UDP-glucuronosyltransferase Activity Research Articles

Potential interactions among myricetin and dietary flavonols through the inhibition of human UDP-glucuronosyltransferase in vitro

Myricetin is a dietary flavonol and possesses multiple bioactivities, which making it an excellent nutritional supplement and a new drug candidate. However, whether myricetin and other homologous dietary flavonols affect the activities of UDP-glucuronosyltransferases (UGT) enzymes and facilitated food-drug interactions remains unclear. Our results demonstrated that myricetin displayed broad-spectrum inhibition against human UGTs. Myricetin exhibited strong inhibitory effects against UGT1A1, 1A3, 1A6, 1A7, 1A10 (IC50 < 10 μM) with non-competitive inhibition type, while serving as a moderate inhibitor against UGT1A9 and 2B7 (IC50 range from 25 to 29 μM) with competitive and mixed inhibition type, respectively. In Silico docking was carried out to explore the binding models and free energies of myricetin towards inhibitory UGTs. The potential risks of food–drug interactions after myricetin consumption were predicted by combining the in vitro inhibitory data and physiological data. The quantitative prediction in vivo of inhibition on gastrointestinal UGTs by myricetin showed that the inhibition against UGT1A1, 1A3, 1A6, 1A7, 1A9, 1A10 and 2B7 would likely occur with high risk. The follow-up findings demonstrated that morin, kaempferol, quercetin and galangin, the four homologous dietary flavonols, shared similar inhibition patterns towards UGTs. These findings altogether demonstrate that myricetin and homologous dietary flavonols have potent and broad-spectrum inhibitory effects against most human UGTs, thus suggest that much caution should be exercised when flavonols-rich foods or supplements are co-administered with UGT substrate drugs.

Antioxidant Extract from Cleistocalyx nervosum var. paniala Pulp Ameliorates Acetaminophen-Induced Acute Hepatotoxicity in Rats.

The indigenous purplish red fruit, Cleistocalyx nervosum var. paniala (CN), is grown in northern Thailand. The aqueous extract of CN pulp is known to exhibit antioxidant and anticarcinogenic properties. To search for an antioxidant fraction separated from CN, various hydroalcoholic extractions were performed. The acidified ethanolic extract of CN obtained from 0.5% (v/v) citric acid in 80% (v/v) ethanol yielded greater polyphenol content and DPPH radical scavenging activity when compared with other hydroethanolic extracts. Cyanidin-3-glucoside is a major anthocyanin present in the acidified ethanolic extract of CN (AECN). At a dose of 5000 mg/kg bw, an anthocyanin-rich extract was found to be safe when given to rats without any acute toxicity. To examine the hepatoprotective properties of AECN, an overdose of acetaminophen (APAP) was induced in a rat model, while silymarin was used as a standard reference. The administration of AECN at a dose of 300 mg/kg bw for 28 days improved hepatocyte architecture and modulated serum alanine aminotransferase levels in APAP-induced rats. Furthermore, it significantly decreased serum and hepatic malondialdehyde levels but increased hepatic glutathione content, as well as glutathione peroxidase and UDP-glucuronosyltransferase activities. In conclusion, AECN may effectively reduce oxidative stress induced acute hepatotoxicity in overdose APAP-treated rats through the suppression of oxidative stress and the enhancement of the antioxidant system in rat livers.

Exploration and application of a liver-on-a-chip device in combination with modelling and simulation for quantitative drug metabolism studies.

