UDP-glucuronosyltransferase Research Articles

Mechanism of dasabuvir inhibition of acetaminophen glucuronidation.

Acetaminophen (APAP) (paracetamol) is a widely used non-prescription drug for pain relief and antipyretic effects. The clearance of APAP is mainly through phase-2 biotransformation catalysed by UDP-glucuronosyl transferases (UGT). Dasabuvir is an anti-hepatitis C drug reported to inhibit several UGT isoforms. The study evaluated the in-vitro inhibitory capacity of dasabuvir versus APAP glucuronidation. Procedures included human liver microsomal incubations with APAP and isoform-selective probe substrates. Dasabuvir inhibited APAP metabolism by a reversible, mixed-type (competitive and non-competitive) partial inhibition, with an inhibition constant Ki = 3.4 µM. The index constant 'a' was 6.7, indicating the relative contribution of competitive and non-competitive inhibition. The enzyme-inhibitor complex was still able to catalyse the reaction by 12% of the control capacity. Dasabuvir produced strong partial inhibition effect of UGT1A1 and UGT1A9 and relatively complete inhibition of UGT1A6. Consistent with previous reports, dasabuvir inhibits the activity of 3 UGT isoforms associated with APAP metabolism. In-vitro to in-vivo scaling by 2 different approaches showed identical results, predicting an increased AUC of APAP by a factor of 1.3-fold with coadministration of dasabuvir. Until the findings are confirmed in clinical drug interaction studies, APAP dosage should not exceed 3 g per day in dasabuvir-treated patients to avoid potentially hepatotoxic APAP exposures.

Nrf2-Dependent Protective Effect of Paeoniflorin on α-Naphthalene Isothiocyanate-Induced Hepatic Injury.

The pathological mechanism of cholestatic hepatic injury is associated with oxidative stress, hepatocyte inflammation, and dysregulation of hepatocyte transporters. Paeonia lactiflora Pall. and its compound can improve hepatic microcirculation, dilate bile duct, and promote bile flow, which is advantageous to ameliorate liver damage. Paeoniflorin (PEA), as the main efficacy component of Paeonia lactiflora Pall., has multiple pharmacological effects. PEA improves liver injury, but it remains obscure whether the protective action on [Formula: see text]-naphthalene isothiocyanate (ANIT)-induced cholestatic liver injury is dependent on the NF-E2 p45-related Factor 2 (Nrf2) signaling pathway. In this study, C57BL/6 mice were administrated with 80 mg⋅kg[Formula: see text]⋅d[Formula: see text] ANIT followed by PEA (75, 150, and 300 mg⋅kg[Formula: see text]⋅d[Formula: see text]) orally for 10 days, respectively. Tissue histology and liver function were detected, including serum enzymes, gallbladder (GB) weight, phenobarbital-induced sleeping time (PEN-induced ST), hepatic uridine di-phosphoglucuronosyltransferase (UDPG-T), malondialdehyde (MDA), and glutathione (GSH). The expressions of protein Nrf2, sodium taurocholate cotransporting polypeptide (Ntcp), and NADPH oxidase 4 (Nox4) were evaluated. Nrf2 plasmid or siRNA-Nrf2 transfection on LO2 cells and Nrf2-/- mice were used to explore the liver protective mechanism of PEA. Compared to ANIT-treated mice, PEA decreased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), direct bilirubin (DBIL), total bile acid (TBA), and phenobarbital-induced sleeping time. The bile secretion, hepatic UDPG-T, MDA, GSH, and liver histology were improved. The expressions of protein Nrf2 and Ntcp in liver tissues increased, but Nox4 decreased. After Nrf2 plasmid or small interfering RNA (siRNA)-Nrf2 transfection, the protective effects of PEA on LO2 cells were, respectively, strengthened or weakened. Moreover, PEA had no significant effects on ANIT-treated Nrf2-/- mice. Our results suggest that Nrf2 is essential for PEA protective effects on ANIT-induced liver injury.

