UDP-glucuronosyltransferase 1A1 Research Articles

Relationship of circulating total bilirubin, UDP-glucuronosyltransferases 1A1 and the development of non-alcoholic fatty liver disease: a cross-sectional study

BackgroundThis study aimed to investigate the correlation of circulating total bilirubin (TB) and UGT1A1 with NAFLD in Chinese Han population.Methods172 adults were enrolled from the Qingdao Municipal Hospital from May 2019 to October 2020. All individuals were examined with MRI-PDFF and divided into no steatosis, mild steatosis, moderate steatosis, and severe steatosis groups according to the MRI-PDFF values. The biochemical indexes and UGT1A1 were measured.ResultsThere was no significant difference of circulating TB and UGT1A1 levels between NAFLD group and controls. In the moderate steatosis and severe steatosis groups, the circulating TB levels were higher than that in control group (all P < 0.05). In addition, circulating TB levels were weak positively associated with liver fat fraction in NAFLD patients (ρ = 0.205, P = 0.001). There was no significant correlation between circulating UGT1A1 levels with liver fat fraction in patients with NAFLD (ρ = 0.080, P = 0.179), but positively correlation was found in patients with severe steatosis (ρ = 0.305, P = 0.026).ConclusionsThe circulating TB levels were significant high in patients with moderate and severe steatosis. Circulating TB levels were weakly associated with liver fat fraction in patients with NAFLD, and the circulating UGT1A1 levels were positively correlated with liver fat fraction in NAFLD patients with severe steatosis.Trial registration: ChiCTR, ChiCTR1900022744. Registered 24 April 2019 – Retrospectively registered, http://www.chictr.org.cn/edit.aspx?pid=38304&htm=4.

Involvement of phase II enzymes and efflux transporters in the metabolism and absorption of naringin, hesperidin and their aglycones in rats

This study examined the effects of phase II metabolism and efflux transportation on the bioavailability of naringin, hesperidin, and their aglycones (naringenin and hesperetin) in rats. Results indicated naringin and hesperidin have a lower oral bioavailability than their aglycones. Of all the phase II enzymes tested, UDP-glucuronosyltransferase (UGT) 1A1, UGT1A2, UGT1A3, UGT1A7 and SULT sulfotransferase (SULT) 1B1 were of minor importance regarding the phase II metabolism of naringenin and hesperetin in the small intestine. Naringin, hesperidin, and their aglycones were all extensively metabolised in the liver. Naringin and hesperidin were more extensively transported by efflux transporters compared to their aglycones. Significant correlations between phase II enzymes and efflux transporters were detected. In conclusion, more extensive metabolism of naringin and hesperidin than their aglycones in the small intestine, and the interplay of phase II enzymes and efflux transporters in the small intestine explain the lower relative oral bioavailability of naringin and hesperidin than their aglycones.

Physiologically-Based Pharmacokinetic Modeling to Investigate the Effect of Maturation on Buprenorphine Pharmacokinetics in Newborns with Neonatal Opioid Withdrawal Syndrome.

Neonatal opioid withdrawal syndrome (NOWS) is a major public health concern whose incidence has paralleled the opioid epidemic in the United States. Sublingual buprenorphine is an emerging treatment for NOWS, but given concerns about long-term adverse effects of perinatal opioid exposure, precision dosing of buprenorphine is needed. Buprenorphine pharmacokinetics (PK) in newborns, however, is highly variable. To evaluate underlying sources of PK variability, a neonatal physiologically-based pharmacokinetic (PBPK) model of sublingual buprenorphine was developed using Simcyp (version 19.1). The PBPK model included metabolism by cytochrome P450 (CYP) 3A4, CYP2C8, UDP-glucuronosyltransferase (UGT) 1A1, UGT1A3, UGT2B7, and UGT2B17, with additional biliary excretion. Maturation of metabolizing enzymes was incorporated, and default CYP2C8 and UGT2B7 ontogeny profiles were updated according to recent literature. A biliary clearance developmental profile was outlined using clinical data from neonates receiving sublingual buprenorphine as NOWS treatment. Extensive PBPK model validation in adults demonstrated good predictability, with geometric mean (95% confidence interval (CI)) predicted/observed ratios (P/O ratios) of area under the curve from zero to infinity (AUC0-∞ ), peak concentration (Cmax ), and time to reach peak concentration (Tmax ) equaling 1.00 (0.74-1.33), 1.04 (0.84-1.29), and 0.95 (0.72-1.26), respectively. In neonates, the geometric mean (95% CI) P/O ratio of whole blood concentrations was 0.75 (95% CI 0.64-0.87). PBPK modeling and simulation demonstrated that variability in biliary clearance, sublingual absorption, and CYP3A4 abundance are likely important drivers of buprenorphine PK variability in neonates. The PBPK model could be used to guide development of improved buprenorphine starting dose regimens for the treatment of NOWS.

