Humanized UGT2 and CYP3A transchromosomic rats for improved prediction of human drug metabolism.

Although "genomically" humanized animals are invaluable tools for generating human disease models as well as for biomedical research, their development has been mainly restricted to mice via established transgenic-based and embryonic stem cell-based technologies. Since rats are widely used for studying human disease and for drug efficacy and toxicity testing, humanized rat models would be preferred over mice for several applications. However, the development of sophisticated humanized rat models has been hampered by the difficulty of complex genetic manipulations in rats. Additionally, several genes and gene clusters, which are megabase range in size, were difficult to introduce into rats with conventional technologies. As a proof of concept, we herein report the generation of genomically humanized rats expressing key human drug-metabolizing enzymes in the absence of their orthologous rat counterparts via the combination of chromosome transfer using mouse artificial chromosome (MAC) and genome editing technologies. About 1.5 Mb and 700 kb of the entire UDP glucuronosyltransferase family 2 and cytochrome P450 family 3 subfamily A genomic regions, respectively, were successfully introduced via the MACs into rats. The transchromosomic rats were combined with rats carrying deletions of the endogenous orthologous genes, achieved by genome editing. In the "transchromosomic humanized" rat strains, the gene expression, pharmacokinetics, and metabolism observed in humans were well reproduced. Thus, the combination of chromosome transfer and genome editing technologies can be used to generate fully humanized rats for improved prediction of the pharmacokinetics and drug-drug interactions in humans, and for basic research, drug discovery, and development.

Enzymatically-crosslinked gelatin hydrogels containing paenipeptin and clarithromycin against carbapenem-resistant pathogen in murine skin wound infection.

BACKGROUND: The recent rise and spread of carbapenem-resistant pathogens pose an urgent threat to public health and has fueled the search for new therapies. Localized delivery of topical antibiotics is an alternative for the treatment of infected wounds caused by drug-resistant pathogens. In this study, we aimed to develop antimicrobial-loaded hydrogels for topical treatment of wound infections in a murine skin wound infection. RESULTS: Paenipeptin analogue 1, a linear lipopeptide, potentiated clarithromycin against multidrug-resistant Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae. Enzymatically-crosslinked gelatin hydrogels were developed to encapsulate paenipeptin analogue 1 and clarithromycin. The encapsulated antimicrobials were gradually released from hydrogels during incubation, reaching 75.43 and 53.66% for paenipeptin and clarithromycin, respectively, at 24 h. The antimicrobial-loaded hydrogels containing paenipeptin and clarithromycin synergistically resulted in 5-log reduction in carbapenem-resistant A. baumannii within 6 h in vitro. Moreover, the antimicrobial-loaded hydrogels reduced 3.6- and 2.5-log of carbapenem-resistant A. baumannii when treated at 4 or 20 h post infection, respectively, in a murine skin wound infection. CONCLUSIONS: Enzymatically-crosslinked gelatin hydrogels loaded with paenipeptin analogue 1 and clarithromycin exhibited potent therapeutic efficacy against carbapenem-resistant A. baumannii in murine skin wound infection.

Systemic regulation of bilirubin homeostasis: Potential benefits of hyperbilirubinemia.

Neurotoxic bilirubin is the end product of heme catabolism in mammals. Bilirubin is solely conjugated by uridine diphospho-glucuronosyltransferase 1A1, which is a membrane-bound enzyme that catalyzes the transfer of glucuronic acid. Due to low function of hepatic and intestinal uridine diphospho-glucuronosyltransferase 1A1 during the neonatal period, human neonates develop mild to severe physiological hyperbilirubinemia. Accumulation of bilirubin in the brain leads to the onset of irreversible brain damage, termed kernicterus. Breastfeeding is one of the most significant factors that increase the risk of developing kernicterus in infants. Why does this most natural way of feeding increase the risk of brain damage or even death? This question leads to the hypothesis that breast milk-induced hyperbilirubinemia might bring certain benefits that outweigh those risks. While bilirubin is neurotoxic and cytotoxic, this compound is also a potent antioxidant. There are studies showing improved clinical conditions in patients with hyperbilirubinemia. Accumulating evidence also shows that genetic polymorphisms linked to hyperbilirubinemia are beneficial against various diseases. In this review article, we first introduce the production, metabolism, and transport of bilirubin. We then discuss the potential benefits of neonatal and adult hyperbilirubinemia. Finally, epigenetic factors as well as metabolomic information associated with hyperbilirubinemia are described. This review article advances the understanding of the physiological importance of the paradoxical compound bilirubin. (Hepatology 2018;67:1609-1619).

