[Show abstract][Hide abstract] ABSTRACT: The human DNAJB3 gene encodes a DNAJ (Heat shock protein 40; Hsp40) homolog, subfamily B, member 3 chaperone protein (DNAJB3), which can be down-regulated in disease conditions, as observed in decreased expression of DNAJB3 mRNA in peripheral blood mononuclear cells (PBMC) of obese patients. Recently, humanized UDP-glucuronosyltransferase (UGT) 1 mice (hUGT1 mice) were developed, in which the introduced human UGT1 gene contained a gene encoding human DNAJB3. In the present study, we analyzed the expression of human DNAJB3 mRNA in hUGT1 mice. Among the examined tissues, the testis had the highest expression of human DNAJB3 mRNA, while the lowest expression was observed in the liver. We found that the pattern of tissue-specific expression of mouse Dnajb3 in hUGT1 mice was very similar to that of human DNAJB3. We further demonstrated that the expression of human DNAJB3 in the liver was significantly reduced in high-fat-diet-fed hUGT1 mice compared to the expression level in the control mice, indicating that the expression of human DNAJB3 in hUGT1 mice could be similarly regulated in disease conditions such as obesity. Humanized UGT1 mice might therefore be useful to investigate the physiological role of human DNAJB3 in vivo.
Preview · Article · Jul 2015 · International Journal of Molecular Sciences
[Show abstract][Hide abstract] ABSTRACT: While UDP-glucuronosyltransferases (UGTs) are important phase II drug metabolizing enzymes, they are also involved in the metabolism of endogenous. Certain substrates of UGTs, such as serotonin and estradiol, play important roles in the brain. However, the expression of UGTs in human brain has not been fully clarified. Recently, humanized UGT1 mice (hUGT1 mice) in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus have been developed. In the present study, the expression pattern of UGT1As in brains from human and hUGT1 mice was examined. We found that UGT1A1, 1A3, 1A6, and 1A10 were expressed in human brains. The expression pattern of UGT1As in hUGT1 mouse brains was similar to that in human brains. In addition, we examined the expression of UGT1A1 and 1A6 in cerebellum, olfactory bulbs, midbrain, hippocampus, and cerebral cortex of hUGT1 mice. UGT1A1 in all brain regions and UGT1A6 in cerebellum and cerebral cortex of 6 month-old hUGT1 mice were expressed significantly higher than those of 2 week-old hUGT1 mice. A difference in expression levels between brain regions was also observed. Brain microsomes exhibited glucuronidation activities toward estradiol and serotonin with the mean values of 0.13 pmol/min/mg and 5.17 pmol/min/mg, respectively. In conclusion, UGT1A1 and UGT1A6 might play important role in function regulation of endogenous compounds in region- and age-dependent manner. Humanized UGT1 mice might be useful to study the importance of brain UGTs in vivo.
The American Society for Pharmacology and Experimental Therapeutics.
No preview · Article · May 2015 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: Inhibition of drug metabolizing enzymes is a major mechanism in drug-drug interactions (DDIs). A number of cases of DDIs via inhibition of UDP-glucuronosyltranseferases (UGTs) have been reported, although the changes in pharmacokinetics are relatively small in comparison to drugs that are metabolized by cytochrome P450s. Most of the past studies have investigated hepatic UGTs, though recent studies have revealed a significant contribution of UGTs in the small intestine to drug clearance. To evaluate potential DDIs caused by inhibition of intestinal UGTs, we assessed inhibitory effects of 578 compounds, including drugs, xenobiotics, and endobiotics, on human UGT1A8 and UGT1A10, which are major contributors to intestinal glucuronidation. We identified 29 inhibitors by monitoring raloxifene glucuronidation with recombinant UGTs. All of the inhibitors potently inhibited UGT1A1 activity, as well. We found that zafirlukast is a potent general inhibitor of UGT1As and a moderate inhibitor of UGT2Bs by monitoring 4-methylumbelliferone glucuronidation by recombinant UGTs. However, zafirlukast did not potently inhibit diclofenac glucuronidation, suggesting that the inhibitory effects might be substrate-specific. Inhibitory effects of zafirlukast on some UGT substrates were further investigated in HLM and HIM to evaluate potential DDIs. R values (ratios of intrinsic clearance with and without an inhibitor) revealed that zafirlukast has potential to cause clinical DDIs in the small intestine. Although we could not identify specific UGT1A8 and UGT1A10 inhibitors, zafirlukast was identified as a general inhibitor for UGTs in vitro. The present study suggests that the inhibition of UGT in the small intestine would be an underlying mechanism for DDIs.
