UDP-glucuronosyltransferase 1A1 Research Articles

Association between UGT1A1*28*28 genotype and lung cancer in the Japanese population.

Lung cancer is the leading cause of cancer death and is closely linked to tobacco smoking. Genetic polymorphisms in genes that encode enzymes involved in metabolizing tobacco carcinogens could affect an individual's risk for lung cancer. While polymorphism of UDP-glucuronosyltransferase1A1 (UGT1A1) is involved in detoxification of benzo(a)pyrene-7,8-dihydrodiol(-), a major tobacco carcinogen, the association between UGT1A1 genotype and lung cancer has not been examined. We retrieved the clinical data of 5,285 patients who underwent systemic chemotherapy at Kyoto University Hospital. A total of 765 patients (194 lung cancer patients and 671 patients with other malignancies) with UGT1A1 genotyping data were included in this analysis. We used logistic regression with recessive, dominant, and additive models to identify differences in genotype frequencies between lung cancer and other malignancies. In the recessive model, UGT1A1*28*28 genotype was significantly associated with lung cancer compared to other malignancies (odds ratio5.3, P=0.0083). Among lung cancer patients with a smoking history, squamous cell carcinoma was significantly predominant in patients with UGT1A1*28*28 compared to those with other UGT1A1 genotypes (P=0.024). This is the first study to demonstrate a significant association between the homozygous UGT1A1*28 genotype and lung cancer.

Relation of Transcriptional Factors to the Expression and Activity of Cytochrome P450 and UDP-Glucuronosyltransferases 1A in Human Liver: Co-Expression Network Analysis.

Cytochrome P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) play important roles in the metabolism of exogenous and endogenous compounds. The gene transcription of CYPs and UGTs can be enhanced or reduced by transcription factors (TFs). This study aims to explore novel TFs involved in the regulatory network of human hepatic UGTs/CYPs. Correlations between the transcription levels of 683 key TFs and CYPs/UGTs in three different human liver expression profiles (n = 640) were calculated first. Supervised weighted correlation network analysis (sWGCNA) was employed to define hub genes among the selected TFs. The relationship among 17 defined TFs, CYPs/UGTs expression, and activity were evaluated in 30 liver samples from Chinese patients. The positive controls (e.g., PPARA, NR1I2, NR1I3) and hub TFs (NFIA, NR3C2, and AR) in the GreysWGCNA Module were significantly and positively associated with CYPs/UGTs expression. And the cancer- or inflammation-related TFs (TEAD4, NFKB2, and NFKB1) were negatively associated with mRNA expression of CYP2C9/CYP2E1/UGT1A9. Furthermore, the effect of NR1I2, NR1I3, AR, TEAD4, and NFKB2 on CYP450/UGT1A gene transcription translated into moderate influences on enzyme activities. To our knowledge, this is the first study to integrate Gene Expression Omnibus (GEO) datasets and supervised weighted correlation network analysis (sWGCNA) for defining TFs potentially related to CYPs/UGTs. We detected several novel TFs involved in the regulatory network of hepatic CYPs and UGTs in humans. Further validation and investigation may reveal their exact mechanism of CYPs/UGTs regulation.

Pharmacogenetic study of the effects of raloxifene on negative symptoms of postmenopausal women with schizophrenia: A double-blind, randomized, placebo-controlled trial

