No description available.

No description available.

Based on reactions with two flavanones, three flavonols, and five flavones the regioselectivities of twelve human UDP-glucuronosyltransferase (UGT) isozymes were elucidated. The various flavonoid glucuronides were differentiated based on LC-MS/MS fragmentation patterns of [Co(II)(flavonoid – H)(4,7-diphenyl-1,10-phenanthroline)2]+ complexes generated upon post-column complexation. Glucuronide distributions were evaluated to allow a systematic assessment of the regioselectivity of each isozyme. The various UGT enzymes, including eight UGT1A and four UGT2B, displayed a remarkable range of selectivities, both in terms of the positions of glucuronidation and relative reactivity with flavanones, flavonols and flavones. The UGT1A enzyme selectivities are affected by the presence of a hydroxyl group at the 3, 6, 4’, or 3’ positions as well as by the presence of a methoxy at the 3’ position. The UGT2B enzymes show poor to no reactivity with the flavonols or flavones. This result implies that the greater planarity of the flavonols and flavones compared to structure of flavanones inhibits interaction with the UGT2 enzymes. For baicalein and scutellarein, three of the UGT1A isozymes (1A8, 1A9, and 1A10) resulted in the formation of 6-O glucuronides, enabling the fragmentation rules for the metal complexation/MS/MS strategy to be expanded. / text

Human uric acid transporter 1 (hURAT1; SLC22A12) is a very important urate anion exchanger. Elevated urate levels are known to play a pivotal role in cardiovascular diseases, chronic renal disease, diabetes, and hypertension. Therefore, the development of potent uric acid transport inhibitors may lead to novel therapeutic agents to combat these human diseases. The current study investigates small molecular weight compounds and their ability to inhibit 14C-urate uptake in oocytes expressing hURAT1. Using the most promising drug candidates generated from our structure-activity relationship fndings, we subsequently conducted in vitro hepatic metabolism and pharmacokinetic (PK) studies in male Sprague-Dawley rats. Compounds were incubated with rat liver microsomes containing cofactors nicotinamide adenine dinucleotide phosphate and uridine 5′-diphosphoglucuronic acid. In vitro metabolism and PK samples were analyzed using liquid chromatography/mass spectrometry-mass spectrometry methods. Independently, six different inhibitors were orally (capsule dosing) or intravenously (orbital sinus) administered to fasting male Sprague-Dawley rats. Blood samples were collected and analyzed; these data were used to compare in vitro and in vivo metabolism and to compute noncompartmental model PK values. Mono-oxidation (Phase I) and glucuronidation (Phase II) pathways were observed in vitro and in vivo. The in vitro data were used to compute hepatic intrinsic clearance, and the in vivo data were used to compute peak blood concentration, time after administration to achieve peak blood concentration, area under the curve, and orally absorbed fraction. The experimental data provide additional insight into the hURAT1 inhibitor structure-activity relationship and in vitro-in vivo correlation. Furthermore, the results illustrate that one may successfully prepare potent inhibitors that exhibit moderate to good oral bioavailability.

Flavonoids are polyphenolics compounds that constitute a major group of plant specialized metabolites, biosynthesized via the phenylpropanoid/polymalonate pathways. The resulting specialized metabolites can be due to decoration of flavonoid compounds with sugars, usually glucose, by the action of regiospecific UDP-glycosyltransferase (UGT) enzymes. In some cases, glycosylation can involve enzymatic attachment of other sugar moieties, such as glucuronic acid, galactose, rhamnose or arabinose. These modifications facilitate or impact the bioactivity, stability, solubility, bioavailability and taste of the resulting flavonoid metabolites. The present work shows the limitations of utilizing mammalian UDP-glucuronosyltransferases (UGATs) for flavonoid glucuronidation, and then proceeds to investigate plant UG(A)T candidates from the model legume Medicago truncatula for glucuronidating brain-targeted flavonoid metabolites that have shown potential in neurological protection. We identified and characterized several UG(A)T candidates from M. truncatula which efficiently glycosylate various flavonoids compounds with different/multiple regiospecificities. Biochemical characterization identified one enzyme, UGT84F9, that efficiently glucuronidates a range of flavonoid compounds in vitro. In addition, examination of the ugt84f9 gene knock-out mutation in M. truncatula indicates that UGT84F9 is the major UG(A)T enzyme that is necessary and sufficient for attaching glucuronic acid to flavonoid aglycones, particularly flavones, in this species. Finally, the identified UG(A)T candidates were analyzed via homology modeling and site-directed mutagenesis towards increasing the repertoire of UG(A)Ts applicable for synthesis of flavonoid glucuronides with potential human health benefits in neurological protection.

No description available.

