UDP-glucuronosyltransferase 1A1 Research Articles

Bictegravir, a novel integrase inhibitor for the treatment of HIV infection.

Bictegravir (BIC), a second-generation integrase strand transfer inhibitor (INSTI) approved for HIV treatment in fixed-dose combination with emtricitabine and tenofovir alafenamide, has potent antiviral activity in vitro to wild-type virus and strains with resistance to first-generation INSTIs. As part of combination therapy, BIC's virologic suppression rates in clinical trials are comparable to those of first-line combination antiretroviral drug regimens. BIC has demonstrated a high genetic barrier to resistance development in vitro, can be administered with or without food, and has a bioavailability of > 70%. A median plasma half-life of 18 hours allows once-daily dosing. Clearance is primarily hepatic through cytochrome P450 3A4 (CYP3A4) oxidation and UDP-glucuronosyltransferase 1A1 (UGT1A1) glucuronidation. Thus, potent inducers of UGT1A1 and CYP3A4 (e.g., rifamycins/anticonvulsants) should be avoided due to significantly decreased BIC serum exposure. Chelation with polyvalent cations can decrease absorption; otherwise, drug-drug interactions are few. BIC is well tolerated; diarrhea, nausea and headache are the main adverse effects associated with its use.

Inhibition of UGT1A1 by natural and synthetic flavonoids

Flavonoids are widely distributed phytochemicals in vegetables, fruits and medicinal plants. Recent studies demonstrate that some natural flavonoids are potent inhibitors of the human UDP-glucuronosyltransferase 1A1 (UGT1A1), a key enzyme in detoxification of endogenous harmful compounds such as bilirubin. In this study, the inhibitory effects of 56 natural and synthetic flavonoids on UGT1A1 were assayed, while the structure–inhibition relationships of flavonoids as UGT1A1 inhibitors were investigated. The results demonstrated that the C-3 and C-7 hydroxyl groups on the flavone skeleton would enhance UGT1A1 inhibition, while flavonoid glycosides displayed weaker inhibitory effects than their corresponding aglycones. Further investigation on inhibition kinetics of two strong flavonoid-type UGT1A1 inhibitors, acacetin and kaempferol, yielded interesting results. Both flavonoids were competitive inhibitors against UGT1A1-mediated NHPN-O-glucuronidation, but were mixed and competitive inhibitors toward UGT1A1-mediated NCHN-O-glucuronidation, respectively. Furthermore, docking simulations showed that the binding areas of NHPN, kaempferol and acacetin on UGT1A1 were highly overlapping, and convergence with the binding area of bilirubin within UGT1A1. In summary, detailed structure-inhibition relationships of flavonoids as UGT1A1 inhibitors were investigated carefully and the findings shed new light on the interactions between flavonoids and UGT1A1, and will contribute considerably to the development of flavonoid-type drugs without strong UGT1A1 inhibition.

Impact of UGT1A1 genetic polymorphism on toxicity in unresectable pancreatic cancer patients undergoing FOLFIRINOX.

