Ming Hu, Ph.D - US grants

[unreadable] DESCRIPTION (provided by applicant): Dietary flavonoids include a broad range of compounds that have been shown to have a variety of health benefits (e.g., antioxidants, antiosteoperosis. anticancers, and anti-aging), which make them attractive for disease prevention and impeding the progress of degenerative diseases in humans. However, these compounds have poor bioavailabilities. The long-term goal of our study is to determine how enteric recycling (Liu and Hu. 2002) contributes to the overall disposition of flavonoids and to the biological activities of flavonoids in humans. The general hypothesis for the present research proposal is that the inhibition of intestinal transporter mediated efflux of conjugated metabolites is more effective than inhibition of intestinal conjugating enzymes in decreasing luminal excretion of flavonoid metabolites and in increasing, local, portal and systemic bioavailability of parent flavonoids. The specific aims are to: (1) determine if intestinal metabolism and subsequent excretion of metabolites is more important than liver metabolism and excretion in a variety of established models using selected flavonoids; (2) determine the main efflux transporters responsible for efflux of hydrophilic flavonoid conjugates using RNA interference or RNAi (e.g., siRNA) and chemical inhibitors, and the rate-limiting step in the cellular excretion of hydrophilic flavonoid conjugates; (3) determine how silencing of a main isoform of UDP-glucuronosyltransferase or sulfotransferases responsible for the metabolism of related representative flavonoids will change the cellular excretion of flavonoid conjugates in the Caco-2 model; and (4) identify chemical inhibitors that are capable of decreasing the efflux transporter activities without affecting the activities of UGT or SULT or vice versa using kinetic methods, and determine if inhibitors of main efflux transporter is more effective than inhibitors of the main conjugating enzyme in enhancing the local, portal and systemic bioavailabilities of selected flavonoids. Through these studies, we will further understand the mechanisms that control the fate of flavonoids following oral ingestion and shed light on the reasons why flavonoids have poor oral bioavailability. These findings may be used to understand the mechanisms of actions of flavonoids and to devise means of increasing their bioavailability in humans. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Dietary flavonoids are known to have a variety of health benefits, ranging from anticancer, antioxidant, antiosteoperosis and cholesterol lowering to anti-aging. However, these compounds are poorly bioavailable which impedes the effort to develop them as drugs. The long-term goal of our study is to determine how coordinated interplay of key elements determines the overall disposition of flavonoids and contributes to their biological fate in vivo. The general hypothesis for the present research proposal is that the flavonoid bioavailability will be improved by interrupting interplays between key components of their disposition. Our previous research has shown that key components of flavonoid disposition are conjugating enzymes (i.e., sulfotransferases and UDP-glucuronosyltransferases) and efflux transporters (e.g., BCRP and MRP2). Our previous research also showed that flavonoids undergo the dual enteric and enterohepatic recycling processes, which increase their conjugate concentrations and apparent biological half- lives in vivo. The conjugated form of flavonoids may be bioavailable since local and systemic hydrolysis of conjugated flavonoids to aglycones by glucuronidases and sulfatases is a viable means of providing aglycone to target organs. Therefore, this present hypothesis is a step beyond the classical hypothesis that bioavailabilities are increased if aglycone AUC is increased. It is also a step beyond our original hypothesis that identification and inhibition of one key element in the disposition processes will lead to higher bioavailabilities, which we found to be untrue when we focused on identifying the enzyme isoform responsible. However, we did find that bioavailability of genistein improved in BCRP-/- (knockout) mice, although we observed a decrease in SULT activities as well. The Specific Aims of this continuing proposal are to: (1) determine the key UGT and SULT isoforms responsible for the metabolism of selected flavonoids, and key efflux transporters responsible for the excretion of phase II conjugates of flavonoids; (2) determine how key UGTs and relevant key efflux transporters identified in aim 1 compensate for each other's functional deficiency; (3) determine how key SULTs and relevant key efflux transporters identified in aim 1 compensate for each other's functional deficiency; and (4) determine how glucuronidation and sulfation pathways will compensate for each other's functional deficiency. Through these new studies, we seek to determine how interplay between key components of flavonoid metabolism can be effectively interrupted to improve their local and systematic bioavailabilities. The success of the proposed studies should lead to the development of dosage forms with improved bioavailability for humans. PUBLIC HEALTH RELEVANCE: Flavonoids have been shown to have a variety of beneficial effects based on in vitro studies using human cells and in vivo studies using animal models. However, there are no definitive clinical data to prove the effectiveness of this class of compounds in humans. A major impediment to definitive clinical studies of flavonoids lies with the facts that clinical trials are expensive (as the result of flavonoid's poor bioavailability) and there is no patentable dosage form that will attract private capital to enable the clinical trials. The proposed basic mechanistic studies should facilitate the development of new dosage forms with improved bioavailability, and benefit the health of American in the long run.

