End-stage renal disease reduces the expression of drug-metabolizing cytochrome P450s.

BACKGROUND: End-stage renal disease is an irreversible status of kidney dysfunction that reduces both renal and non-renal drug clearance. Accumulation of uremic toxins seems to modify the activities of drug-metabolizing cytochrome P450 (CYP) enzymes. The aim of the present work was to refine gene expression analysis for efficient and accurate quantification of CYP mRNAs in patients' leukocytes. METHODS: We compared six liquid-liquid extraction reagents for RNA isolation and five reverse transcriptase kits for RNA-to-cDNA conversion, and developed quantitative polymerase chain reaction methods for duplex measurements of CYP target genes and the reference gene. The expression of CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in patients with end-stage kidney disease (N = 105) and in organ donors with healthy kidney function (N = 110) was compared. RESULTS: Regarding the RNA yield and purity, TRIzol, Trizolate and TRI reagents were equal; however, TRI reagent was the most advantageous in terms of financial cost. Reverse transcription using Maxima First Strand cDNA Synthesis kit appeared to be the most efficient with the widest range for quantification of the target transcript. The refined method with the detection of various CYPs and the reference gene in duplex PCR efficiently quantified even the low-level CYP expression. In leukocytes of patients with end-stage renal disease, all four CYPs were expressed at significantly lower level than in organ donors with normal kidney function (p < 0.0001). CONCLUSIONS: Reduced CYP expression was a direct evidence of transcriptional down-regulation of CYP genes in patients with impaired kidney function.

Personalizing initial calcineurin inhibitor dosing by adjusting to donor CYP3A-status in liver transplant patients.

AIMS: Inter-individual variability in dose requirements of calcineurin inhibitors (CNI) has been linked to genetic polymorphisms of CYP3A enzymes. CYP3A5*3, CYP3A4*1B and CYP3A4*22 alleles of liver grafts may explain about one third of the inter-individual differences in pharmacokinetics of ciclosporin and tacrolimus in recipients. However, non-genetic factors, influencing CYP3A expression, can contribute to the variability of CYP3A function due to phenoconversion. The present study evaluated the association between CYP3A4 expression combined with CYP3A5 genotype of donor livers and recipients' CNI therapy after transplantation. METHODS: The contribution of donors' CYP3A5 genotype and CYP3A4 expression to the blood concentrations and dose requirements of CNIs was evaluated in 131 liver transplant recipients. RESULTS: The recipients with grafts from normal CYP3A4 expresser donors carrying CYP3A5*3/*3 required CNI maintenance doses more or less similar to the bodyweight-controlled starting doses (9.1 mg kg(-1) of ciclosporin and 0.1 mg kg(-1) of tacrolimus). The patients transplanted with grafts from low CYP3A4 expressers required substantial reduction (by about 50%, 4.2 mg kg(-1) of ciclosporin, 0.047 mg kg(-1) of tacrolimus, P < 0.001), while the recipients with grafts from high expressers or with grafts carrying at least one copy of the functional CYP3A5*1 allele required an increase (by about 50% [12.8-13.8 mg kg(-1)] for ciclosporin and 100% [0.21 mg kg(-1) ] for tacrolimus, P < 0.001) of the initial CNI dose for achieving target blood concentrations. CONCLUSIONS: Donor livers' CYP3A-status, taking both CYP3A5 allelic variations and CYP3A4 expression into account, can better identify the risk of CNI over- or underexposure, and may contribute to the avoidance of misdosing-induced graft injury in the early post-operative period.

Estimation of drug-metabolizing capacity by cytochrome P450 genotyping and expression.

Many undesired side effects or therapeutic failures of drugs are the result of differences or changes in drug metabolism, primarily depending on the levels and activities of cytochrome P450 (P450) enzymes. To assess whether P450 expression profiles can reflect hepatic drug metabolism, we compared P450 mRNA levels in the liver or peripheral leukocytes with the corresponding hepatic P450 activities. A preliminary P450 genotyping for the most frequent polymorphisms in white populations (CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2D6*3, CYP2D6*4, CYP2D6*6, and CYP3A5*3) was carried out before P450 phenotyping, excluding the donors with nonfunctional alleles of CYP2C9, CYP2C19, and CYP2D6 and those with a functional CYP3A5*1 allele from a correlation analysis. The hepatic mRNA levels of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 displayed a strong association with P450 activities in the liver, whereas the expression of CYP1A2, CYP2C9, CYP2C19, and CYP3A4 in leukocytes was proven to reflect the hepatic activities of these P450 species. The leukocytes were found to be inappropriate cells for the assessment of hepatic CYP2B6 and CYP2D6 activities. Combining the results of P450 genotyping and phenotyping analyses, patients' drug-metabolizing capacities can be estimated by the P450 expression in the liver and in leukocytes with some limitations. Patients' genetic and nongenetic variations in P450 status can guide the appropriate selection of drugs and the optimal dose, minimizing the risk of harmful side effects and ensuring a successful outcome of drug therapy.

High-resolution melting curve analysis to establish CYP2C19∗2 single nucleotide polymorphism: Comparison with hydrolysis SNP analysis.

Clinically significant consequences of CYP2C19 polymorphism in drug metabolism require reliable genotyping methods for assaying large numbers of blood samples. Hydrolysis single nucleotide polymorphism analysis and high-resolution melting curve (HRM) analysis successfully genotyped the 114 liver donors for CYP2C19∗2. However, the HRM analysis was found to be more efficient, rapid, user- and cost-friendly method.