Intestinal UDP-Glucuronosyltransferase 1A1 and Protection against Irinotecan-Induced Toxicity in a Novel UDP-Glucuronosyltransferase 1A1 Tissue-Specific Humanized Mouse Model.

The human UDP-glucuronosyltransferases (UGTs) represent an important family of drug-metabolizing enzymes, with UGT1A1 targeting the conjugation and detoxification of many exogenous substances, including pharmaceutical drugs. In this study we generated humanized UGT1A1 mice expressing the human UGT1A1 gene in either liver (hUGT1A1HEP ) or intestine (hUGT1A1GI ), enabling experiments to examine tissue-specific properties of UGT1A1-specific glucuronidation. Hepatic and intestinal tissue-specific expression and function of UGT1A1 were demonstrated. Although the liver is considered a major organ for detoxification, intestinal UGT1A1 is an important contributor for drug clearance. Mice were challenged with irinotecan (CPT-11), a prodrug hydrolyzed by carboxylesterases to form the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) and detoxified by UGT1A1. Humanized UGT1A1HEP mice that have no intestinal UGT1A1 displayed a greater lethality rate when exposed to CPT-11 than hUGT1A1GI mice. When exposed to a low dose of CPT-11 (10 mg/kg), hUGT1A1HEP mice displayed greater intestinal inflammatory (IL-1β and IL-6) insult in addition to p53-triggered apoptotic responses. In vitro studies with intestinal crypt organoids exposed to CPT-11 confirmed the results observed in vivo and indicated that CPT-11 impacts stemness, apoptosis, and endoplasmic reticulum (ER) stress in organoids deficient in UGT1A1. When we examined the induction of ER stress in organoids with thapsigargin, an inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase, apoptosis and the caspase surge that occurred in hUGT1A1HEP mice were blocked in hUGT1A1GI organoids. This study reveals the importance of intestinal UGT1A1 in preventing inflammation, apoptosis, and loss of stemness capacity upon systemic challenge with an important chemotherapeutic agent. SIGNIFICANCE STATEMENT: Hepatic and intestinal UGT1A1 play a key role in the metabolism and detoxification of endogenous and exogenous compounds. The use of tissue-specific humanized models expressing UGT1A1 in liver or intestine has confirmed the relevance of the intestinal tract in the detoxification of irinotecan. Mechanistic studies using intestinal organoids highlighted the importance of UGT1A1 in reducing inflammation, apoptosis, and loss of stemness. These new models provide valuable tools for studying tissue-specific glucuronidation of substances that are metabolized by human UGT1A1.

A Molecular-Splicing Strategy for Constructing a Near-Infrared Fluorescent Probe for UDP-Glucuronosyltransferase 1A1.

UDP-glucuronosyltransferase 1A1 (UGT1A1) is a vital metabolic enzyme responsible for the clearance of endogenous substances and drugs. Hitherto, the development of fluorescent probes for UGTs was severely restricted due to the poor isoform selectivity and on-off or blue-shifted fluorescence response. Herein, we established a novel "molecular-splicing" strategy to construct a highly selective near-infrared (NIR) fluorescent probe, HHC, for UGT1A1, which exhibited a NIR signal at 720 nm after UGT1A1 metabolism. HHC was then successfully used for the real-time imaging of endogenous UGT1A1 in living cells and animals and to monitor the bile excretion function. In summary, an isoform-specific NIR fluorescent probe has been developed for monitoring UGT1A1 activity in living systems, high-throughput screening of novel UGT1A1 inhibitors and visual evaluation of bile excretion function.