Microphysiological systems (MPS) are complex and more physiologically realistic cellular in vitro tools that aim to provide more relevant human in vitro data for quantitative prediction of clinical pharmacokinetics while also reducing the need for animal testing. The PhysioMimix liver-on-a-chip integrates medium flow with hepatocyte culture and has the potential to be adopted for in vitro studies investigating the hepatic disposition characteristics of drug candidates. The current study focusses on liver-on-a-chip system exploration for multiple drug metabolism applications. Characterization of cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT) and aldehyde oxidase (AO) activities was performed using 15 drugs and in vitro to in vivo extrapolation (IVIVE) was assessed for 12 of them. Next, the utility of the liver-on-a-chip for estimation of the fraction metabolized (fm) via specific biotransformation pathways of quinidine and diclofenac was established. Finally, the metabolite identification opportunities were also explored using efavirenz as an example drug with complex primary and secondary metabolism involving a combination of CYP, UGT and sulfotransferase enzymes. A key aspect of these investigations was the application of mathematical modelling for improved parameter calculation. Such approaches will be required for quantitative assessment of metabolism and/or transporter processes in systems where medium flow and system compartments result in non-homogeneous drug concentrations. In particular, modelling was used to explore the effect of evaporation from the medium and it was found that the intrinsic clearance (CLint) might be underestimated by up to 40% for low clearance compounds if evaporation is not accounted for. Modelling of liver-on-a-chip in vitro data also enhanced the approach to fm estimation allowing objective assessment of metabolism models of different complexity. The resultant diclofenac fm,UGT of 0.64 was highly comparable with values reported previously in the literature. The current study demonstrates the integration of mathematical modelling with experimental liver-on-a-chip studies and illustrates how this approach supports generation of high quality of data from complex in vitro cellular systems.

Adenine-related compounds modulate UDP-glucuronosyltransferase (UGT) activity in mouse liver microsomes

ABSTRACTAdenine-related compounds are allosteric inhibitors of UDP-glucuronosyltransferase (UGT) in rat liver microsomes (RLM) and human UGT isoforms treated with detergent or pore-forming peptide, alamethicin.To clarify whether the same is true beyond species, the effects of adenine-related compounds on 4-methylumbelliferone (4-MU) glucuronidation were examined using detergent-treated mouse liver microsomes (MLM).Brij-58 treatment of MLM increased the Vmax and the Michaelis constant, Km, of 4-MU. This study was performed using Brij-58-treated MLM as an enzyme source. ATP- and ADP-inhibited 4-MU glucuronidation. In contrast, AMP caused a 1.5-fold increase in glucuronidation. Oxidised forms, NAD+ and NADP+, potently inhibited 4-MU glucuronidation, whereas the reduced forms, NADH and NADPH, did not. Furthermore, the IC50 values of ATP, ADP, NAD+, and NADP+ were approximately 15 μM.In our previous study, ATP was the strongest inhibitor of UGT activity in RLM. However, in this study, the above-mentioned compounds inhibited 4-MU UGT in a comparable and noncompetitive manner. Furthermore, AMP antagonised the inhibitory effects of ATP and ADP.These results suggest that ATP, ADP, NAD+, and NADP+ are common endogenous inhibitors of UGT beyond species.

Синдром Жильбера: термінологія, епідеміологія, генетика, патогенез (частина I)

Мета огляду — аналіз даних літератури щодо поширеності, етіології, генетики, патогенезу синдрому Жильбера. Проведений огляд наукової літератури щодо синдрому Жильбера за ключовими словами «синдром Жильбера», «гіпербілірубінемія», «уридиндифосфатглюкуронілтрансфераза» з використанням як пошукової системи PubMed. Беручи до уваги дослідження, проведені в останні 10 років, проаналізовані тези 75 статей. Більш детально вивчено результати дослідження, висвітлені у 28 статтях.

Inhibition of UDP-Glucuronosyltransferase Enzymes by Major Cannabinoids and Their Metabolites.

The UDP-glucuronosyltransferase (UGT) family of enzymes play a central role in the metabolism and detoxification of a wide range of endogenous and exogenous compounds. UGTs exhibit a high degree of structural similarity and display overlapping substrate specificity, often making estimations of potential drug-drug interactions difficult to fully elucidate. One such interaction yet to be examined may be occurring between UGTs and cannabinoids, as the legalization of recreational and medicinal cannabis and subsequent co-usage of cannabis and therapeutic drugs increases in the United States and internationally. In the present study, the inhibition potential of the major cannabinoids Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN), as well as their major metabolites, was determined in microsomes isolated from HEK293 cells overexpressing individual recombinant UGTs and in microsomes from human liver and kidney specimens. The highest inhibition was seen by CBD against the glucuronidation activity of UGTs 1A9, 2B4, 1A6, and 2B7, with binding-corrected IC50 values of 0.12 ± 0.020 µM, 0.22 ± 0.045 µM, 0.40 ± 0.10 µM, and 0.82 ± 0.15 µM, respectively. Strong inhibition of UGT1A9 was also demonstrated by THC and CBN, with binding-corrected IC50 values of 0.45 ± 0.12 μM and 0.51 ± 0.063 μM, respectively. Strong inhibition of UGT2B7 was also observed for THC and CBN; no or weak inhibition was observed with cannabinoid metabolites. This inhibition of UGT activity suggests that in addition to playing an important role in drug-drug interactions, cannabinoid exposure may have important implications in patients with impaired hepatic or kidney function. SIGNIFICANCE STATEMENT: Major cannabinoids found in the plasma of cannabis users inhibit several UDP-glucuronosyltransferase (UGT) enzymes, including UGT1A6, UGT1A9, UGT2B4, and UGT2B7. This study is the first to show the potential of cannabinoids and their metabolites to inhibit all the major kidney UGTs as well as the two most abundant UGTs present in liver. This study suggests that as all three major kidney UGTs are inhibited by cannabinoids, greater drug-drug interaction effects might be observed from co-use of cannabinods and therapeutics that are cleared renally.