Several double-blind clinical trials have reported improvement in positive, negative and cognitive symptoms of schizophrenia with raloxifene, a selective receptor estrogen modulator. However, there are some inconsistencies in replicating findings between studies of different countries. The failure to replicate these findings may result from genetic factors that could explain some of the variability in the treatment response. However, pharmacogenetic studies exploring this topic in women with schizophrenia are lacking. We aimed to conduct an exploratory pharmacogenetic analysis of a double-blind, randomized, parallel, placebo-controlled study of 24 weeks’ duration of raloxifene aiming to improve negative symptoms in postmenopausal women with schizophrenia. Four single nucleotide polymorphisms (SNPs) were studied: rs9340799, rs2234693 and rs1801132 in the Estrogen Receptor 1 (ESR1) gene, and rs1042597 in the UDP-glucuronosyltransferase 1A8 (UGT1A8) gene. Sixty-five postmenopausal women with schizophrenia (DSM-IV) were randomized to either 60mg/day adjunctive raloxifene (36 women) or adjunctive placebo (29 women). Psychopathological symptoms were assessed at baseline and at weeks 4, 12, and 24 with the Positive and Negative Syndrome Scale (PANSS). Of the four studied SNPs, the rs1042597 variant in the UGT1A8 gene was associated with a different treatment response in negative symptoms with raloxifene treatment, whereas the rs2234693 variant in the ESR1 gene was associated with a distinct response in general psychopathology. In conclusion, our study suggests that genetic variants in UGT1A8 and ESR1 genes modulate the treatment response to adding raloxifene to antipsychotic treatment in postmenopausal women with schizophrenia.

Transcriptome association analysis identifies miR-375 as a major determinant of variable acetaminophen glucuronidation by human liver

Acetaminophen is the leading cause of acute liver failure (ALF) in many countries including the United States. Hepatic glucuronidation by UDP-glucuronosyltransferase (UGT) 1A subfamily enzymes is the major route of acetaminophen elimination. Reduced glucuronidation may predispose some individuals to acetaminophen-induced ALF, but mechanisms underlying reduced glucuronidation are poorly understood. We hypothesized that specific microRNAs (miRNAs) may reduce UGT1A activity by direct effects on the UGT1A 3′-UTR shared by all UGT1A enzyme transcripts, or by indirect effects on transcription factors regulating UGT1A expression. We performed an unbiased miRNA whole transcriptome association analysis using a bank of human livers with known acetaminophen glucuronidation activities. Of 754 miRNAs evaluated, 9 miRNAs were identified that were significantly overexpressed (p<0.05; >2-fold) in livers with low acetaminophen glucuronidation activities compared with those with high activities. miR-375 showed the highest difference (>10-fold), and was chosen for further mechanistic validation. We demonstrated using in silico analysis and luciferase reporter assays that miR-375 has a unique functional binding site in the 3′-UTR of the aryl hydrocarbon receptor (AhR) gene. Furthermore overexpression of miR-375 in LS180 cells demonstrated significant repression of endogenous AhR protein (by 40%) and mRNA (by 10%), as well as enzyme activity and/or mRNA of AhR regulated enzymes including UGT1A1, UGT1A6, and CYP1A2, without affecting UGT2B7, which is not regulated by AhR. Thus miR-375 is identified as a novel repressor of UGT1A-mediated hepatic acetaminophen glucuronidation through reduced AhR expression, which could predispose some individuals to increased risk for acetaminophen-induced ALF.

1-Alpha, 25-dihydroxyvitamin D3 alters the pharmacokinetics of mycophenolic acid in renal transplant recipients by regulating two extrahepatic UDP-glucuronosyltransferases 1A8 and 1A10

Mycophenolic acid (MPA) is an important immunosuppressant broadly used in renal transplantation. However, the large inter-patient variability in mycophenolic acid (MPA) pharmacokinetics (PK) limits its use. We hypothesize that extrahepatic metabolism of MPA may have significant impact on MPA PK variability. Two intestinal UDP-glucuronosyltransferases 1A8 and 1A10 plays critical role in MPA metabolism. Both in silico and previous genome-wide analyses suggested that vitamin D (VD) may regulate intestinal UGT1A expression. We validated the VD response elements (VDREs) across the UGT1A locus with chromatin immunoprecipitation (ChIP) and luciferase reporter assays. The impact of 1-alpha,25-dihydroxyvitamin D3 (D3) on UGT1A8 and UGT1A10 transcription and on MPA glucuronidation was tested in human intestinal cell lines LS180, Caco-2 and HCT-116. The correlation between transcription levels of VD receptor (VDR) and the two UGT genes were examined in human normal colorectal tissue samples (n=73). PK alterations of MPA following the parent drug, mycophenolate mofetil (MMF), and D3 treatment was assessed among renal transplant recipients (n=10). Our ChIP assay validate three VDREs which were further demonstrated as transcriptional enhancers with the luciferase assays. D3 treatment significantly increased transcription of both UGT genes as well as MPA glucuronidation in cells. The VDR mRNA level was highly correlated with that of both UGT1A8 and UGT1A10 in human colorectal tissue. D3 treatment in patients led to about 40% reduction in both AUC0-12 and Cmax while over 70% elevation of total clearance of MPA. Our study suggested a significant regulatory role of VD on MPA metabolism and PK via modulating extrahepatic UGT activity.