Ontogeny of Small Intestinal Drug Transporters and Metabolizing Enzymes Based on Targeted Quantitative Proteomics.

Most drugs are administered to children orally. An information gap remains on the protein abundance of small intestinal drug-metabolizing enzymes (DMEs) and drug transporters (DTs) across the pediatric age range, which hinders precision dosing in children. To explore age-related differences in DMEs and DTs, surgical leftover intestinal tissues from pediatric and adult jejunum and ileum were collected and analyzed by targeted quantitative proteomics for apical sodium-bile acid transporter, breast cancer resistance protein (BCRP), monocarboxylate transporter 1 (MCT1), multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein (MRP) 2, MRP3, organic anion-transporting polypeptide 2B1, organic cation transporter 1, peptide transporter 1 (PEPT1), CYP2C19, CYP3A4, CYP3A5, UDP glucuronosyltransferase (UGT) 1A1, UGT1A10, and UGT2B7. Samples from 58 children (48 ileums, 10 jejunums, age range: 8 weeks to 17 years) and 16 adults (8 ileums, 8 jejunums) were analyzed. When comparing age groups, BCRP, MDR1, PEPT1, and UGT1A1 abundance was significantly higher in adult ileum as compared with the pediatric ileum. Jejunal BCRP, MRP2, UGT1A1, and CYP3A4 abundance was higher in the adults compared with children 0-2 years of age. Examining the data on a continuous age scale showed that PEPT1 and UGT1A1 abundance was significantly higher, whereas MCT1 and UGT2B7 abundance was lower in adult ileum as compared with the pediatric ileum. Our data contribute to the deeper understanding of the ontogeny of small intestinal drug-metabolizing enzymes and drug transporters and shows DME-, DT-, and intestinal location-specific, age-related changes. SIGNIFICANCE STATEMENT: This is the first study that describes the ontogeny of small intestinal DTs and DMEs in human using liquid chromatography with tandem mass spectrometry-based targeted quantitative proteomics. The current analysis provides a detailed picture about the maturation of DT and DME abundances in the human jejunum and ileum. The presented results supply age-related DT and DME abundance data for building more accurate PBPK models that serve to support safer and more efficient drug dosing regimens for the pediatric population.

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

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

The importance of the UGT1A1 variants in the development of osteopenia and osteoporosis in postmenopausal women

The UDP-glucuronosyltransferase 1A1 (UGT1A1) is involved in the process of estrogen conjugation and elimination. The aim of the study was to analyze whether the UGT1A1 genetic variants are associated with the development of osteopenia and osteoporosis in postmenopausal women. The analysis of the rs4148323 (UGT1A1*6) and rs3064744 (UGT1A1*28) variants in the UGT1A1 gene was conducted using real-time PCR. A significant correlation was observed between the genotypes of the rs3064744 (UGT1A1*28) sequence variant and body mass in women with osteoporosis. The analysis of the Z-score values revealed that women with osteoporosis and carrying the 6/6 variant had the lowest Z-score values as compared to women with the 6/7 and the 7/7 variants (− 1.966 ± 0.242 vs. − 1.577 ± 0.125 and − 1.839 ± 0.233). In addition, the odds ratio for the investigated genotypes (6/6, 6/7, 7/7) indicated an increased risk for osteopenia and osteoporosis in women with the 7/7 homozygous genotype. The analysis of the frequencies of the GG, GA and AA genotypes of the rs4148323 UGT1A1 gene showed no statistically significant differences between the groups. Our analysis revealed that the UGT1A1 rs3064744 variant may affect the risk of developing osteoporosis in postmenopausal Polish women. The UGT1A1 rs4148323 variant is not directly associated with the development of osteopenia and osteoporosis.

