Mechanism Investigation On The UGT Metabolism Of Methoxylated Flavonoids

Flavonoids, characterized as polyphenolics, are widely distributed in our daily diets, beverages, medicinal plants and herbal remedies. The diverse biological effects of flavonoids have attracted great interests of scientists. The major reported pharmacological activities of flavonoids include antioxidative effects, protection against cardiovascular disease, and anticancer effects, etc. Despite of the above reported beneficial properties and demonstrated preclinical activities, it's a serious conern that the oral bioavailabilites of flavonoids were reported to be low (<10%), which is a big challenge to develop flavonoids into chemo-preventive and chemo-therapeutic agents. Therefore, a key issue in the development of flavonoids as disease prevention and therapy agents is to find a way to increase their bioavailabilities.The metabolic pathways of flavonoids were diverse and complicated because of their complex structures. It was reported that extensive first-pass metabolism by phaseⅡenzymes including UGTs and SULTs was suggested to be the major causes for their low bioavailabilities; also the glucuronidation of flavonoids is the major metabolic pathway resposnsible for their low bioavailabilities. The majority of UGTs displayed broad and overlapping substrate specificities. Many studies including our own have shown that glucuronidation of flavonoids were strongly influenced by their structures. Furthermore, recent studies demonstrated that the methoxylated flavones, which may have chemopreventive properties superior to the more common unmethylated flavonoids or polyphenols. Therefore, the aim of this thesis is to investigate the mechanism on the UGT metabolism of methoxylated flavonoids.In order to explore the metabolic characterization of methoxylated flavonoids, two 5,7-dihydroxyflavone, wogonin and oroxylin A, as well as three 5-monohydroxyflavone, tectochrysin (5-hydroxy-7-methoxyflavone,5H7MF),5-hydroxy-7,8-dimethoxyflavone (5H7,8MF),5-hydroxy-6,7,8,4'-tetramethoxy-flavone (5H6,7,8,4'MF) were selected as the model compounds. For the first two dihydroxyflavone, there exist minor structural differences of methoxyl position between them. For the later three monohydroxyflavone, the number of methoxyl is increased in order. A set of commercially available expressed human UGTs and human intestinal and liver microsomes were used to make detailed and systematic metabolic profiling studies for the five flavonoids in vitro. Firstly, the objective of the studies is to obtain the UGT-isoform specific metabolic fingerprint (GSMF) for the five model compounds. Another objective is to determine if GSMF and isoform-specific metabolism profiles might be used to predict glucuronidation rates and profiles in microsmes derived from human intestine and liver, two major organs responsible for first-pass metabolism as well as recycling via enteric and enterohepatic schemes. Finally, the third objective is to analyze the effect of methoxyl changes on the characterization of UGT-isoform specific metabolic of model compounds.Methods.1. Enzymatic activities of human expressed UGTs and organ microsomesFor the experiments of GSMF, isoform-specific metabolism profiles and human organ-specific metabolism profiles, the incubation procedures for measuring enzyme's activities using microsomes or UGTs were performed and essentially the same as the previous publications from University of Houston. Briefly, the procedures were as follows:(1) mix microsomes/supersomes (final concentration≈in range of 0.0053-0.053 mg protein per ml as optimum for the reaction), magnesium chloride (0.88 mM), saccharolactone (4.4 mM), alamethicin (0.022 mg/ml); different concentrations of substrates in a 50 mM potassium phosphate buffer (pH 7.4); and UDPGA (3.5 mM, add last) to a final volume of 680μl; (2) separate the 680μl mixture to obtain three equal portions with the volume of 200μl, and incubate them at 37℃simultaneously for a predetermined period of time (10 to 60 min); and (3) stop the reaction by the addition of 100μl of 94%acetonitrile/6%glacial acetic acid containing 90μM acetophenone (propiophenone for 5H7MF,5H7,8MF) as the internal standard. The reaction mixture was centrifuged at 