Understanding substrate selectivity of human UDP-glucuronosyltransferases through QSAR modeling and analysis of homologous enzymes

1. The UDP-glucuronosyltransferase (UGT) enzyme catalyzes the glucuronidation reaction which is a major metabolic and detoxification pathway in humans. Understanding the mechanisms for substrate recognition by UGT assumes great importance in an attempt to predict its contribution to xenobiotic/drug disposition in vivo.

2. Spurred on by this interest, 2D/3D-quantitative structure activity relationships and pharmacophore models have been established in the absence of a complete mammalian UGT crystal structure.

3. This review discusses the recent progress in modeling human UGT substrates including those with multiple sites of glucuronidation. A better understanding of UGT active site contributing to substrate selectivity (and regioselectivity) from the homologous enzymes (i.e. plant and bacterial UGTs, all belong to family 1 of glycosyltransferase (GT1)) is also highlighted, as these enzymes share a common catalytic mechanism and/or overlapping substrate selectivity.

     

Absorption of three wine-related polyphenols in three different matrices by healthy subjects

BACKGROUND: Despite their powerful biologic activities conducive to protection against atherosclerosis, cancer and inflammatory diseases demonstrated in vitro, there is considerable doubt whether the polyphenolic constituents present in red wine and other dietary components are effective in vivo. OBJECTIVE: We have tested the absorptive efficiency of three of these constituents (trans-resveratrol, [+]-catechin and quercetin) when given orally to healthy human subjects in three different media. DESIGN: Twelve healthy males aged 25 to 45 were randomly assigned to three different groups consuming orally one of the following polyphenols: trans-resveratrol, 25 mg/70 kg; [+]-catechin 25 mg/70 kg; quercetin 10 mg/70 kg. Each polyphenol was randomly administered at 4-week intervals in three different matrices: white wine (11.5% ethanol), grape juice, and vegetable juice/homogenate. Blood was collected at zero time and at four intervals over the first four hours after consumption; urine was collected at zero time and for the following 24-h. The sums of free and conjugated polyphenols were measured in blood serum and urine by a gas-chromatographic method. RESULTS: All three polyphenols were present in serum and urine predominantly as glucuronide and sulfate conjugates, reaching peak concentrations in the former around 30-min after consumption. The free polyphenols accounted for 1.7 to 1.9% (trans-resveratrol), 1.1 to 6.5% ([+]-catechin) and 17.2 to 26.9% (quercetin) of the peak serum concentrations. The absorption of trans-resveratrol was the most efficient as judged by peak serum concentration, area-under-the curve (4 h) and urinary 24-h excretion (16-17% of dose consumed). [+]-Catechin was the poorest by these criteria (urine 24-h excretion 1.2%-3.0% of dose consumed), with quercetin being intermediate (urine 24-h excretion 2.9%-7.0% of dose consumed). Some significant matrix effects were observed for the serum polyphenol concentrations, but in the case of urine no matrix promoted significantly higher excretion than the other two. CONCLUSIONS: The absorption of these three polyphenols is broadly equivalent in aqueous and alcoholic matrices but, at peak concentrations of 10 to 40 nmol/L, is inadequate to permit circulating concentrations of 5 to 100 micromol/L consistent with in vitro biologic activity. The voluminous literature reporting powerful in vitro anticancer and antiinflammatory effects of the free polyphenols is irrelevant, given that they are absorbed as conjugates.     

In vitro-in vivo correlation for drugs and other compounds eliminated by glucuronidation in humans: pitfalls and promises

Enzymes of the UDP-glucuronosyltransferase (UGT) superfamily are responsible for the metabolism of many drugs, environmental chemicals and endogenous compounds. Identification of the UGT(s) involved in the metabolism of a given compound ('reaction phenotyping') currently relies on multiple confirmatory approaches, which may be confounded by the dependence of UGT activity on enzyme source, incubation conditions, and the occurrence of atypical glucuronidation kinetics. However, the increasing availability of substrate and inhibitor 'probes' for the individual UGTs provides the prospect for reliable phenotyping of glucuronidation reactions using human liver microsomes or hepatocytes, thereby providing data directly relevant to drug metabolism in humans. While the feasibility of computational prediction of UGT substrate selectivity has been demonstrated, the development of easily interpretable and generalisable models requires further improvement in the datasets available for analysis. Quantitative prediction of the hepatic clearance of glucuronidated drugs and the magnitude of inhibitory interactions based on in vitro kinetic data is more problematic. Intrinsic clearance (CL(int)) values generated using human liver microsomes under-predict in vivo hepatic clearance, typically by an order of magnitude. In vivo clearances of glucuronidated drugs are also generally under-predicted by CL(int) values from human hepatocytes, but to a lesser extent than observed with the microsomal model. While it is anticipated that systematic analysis of the potential causes of under-prediction may provide more reliable in vitro-in vivo scaling strategies, mechanistic interpretation of in vitro-in vivo correlation more broadly awaits further advances in our understanding of the structural and cellular determinants of UGT activity.     

