In Vitro Model for a Drug Assessment of Cytochrome P450 Family 3 Subfamily A Member 4 Substrates Using Human Induced Pluripotent Stem Cells and Genome Editing Technology

Abstract

In drug development, a system for predicting drug metabolism and drug‐induced toxicity is necessary to ensure drug safety. Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) is an important drug‐metabolizing enzyme expressed in the liver and small intestine, and predicting CYP3A4‐mediated drug metabolism and drug‐induced toxicity is essential. We previously developed procedures to differentiate human induced pluripotent stem (iPS) cells into hepatocyte‐like cells (HLCs) or intestinal epithelial‐like cells (IECs) with a fetal phenotype as well as a highly efficient genome editing technology that could enhance the homologous recombination efficiency at any locus, including CYP3A4. By using human iPS cells and our genome editing technology, we generated CYP3A4‐knockout (KO) iPS cell‐derived HLCs and IECs for the evaluation of CYP3A4‐mediated drug metabolism and drug‐induced toxicity. CYP3A4 deficiency did not affect pluripotency and hepatic and intestinal differentiation capacities, and CYP3A4 activity was entirely eradicated by CYP3A4 KO. Off‐target effects (e.g., inhibition of bile acid excretion) were hardly observed in CYP3A4‐KO cells but were observed in CYP3A4 inhibitor‐treated (e.g., ketoconazole) cells. To evaluate whether drug‐induced hepatotoxicity and enterotoxicity could be predicted using our model, we exposed CYP3A4‐KO HLCs and IECs to acetaminophen, amiodarone, desipramine, leflunomide, tacrine, and tolcapone and confirmed that these cells could predict CYP3A4‐mediated toxicity. Finally, we examined whether the therapeutic effects of an anti‐hepatitis C virus (HCV) drug metabolized by CYP3A4 would be predicted using our model. CYP3A4‐KO HLCs were treated with asunaprevir (antiviral drug metabolized by CYP3A4) after HCV infection, and the anti‐viral effect was indeed strengthened by CYP3A4 KO. Conclusion: We succeeded in generating a novel evaluation system for prediction of CYP3A4‐mediated drug metabolism and drug‐induced toxicity.

Toxicological applications of human induced pluripotent stem cell-derived hepatocyte-like cells: an updated review

“…More recently, using the same technology, the group generated a CYP3A4-KO iPSC line, and from which generated HLCs to evaluate CYP3A4-mediated drug metabolism and drug-induced toxicity. Using a small group of model drugs, including acetaminophen, amiodarone, desipramine, leflunomide, tacrine, and tolcapone, the authors confirmed that the CYP3A4-KO HLCs were able to correctly predict CYP3A4-mediated toxicity (Deguchi et al, 2021).…”

“…With the CRISPR/Cas9 system, isogenic cell lines with desired mutations could be generated for a variety of biomedical applications. In several studies that appeared in the last few years, iPSCs were modified by CRISPR/Cas9 and the genetically modified cells were further differentiated into HLCs for toxicity applications (Takayama et al, 2018;Deguchi et al, 2019;Kim et al, 2020;Deguchi et al, 2021). Using the CRISPR/Cas9 system, a CYP3A4-NeoR-EGFP transgenic reporter human iPSC line was established by inserting a neomycin resistant gene (NeoR) and an enhanced green fluorescent protein (EGFP) into the CYP3A4 locus (Takayama et al, 2018).…”

Development of a method of passaging and freezing human iPS cell-derived hepatocytes to improve their functions

“…Human iPS cells derived from individuals with single nucleotide polymorphisms (SNPs) in cytochrome P450 (CYP) 2D6 recapitulated the poor metabolizer phenotype by differentiation into HLCs [ 4 ]. In addition, genome editing technology successfully recapitulated the phenotypes of poor metabolizers in HLCs and revealed the contribution of specific CYP enzymes in pharmacokinetics [ 5 , 6 ]. Moreover, hepatocyte transplantation technologies using HLCs sheets [ 7 ], spheroids [ 8 ], and organoids [ 9 ] have been developed as an alternative to living donor liver transplantation.…”