Grass xylan structural variation suggests functional specialization and distinctive interaction with cellulose and lignin.

Xylan is the most abundant non-cellulosic polysaccharide in grass cell walls, and it has important structural roles. The name glucuronoarabinoxylan (GAX) is used to describe this variable hemicellulose. It has a linear backbone of ß-1,4-xylose (Xyl) residues that may be substituted with α-1,2-linked (4-O-methyl)-glucuronic acid (GlcA), α-1,3-linked arabinofuranose (Araf), and sometimes acetylation at the O-2 and/or O-3 positions. The role of these substitutions remains unclear, although there is increasing evidence that they affect the way xylan interacts with other cell wall components, particularly cellulose and lignin. Here, we used substitution-dependent endo-xylanase enzymes to investigate the variability of xylan substitution in grass culm cell walls. We show that there are at least three different types of xylan: (i) an arabinoxylan with evenly distributed Araf substitutions without GlcA (AXe); (ii) a glucuronoarabinoxylan with clustered GlcA modifications (GAXc); and (iii) a highly substituted glucuronoarabinoxylan (hsGAX). Immunolocalization of AXe and GAXc in Brachypodium distachyon culms revealed that these xylan types are not restricted to a few cell types but are instead widely detected in Brachypodium cell walls. We hypothesize that there are functionally specialized xylan types within the grass cell wall. The even substitutions of AXe may permit folding and binding on the surface of cellulose fibrils, whereas the more complex substitutions of the other xylans may support a role in the matrix and interaction with other cell wall components.

Silencing ScGUX2 reduces xylan glucuronidation and improves biomass saccharification in sugarcane.

There is an increasing need for renewable energy sources to replace part of our fossil fuel-based economy and reduce greenhouse gas emission. Sugarcane bagasse is a prominent feedstock to produce cellulosic bioethanol, but strategies are still needed to improve the cost-effective exploitation of this potential energy source. In model plants, it has been shown that GUX genes are involved in cell wall hemicellulose decoration, adding glucuronic acid substitutions on the xylan backbone. Mutation of GUX genes increases enzyme access to cell wall polysaccharides, reducing biomass recalcitrance in Arabidopsis thaliana. Here, we characterized the sugarcane GUX genes and silenced GUX2 in commercial hybrid sugarcane. The transgenic lines had no penalty in development under greenhouse conditions. The sugarcane GUX1 and GUX2 enzymes generated different patterns of xylan glucuronidation, suggesting they may differently influence the molecular interaction of xylan with cellulose and lignin. Studies using biomass without chemical or steam pretreatment showed that the cell wall polysaccharides, particularly xylan, were less recalcitrant in sugarcane with GUX2 silenced than in WT plants. Our findings suggest that manipulation of GUX in sugarcane can reduce the costs of second-generation ethanol production and enhance the contribution of biofuels to lowering the emission of greenhouse gases.

Altering the substitution and cross-linking of glucuronoarabinoxylans affects cell wall architecture in Brachypodium distachyon.

The Poaceae family of plants provides cereal crops that are critical for human and animal nutrition, and also, they are an important source of biomass. Interacting plant cell wall components give rise to recalcitrance to digestion; thus, understanding the wall molecular architecture is important to improve biomass properties. Xylan is the main hemicellulose in grass cell walls. Recently, we reported structural variation in grass xylans, suggesting functional specialisation and distinct interactions with cellulose and lignin. Here, we investigated the functions of these xylans by perturbing the biosynthesis of specific xylan types. We generated CRISPR/Cas9 knockout mutants in Brachypodium distachyon XAX1 and GUX2 genes involved in xylan substitution. Using carbohydrate gel electrophoresis, we identified biochemical changes in different xylan types. Saccharification, cryo-SEM, subcritical water extraction and ssNMR were used to study wall architecture. BdXAX1A and BdGUX2 enzymes modify different types of grass xylan. Brachypodium mutant walls are likely more porous, suggesting the xylan substitutions directed by both BdXAX1A and GUX2 enzymes influence xylan-xylan and/or xylan-lignin interactions. Since xylan substitutions influence wall architecture and digestibility, our findings open new avenues to improve cereals for food and to use grass biomass for feed and the production of bioenergy and biomaterials.

Identification and Biosynthesis of an N-Glucuronide Metabolite of Camonsertib.

