Establishment Of A Method For Detection Of The Single Nucleotide Polymorphism In Paclitaxel-related Genes And An In Vitro Study Of Paclitaxel Drug Interactions

Background and Aims:1. There has been an increased trends in the occurrence and death of breast and ovarian cancers. The efficay and toxicity is widely different among patients, and that becomes a restricted factor responsible for the lower response rate in cancer therapy. The genetic polymorphisms of drug-related genes including drug-metabolising enzymes, transporters, signal transduction receptors and targets are closely associated with the differences in drug efficacy and toxicity. As a third generation of genetic biomarker, single nucleotide polymorphism(SNP) has been widely used in the association study of gene function with disease, and the SNP information will be valuable for drug usage and disease treatment. Exploitation of method for SNPs detection will be helpfμl to clinical examination, therefore, a SNP detection method by using enzyme agent was established in our lab.2. The metabolic interaction mediated by inhibition of drug metabolising enzymes is the main mechanism for clinical drug-drug interactions. Interactions between paclitaxel and other combined antineoplastic agents has been reported, but the mechanism remains unclear. Paclitaxel is primarily metabolized by CYP2C8 through 6a-hydroxylation, therefore, we attempt to investigate whether the interaction was caused by the inhibition of CYP2C8 enzyme through an in vitro study.Methods:1. A pair of probes was design to detect the SNP, four kinds of enzymes were used including Shrimp alkaline phosphatase, ExonucleaseⅠ,Pfu DNA polymerase, Taq DNA ligase. The specific SNP recognition was attained through a polymerization-ligation reaction by using Pfu DNA polymerase and Taq DNA ligase. Exo. I was added to degradate the residual probes to avoid false positives. Then, a PCR reaction was carried out to amplify polymerization-ligation products, thus leading to the detection signal intensified. 2. Cyclophosphamide, ifosfamide,gemcitabine,topotecan and etoposide was respectively incubated with paclitaxel in the human liver microsomes.With high performance liquid chromatography,6α-hydroxy paclitaxel was detected. The inhibition percentage by different drugs was calcμlated to evaluate their inhibition potency to CYP2C8.Resμlts:1.The genotyping resμlts showed consistent with the direct sequencing resμlts,it could accurately detect heterozygous samples.After optimization, detect costs and time was reduced, operation steps were simplified.2. Cyclophosphamide,ifosfamide,gemcitabine,topotecan and etoposide did not have obvious inhibition on paclitaxel metabolism under the concentration of 10μM, and the further experiments showed their IC50s are all above 100μM.Conclusions:1.The method could meet the rapidness and sensitivity in clinical detection, as well as low cost.2. According to the in vitro inhibition study, clinically observed drug-drug interactions between antineoplastic agents and paclitaxel may not been caused by CYP2C8 inhibition. Other factors such as other drug-metabolising enzymes and transporters should be considered in the further studies to clarify the specific mechanisms.