconazole (CYP3A4 inhibitor)-treated CYP2C19-KO HLCs was significantly enhanced as compared with that in clopidogreland DMSO-treated CYP2C19-KO HLCs. This result suggests that CYP2C19-KO HLCs can predict clopidogrel-induced liver toxicity. We succeeded in generating CYP2C19 PM model cells using human iPS cells and genome-editing technologies for pharmaceutical research. SIGNIFICANCE STATEMENT Although unexpected drug-induced liver injury or decrease of drug efficacy would be caused in CYP2C19 substrate-treated CYP2C19 poor metabolizers, it is difficult to evaluate the safety and effectiveness of drugs and candidate compounds for CYP2C19 poor metabolizers because there is currently no model for this phenotype. Using human iPS cells and our highly efficient genome editing and hepatocyte differentiation technologies, we generated CYP2C19-knockout human iPS cell-derived hepatocyte-like cells as a novel CYP2C19 poor metabolizer model for drug development and research.

Development of immortalized human hepatocyte-like hybrid cells by fusion of multi-lineage progenitor cells with primary hepatocytes

“…isolated from primary hepatocytes was used as a positive control. Primers are described for alpha-fetoprotein (AFP), α-1-antitrypsin (AAT), transthyretin (TTR), cytochrome P450 1A2 (CYP 1A2), cytochrome P450 3A4 (CYP 3A4), cytochrome P450 2C9 (CYP 2C9), hepatocyte nuclear factor 1α (HNF1A), hepatocyte growth factor (HGF), albumin (ALB), and housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [13][14][15][16][17][18]. The PCR primer sequences are listed on S1 Table. Conditions for PCR reactions were initial denaturation at 94˚C for 3 min followed by 30 cycles of denaturation at 94˚C for 1 min, annealing for 1 min at 56˚C, and elongation for 1 min at 72˚C.…”

Stem Cells for Next Level Toxicity Testing in the 21st Century

“…[476][477][478] Along the same line, missense variants of genes encoding retinoic acid receptor-γ, CYP2C19, and multidrug resistance protein-2 have been identified to enhance the risk for anthracycline-induced cardiotoxicity. [479][480][481] Recent studies reporting the generation of genome-edited hiPSC-derived hepatocyte-like cells resembling CYP2C19 poor metabolizers, [482] and CMs carrying the retinoic acid receptor-γ missense variant S427L and exhibiting the associated enhanced sensitivity toward doxorubicin treatment, [481] illustrated the outstanding potential of genome-edited iPSC cells and organotypic models derived thereof for future compound testing in toxicology and pharmacology.…”

<p>In vitro Differentiation of TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells</p>

“…Primers are described for α-fetoprotein (AFP), α-1-antitrypsin (AAT), transthyretin (TTR), cytochrome P450 1A2 (CYP 1A2), cytochrome P450 3A4 (CYP 3A4), cytochrome P450 2C9 (CYP 2C9), hepatocyte nuclear factor 1α (HNF1A), hepatocyte growth factor (HGF), albumin (ALB), and housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). [20][21][22][23][24][25] Primer sequences are shown in Supplemental Table 1. PCR reactions were initiated by denaturation at 94°C for 3 min followed by 30 cycles of denaturation at 94°C for 1 min.…”