Camonsertib is a novel ATR kinase inhibitor in clinical development for advanced cancers targeting sensitizing mutations. This article describes the identification and biosynthesis of an N-glucuronide metabolite of camonsertib. This metabolite was first observed in human hepatocyte incubations and was subsequently isolated to determine the structure, evaluate its stability as part of bioanalytical method development and for use as a standard for estimating its concentration in Phase I samples. The N-glucuronide was scaled-up using a purified bacterial culture preparation and was subsequently isolated using preparative chromatography. The bacterial culture generated sufficient material of the glucuronide to allow for one- and two-dimensional 1H and 13C NMR structural elucidation and further bioanalytical characterization. The NOE data combined with the gradient HMBC experiment and molecular modeling, strongly suggests that the point of attachment of the glucuronide results in the formation of (2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(5-(4-((1R,3r,5S)-3-hydroxy-8-oxabicyclo[3.2.1]octan-3-yl)-6-((R)-3-methylmorpholino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-1H-pyrazol-1-yl)tetrahydro-2H-pyran-2-carboxylic acid. Significance Statement This is the first report of a glucuronide metabolite of camonsertib formed by human hepatocyte incubations. This study reveals the structure of an N-glucuronide metabolite of camonsertib using detailed elucidation by one- and two-dimensional NMR after scale-up using a novel bacterial culture approach yielding significant quantities of a purified metabolite.

Effects of pethoxamid treatment on the disposition of thyroxine in rats.

Pethoxamid (PXA) is a chloroacetamide herbicide that works by inhibiting the germination of target weeds in crops. PXA is not a genotoxic agent, however, in a two-year chronic toxicity study, incidence of thyroid follicular cell hyperplasia was observed in male rats treated at a high dose. Many non-mutagenic chemicals, including agrochemicals are known to produce thyroid hyperplasia in rodents through a hepatic metabolizing enzyme induction mode of action (MoA). In this study, the effects of oral gavage PXA treatment at 300 mg/kg for 7 days on the disposition of intravenously (iv) administered radio-labeled thyroxine ([125I]-T4) was assessed in bile-duct cannulated (BDC) rats. Another group of animals were treated with phenobarbital (PB, 100 mg/kg), a known enzyme inducer, serving as a positive control. The results showed significant increase (p < 0.01) in the mean liver weights in the PB and PXA-treated groups relative to the control group. The serum total T4 radioactivity Cmax and AUC0-4 values for PB and PXA-treated groups were lower than for the control group, suggesting increased clearance from serum. The mean percentages of administered radioactivity excreted in bile were 7.96 ± 0.38%, 16.13 ± 5.46%, and 11.99 ± 2.80% for the control, PB and PXA groups, respectively, indicating increased clearance via the bile in the treated animals. These data indicate that PXA can perturb the thyroid hormone homeostasis in rats by increasing T4 elimination in bile, possibly through enzyme induction mechanism similar to PB. In contrast to humans, the lack of high affinity thyroid binding globulin (TBG) in rats perhaps results in enhanced metabolism of T4 by uridine diphosphate glucuronosyl transferase (UGT). Since this liver enzyme induction MoA for thyroid hyperplasia by PB is known to be rodent specific, PXA effects on thyroid can also be considered not relevant to humans. The data from this study also suggest that incorporating a BDC rat model to determine thyroid hormone disposition using [125I]-T4 is valuable in a thyroid mode of action analysis.

In vitro and in vivo studies on the metabolism and pharmacokinetics of the selective gut microbial ß-glucuronidase targeting compound Inh 1.

In vitro studies using rat, mouse, and human microsomes and hepatocytes on the bacterial ß-glucuronidase inhibitor 1-((6,8-dimethyl-2-oxo-1,2-dihydroquinolin-3-yl)methyl)-3-(4-ethoxyphenyl)-1-(2-hydroxyethyl)thiourea) (Inh 1) revealed extensive metabolism in all species.The intrinsic clearances of Inh 1 in human, mouse, and rat hepatic microsomes were 30.9, 67.8, and 201 µL/min/mg, respectively. For intact hepatocytes intrinsic clearances of 21.6, 96.0, and 129 µL/min/106 cells were seen for human, mouse and rat, respectively.The metabolism of Inh 1 involved an uncommon desulphurisation reaction in addition to oxidation, deethylation, and conjugation reactions at multiple sites. Six metabolites were detected in microsomal incubations in human and rat, and seven for the mouse. With hepatocytes, 18 metabolites were characterised, 9 for human, and 11 for mouse and rat.Following IV administration to mice (3 mg/kg), plasma concentrations of Inh 1 exhibited a monophasic decline with a terminal elimination half-life of 0.91 h and low systemic clearance (11.8% of liver blood flow). After PO dosing to mice (3 mg/kg), peak observed Inh 1 concentrations of 495 ng/mL were measured 0.5 h post dose, declining to under 10 ng/mL at 8 h post dose. The absolute oral bioavailability of Inh 1 in the mouse was ca. 26%.