Studies have indicated an association between UDP‐glucuronosyltransferase‐1A1 (UGT1A1) genetic polymorphisms and irinotecan‐induced toxicity. We undertook this study to investigate the association between UGT1A1 genetic polymorphisms and toxicity in patients treated with the FOLFIRINOX (comprising oxaliplatin, irinotecan, fluorouracil, and leucovorin) chemotherapy regimen in the JASPAC 06 study. Patients screened for UGT1A1*6 and UGT1A1*28, and treated with either the original FOLFIRINOX (oxaliplatin 85 mg/m2, irinotecan 180 mg/m2, leucovorin 200 mg/m2, bolus 5‐fluorouracil [5‐FU] 400 mg/m2, and continuous 5‐FU 2400 mg/m2) or a modified FOLFIRINOX (oxaliplatin 85 mg/m2, irinotecan 150 mg/m2, leucovorin 200 mg/m2, and continuous 5‐FU 2400 mg/m2) as first‐line chemotherapy were included. Of 199 patients eligible for this analysis, 79 patients were treated with the original FOLFIRINOX regimen and 120 patients were treated with the modified FOLFIRINOX regimen. In the original FOLFIRINOX group, 54 were UGT1A1 WT, and 25 were UGT1A1 heterozygous type (−/*6, 12 patients; −/*28, 13 patients). In the modified FOLFIRINOX group, 64 were UGT1A1 WT and 56 were UGT1A1 heterozygous type (−/*6, 33 patients; −/*28, 23 patients).In the original FOLFIRINOX group, the incidence of diarrhea was significantly higher among patients with UGT1A1 heterozygous type than among those with UGT1A1 WT and the incidence of leukopenia and diarrhea was significantly higher among patients with UGT1A1 −/*6 than among those with UGT1A1 −/*28. Patients with UGT1A1 heterozygous type, especially those with UGT1A1 −/*6, tended to show a higher incidence rate of severe adverse events, but this was not statistically significant. However, for patients who received the modified FOLFIRINOX, there was no difference in the frequency of adverse events due to UGT1A1 status. In conclusion, patients with heterozygous UGT1A1 polymorphisms treated with the original FOLFIRINOX regimen experienced severe toxicity more frequently than patients with WT UGT1A1.

Plasma extracellular nanovesicle (exosome)-derived biomarkers for drug metabolism pathways: a novel approach to characterize variability in drug exposure.

Demonstrate the presence of cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) proteins and mRNAs in isolated human plasma exosomes and evaluate the capacity for exosome-derived biomarkers to characterize variability in CYP3A4 activity. The presence of CYP and UGT protein and mRNA in exosomes isolated from human plasma and HepaRG cell culture medium was determined by mass spectrometry and reverse transcription-polymerase chain reaction, respectively. The concordance between exosome-derived CYP3A4 biomarkers and midazolam apparent oral clearance (CL/F) was evaluated in a small proof-of-concept study involving six genotyped (CYP3A4 *1/*1 and CYP3A5 *3/*3) Caucasian males. Exosomes isolated from human plasma contained peptides and mRNA originating from CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4 and 3A5, UGT 1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10 and 2B15, and NADPH-cytochrome P450 reductase. Mean (95% confidence interval) exosome-derived CYP3A4 protein expression pre- and post-rifampicin dosing was 0.24 (0.2-0.28) and 0.42 (0.21-0.65) ngml-1 exosome concentrate. Mean (95% confidence interval) exosome CYP3A4 mRNA expression pre- and post-rifampicin dosing was 6.0 (1.1-32.7) and 48.3 (11.3-104) × 10-11 2-ΔΔCt , respectively. R2 values for correlations of exosome-derived CYP3A4 protein expression, CYP3A4 mRNA expression, and ex vivo CYP3A4 activity with midazolam CL/F were 0.905, 0.787 and 0.832, respectively. Consistent strong concordance was observed between exosome-derived CYP3A4 biomarkers and midazolam CL/F. The significance of these results is that CYP3A4 is the drug-metabolizing enzyme of greatest clinical importance and variability in CYP3A4 activity is poorly described by existing precision dosing strategies.

Cell Cycle and Apoptosis Regulator 1, CCAR1, Regulates Enhancer-Dependent Nuclear Receptor CAR Transactivation.