Inhibition of human UDP-glucuronosyltransferase enzyme by Dabrafenib: Implications for drug-drug interactions.

Dabrafenib is a novel small molecule tyrosine kinase inhibitor (TKI) which is used to treat metastatic melanoma. The aim of this research was to survey the effects of dabrafenib on human UDP-glucuronosyltransferases (UGTs) and to evaluate the risk of drug-drug interactions (DDIs). The formation rates for 4-methylumbelliferone (4-MU) glucuronide and trifluoperazine-glucuronide in 12 recombinant human UGT isoforms with or without dabrafenib were measured and HPLC was used to investigate the inhibitory effects of dabrafenib on UGTs. Inhibition kinetic studies were also conducted. In vitro-in vivo extrapolation approaches were further used to predict the risk of DDI potentials of dabrafenib via inhibition of UGTs. Our data indicated that dabrafenib had a broad inhibitory effect on 4-MU glucuronidation by inhibiting the activities of UGTs, especially on UGT1A1, UGT1A7, UGT1A8, and UGT1A9, and dabrafenib could increase the area under the curve of co-administered drugs. Dabrafenib is a strong inhibitor of several UGTs and the co-administration of dabrafenib with drugs primarily metabolized by UGT1A1, 1A7, 1A8 or 1A9 may induce potential DDIs.

Comparison of the drug-drug interactions potential of ibrutinib and acalabrutinib via inhibition of UDP-glucuronosyltransferase

Ibrutinib and acalabrutinib are two Bruton's tyrosine kinase (BTK) inhibitors which have gained Food and Drug Administration (FDA) approval for the treatment of various B cell malignancies. Herein, we investigated the effects of the two drugs on UDP-glucuronosyltransferase (UGT) activities to evaluate their potential risk for drug-drug interactions (DDIs) via UGT inhibition. Our data indicated that ibrutinib exerted broad inhibition on most of UGTs, including a potent competitive inhibition against UGT1A1 with a Ki value of 0.90 ± 0.03 μM, a noncompetitive inhibition against UGT1A3 and UGT1A7 with Ki values of 0.88 ± 0.03 μM and 2.52 ± 0.23 μM, respectively, while acalabrutinib only exhibited weak UGT inhibition towards all tested UGT isoforms. DDI risk prediction suggested that the inhibition against UGT1A1 and UGT1A3 by ibrutinib might bring a potential DDIs risk, while acalabrutinib was unlikely to trigger clinically significant UGT-mediated DDIs due to its weak effects. Our study raises an alarm bell about potential DDI risk associated with ibrutinib, however, the extrapolation from in vitro data to in vivo drug interactions should be taken with caution, and additional systemic study is needed.

In vitro inhibition of human UDP-glucuronosyltransferase (UGT) 1A1 by osimertinib, and prediction of in vivo drug-drug interactions