Induction of the UDP-Glucuronosyltransferase 1A1 during the Perinatal Period Can Cause Neurodevelopmental Toxicity.

Anticonvulsants can increase the risk of developing neurotoxicity in infants; however, the underlying mechanism has not been elucidated to date. Thyroxine [3,5,3',5'-l-tetraiodothyronine (T4)] plays crucial roles in the development of the central nervous system. In this study, we hypothesized that induction of UDP-glucuronosyltransferase 1A1 (UGT1A1)-an enzyme involved in the metabolism of T4-by anticonvulsants would reduce serum T4 levels and cause neurodevelopmental toxicity. Exposure of mice to phenytoin during both the prenatal and postnatal periods significantly induced UGT1A1 and decreased serum T4 levels on postnatal day 14. In the phenytoin-treated mice, the mRNA levels of synaptophysin and synapsin I in the hippocampus were lower than those in the control mice. The thickness of the external granule cell layer was greater in phenytoin-treated mice, indicating that induction of UGT1A1 during the perinatal period caused neurodevelopmental disorders. Exposure to phenytoin during only the postnatal period also caused these neurodevelopmental disorders. A T4 replacement attenuated the increase in thickness of the external granule cell layer, indicating that the reduced T4 was specifically associated with the phenytoin-induced neurodevelopmental disorder. In addition, these neurodevelopmental disorders were also found in the carbamazepine- and pregnenolone-16-α-carbonitrile-treated mice. Our study is the first to indicate that UGT1A1 can control neurodevelopment by regulating serum T4 levels.

Erratum to: UDP-glucuronosyltransferase 1A1*6 and *28 polymorphisms as indicators of initial dose level of irinotecan to reduce risk of neutropenia in patients receiving FOLFIRI for colorectal cancer.

Background Irinotecan (CPT-11)-induced neutropenia is associated with UDP-glucuronosyltransferase (UGT) 1A1*6 and *28 polymorphisms. This prospective study investigated whether using these polymorphisms to adjust the initial dose of CPT-11 as part of FOLFIRI treatment in colorectal cancer patients might improve safety.

Identification of UDP-glucuronosyltransferases 1A1, 1A3 and 2B15 as the main contributors to glucuronidation of bakuchiol, a natural biologically active compound

1. Bakuchiol, one of the main active compounds of Psoralea corylifolia, possesses a variety of pharmacological activities such as anti-tumor and anti-aging effects. Here, we aimed to characterize the glucuronidation of bakuchiol using human liver microsomes (HLM) and expressed UDP-glucuronosyltransferase (UGT) enzymes.2. The glucuronide of bakuchiol was confirmed by liquid chromatography–mass spectrometry (LC-MS) and β-glucuronidase hydrolysis assay. Glucuronidation rates and kinetic parameters were derived by enzymatic incubation and model fitting. Activity correlation analyses were performed to identify the main UGT isoforms contributing to hepatic metabolism of bakuchiol.3. Among the three UGT enzymes (i.e., UGT1A1, UGT1A3 and UGT2B15) capable of catalyzing bakuchiol glucuronidation, UGT2B15 showed the highest activity with a CLint value of 100 μl/min/nmol. Bakuchiol glucuronidation was strongly correlated with glucuronidation of 5-hydroxyrofecoxib (r = 0.933; p < 0.001), 3-O-glucuronidation of β-estradiol (r = 0.719; p < 0.01) and significantly correlated with 24-O-glucuronidation of CDCA (r = 0.594; p < 0.05). In addition, a marked species difference existed in hepatic glucuronidation of bakuchiol.4. In conclusion, UGT1A1, UGT1A3 and UGT2B15 were identified as the main contributors to glucuronidation of bakuchiol.