Metabolite identification of iridin in rats by using UHPLC-MS/MS and pharmacokinetic study of its metabolite irigenin

Iridin, one of the main bioactive components isolated from Belamcanda chinensis (L.) DC, exerts various pharmacological activities, such as anti-inflammation, antioxidant, and antitumor. However, the metabolism and pharmacokinetics of iridin are still unknown. After 100 mg/kg administration of iridin orally, the plasma, urine, and fecal bio-samples from Sprague–Dawley (SD) rats were collected and detected by ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). The pharmacokinetics of the major metabolite irigenin (aglycon of iridin) and a total of thirteen metabolites of iridin were identified, including five metabolites in plasma, ten metabolites in urine, and six metabolites in feces. The most principal metabolic pathway of iridin was glucuronidation after demethylation and was mediated by UDP-glucuronosyltransferases (UGTs) 1A7, 1A8, 1A9 and 1A10. This study highlights the first-time investigation of the metabolism of iridin in vivo, and the pharmacokinetics of irigenin (the major metabolite of iridin) in rats. These results provide robust evidence for further research and clinical application of iridin.

Upregulation of UGT1A1 expression by ursolic acid and oleanolic acid via the inhibition of the PKC/NF-κB signaling pathway

BackgroundIsomeric ursolic acid (UA) and oleanolic acid (OA) compounds have recently garnered great attention due to their biological effects. Previously, it had been shown that UA and OA can exert important pharmacological action via the protein kinase C (PKC) and nuclear factor-κB (NF-κB) signaling, and that they can induce the expression of UDP-glucuronosyltransferase 1A1 (UGT1A1) in HepG2 cells. This study aims to investigate the role of PKC/NF-κB signaling in regulating the expression of UGT1A1 and examine how UA and OA induce UGT1A1 based on this signaling pathway. MethodsHepG2 cells, hp65-overexpressed HepG2 cell and lentivirus-hp65-shRNA silenced HepG2 cells were stimulated with PKC/NF-κB specific agonists and inhibitors for 24 h in the presence or absence of UA and OA. The expression of UGT1A1, PKC, and NF-κB were determined by qRT-PCR, western blot, and dual-luciferase reporter gene assays. ResultsPKC/NF-κB activation downregulates UGT1A1 expression. This effect is countered by UA and OA treatment. Phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), the agonists of PKC and NF-κB signaling, respectively, significantly inhibit hp65-mediated UGT1A1 luciferase activity. UA, OA, and the PKC/NF-κB inhibitors suppress this effect. PMA and LPS do not affect UGT1A1 activity in p65-silenced HepG2 cells; however, UA and OA mildly influence UGT1A1 expression in these cells. ConclusionThe activation of PKC/NF-κB signaling can significantly downregulate UGT1A1 expression. By inhibiting the PKC/NF-κB signaling pathway, UA and OA promote UGT1A1 expression in HepG2 cells.

Drug Interactions Involving 17α-Ethinylestradiol: Considerations Beyond Cytochrome P450 3A Induction and Inhibition.

It is widely acknowledged that drug-drug interactions (DDIs) involving estrogen (17α-ethinylestradiol (EE))-containing oral contraceptives (OCs) are important. Consequently, sponsors of new molecular entities (NMEs) often conduct clinical studies with priority given to OCs as victims of cytochrome P450 (CYP) 3A (CYP3A) induction and inhibition. Such scenarios are reflected in the US Food and Drug Administration-issued guidance documentation related to OC DDI studies. Although CYP3A is important, OCs such as EE are metabolized by sulfotransferase 1E1 and UDP-glucuronosyltransferase (UGT) 1A1, expressed in the gut and liver, and so both can also serve as loci of victim OC DDI. Therefore, for any NME, one should carefully consider its induction and inhibition profile involving CYP3A4/5, UGT1A1, and SULT1E1. As DDI perpetrators, available clinical DDI data indicate that EE-containing OCs can induce (e.g., UGT1A4 and CYP2A6) and inhibit (CYP1A2 ≥ CYP2C19 > CYP3A4/5 > CYP2C8, CYP2B6, CYP2D6, and CYP2C9) various CYP forms. Although available in vitro CYP inhibition data do not explain such a graded inhibitory effect in vivo, it is hypothesized that EE differentially modulates CYP expression via potent agonism of the estrogen receptor expressed in the gut and liver. From the standpoint of the NME as potential OC DDI victim, therefore, it is important to assess its projected (pre-phase I) or known therapeutic index and pharmacokinetic profile (fraction absorbed, absolute oral bioavailability, clearance/extraction class, fraction metabolized by CYP1A2, CYP2C19, CYP2A6, and UGT1A4). Such information can enable the prioritization, design, and interpretation of NME-OC DDI studies.