Osimertinib is the only third-generation epidermal growth factor receptor tyrosine-kinase inhibitor (EGFR-TKI) approved by Food and Drug Administration (FDA). This study aimed to know the inhibitory effect of osimertinib on human UDP-glucosyltransferases (UGTs) and human liver microsomes (HLMs), as well as to identify its potential to cause drug-drug interaction (DDI) arising from the modulation of UGT activity. High inhibitory effect of osimertinib was shown towards UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A10, 2B7 and 2B15. Especially, osimertinib exhibited competitive inhibition against UGT1A1 with a Ki,u of 0.87 ± 0.12 μM. It also noncompetitively inhibited SN-38 glucuronidation in pooled HLMs with a Ki,u of 3.32 ± 0.25 μM. Results from quantitative prediction study indicated that osimertinib administered at 80 mg/day may result in a 4.83 % increase in the AUC of drugs mainly metabolized by UGT1A1, implying low risk of DDI via liver metabolism. However, the ratios of [I]gut/Ki,u are much higher than 11 in HLMs and recombinant UGT1A1, indicating a risk for interaction in intestine. The effects of osimertinib on intestinal UGT should be paid more attention on to avoid unnecessary clinical DDI risks.

The Effect of Pomegranate Juice on the Expression of Some Murine UDP-Glucuronosyltransferases Genes.

Food-drug interactions may lead to suppression or induction of drug metabolizing enzymes. Pomegranate is a commonly used fruit in folk medicine all over the world. Data concerning the effect of pomegranate on the activity of UDP-glucuronosyltransferases (UGTs) is scarce. The purpose of this work was to investigate the effect of pomegranate juice ingestion on the transcription of ugt2b1, ugt2a3, and ugt1a9 in the liver and small intestine of male mice. Pomegranate juice was administered to 10 male mice for 14 days in drinking bottles instead of water. Ten control mice received water in the drinking bottles. On the 15th day, the mice were sacrificed and the liver and the small intestine were removed. The small intestine was divided into 3 parts. Total mRNA was extracted from samples of these specimens, and cDNA was synthesized by quantitative real-time polymerase chain reaction (RT-PCR) using specific primers for each ugt gene. The ugt1a9 mRNA level was reduced by 2.25-fold in the liver and by 6-, 1.5-, and 3-folds in the first, second and third part of the small intestine, respectively. The ugt2b1 mRNA level in the liver and the third part of the small intestine was not affected, while it was reduced by 3.7- and 3-folds in the first and second parts of the small intestine, respectively. The ugt2a3 mRNA level was not affected in the liver and the 3 parts of the small intestine. Some ugt mRNA levels may be reduced by the ingestion of pomegranate juice, which may reduce the metabolism of their drug substrates. The consequences may be an accumulation of such drugs in the body and enhanced toxicity.

Glucuronidation of Efavirenz and its Cytochrome P450‐dependent Metabolites in Brain Microsomes and Neurons

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) anti-HIV medication that is glucuronidated by UDP-glucuronosyl transferases (UGTs). Brain penetrant anti-HIV drugs, such as EFV, are essential to combatting HIV in the brain. Persons with HIV often suffer from neurocognitive deficits, known as HIV-associated neurocognitive disorder (HAND). In addition to HAND, EFV itself has been shown to lead to severe neurological adverse events that can result the discontinuation of EFV-based therapies. Despite the importance of this drug and the observed adverse side effects, its metabolism in the brain is virtually unknown. Furthermore, UGT expression and activity in the brain remains poorly understood. We tested whether EFV and/or its cytochrome P450-dependent metabolites –8-hydroxyefavirenz (8-OHEFV) and 8,14-dihydroxyefavirenz (8,14-diOHEFV) undergo glucuronidation in the brain. To investigate this, brain microsomes and primary mouse neurons in culture were incubated with EFV, 8-hydroxyefavirenz (8-OHEFV), or 8,14-dihydroxy efavirenz (8,14-diOHEFV). Glucuronidated metabolites were detected using ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC MS/MS). Incubating pooled brain microsomes from mice, cynomolgus macaques, and humans with 10µM EFV or one of its cytochrome P450-dependent metabolites for 30 min, we observed the formation of glucuronidated metabolites of 8-OHEFV and 8,14-diOHEFV for all three species. Interestingly, the cynomolgus macaque microsomes were the only microsomes to yield the EFV N-glucuronide metabolite, indicating interspecies differences in UGT activity toward EFV. In primary C57BL6/J neurons treated with a drug concentration of 50µM for 3-4 hours, we observed glucuronidation of 8-OHEFV, but not EFV. This was commensurate with our findings using mouse brain microsomes. These data lend insight into EFV disposition in brain across humans, mice, and cynomolgus macaques, and can be leveraged to inform understanding of EFV-mediated neurotoxicity.