17β-Estradiol up-regulates UDP-glucuronosyltransferase 1A9 expression via estrogen receptor α

UDP-glucuronosyltransferase 1A9 (UGT1A9) is a major phase II enzyme responsible for elimination of drugs and endogenous molecules. Clinical data have shown increased elimination of UGT1A9 substrates in pregnant women or oral contraceptive users, but the role of estrogen in the regulation of UGT1A9 expression remains unknown. In this study, we investigated the effect of 17β-estradiol (E2) on UGT1A9 expression and the role of ERα in the transcriptional regulation of UGT1A9. E2 significantly increased UGT1A9 promoter activity in HepG2 cells in the presence of ERα. UGT1A9 induction by E2 was abrogated by antiestrogen ICI182,780 in HepG2 cells that constitutively express ERα. Results from transient transfection of ERα mutants into HepG2 cells demonstrated that mutation at DNA-binding domain of ERα abrogates increased UGT1A9 promoter activity by E2. Deletion and mutation assays of UGT1A9 promoter revealed a putative ERE located within −2262/−1987 region. Examination of healthy human liver tissues revealed significantly higher UGT1A9 expression in women as compared to men. Together, these findings provide a mechanistic basis for the previous clinical reports and may shed a light on identifying sources for inter-individual variability in UGT1A9-mediated drug metabolism.

Drug interaction study of natural steroids from herbs specifically toward human UDP-glucuronosyltransferase (UGT) 1A4 and their quantitative structure activity relationship (QSAR) analysis for prediction

The wide application of herbal medicines and foods containing steroids has resulted in the high risk of herb-drug interactions (HDIs). The present study aims to evaluate the inhibition potential of 43 natural steroids from herb medicines toward human UDP- glucuronosyltransferases (UGTs). A remarkable structure-dependent inhibition toward UGT1A4 was observed in vitro. Some natural steroids such as gitogenin, tigogenin, and solasodine were found to be the novel selective inhibitors of UGT1A4, and did not inhibit the activities of major human CYP isoforms. To clarify the possibility of the in vivo interaction of common steroids and clinical drugs, the kinetic inhibition type and related kinetic parameters (Ki) were measured. The target compounds 2–6 and 15, competitively inhibited the UGT1A4-catalyzed trifluoperazine glucuronidation reaction, with Ki values of 0.6, 0.18, 1.1, 0.7, 0.8, and 12.3μM, respectively. And this inhibition of steroids towards UGT1A4 was also verified in human primary hepatocytes. Furthermore, a quantitative structure-activity relationship (QSAR) of steroids with inhibitory effects toward human UGT1A4 isoform was established using the computational methods. Our findings elucidate the potential for in vivo HDI effects of steroids in herbal medicine and foods, with the clinical dr ugs eliminated by UGT1A4, and reveal the vital pharamcophoric requirement of natural steroids for UGT1A4 inhibition activity.

Endocrine Disrupting Effects of Triclosan on the Placenta in Pregnant Rats.

Triclosan (TCS) is a broad-spectrum antimicrobial agent that is frequently used in pharmaceuticals and personal care products. Reports have shown that TCS is a potential endocrine disruptor; however, the potential effects of TCS on placental endocrine function are unclear. The aim of this study was to investigate the endocrine disrupting effects of TCS on the placenta in pregnant rats. Pregnant rats from gestational day (GD) 6 to GD 20 were treated with 0, 30, 100, 300 and 600 mg/kg/d TCS followed by analysis of various biochemical parameters. Of the seven tissues examined, the greatest bioaccumulation of TCS was observed in the placenta. Reduction of gravid uterine weight and the occurrence of abortion were observed in the 600 mg/kg/d TCS-exposed group. Moreover, hormone detection demonstrated that the serum levels of progesterone (P), estradiol (E2), testosterone (T), human chorionic gonadotropin (hCG) and prolactin (PRL) were decreased in groups exposed to higher doses of TCS. Real-time quantitative reverse transcriptase-polymerase chain reaction (Q-RT-PCR) analysis revealed a significant increase in mRNA levels for placental steroid metabolism enzymes, including UDP-glucuronosyltransferase 1A1 (UGT1A1), estrogen sulfotransferase 1E1 (SULT1E1), steroid 5α-reductase 1 (SRD5A1) and steroid 5α-reductase 2 (SRD5A2). Furthermore, the transcriptional expression levels of progesterone receptor (PR), estrogen receptor (ERα) and androgen receptor (AR) were up-regulated. Taken together, these data demonstrated that the placenta was a target tissue of TCS and that TCS induced inhibition of circulating steroid hormone production might be related to the altered expression of hormone metabolism enzyme genes in the placenta. This hormone disruption might subsequently affect fetal development and growth.

