UGT Metabolism Of Luteolin, Orientin And Isoorientin And BCRP Regulation On The UGT Metabolism

Background and ObjectivesIt is well known that natural products are the main sources of chemo-preventive and chemo-therapeutic agents. Flavonoids are widely distributed in plants and Chinese herbs (over20%), which have the excellent efficacy on anti-cancer, anti-oxidation and anti-inflammation. Despite of these claimed healthy benefits and demonstrated preclinical activities, there are significant challenges associated with development of flavones into chemo-preventive and chemo-therapeutic agents. The major challenge currently is their low bioavailability (<10%), as the result of extensive first-pass metabolism by phase Ⅱ enzyme including UGTs. In order to develop and enlarge the usage of flavones, it is necessary to characterise the UGT metabolism and pathway of metabolism. Both efflux transporters (eg. P-gp, BCRP, MRPs) and metabolic enzymes play important roles in flavonoid disposition. In brief, as intracellular flavonoids are metabolized by metabolic enzymes inside the cells, metabolite is too polar to passively diffuse out of cells and the metabolism will be inhibited. Efflux transporters can excrete the metabolite and maintain the metabolism. The inhibition of efflux transporters may lead to different drug pharmacological and/or adverse effect via the reduced metabolite clearance in the intestine/liver and elevated metabolite accumulation in systemic circulation. We believe that a better understand of the factors, which play the key role of drug metabolism can help to overcome their oral bioavailability barriers. It is likely that the interplay between an efflux transporter and a phase II enzyme will be helpful to understand and delineate the complex interplays between multiple phase II enzymes and efflux transporters that determine the drug bioavailability in vivo.We chose a typical flavonoid luteolin and two C-glucosyl flavones to investigate the metabolic mechanism and efflux transporter regulation on the metabolism. Luteolin, a famous flavonoid polyphenolic compound, is widely distributed in many plant groups (Bryophyta, Pteridophyta, Pinophyta, and Magnoliophyta) and food supplements. Dietary sources of luteolin include carrots, peppers, celery, olive oil, peppermint, thyme, rosemary, and oregano. Orientin and isoorientin are two isomeric C-glucosyl flavones, also called luteolin-8-C glucoside and luteolin-6-C glucoside, respectively. Orientin and isoorientin widely distributed in Polygoni orientalis Herba, Erigerontis Herba and Bamboo leaf. They have the diverse beneficial effects, such as cardiovascular protection, antioxidant, anti-inflammatory and anti-cancer function.Currently, most researchers focused on pharmacological effects of orientin and isoorientin while the studies related to their metabolism are still unclear, luteolin has not been developed as chemo-preventive and chemo-therapeutic agent, because of its low bioavailability. Extensive first-pass metabolism by UGTs and efflux by transporters lead to change the pharmacological effect. Our early study found that phase II enzymes caused extensive metabolism and the metabolites were the substrate of BCRP. Clarifing the UGT metabolic and efflux characteristics are conductive to comprehensive understanding of disposition in vivo. They can provide helpful information and guidance for overcoming the oral bioavailability barriers and drug development.In this study, we identified luteolin, orientin and isoorientin glucuronides, deeply elucidate the metabolic mechanism of the three compounds. We investigated the UGT1A9metabolism and metabolite excretion on the UDP-Glucuronosyltransferase (UGT)1A9-Overexpressing Hela cell model in order to better understand the BCRP regulation on the UGT metabolism. Methods and Results1. UGT metabolic mechanism of luteolin, orientin and isoorientin1.1Identification of luteolin, orientin, isoorientin glucuronidesA quadrupole-time of flight (Q-TOF) tandem mass spectrometer with HPLC was used to determine the molecular weight and the structure of the metabolites. For luteolin, six metabolites were formed after eight hours’incubation with UDPGA. There were three di-glucuronides and three mono-glucuronides. The three mono-glucuronides were7-O-,3’-O-, and4’-O-glucuronosyl luteolin. We also identified one of the diglucuronides was3’,4’-O-glucuronosyl luteolin. For orientin and isoorientin, they have two mono-glucuronides, respectively.1.2The UGT1A and UGT2B-mediated metabolism of luteolin, orientin and isoorientinThe present studies represent a detailed and systematic study of UGT-isoform specific metabolism of luteolin, orientin and isoorientin. They were incubated with UGT isform for30min, the concentration was10μM. The rates of metabolism by supersomes were expressed as the amount of metabolites formed (nmol/min/mg). Under such experimental condition, only five metabolites were detected including two di-glucuronides (M-2G, M6) and three mono-glucuronides (M1, M2, M3). M1and M3were the major metabolites for all of the UGT isforms. Recombinant human UGT isform UGT1A9contributed to the formation of di-glucuronide (M-2G) and momo-glucuronides (M1, M2, M3). UGT1A7, UGT1A1, UGT1A8, UGTIA10played a minor role in the formation of di-glucuronide (M-2G). UGT1A1contributed greatly to the formation of di-glucuronide (M6) and UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7played a minor role. UGT2B contributions were very little. Orientin was transformed into two metabolites (M1, M2) in vitro in recombinant human UGT isforms. UGT1A1, UGT1A7, UGT1A8, UGT1A10contributed to the formation of M1, UGT1A8played a minor role in the formation of M2. Isoorientin was also transformed into two metabolites (M1, M2) in vitro in recombinant human UGT isforms. UGT1A10contributed to the formation of M1, UGT1A1and UGT1A10played a minor role in the formation of M2.The primary contributing UGT isoform was UGT1A8and