Stereoselective urinary excretion of S-（-）- and R-（＋）-propranolol glucuronide following oral administration of RS-propranolol in Chinese Han subjects

AIM: To study the stereoselectivity of phase Ⅱ glucuronidation metabolism of side-chain propranolol in Chinese Han population. METHODS: Sixteen adult Chinese Han volunteers with an average age of 20 years were given a single oral dose of 20 mg racemic propranolol. Human urine at indicated time after administration was collected and S-(-)-propranolol glucuronide and R-(+)-propranolol glucuronide were determined simultaneously by using RP-HPLC. RESULTS: The mean values of k were 0.19±0.04 h-1 and 0.28±0.06 h-1, of t1/2 3.56±0.73 h and 2.45±0.50 h, of Tmax 2.21±0.45 and 1.75±0.33 h, and of Xu0-24 5.65±0.98 and 2.95±0.62 μmoL for S-(-)- and R-(+)-propranolol glucuronide, respectively. The cumulative excretion percentages in urine of closes were 14.7±2.46% and 7.68±1.60% for S-(-)-and R-(+)-propranolol glucuronide, respectively. The results showed the elimination rate constant k of S-(-)-propranolol glucuronide was less than that of R-(+)-propranolol glucuronide; and the elimination half-life (t1/2), Tmax and the cumulative excretion amount (Xu0-24) of R-(+)-propranolol glucuronide were significantly less than that of S-(-)-propranolol glucuronide. CONCLUSION: The propranolol glucuronidation of the side-chain undergoes stereoselective excretion in Chinese Han population after an oral administration of racemic propranolol.     

Plasma disposition, metabolism and excretion of the experimental antitumour agent 5,6-dimethylxanthenone-4-acetic acid in the mouse, rat and rabbit

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), an experimental antitumour agent currently undergoing phase I clinical trial, has a maximum tolerated dose (MTD) in male BDF₁ mice of 99 μmol/kg. We have found the male Sprague-Dawley rat and the New Zealand White rabbit to have greater tolerance to DMXAA, with MTDs being 990 and 330 μmol/kg, respectively. To investigate the causes of this difference, we measured plasma and urine DMXAA concentrations by high-performance liquid chromatography (HPLC) after single i.v. bolus injections of 99 and 990 μmol/kg in the rat and following a bolus dose of 99 μmol/kg and a 10-min infusion of 330 μmol/kg in the rabbit. Following administration of DMXAA at the MTD in the mouse, rat and rabbit the maximal concentrations were 600, 2,200 and 1,708 μM, respectively, whereas areas under the concentration-time curves were 2,400, 19,000 and 2,400 μMh, respectively, for unchanged DMXAA. Data obtained for mice and rabbits were satisfactorily fitted to a two-compartment model with Michaelis-Menten kinetics. DMXAA was highly bound to plasma proteins, with the highest degree of binding being found in the rabbit. A small proportion of the total dose (7.8%, 0.6% and 12.4%, respectively) was excreted unchanged in urine over 24 h. This proportion increased (to 11.6%, 3.5% and 72.4%, respectively) following alkaline hydrolysis, suggesting the presence of glucuronide metabolites. Examination of rat and mouse urine by HPLC revealed the presence of two metabolites, which were characterized by mass spectrometry and nuclear magnetic resonance to be the acyl glucuronide of DMXAA and 6-(hydroxymethyl)-5-methylxanthenone-4-acetic acid. Thus, both mice and rats metabolise DMXAA by similar pathways. The results demonstrate considerable interspecies variations in tolerance to DMXAA that cannot be explained by differences in pharmacokinetics. Received: 15 September 1997 / Accepted: 5 August 1998     

Glucuronidation: driving factors and their impact on glucuronide disposition

Glucuronidation is a well-recognized phase II metabolic pathway for a variety of chemicals including drugs and endogenous substances. Although it is usually the secondary metabolic pathway for a compound preceded by phase I hydroxylation, glucuronidation alone could serve as the dominant metabolic pathway for many compounds, including some with high aqueous solubility. Glucuronidation involves the metabolism of parent compound by UDP-glucuronosyltransferases (UGTs) into hydrophilic and negatively charged glucuronides that cannot exit the cell without the aid of efflux transporters. Therefore, elimination of parent compound via glucuronidation in a metabolic active cell is controlled by two driving forces: the formation of glucuronides by UGT enzymes and the (polarized) excretion of these glucuronides by efflux transporters located on the cell surfaces in various drug disposition organs. Contrary to the common assumption that the glucuronides reaching the systemic circulation were destined for urinary excretion, recent evidences suggest that hepatocytes are capable of highly efficient biliary clearance of the gut-generated glucuronides. Furthermore, the biliary- and enteric-eliminated glucuronides participate into recycling schemes involving intestinal microbes, which often prolong their local and systemic exposure, albeit at low systemic concentrations. Taken together, these recent research advances indicate that although UGT determines the rate and extent of glucuronide generation, the efflux and uptake transporters determine the distribution of these glucuronides into blood and then to various organs for elimination. Recycling schemes impact the apparent plasma half-life of parent compounds and their glucuronides that reach intestinal lumen, in addition to prolonging their gut and colon exposure.     