Insight into the pharmacokinetic behavior of tanshinone IIA in the treatment of Crohn’s disease: comparative data for tanshinone IIA and its two glucuronidated metabolites in normal and recurrent colitis models after oral administration

1. Previous reports implied that tanshinone IIA (TSA) may offer potential benefits for Crohn’s disease (CD). However, the detailed pharmacokinetic behavior of TSA in the treatment of colitis remain unclear. Herein, a recurrent trinitrobenzene sulfonic acid (TNBS)-colitis mouse model was used to investigate whether TSA possesses favorable pharmacokinetic and colonic distribution profiles to serve as a candidate drug.2. Although the systemic TSA exposures were low (AUC0–t approximately 330 ng*h/ml) in both the normal and colitis models after oral administration TSA 20 mg/kg, high levels of TSA were found in the gastrointestinal tract (GI). Such a GI exposure of TSA in colitis mice is adequate to exert anti-inflammatory effects as observed in various in vitro studies.3. Interestingly, colonic TSA exposure in the colitis mouse model was much lower than that in the normal mice, which may be explained by a significant upregulation of colonic UDP-glucuronosyltransferase (Ugt)1a9 expression and a higher plasma concentration of TSA glucuronides in the model mice at 0.5, 1 and 2 h after TSA administration.4. Together, these results reveal high accumulation at the site of inflammation and minimal systemic concentration of TSA, which are favorable pharmacokinetic behaviors to meet the requirements for CD treatment.

Population pharmacokinetic analysis of elvitegravir and cobicistat in HIV-1-infected individuals.

Co-formulated elvitegravir, cobicistat, tenofovir disoproxil fumarate and emtricitabine is among the preferred regimens for first-line ART. A population approach was used to characterize the pharmacokinetics of elvitegravir and cobicistat and identify individual factors and co-medications influencing their disposition, taking into consideration the interaction between the two compounds. The study population included 144 HIV-infected individuals who provided 186 and 167 elvitegravir and cobicistat plasma concentrations, respectively. First, distinct NONMEM(®) analyses were conducted for elvitegravir and cobicistat, including individual demographic, clinical and genetic factors as potential covariates. Elvitegravir and cobicistat interaction was then assessed through different inhibitory models. Simulations based on the final model served to compare expected drug concentrations under standard and alternative dosage regimens. Clearance with between-subject variability was 7.6 L/h [coefficient of variation (CV) 16.6%] and volume of distribution 61 L for elvitegravir and 16.0 L/h (CV 41.9%) and 88.3 L, respectively, for cobicistat. Concomitant administration of non-ritonavir-boosted atazanavir decreased elvitegravir clearance by 35%, likely due to UDP-glucuronosyl transferase (UGT) 1A1 inhibition. Concomitant administration of non-ritonavir-boosted atazanavir and ritonavir-boosted darunavir decreased cobicistat clearance by 47% and 27%, respectively. The final interaction model included cobicistat exposure (AUC0-24) on elvitegravir clearance. Simulations confirmed that a reduced elvitegravir dose of 85 mg co-administered with cobicistat and atazanavir produces a concentration-time course comparable to the standard regimen without atazanavir. Elvitegravir and cobicistat pharmacokinetic variability appears to be mainly explained by drug-drug interactions that may be encountered in routine clinical practice. In these cases, therapeutic drug monitoring and surveillance for potential toxicities would be justified.