The American Society for Pharmacology and Experimental Therapeutics.
No preview · Article · Apr 2015 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: Cordycepin, which is an analogue of a nucleoside adenosine, exhibits a wide variety of pharmacological activities including anticancer effects. In this study, ADA1- and ADA2-expressing HEK293 cells were established to determine the major ADA isoform responsible for the deamination of cordycepin. While the metabolic rate of cordycepin deamination was similar between ADA2-expressing and Mock cells, extensive metabolism of cordycepin was observed in the ADA1-expressing cells with Km and Vmax values of 54.9 μmol/L and 45.8 nmole/min/mg protein. Among five natural substances tested in this study (kaempferol, quercetin, myricetin, naringenin, and naringin), naringin strongly inhibited the deamination of cordycepin with Ki values of 58.8 μmol/L in mouse erythrocytes and 168.3 μmol/L in human erythrocytes. A treatment of Jurkat cells with a combination of cordycepin and naringin showed significant cytotoxicity. Our in silico study suggests that not only small molecules such as adenosine derivatives but also bulky molecules like naringin can be a potent ADA1 inhibitor for the clinical usage.
[Show abstract][Hide abstract] ABSTRACT: The major metabolic pathway of vildagliptin in mice, rats, dogs, and humans is hydrolysis at the cyano group to produce a carboxylic acid metabolite M20.7 (LAY151), while the major metabolic enzyme of vildagliptin has not been identified. In the present study, we determined the contribution rate of dipeptidyl peptidase-4 (DPP-4) to the hydrolysis of vildagliptin in the liver. We performed hydrolysis assay of the cyano group of vildagliptin using mouse, rat, and human liver samples. Additionally, DPP-4 activities in each liver sample were assessed by DPP-4 activity assay using the synthetic substrate Gly-Pro-AMC. M20.7 formation rates in liver microsomes were higher than those in liver cytosol. M20.7 formation rate was significantly positively correlated with the DPP-4 activity using Gly-Pro-AMC in liver samples (r = 0.917, P < 0.01). The formation of M20.7 in mouse, rat, and human liver S9 fraction was inhibited by sitagliptin, a selective DPP-4 inhibitor. These findings indicate that DPP-4 is greatly involved in vildagliptin hydrolysis in the liver. Additionally, we established stable single expression systems of human DPP-4 and its R623Q mutant, which is the non-synonymous single nucleotide polymorphism of human DPP-4, in HEK293 cells to investigate the effect of R623Q mutant on vildagliptin-hydrolyzing activity. M20.7 formation rate in HEK293 cells expressing human DPP-4 was significantly higher than that in control HEK293 cells. Interestingly, R623Q mutation resulted in a decrease of the vildagliptin-hydrolyzing activity. Our findings might be useful for the prediction of interindividual variability in vildagliptin pharmacokinetics.
The American Society for Pharmacology and Experimental Therapeutics.
No preview · Article · Jan 2015 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: Cytochrome P450 (CYP) 3A4 is a membrane protein that catalyzes hydroxylation of endogenous and exogenous substrates. Protein–protein interaction is a crucial factor that regulates the function of enzymes. However, protein–protein interactions involving human CYPs have not been fully understood. In this study, extensive protein–protein interactions involving CYP3A4 were determined by a shotgun analysis of immunoprecipitate utilizing anti-CYP3A4 antibody. Our shotgun analysis revealed that 149 proteins were immunoprecipitated with anti-CYP3A4 antibody in human liver microsomes. We further determined that 51 proteins of 149 proteins were specifically immunoprecipitated with the anti-CYP3A4 antibody. Our analysis demonstrated that other CYP isoforms are interacting with CYP3A4, which is in agreement with previous findings. Based on our current and previous findings, we propose that drug-metabolizing enzymes such as CYP3A4 and UDP-glucuronosyltransferase 2B7 form a metabolosome, which is a functional unit of metabolism consisting of multiple metabolism-related proteins.
