| Background:Berberine?BBR?is a kind of isoquinoline alkaloid extracted from the plants of Coptis chinensis and Berberine,which has extensive pharmacological effects such as treating diabetes,arrhythmia,hypertension,anticancer and so on.It has been proved that berberine was mainly metabolized by CYP450 enzyme in vivo,and its primary metabolites berberrubine?BRB?,demethylberberine?DEB?also have a wide range of pharmacological activities.BBR and its CYP450 metabolites were not eliminated until they formed into glucuronicacid conjugates under the catalysis of UGTs.Many data show that such the induced or inhibitory effect of UGTs can lead to drug interactions in clinical practice;furthermore,BBR and berberrubine have also been reported to down-regulate the gene expression of UGTs.It is suggested that BBR and its metabolites may be the substrates of UGTs and may induce or inhibit the metabolic interaction of drugs by UGTs-mediated.However,There were no research reported about the impact on UGTs and the potential risk of drug interactions on major metabolites of BBR and CYP450.Therefore,it is necessary to pay more attention to the effect and mechanism of berberine and its CYP450 metabolites,such as,berberrubine and demethylberberine on the activity of UGTs.In order to predict the potential risks of drug interactions caused by the combined use of BBR,which is of great significance for reducing the risk of rational drug use in clinical practice.Objectives:The inhibitory effects of berberine and its metabolites?Berberrubine,Demethylberberine?on a panel of 12 recombinant human UGT isoforms were systematically investigated in the UGTs enzyme and 50 human mixed liver microsomes?pHLMs?in vitro incubation models,and the mechanism of action was discussed.In addition,in vitro-in vivo extrapolation?IVIVE?was used to predict the potential the risk of UGTs-mediated interactions between clinical drugs.Methods:1.Inhibition screening experiments of BBR,BRB,DEB against 12 UGTs enzymes12 kinds of rhUGTs?UGT1A1,UGT1A3,UGT1A4,UGT1A6,UGT1A7,UGT1A8,UGT1A9,UGT1A10,UGT2B4,UGT2B7,UGT2B15,UGT2B17?were used as the enzyme-sources to characterize the inhibitory effects of BBR,BRB,DEB against UGTs in vitro.4-MU was used as nonselective substrate for all UGT isoforms except for UGT1A4 using trifluorolazide as the probe substrate,respectively.BBR,BRB,DEB with concentrations of 0,10,100?m were added to each reaction system.To explore the glucuronidation of BBR,BRB,DEB on 12 UGTs enzymes in vitro,and screened out the influential UGTs enzyme subtype through LC-MS/MS detection and analysis.2.The inhibition effect of BBR,BRB and DEB on UGT1A1 expressed in vitroThrough the screen-out experiments of BBR,BRB,DEB on 12 kinds of UGT enzymes in vitro,the reasonable substrate concentration was selected according to the km value.A series of drug concentrations?0,1,3,9,18,36,72,100?M?were reasonably designed as inhibitors under the substrate concentration.To calculate the enzyme kinetic parameters IC50 of glucuronidation of BBR,BRB,DEB in vitro.3.The evaluation of enzyme kinetic characteristics of UGT1A1 by BBR,BRB and DEBFour concentration points were selected as the inhibitor concentration corresponding to the IC50 values what we measured before,and another four different substrate concentrations based on the Km value from literature were reasonably selected as the substrate concentration to carry out the glucuronidation of BBR,BRB and DEB in pHLMs and recombinant UGTs enzyme incubation system,respectively.The corresponding kinetic parameters Ki values were calculated by Graphpad prism software,and the type of enzyme kinetic inhibition was discussed.4.Prediction of drug interactions induced by BBR,BRB and DEBBased on the inhibitory kinetics of BBR and CYP450 metabolites on UGTs enzyme kinetics studies above,combined with the relevant literature data,the in vivo/in vitro interaction prediction model was used to predict the potential risk of UGTs-mediated drug interactions in BBR,BRB and DEB in clinical practice.Results:1.Inhibition screening of BBR,BRB,DEB against 12 humanUGTs enzymesBBR,BRB and DEB have certain inhibitory effects