DESCRIPTION (provided by applicant): Dietary flavonoids exert a broad range of biological effects including anticancer, antioxidant, antiosteoperosis, cholesterol lowering, and anti-aging, which make them attractive for preventing cancer and cardiovascular ailments and for impeding the progress of degenerative diseases. However, these compounds are poorly bioavailable, which impedes their development as viable drugs. The long-term goal of our study is to determine how efflux transporters of hydrophilic phase II conjugates control the overall disposition of flavonoids and determine their biological fate in vivo. The central hypothesis for the present research proposal is that the local and systemic bioavailability of a flavonoid will be improved by modulating the function of a critical efflux transporter responsible for its distributin to the local target organs (e.g., colon) or the systemic circulation. Our central hypothesis is a step beyond the classical hypothesis that bioavailabilities of drugs can only be improved if more are absorbed and/or less of the absorbed amount is metabolized. The Specific Aims of this renewal proposal are to: (1) construct precise quantitative cellular metabolic models to describe the kinetics of glucuronide formation and efflux at the molecular level; (2) determine the quantitative structure-efflux relationships (QSERs) for three key efflux transporters of flavonoid glucuronides: BCRP, MRP2, and MRP3; and (3) establish a glucuronidation classification system to map which flavonoid is likely to have good bioavailability by manipulating a particular efflux transporter. Successful completion of our research project will significantly advance the basic sciences as well as practical knowledge that may be used to improve the bioavailability of flavonoids and relevant drugs for human health.

Abstract Irinotecan, a prodrug of SN-38, is used to treat many types of metastatic and drug-resistant cancers, and often represents the therapy of the last resort. Unfortunately, a large percentage (up to 40%) of these patients will experience serious (Grade 2) and severe (Grade 3-4) delayed-onset diarrhea (SDOD), which really downgrade patient?s quality of life. SDOD may lead to prolonged hospitalization and even death in some instances. The long-term goal of our research is to develop experimental therapeutics and/or nutritional supplemental approach to reduce SDOD, so patients can sustain their chemotherapy. Our recent studies have shown that inactivation of intestinal UDP-glucuronosyltransferases (UGTs) by SN-38 is a new mechanism by which SN-38 causes SDOD, and that a Traditional Chinese Medicine, Xiao-Chai-Hu-Tang (XCHT), could attenuate the inactivation of intestinal UGTs in mice. Therefore, the central hypothesis of this current proposal is Therefore, we hypothesize that XCHT will prevent or reduce irinotecan-induced SDOD by attenuating the decline in UGT activities, reducing gut SN-38 exposure, and promoting the recovery of gut UGT activities. We plan to test this hypothesis using four Specific Aims: (1) perform phytochemical, biopharmaceutical and pharmacokinetic characterization of XCHT to enable quality control, systemic and intestinal drug exposure determinations, and to provide bioanalytical methods and pharmacokinetic parameters needed for a clinical study and PK/PD modeling; (2) validate plasma raloxifene-4?-glucuronide levels as a probe to changes in intestinal Ugt/UGT activity; (3) Perform mouse ?co- trial? studies to support human mechanistic trials and to determine the mechanisms of action of XCHT against irinotecan-induced SDOD using both in vitro and in vivo models; and (4) Conduct a mechanistic clinical trial using a randomized double-blind design with a safety ?Run-In? to determine if XCHT can attenuate human intestinal UGT decrease and reduce incidence of Grade 3 or higher diarrhea caused by irinotecan chemotherapy. Aside from these primary outcomes, we will also determine if levels of Ral-4?-G, a probe of intestinal UGT activities is (negatively) correlated with systemic levels of inflammatory cytokines. Success gained through this research will provide a new mechanism by which we can target to treat SDOD caused by irinotecan chemotherapy.