Constructing Stable Expression Cytochrome P450 2D6 Cell Lines And Researching Effects Of The Genetic Polymopyisms On Aripiprazole Metabolism In Vitro

Aims:This study aimed to confirm the effect of CYP2D6 genetic polymopyism on Aripiprazole metabolism in vitro. In vitro, constructed CYP2D6 wild type (WT) and the mutant expression vectors and induced in HepG2 cells to expression. This project researches CYP2D6 genetic polymopyisms on the metabolism of Aripiprazole, by comparing different CYP2D6 genotypes on the metabolism of Aripiprazole to prove that CYP2D6 mutation would affect the process of in vitro metabolism of Aripiprazole.Methods:Extracted mRNA from human liver, reversed to gain cDNA, and PCR amplificated the gene CYP2D6*1 (wild type) sequence as a template and by site-directed mutagenesis PCR to construct CYP2D6*10 (mutant) cDNA in vitro. The CYP2D6*1 cDNA and CYP2D6*10 cDNA connected to the pcDNA3.1 vector and induced in HepG2 cells to expression, then extractting the recombinant enzyme proteins, using Western blotting to validate its expression, and using the probe drug Bufuralol for CYP2D6*1 and CYP2D6*10 for enzyme kinetic analysis and verifying the metabolic activity. At the same time using the inhibitor Quinidine for the inhibition experiments to verify the model is correct. Substrate Aripiprazole and the cell lines were incubated, then absorbing the supernatant, and using HPLC techniques to measured the substrate concentration, to calculated the enzyme metabolism of Aripiprazole in the Michaelis-Menten constant (Km) and Maximum velocity (Vmax), and to calculated the intrinsic clearance rate (CLint).Results:Real-time PCR showed good amplification of target gene CYP2D6, screened cell lines showed the expression of CYP2D6 and Western-blotting showed that CYP2D6*1 and *10 have been well expressed. In the metabolism of Bufuralol, the Km value of CYP2D6*1 is 18.93±1.094, the Km value of CYP2D6*10 is 14.21±2.576, so we can derived that CYP2D6*1 and CYP2D6*10 for Bufuralol shows no significant difference in affinity, and the Vmax value of CYP2D6*1 is 160.8±3.518, the Vmax value of CYP2D6*10 is 26.11±1.642, so we can derived that the Maximum velocity of CYP2D6*10 for Bufuralol is about 1/6 of CYP2D6*1, and the CLint value of CYP2D6*1 is 8.494±1.143, the CLint value of CYP2D6*10 is 1.837±0.279, so we can derived that the intrinsic clearance rate of CYP2D6*10 for Bufuralol is about 1/5 of CYP2D6*1. In the metabolism of Aripiprazole, the Km value of CYP2D6*1 is 24.23±3.027, the Km value of CYP2D6*10 is 10.28±1.301, so we can derived that the affinity of CYP2D6*10 for Aripiprazole is higher than CYP2D6*1, and the Vmax value of CYP2D6*1 is 42.18±2.156, the Vmax value of CYP2D6*10 is 7.844±0.313, so we can derived that the Maximum velocity of CYP2D6*10 for Aripiprazole is about 1/6 of CYP2D6*1, and the CLint value of CYP2D6*1 is 1.741±0.196, the CLint value of CYP2D6*10 is 0.763±0.087, so we can derived that the intrinsic clearance rate of CYP2D6*10 for Aripiprazole is about 1/2 of CYP2D6*1.Conclusions: 1. Successfuly constructed a stable expression of CYP2D6 wild type and CYP2D6*10 mutant cell lines.2. CYP2D6*10 mutations in the CYP2D6 on the metabolism of Aripiprazole, decreased activity, and CYP2D6 enzyme activity of the mutant is lower than the wild type,but the affinity of * 10 to Aripiprazole increased in contrast.