UGT1A1 Activity Research Articles

Inhibitory potential of three Yin-tonification herbal formulas on the activities of human major cytochrome P450 and UDP- glucuronosyltransferases isozymes in vitro

OBJECTIVETo investigate the influence of Yin-tonification herbal formulas Jaeumganghwa-tang (Ziyin Jianghuo Tang, JEGHT), Ssanghwa-tang (Shuanghe Tang, SHT) and Yukmijihwang-tang (Liuwei Di huang Tang, YMJHT) on the activities of human major cytochrome P450 (CYP450s) and UDP-glucuronosyltransferases isozymes (UGTs) in vitro. METHODSThe activities of CYP450s (CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) and UGTs (UGT1A1, UGT1A4 and UGT2B7) were assessed using in vitro fluorescence- and luminescence-based enzyme assays, respectively. The effects of herbal formulas on the activities of CYP450s and UGTs were presented as IC50 values. RESULTSJEGHT showed the potent inhibition of the CYP2D6 activity, with weak inhibition on the activities of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2E1, CYP3A4, UGT1A1, UGT1A4 and UGT2B7. SHT inhibited the activities of CYP1A2 and CYP2E1, whereas the negligible inhibition of the activities of CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A4 and UGT2B7 through SHT was observed. YMJHT inhibited CYP2E1 activity, with a negligible inhibition on the activities of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A4 and UGT2B7. CONCLUSIONThese findings provide information about the potential interactions between three Yin-tonification herbal formulas and conventional drugs.

Identification and characterization of in vitro inhibitors against UDP-glucuronosyltransferase 1A1 in uva-ursi extracts and evaluation of in vivo uva-ursi-drug interactions

Uva-ursi leaf is widely used to treat symptoms of lower urinary tract infections. Here, we evaluated the in vitro inhibitory effects of uva-ursi extracts on 10 major human UDP-glucuronosyltransferases (UGT) isoforms. Of the 10 tested UGT isoforms, uva-ursi extracts exerted the strongest inhibitory effect on UGT1A1-mediated β-estradiol 3-glucuronidation with the lowest IC50 value of 8.45 ± 1.56 μg/mL. To identify the components of uva-ursi extracts showing strong inhibitory effects against UGT1A1, the inhibitory effects of nine major constituents of the extracts were assessed. Among the tested compounds, gallotannin exerted the most potent inhibition on UGT1A1, followed by 1,2,3,6-tetragalloylglucose; both demonstrated competitive inhibition, with Ki values of 1.68 ± 0.150 μM and 3.55 ± 0.418 μM. We found that gallotannin and 1,2,3,6-tetragalloylglucose also inhibited another UGT1A1-specific biotransformation, SN-38-glucuronidation, showing the same order of inhibition. Thus, in vitro UGT1A1 inhibitory potentials of uva-ursi extracts might primarily result from the inhibitory activities of gallotannin and 1,2,3,6-tetragalloylglucose present in the extracts. However, in rats, co-administration with uva-ursi extracts did not alter the in vivo marker for UGT1A1 activity, expressed as the molar ratio of AUCSN-38 glucuronide/AUCSN-38, because plasma concentrations of gallotannin and 1,2,3,6-tetragalloylglucose may be too low to inhibit the UGT1A1-mediated metabolism of SN-38 in vivo. The poor oral absorption of gallotannin and 1,2,3,6-tetragalloylglucose in uva-ursi extracts might cause the poor in vitro-in vivo correlation. These findings will be helpful for the safe and effective use of uva-ursi extracts in clinical practice.

Influence of UGT1A1 polymorphisms on the outcome of acute myeloid leukemia patients treated with cytarabine-base regimens

BackgroundsUDP-glucuronosyltransferase 1A subfamily (UGT1A) enzymes can inactivate cytarabine (Ara-C) by glucuronidation, and thus serves as candidate genes for interindividual difference in Ara-C response. UGT1A1 is a major UGT1A isoform expressed in human liver.MethodsUGT1A1*6 and *28 polymorphisms resulting in reduced UGT1A1 activity were genotyped in 726 adult acute myeloid leukemia (AML) patients treated with Ara-C based regimens. Influences of both polymorphisms on chemosensitivity and disease prognosis of the patients were evaluated.ResultsAfter one or two courses of Ara-C based induction chemotherapy, the complete remission (CR) rate was significantly higher in patients carrying the UGT1A1*6 (77.0%) or the UGT1A1*28 (76.4%) alleles as compared with corresponding wild-type homozygotes (66.9 and 68.5%, respectively). Carriers of the UGT1A1*6 or *28 alleles showed significantly decreased risk of non-CR (OR = 0.528, 95% CI 0.379–0.737, P = 1.7 × 10−4) and better overall survival (HR = 0.787, 95% CI 0.627–0.990, P = 0.040) as compared with homozygotes for both polymorphisms.ConclusionOur results suggest that UGT1A1*28 and UGT1A1*6 are associated with improved clinical outcomes in Chinese AML patients treated with Ara-C.