Enzymatic analysis of glucuronidation of synthetic cannabinoid 1-naphthyl 1-(4-fluorobenzyl)-1H-indole-3-carboxylate (FDU-PB-22).

Recently, there has been a rise in abuse of synthetic cannabinoids (SCBs). The consumption of SCBs results in various effects and can induce toxic reactions, including paranoia, seizures, tachycardia and even death. 1-Naphthyl 1-(4-fluorobenzyl)-1H-indole-3-carboxylate (FDU-PB-22) is a third generation SCB whose metabolic pathway has not been fully characterized. In this study, we conducted in vitro pharmacokinetic analysis of FDU-PB-22 metabolism. Metabolic reactions containing FDU-PB-22 and human liver microsomes (HLMs) were independent of NADPH but not UDP-glucuronic acid (UDPGA), suggesting that UDP-glucuronosyltransferases (UGTs) are the primary enzymes involved in this metabolism. It was further determined that the metabolite extensively formed after incubating FDU-PB-22 with UDPGA in HLMs was the glucuronide of FDU-PB-22 3-carboxyindole (FBI-COOH). Various hepatic UGTs showed enzymatic activity for FBI-COOH. A series of UGT inhibitors showed moderate to strong inhibition of FBI-COOH-glucuronidation in HLMs, suggesting that multiple UGT isoforms are involved in FBI-COOH-glucuronidation in the liver. Interestingly, an extra-hepatic isoform, UGT1A10, exhibited the highest activity with a Km value of 38 µM and a Vmax value of 5.90 nmol/min/mg. Collectively, these results suggest that both genetic mutations of and the co-administration of inhibitors for FDU-PB-22-metabolizing UGTs will likely increase the risk of FDU-PB-22-induced toxicity.

Altered metabolism of synthetic cannabinoid JWH-018 by human cytochrome P450 2C9 and variants.

Synthetic cannabinoids (SCBs), synonymous with 'K2', 'Spice' or 'synthetic marijuana', are psychoactive drugs of abuse that frequently result in clinical effects and toxicity more severe than those classically associated with Δ9-tetrahydrocannabinol such as extreme agitation, hallucinations, supraventricular tachycardia, syncope, and seizures. JWH-018 is one of the earliest compounds identified in various SCB products, and our laboratory previously demonstrated that JWH-018 undergoes extensive metabolism by cytochromes P450 (P450), binds to, and activates cannabinoid receptors (CBRs). The major enzyme involved in the metabolism of JWH-018 is CYP2C9, a highly polymorphic enzyme found largely in the intestines and liver, with *1 being designated as the wild type, and *2 and *3 as the two most common variants. Three different major products have been identified in human urine and plasma: JWH-018 (ω)-OH, JWH-018 (ω-1)-OH(R), and JWH-018 (ω-1)-OH(S). The (ω-1)-OH metabolite of JWH-018 is a chiral molecule, and is thus designated as either (ω-1)-OH(R) or (ω-1)-OH(S). Here, in vitro enzyme kinetic assays performed with human recombinant CYP2C9 variants (*1, *2, and *3) revealed that oxidative metabolism by CYP2C9*3 resulted in significantly less formation of (ω)-OH and (ω-1)-OH metabolites. Surprisingly, CYP2C9*2 was roughly 3.6-fold more efficient as the CYP2C9*1 enzyme based on Vmax/Km, increasing the rate of JWH-018 metabolism and allowed for a much more rapid elimination. These results suggest that genetic polymorphisms of P450 enzymes result in the production of varying levels of biologically active JWH-018 metabolites in some individuals, offering a mechanistic explanation for the diverse clinical toxicity often observed following JWH-018 abuse.