Hepatic glucuronidation of tetrabromobisphenol A and tetrachlorobisphenol A: interspecies differences in humans and laboratory animals and responsible UDP-glucuronosyltransferase isoforms in humans.

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA), bisphenol A (BPA) analogs, are endocrine-disrupting chemicals predominantly metabolized into glucuronides by UDP-glucuronosyltransferase (UGT) enzymes in humans and rats. In the present study, TBBPA and TCBPA glucuronidation by the liver microsomes of humans and laboratory animals (monkeys, dogs, minipigs, rats, mice, and hamsters) and recombinant human hepatic UGTs (10 isoforms) were examined. TBBPA glucuronidation by the liver microsomes followed the Michaelis-Menten model kinetics in humans, rats, and hamsters and the biphasic model in monkeys, dogs, minipigs, and mice. The CLint values based on the Eadie-Hofstee plots were mice (147) > monkeys (122) > minipigs (108) > humans (100) and rats (98) > dogs (81) > hamsters (47). TCBPA glucuronidation kinetics by the liver microsomes followed the biphasic model in all species except for minipigs, which followed the Michaelis-Menten model. The CLint values were monkeys (172) > rats (151) > mice (134) > minipigs (104), dogs (102), and humans (100) > hamsters (88). Among recombinant human UGTs examined, UGT1A1 and UGT1A9 showed higher TBBPA and TCBPA glucuronidation abilities. The kinetics of TBBPA and TCBPA glucuronidation followed the substrate inhibition model in UGT1A1 and the Michaelis-Menten model in UGT1A9. The CLint values were UGT1A1 (100) > UGT1A9 (42) for TBBPA glucuronidation and UGT1A1 (100) > UGT1A9 (53) for TCBPA glucuronidation, and the activities at high substrate concentration ranges were higher in UGT1A9 than in UGT1A1 for both TBBPA and TCBPA. These results suggest that the glucuronidation abilities toward TBBPA and TCBPA in the liver differ extensively across species, and that UGT1A1 and UGT1A9 expressed in the liver mainly contribute to the metabolism and detoxification of TBBPA and TCBPA in humans.

Identification of Bromophenols' glucuronidation and its induction on UDP- glucuronosyltransferases isoforms.

Bromophenols (BPs) are prominent environmental pollutants extensively utilized in aquaculture, pharmaceuticals, and chemical manufacturing. This study aims to identify UDP- glucuronosyltransferases (UGTs) isoforms involved in the metabolic elimination of BPs. Mono-glucuronides of BPs were detected in human liver microsomes (HLMs) incubated with the co-factor uridine-diphosphate glucuronic acid (UDPGA). The glucuronidation metabolism reactions catalyzed by HLMs followed Michaelis-Menten or substrate inhibition kinetics. Recombinant enzymes and inhibition experiments with chemical reagents were employed to phenotype the principal UGT isoforms participating in BP glucuronidation. UGT1A6 emerged as the major enzyme in the glucuronidation of 4-Bromophenol (4-BP), while UGT1A1, UGT1A6, and UGT1A8 were identified as the most essential isoforms for metabolizing 2,4-dibromophenol (2,4-DBP). UGT1A1, UGT1A8, and UGT2B4 were deemed the most critical isoforms in the catalysis of 2,4,6-tribromophenol (2,4,6-TBP) glucuronidation. Species differences were investigated using the liver microsomes of pig (PLM), rat (RLM), monkey (MyLM), and dog (DLM). Additionally, 2,4,6-TBP effects on the expression of UGT1A1 and UGT2B7 in HepG2 cells were evaluated. The results demonstrated potential induction of UGT1A1 and UGT2B7 upon exposure to 2,4,6-TBP at a concentration of 50 µM. Collectively, these findings contribute to elucidating the metabolic elimination and toxicity of BPs.