Physiologically-based pharmacokinetic modelling of a CYP2C19 substrate, BMS-823778, utilizing pharmacogenetic data.

Previous studies demonstrated direct correlation between CYP2C19 genotype and BMS-823778 clearance in healthy volunteers. The objective of the present study was to develop a physiologically-based pharmacokinetic (PBPK) model for BMS-823778 and use the model to predict PK and drug-drug interaction (DDI) in virtual populations with multiple polymorphic genes. The PBPK model was built and verified using existing clinical data. The verified model was simulated to predict PK of BMS-823778 and significance of DDI with a strong CYP3A4 inhibitor in subjects with various CYP2C19 and UGT1A4 genotypes. The verified PBPK model of BMS-823778 accurately recovered observed PK in different populations. In addition, the model was able to capture the exposure differences between subjects with different CYP2C19 genotypes. PK simulation indicated higher exposures of BMS-823778 in CYP2C19 poor metabolizers who were also devoid of UGT1A4 activity, compared to those with normal UGT1A4 functionality. Moderate DDI with itraconazole was predicted in subjects with wild-type CYP2C19 or UGT1A4. However, in subjects without CYP2C19 or UGT1A4 functionality, significant DDI was predicted when BMS-823778 was coadministered with itraconazole. A PBPK model was developed using clinical data that accurately predicted human PK in different population with various CYP2C19 phenotypes. Simulations with the verified PBPK model indicated that UGT1A4 was probably an important clearance pathway in CYP2C19 poor metabolizers. DDI with itraconazole is likely to be dependent on the genotypes of CYP2C19 and UGT1A4.

Confounding effect of UGT1A1 interindividual variability on inhibition of its in vitro activity by kinase inhibitors.

Background and ObjectiveInhibition of UGT1A1 by kinase inhibitors (KIs) has been shown to cause mild to serve hyperbilirubinemia in cancer patients1. UGT1A1 is highly polymorphic and displays variable expression. The specific aims of this study were i) to stratify human liver microsomes (HLMs) based on UGT1A1 abundance, activity and genotype, ii) to screen 10 clinically used KIs for their UGT1A1 inhibition potential, and iii) to determine inhibition potential (IC50) of the top three potent KIs in high and low UGT1A1 expressing HLM samples.MethodsTargeted genotyping of liver tissue donors (N=445) was performed; and UGT1A1 protein abundance and activity in HLM samples were quantified using a validated proteomics method and using bilirubin or estradiol as probe substrates, respectively. The mono and di‐bilirubin glucuronides and estradiol 3‐β‐D‐glucuronide (E3βG) were monitored by LC‐MS/MS. Association analysis of UGT1A1 genotype with protein abundance or activity was performed. Screening of UGT1A1 inhibition by KIs (at 1 and 20 uM) was performed using a high UGT1A1 expressing (46.7 ± 4.6 pmol/mg) HLM sample. IC50 values of KIs for UGT1A1 were determined using above mentioned high and a low (10.1 ± 1.6 pmol/mg) expressing HLM samples.ResultsUGT1A1 abundance and activity exhibited age‐dependent maturation with 8‐fold increase from neonates to adults. UGT1A1 SNPs rs10929302, rs887829, rs111741722 and rs4124874, are associated with decreased UGT1A1 abundance and activity, whereas rs8175347 was only associated with decreased abundance. Glucuronidation of both substrates was inhibited by the KIs in a substrate‐independent manner in the following order (% inhibition of E3βG activity at 1 and 20 μM in parenthesis): dasatanib (46 and 91)> sorafenib (11 and 74)> temsirolimus (50 and 71)> sirolimus (0 and 63) > sunitinib (21 and 38) > nilotinib (22 and 34) > gefitinib (0 and 29) > pazopanib = imatinib = lapatinib (0). The top three KIs showed differential IC50 values for E3βG inhibition in high and low UGT1A1 expressers, i.e., dasatanib (2.0, and 4.0), sirolimus (IC50= 7.6, and 32.6), and sorafenib (0.95, and 18.0), respectively.DiscussionUGT1A1 abundance is highly variable with significant effect of age and genotype. Inter‐individual variability in UGT1A1 plays a confounding role in predicting drug‐endobiotic or drug‐drug interactions (DDIs) mediated by this enzyme. Identification of genetic or non‐genetic factors associated with UGT1A1 activity will allow better prediction of UGT1A1 mediated drug metabolism and toxicity.Support or Funding InformationThis work was primarily supported by the National Institutes of Health [Grant no. R01 HD081299]. NA and AB contributed equally to this work. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibitory Effect of Sauchinone on UDP-Glucuronosyltransferase (UGT) 2B7 Activity