The constitutive active/androstane receptor (CAR) controls genes involved in xenochemical metabolism. Although numerous cofactors have been reported to be involved in CAR-mediated transactivation, unknown and poorly defined proteins recruited by CAR have yet to be characterized. In this study, a novel CAR-interacting protein, cell cycle and apoptosis regulator 1 (CCAR1), was identified by coimmunoprecipitation analysis using human hepatocarcinoma HepG2 cells expressing FLAG epitope-tagged CAR. We demonstrated that CCAR1 can act as an enhancer-dependent coactivator of CAR. First, we showed that overexpression of CCAR1 enhanced CAR-induced reporter gene activity with triplicate consensus direct repeat 4 motif (DR4-Luc), xenobiotic-responsive enhancer module (XREM)-enhancer of CYP3A4 (XREM-Luc), and phenobarbital-responsive enhancer module of UDP-glucuronosyltransferases 1A1 (UGT1A1) (gtPBREM)-enhancer of UGT1A1 (gtPBREM-Luc)-driven reporter plasmids but not PBREM-enhancer of CYP2B6 (PBREM-Luc)-driven reporter activity. Furthermore, we showed that knockdown of CCAR1 suppressed CAR-induced UGT1A1 mRNA expression but did not affect CAR-induced CYP2B6 mRNA expression in HepTR/CAR and HepaRG cells. Moreover, CCAR1 could be recruited to the gtPBREM of the UGT1A1 enhancer by CAR but not to the PBREM of the CYP2B6 enhancer. Moreover, we showed that CCAR1 can act as a secondary coactivator by cooperating with the p160 family of steroid receptor coactivators (SRCs). These findings demonstrated CCAR1 to be a novel transcriptional cofactor for CAR and provided insight regarding the mechanism of CAR-mediated gene-selective transactivation.

UGT1A1 polymorphisms in rectal cancer associated with the efficacy and toxicity of preoperative chemoradiotherapy using irinotecan.

The purpose of the present study was to assess the efficacy and toxicity of preoperative chemoradiotherapy using irinotecan against locally advanced lower rectal cancer according to UDP‐glucuronosyltransferase 1A1 (UGT1A1) polymorphisms. Between 2009 and 2016, 46 patients with resectable rectal cancer (T3‐T4, N0‐N2, M0) received preoperative chemoradiotherapy consisting of 80 mg/m2 per day tegafur/gimeracil/oteracil (S‐1; days 1‐5, 8‐12, 22‐26, and 29‐33), 60 mg/m2 per day irinotecan (days 1, 8, 22, and 29), and 45 Gy radiation (1.8 Gy/day, 5 days per week for 5 weeks). Six to eight weeks after completing chemoradiotherapy, total mesorectal excision was carried out. Patients with UGT1A1 polymorphisms were divided into WT (n = 26), heterozygous (n = 15), and homozygous (n = 5) groups, the latter including double heterozygosities. We evaluated associations between clinical characteristics, including UGT1A1 polymorphisms, and chemoradiotherapy efficacy and toxicity. Incidence rates of grade 3+ neutropenia and diarrhea were 17.0% and 30.4%, respectively. Relative dose intensity was 89.3%. Pathological complete response rate (grade 3) was 26.1%, and the good response (grade 2/3) rate was 84.8%. UGT1A1 polymorphisms were significantly associated with neutropenia and pathological good responses, but not with diarrhea. UGT1A1 polymorphism was the only predictive factor for pathological good responses. Our results indicate that UGT1A1 polymorphism is a predictive factor to determine the clinical efficacy of preoperative chemoradiotherapy and hematological toxicity induced by chemoradiotherapy using irinotecan in locally advanced rectal cancer patients.

Bilirubin, a Cardiometabolic Signaling Molecule

Mostly considered as a marker for hepatic disease, bilirubin, has been revealed as a cardio and metabolic protective factor that has implications for therapy. Reports of bilirubin date back to ancient Greek medicine with Hippocrates and the four humors of human health, which metabolic agents included Choleric-yellow bile, Melancholic-black bile, Sanguine-blood, and Phlegmatic-phlegm. The yellow bile was associated with the gallbladder and black bile the spleen. These intricacies are still under consideration today. Serum levels of bilirubin are thought to be derived from the breakdown of red blood cells that mostly occurs in the spleen. Bilirubin is a naturally occurring product from the catabolism of heme by heme oxygenase (HO), which releases biliverdin, carbon monoxide (CO), and iron1, 2. Then, biliverdin is reduced to bilirubin via biliverdin reductase (BVR). Bilirubin is released into the blood and binds albumin where it travels to other organs or is deposited in the liver and conjugated with glucuronic acid by the UDP-glucuronosyltransferase 1A1 (UGT1A1) enzyme 3, 4 Conjugated bilirubin (CBR) is excreted from the liver to bile in the gallbladder and eventually released into the intestine where it is broken down by the gut microbiota into urobilinoids, mainly stercobilin, which is the predominant pigment in feces 3. High blood levels of unconjugated bilirubin (UCB) is most widely known as the causative agent of jaundice, a yellowing of the skin and eyes. Neonatal jaundice has been described in the medical literature since the late eighteenth century and is a condition reflective of very high levels of UCB in the serum of newborn infants. Severe cases of neonatal jaundice have resulted in a bilirubin-induced neurological condition in newborns known as kernicterus, which can cause brain damage. Despite its role in jaundice, UCB has several beneficial effects on the body 5. The results of numerous large-scale population and epidemiological studies have correlated a protective effect of serum bilirubin levels against cardiovascular and metabolic disease. However, the molecular mechanism by which this occurs is not fully understood. The objective of this review is to highlight and discuss the unique signaling actions of BVR generation of bilirubin and how they signal to protect against cardiovascular and metabolic disease, which may lead to new therapeutics.