A highly selective fluorescent probe for real-time imaging of UDP-glucuronosyltransferase 1A8 in living cells and tissues

A highly selective fluorescent probe for real-time imaging of UDP-glucuronosyltransferase 1A8 in living cells and tissues

Yinzhihuang Oral Liquid Ameliorates Hyperbilirubinemia Induced by δ-Aminolevulinic Acid and Novobiocin in Neonatal Rats.

Yinzhihuang oral liquid (YZH) is a traditional Chinese medicine that has been widely used in Asia to prevent and treat neonatal hyperbilirubinemia, but the published preclinical studies on its anti-hyperbilirubinemia effect are conducted in adult animals, partly due to the lack of preclinical neonatal hyperbilirubinemia animal models. In the present study, we tested six reagents to induce hyperbilirubinemia in neonatal rats, and established two appropriate neonatal hyperbilirubinemia rat models by subcutaneous injection of δ-Aminolevulinic acid (ALA, 200 mg/kg) or novobiocin (NOVO, 200 mg/kg). Oral treatment of YZH (80, 160 and 320 mg/kg) significantly decreased serum conjugated bilirubin levels in ALA-treated neonatal rats and serum unconjugated bilirubin levels in NOVO-treated neonatal rats, respectively. Additionally, pre-treatment of YZH also prevented the increase of serum bilirubin levels in both ALA- and NOVO-treated rats. Mechanistically, YZH significantly up-regulated the mRNA expression of genes involved in hepatic bilirubin disposition (organic anion-transporting polypeptide 1b2, Oatp1b2; multidrug resistance-associated protein 2, Mrp2) and bilirubin conjugation (UDP-glucuronosyltransferase 1a1, Ugt1a1). Additionally, YZH up-regulated the mRNA expression of cytochrome P450 1A1 (Cyp1a1), the target gene of aryl hydrocarbon receptor (AhR), and increased the nuclear protein levels of AhR in livers of neonatal rats. YZH and its two active ingredients, namely baicalin (BCL) and 4'-hydroxyacetophenone (4-HT), up-regulated the mRNA expression of AhR target genes (CYP1A1 and UGT1A1) and increased nuclear protein levels of AhR in HepG2 cells. In conclusion, the present study provides two neonatal hyperbilirubinemia animal models and evaluates the anti-hyperbilirubinemia effect and mechanisms of YZH in neonatal animals.

Metabolism of 2,3-Dehydrosilybin A and 2,3-Dehydrosilybin B: A Study with Human Hepatocytes and Recombinant UDP-Glucuronosyltransferases and Sulfotransferases.

2,3-Dehydrosilybin A and 2,3-dehydrosilybin B are a pair of enantiomers formed by the oxidation of the natural flavonolignans silybin A and silybin B, respectively. However, the antioxidant activity of 2,3-dehydrosilybin molecules is much stronger than that of their precursors. Here, we investigated the biotransformation of pure 2,3-dehydrosilybin A and 2,3-dehydrosilybin B in isolated human hepatocytes, and we also aimed to identify human UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) with activity toward their respective enantiomers. After incubation with hepatocytes, both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B were converted to hydroxyl derivatives, methylated hydroxyl derivatives, methyl derivatives, sulfates, and glucuronides. The products of direct conjugations predominated over those of oxidative metabolism, and glucuronides were the most abundant metabolites. Furthermore, we found that recombinant human UGTs 1A1, 1A3, 1A7, 1A8, 1A9, and 1A10 were capable of catalyzing the glucuronidation of both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B. UGTs 1A1 and 1A7 showed the highest activity toward 2,3-dehydrosilybin A, and UGT1A9 showed the highest activity toward 2,3-dehydrosilybin B. The sulfation of 2,3-dehydrosilybin A and B was catalyzed by SULTs 1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1C2, 1C4, and 1E1, of which SULT1A3 exhibited the highest activity toward both enantiomers. We conclude that 2,3-dehydrosilybin A and B are preferentially metabolized by conjugation reactions, and that several human UGT and SULT enzymes may play a role in these conjugations.