Herb–drug interaction (HDI) limits clinical application of herbs and drugs, and inhibition of herbs towards uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) has gained attention as one of the important reasons to cause HDIs. Sauchinone, an active lignan isolated from aerial parts of Saururus chinensis (Saururacease), possesses anti-oxidant, anti-inflammatory, and anti-viral activities. In pharmacokinetics of sauchinone, sauchinone is highly distributed to the liver, forming extensive metabolites of sauchinone via UGTs in the liver. Thus, we investigated whether sauchinone inhibited UGTs to explore potential of sauchinone–drug interactions. In human liver microsomes (HLMs), sauchinone inhibited activities of UGT1A1, 1A3, 1A6, and 2B7 with IC50 values of 8.83, 43.9, 0.758, and 0.279 μM, respectively. Sauchinone also noncompetitively inhibited UGT1A6 and 2B7 with Ki values of 1.08 and 0.524 μM, respectively. In in vivo interaction study using mice, sauchinone inhibited UGT2B7-mediated zidovudine metabolism, resulting in increased systemic exposure of zidovudine when sauchinone and zidovudine were co-administered together. Our results indicated that there is potential HDI between sauchinone and drugs undergoing UGT2B7-mediated metabolism, possibly contributing to the safe use of sauchinone and drug combinations.

Inhibition of UDP-glucuronosyltransferases (UGTs) by phthalate monoesters

Phthalate monoesters are important metabolites of phthalate esters (PAEs) which have been extensively utilized in industry. This study aims to investigate the inhibition of phthalate monoesters on the activity of various isoforms of UDP-glucuronosyltransferases (UGTs), trying to elucidate the toxicity mechanism of environmental endocrine disruptors from the new perspectives. In vitro recombinant UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU) was employed to evaluate 8 kinds of phthalate monoesters on 11 sorts of main human UGT isoforms. 100 μM phthalate monoesters exhibited negligible inhibition towards the activity of UGT1A1, UGT1A3, UGT1A6, UGT1A8, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17. The activity of UGT1A7 was strongly inhibited by monoethylhexyl phthalate (MEHP), but slightly inhibited by all the other phthalate monoesters. UGT1A9 was broadly inhibited by monobenzyl phthalate (MBZP), monocyclohexyl phthalate (MCHP), MEHP, monohexyl phthalate (MHP) and monooctyl phthalate (MOP), respectively. MEHP exhibited competitive inhibition towards UGT1A7, and MBZP, MCHP, MEHP, MHP and MOP showed competitive inhibition towards UGT1A9. The inhibition kinetic parameters (Ki) were calculated to be 11.25 μM for MEHP-UGT1A7, and 2.13, 0.09, 1.17, 7.47, 0.16 μM for MBZP-UGT1A9, MCHP-UGT1A9, MEHP-UGT1A9, MHP-UGT1A9, MOP-UGT1A9, respectively. Molecular docking indicated that both hydrogen bonds formation and hydrophobic interactions significantly contributed to the interaction between phthalate monoesters and UGT isoforms. All these information will be beneficial for understanding the adverse effects of PAEs.