Effect of 1,4-Dioxane Solvent on ß-Glucuronidation Using Methyl 1,2,3,4-Tetra-O-acetyl-ß-D-glucuronate as the Glycosyl Donor.

A facile and selective ß-D-glucuronidation of alcohols, such as (-)-menthol, cholestanol, (+)- and (-)-borneols, and 2-adamantanol, using commercially available methyl 1,2,3,4-tetra-O-acetyl-ß-D-glucuronate as the glycosyl donor and trimethylsilyl bis(trifluoromethanesulfonyl)imide (Tf2NTMS) (0.5 equivalent) as the activator in 1,4-dioxane at 60 °C gave products in moderate yields. The addition of MS4A increased the ß : α ratios of D-glucuronides when cholestanol, (+)-borneol, and 2-adamantanol were used as the acceptor substrate.

Maturation of Paracetamol Elimination Routes in Preterm Neonates Born Below 32 Weeks of Gestation.

PURPOSE: Despite being off-label, intravenous paracetamol (PCM) is increasingly used to control mild-to-moderate pain in preterm neonates. Here we aim to quantify the maturation of paracetamol elimination pathways in preterm neonates born below 32 weeks of gestation. METHODS: Datasets after single dose (rich data) or multiple doses (sparse data) of intravenous PCM dose (median (range)) 9 (3-25) mg/kg were pooled, containing 534 plasma and 44 urine samples of PCM and metabolites (PCM-glucuronide, PCM-sulfate, PCM-cysteine, and PCM-mercapturate) from 143 preterm neonates (gestational age 27.7 (24.0-31.9) weeks, birthweight 985 (462-1,925) g, postnatal age (PNA) 5 (0-30) days, current weight 1,012 (462-1,959) g. Population pharmacokinetic analysis was performed using NONMEM® 7.4. RESULTS: For a typical preterm neonate (birthweight 985 g; PNA 5 days), PCM clearance was 0.137 L/h, with glucuronidation, sulfation, oxidation and unchanged renal clearance accounting for 5.3%, 73.7%, 16.3% and 4.6%, respectively. Maturational changes in total PCM clearance and its elimination pathways were best described by birthweight and PNA. Between 500-1,500 g birthweight, total PCM clearance increases by 169%, with glucuronidation, sulfation and oxidation clearance increasing by 347%, 164% and 164%. From 1-30 days PNA for 985 g birthweight neonate, total PCM clearance increases by 167%, with clearance via glucuronidation and oxidation increasing by 551%, and sulfation by 69%. CONCLUSION: Birthweight and PNA are the most important predictors for maturational changes in paracetamol clearance and its glucuronidation, sulfation and oxidation. As a result, dosing based on bodyweight alone will not lead to consistent paracetamol concentrations among preterm neonates.

Discovery of novel pyridinones as MGAT2 inhibitors for the treatment of metabolic disorders.

Inhibition of monoacylglycerol transferase 2 (MGAT2) has recently emerged as a potential therapeutic strategy for the treatment of metabolic diseases such as obesity, diabetes and non-alcoholic steatohepatitis (NASH). Metabolism studies with our clinical lead (1) suggested variability in in vitro glucuronidation rates in liver microsomes across species, which made projection of human doses challenging. In addition, the observation of deconjugation of the C3-C4 double bond in the dihydropyridinone ring of 1 in solution had the potential to complicate its clinical development. This report describes our lead optimization efforts in a novel pyridinone series, exemplified by compound 33, which successfully addressed both of these potential issues.

Chemical metabolite synthesis and profiling: Mimicking in vivo biotransformation reactions.

