The Construction of CYP2C8 and CYP3A4,and the Inhibition of Paclitaxel Metabolism by Small Kinase Inhibitor

doi:10.13560/j.cnki.biotech.bull.1985.2016.07.032

Abstract

Abstract: The aims of this work are to establish the cell expression system of CYP2C8 and its 3 mutants,to study the effects of gene polymorphisms on its enzymatic activities,and to investigate the effects of small molecular kinase inhibitors on paclitaxel metabolism with the established CYP2C8 and CYP3A4 co-transfected cell lines. The gene fragments of CYP3A4,CYP2C8 and its three mutants（CYP2C8*2（805A>T）,CYP2C8*3（416G>A,1196A>G）,and CYP2C8*4（792C>G））were synthesized based on gene bank. Then those fragments were ligated to expression plasmid PEGFP-N1 for sequencing evaluation. Twenty-four hours after the wild and mutant plasmids were transfected into HepG2 cells,paclitaxel was added and incubation proceeded,and then the metabolites were quantitatively detected by well-constructed LC-MS/MS. Concurrently,the wild CYP2C8 and CYP3A4 plasmids in certain concentration ratios were transfected into HepG2 cells,to establish a co-expression system. Screening the plasmids with proper concentration ratio and transfecting them to the cells,adding small molecule kinase inhibitor while adding paclitaxel for incubation,the inhibition of paclitaxel metabolism by small molecule kinase inhibitors was investigated. The results showed that the metabolic enzyme of different CYP2C8 caused the significant difference of paclitaxel metabolic activity,of which the metabolic activity of CYP2C8*2,CYP2C8*3,CYP2C8*4 remained at about 80%（P < 0.05）,87%（P < 0.05）,and 65%（P < 0.01）,respectively,as compared to the wild type. Nilotinib completely inhibited the metabolism of paclitaxel and axitinib showed a 30% inhibition,while imatinib didn’t have any inhibitory effect. As conclusion,different genotypes of CYP2C8 differentially affect the overall paclitaxel metabolism,which might be the reason that results in the varied treatment effect in clinic. Small molecule kinase inhibitors while combined use with paclitaxel may inhibit the metabolism of paclitaxel to different extents.

Key words: paclitaxel, metabolism enzyme, gene polymorphism, small molecule kinase inhibitors, drug-drug interaction

LI Yuan HUANG Jie-qiong LI Jia-jun WANG Wei-peng ZHANG Hong-jian. The Construction of CYP2C8 and CYP3A4,and the Inhibition of Paclitaxel Metabolism by Small Kinase Inhibitor[J]. Biotechnology Bulletin, 2016, 32(7): 227-233.

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