Construction Of UGT1A1*1 And UGT1A1*6 Activity Evaluation System And Its Application In In Vitro Safety Study Of Irinotecan

UGT1A1 is an important phase II drug-metabolizing enzyme expressed on the smooth endoplasmic reticulum membrane of the liver.It is involved in the metabolism of various drugs and endogenous compounds,including SN-38(active metabolite of irinotecan)and bilirubin.Its gene is highly polymorphic,and more than 100 variants have been found.SN-38 is the active product of the chemotherapeutic drug irinotecan,which is catalyzed by UGT1A1 to inactive SN-38G.The combination of irinotecan and UGT1A1 inhibitors or UGT1A1 gene polymorphism may affect the blood concentration of SN-38 in vivo,leading to adverse drug reactions.Patients who use irinotecan may also take Chinese herbal medicine or dietary supplements,such as liver-protective Silymarin,anxiolytic Hypericum perforatum extract and antioxidant resveratrol.If the components of dietary supplements derived from these natural products affect the UGT1A1 activity,herb-drug interaction(HDI)may occur,which may cause adverse reactions of irinotecan.Especially in the presence of UGT1A1 gene polymorphism,the interaction may become more complex.Therefore,it is of great clinical significance for the safe use of irinotecan to explore the effects of the main active components of the above natural products on the metabolism of SN38,especially in the presence of common variant UGT1A1*6 in Asian population.However,there is no quantitative study on the effect of active ingredients such as silybins and amentoflavone on the metabolism of SN-38 catalyzed by UGT1A1*1 and UGT1A1*6.In view of this,this study first used insect Sf9 cells and baculovirus system to express and prepare microsomes containing UGT1A1*1(wild type)or UGT1A1*6.In vitro enzyme activity experiments verified that UGT1A1*6 showed lower metabolic clearance ability for SN-38 than UGT1A1*1.Secondly,we evaluated the risk of HDI induced by several active ingredients that may be taken with irinotecan based on inhibiting UGT1A1*1 and UGT1A1*6.Silybins and amentoflavone showed the strongest inhibitory effect on SN-38 glucuronidation.Further quantitative analysis suggested that the combination of irinotecan and silybinphosphatidylcholine complex,two Legalon capsules,four Silymarin tablets,four Liverman capsules or natural products containing amentoflavone may lead to significant HDI by inhibiting UGT1A1.Therefore,the combination of hydrous silybins and amentoflavone with irinotecan should be avoided.Finally,we prepared UGT1A1*1 and UGT1A1*6 immobilized enzyme systems based on nano-silica materials,respectively.While applying them to UGT1A1 metabolic activity and inhibitory activity,we tried to achieve enzyme repetition to solve the problems of limited source of metabolic enzymes and high experimental cost.This paper mainly includes the following four chapters:Chapter 1 Preparation of Microsomes Containing Recombinant Human UGT1A1*1 and UGT1A1*6 and Study on SN-38 MetabolismObjective:Microsomes expressing human UGT1A1*1 and UGT1A1*6 were obtained.The differences in the metabolic scavenging ability of SN-38 were evaluated,and the inhibitory effects of active components in several natural products on UGT1A1*1 and UGT1A1*6 catalyzing SN-38 metabolism were screened.Methods:1.The microsomes expressing human UGT1A1*1 and UGT1A1*6 were prepared using insect Sf9 cell-baculovirus expression system.The recombinant enzyme was qualitatively and quantitatively evaluated by Bradford,Western Blot and ELISA assay.2.Using SN-38 as the substrate,the formation rate of SN-38 glucuronate(SN-38G)catalyzed by UGT1A1 was detected by ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS),and the differences in kinetic parameters of UGT1A1*1 and UGT1A1*6 metabolizing SN-38 were evaluated.3.The inhibitory effects of several compounds from natural products on UGT1A1*1 and UGT1A1*6 catalyzed SN-38G formation were screened.Results:Microsomes containing recombinant human UGT1A1*1 and UGT1A1*6 were successfully prepared.The intrinsic clearance of SN-38 metabolized by UGT1A1*1 and UGT1A1*6 were 0.055 and 0.016 pL/min/pmol UGT,respectively.Several active components of traditional Chinese medicine including shikonin,resveratrol,piceatannol,silybins and amentoflavone can inhibit the glucuronidation of SN-38 catalyzed by UGT1A1*1 and UGT1A1*6.Among them,silybins and amentoflavone showed the strongest inhibitory effect.Conclusion:Microsomes containing recombinant human UGT1A1*1 and UGT1A1*6 were successfully prepared.The intrinsic clearance of UGT1A1*6 to SN-38 was lower than that of UGT1A1*1,which was a weak metabolizer.Silybins and amentoflavone showed strong inhibitory effects,and further studies should be performed to quantitatively evaluate the inhibitory effects.Chapter 2 Inhibition of UGT1A1*1 and UGT1A1*6 catalyzed glucuronidation of SN-38 by silybinsObjective:Quantitative evaluation of the inhibitory effect of silybin A and silybin B on UGT1A1*1 and UGT1A1*6 catalyzed SN-38 metabolism.The risk of adverse reactions of irinotecan based on UGT1A1*1 or UGT1A1*6 inhibition was predicted when silybins was taken with irinotecan.Methods:Using the UGT1A1*1 or UGT1A1*6 microsomes obtained in the first chapter,silybin A and silybin B were incubated with SN-38,respectively.The