Echinacoside alleviates acetaminophen-induced liver injury by attenuating oxidative stress and inflammatory cytokines in mice.

This study evaluates the protective effect of Echinacoside on acute liver toxicity induced by acetaminophen in mice and the mechanism behind it. Echinacoside and N-Acetyl Cysteine were intragastrically administrated for 7 days, and acetaminophen was intraperitoneally injected into mice 1 h after the last treatment on day 7. At the end of the experimental period, histological examination, parameters for the level of oxidative damage, hepatic malondialdehyde, serum pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6, and interleukin-1β), UDP-glucuronosyltransferases, and sulfotransferases changes were examined using enzyme-linked immunosorbent assay and standard biochemical procedures. The expression of cytochrome P450 2E1 protein was assessed by western blot, followed by in silico molecular docking. Acetaminophen treatment obviously increased the levels of ALT and AST, changed hepatic histopathology, promoted oxidative stress, decreased antioxidant enzyme activities, and elevated the pro-inflammatory cytokines. Echinacoside significantly attenuated Acetaminophen-induced liver damage in a dose-dependent manner, with the most effective dose at 100 mg/kg. The pretreatments of Echinacoside in different concentrations altered the Acetaminophen-induced hepatotoxicity levels by decreasing the level of liver enzymes, reducing the liver necrosis with vacuolization, decreasing the hepatic malondialdehyde formation, increasing hepatic antioxidants activities, suppressing the pro-inflammatory cytokines (Tumor Necrosis Factor, Interleukin-6 and Interleukin-1beta), inhibiting Nitric Oxide production, enhancing sulfotransferases and UDP-glucuronosyltransferases activities. Notably, the expression of cytochrome P450 2E1 was inhibited by Echinacoside in a dose-dependent manner and the binding energy was -214.3 MeV. Echinacoside showed a significant protective effect against Acetaminophen-induced hepatotoxicity through the inhibition of oxidative stress, the expression of pro-inflammatory cytokines and cytochrome P450 2E1 protein expression.

The odorant metabolizing enzyme UGT2A1: Immunolocalization and impact of the modulation of its activity on the olfactory response.

Odorant metabolizing enzymes (OMEs) are expressed in the olfactory epithelium (OE) where they play a significant role in the peripheral olfactory process by catalyzing the fast biotransformation of odorants leading either to their elimination or to the synthesis of new odorant stimuli. The large family of OMEs gathers different classes which interact with a myriad of odorants alike and complementary to olfactory receptors. Thus, it is necessary to increase our knowledge on OMEs to better understand their function in the physiological process of olfaction. This study focused on a major olfactory UDP-glucuronosyltransferase (UGT): UGT2A1. Immunohistochemistry and immunogold electronic microscopy allowed to localize its expression in the apical part of the sustentacular cells and originally at the plasma membrane of the olfactory cilia of the olfactory sensory neurons, both locations in close vicinity with olfactory receptors. Moreover, using electroolfactogram, we showed that a treatment of the OE with beta-glucuronidase, an enzyme which counterbalance the UGTs activity, increased the response to eugenol which is a strong odorant UGT substrate. Altogether, the results supported the function of the olfactory UGTs in the vertebrate olfactory perireceptor process.

Effects of Shengmai San on key enzymes involved in hepatic and intestinal drug metabolism in rats

Ethnopharmacological relevanceShengmai San (SMS) has been commonly used as a traditional Chinese medicine for the treatment of cardiovascular disorders, of which drug interactions need to be assessed for the safety concern. There is little evidence for the alterations of hepatic and intestinal drug-metabolizing enzymes after repeated SMS treatments to assess drug interactions. Aim of the studyThe studies aim to illustrate the effects of repeated treatments with SMS on cytochrome P450s (CYPs), reduced nicotinamide adenine dinucleotide (phosphate)-quinone oxidoreductase (NQO), uridine diphosphate-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) using in vivo rat model. Materials and methodsThe SMS was prepared using Schisandrae Fructus, Ginseng Radix, and Ophiopogonis Radix (OR) (1:2:2). Chromatographic analyses of decoctions were performed using ultra-performance liquid chromatography (UPLC) and LC-mass spectrometry. Sprague-Dawley rats were orally treated with the SMS and its component herbal decoctions for 2 or 3 weeks. Hepatic and intestinal enzyme activities were determined. CYP3A expression and the kinetics of intestinal nifedipine oxidation (NFO, a CYP3A marker reaction) were determined. ResultsSchisandrol A, schisandrin B, ginsenoside Rb1 and ophiopogonin D were identified in SMS. SMS selectively suppressed intestinal, but not hepatic, NFO activity in a dose- and time-dependent manner. Hepatic and intestinal UGT, NQO and GST activities were not affected. A 3-week SMS treatment decreased the maximal velocity of intestinal NFO by 50%, while the CYP3A protein level remained unchanged. Among SMS component herbs, the decoction of OR decreased intestinal NFO activity. ConclusionsThese findings demonstrate that 3-week treatment with SMS and OR suppress intestinal, but not hepatic CYP3A function. It suggested that the potential interactions of SMS with CYP 3A drug substrates should be noticed, especially the drugs whose bioavailability depends heavily on intestinal CYP3A.