Reserpine Inhibit the JB6 P+ Cell Transformation Through Epigenetic Reactivation of Nrf2-Mediated Anti-oxidative Stress Pathway.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a crucial transcription factor that regulates the expression of defensive antioxidants and detoxification enzymes in cells. In a previous study, we showed that expression of the Nrf2 gene is regulated by an epigenetic modification. Rauvolfia verticillata, a traditional Chinese herbal medicine widely used in China, possesses anticancer and antioxidant effects. In this study, we investigated how Nrf2 is epigenetically regulated by reserpine, the main active component in R. verticillata, in mouse skin epidermal JB6 P+ cells. Reserpine induced ARE (antioxidant response element)-luciferase activity in HepG2-C8 cells. Accordingly, in JB6 P+ cells, it upregulated the mRNA and protein levels of Nrf2 and its downstream target genes heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1), while it only increased the protein level of UDP-glucuronosyltransferase 1A1 (UGT1A1). Furthermore, reserpine decreased the TPA (12-O-tetradecanoylphorbol-13-acetate)-induced colony formation of JB6 cells in a dose-dependent manner. DNA sequencing and methylated DNA immunoprecipitation further demonstrated the demethylation effect of reserpine on the first 15 CpGs of the Nrf2 promoter in JB6 P+ cells. Reserpine also reduced the mRNA and protein expression of DNMT1 (DNA methyltransferase 1), DNMT3a (DNA methyltransferases 3a), and DNMT3b (DNA methyltransferases 3b). Moreover, reserpine induced Nrf2 expression via an epigenetic pathway in skin epidermal JB6 P+ cells, enhancing the protective antioxidant activity and decreasing TPA-induced cell transformation. These results suggest that reserpine exhibits a cancer preventive effect by reactivating Nrf2 and inducing the expression of target genes involved in cellular protection, potentially providing new insight into the chemoprevention of skin cancer using reserpine.

Identification of Human UDP-Glucuronosyltransferase 1A4 as the Major Isozyme Responsible for the Glucuronidation of 20(S)-Protopanaxadiol in Human Liver Microsomes.

20(S)-protopanaxadiol (PPD), one of the representative aglycones of ginsenosides, has a broad spectrum of pharmacological activities. Although phase I metabolism has been investigated extensively, information regarding phase II metabolism of this compound remains to be elucidated. Here, a glucuronidated metabolite of PPD in human liver microsomes (HLMs) and rat liver microsomes (RLMs) was unambiguously identified as PPD-3-O-β-d-glucuronide by nuclear magnetic resonance spectroscopy and high resolution mass spectrometry. The chemical inhibition and recombinant human UDP-Glucuronosyltransferase (UGT) isoforms assay showed that the PPD glucuronidation was mainly catalyzed by UGT1A4 in HLM, whereas UGT1A3 showed weak catalytic activity. In conclusion, PPD-3-O-β-d-glucuronide was first identified as the principal glucuronidation metabolite of PPD in HLMs, which was catalyzed by UGT1A4.

Relationship between plasma bilirubin level and oxidative stress markers in HIV-infected patients on atazanavir- vs. efavirenz-based antiretroviral therapy.

