Studies On N-glucuronidation Of Tertiary Alkaloid And Its Regulatory Mechanism

Objective:Alkaloids are a group of organic compounds that contain mitrogen atoms.Many natural products?e.g.,camptothecin,ephedrine,aconitine,and strychnine?and synthetic drugs?e.g.,procaine,trifluoperazine,and amitriptyline?belong to the alkaloid class.Alkaloids possess many pharmacological activities,including antiviral,antibiosis,anticancer,anti-inflammatory and anti-depression activities.N-glucuronidation is an important pathway for the metabolism and disposition of some alkaloids in humans.N-glucuronidation regulates the concentrations of many tertiary alkaloids in biofluids,thus influencing the efficiency/toxicity of tertiary alkaloids.However,the metabolic patterns of N-glucuronidation are not clear,and the factors influencing N-glucuronidation remain to be determined.Therefore,the overall goal of this thesis is to clarify the roles of UDP-glucuronosyltransferases?UGTs?in the N-glucuronidation of tertiary alkaloids,and to evaluate the regulatory effects and underlying mechanisms of efflux transporters and nuclear receptors in regulating N-glucuronidation of alkaloids.To approach this goal,four specific aims are?1?to develop an in vitro assay system for N-glucuronidation,and identify tertiary alkaloids that can undergo N-glucuronidation;?2?to evaluate the roles of human UGT1A4 and UGT2B10 enzymes in catalyzing N-glucuronidation of tertiary alkaloids,and to investigate the species differences of N-glucuronidation;?3?to investigate the roles of efflux transporters in the efflux transport of N-glucuronides,and to evaluate the potential regulatory effects of efflux transporters on N-glucuronidation;and?4?to identify the key nuclear receptor involved in regulating N-glucuronidation and to determine the underlying mechanism.Methods:1. Specific substrates?trifluoperazine for UGT1A4,cotinine and amitriptyline for UGT2B10?were used to develop the in vitro assay system for N-glucuronidation.We evaluated several factors that affect N-glucuronidation.These factors include:buffer types,p H values,pore-forming agents,concentrations of Mg Cl₂ and UDPGA,and inhibitor of?-glucuronidase.Then the N-glucuronidation of tertiary alkaloids were evaluated using the optimized metabolic conditions,the generated N-glucuronides were detected and confirmed by LC-MS/MS analysis.Furthermore,recombinant UGT1A4 and UGT2B10enzymes were used to evaluate their abilities in catalyzing N-glucuronidation of tertiary alkaloids.2.The N-glucuronidation of six alkaloids?i.e.,mirazapine,mianserin,cyclizine,chlorcyclizine,strychnine and brucine?was systematacially evaluated using several methods,including reaction phenotyping,kinetic analysis,correlation analysis,and inhibition experiment.The contributions of UGT1A4 and UGT2B10 in the N-glucuronidation of these alkaloids were clarified.3.The c DNA of UGT2B10 was introduced into HEK293 cells?referred to as HEK2B10cells?using the lentiviral transfection method.A combination of q PCR,Western blotting and activity assays were used to confirm the successful construction of the HEK2B10 cells.The metabolism and metabolites efflux of three alkaloids?i.e.,amitriptyline,mianserin,and cyclizine?were evaluated using the constructed HEK2B10 cells.The regulatory effects of efflux transporters?i.e.,BCRP and MRP4?on N-glucuronidation were determined by using combined approaches of chemical inhibition?MK-571 for MRP4 and Ko143 for BCRP?and biological inhibition?sh RNA-mediated gene silencing?.4.The m RNA expression levels of several nuclear receptors involved in regulating drug-metabolizing enzymes in human hepatoma Hep G2 cells were determined by q PCR assay.Specific agonists were used to identify the key nuclear receptors in regulating UGT2B10 expression in Hep G2 cells.The regulatory mechanism of the key receptor on UGT2B10 was explored by using combined approaches of luciferase repoeter assay,electrophoretic mobility shift assay?EMSA?and chromatin immunoprecipitation assay?CHIP?.Results:1. The in vitro assay system for N-glucuronidation was developed According to the metabolic velocities of probe substrates in the assay system,we optimized the contents of several factors affecting N-glucuronidation.The optimized assay system consists of 100 m M Tris-HCl buffer?p H 7.4?,4 m M Mg Cl₂,20?g/m L alamethicin,and 8 m M UDPGA.Under the optimized condition,we determined the metabolic velocities of three alkaloids?trifluoperazine,cotinine and amitriptyline?in individual human liver microsomes.The N-glucuronidation rates of amitriptyline?5 and 100?M?were well correlated with