Studies On Glucuronidation Of Natural Phenolic Compounds And Efflux Transport Of Glucuronide Metabolites

Objective: The natural phenolic compounds possess a variety of pharmacological activities, such as anti-oxidation, anti-inflammation and anti-cancer effects. However, the effective forms of this type of compounds and their mechanisms of action in vivo have remained to be uncertern. It is necessary to elucidate the metabolism and disposition of these compounds. Therefore, the overall goal of this thesis is to clarify the role of UDP-glucuronosyltransferase(UGTs) in the disposition of natural phenolic compounds, and its inherent mechanisms. To approach this goal, four specific aims are(1) to characterize the glucuronidation of two natural phenolic compounds(capsaicin and psoralidin);(2) to identify the role of breast cancer resistance protein(BCRP) and multidrug resistance-associated proteins(MRPs) in the efflux transport of glucuronides;(3) to evaluate the modulatory effect of efflux transporters in cellular glucuronidation; and(4) to determine the mechanism involved in the transporter-dependent glucuronidation of natural phenolic compounds.Methods: 1. The glucuronidation rate of capsincin and psoralidin were determined using liver microsomes and expressed UGT isoforms. The relative activity factors approach(RAF) and correlation analysis were performed to identify the main UGT isoforms contributing to glucuronidation of capsaicin and psoralidin. 2. The c DNA of UGT1A1 was introduced into He La cells using the lentiviral transfection method(referred to as He La1A1 cells). The contributions of efflux transporter(i.e., BCRP, MRP1, MRP3, or MRP4) on glucuronide excretion were determined by using combined approaches of chemical inhibition(Ko143 for BCRP and MK-571 for MRPs) and biological inhibition(sh RNA-mediated gene silencing). 3. Single efflux transporter(BCRP, MRP1, MRP3, or MRP4) was stably knocked-down in He La1A1 cells. Knock-down of transporters was performed by stable transfection of shorthairpin RNA(sh RNA) using lentiviral vectors(referred to as He La1A1-BCRP-sh RNA cells, He La1A1-MRP1-sh RNA cells, He La1A1-MRP3-sh RNA cells and He La1A1-MRP4-sh RNA cells). Glucuronidation and glucuronide transport in the cells were characterized using three different phenolic compounds(i.e., genistein, apigenin,and emodin) with distinct metabolic activities. The fraction of dose metabolized(fmet), an indicator of the extent of drug glucuronidation, was calculated to identify the regulation role of transporters in cellular glucuronidation. 4. Glucuronide excretion and total glucuronidation of genistein and apigenin were determined in He La1A1-MRP4-sh RNA cells compared to in He La1A1 cells at different time points. And then a two-compartment pharmacokinetic model, incorporating the process of glucuronidation, glucuronide excretion and deglucuronidation(futile recycling), was used to describe the alterations in glucuronide excretion and cellular glucuronidation.Results: 1. Capsaicin and psoralidin were subjected to efficient glucuronidatinon.(a) Capsaicin was efficiently glucuronidated in pooled human liver microsomes(p HLM). Eight UGT isoforms(i.e., UGT1A1, 1A3, 1A7, 1A8, 1A9, UGT2B7, 2B15 and 2B17) were able to generate glucuronide from capsaicin. Capsaicin glucuronidation was strongly correlated with 3-O-glucuronidation of β-estradiol(a probe substrate for UGT1A1) and UGT1A1 protein levels(p < 0.05). Also, capsaicin glucuronidation was significantly correlated with zidovudine(a probe substrate for UGT2B7) glucuronidation and UGT2B7 protein levels(p < 0.01). UGT1A1 and 2B7 contributed 30.3%, and 49.0% of total glucuronidation of capsaicin in p HLM, respectively. The results showed that UGT1A1 and 2B7 were the main contributing enzymes for capsaicin glucuronidation.(b) HLM generated two monoglucuronides(9-O-G and 3-O-G) from psoralidin. UGT1A1, 1A7, 1A8 and 1A9 exhibited catalytic activity toward psoralidin. Of these enzymes, only UGT1A9 could catalyze the formation of 3-O-G. This result suggested that UGT1A9 was solely responsible for the glucuronidation of psoralidin at 3-OH group. Further experiment showed that formation of 3-O-G in HLM was markedly inhibited by the UGT1A9-selective inhibitors. And psoralidin-3-O-glucuronidation was strongly correlated with the UGT1A9 protein levels and propofol(a probe substrate of UGT1A9) glucuronidation(p < 0.001). Moreover, UGT1A9 was responsible for 99.6% of psoralidin-3-O-glucuronidation in HLM based on the RAF approach. Taken together, psoralidin-3-O-glucuronidation was an excellent in vitro marker for UGT1A9. 2. Efflux transporters(BCRP and MRPs) were significant contributors to cellular excretion of glucuronide. The modified cells were functional in generation of the glucuronide from chrysin. Reduced activity of BCRP and MRPs caused significantly decrease in the excretion rate and efflux clearance of glucuronide(P < 0.01). Silencing of a target transporter(BCRP, MRP1, MRP3 and MRP4) also led to a marked reduction in the excretion rate and efflux clearance of glucuronide(P < 0.01). The results suggested that cellular excretion of glucuronide was contributed by BCRP, MRP1, MRP3, and MRP4. 3. Efflux transporters(BCRP and MRPs) were involved in the total intracellular glucuronidation. He La1A1-BCRP-sh RNA, He La1A1-MRP1-sh RNA, He La1A1-MRP3-sh RNA and He La1A1-MRP4-sh RNA cells were successfully established. Knock-down of a target transporter(BCRP, MRP1, MRP3 and MPR4) resulted in significant reductions in cellular glucuronidation and excretion rate of glucuronides. The result showed that cellular glucuronidation was significantly regulated by BCRP, MRP1, MRP3 and MPR4. 4. The futile recycling played an important role in the transport-glucuronidation interplay. Glucuronide excretion and total glucuronidation of genistein and apigenin were significantly reduced in MRP4 knocked-down cells compared to control cells. The alterations were adequately characterized by a two-compartment pharmacokinetic model incorporating the process of futile recycling. This study provided the first experimental evidence that the effect of glucuronide efflux on total glucuronidation was through a futile recycling mechanism.Conclusions: 1. Capsaicin and psoralidin were subjected to significant glucuronidation. UGT1A1 and 2B7 were the main contributing enzymes for capsaicin. UGT1A1, 1A7, 1A8 and 1A9 were the main contributing enzymes for psoralidin. Further, psoralidin-3-O-glucuronidation was an excellent in vitro marker for UGT1A9. 2. Multiple efflux transporters(including BCRP, MRP1, MRP3, and MRP4) potentially mediated the glucuronide excretion and the total glucuronidation in cells. Further, we provide evidence that the modulation of transporters on total glucuronidation was through a futile recycling mechanism.