Effects Of CYP2C9 Single Nucleotide Polymorphism On The Safety Of Silybin Combined With Phenytoin

Phenytoin is an important second-line drug in the treatment of epilepsy.Cytochrome P4502C9（CYP2C9）is responsible for 90%of the metabolism.The adverse reactions of phenytoin are corelated with the increase of its plasma concentration,either caused by co-administered CYP2C9 inhibitors or CYP2C9 genetic polymorphisms.Silybin is a commonly used herbal dietary supplement for liver protection and antioxidant purpose.Patients with epilepsy may co-administered silybin and phenytoin,which may cause herb-drug interaction（HDI）.Genetic polymorphisms may complicate HDI.Therefore,it is of great clinical significance to explore the effect of CYP2C9 variants on the safety of silybin combined with phenytoin.However,the HDI potential due to CYP2C9 inhibition was not evaluated.Meanwhile,whether genetic polymorphisms of CYP2C9 affect the drug-drug interaction was still unknown.Firstly,our research prepared the wild type of CYP2C9（CYP2C9*1）,and its four common variants（CYP2C9*2,*3,*8 and*27）.In vitro experiments showed that the metabolic clearance of phenytoin in CYP2C9*2,CYP2C9*3 and CYP2C9*8 was lower than that of CYP2C9*1.These results indicated that these variants are weak metabolizers.The metabolic clearance was higher in CYP2C9*27 than in CYP2C9*1.Secondly,this study evaluated the risk of HDI due to CYP2C9 inhibition when several main components of Chinese traditional medicine was taken together with phenytoin.Our study found that silybin significantly inhibited CYP2C9 catalyzed formation of phenytoin’s metabolite,i.e.,5-（4-hydroxyphenyl）-5phenylacetonylureas（P-HPPH）.When four Liverman capsules or silybin-phosphatidylcholine complex was co-administrated,phenytoin AUC ratio may exceed 1.45,and may cause HDI and subsequent phenytoin toxicity.Finally,the risk of drug interaction between silybin and phenytoin was quantitatively evaluated in CYP2C9 variants.The results showed that both silybin A and silybin B strongly inhibited CYP2C9 catalyzed phenytoin metabolism in vitro.In addition,the risk of HDI changes in different CYP2C9 variants.Individuals carrying CYP2C9*1 and CYP2C9*8 would show higher AUC elevation than CYP2C9*2,*3 and*27.Co-administration of silybin and phenytoin may cause HDI which could affect safety of phenytoin.The main content of this research includes the following three parts.SECTION 1 Co-expression of CYP2C9*1,*2,*3,*8 and*27 with POR and the different clearance activity of these variants on phenytoinObjective:Co-express CYP2C9*1,*2,*3,*8 and*27 with POR using recombinant baculovirus in Sf9 cells to obtain functional wild-type CYP2C9 and its common variants and to evaluate the metabolism of phenytoin in these five recombinant CYP2C9.Methods:1.E.coli containing CYP2C9*1,*2,*3,*8,*27 and POR Bacmid were cultured,and then CYP2C9*1,*2,*3,*8,*27 and POR Bacmid DNA were extracted.The five CYP2C9 variants and POR were transfected into Sf9 insect cells,respectively,to obtain P1 generation baculovirus.The P1 baculovirus was amplified continuously to obtain P2 and P3 generations.The P3 CYP2C9 variant baculovirus was co-infected with P3 POR baculovirus into Sf9 cells.Supersomes was extracted and recombinant CYP2C9 variants co-expressed with POR was obtained.The protein concentration of these recombinant enzyme was determined by BCA protein quantitative method.The concentration of P450 was determined by CO differential spectroscopy.The concentration of POR was determined by cytochrome c reduction assay.2.A series of phenytoin concentrations（0.0005-0.05 mM）were incubated with the five CYP2C9 variants prepared above.Ultra-performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）was used to determine the formation rate of 5-（4hydroxyphenyl）-5-phenylacetonitroide（P-HPPH）,a metabolite of phenytoin.Kinetic parameters were calculated and the effects of the CYP2C9 variants on phenytoin metabolic clearance were evaluated.Results:The above five recombinant CYP2C9 were obtained.The intrinsic clearance of CYP2C9*1,*2,*3,*8 and*27 was 2.8,1.4,0.15,1.8 and 3.5 nL/min/pmol P450,respectively.Conclusion:CYP2C9*1,*2,*3,*8 and*27 was obtained.The intrinsic clearance of CYP2C9*2,CYP2C9*3 and CYP2C9*8 was lower than that of CYP2C9*1,indicating that CYP2C9*2,*3 and*8 are weak metabolizers.The metabolic clearance rate of CYP2C9*27 was higher than that of CYP2C9*1.SECTION 2 Inhibitory effect of the main active components of Brucea javanica and Silythistle on the metabolism of PhenytoinObjective:To investigate whether the main components of Brucea and Milk thistle will inhibit Phenytoin metabolism in human