Development of a novel method to determine UGT1A1 and MRP2 activities in primary hepatocytes using bilirubin as substrate

Development of a novel method to determine UGT1A1 and MRP2 activities in primary hepatocytes using bilirubin as substrate

Impact of edaphic factors and nutrient management on the hepatoprotective efficiency of Carlinoside purified from pigeon pea leaves: An evaluation of UGT1A1 activity in hepatitis induced organelles

Carlinoside is a unique compound well-known for its excellent curative potential in hepatitis. There is a substantial research gap regarding the medicinal use of carlinoside, as its concentrations are greatly variable (depending on locality). We cultivated Cajanus cajan using vermicompost as a major organic amendment at two locations (Sonitpur and Birbhum) with different soil types, but identical climate conditions. Sonitpur soils were richer in soil organic C (SOC), enzyme activation, and N/P content than Birbhum. However, vermi-treatment improved many soil properties (bulk density, water retention, pH, N/P/K, and enzyme activity) to narrow the locational gap in soil quality by 15–28%. We also recorded a many-fold increment in SOC storage capacities in both locations, which was significantly correlated with carlinoside, total phenol, and flavonoid contents in Cajanus leaves. This significantly up-regulated the carlinoside induced expression of the bilirubin-solubilizing UGT1A1enzyme in HepG2 cell and rat liver. Leaf extracts of vermicompost-aided plants could cure hepatitis in affected rat livers and in the HepG2 cell line. Accordingly, vermi-treatment is an effective route for the growth of Cajanus as a cash crop for biomedical applications and can produce a concurrent improvement in soil quality.

Development of a LC–MS/MS method to investigate the interference of pharmacokinetics of the main constituents in Saxifraga stolonifera: Involvement of drug metabolism enzymes

A specific and sensitive LC–MS/MS method was established for the simultaneous determination of bergenin, protocatechuic acid and gallic acid, the main active constituents of Saxifraga stolonifera (L.) Meerb. herb, in rat plasma. After fully validated, the method was applied to the comparative pharmacokinetic studies of the three compounds orally administered alone and in combination in the S. stolonifera extract, respectively. The results showed that the pharmacokinetic parameters, including Cmax, Tmax, AUC, CLz/F, MRT0-∞, were significantly different for both bergenin and protocatechuic acid in the extract as compared to the corresponding compounds administered alone. However, the pharmacokinetic behavior of gallic acid in the extract did not differ from that administered alone. Further studies found that quercetin, coexisting in the herb extract, significantly decreased the glucuronidation of bergenin through inhibiting the activities of UGT1A1 and UGT1A3, and reduced the metabolism of protocatechuic acid by inhibiting the activity of catechol-O-methyltransferase. Quercetin and other flavonoids occurring in the S. stolonifera extract might increase the absorption and improve the bioavailability of bergenin and protocatechuic acid by slowing down the liver metabolism. The findings provide a good guidance for the development and clinical application of S. stolonifera.

Direct comparison of UDP-glucuronosyltransferase and cytochrome P450 activities in human liver microsomes, plated and suspended primary human hepatocytes from five liver donors

UDP-glucuronosyltransferases (UGTs) and cytochrome P450s (CYPs) are the major enzymes involved in hepatic metabolism of drugs. Hepatic drug metabolism is commonly investigated using human liver microsomes (HLM) or primary human hepatocytes (PHH). We describe the development of a sensitive assay to phenotype activities of six major hepatic UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7) in intact PHH by analysis of glucuronidation of selective probe substrates. The non-selective, general substrate 7-hydroxycoumarin was included for comparison. For each liver donor preparation (five donors) UGT activities in cryopreserved suspended and plated PHH were compared to HLM prepared from the same donors. Standard CYP reaction phenotyping of seven major isoforms was performed in parallel. For all donors, CYP- and UGT-isoforms activity profiles were comparable in PHH and HLM, indicating that reaction phenotyping with selective probe substrates in intact cells primarily reflects respective CYP or UGT activity. System-dependent effects on UGT and CYP isoform activity were still found. While UGT activity of UGT1A1 was equivalent in plated and suspended PHH, UGT1A3, UGT1A6 and UGT2B7 activity was higher in suspended PHH and UGT1A9 and UGT1A4 activity was higher in plated PHH. The well-known decrease in activity of most CYP isoforms in plated compared to suspended PHH was confirmed. Importantly, we found a significant loss in CYP2C19 and CYP2B6 in HLM, activity being lower than in intact cells. Taken together, these findings implicate that, dependent on the UGT or CYP isoforms involved in the metabolism of a given compound, the outcome of metabolic assays is strongly dependent on the choice of the in vitro system. The currently described UGT- and CYP- activity profiling method can be used as a standard assay in intact cells and can especially aid in reaction phenotyping of in vitro systems for which a limited number of cells are available.