Biotransformation was previously viewed as merely the structural characterization of drug metabolites, and it was performed only when drug candidates entered clinical development. The synthesis of drug metabolites is crucial to the drug development process because it generates either pharmacologically active, inactive, or reactive molecules and hence their characterization and comprehensive pharmacological evaluation is necessary. The chemical metabolite synthesis is very challenging due to the complex structures of many drug molecules, presence of multiple stereocenters, poor reaction yields, and the formation of unwanted by-products. Drug metabolites and their chemical synthesis have immense significance in the drug discovery process. The chemical synthesis of metabolites facilitates on- or off-target pharmacological and toxicological evaluations at the easiest. In a broader view metabolite could be a target lead molecule for drug design, toxic reactive metabolites, pharmaceutical standards for bioanalytical methods, etc. Collectively these metabolite information dossiers will aid regulatory agencies such as the EMA and FDA in maintaining strict vigilance over drug manufacturers with regard to the safety of NCE's and their hidden metabolites. Herein, we are presenting a systematic compilation of chemical and biocatalytic strategies reported to date for pharmaceutical drug metabolite synthesis. This review report is very useful for the laboratory synthesis of new drug metabolites, and their preclinical biological evaluation could aid in the detection of early threats (alerts) in drug discovery, eliminate the toxicity profile, explore newer pharmacology, and delivering a promising and safe drug candidate to humankind.

Preclinical safety, toxicokinetics and metabolism of BIIB131, a novel prothrombolytic agent for acute stroke.

BIIB131, a small molecule, is currently in Phase 2 for the treatment of acute ischemic stroke. Safety and metabolism of BIIB131 were evaluated following intravenous administration to rats and monkeys. Exposure increased dose-proportionally in rats up to 60 mg/kg and more than dose-proportionally in monkeys at greater than 10 mg/kg accompanied by prolonged half-life and safety findings. The BIIB131 was poorly metabolized in microsomes with no inhibition of CYPs. BIIB131-glucuronide, formed by UGT1A1, accounted for 21.5% metabolism in human hepatocytes and 28-40% in rat bile. In rats, excretion was primarily via the bile. BIIB131 inhibited the hERG and Nav1.5 cardiac channels by 39% but showed no effect on cardiovascular parameters in monkeys. Toxicology findings were limited to reversable hematuria, changes in urinary parameters and local effects. A MTD of 30 mg/kg was established in monkeys, the most sensitive species, at total plasma Cmax and AUC of 6- and 14-fold, respectively, greater than the NOAEL. The Phase 1 study started with intravenous 0.05 mg/kg and ascended to 6.0 mg/kg which corresponded to safety margins of 147- to 0.9-fold (for Cmax) within the linear drug exposure. Thus, the preclinical profile of BIIB131 has been appropriately characterized and supports its further clinical development.

Boscalid shows increased thyroxin-glucuronidation in rat but not in human hepatocytes in vitro.

The fungicide boscalid induces thyroid histopathological and hormone effects in the rat, secondary to liver enzyme induction. To assess the human relevance of liver enzyme induction presumably leading to thyroid hormone disruption, a species comparative in vitro study on T4-glucuronidation was conducted. Currently, no guidelines how to evaluate Phase II induction are in place. Therefore, we investigated the optimal conditions to evaluate Phase I and Phase II induction potential of boscalid in primary rat (PRH) and human (PHH) hepatocytes. Endpoints included mRNA gene expression and enzyme activities (cytochrome P450 isozymes [CYPs] and uridine diphosphate-glucuronosyltransferases [UGTs]), measured after 3 (D3) and 7 (D7) days of exposure to reference compounds and to 5, 10, and 20 µM boscalid, focusing on T4-glucuronidation. Basal CYP activities and T4 glucuronidation were similar or higher on D7 than D3. The highest induction responses of CYPs were on D3, whereas UGT induction and T4-glucuronidation increases were highest on D7. Boscalid induced CYP1A, CYP2B, and CYP3A mRNA and/or increased related activities in PRH and PHH. Species differences in the induction pattern of UGT genes by reference inducers (ß-naphthoflavone [BNF], 5-pregnen-3ß-ol-20-one-16α-carbonitirile [PCN], rifampicin [RIF], and phenobarbital [PB]) and boscalid were seen: UGT1A1, UGT1A3, and UGT1A9 were predominantly induced in PHH, while UGT2B1 was predominantly induced in PRH. Basal activity levels for T4-glucuronidation were very low in humans and an order of magnitude higher in rat, for this reason increases in activities were assessed as delta activity to the control. Significant increases in T4-glucuronidation occurred with boscalid in rat but not in human hepatocytes.

Investigation of the factors responsible for the low oral bioavailability of alizarin using a sensitive LC-MS/MS method: In vitro, in situ, and in vivo evaluations.