UGTIAI, UGT1A7, UGT1A8, UGT1A10were secondary contributors. Isoorientin was also transformed into two metabolites (M1, M2), the primary contributing UGT isoforms were UGT1A10and UGT1A1.1.3Kinetic characterization of luteolin, orientin and isoorientin glucuronidation in Human Liver Microsomes and Rat Liver MicrosomesIn order to study the UGT metabolic mechanism of luteolin, orientin and isoorientin, we characterized the glucuronidation of luteolin and the two C-glycosyl flavones using human liver microsomes as well as rat liver microsomes. The results indicated that the metabolism of the three flavones followed the classic Michaelis-Menten equation both in human liver and rat liver microsomes. Luteolin was metabolized faster than orientin and isoorientin. The Vmax value for7-0-glucuronosyl luteolin is the highest. The Vmax value of orientin in rat liver microsomes was higer than that in human liver microsomes. While for isoorientin, the Vmax values were similar.2. The mechanism of BCRP regulation on the UGT metabolism of luteolin2.1. UGT1A9inhibitor experiment in HLMs and UGT1A9and Time-dependent of di-glucuronide (M-2G)Carvacrol was used as the inhibitor of UGT1A9in human liver microsomes and expressed UGT1A9to determine whether UGT1A9is a major isoform for metabolism contribution in the phase Ⅱ reaction system in HLMs. Approximately200μM of carvacrol inhibited the activities of M-2G, M1, M2, M3by27.8%,44.3%,17.6%and53.2%, respectively, in the phase Ⅱ reaction system in HLMs. Approximately200μM of carvacrol inhibited the activitied of M-2G, M1, M2, M3by68.9%,59.8%,34.6%and79.2%, respectively, in the phase Ⅱ reaction system in UGT1A9. The inhibition effect increased in a concentration-dependent manner in HLMs and UGT1A9. UGT1A9has the most important role in metabolizing luteolin in human liver.In order to study the formation of di-glucuronide (M-2G), luteolin was incubated for different time at a concentration of10μM with UGT1A9(final concentration,0.265mg/ml). The amount of M-2G was linear with time. 2.2Western blot analysis of UGT1A9protein expression in Hela-UGT1A9cells and enzyme kinetics study using Hela-UGT1A9cell lysate and UGT1A9isoformUDP-Glucuronosyltransferase (UGT)1A9-Overexpressing HeLa (Hela-UGT1A9) was the stably transfected with UGT1A9Hela cells. BCRP had a relatively higher mRNA expression level in this Hela cells. This cells model is an appropriate model to study the kinetic of glucuronide exfflux by Breast Cancer Resistance Protein (BCRP). Western blot analysis showed that UGT1A9was well expressed at protein levels in Hela cells with stable transfection. The kinetic profiles of Hela-UGT1A9cell lysate were determined and compared with commercially available human UGT1A9Supersomes. In general, Km values of M1, M2and M3were similar in Hela-UGTl A9and UGT1A9isoform. Vmax values from Hela-UGT1A9cells were also similar in the two sources.2.3Investigating the metabolism and excretion characteristics of luteolin in Hela-UGT1A9cellsIn our laboratory culture conditions, the Hela-UGT1A9cells were grown on6-well plates for approximately3to4days. We tested six concentrations of luteolin including1μM、2μM、5μM、10μM、20μM and40μM in the Hela-UGT1A9cells model to study the metabolism and excretion characteristics. In Hela-UGT1A9cells, luteolin was metabolized into glucuronides and excreted quickly. From1to5μM, the rates of excretion of three glucuronides increased, at5μM, they reached a plateau and decreased from20μM. By contrast, the amount of intracellular glucuronides increased from1μM to40μM. The concentration of intracellular glucuronides increased faster than the changes in the loading concentration. As a result, the cellular clearance of the three glucuronides significantly decreased. In addition, Fmet decreased with increasing concentration.2.4The mechanism of BCRP regulation on the UGT metabolism of luteolin in Hela-UGT1A9cellsA specific and potent chemical inhibitor Ko143was used to determine the role of BCRP in excretion of glucuronides in Hela-UGT1A9cells. The concentrations of Ko143(5and10μM) were selected and the concentrations of luteolin were2,10,40 . The date showed that the excretion rates of glucuronides were reduced significantly in the presence of Ko143. Compared with the control, the intracellular amount of glucuronides increased significantly. At10μM substrate concentration, di-glucuronide was detectable with5μM Ko143. At40μM substrate concentration, di-glucuronide was detectable with or without Ko143. Compared with the control, the intracellular amount of di-glucuronide increased significantly. The total intracellular amounts of mono-glucuronides were increased with Ko143. The total amounts of intracellular and extracellular mono-glucuronides reduced with Ko143. Additional analysis of the results indicated that Ko143limited or even did not affect glucuronidation, as evidenced by the moderate decrease in Fmet. Compare with the limited reduction of Fmet, Ko143drastically inhibited the cellular clearance of the three momo-glucuronides. At10μM substrate concentration, the cellular clearance of the three momo-glucuronides reduced in the presence of5or10μM Ko143. At40μM substrate concentration, the cellular clearance of the three momo-glucuronides reduced in the presence of5μM Ko143.In summary, this thesis investigated the glucuronidation mechanism of luteolin, orientin and isoorientin. We found three novel di-glucuronides of luteolin in vitro and one was determined as3’,4’-luteolin glucuronides for the first time. The interplay between phase Ⅱ enzymes and efflux transporters cause extensive metabolism and low bioavailability for luteolin. When the function of BCRP was blocked or mono-glucuronide concentration was sufficient, the cell system promotes a compensatory pathway for the clearance of luteolin to further metabolize mono-glucuronides to di-glucuronides. All of this can provide guidance for improving their bioavailability and the pre-clinical studies.