Establishment And Application Of An Assay System For Evaluating The Effects Of Cytochrome P450 Non-synonymous Single-nucleotide Polymorphisms On Drug Safety

CYPs are the most important drug-metabolizing enzymes, being responsible for 70-80% of all phase I-dependent metabolism of clinically useful drugs. More than 1000 single-nucleotide polymorphisms (SNPs) have been discovered in CYP genes, of which the most important ones are the non-synonymous SNPs (nsSNPs). nsSNPs lead to the expression of variant CYP enzymes that may possess altered metabolic activities and increase the risk of ADRs for individuals carrying the nsSNPs. Pharmacogenomic studies also indicate that CYP nsSNPs can have profound effects on the ability and potency of drugs to inhibit CYPs, thus resulting in unexpected drug-drug interactions (DDIs) in individuals carrying polymorphic genotypes.CYP genetic polymorphisms and CYP-mediated DDIs are both important in the assessment of drug safety and efficacy. However, the effects of CYP nsSNPs on DDIs are still underappreciated in drug development because methods are lacking to study the effects of a large number of SNPs.To address this deficiency, we established and experimentally validated a homogeneous yeast assay system consisting of eight human CYP isoforms and 17 CYP3A4 nsSNP variants. We tested the metabolic activities of those enzymes and their abilities to alter the drug's inhibitory potencies. The results suggested that 9 of the 17 CYP3A4 nsSNP variants can lead to deceased metabolic activities. Those variants also can weaken the inhibitory potencies of most tested drugs to CYP3A4.Furthermore, we tested a new chemical entity, tanshinol borneol ester (DBZ), in this system to systematically evaluate the effects of CYP nsSNPs on both the CYP-inhibition potential of DBZ and on DBZ metabolism. The inhibitory potency of DBZ toward three CYP3A4 allelic variants, CYP3A4.2,3A4.12, and 3A4.16, was reduced by 2-10 folds relative to prototype CYP3A4, which implies that DBZ may have less potential to interact with CYP3A4-metabolised drugs in patients carrying these three alleles. Furthermore, compared with prototypic CYP2C8, the allelic variant, CYP2C8.3, produced a 54% decrease in the intrinsic clearance of DBZ, which suggests a dosage adjustment of DBZ may be needed in patients carrying the CYP2C8*3 allele. This pharmacogenomic information could help to rationalize clinical trials of DBZ, including improving the selection of drug doses, drug combinations and volunteers. Our results suggest applications for this in vitro CYP assay system both for basic research in pharmacogenomics and for drug development.