Inhibition of UDP-glucuronosyltransferase (UGT)-mediated glycyrrhetinic acid 3-O-glucuronidation by polyphenols and triterpenoids

Glycyrrhetinic acid (GA) is an active metabolite of glycyrrhizin, which is a main constituent in licorice (Glycyrrhiza glabra). While GA exhibits a wide variety of pharmacological activities in the body, it is converted to a toxic metabolite GA 3-O-glucuronide by hepatic UDP-glucuronosyltransferases (UGTs). To avoid the development of the toxic metabolite-induced pseudohyperaldosteronism (pseudoaldosteronism), there is a limitation in maximum daily dosage of licorice and in combined usage of other glycyrrhizin-containing natural medicine. In this study, we investigated the inhibitory effects of various polyphenols and triterpenoids on the UGT-mediated GA 3-O-glucuronidation. In human liver microsomes, UGT-mediated GA glucuronidation was significantly inhibited by protopanaxadiol with an IC₅₀ value of 59.2 μM. Isoliquiritigenin, rosmarinic acid, alisol B, alisol acetate, and catechin moderately inhibited the GA glucuronidation with IC₅₀ values of 96.4 μM, 125 μM, 160 μM, 163 μM, and 164 μM. Other tested 19 polyphenols and triterpenoids, including liquiritigenin, did not inhibit UGT-mediated GA glucuronidation in human liver microsomes. Our data indicate that relatively higher dosage of licorice can be used without a risk of developing pseudohyperaldosteronism in combination of natural medicine containing protopanaxadiol such as Panax ginseng. Furthermore, supplemental protopanaxadiol and isoliquiritigenin might be useful in preventing licorice-inducing pseudoaldosteronism.     

Glucuronidation in rat intestinal epithelial cells

Glucuronidation of 1-naphthol was studied in mucosal cells isolated from the rat intestine. Glucuronidation was directly dependent on the supply of extracellular carbohydrates. Basal glucuronidation (ca. 0.3 nmoles/min · mg cell protein) was increased 2- to 3-fold by adding glucose or fructose to the incubation medium. Saturation of glucuroniation was achieved by adding 0.3 mM glucose, while saturation by fructose was not reached at concentrations below 2 mM. No carbohydrate reserves able to support glucuronidation appear to be present in intestinal cells, since no difference in glucuronidation was observed between cells prepared from fasted (18 or 42 h) and control rats. Glucuronidation was decreased by adding d-galactosamine to the incubation medium, but only when extracellular glucose was present. Various chemicals which are known to inhibit glucuronidation in hepatocytes (ethanol, diethyl ether, sorbitol) did not influence the glucuronidation of 1-naphthol in isolated intestinal cells. Only when ethanol was added to mucosal cells in the absence of extracellular glucose was a small decrease in glucuronidation observed.     

Glucuronidation of flavonoids by recombinant UGT1A3 and UGT1A9

Flavonoids are highlighted for their potential roles in the prevention of oxidative stress-associated diseases. Their metabolisms in vivo, such as glucuronidation, are the key points to determine their health beneficial properties. In this paper, we tested the glucuronidation of nineteen flavonoids by both recombinant human UGT1A3 and UGT1A9. Eleven compounds could be catalyzed by both enzymes. In general, both enzymes showed moderate to high catalyzing activity to most flavonoid aglycones, while the catalyzing efficiency changed with structures. Each flavonoid produced more than one monoglucuronide with no diglucuronide detected by liquid chromatography-mass spectrometry (LC-MS). Enzymatic kinetic analysis indicated that the catalyzing efficiency (Vmax/Km) of UGT1A9 was higher than that of UGT1A3, suggesting its important role in flavonoid glucuronidation. Both human UGT1A3 and UGT1A9 preferred flavonoid aglycone to flavonoid glycoside, and their metabolism to arabinoside was stronger than to other glycosides. Of the flavonoids studied, it is the first time to report isorhamnetin, morin, silybin, kaempferol, daidzein, quercetin-3',4'-OCHO-, quercetin xylopyranoside and avicularin as substrates of UGT1A3. Apigenin, morin, daidzein, quercetin-3',4'-OCHO-, quercetin xylopyranoside and avicularin were the newly reported substrates of UGT1A9.