Chronic oxidative stress (OS) may play a role in cardiovascular disease in HIV-infected patients, and increased bilirubin levels may have a beneficial role in counteracting OS. Atazanavir (ATV) inhibits UDP-glucuronosyl-transferase 1A1 (UGT1A1), thus increasing unconjugated bilirubin levels. We aimed to compare changes in OS markers in patients on ATV/ritonavir (ATV/r)- vs. efavirenz (EFV)-based first-line antiretroviral therapy (ART). A multicentre, prospective cohort study of HIV-infected patients who started first-line ART with either ATV/r or EFV was conducted. Lipoprotein-associated phospholipase A2 (Lp-PLA2), myeloperoxidase (MPO) and oxidized low-density lipoprotein (oxLDL) were measured for 145 patients in samples obtained at baseline and after at least 9 months of ART during which the initial regimen was maintained and the patient was virologically suppressed. The change in OS markers was modelled using multiple linear regressions adjusting for baseline values and confounders. After adjustment for baseline variables, patients on ATV/r had a significantly greater decrease in Lp-PLA2 [estimated difference -16.3; 95% confidence interval (CI) -31.4, -1.25; P = 0.03] and a significantly smaller increase in OxLDL (estimated difference -21.8; 95% CI -38.0, -5.6; P < 0.01) relative to those on EFV, whereas changes in MPO were not significantly different (estimated difference 1.2; 95% CI -14.3, 16.7; P = 0.88). Adjusted changes in bilirubin were significantly greater for the ATV/r group than for the EFV group (estimated difference 1.33 mg/dL; 95% CI 1.03, 1.52 mg/dL; P < 0.01). Changes in bilirubin and changes in OS markers were significantly correlated. When compared with EFV, ATV/r-based therapy was associated with lower levels of oxidative stress biomarkers, which was in part attributable to increased bilirubin levels.

Efflux Transport Characterization of Resveratrol Glucuronides in UDP-Glucuronosyltransferase 1A1 Transfected HeLa Cells: Application of a Cellular Pharmacokinetic Model to Decipher the Contribution of Multidrug Resistance-Associated Protein 4.

Resveratrol undergoes extensive metabolism to form biologically active glucuronides in humans. However, the transport mechanisms for resveratrol glucuronides are not fully established. Here, we aimed to characterize the efflux transport of resveratrol glucuronides using UGT1A1-overexpressing HeLa cells (HeLa1A1 cells), and to determine the contribution of multidrug resistance-associated protein (MRP) 4 to cellular excretion of the glucuronides. Two glucuronide isomers [i.e., resveratrol 3-O-glucuronide (R3G) and resveratrol 4'-O-glucuronide (R4'G)] were excreted into the extracellular compartment after incubation of resveratrol (1-100 μM) with HeLa1A1 cells. The excretion rate was linearly related to the level of intracellular glucuronide, indicating that glucuronide efflux was a nonsaturable process. MK-571 (a dual inhibitor of UGT1A1 and MRPs) significantly decreased the excretion rates of R3G and R4'G while increasing their intracellular levels. Likewise, short-hairpin RNA (shRNA)-mediated silencing of MRP4 caused a significant reduction in glucuronide excretion but an elevation in glucuronide accumulation. Furthermore, β-glucuronidase expressed in the cells catalyzed the hydrolysis of the glucuronides back to the parent compound. A cellular pharmacokinetic model integrating resveratrol transport/metabolism with glucuronide hydrolysis/excretion was well fitted to the experimental data, allowing derivation of the efflux rate constant values in the absence or presence of shRNA targeting MRP4. It was found that a large percentage of glucuronide excretion (43%-46%) was attributed to MRP4. In conclusion, MRP4 participated in cellular excretion of R3G and R4'G. Integration of mechanistic pharmacokinetic modeling with transporter knockdown was a useful method to derive the contribution percentage of an exporter to overall glucuronide excretion.

Three-dimensional polyacrylamide gel-based DNA microarray method effectively identifies UDP-glucuronosyltransferase 1A1 gene polymorphisms for the correct diagnosis of Gilbert's syndrome.

Gilbert's syndrome is a mild genetic liver disorder characterized by unconjugated hyperbilirubinemia due to defects in the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene. The T-3279G mutation in the phenobarbital responsive enhancer module (PBREM), the TA-insertion in the TATA box, creating the A(TA)7TAA motif instead of A(TA)6TAA and the G211A mutation in coding exon 1, particularly in Asian populations, of the human UGT1A1 gene are the three common genotypes found in patients with Gilbert's syndrome. Different approaches for detecting the T-3279G, A(TA)6/7TAA and G211A mutations of the UGT1A1 gene have been described. In this study, to the best of our knowledge, we established a three-dimensional polyacrylamide gel-based DNA microarray method for the first time, in order to study UGT1A1 gene polymorphisms. This method, based on a step-by-step three-dimensional polyacrylamide gel-based DNA microarray protocol, successfully identified all possible genotypes of T-3279G, A(TA)6/7TAA and G211A in 20 patients with hyperbilirubinemia. In addition, sequencing was performed to confirm these results. The data from the current study demonstrate that the three-dimensional polyacrylamide gel microarray method has the potential to be applied as a useful, reliable and cost-effective tool to detect the T-3279G, the A(TA)6/7TAA and the G211A mutations of the UGT1A1 gene in patients with hyperbilirubinemia and thereby aid in the diagnosis of Gilbert's syndrome.