Assessment of susceptibility to phthalate and DINCH exposure through CYP and UGT single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs) of cytochrome P450 (CYPs) and UDP-glucuronosyltransferase (UGTs) genes have been proposed to influence phthalates and 1,2-cyclo-hexanedicarboxylic acid diisononyl ester (DINCH) biotransformation but have not been investigated on a populational level. We investigated the role of SNPs in CYP2C9, CYP2C19, CYP2D6, UGT2B15, and UGT1A7 genes in the biotransformation of phthalates (DEHP, DEP, DiBP, DnBP, BBzP, DiNP, DidP) and DINCH by determining their urine metabolites. From the Slovenian study population of 274 men and 289 lactating primiparous women we obtained data on phthalate and DINCH urine metabolite levels (MEHP, 5OH-MEHP, 5oxo-MEHP, 5cx-MEPP, MEP, MiBP, MnBP, MBzP, cx-MINP, OH-MiDP, MCHP, MnPeP, MnOP, 5OH-MINCH, 5oxo-MINCH), SNP genotypes (rs1057910=CYP2C9*3, rs1799853=CYP2C9*2, rs4244285=CYP2C19*2, rs12248560=CYP2C19*17, rs3892097=CYP2D6*4, rs1902023=UGT2B15*2, and rs11692021=UGT1A7*3) and questionnaires. Associations of SNPs with levels of metabolites and their ratios were assessed by multiple linear regression and ordinary logistic regression analyses. Significant associations were observed for CYP2C9*2, CYP2C9*3, CYP2C19*17, and UGT1A7*3 SNPs. The most pronounced was the influence of CYP2C9*2 and *3 on the reduced DEHP biotransformation, with lower levels of metabolites and their ratios in men and women. In contrast, carriers of CYP2C19*17 showed higher urine levels of DEHP metabolites in both genders, and in women also in higher DiNP, DiDP, and DINCH metabolite levels. The presence of UGT1A7*3 was associated with increased metabolite levels of DINCH in men and of DiBP and DBzP in women. Statistical models explained up to 27% of variability in metabolite levels or their ratios. Our observations confirm the effect of CYP2C9*2 and *3 SNPs towards reduced DEHP biotransformation. We show that CYP2C9*2, CYP2C9*3, CYP2C19*17, and UGT1A7*3 SNPs might represent biomarkers of susceptibility or resilience in phthalates and DINCH exposure that have been so far unrecognised.

The Effects of Clofibrate on Neonatal Jaundice: A Systematic Review.

Background:Neonatal jaundice is a prevalent disease that causes many complications, including kernicterus and even death. Previous studies have shown that clofibrate as an aryloxy isobutyric acid derivate can be effectively applied for the treatment of neonatal jaundice. Thus, this review was carried out to investigate the effects and mechanism of action of clofibrate on neonatal jaundice.Methods:The keywords such as “Clofibrate” in combination with “Neonatal jaundice” or “Neonatal hyperbilirubinemia” or “Newborn Jaundice” were used to search for relevant publications indexed in the Institute for Scientific Information (ISI), Scopus, PubMed, and Google Scholar databases. Finally, after reviewing the studies, 24 papers were included in this study.Results:Results showed that the processes of albumin-bound bilirubin transfer to the hepatocytes, hepatic uptake, and storage via ligandin, hepatic conjugation via uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), conjugation into the bile via MRP2 represent the main action mechanism of clofibrate that turns it into the bilirubin conjugates and expels it from the bile. Besides, clofibrate has been shown to reduce the level of Total Serum Bilirubin (TSB) in infants even at a dosage of 25 mg/kg without leaving side effects.Conclusions:The results of this review revealed that clofibrate effectively reduces TSB in short-term usage and can even have a promising effect at the dosage of 25 mg/kg in full-term infants. Most studies have shown this property over a short period in term infants, and there is no evidence about long-term usage in this regard.

A systemic review of association between UDP glucuronosyltransferase family 1 member A1 (UGT1A1) polymorphisms in Gilbert's syndrome in Sickle Cell Disease