Potent inhibition of tributyltin (TBT) and triphenyltin (TPT) against multiple UDP-glucuronosyltransferases (UGT): A new potential mechanism underlying endocrine disrupting actions

Organotin compounds (OTs) act as potent endocrine disruptors that are often found in polluted food and water. UDP-glucuronosyltransferases (UGTs) are responsible for termination of multiple endogenous hormones. This study was conducted to investigate the inhibitory effects of two tri-submitted OTs tributyltin (TBT) and triphenyltin (TPT), against activities of UGTs. It is revealed that TBT and TPT act as two potent inhibitors for multiple UGTs. UGT1A8 and -2B15 were coinhibited by the two OTs. UGT1A1 and -1A10 were inhibited by TPT, whereas UGT 2B4 and -2B7 were inhibited by TBT. Kinetic analyses further indicated that TBT and TPT are two competitive nanomolar inhibitors of UGT2B15, with Ki values of 0.45 and 0.46 μM, respectively. Ki values for the other UGTs are determined to be a few micromolars. In addition, the two OTs displayed effective inhibition against UGT2B15 in catalyzing dihydrotestosterone glucuronidation, with IC50 values both in nano-molar range. TPT can additionally inhibit activities of UGT1A1 and -1A10 in estradiol-3-O-glucuronidation, with IC50 values of a few micro-molars. These results indicated that the two OTs can extensively interfere with glucuronidation of endogenous hormones, which may act as a new potential mechanism resulting in endocrine disrupting actions.

Effect of resveratrol, carnitin, quercetin and aromatic amino acids on the xenobiotic metabolising and antioxidant enzymes in the liver during obesity in rats with different genotypes

The use of minor biologically active substances (BAS) in specialized products is one of the promising areas in the diet therapy for obesity and other alimentary-dependent diseases (metabolic syndrome, type 2 diabetes mellitus, etc.). The effects of the BAS in patients are often ambiguous, depending on a number of factors, one of which is the state of the organism enzyme systems (enzymes of xenobiotic metabolism and antioxidant defense), the patient's genotype, and many others. The aim was to study the effect of BAS [quercetin (Q), L-carnitine (L-Car), resveratrol (Res), aromatic amino acids tyrosine (Tyr) and tryptophan (Trp)] on the activities of phase I and II of xenobiotic-metabolising enzymes and antioxidant enzymes in rats using various in vivo models of obesity and with impaired dopamine transport. Material and methods. The activities of cytochrome P450 enzymes (CYP1A1 and CYP3A), glutathione transferase (GT), UDPglucuronosyltransferase (UDP-GT), hemoxygenase-1 (HO-1), and quinone reductase (QR) were determined by fluorimetric, spectrophotometric methods and HPLC in microsomes and cytosolic fraction of rat's liver. We used rats of outbred Wistar line, Zucker ZF line with hereditarily determined obesity and DAT-KO line with a knockout of the DAT dopamine transporter gene, which for 62 days were fed a standard balanced control or high-carbohydrate high-fat diet (30% fat by weight and 20% fructose solution instead of water) supplemented with BAS, such as Q, Res, L-Car, Tyr and Trp in doses 50, 25, 300, 1250 and 250 mg/kg of body weight respectively. Results and discussion. The presence of a DAT knockout led to a small but statistically significant decrease in the activity of GT in the liver in both homozygous and heterozygous animals. The CYP1A1 activity was significantly decreased in all carriers of the DAT knockout gene, while HO-1 activity, on the contrary, was increased, independently of the composition of the diet used. In Zucker ZF rats of all groups, in comparison with Wistar rats fed the corresponding diets, the activities of GT, UDP-GT, CYP1A1, CYP3A and QR were significantly reduced in terms of the total protein content. HO-1 activity was reduced in Zucker ZF rats in comparison with Wistar rats to a lesser extent, however, the addition of Q significantly influenced the difference between the two lines. Trp consumption led to a significant increase in GT activity in Wistar rats. In DAT-KO homozygotes this effect was insignificant, while in heterozygotes it was absent. Similarly, consumption of Trp resulted in a significant increase in CYP1A1 activity only in Wistar rats, but not in DAT-KO rats. The activity of UDP-GT under Trp intake increased only in DAT heterozygotes. The genotype significantly influenced the response of QR activity to Trp consumption, but in an ambiguous way - there was an increase in activity in heterozygotes and a decrease in homozygotes for DAT knockout. CYP1A1 activity was significantly increased in rats treated with Tyr. Conclusion. The data obtained indicate that the effect of various dietary supplements used in the treatment of obesity and metabolic syndrome on the xenobiotic-metabolising enzymes and antioxidant enzymes can have a different nature and direction depending on the genotype and the level of spontaneous physical activity and energy expenditure determined by it, which should be taken into account when approaches to personalized diet therapy of alimentary-dependent diseases are developing.

