Study On The Inhibitions And Expressions Of Imrecoxib On CYP2C9 Enzyme

The occurrence of many diseases are accompanied by inflammation.Hundreds of millions of patients suffer from inflammation each year.Imrecoxib is a new selective inhibitor of COX-2,which has a good therapeutic effect on acute and chronic inflammation.Recent studies showed that imrecoxib combined with CYP2C9 substrate drug changed the metabolism of imrecoxib,however,the effect on CYP2C9enzyme was still uncertain,and conclusions of related literature reports on in-vivo and in-vitro experiments were inconsistent.It was reported in literature that the successive perfusion of imrecoxib accelerated the metabolism of co-administered tolbutamide in rats,and an induction effect on CYP2C9 by imrecoxib showed.While another in-vitro liver microsomes experiment showed that imrecoxib had a slight inhibition effects on CYP2C9.Then,whether imrecoxib has an induction effects or an inhibition effects on CYP2C9,and whether long-term administration of imrecoxib affects the medicine metabolized through CYP2C9 enzyme are the questions need to be clarified now.This experiment probed the effects of imrecoxib on CYP2C9 enzyme through the in-vivo and in-vitro experiments and carried out the preliminary study on the effects and mechanisms,which aimed to provide the theoretical basis for the reasonable clinical application of imrecoxib and the prevention of adverse drug interactions.Part 1 The effects of imrecoxib on CYP2C9 enzyme in liver microsomes incubation systemObjective:To establish a UPLC method for determining the content of4-Hydroxy-tolbutamide in liver microsomes,and probe the effects of imrecoxib on the activity of CYP2C9 enzyme by measuring the content of4-Hydroxy-tolbutamide in CYP2C9 liver microsomes incubation system.Methods:UPLC chromatographic conditions:ACQUITY UPLC?BEH-C18;chromatographic column（2.1×50 mm,1.7 m）;column temperature was 30°C;the mobile phase was water-acetonitrile（V/V=76:24）;the flow rate was 0.4 mL/min;the detection wavelength was 235 nm,the sample volume was 2μL;and the internal standard was carbamazepine.500μL of liver microsomes incubation system was added with 50μM of tolbutamide and stopped reactions after the incubation in water at 37°C,then,5μL of carbamazepine fluid was added and mixed together,then extracted with 1500μL of ethyl acetate,removed the supernatant of 1250μL and dried,next it was redissolved in 50μL of methanol and removed the supernatant.Then UPLC method was adopted to measure the content of4-Hydroxy-tolbutamide,the metabolin of tolbutamide,in liver microsomes of rats.The single-factor method was adopted to optimize the incubation protein concentration and incubation time of liver microsomes.Then analyzed the effects of imrecoxib on the function of CYP2C9 enzyme through comparing the changes of 4-Hydroxy-tolbutamide content in liver microsomes incubation system before and after the combination of imrecoxib.Results:1.The UPLC method for determining the content of 4-Hydroxy-tolbutamide in liver microsomes incubation system was established.The standard curve equation of this method is:Y=0.1024X-0.0107.The linear relationship was good in the range of 0.3125 to 20μg/mL and specificity,lower limit of quantitation,precision,recovery rate and stability met the requirements of detection on biological samples.2.The incubation conditions for liver microsomes were screened and optimized:1 mg/mL was selected as incubation protein concentration of liver microsomes finally and 60 min was set as the incubation time for liver microsomes.3.The in-vitro liver microsomes incubation experiment showed that with the increase of imrecoxib concentration,the production of 4-Hydroxy-tolbutamide was lower compared with the control group.The fitting result indicated that the inhibition effects of imrecoxib on the activity of CYP2C9enzyme was increased dose-dependently.The IC₅₀ was 74.77μM.Summary:This study established the method for determining the content of 4-Hydroxy-tolbutamide in rat liver microsomes incubation system.The liver microsomes incubation experiment results showed that imrecoxib had an inhibition effects on CYP2C9 enzyme in rat liver microsomes,but IC50 was far higher than clinical blood concentration.It was speculated that imrecoxib has a weak direct inhibition effects on CYP2C9 enzyme under the clinical dosage.Part 2 The expressions of imrecoxib on CYP2C9 enzyme in rat liverObjective:To determine the changes in the protein and mRNA expression of CYP2C9 enzyme in liver after successive intragastric administration of imrecoxib to rats,and to explore the effects and mechanisms of imrecoxib on CYP2C9 enzyme.Methods:The rats were randomLy divided into groups.Successive intragastric administration of imrecoxib（10mg/kg）or equal volume of blank solvent for different days.Removed livers after the completion of the last intragastric administration and probe the effects of imrecoxib on CYP2C9enzyme protein and mRNA expression of rat livers by the methods of Western Blot and RT-PCR.Results:The results of Western Blot experiment showed that with the extension of the time for intragastric administration of imrecoxib,the rat livers’CYP2C9 strips gradually became lighter and the CYP2C9 protein expressions on the 1st,7th and 14th day of intragastric administration were80%,37%,and 34%compared with the control group respectively,there was a significant statistical differences（P<0.05）.PCR experiment results showed that with the extension of the time for intragastric administration of imrecoxib,the CYP2C9 enzyme mRNA expression of rat liver significantly decreased and the CYP2C9 enzyme mRNA expressions on the 1st,7th and 14th day of intragastric administration were 65%,35%,and 34%compared with the control group respectively,there was a significant statistical differences（P<0.05）.Summary:With the extension of the time for intragastric administration of imrecoxib,both CYP2C9 enzyme protein expression and mRNA expression in rat liver decreased gradually,and the results of protein and gene expression were consistent,indicated that imrecoxib reduced protein synthesis by regulating the expression of mRNA possibly.Conclusion:This study established UPLC method for measuring the content of 4-Hydroxy-tolbutamide in the incubation system of rat liver microsomes and obtained an appropriate incubation system of liver microsomes through screening and optimization.The in-vitro liver microsomes experiment showed that imrecoxib had a certain inhibition effects on CYP2C9 enzyme in the rat liver microsomes,but IC50 was far higher than the clinical blood concentration under the clinical dosage.Therefore,it is believed that imrecoxib has a weak direct inhibition effects on CYP2C9enzyme.When the animals were administered as a whole,,the CYP2C9enzyme protein expression and mRNA expression of rat liver gradually decreased with the extension of time for intragastric administration of imrecoxib,and there was consistency in the regulation of both expressions,which indicated that imrecoxib affected the expression of mRNA in the process of transcription and caused the reduction of CYP2C9 enzyme protein synthesis.