Puromycin-based purification of cells with high expression of the cytochrome P450 CYP3A4 gene from a patient with drug-induced liver injury (DILI)

Background Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be accurately evaluated for drug-mediated CYP3A4 induction; this is the gold standard for in vitro hepatic toxicology testing. However, the variation from lot to lot is an issue that needs to be addressed. Only a limited number of immortalized hepatocyte cell lines have been reported. In this study, immortalized cells expressing CYP3A4 were generated from a patient with drug-induced liver injury (DILI). Methods To generate DILI-derived cells with high expression of CYP3A4, a three-step approach was employed: (1) Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); (2) Immortalization of the differentiated cells; (3) Selection of the cells by puromycin. It was hypothesized that cells with high cytochrome P450 gene expression would be able to survive exposure to cytotoxic antibiotics because of their increased drug-metabolizing activity. Puromycin, a cytotoxic antibiotic, was used in this study because of its rapid cytocidal effect at low concentrations. Results The hepatocyte-like cells differentiated from DILI-iPSCs were purified by exposure to puromycin. The puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at extremely high levels and exhibited hepatocytic features over time. However, unlike primary hepatocytes, the established cells did not produce bile or accumulate glycogen. Conclusions iPSC-derived hepatocyte-like cells with intrinsic drug-metabolizing enzymes can be purified from non-hepatocytes and undifferentiated iPSCs using the cytocidal antibiotic puromycin. The puromycin-selected hepatocyte-like cells exhibited characteristics of hepatocytes after immortalization and may serve as another useful source for in vitro hepatotoxicity testing of low molecular weight drugs.

The intracellular mechanism of Berberine-induced inhibition of CYP3A4 activity

Background: Berberine (BBR) is an isoquinoline alkaloid extracted from Chinese medicine, exerting various pharmacological effects. BBR is partially metabolized by cytochrome 3A4 (CYP3A4) in vivo. Some reports indicated that BBR could inhibit the activity of CYP3A4. However, the underlying mechanisms are not entirely understood. CYP3A4 is transcriptionally regulated by two nuclear receptors: nuclear transcription X receptor (PXR) and constitutive androstane receptor (CAR). It degraded via the ubiquitin-proteasome system. Hence, we tried to explore the mechanisms of CYP3A4 inhibition on both transcriptive and protein levels. Methods: Western Blot, RT-PCR, and Co-immunoprecipitation were used to perform the experiments. Results: Our results showed that BBR inhibited the transcription of CYP3A4 gene by downregulating PXR. In addition, BBR accelerated the degradation of CYP3A4 protein via the polyubiquitination pathway. Conclusion: These findings may lead to the determination of novel drug-drug interactions with BBR and contribute to the future clinical application of BBR.

Implementation of a next-generation sequencing-based targeted approach for full-length CYP3A4 gene sequencing

Aim: To evaluate the application of next-generation sequencing-based targeted protocol for full-length CYP3A4 gene sequencing analysis. Materials & methods: The developed sequencing protocol was applied to analyze human DNA samples (n = 7) obtained from tuberculosis patients admitted to the Riga East University Hospital, Center of Tuberculosis and Lung diseases. Results: The sequencing data quality was sufficient for the detection of already known genetic variants, as well as for identifying rare and novel variants dispersed throughout the CYP3A4 gene with a high degree of confidence. Conclusion: Developed protocol can be applied in subpopulation level association studies to determine whether specific genetic variants or variant combinations from multiple regions of the CYP3A4 gene are of clinical significance.