Enzyme Kinetics of Uridine Diphosphate Glucuronosyltransferases (UGTs).

Glucuronidation, catalyzed by uridine diphosphate glucuronosyltransferases (UGTs), is an important process for the metabolism and clearance of many lipophilic chemicals, including drugs, environmental chemicals, and endogenous compounds. Glucuronidation is a bisubstrate reaction that requires the aglycone and the cofactor, UDP-GlcUA. Accumulating evidence suggests that the bisubstrate reaction follows a compulsory-order ternary mechanism. To simplify the kinetic modeling of glucuronidation reactions in vitro, UDP-GlcUA is usually added to incubations in large excess. Many factors have been shown to influence UGT activity and kinetics in vitro, and these must be accounted for during experimental design and data interpretation. While the assessment of drug-drug interactions resulting from UGT inhibition has been challenging in the past, the increasing availability of UGT enzyme-selective substrate and inhibitor "probes" provides the prospect for more reliable reaction phenotyping and assessment of drug-drug interaction potential. Although extrapolation of the in vitro intrinsic clearance of a glucuronidated drug often underpredicts in vivo clearance, careful selection of in vitro experimental conditions and inclusion of extrahepatic glucuronidation may improve the predictivity of in vitro-in vivo extrapolation. Physiologically based pharmacokinetic (PBPK) modeling has also shown to be of value for predicting PK of drugs eliminated by glucuronidation.

Drug glucuronidation assays on human liver microsomes immobilized on microfluidic flow-through reactors

UDP-glucuronosyltransferases (UGTs), located in the endoplasmic reticulum of liver cells, are an important family of enzymes, responsible for the biotransformation of several endogenous and exogenous chemicals, including therapeutic drugs. However, the phenomenon of ‘latency’, i.e., full UGT activity revealed by disruption of the microsomal membrane, poses substantial challenges for predicting drug clearance based on in vitro glucuronidation assays. This work introduces a microfluidic reactor design comprising immobilized human liver microsomes to facilitate the study of UGT-mediated drug clearance under flow-through conditions. The performance of the microreactor is characterized using glucuronidation of 8-hydroxyquinoline (via multiple UGTs) and zidovudine (via UGT2B7) as the model reactions. With the help of alamethicin and albumin effects, we show that conducting UGT metabolism assays under flow conditions facilitates in-depth mechanistic studies, which may also shed light on UGT latency.