Effect of Celecoxib on Differentiation of Human Induced Pluripotent Stem Cells into Hepatocytes Involves STAT5 Activation.

The liver abundantly expresses various drug-metabolizing enzymes and, thus, plays a central role in drug metabolism. In this regard, cytochrome P450 (CYP) is responsible for drug metabolism in the liver. Therefore, since CYP3A4 accounts for approximately 30% of the CYPs, the prediction of hepatic CYP3A4-mediated pharmacokinetics is essential for drug development. Human induced pluripotent stem cell-derived hepatocytes (hiHep) have become a major model of drug metabolism in drug development studies. However, drug metabolizing activities, such as those involving CYP3A4, are lower in hiHep than in human primary hepatocytes (HPHs). Recently, it was revealed that celecoxib upregulates the expression of CYPs to normal levels through the activation of signal transducer and transcriptional activation factor 5 (STAT5). Therefore, we investigated whether celecoxib treatment could normalize the low drug metabolism activities in hiHep. The mRNA expression levels of hepatic markers [asialoglycoprotein receptor 1 (ASGR1) and tyrosine aminotransferase (TAT)] and metabolic enzymes (UDP-glucuronosyltransferase 1A1 and CYP3A4) in hiHep significantly increased after celecoxib treatment. These mRNA expression levels were 7-, 1/3-, 1/2-, and 1/10-fold of the HPHs cultured for 48 hours, respectively. Furthermore, CYP3A4 activity significantly increased. To investigate the mechanism of CYP3A4 mRNA upregulation, we analyzed the phosphorylation of STAT5 after celecoxib treatment and found it to be significantly increased. Moreover, the increase in CYP3A4 mRNA expression was attenuated by cotreatment with STAT5 inhibitor. These results suggest that celecoxib promotes hepatocyte differentiation of hiHep by activating STAT5 and is useful for the generation of functional hiHep.

Oligomerization and Catalytic Parameters of Human UDP-Glucuronosyltransferase 1A10: Expression and Characterization of the Recombinant Protein.

UDP-glucuronosyltransferase (UGT), as an integral membrane protein localized in the endoplasmic reticulum, has the ability to detoxify potentially hazardous xenobiotic substances. Most UGTs are expressed in liver, but UGT1A10 has proven to be an extrahepatic enzyme considerably expressed throughout the gastrointestinal tract. Earlier studies indicated that different UGT isoforms could exist in higher-order homo-oligomers or at least dimers within the membrane, but the formation of intermolecular disulfide bridges between UGT molecules was not often observed. In this study, we expressed recombinant human UGT1A10 in human embryonic kidney (HEK)293 and Chinese hamster ovary (CHO) cells to examine its oligomeric states and characterize its enzymatic activities against two therapeutically interesting substrates, morphine and entacapone, including determination of the catalytic rate constant (kcat) values for the first time. It was observed that a majority of the UGT1A10 protein expressed in HEK293 cells existed in covalently crosslinked higher-order oligomers via formation of intermolecular disulfide bonds, whereas formation of the intermolecular disulfide bonds was not observed in the UGT1A10 protein expressed in CHO cells. Owing to the formation of the covalently crosslinked higher-order oligomers, the UGT1A10 protein expressed in HEK293 cells had much lower catalytic activity (particularly the catalytic rate constant kcat) against both morphine and entacapone, compared with the UGT1A10 protein form expressed in CHO cells against the same substrates.

