| Background:Oleanolic acid(OA)has a protective effect on liver injury,and it is widely used in adjuvant treatment of acute and chronic hepatitis.Ursolic acid(UA)as an isomer of oleanolic acid,for which the structure and pharmacological effects are similar to oleanolic acid.Moreover,it also has good clinical application prospect.The previous study of our research group confirmed that the in vivo metabolism of ursolic acid is related to the phase II metabolic enzyme UGT1A3,UGT1A4.However,it is reamins unclear that whether ro not ursolic acid and oleanolic acid have an effect on the expression and activity of UGTs,and whether or not OA/UA and UGTs-mediated drugs interactions will occur when co-administered with one another.Thus,it is worth our attention to study it.Objectives:To systematically study the effects of UA and OA on the enzymatic activity and expression of serial UGTs using the UGTs recombinant enzyme and HepG2 cell model.To predict the potential drug-drug interaction among UGTs-mediated drugs via in vivo-in vitro extrapolation prediction model.Methods:(1)The effects of UA and OA on the activity of UGTs recombinase and the prediction of the risk for drug-drug interaction(1)The effects of UA and OA on the activity of 12 UGTs(UGT1A1,UGT1A3,UGT1A4,UGT1A6,UGT1A7,UGT1A8,UGT1A9,UGT1A10,UGT2B4,UGT2B7,UGT2B15,UGT2B17)were systematically studied in-vitro UGTs recombinase incubation system.4-methylumbelliferone(4-MU)was used as probe substrate for all UGTs,except for UGT1A4 for which trifluoperazine was used as probe substrate.(2)Based on the enzyme kinetic study,the enzyme inhibition kinetic parameters—IC50 and Ki value were calculated and the inhibition type and mechanism were explored and discussed.Based on the enzyme kinetic study,the potential risks for drug-drug interaction between UA/OA and UGTs--catalyzed drugs were characterized by in vitro-in vivo extrapolation prediction model and relevant data from previous researches.(2)The effects of UA and OA on the expression of UGTs mRNA in HepG2 cellsThe effects of UA/OA on the expression of 8 common UGTs mRNA in HepG2 cells were studied with the help of RT-qPCR technology,followed by predicting the potential risks of UGTs-mediated drug-drug interaction when UA/OA was co-administered with other drugs for a long-term use.Results:(1)The effects of UA and OA on the activity of UGTs recombinase and the prediction of potential risks for drug-drug interaction.Among 12 UGTs recombinases,UA and OA only exhibited significant inhibitory effects on the activity of UGT1A3 and UGT1A4.UA competitively inhibited UGT1A3,for which the IC50 was 0.391 ± 0.013 ?M and Ki was 0.185 ± 0.015 ?M.What's more,UA also competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation,for which the IC50 was 2.651 ± 0.201 ?M and Ki was 1.334 ± 0.146 ?M.OA showed mixed inhibition(coexistence of competitive inhibition and noncompetitive inhibition)on UGT1A3-mediated 4-MU-?-Dglucuronidation with IC50 and Ki value calculated to be 0.336 ± 0.013 ?M and 0.176 ± 0.007 ?M,respectively.However,OA showed competitive inhibition on UGT1A4-mediated trifluoperazine-N-glucuronidation with IC50 and Ki value calculated to be 5.468 ± 0.697 ?M and 6.298 ± 0.891 ?M,respectively.It was shown that UA and OA has a strong inhibitory effect on the activity of UGT1A3 via the prediction model of in vitro-in vivo extrapolation and the [I]in,u/Ki value for UA and OA was 3.054 and 1.489(greater than 1)respectively,which meant highly possible drug-drug interaction between UA/OA and UGT1A3-catalysed drugs might occur.UA showed a ralatively strong inhibition on UGT1A4 and the [I]in,u/Ki value for UA was 0.424(within the range of 0.1~1),which meant medium risk of drug-drug interaction between UA/OA and UGT1A4-catalysed drugs.OA showed a relatively weak inhibition on UGT1A4 and its [I]in,u/Ki value was 0.042(less than 0.1),which stood for low risk of drug-drug interactions between UA/OA and UGT1A4-catalysed drugs.(2)The effects of UA and OA on the expression of UGTs mRNA in HepG2 cellsThe addition of UA(10 ?M,20 ?M,40 ?M)showed no significant effects(P > 0.05)on the expression of UGT1A1,UGT1A3,UGT1A4,UGT1A6,UGT1A9,UGT2B4 and UGT2B15 mRNA in HepG2 cells.OA(10?M)exhibited no significant effect on the expression of these 8 common UGTs mRNA(P>0.05)in HepG2 cells.However,the induced efficiency of OA at 20?M for UGT1A1,UGT1A3,UGT1A4,UGT1A6,UGT1A9,UGT2B4,UGT2B7 and UGT2B15 mRNA was 1.55(P < 0.001),1.49(P < 0.001),2.40(P < 0.001),3.36(P < 0.001),1.30(P < 0.05),1.26(P < 0.01),1.53(P < 0.001)and 1.29(P < 0.01)respectively.The induced efficiency of OA at 40?M for UGT1A1,UGT1A3,UGT1A6,UGT1A9 and UGT2B15 mRNA was 1.41(P < 0.001),1.32(P < 0.01),1.56(P < 0.01),1.69(P < 0.001)and 1.20(P < 0.05)respectively.Conclusions:(1)UA showed a competitive inhibitory effect on the activity of UGT1A3 and UGT1A4.OA showed a mixed competitive inhibition on UGT1A3 and a competitive inhibition on UGT1A4.(2)UA and OA both showed strong inhibitory effects on the activity of UGT1A3,which indicated that the occurence of drug-drug interactions when co-administered the UGT1A3-catalysed drugs was highly possible.UA showed a relatively strong inhibition on UGT1A4,which meant medium risk of drug-drug interaction between UA and UGT1A4-catalysed drugs.OA showed a relatively weak inhibition on UGT1A4,which suggested that the drug-drug interactions between OA and UGT1A4-catalysed drugs were not likely to occur.(3)OA showed strong induction on the expression of UGT1A1,UGT1A3,UGT1A4,UGT1A6,UGT1A9,UGT2B4,UGT2B7,and UGT2B15 mRNA in HepG2 cells. |
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