| Background:Hesperetin(HET)and naringenin(NGR),aglycones of the flavanone glycoside hesperidin and naringin,which were found to be abundant in the plants of rutaceae family such as oranges and grapefruits.They also were bioactive components of Aurantii Fructus Immaturus,which is used as traditional Chinese medicine.Several researchs have shown that hesperetin and naringenin possessed pharmacological effects such as anti-inflammation,anti-oxidation,anti-tumour,cardiovascular protection and so on.In the past decades,the inhibition of HET and NGR on CYP450 has been widely studied.But the effects of HET and NGR on UGTs activity and the interaction among UGTs-metabolised drugs have not been fully characterized yet.Thus,this research is pivotal for providing guidance on the co-administration of HET/NGR and drugs metabolized by UGTs and for promoting the clinical rational use of drugs.Objective:A panel of recombinant human UGT isoforms as well as substrates including trifluoperazine as a specific probe substrate for UGT1A4 and 4-methylumbelliferone for other 11 UGT isoforms were chosen to characterize the inhibitory effects of HET and NGR on 12 human UGTs in the in-vitro incubation system,and through the in vitro-in vivo exploration to predict the risk of drug-drug interactions,it provide theoretical and experimental basis for clinical rational drug use.Method:1、The screening of HET/ NGR-inhibited UGTs subtypeTo incubate 4-MU/TFP with 12 recombinant UGTs in the presence of HET/NGR at three different concentrations(1,10,100μM)in the incubation system and to calculate the residual enzyme activity,preliminary evaluation of HET/NGR-inhibited UGTs subtype.2、Enzyme kinetics analyses for HET/NGR-associated inhibition on recombinant human UGTsAccording to the results of screening,a series concentration of HET/NGR were incubated in UGTs system.To calculate the residual enzyme activity and IC50 values by Graphpad Prism 5.0.Based on the UGTs subtypes of IC50<10μM,inhibitors at 3different concentrations and substrate at 3 different concentrations were slected further research.The inhibition type and Ki values were determined by Dixon and Lineweaver plots(and plot slope).3、Prediction for the risk of clinical drug interactionTo calculate the value of [I]in vivo/Ki based on the HET/NGR’ clearance,Cmax,dissociation constant of HET/NGR and the kinetic parameters of UGTs from previous study.As per the evaluation standard,[I]in vivo/Ki <0.1 means not possible;0.1<[I]in vivo/Ki <1 means possible;[I]in vivo/Ki>1 means highly possible,which were used to predict the risk of interaction among clinical drugs.Results:1.The screening results of HET and NGR-inhibited UGTs subtype(1)The addition of HET(100μM,final concentration)exhibited inhibition on UGT1A1,UGT1A3,UGT1A4,UGT1A7,UGT1A8,UGT1A9,UGT2B4 and the corresponding residual activities of UGT1A1,UGT1A3,UGT1A4,UGT1A7,UGT1A8,UGT1A9,UGT2B4 were less than 50%.(2)The addition of NGR(100μM,final concentration)exhibited inhibition on UGT1A1,UGT1A3,UGT1A4,UGT1A7,UGT1A8,UGT1A9,UGT1A10,UGT2B7,UGT2B15 and the corresponding residual activities of UGT1A1,UGT1A3,UGT1A4,UGT1A7,UGT1A8,UGT1A9,UGT1A10,UGT2B7,UGT2B15 were less than 50%.2.Kinetic analyses for HET/NGR-associated inhibition on recombinant human UGTs(1)Hesperetin inhibited UGT1A1,UGT1A3,UGT1A4,UGT1A7,UGT1A8,UGT1A9,UGT2B4,respectively,and the corresponding IC50 values were4.72±0.52μM,9.19±0.37μM,63.87±3.67μM,29.68±1.87μM,74.75±4.73μM,3.94±0.17μM,32.73±1.67μM.Based on above IC50 values(<10μM),HET competitively inhibited UGT1A1-catalyzed 4-MU glucuronidation,and exertedmixed inhibition towards UGT1A3 and 1A9-catalyzed 4-MU glucuronidation,the Ki values were calculated to be 9.63±0.87μM,0.99±0.08μM,3.41±0.35μM,respectively.(2)Hesperetin inhibited UGT1A1,UGT1A3,UGT1A4,UGT1A7,UGT1A8,UGT1A9,UGT1A10,UGT2B7 and UGT2B15,respectively,and the corresponding IC50 values were 8.577±0.65μM,10.85±0.37μM,55.21±1.54μM,31.91±1.87μM,26.77±0.56μM,15.34±0.39μM,38.35±1.05μM,3.47±0.52μM,32.51±2.03μM.Based on above IC50 values(<10μM),NGR was found to be a strong competitive inhibitor of UGT2B7 with Ki of 1.39±0.21μM,and it also exerted intermediate non-competitive inhibition against UGT1A1 with Ki of 7.61±0.56μM,as well as an intermediate un-competitive inhibition against UGT1A3 with Ki of 85.76±1.67μM.3.The risk prediction analysis of clinical drug interactionsThe results showed that the values of [I]in vivo/Ki for HET-inibited UGT1A3 and NGR-inhibited UGT2B7 were within the range 0.1~1,which indicated that highly possible HDI between HET/NGR and drugs mainly undergoing UGT1A3/UGT2B7-catalysed metabolism might occur.Therefore,we should pay more attention with repsect to clinical co-administration of HET/NGR and UGT1A3/UGT2B7-catalysed drugs.Conclusion:1.HET competitively inhibited UGT1A1-catalyzed 4-MU glucuronidation,and exerted mixed inhibition towards UGT1A3 and 1A9-catalyzed 4-MU glucuronidation,NGR non-competitively inhibited UGT1A1 and exerted un-competitive towards UGT1A3 and competitively inhibited 2B7-catalyzed 4-MU glucuronidation.2.Through the in vitro-in vivo extrapolation,we concluded that HET and NGR inhibited the activity of UGT1A3 and UGT2B7 respectively and it was highly possible that drug interation will occur among HET/NGR and UGT1A3/UGT2B7-catalysed drugs.Thus,close attention should be paid to monitor the drug interaction when UGT1A3/UGT2B7-mediated drugs were co-administered with one another. |
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