Gilbert's syndrome (GS) is a benign hereditary disorder of bilirubin metabolism due to a mutation in the UDP glucuronosyltransferase family 1 member A1 (UGT1A1) gene which results in hyperbilirubinaemia and related complications mainly cholelithiasis. It can be co-inherited along with sickle cell anaemia, thalassaemias and other haemoglobinopathies including glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis and cystic fibrosis. More than 100 mutations have been reported in UGT1A1 gene and the most common as insertion of extra (TA) nucleotides in the promoter region of TATA box. The more the number of TA repeats, the higher is the bilirubin levels. These mutations result in a 10%–35% reduction in the UGT1A1 enzyme activity resulting in mild to moderate unconjugated hyperbilirubinaemia and related complications. For diagnosis the mode of inheritance is more important than testing in the patients. However; the inheritance pattern of GS differs in ethnicities. For early diagnosis to prevent worsening of the symptoms and for timely management one should be aware of the inheritance pattern in patient. In this systemic analysis we studied the association between complications in GS with the genotypes and complications. It was found that TA7/7 is more significant in GS with sickle cell disease (SCD) group when compared to healthy controls with 2.2% chances of having this genotype in GS with SCD than healthy controls. The significance of having TA7/7 genotype is similar in GS with SCD and α-thalasaemia group. However, there is a high recommendation to carry out multicentre studies and conduct meta-analyses for establishing universal recommendations.

The potency of red onion skin flavonoids in acetaminophen-induced liver injury management: A biomolecular review

Acetaminophen is widely used as a fever and pain reliever drug. However, acetaminophen intoxication was common and responsible for thousands of acetaminophen-induced liver injury (AILI) cases worldwide. It was understood that glutathione (GSH) depletion causes a deposit of acetaminophen metabolites which are toxic to liver cells. N-acetylcysteine (NAC), used as acetaminophen intoxication therapy, has been reported to cause side effects such as nausea, vomiting, and anaphylactic reaction. This narrative review aimed to discuss other potential candidates in AILI management. The biomolecular approach was used to investigate onion skin in-depth and holistic discussion. According to the results, onion peel extract is rich in flavonoids (quercetin and kaempferol) and potentially be a candidate in the management of AILI through proposed mechanisms such as restoration of reduced glutathione (GSH), inhibition of cytochrome P450 (CYP) enzymes, upregulation of uridine 5'-diphospho-glucuronosyltransferase (UGT) enzyme, and inhibition of TLR4 activity.

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.

Comparison of Tissue Abundance of Non-Cytochrome P450 Drug-Metabolizing Enzymes by Quantitative Proteomics between Humans and Laboratory Animal Species.

The use of animal pharmacokinetic models as surrogates for humans relies on the assumption that the drug disposition mechanisms are similar between preclinical species and humans. However, significant cross-species differences exist in the tissue distribution and protein abundance of drug-metabolizing enzymes (DMEs) and transporters. We quantified non-cytochrome P450 (non-CYP) DMEs across commonly used preclinical species (cynomolgus and rhesus monkeys, beagle dog, Sprague Dawley and Wistar Han rats, and CD1 mouse) and compared these data with previously obtained human data. Aldehyde oxidase was abundant in humans and monkeys while poorly expressed in rodents, and not expressed in dogs. Carboxylesterase (CES) 1 abundance was highest in the liver while CES2 was primarily expressed in the intestine in all species with notable species differences. For example, hepatic CES1 was 3× higher in humans than in monkeys, but hepatic CES2 was 3-5× higher in monkeys than in humans. Hepatic UDP-glucuronosyltransferase (UGT) 1A2 abundance was ∼4× higher in dogs compared with rats, whereas UGT1A3 abundance was 3-5× higher in dog livers than its ortholog in human and monkey livers. UGT1A6 abundance was 5-6× higher in human livers compared with monkey and dog livers. Hepatic sulfotransferase 1B1 abundance was 5-7× higher in rats compared with the rest of the species. These quantitative non-CYP proteomics data can be used to explain unique toxicological profiles across species and can be integrated into physiologically based pharmacokinetic models for the mechanistic explanation of pharmacokinetics and tissue distribution of xenobiotics in animal species. SIGNIFICANCE STATEMENT: We characterized the quantitative differences in non-cytochrome P450 (non-CYP) drug-metabolizing enzymes across commonly used preclinical species (cynomolgus and rhesus monkeys, beagle dogs, Sprague Dawley and Wistar Han rats, and CD1 mice) and compared these data with previously obtained human data. Unique differences in non-CYP enzymes across species were observed, which can be used to explain significant pharmacokinetic and toxicokinetic differences between experimental animals and humans.