Alizarin (1,2-dihydroxyanthraquinone) is an anthraquinone reddish dye widely used for painting and textile dyeing. As the biological activity of alizarin has recently attracted increasing attention from researchers, its therapeutic potential as complementary and alternative medicine is of interest. However, no systematic research has been conducted on the biopharmaceutical and pharmacokinetic aspects of alizarin. Therefore, this study aimed to comprehensively investigate the oral absorption and intestinal/hepatic metabolism of alizarin using a simple and sensitive tandem mass spectrometry method developed and validated in-house. The present method for the bioanalysis of alizarin has merits, including a simple pretreatment procedure, small sample volume, and adequate sensitivity. Alizarin exhibited pH-dependent moderate lipophilicity and low solubility with limited intestinal luminal stability. Based on the in vivo pharmacokinetic data, the hepatic extraction ratio of alizarin was estimated to be 0.165-0.264, classified as a low level of hepatic extraction. In an in situ loop study, considerable fractions (28.2%-56.4%) of the alizarin dose were significantly absorbed in gut segments from the duodenum to ileum, suggesting that alizarin may be classified as the Biopharmaceutical Classification System class II. An in vitro metabolism study using rat and human hepatic S9 fractions revealed that glucuronidation and sulfation, but not NADPH-mediated phase I reactions and methylation, are significantly involved in the hepatic metabolism of alizarin. Taken together, it can be estimated that the fractions of oral alizarin dose unabsorbed from the gut lumen and eliminated by the gut and liver before reaching the systemic circulation are 43.6%-76.7%, 0.474%-36.3%, and 3.77%-5.31% of the dose, respectively, resulting in a low oral bioavailability of 16.8%. Therefore, the oral bioavailability of alizarin depends primarily on its chemical degradation in the gut lumen and secondarily on first-pass metabolism.

Identification of Human UDP-Glucuronosyltransferase Involved in Gypensapogenin C Glucuronidation and Species Differences.

Gypensapogenin C (GPC) is one of the important aglycones of Gynostemma pentaphyllum (GP), which is structurally glucuronidated and is highly likely to bind to UGT enzymes in vivo. Due to the important role of glucuronidation in the metabolism of GPC, the UDP-glucuronosyltransferase metabolic pathway of GPC in human and other species' liver microsomes is investigated in this study. In the present study, metabolites were detected using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results show that GPC could generate a metabolite through glucuronidation in the human liver microsomes (HLMs). Additionally, chemical inhibitors combined with recombinant human UGT enzymes clarified that UGT1A4 is the primary metabolic enzyme for GPC glucuronidation in HLMs according to the kinetic analysis of the enzyme. Metabolic differential analysis in seven other species indicated that rats exhibited the most similar metabolic rate to that of humans. In conclusion, UGT1A4 is a major enzyme responsible for the glucuronidation of GPC in HLMs, and rats may be an appropriate animal model to evaluate the GPC metabolism.

Glycogen-Endoplasmic Reticulum Connection in the Liver.

Glycogen, the branched polymer of glucose is found mainly in the liver and muscle in mammals. Along with several other proteins, glycogen forms separate cellular organelles, and particles in cells. Glycogen particles in the liver have a special metabolic and also regulatory connection to the intracellular endomembrane system, particularly the endoplasmic reticulum. This connection is part of the organelle homeostasis in hepatocytes and forms a "glycogenoreticular system". The actual size of hepatic glycogen stores and the rate of glycogenolysis determines several essential liver-specific metabolic processes, such as glucose secretion for the maintenance of blood glucose levels or the glucuronidation of certain vital endo-, and xenobiotics, and are also related to liver antioxidant defense. In starvation, and in certain physiological and pathological states, where glycogen stores are depleted, functions of the glycogenoreticular system are altered. The starvation-induced depletion of hepatic glycogen content changes the biotransformation of various endo- and xenobiotics. This can be observed especially in acute DILI (drug-induced liver injury) due to paracetamol overdose, which is the most common cause of acute liver failure in the West.

Glucuronidation Pathways of 5- and 7-Hydroxypropranolol: Determination of Glucuronide Structures and Enzyme Selectivity.

