ABSTRACT
BACKGROUND: Cytochrome P450 3A enzymes exhibit a variety of physiological roles and have been reported to be the most predominant enzymes involved in drugs metabolism. Single nucleotide polymorphisms (SNPs) in the genes that code for these enzymes may result in functional changes that affect enzyme activity. CYP3A4 is an important enzyme in the metabolism of many important drugs used in the treatment of breast cancer. METHODS: A total of 94 post-menopausal breast cancer patients were recruited for the study and their DNA was isolated for polymerase chain reaction (PCR). The primers were designed using Primer3 software with primer specificities checked via the Basic Local Alignment Tool (BLAST) database. The primer specificity, functionality and annealing temperature were first investigated using uniplex PCR protocols, followed by a single multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The digested amplification fragments were analysed by gel electrophoresis and subsequently validated by sequencing. RESULTS: A multiplex PCR-RFLP method was successfully developed for simultaneous detection of CYP3A4*4, CYP3A4*18B and CYP3A4*22 in a population of post-menopausal breast cancer patients. CONCLUSION: The technique is simple, cost-effective, time-saving and can be routinely applied in the identification of SNPs and determination of allelic and genotypic frequencies of CYP3A4*4, CYP3A4*18B and CYP3A4*22.
ABSTRACT
OBJECTIVE: This study aimed to investigate the effects of clarithromycin and ketoconazole on the pharmacokinetic properties of tacrolimus in different CYP3A4 genotype recombinant metabolic enzyme systems, so as to understand the drug interactions and their mechanisms further. METHOD: The experiment was divided into three groups: a blank control group, CYP3A4*1 group and CYP3A4*18 recombinant enzyme group. Each group was added with tacrolimus (FK506) of a series of concentrations. Then 1 umol/L clarithromycin or ketoconazole was added to the recombinant enzyme group and incubated in the NADPH system for 30 minutes to examine the effects of clarithromycin and ketoconazole on the metabolizing enzymes' activity of different genotypes. The remaining concentration of FK506 in the reaction system was determined using UPLC-MS/MS, and the enzyme kinetic parameters were calculated using the software. RESULTS: The metabolism of CYP3A4*18 to FK506 was greater than that of CyP3Ð4*1B. Compared with the CYP3A4*1 group, the metabolic rate and clearance of FK506 in the CYP3A4*18 group significantly increased, with Km decreasing. Clarithromycin and ketoconazole inhibit the metabolism of FK506 by affecting the enzyme activity of CYP3A4*1B and CYP3A4*18B. After adding clarithromycin or ketoconazole, the metabolic rate of FK506 significantly decreased in CYP3A4*1 and CYP3A4*18, with Km increasing, Vmax and Clint decreasing. CONCLUSION: Compared with CYP3A4*1, CYP3A4*18 has a greater metabolism of FK506, clarithromycin and ketoconazole can inhibit both the enzymatic activities of CYP3A4*1 and CYP3A4*18, consequently affecting the metabolism of FK506 and the inhibitory on CYP3A4*1 is stronger.