The purported chemopreventive and chemotherapeutic properties of the dietary phytochemical resveratrol continue to undergo active investigations. Systemic pharmacokinetics of this compound revealed that it was rapidly and extensively metabolized into its sulfate and glucuronide conjugates. This extensive metabolism leads to high plasma levels of resveratrol sulfates and glucuronides and very low levels of the parent compound (low bioavailability). These observations raised many questions, some of which this body of work examined and has helped to explain. Chapter 1 presents a detailed introduction to resveratrol and its role in colorectal cancer chemoprevention. It also lays the foundation for the hypotheses generated and the studies presented in succeeding chapters. In chapter 2, we explored the possibility that resveratrol metabolites possess intrinsic activity and thus contribute to the observed effects of the parent. The mono-sulfated and glucuronidated conjugates of trans-resveratrol were synthesized and tested for antiproliferative activity in a panel of mammalian cell lines. Their activity was then compared with the parent compound. Resveratrol was shown to be antiproliferative in all cell lines studied while no discernible antiproliferative activity was observed for the metabolites. Chapter 3 details the results of the glucuronidation kinetics of cis and trans-resveratrol isomers across a wide concentration range chosen to mimic blood levels following high dose consumption. Human tissue microsomes and recombinant supersomes over-expressing the enzymes (UGTs) of interest were used for these studies. Our results show the presence of atypical kinetics for the formation of resveratrol glucuronides across most of the protein sources used. Prior to this study, the full glucuronidation kinetics of total resveratrol had not been conducted. In chapter 4, we examined the association between genetic polymorphisms in the major enzymes (UGT1A1 and UGT1A6) and rates of glucuronidation of trans and cis-resveratrol. We set out to correlate functional genetic variations in these UGTs with their catalytic rates and a positive association was made for cis-resveratrol and UGT1A6 where the UGT1A6 variants mediated higher glucuronidation rates compared to the reference genotype. Chapter 5 explored the inherent ability of resveratrol to induce its own glucuronidation upon chronic dosing. Enzyme induction has been proposed as a mechanism that may contribute to the low bioavailability of resveratrol. Since dietary polyphenols like resveratrol are not consumed in isolation, we also studied the effects of combining resveratrol with two dietary polyphenols (curcumin and chrysin) on two chemoprevention endpoints - i) antiproliferation and ii) UGT enzyme induction. Our results indicate that resveratrol is capable of inducing UGT1A1 expression and activity in a non-concentration dependent manner and this induction as well as its antiproliferative effects are enhanced by both curcumin and chrysin. In summary, en route to probing the activity of resveratrol metabolites, we optimized two synthetic routes and generated measurable quantities of these compounds for future use. While the in vitro kinetics of resveratrol did not allow for any in vivo predictions, we were able to show alterations in resveratrol metabolism with respect to genotypic differences and enzyme induction that may contribute to the observed low bioavailability profile. / Pharmaceutical Sciences

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Metabolomics is an emerging field that entails the detailed characterization of the ensemble of metabolites produced by living organisms; subfields include drug metabolism and natural environmental toxin production. The first part of the dissertation pursued metabolism of glyceollins, i.e., isoflavones produced by soybeans, that are potential cancer therapy agents. In vivo glyceollin metabolites produced in rats were investigated by on-line Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry. An odd-electron fragment ion at m/z 148, formed in violation of the even-electron rule, and diagnostic of the glyceollin backbone, was discovered. Based on this finding, a negative mode precursor ion scanning method was developed to screen for glyceollins and their metabolites from biological samples. Products of both Phase I and Phase II metabolism were identified, none of which have been previously reported. Sulfated metabolites were confirmed by accurate mass measurement, while glucuronide conjugation was confirmed by enzyme-assisted glucuronidation by rat liver microsomes. Intact GSH-glyceollin conjugates were not observed, but breakdown products of the GSH pathway, i.e., cysteinylglyceine, cysteine, and acetylated cysteine, were identified as conjugates of oxygenated glyceollins. The identification of GSH by-product conjugates was confirmed in product ion spectra acquired in the negative mode (where peptide anions, and glyceollin-bearing cleaved peptide portions were observed), as well as in the positive mode (where intact oxygenated glyceollin fragments appeared without the initially-present peptide portion). Mass spectral evidence strongly supports a metabolic pathway involving initial epoxidation of glyceollins followed by GSH addition at the epoxidation site. The second part of the dissertation undertook the investigation of secondary metabolites called microbial volatile organic compounds (MVOCs) produced by fungi (mold) that have been reported to have adverse human health effects. MVOCs were collected onto different sorbent materials and analyzed by Thermal Desorption Analysis coupled with on-line Gas Chromatography-Mass Spectrometry. Fungal MVOCs were characterized from various simulated flooding conditions (brackish, freshwater, and saltwater) and different substrates (nutrient rich vs. low nutrient) to determine diagnostic MVOCs. Ten fungi from simulated environments were identified by genetic sequencing. Cladosporium sp. and Chaetomium sp. were cultivated and their emitted MVOCs, 3-furaldehyde and 3-(4-hydroxy-3-methoxyphenyl)-2-propenal, were proposed as diagnostic indicators of these fungi.