Correction to "Metabolism and Disposition of a Novel Selective α7 Neuronal Acetylcholine Receptor Agonist ABT-126 in Humans: Characterization of the Major Roles for Flavin-Containing Monooxygenases and UDP-Glucuronosyl Transferase 1A4 and 2B10 in Catalysis".

Correction to "Metabolism and Disposition of a Novel Selective α7 Neuronal Acetylcholine Receptor Agonist ABT-126 in Humans: Characterization of the Major Roles for Flavin-Containing Monooxygenases and UDP-Glucuronosyl Transferase 1A4 and 2B10 in Catalysis".

Effect of sex and polymorphisms of CYP2B6 and UGT1A9 on the difference between the target-controlled infusion predicted and measured plasma propofol concentration

IntroductionTo examine whether sex and polymorphisms of cytochrome P450 (CYP) 2B6 and UDP-glucuronosyltransferase (UGT) 1A9 affect the difference between predicted and measured plasma propofol concentration during continuous infusion by target-controlled infusion.ResultsBlood samples of 69 patients (48 men and 21 women) were obtained at 4 h after initial propofol infusion. Percentage performance error (PE) was calculated to assess the difference between measured and predicted propofol concentration. Regression coefficients (β) and 95% confidence intervals (CI) of sex and the polymorphisms of CYP2B6 and UGT1A9 for PE were, separately and mutually, estimated with linear regression. Covariates included age and body mass index in the minimal adjusted model, and additionally included clinical factors (mean blood pressure, heart rate, volume of intravenous fluid, surgical site, surgical position, and pneumoperitoneum) in the full adjusted model. PE was higher in men than in women (28.7% versus 10.5%, p = 0.015). Female sex was inversely associated with PE: the minimal adjusted β = − 8.84 (95% CI, − 16.26 to − 1.43); however, the fully adjusted β with clinical factors became not significant. The average of PE did not differ between polymorphisms of CYP2B6 and UGT1A9, and β of CYP2B6 516G>T polymorphisms mutually adjusted with female sex was not significant. Mean blood pressure, heart rate, and volume of intravenous fluid were independently associated with PE in the full adjusted model.ConclusionsUnder 4 h anesthesia with propofol target-controlled infusion in our population, sex differences appeared to exist in the propofol concentration, which might be largely mediated by clinical factors, such as hemodynamic status.Trial registrationUMIN-CTR UMIN000009015, Registered 1 October 2012

Humanized UGT1 Mice, Regulation of UGT1A1, and the Role of the Intestinal Tract in Neonatal Hyperbilirubinemia and Breast Milk-Induced Jaundice.

Neonatal hyperbilirubinemia and the onset of bilirubin encephalopathy and kernicterus result in part from delayed expression of UDP-glucuronosyltransferase 1A1 (UGT1A1) and the ability to metabolize bilirubin. It is generally believed that acute neonatal forms of hyperbilirubinemia develop due to an inability of hepatic UGT1A1 to metabolize efficiently bilirubin for clearance through the hepatobiliary tract. Newly developed mouse models designed to study bilirubin metabolism have led to new insight into the role of the intestinal tract in controlling neonatal hyperbilirubinemia. Humanization of mice with the UGT1 locus (hUGT1 mice) and the UGT1A1 gene provide a unique tool to study the onset of hyperbilirubinemia since the human UGT1A1 gene is developmentally regulated during the neonatal period in hUGT1 mice. A new mechanism outlying developmental expression of intestinal UGT1A1 is presented and its implications in the control of neonatal hyperbilirubinemia discussed. New findings linking breast milk protection against necrotizing enterocolitis and intestinal control of UGT1A1 may help explain the contribution of breast milk toward the development of neonatal hyperbilirubinemia. Our findings outline a new model that includes an active intestinal ROS /IκB kinase/nuclear receptor corepressor 1 loop that can be applied to an understanding of breast milk-induced jaundice.