Association of Serum Bilirubin and Functional Variants of Heme Oxygenase 1 and Bilirubin UDP-Glucuronosyl Transferase Genes in Czech Adult Patients with Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disorder worldwide. The aim of our study was to assess the role of bilirubin, and the heme oxygenase 1 (HMOX1) and bilirubin UDP-glucuronosyl transferase (UGT1A1) promoter gene variants, which are involved in bilirubin homeostasis, in the NAFLD development in adult patients. The study was performed on 84 patients with NAFLD and 103 age/sex-matched controls. Routine biochemistry, inflammatory markers, adipokines, and the fibrosis/steatohepatitis stage were determined in the NAFLD patients. The (GT)n/(TA)n dinucleotide variations in HMOX1/UGT1A1 gene promoters, respectively, were analyzed by fragment analysis. Compared to controls, serum bilirubin concentrations in NAFLD patients tended to be decreased, while the prevalence of phenotypic Gilbert syndrome was significantly low. Genetic variations in HMOX1 and UGT1A1 gene promoters did not differ between NAFLD patients and controls, and no relationship was found in the NAFLD patients between these gene variants and any of the laboratory or histological parameters. In conclusion, metabolism of bilirubin is dysregulated in NAFLD patients, most likely due to increased oxidative stress, since frequencies of the major functional variants in the HMOX1 or UGT1A1 gene promoters did not have any effect on development of NAFLD in adult patients.

Inhibition of cytochrome P450 enzymes and uridine 5′-diphospho-glucuronosyltransferases by vicagrel in human liver microsomes: A prediction of potential drug-drug interactions

Vicagrel, an antiplatelet drug candidate targeting platelet P2Y12 receptor and has finished its phase II clinical trial. The inhibition of six major cytochrome P450 enzymes (P450) (CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and six UDP-glucuronosyltransferases (UGT) (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) by vicagrel was evaluated using pooled human liver microsomes and specific probe substrates. Physiology-based pharmacokinetic (PBPK) simulation was further applied to predict the in vivo drug-drug interaction (DDI) potential between vicagrel and bupropion as well as S-mephenytoin. The results suggested that vicagrel inhibited CYP2B6 and CYP2C19 potently with apparent IC50 values of 1.6 and 2.0 μM, respectively. In terms of mode of reversible inhibition, vicagrel exhibited mixed-type inhibition of CYP2B6-catalyzed bupropion hydroxylation and noncompetitive inhibition of CYP2C19-mediated S-mephenytoin 4′-hydroxylation with Ki values of 0.19 μM and 1.2 μM, respectively. Vicagrel displayed profound time-dependent inhibition towards CYP2B6 with maximal rate constant of inactivation (kinact) and half-maximal inactivator concentration (KI) values of 0.062 min−1 and 1.52 μM, respectively. No time-dependent inhibition by vicagrel was noted for CYP2C19. For UGT, negligible to moderate inhibition by vicagrel was observed with IC50 values of >50.0, >50.0, 28.2, 8.7, >50.0 and 28.2 μM for UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7, respectively. In terms of mode of reversible inhibition, vicagrel exhibited mixed-type inhibition of UGT1A6-catalyzed N-Acetylserotonin β-D-glucuronidation with a Ki value of 5.6 μM. No time-dependent inhibition by vicagrel was noted for UGT1A6. PBPK simulation indicated that neither altered AUC nor Cmax of bupropion and S-mephenytoin was observed in the presence of vicagrel. Our study provides inhibitory constants for future DDI prediction between vicagrel and drug substrates of CYP2B6, CYP2C19 and UGT1A6. In addition, our simulation suggests the lack of clinically important DDI between vicagrel and bupropion or S-mephenytoin.

Pharmacokinetics, Mass Balance, and Metabolism of the Novel Urate Transporter 1 Inhibitor [14C]HR011303 in Humans: Metabolism Is Mediated Predominantly by UDP-Glucuronosyltransferase.