Hepatic Dipeptidyl Peptidase-4 Controls Pharmacokinetics of Vildagliptin In Vivo.

The main route of elimination of vildagliptin, which is an inhibitor of dipeptidyl peptidase-4 (DPP-4), in humans is cyano group hydrolysis to produce a carboxylic acid metabolite M20.7. Our in vitro study previously demonstrated that DPP-4 itself greatly contributed to the hydrolysis of vildagliptin in mouse, rat, and human livers. To investigate whether hepatic DPP-4 contributes to the hydrolysis of vildagliptin in vivo, in the present study, we conducted in vivo pharmacokinetics studies of vildagliptin in mice coadministered with vildagliptin and sitagliptin, which is another DPP-4 inhibitor, and also in streptozotocin (STZ)-induced diabetic mice. The area under the plasma concentration-time curve (AUC) value of M20.7 in mice coadministered with vildagliptin and sitagliptin was significantly lower than that in mice administered vildagliptin alone (P < 0.01). Although plasma DPP-4 expression level was increased 1.9-fold, hepatic DPP-4 activity was decreased in STZ-induced diabetic mice. The AUC values of M20.7 in STZ-induced diabetic mice were lower than those in control mice (P < 0.01). Additionally, the AUC values of M20.7 significantly positively correlated with hepatic DPP-4 activities in the individual mice (Rs = 0.943, P < 0.05). These findings indicated that DPP-4 greatly contributed to the hydrolysis of vildagliptin in vivo and that not plasma, but hepatic DPP-4 controlled pharmacokinetics of vildagliptin. Furthermore, enzyme assays of 23 individual human liver samples showed that there was a 3.6-fold interindividual variability in vildagliptin-hydrolyzing activities. Predetermination of the interindividual variability of hepatic vildagliptin-hydrolyzing activity might be useful for the prediction of blood vildagliptin concentrations in vivo.

Prediction of drug-induced liver injury using keratinocytes.

Drug-induced liver injury (DILI) is one of the most common adverse drug reactions. DILI is often accompanied by skin reactions, including rash and pruritus. However, it is still unknown whether DILI-associated genes such as S100 calcium-binding protein A and interleukin (IL)-1ß are involved in drug-induced skin toxicity. In the present study, most of the tested hepatotoxic drugs such as pioglitazone and diclofenac induced DILI-associated genes in human and mouse keratinocytes. Keratinocytes of mice at higher risk for DILI exhibited an increased IL-1ß basal expression. They also showed a higher inducibility of IL-1ß when treated by pioglitazone. Mice at higher risk for DILI showed even higher sums of DILI-associated gene basal expression levels and induction rates in keratinocytes. Our data suggest that DILI-associated genes might be involved in the onset and progression of drug-induced skin toxicity. Furthermore, we might be able to identify individuals at higher risk of developing DILI less invasively by examining gene expression patterns in keratinocytes. Copyright © 2017 John Wiley & Sons, Ltd.

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

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

Acyl-glucuronide as a Possible Cause of Trovafloxacin-Induced Liver Toxicity: Induction of Chemokine (C-X-C Motif) Ligand 2 by Trovafloxacin Acyl-glucuronide.

Trovafloxacin is an antibiotic that was withdrawn from the market relatively soon after its release due to the risk of hepatotoxicity. Trovafloxacin is mainly metabolized to its acyl-glucuronide by uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) 1A1. In this study, we examined whether the acyl-glucuronide is involved in the development of hepatotoxicity. A UGT1A1-induced cell model was developed and the toxicity of trovafloxacin acyl-glucuronide was evaluated. The UGT1A1-induced cell model was developed by treating HepG2 cells with chrysin for 48 h. Chemokine (C-X-C motif) ligand 2, a cytokine involved in drug-induced liver injury, was uniquely induced by trovafloxacin in the UGT1A1-induced HepG2 cells. Induction of UGT1A1 resulted in a decrease in cell viability. An in vivo animal study further demonstrated the importance of UGT1A1 in the trovafloxacin-induced liver toxicity. Although the complete mechanism of trovafloxacin-induced liver injury is still unknown, trovafloxacin acyl-glucuronide can be involved in the development of toxic reactions in vitro and in vivo.