Stereoselective glucuronidation metabolism, pharmacokinetics, anti-amnesic pharmacodynamics, and toxic properties of vasicine enantiomers in vitro and in vivo

Vasicine (VAS) is a potential natural cholinesterase inhibitor for treatment of Alzheimer's disease. Due to one chiral centre (C-3) presenting in molecule, VAS has two enantiomers, d-vasicine (d-VAS) and l-vasicine (l-VAS). The study was undertaken to investigate the stereoselective glucuronidation metabolism, pharmacokinetics, anti-amnesic effect and acute toxicity of VAS enantiomers. In results, the glucuronidation metabolic rate of l-VAS was faster than d-VAS in human liver microsomes and isoenzymes tests, and it was proved that the UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B15 were the major metabolic enzymes for glucuronidation of l-VAS, while only UGT1A9 for d-VAS, which take responsibility of the significantly less metabolic affinity of d-VAS than l-VAS in HLM and rhUGT1A9. The plasma exposure of d-VAS in rats was 1.3-fold and 1.6-fold higher than that of l-VAS after intravenous and oral administration of d-VAS and l-VAS, respectively. And the plasma exposure of the major glucuronidation metabolite d-VASG was one of tenth of l-VASG or more less, no matter by intravenous or oral administration. Both d-VAS and l-VAS were exhibited promising acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities, and the BChE inhibitory activity of d-VAS with IC50 of 0.03 ± 0.001 μM was significantly stronger than that of l-VAS with IC50 of 0.98 ± 0.19 μM. The molecular docking results indicated that d-VAS and l-VAS could bind to the catalytic active site (CAS position) either of human AChE and BChE, and the BChE combing ability of d-VAS (the score of GBI/WAS dG −7.398) was stronger than that of l-VAS (the score of GBI/WAS dG −7.135). Both d-VAS and l-VAS could improving the learning and memory on scopolamine-induced memory deficits in mice. The content of acetylcholine (ACh) after oral administration d-VAS increased more than that of l-VAS in mice cortex, through inhibiting cholinesterase (ChE) and increasing choline acetyltransferase (ChAT). In addition, the LD50 value of d-VAS (282.51 mg·kg−1) was slight lower than l-VAS (319.75 mg·kg−1). These results indicated that VAS enantiomers displayed significantly stereoselective metabolic, pharmacokinetics, anti-amnesic effect and toxic properties in vitro and in vivo. The d-VAS might be the dominant configuration for treating Alzheimer's disease.

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.

Resveratrol represses estrogen-induced mammary carcinogenesis through NRF2-UGT1A8-estrogen metabolic axis activation

Estrogen plays a pivotal role in the pathological development of breast cancer. Resveratrol has chemo-preventive effects against breast cancer, whereas, the mechanism of antitumor activities of resveratrol remains unanswered. In this study, we showed that estrogen homeostasis profile was disturbed in both breast cancer patients and in experimental breast cancer model rats, with carcinogenic catechol estrogens significantly accumulated in the mammary tissues. UDP-glucuronosyltransferase 1A8 (UGT1A8) is an important phase II drug-metabolizing enzymes which involved in the metabolism of catechol estrogens. Here we found that the mammary nuclear factor erythroid 2-related factor 2 (NRF2) – UGT1A8 signaling was down-regulated in breast cancer rats, whereas treatment with resveratrol could upregulate the expression of NRF2 and UGT1A8, accelerate metabolic elimination of catechol estrogens, inhibit estrogen-induced DNA damage and suppress the pathological development of breast cancer. In addition, luciferase reporter assay suggested that resveratrol activated the expression of UGT1A8 by up-regulating the transcriptional activity of NRF2. Small-interfering RNA-mediated silencing of NRF2 abolished resveratrol-mediated preventive effects indicated that the antitumor effect of resveratrol is based on NRF2-UGT1A8-estrogen metabolism axis. Taken together, we established the resveratrol regulating potential on estrogen homeostasis based on NRF2-UGT1A8 signaling pathway, and also provided a novel link between estrogen glucuronidation metabolism and breast cancer pathological development.