Effect of Vitamin K-Mediated PXR Activation on Drug-Metabolizing Gene Expression in Human Intestinal Carcinoma LS180 Cell Line

The pregnane X receptor (PXR) is the key regulator of our defense mechanism against foreign substances such as drugs, dietary nutrients, or environmental pollutants. Because of increased health consciousness, the use of dietary supplements has gradually increased, and most of them can activate PXR. Therefore, an analysis of the interaction between drugs and nutrients is important because altered levels of drug-metabolizing enzymes or transporters can remarkably affect the efficiency of a co-administered drug. In the present study, we analyzed the effect of vitamin K-mediated PXR activation on drug metabolism-related gene expression in intestine-derived LS180 cells via gene expression studies and western blotting analyses. We demonstrated that menaquinone 4 (MK-4), along with other vitamin Ks, including vitamin K1, has the potential to induce MDR1 and CYP3A4 gene expression. We showed that PXR knockdown reversed MK-4-mediated stimulation of these genes, indicating the involvement of PXR in this effect. In addition, we showed that the expression of MDR1 and CYP3A4 genes increased synergistically after 24 h of rifampicin and MK-4 co-treatment. Our study thus elucidates the importance of drug–nutrient interaction mediated via PXR.

Impact of Cytochromes P450 3A4 and 2B6 gene polymorphisms on predisposition and prognosis of acute myeloid leukemia: an Egyptian case-control study

Background It has been postulated that the interaction between environmental risk factors and genetic susceptibility is a possible cause for the development of acute myeloid leukemia (AML). Cytochrome P450 (CYP) detoxification enzymes are responsible for the elimination of oxidative stress. Genetic polymorphisms in these enzymes may cause AML due to enhanced accumulation of reactive oxygen species. To study the association between CYP3A4 (A290G) and CYP2B6 (G516T) gene polymorphisms and the predisposition and prognosis of AML, 50 upfront AML patients and 50 healthy individuals were genotyped for CYP2B6 (G516T) and CYP3A4 (A290G) single-nucleotide polymorphisms (SNPs) using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) technique. The polymorphisms were evaluated in relation to the response to chemotherapy and survival. Results CYP2B6 gene mutation carries a threefold risk of developing AML (odds ratio [OR], 3.0; 95% confidence interval [CI], 1.3–6.9), whereas CYP3A4 gene mutation carries approximately fourfold risk (OR, 3.8; 95% CI, 1.4–10.1). The presence of combined gene mutation conferred about 15-fold increased risk of developing AML compared with the presence of a single gene mutation (OR, 14.8; 95% CI, 1.8–124.2). CYP3A4 gene mutation is associated with worse overall survival (P = 0.030). Conclusion CYP enzyme gene polymorphisms are associated with the development of AML. Elimination of oxidative stress in genetically susceptible individuals may decrease the risk of AML and may improve survival.

The effect of CYP3A4 genetic polymorphisms on the pharmacokinetics of calcineurin inhibitors in renal transplant recipients: a systematic review and meta-analysis protocol

Background: Calcineurin inhibitors (CNIs) are metabolized by CYP3A4. Polymorphisms in the CYP3A4 gene alter the activity of CYP3A4 protein and therefore affect the CNIs concentrations. Results of studies that investigated the association between CYP3A4 polymorphisms and CNIs pharmacokinetics are controversial. Therefore, this systematic review and meta-analysis will evaluate the effect of CYP3A4 polymorphisms on the pharmacokinetics of CNIs in renal transplant recipients. Method: This protocol is developed according to the PRISMA-P guideline and registered in PROSPERO (CRD42020145219). The MeSH/Emtree terms of CYP3A4 polymorphisms and CNIs pharmacokinetics in PECO-based question will be obtained from the comprehensive literature search on PubMed/MEDLINE, Scopus, Web of Science, Embase, CENTRAL, and ProQuest without any language limitation from 1 January 1998 to 31 March 2021. Google Scholar search engine, registries, conference papers, and key journals will also be searched. Screening, selection, quality assessment, and data extraction will be performed by two independent reviewers. Statistical heterogeneity will be calculated by the Q Cochrane test and I2 statistic. Publication bias and sensitivity analysis will be evaluated by appropriate tests. Results and conclusion: According to the meta-analysis of the aggregated data from the relevant primary studies, the association between CYP3A4 polymorphisms and CNIs pharmacokinetics will be reported, which possibly help the pharmacogenetic-guided dosing approach.

