Effects Of Berberine On Metabolism Of Rat Hepatic CYP3A4

Drug interactions have became the main content of drug safety evaluation.CYP3A4 is one of the largest subfamilies in the human CYP450 enzyme families and is widely involved in the metabolism of clinical drugs.CYP3A4 can be induced or inhibited by other drugs and compounds,leading to serious adverse reactions and drug interactions.Berberine widely exists in a variety of Chinese herbal medicines,food and vegetables,it can induct or inhibit human and animal liver CYP450 enzyme in vitro and in vivo.With the widespread application of berberine and its preparations in daily life,the problem of its interaction with CYP3A4 has also become increasingly prominent.ObjectivesIn this study,we studied the effect of berberine on CYP3A4 in rats in vivo and in vitro,and to elucidate the pharmacokinetics of midazolam in rats and its mechanism for the study of clinical rational drug and drug interactions.Methods1 Determination of CYP3A4 inhibitory constant（K_i）in rat liver by berberine Blank rat liver microsomes were prepared by calcium precipitation.Midazolam,potassium phosphate buffer and microsome suspension were added into rat liver microsomes,NADPH was added to initiate the reaction after pre-incubation.After incubation,ice was added The reaction was stopped by acetonitrile.Then vortexed,centrifuged,and the supernatant was analyzed for midazolam concentration.The conversion of midazolam in rat liver microsomes was used as an indicator of enzyme activity.In the above incubation system,the effect of different concentrations of berberine on the conversion of midazolam was determined,and the inhibition constant of the activity of berberine on CYP3A4 was calculated.2 Effects of berberine on liver CYP3A4 in rats at different doses and different time Rats were administrated with gavage berberine at low dose(75mg·kg^-1·d^-1),middle dose(150 mg·kg^-1·d^-1)and high dose(300 mg·kg^-1·d^-1)for 8days to prepare liver microsomes.The conversion rate of midazolam in liver microsomes was used as an index of enzyme activity to investigate the effect of different doses of berberine on liver CYP3A4 in rats.The rats were administrated with berberine 150 mg·kg^-1·d^-1 for 4 days,8 days and 12 days,respectively.The liver microsomes were prepared and the conversion of midazolam in rat liver microsomes.The conversion rate of midazolam in liver microsomes was used as an index of enzyme activity to investigate the effect of different time periods of berberine on liver CYP3A4 in rats.3 Effects of berberine on CYP3A4 in rats Twelve healthy maleSprague-Dawley rats were randomly divided into control group（n=6）and test group（n=6）.After 3 days of continuous administration of saline,the rats in the control group were treated with 150mg·kg^-1·d^-1 berberine 3d,the two groups were immediately after the last gavage,intravenous injection of 10mg·kg^-1 midazolam injection,and at different time points after administration by orbital canthus blood0.5ml,after centrifugation and efficient.Midazolam concentrations in rat plasma were determination by liquid chromatography（HPLC）.Data were processed using DAS 2.0 software to calculate midazolam pharmacokinetic parameters,the peak concentration（Cmax）of the measured value to investigate the effect of berberine on midazolam pharmacokinetics.Result1 Determination of CYP3A4 inhibitory constant（K_i）in rat liver by berberine The K_m of CYP3A4 in rat liver CYP3A4 was 28.67μM,the V_max was3.06 nmol·min^-1·mg protein^-1,and the internal clearance rate CLint was 0.11ml·min^-1·mg^-1.Berberine competitive inhibition of rat liver microsomal CYP3A4 on the metabolism of midazolam,the inhibition of K_i is 116.4μM.2 Effects of berberine on liver CYP3A4 in rats at different doses and different time The low dose of berberine and the control group had no significant difference.The middle and high dose of berberine could significantly inhibit the activity of CYP3A4 in rats and decrease the metabolism of midazolam in vitro,The results showed that K_m were 28.66±7.89,28.88±8.11,30.99±7.78 and 36.37±8.22μM,respectively;V_max were 3.96±0.30,3.05±0.30,2.89±0.26 and 2.54±0.82nmol·min^-1·mg protein^-1,respectively;CL_int were 138.17±5.29、105.61±3.65、93.26±1.47 and 69.84±1.08 ml·min^-1·mg^-1,respectively.There was no significant difference between the three groups and the control group（P<0.05）.Berberine could significantly inhibit the activity of CYP3A4 in rats and decrease the metabolism of midazolam in vitro.The results showed that K_m were28.66±7.89,28.88±8.11,30.99±7.78 and 36.37±8.22μM,respectively;V_max were3.96±0.30,3.05±0.30,2.89±0.26 and 2.54±0.82 nmol·min^-1·mg protein^-1,respectively;CLint were138.17±5.29,105.61±3.65,93.26±1.47 and 69.84±1.08ml·min^-1·mg^-1,respectively.3 Effects of berberine on CYP3A4 in rats in vivo Compared with the midazolam group and midazolam combined with berberine solution group.The main pharmacokinetic parameters C_max were 6.53±0.58 and 8.82±1.05μg·ml^-1,respectively,and T1/2 were30.00±8.75 and 27.42±5.08 min,respectively,Vd were2.85±1.35 and1.92±0.43 L·kg^-1,respectively,and CL were4.03±1.24 and 2.98±0.76 L·h^-1·kg^-1,respectively,and AUC_0-∞were 2.81±1.11 and 3.59±0.95mg·h·L^-1.Conclusion1.Berberine competitively inhibits the metabolism of rat liver microsomes CYP3A4 on midazolam.2.The inhibitory effect of berberine on CYP3A4 in rats increased with the increase of dose,and increased with the prolongation of the time.3.Berberine can significantly change the pharmacokinetics of midazolam in rats,the Cmax and AUC significantly increased,while CL significantly reduced.The mechanism may be related to the competitive inhibition of berberine in rat liver CYP3A4 enzyme.