Propranolol, a non-selective beta-blocker medication, has been utilized in the treatment of cardiovascular diseases for several decades. Its hydroxynaphthyl metabolites have been recognized to possess varying degrees of beta-blocker activity due to the unaltered side-chain. This study achieved the successful separation and identification of diastereomeric glucuronic metabolites derived from 4-, 5-, and 7-hydroxypropranolol (4-OHP, 5-OHP, and 7-OHP) in human urine. Subsequently, reaction phenotyping of 5- and 7-hydroxypropranolol by different uridine 5'-diphospho-glucuronosyltransferases (UGTs) was carried out, with a comparison to the glucuronidation of 4-hydroxypropranolol (4-OHP). Among the 19 UGT enzymes examined, UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2A1, and UGT2A2 were found to be involved in the glucuronidation of 5-OHP. Furthermore, UGT1A6 exhibited glucuronidation activity towards 7-OHP, along with the aforementioned eight UGTs. Results obtained by glucuronidation of corresponding methoxypropranolols and MS/MS analysis of 1,2-dimethylimidazole-4-sulfonyl (DMIS) derivatives of hydroxypropranolol glucuronides suggest that both the aromatic and aliphatic hydroxy groups of the hydroxypropranolols may be glucuronidated in vitro. However, the analysis of human urine samples collected after the administration of propranolol leads us to conclude that aromatic-linked glucuronidation is the preferred pathway under physiological conditions.

Effects of common genetic variants of human uridine diphosphate glucuronosyltransferase subfamilies on irinotecan glucuronidation.

The adverse effects (diarrhea and neutropenia) of irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) are associated with genetic variants of uridine diphosphate glucuronosyltransferase 1A subfamilies (UGT1As). UGT1As are enzymes that metabolize the active form of irinotecan, 7-ethyl-10 hydroxycamptothecin (SN-38), by glucuronidation in the liver. They are widely known as predictive factors of severe adverse effects, such as neutropenia and diarrhea. Some studies have suggested that variants of UGT1As affect SN-38 glucuronidation activities, thus exerting severe adverse effects. We aimed to identify UGT1A isoforms that show SN-38 glucuronidation activity and determine the relationship between UGT1A variants and SN-38 glucuronidation in vitro. We found that UGT1A1 and UGT1A6-UGT1A10 displayed SN-38 glucuronidation activity. Among these, UGT1A1 was the most active. Furthermore, the variants of these isoforms showed decreased SN-38 glucuronidation activity. In our study, we compared the different variants of UGT1As, such as UGT1A1.6, UGT1A1.7, UGT1A1.27, UGT1A1.35, UGT1A7.3, UGT1A8.4, UGT1A10M59I, and UGT1A10T202I, to determine the differences in the reduction of glucuronidation. Our study elucidates the relationship between UGT1A variants and the level of glucuronidation associated with each variant. Therefore, testing can be done before the initiation of irinotecan treatment to predict potential toxicities and adverse effects.

Regioselective Glucuronidation of Flavones at C5, C7, and C4' Positions in Human Liver and Intestinal Microsomes: Comparison among Apigenin, Acacetin, and Genkwanin.

Flavones, which are distributed in a variety of plants and foods in nature, possess significant biological activities, including antitumor and anti-inflammatory effects, and are metabolized into glucuronides by uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes in humans. In this study, apigenin, acacetin, and genkwanin, flavones having hydroxyl groups at C5, C7, and/or C4'positions were focused on, and the regioselective glucuronidation in human liver and intestinal microsomes was examined. Two glucuronides (namely, AP-7G and AP-4'G for apigenin, AC-5G and AC-7G for acacetin, and GE-5G and GE-4'G for genkwanin) were formed from each flavone by liver and intestinal microsomes, except for only GE-4'G formation from genkwanin by intestinal microsomes. The order of total glucuronidation activities was liver microsomes > intestinal microsomes for apigenin and acacetin, and liver microsomes < intestinal microsomes for genkwanin. The order of CLint values (x-intercept) based on v versus V/[S] plots for apigenin glucuronidation was AP-7G > AP-4'G in liver microsomes and AP-7G < AP-4'G in intestinal microsomes. The order of CLint values was AC-5G < AC-7G for acacetin and GE-5G < GE-4'G genkwanin glucuronidation in both liver and intestinal microsomes. This suggests that the abilities and roles of UGT enzymes in the glucuronidation of apigenin, acacetin, and genkwanin in humans differ depending on the chemical structure of flavones.