[Show abstract][Hide abstract] ABSTRACT: Inadequate calorie intake or starvation has been suggested as a cause of neonatal jaundice, which can further cause permanent brain damage, kernicterus. This study experimentally investigated whether additional glucose treatments induce the bilirubin-metabolizing enzyme - UDP-glucuronosyltransferase (UGT) 1A1 - to prevent the onset of neonatal hyperbilirubinemia. Neonatal humanized UGT1 (hUGT1) mice physiologically develop jaundice. In this study, UGT1A1 expression levels were determined in the liver and small intestine of neonatal hUGT1 mice that were orally treated with glucose. In the hUGT1 mice, glucose induced UGT1A1 in the small intestine, while it did not affect the expression of UGT1A1 in the liver. UGT1A1 was also induced in the human intestinal Caco-2 cells when the cells were cultured in the presence of glucose. Luciferase assays demonstrated that not only the proximal region (-1300/-7) of the UGT1A1 promoter, but also distal region (-6500/-4050) were responsible for the induction of UGT1A1 in the intestinal cells. Adequate calorie intake would lead to the sufficient expression of UGT1A1 in the small intestine to reduce serum bilirubin levels. Supplemental treatment of newborns with glucose solution can be a convenient and efficient method to treat neonatal jaundice while allowing continuous breastfeeding.
[Show abstract][Hide abstract] ABSTRACT: Nitrilase, which is found in plants and many types of bacteria, is known as the enzyme that catalyzes hydrolysis of a wide variety of nitrile compounds. While human nitrilase-like protein (NIT), which is a member of the nitrilase superfamily, has two distinct isozymes, NIT1 and NIT2, their function has not been well understood. In this study, we investigated whether human NIT1 and NIT2 are involved in the hydrolysis of drugs using vildagliptin as a substrate. We performed Western blot analysis using human liver samples to examine protein expression of human NIT in the liver, finding that human NIT1 and NIT2 were highly expressed in the liver cytosol. We established stable single expression systems of human NIT1 and NIT2 in HEK293 cells to clarify the contribution of human NIT to hydrolysis of vildagliptin. Although the formation of a carboxylic acid metabolite of vildagliptin (M20.7) was observed in human liver samples, M20.7 was not formed by incubating vildagliptin with HEK293 cells expressing human NIT1 or NIT2. This suggests that human NIT1 or NIT2 is not involved in the metabolism of vildagliptin. Further investigation using other drugs is needed to clarify the contribution of human NIT to drug metabolism.
No preview · Article · Jul 2014 · Drug Metabolism and Pharmacokinetics
[Show abstract][Hide abstract] ABSTRACT: Solute carrier (SLC) transporters play important roles in absorption and disposition of drugs in cells; however, the expression pattern of human SLC transporters in the skin has not been determined. In the present study, the expression patterns of 28 human SLC transporters were determined in the human skin. Most of the SLC transporter family members were either highly or moderately expressed in the liver, while their expression was limited in the skin and small intestine. Treatment of human keratinocytes with a reactive metabolite of ibuprofen significantly reduced cell viability. Expression array analysis revealed that S100 calcium binding protein A7A (S100A7A) was induced nearly 50-fold in dermal cells treated with ibuprofen acyl-glucuronide. Determination of the expression of drug-metabolizing enzymes as well as drug transporters prior to the administration of drugs would make it possible to avoid the development of idiosyncratic skin diseases in individuals.
[Show abstract][Hide abstract] ABSTRACT: UDP-glucuronosyltransferases (UGTs) are phase II drug-metabolizing enzymes that catalyze glucuronidation of various drugs. Although experimental rodents are used in preclinical studies to predict glucuronidation and toxicity of drugs in humans, species differences in glucuronidation and drug-induced toxicity have been reported. Recently, humanized UGT1 mice in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus (hUGT1 mice) have been developed. In this study, acyl-glucuronidations of etodolac, diclofenac, and ibuprofen in liver microsomes of hUGT1 mice were examined and compared with those of human and regular mice. The kinetics of etodolac, diclofenac, and ibuprofen acyl-glucuronidation in hUGT1 mice were almost comparable to those in humans, rather than in mice. We further investigated hepatotoxicity of ibuprofen in hUGT1 mice and regular mice by measuring serum alanine amino transferase (ALT) levels. As ALT levels were increased at 6 h after dosing in hUGT1 mice and at 24 h after dosing in regular mice, the onset pattern of ibuprofen-induced liver toxicity in hUGT1 mice was different from that in regular mice. These data suggest that hUGT1 mice can be valuable tools for understanding glucuronidations of drugs and drug-induced toxicity in humans.
Preview · Article · Apr 2014 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: Folic acid (FA) is a water-soluble vitamin, and orally ingested FA is absorbed from the small intestine by the proton-coupled folate transporter (PCFT). In the present study, we investigated whether epigallocatechin gallate (EGCG), one of the tea catechins, affects the transport of FA by PCFT. EGCG inhibited the uptake of FA into Caco-2 cells and human PCFT-expressing HEK293 cells (PCFT-HEK293 cells). The initial rate of uptake of FA into PCFT-HEK293 cells followed Michaelis-Menten kinetics (Km = 1.9 µM). Dixon plots revealed that PCFT-mediated FA uptake was competitively inhibited by EGCG (Ki ~ 9 µM). The uptake of the PCFT substrate methotrexate (MTX) was competitively inhibited by EGCG as well (Ki ~ 15 µM). In conclusion, it is suggested that when FA or MTX is ingested with tea, it is likely that the intestinal absorption of these compounds by PCFT is inhibited, which could result in insufficient efficacy.