formation rate of SN-38G was detected by UPLC-MS/MS.The inhibitory effect of silybin A and silybin B on UGT1A1*1 and UGT1A1*6 catalyzed SN-38 metabolism was evaluated by the change of residual activity.IC25,IC50 and IC75 were calculated by nonlinear regression.Isobolographics was used to illustrate the additive,synergistic,or antagonistic effect of the co-occurrence of silybin A and silybin B on SN-38 glucuronidation.Finally,the potential of HDI in vivo was predicted by calculating AUCi/AUC ratios(the ratio of the area under the plasma concentration-time curve of SN-38 in the presence and absence of silybins).Results:Silybin A inhibits SN-38 metabolism catalyzed by UGT1A1*1 and UGT1A1*6 with IC50 values of 0.512 μM and 0.999 μM,respectively.Silybin B inhibits SN-38 metabolism catalyzed by UGT1A1*1 and UGT1A1*6 with IC50 values of 0.747 μM and 2.599 μM,respectively.At the concentration around IC50,silybin A and silybin B showed synergistic effect in UGT1A1*1 microsomes,while additive effect on UGT1A1*6 was observed.When the concentration reached IC95,antagonistic effect was observed in both UGT1A1*1 and UGT1A1*6 groups.Through calculation and prediction,it was found that silybinphosphatidylcholine complex,two Legalon capsules,four Silymarin tablets or four Liverman capsules may increase the AUCi/AUC by more than 1.23 in UGT1A1*1 group.Meanwhile,the predicted Rgut values of all groups increased by 11 times.Conclusion:Silybin A and silybin B inhibited UGT1A1*1 and UGT1A1*6 mediated SN-38 metabolism in vitro.The prediction results suggest that when silymarin products are taken with irinotecan,HDI may occur.Chapter 3 Inhibition of UGT1A1*1 and UGT1A1*6 catalyzed glucuronidation of SN-38 by AmentoflavoneObjective:The purpose of this chapter was to evaluate the inhibition kinetics of amentoflavone on UGT1A1*1 and UGT1A1*6 catalyzed SN-38 glucuronidation was evaluated.The risk of HDI induced by inhibiting UGT1A1*1 and UGT1A1*6 was also quantitatively evaluated.Methods:Amentoflavone was co-incubated with SN-38 using UGT1A1*1 and UGT1A1*6 recombinases obtained in the first chapter.Using UPLC-MS/MS method,the formation rate of SN-38G was detected.The inhibitory effect of amentoflavone on SN-38 metabolism catalyzed by UGT1A1*1 and UGT1A1*6 was evaluated.The inhibition kinetic parameters of amentoflavone on SN-38 metabolism catalyzed by UGT1A1*1 and UGT1A1*6 were obtained by kinetic analysis.Finally,the possibility of HDI in vivo was predicted by calculating AUCi/AUC ratios(the ratio of the area under the plasma concentration-time curve of SN-38 in the presence and absence of amentoflavone).Results:The IC50 values of amentoflavone on UGT1A1*1 and UGT1A1*6 catalyzed SN-38 metabolism inhibition were 0.030 μM and 0.062 μM,respectively.Further kinetic studies showed that amentoflavone was a non-competitive inhibitor toward UGT1A1*1 and UGT1A1*6 mediated SN-38 metabolism,with Ki values of 0.012 μM and 0.043 μM,respectively.When the concentration of amentoflavone in vivo reached 15 ng/ml,the predicted AUCi/AUC was higher than 1.23.At the same time,according to our prediction,Rgut values were much higher than 11 in all the groups.Conclusion:The combination of the products containing amentoflavone and irinotecan may lead to HDI,and there is a risk of adverse reactions of irinotecan.Chapter 4 Immobilization of UGT1A1 by nano-silica material and its applicationObjective:In order to save the experimental cost and solve the problem of limited enzyme source,the immobilization of enzyme was used to realize the reuse of enzyme.The stability and repeatability of the immobilized enzyme and its inhibitory effect were evaluated.Methods:1.In this part,UGT1A1*1 and UGT1A1*6 were immobilized by using nano-silica material as carrier base on the adsorption force between silica and phospholipid in microsome membrane.2.The activity of immobilized and unimmobilized UGT1A1 were compared after they were stored at 4 ℃ for 1,2,10,and 15 days,respectively.3.The catalytic activities of UGT1A1-nano silica complex after 5 times recycling for were determined to evaluate the recovery stability of the immobilized enzymes.4.Using silybin A and silybin B as inhibitors,the application of UGT1A1 nano-silica complex in inhibitor screening field was evaluated.Results:After 15 days of storage,UGT1A1 nano-silica complex still retains the ability to catalyze the formation of SN-38G,and its residual activity is close to or higher than 4℃ preserved microsome group.The results show that the catalytic activity of UGT1 A1-nano silica complex is stable.The UGT1Al-nano silica composite was reused for 5 times,and the residual activity was still above 50%,indicating that the composite could be reused for many times.The inhibition trend was similar in the UGT1 A1 microsome and the UGT1A1-nano silica complex group,indicating that the UGT1A1-nano silica complex is suitable for UGT1A1 inhibitors screening.Conclusion:The UGT1A1 nano-silica complex prepared in this part can be used for the evaluation of enzyme activity and inhibitors screening,and showed good stability and repeatability.To a certain extent,it can alleviate the problem of limited source of metabolic enzymes and reduce the cost of experimental research.However,the immobilization method still needs to be improved to obtain better stability and reusability.