Age-related changes in hepatic expression and activity of drug metabolizing enzymes in male wild-type and breast cancer resistance protein knockout mice.

This study aimed to reveal age-related changes in the expression and activity of seven hepatic drug metabolizing enzymes (DMEs) in male wild-type and breast cancer resistance protein knockout (Bcrp1-/- ) FVB mice. The protein expression of four cytochrome P450 (Cyps) (Cyp3a11, 2d22, 2e1, and 1a2), and three UDP-glucuronosyltransferases (Ugts) (Ugt1a1, 1a6a, and 1a9) in liver microsomes of wild-type and Bcrp1-/- FVB mice at different ages were determined using a validated ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method. The activities and mRNA levels of these DMEs were measured using the probe substrates method and real-time PCR, respectively. In the liver of wild-type FVB mice, Cyp3a11, 2d22, 2e1, 1a2, Ugt1a1, and 1a6a displayed maximum protein levels at 6-9weeks of age. Cyp1a2, Ugt1a1, 1a6a, and 1a9 showed maximum activities at 6-9weeks of age, whereas Cyp3a11, 2d22, and 2e1 showed maximum activities in 1-3-week-old mice. Additionally, most of the DMEs showed maximum mRNA levels in 17-week-old mice liver. Compared with wild-type FVB mice, the protein levels of these DMEs showed no significant changes in Bcrp1-/- FVB mice liver. However, the activity of Cyp2e1 was increased and that of Cyp2d22 was decreased. In conclusion, the seven hepatic DMEs in FVB mice liver showed significant alterations in an isoform-specific manner with increased age. Although the protein levels of these DMEs showed no significant changes, the activities of Cyp2e1 and 2d22 were changed in Bcrp1-/- mice.

Hepatic Metabolism of Sakuranetin and Its Modulating Effects on Cytochrome P450s and UDP-Glucuronosyltransferases.

Sakuranetin (SKN), found in cherry trees and rice, is a flavanone with various pharmacological activities. It is biosynthesized from naringenin in rice or cherry trees, and the metabolism of SKN has been studied in non-human species. The present study aimed to investigate the metabolic pathways of SKN in human liver microsomes and identify the phase I and phase II metabolites, as well as evaluate the potential for drug–herb interactions through the modulation of drug metabolizing enzymes (DMEs). HPLC-DAD and HPLC-electrospray mass spectrometry were used to study the metabolic stability and identify the metabolites from human liver microsomes incubated with SKN. The potential of SKN to inhibit the DMEs was evaluated by monitoring the formation of a DME-specific product. The cytochrome P450 2B6 and 3A4-inductive effects were studied using promoter reporter assays in human hepatocarcinoma cells. The major pathways for SKN metabolism include B-ring hydroxylation, 5-O-demethylation, and conjugation with glutathione or glucuronic acid. The phase I metabolites were identified as naringenin and eriodictyol. SKN was found to be a UDP-glucuronosyltransferases (UGT) 1A9 inhibitor, whereas it induced transactivation of the human pregnane X receptor-mediated cytochrome P450 (CYP) 3A4 gene.

Alteration of UDP-glucuronosyltransferase 1a1, 1a7 and P-glycoprotein expression in hepatic fibrosis rats and the impact on pharmacokinetics of puerarin