13,000 rpm for 15min and the supernatant was directly subjected to UPLC for analysis. To profile UGT's activities, three substrate concentrations,2.5,10 and 35μM were used. To profile kinetics of wogonin and oroxylin A by UGT 1A1,1A3 and 1A7-1A10,9-11 substrate concentrations in the range of 1.25-35μM (0.5-35μM) were used.2. UPLC analysis of five flavones and their glucuronidesTo make precise analysis for all model compounds and their metabolites, a common method was applied for wogonin, oroxylin A as well as their corresponding glucuronides:system, Waters Acquity UPLC with photodiode array detector and Empower software; column, BEH C18,1.7μm,2.1×50 mm; mobile phase B,100% acetonitrile, mobile phase A,100%aqueous buffer (0.1%,v/v formic acid, pH 2.5); flow rate 0.4 ml/min; gradient,0 to 1.5min,30-40%B,1.5 to 2.5 min,40-70%B, 2.50 to 3.0 min,70-30%B, wavelength,280 nm for flavones and their respective glucuronides and acetophenone; and injection volume,10μl. For 5H7MF,5H7,8MF, 5H6,7,8,4'MF as well as their corresponding glucuronides, analysis method was similar to that of wogonin, oroxylin A with minor modification as follows:gradient,0 to 1.5min,30-40%B,1.5 to 3.0 min,40-90%B,3.0 to 4.0 min,90-30%B.Stability studies were made to ensure all model compounds keep stable during the course of experiments. Analytical methods for each compound were validated for inter-day and intra-day variation using 6 samples at three concentrations (40,10 and 1.25μM).3. Determination of conversion factor for four flavone glucuronidesTo quanify glucuronides more precisely, following experiment procedures were operated to obtain the conversion factor (K) for the glucuronides of the four flavones: (1) glucuronidate substrates with the most active UGT isoform using the incubation procedures described previously; for 5H7MF,5H7,8MF with only one phenolic hydroxyl group, rat liver microsomes were used; (2) extract the aqueous samples containing glucuronides with dichloromethane (sample/dichloromethane=2:5, v/v) twice to remove the aglycones; (3) divide one extracted aqueous sample into two equal portions, where one portion was subjected to UPLC for analysis directly, and the other was analyzed after hydrolysis by P-glucuronidase (800 units/ml) at 37℃for 1 h (10 h for oroxylin A,12 h for 5H7MF,5H7,8MF). The conversion factor of wogonin was also directly derived using the standard curve of both wogonin and wogonoside.4. Effects of UPLC analysis conditions on the conversion factorIn order to observe whether various analysis conditions, such as pH value and ionic strength of mobile phase, detection wavelength as well as pH value of sample medium, have influences on the conversion factor, a series of standard working solutions with seven concentrations in the range of 0.625 to 40μM for wogonin and its glucuronide were prepared. Then, analysis conditions were altered respectively and observed the variance of AWG:AW.When one of above analysis conditions changed, other analysis conditions were consistent with described in common method. Briefly, the procedures of varying analysis conditions were performed as follows:(1) prepare a series of mobile phase A with different pH value:3.5,4.5,6.5 and 8.5, via adding 0.1%aqueous ammonia into 0.1%methanoic acid solution (pH 2.5); (2) adjuste pH value of the series of solutions back to 2.5 by 1%methanoic acid, then ionic strength of these solutions were different from one another notwithstanding their pH values were same; (3) change the detection wavelentgh to 254,274,342nm; (4) pH value of sample medium were changed from 2.5 to 3.5,6.0,7.4 with 0.1% phosphoric acid.5. Confirmation of flavone glucuronide structure by LC-MS/MSTo further confirm the identity of the metabolite as mono-glucuronides of their parent compounds, LC-MS/MS was applied. The separation, detection and analysis of flavones and their glucuronides were achieved by Waters Micromass Quattro Premier XE, operated in the positive ion mode. The main mass working parameters for the mass spectrometers were set as follows:capillary voltage,3KV; cone voltage,35V; ion source temperature,100℃; desolvation temperature,350℃; cone gas flow,501/hr; desolvation temperature gas flow,6001/hr. Data acquisition and analysis were performed using a MassLynx V4.1 