No preview · Article · Apr 2014 · Drug Metabolism and Pharmacokinetics
[Show abstract][Hide abstract] ABSTRACT: The stereoselective transport of methotrexate (L-amethopterin, L-MTX) and its antipode (D-amethopterin, D-MTX) by human reduced folate carrier (hRFC) has been examined in HEK293 cells expressing H27-hRFC and R27-hRFC. The uptake of both L-MTX and D-MTX increased as the extracellular pH increased from 6.0 to 7.4. The initial uptake rate of L-MTX into the H27- and R27-hRFCs of the HEK293 cells followed Michaelis-Menten kinetics with Km values of approximately 0.24 and 0.47 µM, respectively. Dixon plots revealed that the [(3)H]-L-MTX uptake mediated by the H27- and R27-hRFCs was inhibited competitively by unlabeled L-MTX with Ki values of approximately 0.1 and 0.5 µM, respectively. D-MTX also competitively inhibited the H27- and R27-hRFC mediated uptake of [(3)H]-L-MTX with Ki values of approximately 3.4 and 3.2 µM, respectively. The RFC-mediated uptake clearance of L-MTX was approximately 15-fold greater than that of D-MTX in the H27-hRFC-HEK293 cells, and was more than 30-fold greater than that of D-MTX in the R27-hRFC-HEK293 cells. The results of the current study have revealed that the enantiomers of MTX enantiomers can be transported in a stereoselective manner by the H27- and R27-hRFCs because of significant differences in the affinities of the enantiomers to the hRFC.
No preview · Article · Mar 2014 · Biological & Pharmaceutical Bulletin
[Show abstract][Hide abstract] ABSTRACT: UDP-glucuronosyltransferase (UGT) 2B7 is a membrane protein that catalyzes glucuronidation of endogenous and exogenous substrates. Because UGTs are expressed in endoplasmic reticulum (ER), their substrates and metabolites need to be transported through the ER membrane. However, insight into the mechanism underlying the transport of substrates/metabolites of UGTs through the ER membrane has not been elucidated. Metabolosome is a functional unit of metabolism consisting of multiple metabolism-related proteins. UGTs might form a metabolosome to facilitate the transport of their substrates and/or metabolites through the ER membrane. In the present study, therefore, extensive protein-protein interactions involving UGT2B7 were determined by a shotgun analysis of immunoprecipitate. Our shotgun analysis revealed that 92 proteins were immunoprecipitated with anti-UGT2B7 antibody in human liver microsomes. We further determined that 42 proteins out of 92 proteins were specifically immunoprecipitated with the anti-UGT2B7 antibody. In addition to UGT2B7, other microsomal enzymes such as UGT1A, CYP3A4, CYP1A2, and a monoamine oxidase, were included in the list of proteins immunoprecipitated with the anti-UGT2B7 antibody, suggesting that these proteins might form a metabolosome to regulate their functions in the liver. Further analyses are needed to elucidate the roles of those proteins in the enzymatic activity of human UGTs.
No preview · Article · Dec 2013 · Drug Metabolism and Pharmacokinetics
[Show abstract][Hide abstract] ABSTRACT: Hepatic intrinsic clearance (CLint) of drugs is often predicted based on in vitro data that are obtained from the Michaelis-Menten analysis. While most of the metabolic rate-substrate concentration kinetic curves fit to the Michaelis-Menten equation, cytochrome P450 (CYP) and uridine 5'-diphosphate (UDP)-glucuronosyltransferases exhibit sigmoidal kinetics for certain drugs. In our study, the kinetics of CYP3A4-catalyzed carbamazepine 10,11-epoxidation in human liver microsomes was sigmoidal and fitted to the Hill equation, revealing the S50 value of 358 µM, n of 2.0, and the Vmax value of 463 pmol/min/mg. While the intrinsic clearance calculated from Michaelis-Menten parameters (CLint) overestimated the observed in vivo intrinsic clearance (CLint, in vivo), the maximum intrinsic clearance calculated based on the Hill equation (CLmax) exhibited better predictions of CLint, in vivo. Such better prediction using the CLmax was also observed for other four drugs, all of which also exhibited sigmoidal metabolic rate-concentration curves, according to the literature data. However, even if we assume such Hill equation, intrinsic clearances predicted at their therapeutic concentrations from in vitro data were still much lower than their CLint, in vivo, suggesting the existence of unknown factors causing discrepancy between in vitro intrinsic clearance in human liver microsomes and in vivo data. Thus, even if we assume sigmoidal kinetics, that would not be enough for accurate prediction of CLint, in vivo, and it would be preferable to use CLmax to quantitatively extrapolate the in vitro data to in vivo clearance.