HR011303, a promising selective urate transporter 1 inhibitor, is currently being studied in a phase III clinical trial in China for the treatment of hyperuricemia and gout. In the current study, the pharmacokinetics, mass balance, and metabolism of HR011303 were examined in six healthy Chinese male subjects who received a single oral dose of 10 mg of [14C]HR011303 (80 µCi). The results showed that HR011303 was rapidly absorbed with a median time to reach C max of 1.50 hours postdose, and the arithmetic mean half-life of total radioactivity was approximately 24.2 hours in plasma. The mean blood-to-plasma radioactivity concentration ratio was 0.66, suggesting the preferential distribution of drug-related components in plasma. At 216 hours postdose, the mean cumulative excreted radioactivity was 91.75% of the dose, including 81.50% in urine and 10.26% in feces. Six metabolites were identified, and the parent drug HR011303 was the most abundant component in plasma and feces, but a minor component in urine. Glucuronidation of the carboxylic acid moiety of HR011303 was the primary metabolic pathway in humans, amounting to 69.63% of the dose (M5, 51.57% of the dose; M5/2, 18.06% of the dose) in the urine; however, it was not detected in plasma. UDP-glucuronosyltransferase (UGT) 2B7 was responsible for the formation of M5. Overall, after a single oral dose of 10 mg of [14C]HR011303 (80 µCi), HR011303 and its main metabolites were eliminated via renal excretion. The major metabolic pathway was carboxylic acid glucuronidation, which was catalyzed predominantly by UGT2B7. SIGNIFICANCE STATEMENT: This study determined the absorption and disposition of HR011303, a selective urate transporter (URAT) 1 inhibitor currently in development for the treatment of hyperuricemia and gout. This work helps to characterize the major metabolic pathways of new URAT inhibitors and identify the absorption and clearance mechanism.

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Announcement of the 2022 winners of the Spychala and Women in Science Awards

The role of AKR1 family in tamoxifen resistant invasive lobular breast cancer based on data mining

BackgroundTamoxifen (TAM) resistance to invasive lobular cell carcinoma is a challenge for breast cancer treatment. This study explored the role of Aldo-keto reductase family 1 (AKR1) family in tamoxifen-resistant aggressive lobular breast cancer based on data mining.MethodsTAM-resistant invasive lobular breast cancer gene chip was downloaded from the Gene Expression Omnibus (GEO) database (accession-numbered as GSE96670). The online analytical tool GEO2R was used to screen for differentially expressed genes in TAM-resistant invasive lobular breast cancer cells and TAM-sensitive counterparts. A protein-protein interaction (PPI) networks were constructed using the STRING online platform and the Cytoscape software. GeneMANIA and GSCALite online tools were used to reveal the potential role of these hub genes in breast cancer progression and TAM resistance development. And the used the GSE67916 microarray data set to verify the differentially expression of these hub genes in breast cancer. The protein expression levels of AKR1C1, AKR1C2 and AKR1C3 in TAM-sensitive and resistant breast cancer cells were compared. The TAM sensitivity of breast cancer cells with or without AKR1C1, AKR1C2 or AKR1C3 gene manipulation was evaluated by cell viability assay.ResultsA total of 184 differentially expressed genes were screened. Compared with TAM sensitive breast cancer cells, 162 were up-regulated and 22 were down-regulated. The study identified several hub genes in the PPI network that may be involved in the development of TAM resistance of breast cancer, including signal transducer and activator of transcription 1 (STAT1), estrogen receptor alpha (ESR1), fibronectin1 (FN1), cytochrome P4501B1 (CYP1B1), AKR1C1, AKR1C2, AKR1C3 and uridine diphosphate glucuronosyltransferase (UGT) 1A family genes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10). Compared with TAM-sensitive counterparts, the expression levels of AKR1C1, AKR1C2, and AKR1C3 were up-regulated in TAM-resistant breast cancer cells.ConclusionsOverexpression of each of these three genes significantly increased the resistance of breast cancer cells to TAM treatment, while their knockdown showed opposite effects, indicating that they are potential therapeutic target for the treatment of TAM-resistant breast cancer.

UDP-glucuronosyltransferases mediate coffee-associated reduction of liver fibrosis in bile duct ligated humanized transgenic UGT1A mice.

