Study On The Impact Of Warfarin On The Activity Of CYP2C9 Genetic Polymorphisms And The Pharmacokinetics Of Gomisin G

ObjectTo investigate the correlation between CYP2C9 gene polymorphism and the maintenance dose of warfarin anticoagulation, predict binding sites between Gomisin G and CYP2C9, warfarin and CYP2C9, respectively, then observe the effects of Gomisin G to different genotypes of CYP2C9 and its influence to pharmacokinetic process of warfarin in vivo.MethodsWe collected 270 cases undergoing heart valve replacement and patients taking warfarin accordance with access standards Jan 1 st 2013-Dec 31 st 2015 in The First Hospital of Jilin University,3-5 mL venous blood were collected, with EDTA anticoagulant. We extracted the DNA sample with a saturated phenol-chloroform method, we detected three candidate sites in the sample CYP2C9 gene (CYP2C9 * 2, CYP2C9 * 3, CYP2C9 * c 65) genotype and allele frequencies using polymerization chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing.The crystal structure of CYP2C9 was obtained from protein database (http://www.rcsb.org/pdb), and the protein code is 1OG2 for which the substrate warfarin was bound. The chemical structure of gomisin G was drawn using chemdraw software.We have detected Gomisin G of CYP2C9 * 2, CYP2C9 * 3, CYP2C9 * c 65 action of enzymes, IC50 and time-dependent inhibition in vitro by high performance liquid chromatography, to establish the content of plasma samples warfarin measuring method to detect the influence of Gomisin G warfarin pharmacokinetics in SD rats.Results(1) We did not detect the CYP2C9 * 2 (rs 1799853) mutation in all samples, detected only one kind of allele C, genotype CYP2C9 * 2 of all C/C wild-type. Both alleles detected in the CYP2C9 * 3 (rs 1057910) in all samples, A and C, respectively. We detected three genotypes, A/A type, A/C type, and C/C type, where A/A type 231 cases, accounting for 85.6%, A/C heterozygous mutant of 25 cases, accounting for 9.26%, C/C homozygous mutant 14 cases, accounting for 5.19%. We analyzed allele results showed that the frequency of allele A was 94.3%, C allele frequency of 5.7%, there is a correlation between gene mutations and medication maintenance dose (P<0.05), A/C patients with medication dose reduction of 18.46% compared with the A/A type patients, and medication dose in C/C patients with reduction by 76.0% compared with the A/A type, indicating that inCYP2C9 * 3 mutation site C patients, the dose of warfarin was reduced. We have detected G and C from both alleles CYP2C9 * c 65 (rs9332127) sites, including G/G type and G/C type two genotypes. Our statistics G/G wild type, resulting in 246 cases, accounting for 91.1%; G/C heterozygous mutation type in 24 cases (8.9%), There is no significant correlation between warfarin maintenance dose and both two gene mutations patients(2) Several X-ray resolutions of the structures of CYP2C9 can be found in the protein database, including the crystal structure bound with warfarin and the crystal structure bound with the inhibitor. Given that the metabolic prediction of gomisin G by CYP2C9 was performed in the present study, the substrate binding crystal structure of CYP2C9 was selected. The substrate warfarin was firstly extracted from the metabolic active site, and then gomisin G was docked into the activity cavity of CYP2C9. As shown in Figure2-1, Gomisin G can be well docked into the activity site of CYP2C9, and gomisin g can form hydrogen bonds with Phe476 and Gln214. To demonstrate that gomisin G is a good substrate of CYP2C9, co-docking process was performed to dock both gomisin G and warfarin into the active site of CYP2C9. As shown in Figure 2-2, closer distance between gomisin G and the active site than warfarin was found, indicating the good substance of gomisin G for CYP2C9.(3) Gomisin G inhibition of the CYP2C9 enzyme in the three genotypes were CYP2C9 * 3> CYP2C9 * 2> CYP2C9 * 1, indicates that there are individual differences in the inhibition of the CYP2C9 enzyme Gomisin G We handle SD rats with warfarin and Gomisin G The results showed that:compared with the control group, warfarin AUC, Cmax and CL/F did not change in the administration group, and Tmax and T1/2 will be extended, indicating an impact in vivo pharmacokinetics in Gomis oct-G and warfarin, the reason may be related to Gomisin G capable of active sites warfarin competition CYP2C9 enzyme, inhibiting the effect of warfarin in the CYP2C9 enzyme.Conclusion(1) The CYP2C9 * 2 and CYP2C9 * c 65 gene sites do not significantly correlated with warfarin anticoagulant therapy, while CYP2C9 * 3 polymorphism significantly correlated with warfarin anticoagulation therapy, and mutation C patients in this site taking least warfarin.(2) The metabolic behavior of gomisin G by CYP2C9 was predicted in the present study using molecular docking method, and the results showed that gomisin G is a good substrate of CYP2C9.(3) Gomisin G inhibition of CYP2C9 enzyme, and has a difference in genotype; Gomisin G affect pharmacokinetics of warfarin, which may due to compete with warfarin CYP2C9 enzyme active site thus inhibiting themetabolism of CYP2C9 to warfarin.