on 12 UGTs enzymes under the concentrations of 0,10,100?M.When the drug concentration is 100?M,the residual activity of UGT1A1 enzyme is 27%,15%and 29%respectively.However,the residual activity of inhibition of other 11 enzyme subtypes except UGT1A1 was all over 50%,and the inhibition efficiency was relatively weak.2.The inhibitory effect of BBR,BRB and DEB on UGT1A1 expressed in vitroThe IC50 values of BBR,BRB and DEB for 4-MU-O-gluconylation mediated by UGT1A1 were 12.44±0.072?M,6.239±0.05?M and 15.00±0.62?M,respectively.The IC50 values of BBR,BRB and DEB for bilirubin-O-gluconylation mediated by UGT1A1 were 13.16±0.59?M,5.764±0.73?Mand 16.04±0.36?M,respectively.BRB,the main CYP450 metabolite of BBR,showed strong inhibition on UGT1A1?IC50<10?M?,and its IC50 value was about 0.5 times and0.44 times of the original BBR.3.The value of Ki and inhibitory type of BBR,BRB and DEB on UGT1A13.1 BBR showed mixed competitive inhibition on UGT1A1-mediated 4-MU-O-glucuronidation,with Ki value of 11.334±0.146?M.Both of BRB and DEB showed non-competitive inhibition on UGT1A1-mediated 4-MU-O-glucosaldehyde with Ki value calculated to be 5.125±0.613?M,14.343±0.816?M.suggesting that in UGT1A1 incubation system in vitro,UGT1A1 enzyme combined with 4-MU first to form its complex,and then combined with BRB and DEB to prevent the formation of 4-MUG,thereby inhibiting the enzyme activity of UGT1A1.3.2 It was shown that BBR has a mixed competitive inhibition to UGT1A1 and HLM-mediated bilirubin-O-glucuronidation,for which Ki values was 10.352±0.276?M and 13.247±0.017?M,respectively.DEB showed mixed competitive inhibition on UGT1A1 and HLM-mediated bilirubin-O-glucoaldehyde,for which Ki values was 12.025±0.131?M and 16.333±0.616?M,respectively.The responses of BRB to UGT1A1 and HLM-mediated bilirubin-O-glucuronidation were mixed competitive inhibition and competitive inhibition,respectively,for which Ki values was4.681±0.746?M and 6.421±0.153?M.The results showed that in rhUGT1A1and pHLMs systems,BBR,BRB,and DEB could compete with bilirubin for the active centers of UGT1A1 enzyme,and they were successively arranged with Ki?BRB?>Ki?BBR?>Ki?DEB?.The enzyme binding ability of UGT1A1 was shown to be BRB>BBR>DEB,which interfered with the binding of UGT1A1 and bilirubin,and reduced and inhibited the activity of UGT1A1 enzyme to metabolize bilirubin.4.The prediction of potential risks for drug-drug interaction induced by BBR,BRB and DEBBBR has a weak inhibition on UGT1A1-mediated bilirubin-glucuronidation via the prediction model of in vitro-in vivo extrapolation with the[I]in,u/Ki value0.1133?between 0.1 and 1?,which stood for low risk of drug-drug interactions between BBR and UGT1A1-catalysed drugs,while its[I]in,u/Ki value to pHLMs-mediated bilirubin-glucuronidation within the range of 0.11,suggesting that it has a low risk of drug-drug interactions.The[I]in,u/Ki value of DEB was also range from 0.1 to 1,which meant medium risk of drug-drug interaction between DEB and UGT1A1-catalysed drugs.The[I]in,u/Ki value of BRB was 1.812 and 1.321?greater than 1?respectively,which was about 14-15 times than BBR's prediction value,It was suggested that BBR's CYP450 metabolite BRB has a stronger inhibitory effect on UGT1A1 than the original drug,The above data may support that the drug and its metabolites may interact between the CYP450 and UGTs metabolic systems.Conclusions:BBR,BRB and DEB all showed dose-independent inhibitory effects on UGT1A1,and BRB had the strongest inhibitory effect on UGT1A1.The prediction of in vitro/in vivo drug interaction model showed that the risk of BBR and DEB inhibiting drug interaction in UGT1A1 was lower,while its'main CYP450 metabolites,BRB,inhibited the activity of UGT1A1 was significantly higher than that of berberine among UGT1A1-catalysed drugs.This study has shown that when BBR is used combination with UGT1A1 metabolized drugs in clinical practice,it is possible to induce potential drug-drug interactions due to the inhibition of UGT1A1 by its CYP450 metabolites,which should be paid great attention to in clinical combination. |
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