Coffee consumption has been shown to reduce the risk of liver fibrosis and is capable of inducing human UDP-glucuronosyltransferase (UGT) 1A genes. UGT1A enzymes act as indirect antioxidants catalyzing the elimination of reactive metabolites, which in turn are potent initiators of profibrotic mechanisms. The aim of this study was to analyze the role of UGT1A genes as effectors of the protective properties of coffee in bile duct ligation (BDL) induced liver fibrosis. Fourteen days BDL with and without coffee pre- and co-treatment was performed in htgUGT1A-WT and htgUGT1A-SNP mice. Hepatic UGT1A mRNA expression levels, serum bilirubin and aminotransferase activities were determined. Liver fibrosis was assessed by collagen deposition, computational analysis of Sirius red tissue staining and expression of profibrotic marker genes. Oxidative stress was measured by hepatic peroxidase concentrations and immunofluorescence staining. UGT1A transcription was differentially activated in the livers of htgUGT1A-WT mice after BDL, in contrast to a reduced or absent induction in the presence of SNPs. Co-treated (coffee + BDL) htgUGT1A-WT-mice showed significantly increased UGT1A expression and protein levels and a considerably higher induction compared to water drinking WT mice (BDL), whereas in co-treated htgUGT1A-SNP mice absolute expression levels remained below those observed in htgUGT1A-WT mice. Collagen deposition, oxidative stress and the expression of profibrotic markers inversely correlated with UGT1A expression levels in htgUGT1A-WT and SNP mice after BDL and coffee + BDL co-treatment. Coffee exerts hepatoprotective and antioxidative effects via activation of UGT1A enzymes. Attenuated hepatic fibrosis as a result of coffee-mediated UGT1A induction during cholestasis was detected, while the protective action of coffee was lower in a common low-function UGT1A SNP haplotype present in 10% of the Caucasian population. This study suggests that coffee consumption might constitute a potential strategy to support the conventional treatment of cholestasis-related liver diseases.

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.

Simultaneous Quantification of Propylthiouracil and Its N-β-d Glucuronide by HPLC-MS/MS: Application to a Metabolic Study.

Propylthiouracil (PTU) is commonly prescribed for the management of hyperthyroidism and thyrotoxicosis. Although the exact mechanism of action is not fully understood, PTU is associated with hepatoxicity in pediatric population. Glucuronidation mediated by uridine 5′-diphospho-glucuronosyltransferases (UGTs), which possess age-dependent expression, has been proposed as an important metabolic pathway of PTU. To further examine the metabolism of PTU, a reliable HPLC-MS/MS method for the simultaneous quantification of PTU and its N-β-D glucuronide (PTU-GLU) was developed and validated. The chromatographic separation was achieved on a ZORBAX Extend-C18 column (2.1 × 50 mm, 1.8 μm) through gradient delivery of a mixture of formic acid, methanol and acetonitrile. The electrospray ionization (ESI) was operated in its negative ion mode while PTU and PTU-GLU were detected by multiple reaction monitoring (MRM). This analytical method displayed excellent linearity, sensitivity, accuracy, precision, recovery and stability while its matrix effect and carry-over were insignificant. Subsequently, the in vitro metabolism of PTU was assessed and UGT1A9 was identified as an important UGT isoform responsible for the glucuronidation of PTU. The information obtained from this study will facilitate future mechanistic investigation on the hepatoxicity of PTU and may optimize its clinical application.

Gene expression changes related to bone mineralization, blood pressure and lipid metabolism in mouse kidneys after space travel

Space travel burdens health by imposing considerable environmental stress associated with radioactivity and microgravity. In particular, gravity change predominantly impacts blood pressure and bone homeostasis, both of which are controlled mainly by the kidneys. Nuclear factor erythroid-2-related transcription factor 2 (Nrf2) plays essential roles in protecting the kidneys from various environmental stresses and injuries. To elucidate the effects of space travel on mammals in preparation for the upcoming space era, our study investigated the contribution of Nrf2 to kidney function in mice two days after their return from a 31-day stay in the International Space Station using Nrf2 knockout mice. Meaningfully, expression levels of genes regulating bone mineralization, blood pressure and lipid metabolism were found to be significantly altered in the kidneys after space travel in an Nrf2-independent manner. In particular, uridine diphosphate-glucuronosyltransferase 1A (Ugt1a) isoform genes were found to be expressed in an Nrf2-dependent manner and induced exclusively in the kidneys after return to Earth. Since spaceflight elevated the concentrations of fatty acids in the mouse plasma, we suggest that Ugt1a isoform expression in the kidneys was induced to promote glucuronidation of excessively accumulated lipids and excrete them into urine after the return from space. Thus, the kidneys were proven to play central roles in adaptation to gravity changes caused by going to and returning from space by controlling blood pressure and bone mineralization. Additionally, kidney Ugt1a isoform induction after space travel implies a significant role of the kidneys for space travelers in the excretion of excessive lipids.

Permeabilized Cryopreserved Human Hepatocytes as an Exogenous Metabolic System in a Novel Metabolism-Dependent Cytotoxicity Assay for the Evaluation of Metabolic Activation and Detoxification of Drugs Associated with Drug-Induced Liver Injuries: Results with Acetaminophen, Amiodarone, Cyclophosphamide, Ketoconazole, Nefazodone, and Troglitazone.