BackgroundPuerarin, derived from a traditional Chinese herb Pueraria lobata (Willd.) Ohwi which was distributed globally and planted in most parts of China, has been extensively applied in patients with cardiovascular diseases in China. Yet a considerable proportion of the patients were accompanied with liver illnesses simultaneously because of all sorts of reasons. Hypothesis/PurposeIt had been implied by some previous research that the absorption and the metabolism of puerarin were susceptible to liver issues due to changed P-gp and Ugt1a level, but pharmacokinetics of puerarin under such conditions were few concerned. Our study aimed to make sure whether and how much the behavior of puerarin in vivo was affected by hepatic diseases, and to explore the potential mechanisms. MethodsA CCl4 induced rat model of hepatic fibrosis (HF) was prepared and verified. Single low/high doses of oral and intravenous administration of puerarin to HF and normal rats were performed. Pharmacokinetics of puerarin were determined by a validated HPLC method. The expression of P-gp, Ugt1a1, and Ugt1a7 in both liver and intestines were determined by quantitative RT-PCR and Western blot analysis respectively. ResultsThe systemic exposure of puerarin in HF rats of experimental groups were found decreased remarkably except for that of the high dose intravenous group. Moreover, the expression of P-gp, Ugt1a1, and Ugt1a7 in liver and intestines of HF rats were figured out increased. ConclusionThe results indicated that the HF originated overexpression of Ugt1a1, Ugt1a7, and P-gp level played important roles in pharmacokinetics of puerarin, suggested the clinical regimen of puerarin based on normal populations might be inappropriate for patients with chronic liver diseases. It was implied drugs whose absorption or elimination were related to P-gp, Ugt1a1, or Ugt1a7 might also be affected by hepatic illnesses.

Molecular and functional characterization of UDP-glucuronosyltransferase 1A in cynomolgus macaques

UDP-glucuronosyltransferases (UGTs) are drug-metabolizing enzymes essential for the metabolism of endogenous substrates and xenobiotics. Molecular characteristics of UGTs have been extensively investigated in humans, but in cynomolgus macaques, a non-human primate species widely used in drug metabolism studies, remain to be investigated. In this study, 12 UGT1A cDNAs (UGT1A1, 1A2, 1A4A, 1A4B, 1A5A, 1A5B, 1A5C, 1A6, 1A7, 1A8, 1A9, and 1A10) were isolated and characterized in cynomolgus macaques. UGT1A5C cDNA did not contain a complete coding region due to nonsense mutations, and was excluded from further analysis. Amino acid sequences of all 11 cynomolgus UGT1As had high sequence identities (92–95%) with human UGT1As and were phylogenetically close to human UGT1As. These cynomolgus UGT1A genes shared exons 2–5, and contained a variable exon 1 unique to each gene, similar to human UGT1A genes. Moreover, cynomolgus and human UGT1A gene clusters were located in corresponding regions in the genome. Among the 10 tissue types analyzed, cynomolgus UGT1A mRNAs were most abundantly expressed in the liver, jejunum, and/or kidney, the drug-metabolizing organs, similar to human UGT1As. Among these 11 cynomolgus UGT1A mRNAs, cynomolgus UGT1A2, UGT1A9, and UGT1A10 mRNAs were most abundantly expressed in the liver, kidney, and jejunum, respectively. Cynomolgus liver microsomes and UGT1A proteins catalyzed glucuronidation of the substrates human UGT1As catalyze, including 4-methylumbelliferone, 4-nitrophenol, estradiol, trifluoperazine, serotonin, and propofol, although trifluoperazine glucuronidation was not catalyzed by any cynomolgus UGT1A proteins. These results suggest that cynomolgus UGT1As are functional enzymes with molecular similarities to human UGT1As.

Backbone assignment of the apo-form of the human C-terminal domain of UDP-glucuronosyltransferase 1A (UGT1A).

A major component of phase II drug metabolism is the covalent addition of glucuronic acid to metabolites and xenobiotics. This activity is carried out by UDP-glucuronosyltransferases (UGT) which bind the UDP-glucuronic acid donor and catalyze the covalent addition of glucuronic acid sugar moieties onto a wide variety of substrates. UGTs play important roles in drug detoxification and were recently shown to act in an inducible form of multi-drug resistance in cancer patients. Despite their biological importance, structural understanding of these enzymes is limited. The C-terminal domain is identical for all UGT1A family members and required for binding to UDP-glucuronic acid as well as involved in contacts with substrates. Here, we report the backbone assignments for the C-terminal domain of UGT1A. These assignments are a critical tool for the development of a deeper biochemical understanding of substrate specificity and enzymatic activity.