software (Waters Corp, Milford, MA, USA).6. Kinetic of glucuronidationTo obtain the kinetics of wogonin and oroxylin A glucuronidation by major UGT isoforms, human liver and intestinal microsomes, data from incubation experiments were used for analysis. Rates of flavone metabolism by expressed human UGT isoforms, human liver and intestine microsomes were expressed as amounts of metabolites formed per min per mg protein (nmol/min/mg). Kinetic parameters were then obtained according to the profile of Eadie-Hofstee plots. If the Eadie-Hofstee plot was linear or showed characteristic profiles of atypical kinetics, formation rates (V) of flavone glucuronides at respective substrate concentrations (C) were fit to the standard Michaelis-Menten equation or other atypical kinetics equations, using the ADAPTⅡprogram. To determine the best-fit model, the model candidates were discriminated using the Akaike's information criterion (AIC), and the rule of parsimony was applied.7. Use of expressed UGTs to predict flavone glucuronidation in human intestinal and liver microsomesTo predict flavone glucuronidation in human intestinal and liver microsomes, tissue expression of different UGTs and the contribution of isoforms to flavone glucuronidation were considered. Because each tissue expressed different UGTs and/or different quantities of the same UGTs, we first determined the main isoforms responsible for the metabolism of each flavone and then use a combination of several major active isoforms to predict substrate metabolism profiles in human intestinal and liver microsomes. The combination was achieved based on weighted mean average expression levels. Subsequently, the glucuronidation profiles obtained using the combined glucuronidation rates versus flavone concentrations were used to obtain the apparent kinetic parameters after corresponding Eadie-Hofstee plots were generated. Moreover, linear regression was applied to derive apparent correlations between rates of reaction obtained using a combination of main UGT isoforms and those obtained using human intestinal and liver microsomes.8. Statistical AnalysisOne-way ANOVA with or without Tukey-Kramer multiple comparison (posthoc) tests were used to evaluate statistical differences. Differences were considered significant when p values were less than 0.05.Results1. For wogonin and oroxylin A, their metabolites were found to be wogonin-7-glucuronide and oroxylin A-7-glucuronide respectively, GSMF experiments indicated that they were metabolized mainly by UGTIAs, with major contributions from UGT1A3 and UGT1A7-1A10. Isoform-specific metabolism showed that kinetic profiles obtained using expressed UGT1A3 and UGT1A7-1A10 could fit to known kinetic models. Glucuronidation of both flavonoids in human intestinal and liver microsomes followed simple Michaelis-Menten kinetics. A comparison of the kinetic parameters and profiles suggests that UGT1A9 is likely the main isoform responsible for liver metabolism. In contrast, a combination of UGT1As with a major contribution from UGT1A10 contributed to their intestinal metabolism. Correlation studies clearly showed that UGT isoforms could predict metabolism of these two compounds in human intestinal and liver microsomes. With the change of methoxyl from 8-position to 6-position, the glucuronidation rates decreased for most isoforms, especially for UGT1 A3,1A8,1A9.2. No metabolite was found for 5H6,7,8,4'MF. For 5H7MF and 5H7,8MF, metabolites were both found to be mono-glucuronides, almost all isoforms contribute to their glucuronidation at a low level. GSMF experiments indicated that UGT1A1 was found to be the most important isoform for 5H7MF, followed by UGT1A3. For 5H7,8MF, UGT1A3 was found to be the most important, followed by UGT1A7-1A10 and UGT1A1, especially UGT1A9 (at substrate concentration of 2.5μM,10μM) or UGT1A8 (at substrate concentration of 35μM). Correlation studies clearly showed that the glucuronidation rates of human liver microsomes have an apparent correlation with those derived from a combination of UGT 1A1,1A3 and 1A9. In addition, the UGT isoform-specific metabolic pattern changed with the increase of methoxyl number.3. Analytical methods for each compound were validated for inter-day and intra-day variation, and found