A translationally optimized AAV-UGT1A1 vector drives safe and long-lasting correction of Crigler-Najjar syndrome

Crigler-Najjar syndrome is a severe metabolic disease of the liver due to a reduced activity of the UDP Glucuronosyltransferase 1A1 (UGT1A1) enzyme. In an effort to translate to the clinic an adeno-associated virus vector mediated liver gene transfer approach to treat Crigler-Najjar syndrome, we developed and optimized a vector expressing the UGT1A1 transgene. For this purpose, we designed and tested in vitro and in vivo multiple codon-optimized UGT1A1 transgene cDNAs. We also optimized noncoding sequences in the transgene expression cassette. Our results indicate that transgene codon-optimization is a strategy that can improve efficacy of gene transfer but needs to be carefully tested in vitro and in vivo. Additionally, while inclusion of introns can enhance gene expression, optimization of these introns, and in particular removal of cryptic ATGs and splice sites, is an important maneuver to enhance safety and efficacy of gene transfer. Finally, using a translationally optimized adeno-associated virus vector expressing the UGT1A1 transgene, we demonstrated rescue of the phenotype of Crigler-Najjar syndrome in two animal models of the disease, Gunn rats and Ugt1a1-/- mice. We also showed long-term (>1 year) correction of the disease in Gunn rats. These results support further translation of the approach to humans.

Guest Editorial: Breast Cancer Epigenetics in the Era of Precision Medicine

The Precision Medicine Initiative (PMI) was officially unveiled on January 20, 2015 by President Obama during his State of the Union address. This initiative will transform healthcare by providing health professionals with new and improved strategies to identify individuals at risk for diseases like breast cancer and to determine which treatments and therapies are best suited for the patient. The term “precision medicine” which most researchers often refer to as “personalized or individualized medicine” considers the approach that one-size-does-not-fit-all [1]. The phrase “onesize-does-not-fit-all” refers to a treatment or drug that may work well for some individuals but not for others. A nice example is the anti-estrogen breast cancer drug tamoxifen (TAM) which is used to prevent breast cancer in high-risk populations and to treat postmenopausal estrogen receptor (ER) – positive breast cancers [2,3]. Breast cancer is the most common cancer and one of the leading causes of cancer death among women [4]. Close to one-third of postmenopausal breast cancer patients experience a relapse after adjuvant treatment with TAM [5,6]. It is unclear as to why some patients experience a more favorable response to certain drugs, such as TAM, while other patients with the same diagnosis experience multiple adverse side effects from taking TAM. Recent advances in the field of breast cancer pharmacogenomics provide strong evidence that acquired or inherited genetic differences in drug metabolic pathways can affect an individual’s response to TAM and other chemotherapeutic drugs [7-10]. In support of these observations, our pharmacogenetic study showed that a single nucleotide polymorphism in the promoter of the TAM metabolizing gene UDP-glucuronosyltransferase 1A4 (UGT1A4) significantly decreases how TAM and its derivatives are metabolized in the human liver [11]. These studies, along with countless other pharmacogenomic studies, demonstrate the influence of pharmacogenomics on policy implementation and efforts that guide the development of more effective medical treatments tailored to an individual’s genetic makeup. Currently, more than 100 pharmacogenomic biomarkers have been added to drug labels that may help clinicians take appropriate actions to manage their patient’s health based on their biomarker information [12]. Because the PMI has directed most of its efforts toward research that seeks to provide new knowledge about how an individual’s genetic makeup, environment, and behavior can impact disease risk and response to treatment, I expect that this initiative will require continuous and effective collaborations between clinicians, scientists, health professionals and policy makers.