No preview · Article · Dec 2013 · Biological & Pharmaceutical Bulletin
[Show abstract][Hide abstract] ABSTRACT: While breast milk has been known as a cause of neonatal hyperbilirubinemia, the underlying mechanism of breast milk-induced jaundice has not been clarified. Here, the impact of fatty acids on human UDP-glucuronosyltransferase (UGT) 1A1 - the sole enzyme that can metabolize bilirubin - were examined. Oleic acid, linoleic acid, and docosahexaenoic acid (DHA) strongly inhibited UGT1A1 activity. Forty-eight hours after a treatment with a lower concentration of DHA (10 mg/kg), total bilirubin significantly increased in neonatal hUGT1 mice, which are human neonatal jaundice models. In contrast, treatments with higher concentrations of fatty acids (0.1-10 g/kg) resulted in a decrease in serum bilirubin in hUGT1 mice. It was further demonstrated that the treatment with higher concentrations of fatty acids induced UGT1A1, possibly by activation of peroxisome proliferator-activated receptors. Our data indicates that activation of peroxisome proliferator-activated receptors would increase UGT1A1 expression, resulting in reduction of serum bilirubin levels in human infants.
[Show abstract][Hide abstract] ABSTRACT: ATP-binding cassette (ABC) transporters transport a variety of substrates across cellular membranes coupled with hydrolysis of ATP. Currently 49 ABC transporters consisting of seven subfamilies, ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, and ABCG, have been identified in humans and they are extensively expressed in various tissues. Skin can develop a number of drug-induced toxicities' such as Stevens–Johnson syndrome and psoriasis. Concentration of drugs in the skin cells is associated with the development of adverse drug reactions. ABC transporters play important roles in absorption and disposition of drugs in the cells; however, the expression pattern of human ABC transporters in the skin has not been determined. In this study, the expression patterns of 48 human ABC transporters were determined in the human skin as well as in the liver and small intestine. Most of the ABCA, ABCB, ABCC, ABCD, ABCE, and ABCF family members were highly or moderately expressed in the skin, while ABCG family members were slightly expressed in the skin. Significant interindividual variability was also observed in the expression levels of those ATP transporters in the skin, except for ABCA5 and ABCF1, which were found to be expressed in all of the human skin samples tested in this study. In conclusion, this is the first study to identify the expression pattern of the whole human ABC family of transporters in the skin. The interindividual variability in the expression levels of ABC transporters in the human skin might be associated with drug-induced skin diseases.
[Show abstract][Hide abstract] ABSTRACT: Uridine 5′-diphosphate-glucuronosyltransferases (UGTs) are phase II drug-metabolizing enzymes that catalyze glucuronidation of various endogenous and exogenous substrates. Among 19 functional human UGTs, UGT1A family enzymes largely contribute to the metabolism of clinically used drugs. While the UGT1A locus is conserved in mammals such as humans, mice, and rats, species differences in drug glucuronidation have been reported. Recently, humanized UGT1 mice in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus (hUGT1 mice) have been developed. To evaluate the usefulness of hUGT1 mice to predict human glucuronidation of drugs, UGT activities, and inhibitory effects on UGTs were examined in liver microsomes of hUGT1 mice as well as in those of wild-type mice and humans. Furosemide acyl-glucuronidation was sigmoidal and best fitted to the Hill equation in hUGT1 mice and human liver microsomes, while it was fitted to the substrate inhibition equation in mouse liver microsomes. Kinetic parameters of furosemide glucuronidation were very similar between hUGT1 mice and human liver microsomes. The kinetics of S-naproxen acyl-glucuronidation and inhibitory effects of compounds on furosemide glucuronidation in hUGT1 liver microsomes were also slightly, but similar to those in human liver microsomes, rather than in wild-type mice. While wild-type mice lack imipramine and trifluoperazine N-glucuronidation potential, hUGT1 mice showed comparable N-glucuronidation activity to that of humans. Our data indicate that hUGT1 mice are promising tools to predict not only in vivo human drug glucuronidation but also potential drug-drug interactions.