the glucuronidation rates of cotinine?2 m M??r>0.8,p<0.001?,while they were not correlated with the glucuronidation rates of trifluoperazine?40?M?.These results suggested that amitriptyline at low concentrations could be used as probe substrate for evaluating UGT2B10 activity in human liver.2.UGT1A4 and UGT2B10 catalyzed N-glucuronidation of many tertiary alkaloids We identified 17 tertiary alkaloids that could undergo N-glucuronidation in human liver microsomes.These alkaloids included asenapine,loxapine,clozapine,chlorpromazine,dothiepin,doxepin,mirtazapine,mianserin,chlorcyclizine,cyclizine,promathazine,cyclobenzaprine,imatinib,retrorsine,strychnine,trazodone and brucine.Among these substrates,trazodone was metabolized neither by UGT1A4 nor by UGT2B10,retrorsine and brucine were only metabolized by UGT1A4,while other alkaloids were glucuronidated by both UGT1A4 and UGT2B10.3.UGT2B10 was a key enzyme in catalyzing N-glucuronidation of tertiary akaloids UGT2B10 played key roles in the N-glucuronidation of five alkaloids?mirtazapine,mianserin,cyclizine,chlorcyclizine and brucine?,while it was not involved in the N-glucuronidation of brucine.Compared with UGT1A4,UGT2B10 showed high affinities for all alkaloids as indicated from the results of kinetic analysis,indicating that UGT2B10might play a pivotal role in the in vivo N-glucuronidaton reaction.4.Significant species differences were observed in the N-glcuronidation of alkaloids.The N-glucuronidation of nine alkaloids?i.e.,trifluoperazine,cotinine,amitriptyline,mirtazapine,mianserin,cyclizine,chlorcyclizine,strychnine and brucine?in livers from human and animals?i.e.,monkey,mouse,rat,rabbit,dog and guinea pig?were quite different.It was noteworthy that these alkaloids was well glucuronidated in humans,whereas they were hardly glucuronidated in mouse and rat?two commonly used animal models?.The results demonstrated that conventional animals cannot be used to study N-glucuronidation of tertiary alkaloids..5.Efflux transporters?BCRP and MRP4?regulated N-glucuronidation.The HEK2B10 cells were successfully constructed and validated.Three alkaloids?amitriptyline,mianserin and cyclizine?respectively generated one N-glucuronide when they were incubated with HEK2B10 cells.Chemical inhibition or biological inhibition of BCRP and MRP4 led to significant reduction in the excretion of N-glucuronides,while they promoted the accumulation of N-glucuronides in cells,indicating that BCRP and MRP4 were responsible for the excretion of these N-glucuronides.In addition,inhibition of BCRP or MRP4 decreased the generation of N-glucuronides in HEK2B10 cells,while it promoted the generation of N-glucosides.Based on these results,it can be concluded that the efficiency of UGT2B10-mediated N-glucuronidation was dependent on the function of efflux transporter?the so called“efflux transporter-UGT enzyme interplay”?.Our results also indicated that N-glucosidation can serve as a compensating pathway for N-glucuronidation.6.The bile acid nuclear receptor FXR regulated the expression of UGT2B10 We uncovered that UGT2B10 was transcriptionally regulated by FXR in Hep G2 cells.GW4064 and CDCA treatment of Hep G2 cells led to an increase in the m RNA level of UGT2B10.The treated cells also showed enhanced glucuronidation activities toward amitriptyline?an UGT2B10 probe substrate?.A combination of reporter gene assay,EMSA,and CHIP demonstrated that the FXR receptor trans-activated UGT2B10 through its specific binding to the-209-to-197-bp region?an IR1 element?of the UGT2B10 promoter.In addition,the identified UGT2B10-IR1 element was crucial for the basal expression of UGT2B10 gene.These results indicated that FXR was a key regulator for UGT2B10expression,and it may contribute to the significant inter-individual differences of UGT2B10 expression in human livers.Conclusion:1. This study discovers that N-glucuronidation is a common metabolic pathway for many tertiary alkaloids,contributing to a better understanding of the metabolic pathway of alkaloid in human.2.This study unreveals that N-glucuronidation show significant species differences,providing forceful guidance.for the proper selection of model animal.3.This study indentifies that UGT1A4 and UGT2B10 are main metabolizing enzymes for N-glucuronidation of tertiary alkaloids,and UGT2B10 may play a pivotal role in N-glucuronidaton in human,providing scientific basis for the functional study of UGT2B10.4.This study finds that N-glucuronidation can be regulated by efflux transporters?i.e.,BCRP and MRP4?and nuclear receptor FXR,revealing the complexity of N-glucuronidation in vivo.