Inhibitory effects of quercetin and its major metabolite quercetin-3-O-β-D-glucoside on human UDP-glucuronosyltransferase 1A isoforms by liquid chromatography-tandem mass spectrometry.

Quercetin is a flavonoid that is widely present in plant-derived food. Quercetin-3-O-β-D-glucoside (Q3GA) is a predominant metabolite of quercetin in animal and human plasma. The inhibitory effects of the UDP-glucuronosyl transferases (UGTs) caused by herbal components may be a key factor for the clinical assessment of herb-drug interactions (HDIs). The present study aimed to investigate the inhibitory profile of quercetin and Q3GA on recombinant UGT1A isoforms in vitro. The metabolism of the nonspecific substrate 4-methylumbelliferone (4-MU) by the UGT1A isoforms was assessed by liquid chromatography-tandem mass spectrometry. Preliminary screening experiments indicated that quercetin exhibited stronger inhibitory effects on UGT1A1, UGT1A3, UGT1A6 and UGT1A9 enzymes than Q3GA. Kinetic experiments were performed to characterize the type of inhibition caused by quercetin and Q3GA towards these UGT isoforms. Quercetin exerted non-competitive inhibition on UGT1A1 and UGT1A6, with half maximal inhibitory concentration (IC50) values of 7.47 and 7.07 µM and inhibition kinetic parameter (Ki) values of 2.18 and 28.87 µM, respectively. Quercetin also exhibited competitive inhibition on UGT1A3 and UGT1A9, with IC50 values of 10.58 and 2.81 µM and Ki values of 1.60 and 0.51 µM, respectively. However, Q3GA displayed weak inhibition on UGT1A1, UGT1A3 and UGT1A6 enzymes with IC50 values of 45.21, 106.5 and 51.37 µM, respectively. In the present study, quercetin was a moderate inhibitor of UGT1A1 and UGT1A3, a weak inhibitor of UGT1A6, and a strong inhibitor on UGT1A9. The results of the present study suggested potential HDIs that may occur following quercetin co-administration with drugs that are mainly metabolized by UGT1A1, UGT1A3 and UGT1A9 enzymes.

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

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

TaqMan real time PCR for the Detection of the Gilbert's Syndrome Markers UGT1A1*28; UGT1A1*36 and UGT1A1*37.

Gilbert's syndrome is characterized by mild unconjugated hyperbilirubinemia. The key of this disease is a diminished activity of UDP-glucuronosyltransferase 1A1 (UGT1A1). TA insertion into the TATA box promoter region of the UGT1A1 gene on chromosome 2 is the genetic basis of Gilbert's syndrome (UGT1A1*28). An extra TA insert leads to eight (TA)8 repeats (UGT1A1*37) resulting in a further reduction of glucuronidation activity. A variant lacking one TA repeat (TA)5 (UGT1A1*36) has been identified. (TA)8 repeats (UGT1A1*37) and (TA)5 (UGT1A1*36) have been detected in Africans (frequency up to 0.07 and 0.08 respectively). We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR on 7500 and cfx96 Real-Time PCR System. We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR. About clinical validation, all 53 samples collected from patients referred for suspected Gilbert's syndrome were analyzed. We found 21 on the 53 patients (39.6%) were homozygotes (UGT1A1-TATA (TA)6) and referred as wild-type, 13 on the 53 patients (24.5%) were homozygotes (UGT1A1-TATA (TA)7) and referred as mutated, 1 on the 53 patients (1.9%) were homozygotes (UGT1A1-TATA (TA)8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)7/8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)5/6) and referred as mutated, and 16 on the 53 patients (30.2%) were heterozygotes (UGT1A1-TATA (TA)6/7). None were homozygotes UGT1A1-TATA (TA)5, homozygotes UGT1A1-TATA (TA)8, or heterozygotes with (TA)5 or (TA)8 alleles. The newly described technique represents a valid alternative method to sequencing, mainly due to its rapidity, easiness, and minor costs.