liver microsomes.To evaluate the potential risk of herb-drug interaction when the above traditional Chinese medicine preparations were taken together with phenytoin.Methods:Ultra-performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）was used to determine the formation rate of 5-（4-hydroxyphenyl）-5-phenylacetonitroide（P-HPPH）,a metabolite of phenytoin,in ESI positive mode.The alteration of p-HPPH formation rate was employed to evaluate the inhibitory effect of brusatol,bruceine D,silybin A and silybin B on Phenytoin metabolism by human liver microsomes.Further enzymatic Kinetic analysis was conducted to study the inhibitory Kinetic parameters of silybin A and silybin B on phenytoin metabolism catalyzed by human liver microsomes.The potential for HDI between silybin and phenytoin in vivo was also evaluated using area under the curve（AUC）ratios.Results:Silybin A potently inhibited human liver microsomes catalyzed phenytoin metabolism with an IC50 values of 7.67 μM.Silybin B was a moderate inhibitor with an IC50 values of 15.49μM.Brusatol and bruceine D showed no significant inhibitory effect.Further studies of inhibition Kinetics showed that,the inhibition of p-HPPH formation by silybin A followed mixed-type inhibition model with a Ki value of 3.85 μM,while silybin B competitively inhibited the metabolism of phenytoin with a Ki value of 8.36 μM in HLMs.According to the calculated AUCi/AUC value,four Liverman capsules and silybin-phosphatidylcholine complex may lead to clinically significant HDI.Conclusion:In order to avoid drug interaction caused phenytoin adverse reaction,the combination of silybin A and silybin B with phenytoin should be avoided.SECTION 3 Effects of CYP2C9 single nucleotide polymorphism on silybin inhibition of phenytoin metabolismObjective:The second part of the study found that silybin A and silybin B inhibited phenytoin metabolism and would result in HDI.The prevalence of CYP2C9 genetic polymorphisms would complicate HDI.Therefore,in this part,the recombinant wild-type CYP2C9（CYP2C9*1）and its common single nucleotide variants（CYP2C9*2,*3,*8 and*27）were used to evaluate the inhibitory effect of silybin A and silybin B on phenytoin metabolism.The risks of HDI were also quantitatively predicted.Methods:Silybin A and silybin B were co-incubated with phenytoin in recombinant CYP2C9*1,2,*3,*8,or*27,respectively.Ultra-performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）was used to determine the formation rate of 5-（4hydroxyphenyl）-5-phenylacetonitroide（P-HPPH）,a metabolite of phenytoin.The alteration of p-HPPH formation rate was employed to evaluate the inhibitory effect of silybin A and silybin B on phenytoin metabolism catalyzed by CYP2C9 variants.Further enzymatic kinetic analysis was conducted to study the inhibitory kinetic parameters of silybin A and silybin B on phenytoin metabolism catalyzed by CYP2C9 variants.The potential HDI risks between silybin and phenytoin was also evaluated using area under the curve（AUC）ratios.Results:Silybin A strongly inhibited phenytoin metabolism catalyzed by CYP2C9*1,*2,*3,*8 or*27,with IC50 values of 5.16,16.52,6.70,8.20 and 11.04 μM,respectively.The IC50 values of silybin B on phenytoin metabolism catalyzed by CYP2C9*1,*2,*3,*8 and*27 was 7.90,24.68,8.06,17.82 and 11.94 μM,respectively.Silybin A seemed to be a more potent inhibitor against all the CYP2C9 variants compared with silybin B.Further Kinetic studies showed that silybin A competitively inhibited phenytoin metabolism mediated by CYP2C9*1 and*3 with Ki of 5.12 μM and 6.72 μM,respectively.Silybin A was a non-competitive inhibitor toward CYP2C9*8 and*27 catalyzed phenytoin metabolism,and the Ki was 6.08 μM and 10.69 μM,respectively.Silybin A inhibited CYP2C9*2 catalyzed phenytoin metabolism in a mixed inhibitory model with the Ki value of 9.30 μM.Silybin B competitively inhibited phenytoin metabolism mediated by CYP2C9*1 and*8 with Ki of 7.93 pM and 6.21 μM,respectively.In CYP2C9*2,*3 and*27,the inhibition type was mixed,mixed and competitive,respectively.The Ki values was 22.36 μM,8.56 μM and 12.42 μM,respectively.When four Liverman capsules or silybin-phosphatidylcholine complex was co-administrated,the AUC ratio may exceed 1.45 in individuals carrying homozygous CYP2C9*1 and*8.In individuals carrying homozygous CYP2C9*2,*3 or*27,the AUC ratio would be lower.Conclusion:Silybin A and silybin B both inhibited phenytoin metabolism mediated by CYP2C9 variants in vitro.CYP2C9*1 and*8 would contribute to higher phenytoin AUC change when silybin was co-administrated than CYP2C9*2,*3 and*27.Dose should be adjusted appropriately according to the variants.