Preliminarily Pharmacokinetic Study Of The Flavonoid Derivative Fla-CN

Objective:In the study of anti-diabetes active ingredients from Potentilla chinensis,we have got a main compound tiliroside which could significantly reduce the blood glucose.Then in the preliminarily pharmacokinetic study of tiliroside,we find that this compound can be decomposed mostly by carboxylesterases in vivo,its half-life is short and its bioavailability is low,all of this lower its drug efficacy.Then we find a more active flavonoid derivative Fla-CN by further synthesis and activity screening,the anti-diabetic effection of which is better than Metformin.In our experiment,we studied the pharmacokinetic properties of Fla-CN preliminarily.In one hand,we investigated the impact of oralling Fla-CN initially on rat cytochrome P450(CYP450)enzymes(CYPlA2,CYP2C9,CYP2C19,CYP2D6,CYP2E1 and CYP3A4)activity in vivo metabolism.In the other hand,we established a LC-MS/MS method to determinate the rat plasma drug concentration of Fla-CN and its metabolites,and then preliminarily invesgated its pharmacokinetics to provide a reference for the further study.Methods:1.We used a Cocktail approach including six probe drugs,namely caffeine,metoprolol,omeprazole,tolbutamide,chlorzoxazone and midazolam to evaluate the metabolic activities of CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP2E1 and CYP3A4 in vivo in rats by the changes of the six probe drugs’ pharmacokinetic parameters.The rats were divided into six groups:blank control group,low-dose Fla-CN group,medium-dose Fla-CN group,high-dose Fla-CN group,inhibitor control group,inducer control group.After continuous administration of 14 days,the rats were given intraperitoneal injection of Cocktail substrates solution,and then we collected the angular vein blood in the eye before administration and at different time points after administration of Fla-CN.After that,we used HPLC-UV detection method to determinate the plasma drug concentrations of each probe drug.Lastly,we assessed the impact of Fla-CN to the six CYP450 isoforms in rat in vivo metabolic activity by comparing the pharmacokinetics parameters of the probe drugs between the control group and the experimental group.2.Healthy male SD rats were orally administrated of the test drug Fla-CN at the dose of 140 mg/kg,and then we collected the angular vein blood in the eye at different time points to prepare analytical samples.Tiliroside from Potentilla chinensis was used as the intermal standard,analytical samples were analyzed by HPLC-MS/MS method in which samples were separated on Agilent XDB-Cls column by using acetonitrile-water(65:35)as mobile phase at the flow of 0.4 mL·min-1.The quantitation was performed on an LC-MS/MS with negative electrospray ionization in multiple reaction monitoring(MRM)mode.After that we speculated the possible metabolites,and then synthesized the corresponding reference drugs for further confirmation.Finally,we performed a quantitative analysis to plot the concentration-time curves and fitted the pharmacokinetic parameters in vivo by DAS 2.0 pharmacokinetic software.Results:1.Compared with the blank control group,in low-dose Fla-CN group AUC and MRT of caffeine reduced,CL of caffeine increased(P<0.05);in high-dose Fla-CN group AUC and MRT of caffeine reduced(P<0.05);in high-dose Fla-CN group AUC and MRT of metoprolol increased,CL of metoprolol reduced(P<0.05),t1/2 of metoprolol did not change significantly;in medium and high dose Fla-CN groups AUC of midazolam increased,CL of midazolam reduced(P<0.05),Ua of midazolam did not change significantly.There were no significant differences between the main pharmacokinetic parameters of chlorzoxazone and tolbutamide in each group(P>0.05).2.We identified two Fla-CN metabolites M1 and M2 in the plasma of rats after administration of Fla-CN through the analysis of rat plasma using LC-MS/MS means.At the same time,we established a LC-MS/MS method to performe quantitative analysis for Fla-CN and the two metabolites M1 and M2.The linear calibration curves were obtained at the concentration range of 0.5-1000 ng mL-1 for Fla-CN,0.1-1000 ng·mL-1 for M1,and 0.2-1000 ng·mL-1 for M2.The inter-day and intra-day precision values were below 4.9%,and the accuracy(relative error)was with in ±6.2%.AUC0-∞ of Fla-CN,M and M2 were 173.982,24.271 and 0.557μg·L-1.h,Tmax of which were 1.500,10.375 and 0.500 h,Cmax of which were 22.117,0.722 and 0.077 μg·L-1,t1/2Z of which were 8.516,6.055 and 9.146 h after administration.Conclusion:Our experiment has performed a preliminary assessment to the impact of Fla-CN on the activity of CYP450,and the results suggest that Fla-CN could inhibit metabolic activity of CYP2D6 and CYP3A4 in vivo,and induct metabolic activity of CYP1A2 in vivo,but had no significant effect on metabolic activity of CYP2C9 and CYP2E1 in vivo.The pharmacokinetic parameters of rats had been administered Fla-CN at the dose of 140 mg/kg have showed that Fla-CN distribute in vivo mainly in the form of Fla-CN,and its metabolites distribute in vivo narrowly.Cmax of Fla-CN and its metabolites Ml and M2 were lower,indicating a lower bioavailability of oralling administration Fla-CN.The reason may be of low solubility in water,not completely being absorbed or the affect of the metabolic enzymes in gastrointestinal tract and so on,which required further study.