Inhibition of human UDP-glucuronosyltransferase enzyme by belinostat: Implications for drug-drug interactions

Belinostat is a pan-histone deacetylase (HDAC) inhibitor which recently approved for the treatment of relapsed/refractory Peripheral T-cell lymphomas (PTCL). To assess drug-drug interactions (DDIs) potential of belinostat via inhibition of UDP-glucuronosyltransferases (UGTs), the effects of belinostat on UGTs activities were investigated using the non-selective probe substrate 4-methylumbelliferone (4-MU) and trifluoperazine (TFP) by UPLC–MS/MS. Belinostat exhibited a wide range of inhibition against UGTs activities, particularly a potent non-competitive inhibition against UGT1A3, and weak inhibition against UGT1A1, 1A7, 1A8, 2B4 and 2B7. Further, in vitro–in vivo extrapolation (IVIVE) approaches were used to predict the risk of DDI arising from inhibition of UGTs. Our data indicate that the intravenous infusion of belinostat at clinical available dose can contribute a significant increase to the AUC of co-administrated drugs primarily cleared by UGT1A3 or UGT1A1, which will result in potential DDIs. In contrast, oral administrated belinostat is unlikely to cause significant DDIs through inhibition of glucuronidation.

Gene mutation pattern of Gilbert's syndrome combined with viral hepatitis and its relationship with the exploration of clinical data

Objective: To study whether gene mutation pattern of Gilbert's syndrome (GS) is combined with viral hepatitis and its relationship with relevant clinical data. Methods: Clinical data of GS patients combined with viral hepatitis who was admitted to the Department of Infectious Diseases of Henan Provincial People's Hospital from August 2013 to December 2018 was retrospectively analyzed. The relationship between gene mutation pattern, general data (age, gender, etc.) and liver biochemical indexes was analyzed. The differences of the above data in patients with or without combined viral hepatitis were analyzed. The measurement data were compared by t-test. The categorical data was compared by the χ (2) test. The median and interquartile range of non-normally distributed data was used to indicate the central and discrete tendency. Results: A total of 107 GS eligible cases data were collected. The male to female ratio was 4.94:1 (89:18). The average age of onset was (36.36 ± 12.51) years. Alanine aminotransferase and total bilirubin levels were normal or slightly elevated, while aspartate aminotransferase, alkaline phosphatase, and γ-glutamyltransferase were all within the normal range. There were 49 cases in the combined viral hepatitis group (36 cases with HBV and 13 cases with HCV), and 58 cases in the GS alone group. Total bilirubin level in GS alone group was higher than the combined viral hepatitis group (z = 0.035, P < 0.05), and there were no statistically significant differences in gender, age, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and gamma glutamyltransferase (P > 0.05). Uridine diphosphate glucuronide transferase 1A1 (UGT1A1), specifically encoded by GS was detected in all 107 cases. Mutations was mainly occurred in the upstream promoter PBREM-3263 (-3279) (86 cases) and TATA box TA insertion mutation (71 cases), and GGA-AGA Gly71Arg (57 cases) mutation in EXON1 of the coding region. All mutation forms had manifestations of homozygous and heterozygous abnormalities. The combined incidence of main mutation forms in the genetic testing data were sequenced as: A2 + B2 + C2 (17 cases, 25.23%), A1 + B1 (17 cases, 15.89%), A2 (11 cases, 10.28%), C2 (10 Cases, 9.34%), A2 + B2 (7 cases, 6.54%), A1 + B2 (7 cases, 6.54%), C1 (7 cases, 6.54%), and there was no statistically significant difference between different mutation combinations in patients with or without hepatitis (P > 0.05). The results of total data analysis showed that the total bilirubin level in the single-site mutation group was higher than the multi-site mutation group (Z=2.019, P = 0.043), and other biochemical indicators had no effect (P > 0.05) and the differences were not statistically significant. Further analysis showed that the total bilirubin level of the single-site mutation subgroup in the GS alone group was higher than the multi-site mutation subgroup (Z = 1.999, P = 0.046), and the statistical difference was similar to the combined viral hepatitis group (P > 0.05). Different mutation combinations had no effect on biochemical indexes, and had no relationship with combined viral hepatitis (P > 0.05). Conclusion: GS is common in patients with combined viral hepatitis, and there is no significant difference between the incidence of gene mutation, mutation forms, biochemical indexes, and non-hepatitis group. The increase in the number of GS mutation sites does not aggravate the deterioration of bilirubin levels due to the decrease in the content and activity of uridine diphosphate glucuronosyltransferase, and the combination of different mutation sites does not affect the changes of various biochemical indexes, and at the same time it is not related to hepatitis.