The inhibition of UDP-glucuronosyltransferases (UGTs) by tetraiodothyronine (T4) and triiodothyronine (T3)

1. UDP-glucuronosyltransferases (UGTs) are important drug-metabolizing enzymes (DMEs) catalyzing the glucuronidation elimination of various xenobiotics and endogenous substances. Endogenous substances are important regulators for the activity of various UGT isoforms. Triiodothyronine (T3) and thyroxine (T4) are important thyroid hormones essential for normal cellular differentiation and growth. The present study aims to elucidate the inhibition behavior of T3 and T4 on the activity of UGT isoforms.2. In vitro recombinant UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU) was used to screen the inhibition potential of T3 and T4 on the activity of various UGT isoforms. Initial screening results showed that T4 exerted stronger inhibition potential than T3 on the activity of various UGT isoforms at 100 μM. Inhibition kinetics was determined for the inhibition of T4 on the representative UGT isoforms, including UGT1A1, -1A3, -1A7, -1A8, -1A10 and -2B7. The results showed that T4 competitively inhibited the activity of UGT1A1, -1A3, -1A7, 1A10 and -2B7, and noncompetitively inhibited the activity of UGT1A8. The inhibition kinetic parameters were calculated to be 1.5, 2.4, 11, 9.6, 4.8 and 3.0 μM for UGT1A1, -1A3, -1A7, -1A8, -1A10 and -2B7, respectively. In silico docking method was employed to demonstrate why T4 exerted stronger inhibition than T3 towards UGT1A1. Stronger hydrogen bonds and hydrophobic interaction between T4 and activity cavity of UGT1A1 than T3 contributed to stronger inhibition of T4 towards UGT1A1.3. In conclusion, more clinical monitoring should be given for the patients with the elevation of T4 level due to stronger inhibition of UGT isoforms-catalyzed metabolism of drugs or endogenous substances by T4.

Identification and validation of microRNAs directly regulating the UDP-glucuronosyltransferase 1A subfamily enzymes by a functional genomics approach

Posttranscriptional repression of UDP-glucuronosyltransferase (UGT) 1A expression by microRNAs (miRNAs) may be an important mechanism underlying interindividual variability in drug glucuronidation. Furthermore, the UGT1A 3′-UTR shared by all UGT1A enzymes is polymorphic, containing three linked SNPs (rs10929303, rs1042640, and rs8330) that could influence miRNA binding. The aim of this study was to identify the complete complement of miRNAs that could regulate UGT1A expression through binding to the reference and/or common variant UGT1A 3′-UTR. Luciferase reporter plasmids containing either the reference or variant UGT1A 3′-UTR were screened against a 2048 human miRNA library to identify those miRNAs that decrease luciferase activity by at least 30% when co-transfected into HEK293 cells. Four novel miRNAs (miR-103b, miR-141-3p, miR-200a-3p, and miR-376b-3p) were identified that repressed both reference and variant UGT1A 3′-UTR, while two other miRNAs selectively repressed the reference (miR-1286) or variant (miR-21-3p) 3′-UTR. Deletion and mutagenesis studies confirmed the binding site location for each miRNA. rs8330 disrupted miR-1286 binding to the reference UGT1A 3′-UTR, while rs10929303 enhanced miR-21-3p binding to the variant 3′-UTR. Transfection of miR-21-3p, miR-103b, miR-141-3p, miR-200a-3p, and miR-376b-3p mimics into LS180 human intestinal cells showed repression of UGT1A1 and UGT1A6 mediated glucuronidation and mRNA without affecting UGT2B7 activity or mRNA. Furthermore, transfection of miR-21-3p, miR-141-3p, and miR-200a-3p into primary human hepatocytes, repressed UGT1A1 activity and mRNA without affecting CYP3A activity. Finally, miR-21-3p and miR-200a-3p expression were negatively correlated with UGT1A6 activity and mRNA in human liver samples. Thus, UGT1A is regulated by multiple miRNAs with some showing allele-dependent effects.

AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5'-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes.

AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP) or uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) enzymes in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 µM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 µM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

Frequency of the UGT1A1*28 Polymorphism in a Romanian Cohort of Gilbert Syndrome Individuals.