Yes-Associated Protein Is Crucial for Constitutive Androstane Receptor-Driven Hepatocyte Proliferation But Not for Induction of Drug Metabolism Genes in Mice.

Background and AimsConstitutive androstane receptor (CAR) agonists, such as 1,4‐bis [2‐(3,5‐dichloropyridyloxy)] benzene (TCPOBOP), are known to cause robust hepatocyte proliferation and hepatomegaly in mice along with induction of drug metabolism genes without any associated liver injury. Yes‐associated protein (Yap) is a key transcription regulator that tightly controls organ size, including that of liver. Our and other previous studies suggested increased nuclear localization and activation of Yap after TCPOBOP treatment in mice and the potential role of Yap in CAR‐driven proliferative response. Here, we investigated a direct role of Yap in CAR‐driven hepatomegaly and hepatocyte proliferation using hepatocyte‐specific Yap‐knockout (KO) mice.Approach and ResultsAdeno‐associated virus 8‐thyroxine binding globulin promoter‐Cre recombinase vector was injected to Yap‐floxed mice for achieving hepatocyte‐specific Yap deletion followed by TCPOBOP treatment. Yap deletion did not decrease protein expression of CAR or CAR‐driven induction of drug metabolism genes (including cytochrome P450 [Cyp] 2b10, Cyp2c55, and UDP‐glucuronosyltransferase 1a1 [Ugt1a1]). However, Yap deletion substantially reduced TCPOBOP‐induced hepatocyte proliferation. TCPOBOP‐driven cell cycle activation was disrupted in Yap‐KO mice because of delayed (and decreased) induction of cyclin D1 and higher expression of p21, resulting in decreased phosphorylation of retinoblastoma protein. Furthermore, the induction of other cyclins, which are sequentially involved in progression through cell cycle (including cyclin E1, A2, and B1), and important mitotic regulators (such as Aurora B kinase and polo‐like kinase 1) was remarkably reduced in Yap‐KO mice. Microarray analysis revealed that 26% of TCPOBOP‐responsive genes that were mainly related to proliferation, but not to drug metabolism, were altered by Yap deletion. Yap regulated these proliferation genes through alerting expression of Myc and forkhead box protein M1, two critical transcriptional regulators of CAR‐mediated hepatocyte proliferation.ConclusionsOur study revealed an important role of Yap signaling in CAR‐driven hepatocyte proliferation; however, CAR‐driven induction of drug metabolism genes was independent of Yap.

UGT1A1 Gene Polymorphisms in Patients with Small Cell Lung Cancer Treated with Irinotecan-Platinum Doublet Chemotherapy and Their Association with Gastrointestinal Toxicity and Overall Survival.