that precision and accuracy for five compounds were in the acceptable range of 0.02%-6.75%and 86.73%-110.84%respectively. The result suggests that the analytical methods for model compounds were credible, and all model flavones keep stable during the course of experiments.4. The list of the conversion factor measured was as follows:wogonin,1.09±0.04; oroxylin A,1.29±0.05; 5H7MF,2.05±0.18; 5H7,8MF,1.25±0.09. The conversion factor of wogonoside was also derived directly using the standard curve of both wogonin and wogonoside, and the result was 1.03±0.02. Therefore, the two conversion factors of wogonoside derived from different methods were similar, showing that the accuracy of the conversion factor obtained for all glucuronides is acceptable.5. When the effects of various analysis conditions on the deterimination of conversion factor were investigated, it was found that the retention time of wogonin and wogonoside moved forward 39.43%,41.47%respectively, when the pH value of mobile phase A increased to 8.5, and the ratio of AWG:AW obtained under pH 3.5,4.5, 6.5 was individually 10.7-14.6%higher than that obtained under pH 2.5, whereas the ratios determined under pH 2.5,8.5 were close to each other. With the variance of ionic strength of mobile phase A and pH value of sample medium, the ratios determined were similar. Under the predetermined detection wavelength of 254,274, 280 and 342 nm, the ratios of AWG:AW obtained were found similar, and the ratio obtained under 342 nm deviated 9.71%from those obtained under 280 nm.Conclusions1. For wogonin and oroxylinA, GSMF experiments indicated that both flavonoids were metabolized mainly by UGT1As, with major contributions from UGT1A3 and UGT1A7-1A10. GSMF and isoform-specific metabolism profiles can be used to predict glucuronidation rates and profiles in human tissue microsomes. Minor structural differences of methoxyl position between wogonin and oroxylin A significantly impacted their metabolism 2. For 5H7MF,5H7,8MF,5H6,7,8,4'MF, GSMF experiments indicated that no metabolite was found for 5H6,7,8,4'MF, other flavonoids were metabolized mainly by UGT1 As, with major contributions from UGT1A1, UGT1A3 and UGT1A8-1A10. GSMF could be used to predict the three methoxylated flavones metabolism in liver microsomes. Structural differences of methoxyl number significantly impacted their UGT isoform-specific metabolic pattern.3. The selected five flavones, wogonin, oroxylin A as well as 5H7MF,5H7,8MF, 5H6,7,8,4'MF could be analyzed accurately, and keep their stability during the course of experiments.4. For wogonin, the conversion factor derived directly from the standard curve of both wogonin and wogonoside, was very similar with that from the determination method used for all glucuronides of model flavone, so the accuracy of the conversion factor obtained for all glucuronides is acceptable.5. Among all UPLC analysis conditions, pH value variance of mobile phase would lead to the change of retention time of wogonin, wogonoside and impact the determination of conversion factor, and pH values<7 were more suitable for the determination. In addition, results measured under various detection wavelength were found similar, ionic strength variance of mobile phase, pH value variance of the sample medium was found had no influences on the determination of conversion factor.In conclusion, the GSMF were obtained for five methoxylated flavonoids. For two 5,7-dihydroxyflavone, wogonin and oroxylin A, they were metabolized mainly by UGT 1 A3 and UGT1A7-1A10. For three 5-monohydroxyflavone, no metabolite was found for 5H6,7,8,4'MF, UGT1A1, UGT1A3 and UGT1A7-1A10 were found to be the main isoforms contributing to the glucuronidation of 5H7MF and 5H7,8MF. GSMF and isoform-specific metabolism profiles, could be used to predict glucuronidation rates and profiles of wogonin, oroxylin A in human liver and intestine microsomes and predict glucuronidation rates of 5H7MF,5H7,8MF and 5H6,7,8,4'MF in human liver microsomes. For wogonin and oroxylin A, the glucuronidation rates of most isoforms were found decreased with the change of methoxyl from 8-position to 6-position. For 5H7MF,5H7,8MF and 5H6,7,8,4'MF, the UGT isoform-specific metabolic pattern were found changed with the vaiance of methoxyl number.