Evaluation of the individual detoxification potential in the firefighters and rescuers of emercom of Russia

Relevance. Professional activities of firefighters and rescuers are usually associated with aggressive chemical exposure with increased physical exertion and psychological stress. Therefore, for professional selection and monitoring the health status of firefighters and rescuers of EMERCOM of Russia it is necessary to evaluate the activity of cytochrome P450 isoenzymes that are directly involved in detoxification pathways in the liver.Intention. To evaluate the activity of the CYP3A4 enzyme by phenotyping methods and to establish the frequency of allelic variants of the gene of this enzyme (rs2740574 and rs4987161 polymorphisms) in firefighters and rescuers of EMERCOM of Russia for targeted treatment, rehabilitation and prevention.Methodology. Polymorphisms (rs2740574 and rs4987161) of the CYP3A4 gene and also CYP3A4 activity by the ratio of 6-β-hydroxycortisol / cortisol in the urine were determined during routine periodical medical examination of 64 rescuers and firefighters of rescue units of EMERCOM of Russia. The average age of examined persons was (29.8 ± 5.5) years; 30 of them are rescuers of the North-West Regional Search and Rescue Squad and 34 are firefighters of the territorial fire departments of St. Petersburg.Results and Discussion. According to the results of genotyping of rs2740574 polymorphism of the CYP3A4 gene, EMERCOM employees were divided into 2 groups depending on the presence or absence of a minor allele: “poor” and “rapid” metabolizers (9.4% and 80.6%, respectively). According to the results of genotyping of the rs4987161 polymorphism of the CYP3A4 gene, the examined were classified as “rapid” metabolizers, because patients with a minor allele were not identified. Depending on the ratio of 6-β-hydroxycortisol / cortisol in the urine, the activity level of CYP3A4 was “normal” in 67 %, “poor” in 13 % and “rapid” in 20 % of cases. The ratio of 6-β-hydroxycortisol / cortisol in the urine tended to increase with an increase in work experience and age. No relationship between the studied polymorphic variants of the CYP3A4 gene and the established enzyme activity was observed.Conclusion. Genotyping methods made it possible to identify allelic variants of the CYP3A4 gene that could affect the functionality of the enzyme; however, no association of the studied polymorphisms with enzyme activity was found. In such cases, in the absence of informative genetic markers, it is recommended to evaluate the enzyme activity by phenotyping methods.

New Pharmacogenetic Markers to Predict the Risk of Bleeding During Taking of Direct Oral Anticoagulants

Aim. To search for new pharmacogenetic biomarkers of bleeding risk in patients taking rivaroxaban and dabigatran for different indications: atrial fibrillation, endoprosthesis of large joints of lower limbs.Material and methods. The study enrolled 29 patients (17 patients received dabigatran and 12 –rivaroxaban), who had hemorrhagic complications during taking direct oral anticoagulants. To find new pharmacogenetic biomarkers of bleeding risk, a next generation sequencing (NGS) was performed for selected candidate genes.Results. Among the patients with bleeding who received dabigatran, 13 variants of the nucleotide sequence showed statistically significant deviation from the population values: 11 in the CES1 gene and 2 in the ABCB1 gene. Among the patients with bleeding who received rivaroxaban, 7 variants of nucleotide sequence showed significant deviation: 4 in the ABCG2 gene, 2 in the CYP3A4 gene, and 1 in the ABCB1 gene.Conclusion. The identified in this study polymorphisms of candidate genes ABCB1, ABCG2, CES1, CYP3A4 were associated with the risk of bleeding in patients taking rivaroxaban and dabigatran. It makes an important contribution to the pharmacogenetics of direct oral anticoagulants and require additional assessment of clinical significance in further studies.