Dipeptidyl peptidase-4 greatly contributes to the hydrolysis of vildagliptin in human liver.

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), whereas 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 H-glycyl-prolyl-7-amino-4-methylcoumarin (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 nonsynonymous single-nucleotide polymorphism of human DPP-4, in human embryonic kidney 293 (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.

Expression of UDP-Glucuronosyltransferase 1 (UGT1) and Glucuronidation Activity toward Endogenous Substances in Humanized UGT1 Mouse Brain.

Although UDP-glucuronosyltransferases (UGTs) are important phase II drug-metabolizing enzymes, they are also involved in the metabolism of endogenous compounds. Certain substrates of UGTs, such as serotonin and estradiol, play important roles in the brain. However, the expression of UGTs in the 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 humans 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 the cerebellum, olfactory bulbs, midbrain, hippocampus, and cerebral cortex of hUGT1 mice. UGT1A1 in all brain regions and UGT1A6 in the cerebellum and cerebral cortex of 6-month-old hUGT1 mice were expressed at a significantly higher rate 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 mean values of 0.13 and 5.17 pmol/min/mg, respectively. In conclusion, UGT1A1 and UGT1A6 might play an important role in function regulation of endogenous compounds in a region- and age-dependent manner. Humanized UGT1 mice might be useful to study the importance of brain UGTs in vivo.

Targeted screen for human UDP-glucuronosyltransferases inhibitors and the evaluation of potential drug-drug interactions with zafirlukast.

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 with drugs that are metabolized by cytochrome P450s. Most of the past studies have investigated hepatic UGTs, although 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 because it monitors 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 human liver and human small intestine microsomes in order to evaluate potential DDIs. The R values (the 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.

Role of extrahepatic UDP-glucuronosyltransferase 1A1: Advances in understanding breast milk-induced neonatal hyperbilirubinemia.

Newborns commonly develop physiological hyperbilirubinemia (also known as jaundice). With increased bilirubin levels being observed in breast-fed infants, breast-feeding has been recognized as a contributing factor for the development of neonatal hyperbilirubinemia. Bilirubin undergoes selective metabolism by UDP-glucuronosyltransferase (UGT) 1A1 and becomes a water soluble glucuronide. Although several factors such as gestational age, dehydration and weight loss, and increased enterohepatic circulation have been associated with breast milk-induced jaundice (BMJ), deficiency in UGT1A1 expression is a known cause of BMJ. It is currently believed that unconjugated bilirubin is metabolized mainly in the liver. However, recent findings support the concept that extrahepatic tissues, such as small intestine and skin, contribute to bilirubin glucuronidation during the neonatal period. We will review the recent advances made towards understanding biological and molecular events impacting BMJ, especially regarding the role of extrahepatic UGT1A1 expression.

Expression of Human DNAJ (Heat Shock Protein-40) B3 in Humanized UDP-glucuronosyltransferase 1 Mice.

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.

Glucuronidation of drugs and drug-induced toxicity in humanized UDP-glucuronosyltransferase 1 mice.

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. Humanized UGT1 mice in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus (hUGT1 mice) were recently developed. In this study, acyl-glucuronidations of etodolac, diclofenac, and ibuprofen in liver microsomes of hUGT1 mice were examined and compared with those of humans 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 the hepatotoxicity of ibuprofen in hUGT1 mice and regular mice by measuring serum alanine amino transferase (ALT) levels. Because ALT levels were increased at 6 hours after dosing in hUGT1 mice and at 24 hours 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.

Importance of UDP-glucuronosyltransferase 1A1 expression in skin and its induction by UVB in neonatal hyperbilirubinemia.