We report here a novel in vitro experimental system, the metabolism-dependent cytotoxicity assay (MDCA), for the definition of the roles of hepatic drug metabolism in toxicity. MDCA employs permeabilized cofactor-supplemented cryopreserved human hepatocytes (MetMax Human Hepatocytes, MMHH), as an exogenous metabolic activating system, and human embryonic kidney 293 (HEK293) cells, a cell line devoid of drug-metabolizing enzyme activity, as target cells for the quantification of drug toxicity. The assay was performed in the presence and absence of cofactors for key drug metabolism pathways known to play key roles in drug toxicity: NADPH/NAD+ for phase 1 oxidation, uridine 5'-diphosphoglucuronic acid (UDPGA) for uridine 5'-diphospho-glucuronosyltransferase (UGT) mediated glucuronidation, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for cytosolic sulfotransferase (SULT) mediated sulfation, and glutathione (GSH) for glutathione S-transferase (GST) mediated GSH conjugation. Six drugs with clinically significant hepatoxicity, resulting in liver failure or a need for liver transplantation: acetaminophen, amiodarone, cyclophosphamide, ketoconazole, nefazodone, and troglitazone were evaluated. All six drugs exhibited cytotoxicity enhancement by NADPH/NAD+, suggesting metabolic activation via phase 1 oxidation. Attenuation of cytotoxicity by UDPGA was observed for acetaminophen, ketoconazole, and troglitazone, by PAPS for acetaminophen, ketoconazole, and troglitazone, and by GSH for all six drugs. Our results suggest that MDCA can be applied toward the elucidation of metabolic activation and detoxification pathways, providing information that can be applied in drug development to guide structure optimization to reduce toxicity and to aid the assessment of metabolism-based risk factors for drug toxicity. GSH detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites, a key property of drugs with idiosyncratic hepatotoxicity. SIGNIFICANCE STATEMENT: Application of the metabolism-dependent cytotoxicity assay (MDCA) for the elucidation of the roles of metabolic activation and detoxification pathways in drug toxicity may provide information to guide structure optimization in drug development to reduce hepatotoxic potential and to aid the assessment of metabolism-based risk factors. Glutathione (GSH) detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites that may be applied toward the evaluation of idiosyncratic hepatotoxicity.

Metabolite Diversity and Metabolic Genome-Wide Marker Association Studies (Mgwas) for Health Benefiting Nutritional Traits in Pearl Millet Grains.

As efforts are made to increase food security, millets are gaining increasing importance due to their excellent nutritional credentials. Among the millets, pearl millet is the predominant species possessing several health benefiting nutritional traits in its grain that are helpful in mitigating chronic illnesses such as type−2 diabetes and obesity. In this paper, we conducted metabolomic fingerprinting of 197 pearl millet inbred lines drawn randomly from within the world collection of pearl millet germplasm and report the extent of genetic variation for health benefitting metabolites in these genotypes. Metabolites were extracted from seeds and assessed using flow infusion high-resolution mass spectrometry (FIE-HRMS). Metabolite features (m/z), whose levels significantly differed among the germplasm inbred lines, were identified by ANOVA corrected for FDR and subjected to functional pathway analysis. A number of health-benefiting metabolites linked to dietary starch, antioxidants, vitamins, and lipid metabolism-related compounds were identified. Metabolic genome-wide association analysis (mGWAS) performed using the 396 m/z as phenotypic traits and the 76 K SNP as genotypic variants identified a total of 897 SNPs associated with health benefiting nutritional metabolite at the -log p-value ≤ 4.0. From these associations, 738 probable candidate genes were predicted to have an important role in starch, antioxidants, vitamins, and lipid metabolism. The mGWAS analysis focused on genes involved in starch branching (α-amylase, β-amylase), vitamin-K reductase, UDP-glucuronosyl, and UDP-glucosyl transferase (UGTs), L-ascorbate oxidase, and isoflavone 2′-monooxygenase genes, which are known to be linked to increases in human health benefiting metabolites. We demonstrate how metabolomic, genomic, and statistical approaches can be utilized to pinpoint genetic variations and their functions linked to key nutritional properties in pearl millet, which in turn can be bred into millets and other cereals crops using plant breeding methods.

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

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