Gilbert syndrome (GS) is characterized by unconjugated hyperbilirubinemia without liver disease or overt hemolysis and it is found in 3-10% of the general population. Inherited hyperbilirubinaemia is attributable to a reduced UGT1A1 activity. The UGT1A1 promoter (TA) repeats variants are documented of being involved in abnormally elevated bilirubin levels. The aim of the present study is to analyze the impact of UGT1A1 promoter variants on bilirubin levels in Romanian patients clinically supected with GS. The study group included 897 subjects: 292 GS patients and 605 healthy controls. Genomic DNA was extracted from the peripheral blood leukocytes. All individuals were screened for the presence of the (TA) insertion in the TATA box region of UGT1A1 gene by PCR amplification. This case-control study was conducted at the Department of Medical Genetics, Synevo, Romania. UGT1A1*28 (7TA) revealed the highest frequency (61.87%) of all individuals, while the UGT1A1*1 (6TA) allele was found in 36.79%. We identified two other variants of the UGT1A1 gene, depending on the number of TA repeats in the promoter: 5TA (0.61%) and 8TA (0.72%). The (TA)7/7 homozygous genotype was identified in 32.33% of all individuals, while the (TA)6/7 heterozygous genotype was the most prevalent (57.64%). The wild type (TA)6/6 was identified in 7.36% of the whole cohort. Because other polymorphisms have been associated with GS, the absence of the UGT1A1*28 allele does not rule out this condition. The results suggest that in the Romanian population there is a strong correlation between the UGT1A1*28 polymorphism and hyperbilirubinemia in patients with GS.

Study on the association of UGT1A9 gene c.98T>C polymorphism and mycophenolic acid plasma levels in renal transplant patients.

Mycophenolate mofetil (MMF) is a prodrug active only after its hydrolysis to mycophenolic acid (MPA). The UGT1A9 enzyme is of special interest since it is the main enzyme involved in the glucuronidation of MPA. Single nucleotide polymorphisms (SNPs) in the UGT1A9 gene may be responsible for individual differences in the pharmacokinetics of MMF. Expression levels and the activity of UGT1A9 may depend on the presence of some SNPs located in the gene promoter region (-2152C>T and -275T>A), as well as changes in the coding region (c.98T>C). The objective of this study was to evaluate the effect of allelic variants of the UGT1A9 c.98T>C polymorphism (rs72551330; g. 87289T>C) on MMF metabolism in renal transplant patients. MPA and MPA 7-O glucuronide (MPAG) levels were determined on plasma samples of kidney transplant patients (N = 39) by high-performance liquid chromatography using ultraviolet detection. DNA was isolated from leukocytes and stored at -20°C. The presence of SNPs was investigated using polymerase chain reaction, followed by amplicon sequencing. The analysis of the UGT1A9 c.98T>C polymorphism revealed that all study patients presented the TT genotype. Diverse MPA and MPAG plasma concentrations were detected, including therapeutic, subtherapeutic, and toxic levels. A standardized molecular method permitted identification of UGT1A9 c.98T>C polymorphism genotypes in the examined renal transplant patients. All individuals of the study group presented the same genotype (c.98TT) for that polymorphism. Thereby, no association between the c.98T>C polymorphism and MPA and MPAG plasma levels could be evaluated, despite different levels of these compounds being observed.

Isochlorogenic acid A affects P450 and UGT enzymes in vitro and in vivo

Isochlorogenic acid A (ICQA), which has anti-inflammatory, hepatoprotective, and antiviral properties, is commonly presented in fruits, vegetables, coffee, plant-based food products, and herbal medicines. These herbal medicines are usually used in combination with other medicines in the clinic. However, little is known about the regulatory effects of ICQA on drug-metabolizing enzymes and the herb-drug interactions. In the present study, we evaluated the inhibitory potentials of ICQA on CYP1A2, CYP2C9, CYP2C19, CYP3A4, CYP2D6, and CYP2E1 in vitro based on a cocktail approach. The P450 and UGT activities in mice treated with ICQA for a prolonged period were also determined. Our results demonstrated that ICQA exhibited a weak inhibitory effect on CYP2C9 in human liver microsomes with IC50 being 57.25 μmol·L−1 and Ki being 26.77 μmol·L−1. In addition, ICQA inhibited UGT1A6 activity by 25%, in the mice treated with ICQA (i.p.) at 30 mg·kg−1 for 14 d, compared with the control group. Moreover, ICQA showed no mechanism-based inhibition on CYP2C9 or UGT1A6. In conclusion, our results further confirm a safe use of ICQA in clinical practice.