Irinotecan (CPT11) is an important drug for small cell lung cancer (SCLC) chemotherapy (CTx). UDP-glucuronosyltransferase 1A1 (UGT1A1) polymorphisms can influence CPT11-related toxicity. This study aimed to assess prevalence of UGT1A1 polymorphisms and their association with clinical outcomes in patients with SCLC on CPT11-CTx. An observational cohort of treatment-naïve patients with SCLC was given CPT11-platinum doublet at a referral center in North India over 3 years. Clinical outcomes assessed were hematological and gastrointestinal toxicity (Common Terminology Criteria for Adverse Events, version 3.0), response rates (RECIST), and overall survival (OS). Peripheral blood was drawn from all enrolled patients just prior to CPT11-CTx initiation, and genomic DNA was isolated. Genotyping was done by polymerase chain reaction (PCR)-restriction fragment length polymorphism for UGT1A1*7, UGT1A1*6, and UGT1A1*27 and by PCR-DNA sequencing for UGT1A1*28. Patients were classified as homozygous wild-type (WT/WT), heterozygous mutant (WT/M), or homozygous mutant (M/M) for each polymorphism assessed. Of 140 patients enrolled, no mutant alleles were found for UGT1A1*27 or UGT1A1*7. Frequency of UGT1A1*6 polymorphisms (n = 111) was 89.2% for WT/WT, 8.1% for WT/M, and 2.7% for M/M. For UGT1A1*28 (n = 102), this was 41.2% for WT/WT, 43.1% for WT/M, and 15.7% for M/M. UGT1A1*6 WT/WT patients tolerated >95% predicted CPT11 dose more frequently (59.6% vs. 25.0% in WT/M-M/M combined group; p = .026). The UGT1A1*6 WT/M-M/M group also experienced severe (grade ≥3) diarrhea (41.7% vs. 17.2% in WT/WT; p = .044) and mucositis (41.7% vs. 8.1% in WT/WT; p = .005) more frequently. On multivariate logistic regression analysis, UGT1A1*6 WT/M-M/M status was the only variable associated with occurrence of both mucositis (odds ratio [OR], 10.4) and severe diarrhea (OR, 4.8). UGT1A1*28 WT/M-M/M patients had better OS (320 days [95% confidence interval, 203-437] vs. 216 days [95% confidence interval, 140-292] in WT/WT group; p = .047). On multivariate Cox proportional hazards analysis, UGT1A1*28 WT/M-M/M status was independently associated with better OS (hazard ratio, 0.35), whereas lack of objective radiological response, moderate/heavy smoking, and increasing age were associated with worse OS. UGT1A1*6 and UGT1A1*28 polymorphisms were associated with increased gastrointestinal toxicity and improved OS, respectively, in North Indian patients with SCLC receiving CPT11-CTx. UGT1A1 gene polymorphisms are known to influence irinotecan-related toxicity. In this prospective cohort study of patients with small cell lung cancer receiving first-line irinotecan-platinum chemotherapy, the prevalence of UGT1A1*6 polymorphisms was found to be 10.8% UGT1A1*6 and 58.8% UGT1A1*28 homo/heterozygous mutant, respectively. UGT1A1*6 homo/heterozygous mutant status was associated with severe diarrhea and mucositis. UGT1A1*28 homo/heterozygous mutant status was independently associated with improved overall survival.

In vitro evidence suggesting that the toll-like receptor 7 and 8 agonist resiquimod (R-848) unlikely affects drug levels of co-administered compounds

Resiquimod (R-848) is an immune response modifier activating toll-like receptor 7 and 8. Its potential to cause pharmacokinetic interactions with concurrently administered drugs is unknown.To study the time course of the effect of resiquimod in LS180 cells as a model for intestinal tissue, luciferase-based reporter gene assays and reverse transcription polymerase chain reaction were used to investigate whether resiquimod affects the activities of nuclear factor kappa B (NF-ĸB), pregnane x receptor (PXR) or the transcription of selected central genes for drug disposition (cytochrome P-450 isozyme 3A4 (CYP3A4), CYP1A1, UDP-glucuronosyltransferase 1A1 (UGT1A1), ATP-binding cassette transporters ABCC2, ABCB1). Its impact on the activities of organic anion transporting polypeptides 1 or 3 (OATP1B1/3), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp) or CYP3A4 was evaluated using fluorescence- or luminescence-based activity assays.Resiquimod irrelevantly increased NF-ĸB activity after 2 h (1 µM: 1.07-fold, P = 0.0188; 10 µM: 1.09-fold, P = 0.0142), and diminished it after 24 h (1 µM: 0.64-fold, P < 0.0001; 10 µM: 0.68-fold, P < 0.0001) and 30 h (10 µM: 0.68-fold, P = 0.0003). Concurrently, PXR activity after 24 h was marginally increased by 10 µM (1.05-fold, P = 0.0019). Resiquimod did not alter mRNA expression levels, activities of uptake or efflux transporters, or CYP3A4 activity.Given the marginal effects on NF-ĸB, PXR, expression levels of selected PXR target genes, and activities of important drug transporters and CYP3A4 in vitro, resiquimod is not expected to cause major pharmacokinetic drug-drug interactions in vivo.