In vitro inhibition of UGT1A3, UGT1A4 by ursolic acid and oleanolic acid and drug–drug interaction risk prediction

1. Ursolic acid (UA) and oleanolic acid (OA) may have important activity relevant to health and disease prevention. Thus, we studied the activity of UA and OA on UDP-glucuronosyltransferases (UGTs) and used trifluoperazine as a probe substrate to test UGT1A4 activity. Recombinant UGT-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as a probe reaction for other UGT isoforms.2. UA and OA inhibited UGT1A3 and UGT1A4 activity but did not inhibit other tested UGT isoforms.3. UA-mediated inhibition of UGT1A3 catalyzed 4-MU-β-d-glucuronidation was via competitive inhibition (IC50 0.391 ± 0.013 μM; Ki 0.185 ± 0.015 μM). UA also competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC50 2.651 ± 0.201 μM; Ki 1.334 ± 0.146 μM).4. OA offered mixed inhibition of UGT1A3-mediated 4-MU-β-d-glucuronidation (IC50 0.336 ± 0.013 μM; Ki 0.176 ± 0.007 μM) and competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC50 5.468 ± 0.697 μM; Ki 6.298 ± 0.891 μM).5. Co-administering OA or UA with drugs or products that are substrates of UGT1A3 or UGT1A4 may produce drug-mediated side effects.

Cyp3a11-mediated testosterone-6β-hydroxylation decreased, while UGT1a9-mediated propofol O-glucuronidation increased, in mice with diabetes mellitus.

The db/db mouse is one of the most popular animal models for type 2 diabetes mellitus, but changes in the activities of important P450s and UGTs are still not completely clear. This study was designed to investigate the alterations of major hepatic cytochrome P450s and UDP-glucuronyltransferase enzymes in db/db mice. Mouse liver microsomes (MLMs) were obtained from male db/db mice and their wild type littermates. After incubation of the substrates separately with MLMs, the samples were pooled and analysed by high-throughput liquid chromatography-tandem mass spectrometry system for the simultaneous study of nine phase I metabolic reactions and three glucuronidation conjugation reactions to determine the activity of the metabolic enzymes. Compared with normal controls, the Clint estimate for testosterone-6β-hydroxylation was lower (46%) (p<0.05), while the Vmax and Clint estimates for propofol O-glucuronidation were 5-fold higher (p<0.01) in the liver microsomes from db/db mice. There was no significant difference in phase I metabolic reactions of phenacetin-O-deethylation, coumarin-7-hydroxylation, bupropion-hydroxylation, omeprazole-5-hydroxylation, dextromethorphan-O-demethylation, tolbutamide-4-hydroxylation, chlorzoxazone-6-hydroxylation and midazolam-1-hydroxylation and in glucuronidation reactions of estradiol 3-O-glucuronidation, and 3-azido-3-deoxythymidine glucuronidation. The data suggest that, in db/db mice, the activity of Cyp3a11, catalysing testosterone-6β-hydroxylation, decreased, while the activity of UGT1a9, catalysing propofol O-glucuronidation, increased. Copyright © 2016 John Wiley & Sons, Ltd.

In vitro effects of Genkwa Flos chloroform extract on activity of human liver microsomes UGTs and UGT1A1

To predict the mechanism of liver injury induced by Genkwa Flos, we investigated the effect of chloroform extract on UGTs and UGT1A1 activities of the liver microsomes in rat and human. In the present study, 4-nitrophenol(4-NP) and β-estradiol were elected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC. The results showed that there were 1.00% of apigenin, 6.40% of hydroxygenkwanin and 18.38% of genkwanin in chloroform extract; and total diterpene mass fraction was 31.40%. Compared with the control group, chloroform extract could significantly inhibit the activity of UGTs in rat liver microsomes(RLM) system, while the inhibitory effect was not obvious in human liver microsomes(HLM) system. UGT1A1 activity was inhibited by chloroform extract in rat liver microsomes and human liver microsomes (based on genkwanin, IC₅₀=8.76, 10.36 μmol•L⁻¹). The inhibition types were non-competitive inhibition(RLM) and uncompetitive inhibition(HLM). In conclusion, the results indicated that chloroform extract showed different inhibitory effects on UGTs and UGT1A1 activity, which may be one of the mechanisms of liver injury induced by Genkwa Flos.