UDP-glucuronosyltransferase (UGT) 1A1 is the sole enzyme that can metabolize bilirubin. Human infants physiologically develop hyperbilirubinemia as the result of inadequate expression of UGT1A1 in the liver. Although phototherapy using blue light is effective in preventing jaundice, sunlight has also been suggested, but without conclusive evidence, to reduce serum bilirubin levels. We investigated the mRNA expression pattern of human UGT1A1 in human skin, human skin keratinocyte (HaCaT) cells, and skin of humanized UGT1 mice. The effects of UVB irradiation on the expression of UGT1A1 in the HaCaT cells were also examined. Multiple UGT1A isoforms, including UGT1A1, were expressed in human skin and HaCaT cells. When HaCaT cells were treated with UVB-exposed tryptophan, UGT1A1 mRNA and activity were significantly induced. Treatment of the HaCaT cells with 6-formylindolo[3,2-b]carbazole, which is one of the tryptophan derivatives formed by UVB, resulted in an induction of UGT1A1 mRNA and activity. In neonates, the expression of UGT1A1 was greater in the skin; in adults, UGT1A1 was expressed mainly in the liver. Treatment of humanized UGT1 mice with UVB resulted in a reduction of serum bilirubin levels, along with increased UGT1A1 expression and activity in the skin. Our data revealed a protective role of UGT1A1 expressed in the skin against neonatal hyperbilirubinemia. Sunlight, a natural and free source of light, makes it possible to treat neonatal jaundice while allowing mothers to breast-feed neonates.

Reduced expression of UGT1A1 in intestines of humanized UGT1 mice via inactivation of NF-κB leads to hyperbilirubinemia.

BACKGROUND & AIMS: Bilirubin is a natural and potent antioxidant that accumulates in the blood of newborn children and leads to physiological jaundice. Breastfed infants have higher serum levels of bilirubin than formula-fed infants and are at risk for bilirubin-induced neurological dysfunction (BIND). Clearance of bilirubin requires the expression of uridine diphosphate glucuronosyltransferase (UGT) 1A1; we investigated its role in the association between breast feeding with jaundice in mice. METHODS: We studied mice in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus (hUGT1 mice); these mice spontaneously develop neonatal hyperbilirubinemia and BIND. We fed human breast milk or formula to neonatal hUGT1 mice and examined activation of the intestinal xenobiotic receptors pregnane X receptor and constitutive androstane receptor. We also examined inflammatory signaling pathways in mice with disruptions in IκB-kinase-α and IκB kinase-ß in the intestinal epithelium. RESULTS: hUGT1 mice that were fed breast milk developed severe hyperbilirubinemia because of suppression of UGT1A1 in the gastrointestinal tract. Formula-fed hUGT1 mice had lower serum levels of bilirubin, which resulted from induction of UGT1A1 in the gastrointestinal tract. hUGT1/Pxr-null mice did not develop severe hyperbilirubinemia, whereas hUGT1/Car-null mice were susceptible to BIND when they were fed breast milk. Breast milk appeared to suppress intestinal IκB kinase α and ß, resulting in inactivation of nuclear factor-κB and loss of expression of UGT1A1, leading to hyperbilirubinemia. CONCLUSIONS: Breast milk reduces expression of intestinal UGT1A1, which leads to hyperbilirubinemia and BIND; suppression of this gene appears to involve inactivation of nuclear factor-κB. Hyperbilirubinemia can be reduced by activation of pregnane X receptor, constitutive androstane receptor, or nuclear factor-κB.

Prediction of in vivo carbamazepine 10,11-epoxidation from in vitro metabolic studies with human liver microsomes: importance of its sigmoidal kinetics.

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.

Developmental hyperbilirubinemia and CNS toxicity in mice humanized with the UDP glucuronosyltransferase 1 (UGT1) locus.

High levels of unconjugated bilirubin (UCB) in newborn children is associated with a reduction in hepatic UDP glucuronosyltransferase (UGT) 1A1 activity that can lead to CNS toxicity, brain damage, and even death. Little is known regarding those events that lead to UCB accumulation in brain tissue, and therefore, we sought to duplicate this condition in mice. The human UGT1 locus, encoding all 9-UGT1A genes including UGT1A1, was expressed in Ugt1(-/-) mice. Because the most common clinical condition associated with jaundice in adults is Gilbert's syndrome, which is characterized by an allelic polymorphism in the UGT1A1 promoter, hyperbilirubinemia was monitored in humanized UGT1 mice that expressed either the Gilbert's UGT1A1*28 allele [Tg(UGT1(A1*28))Ugt1(-/-) mice] or the normal UGT1A1*1 allele [Tg(UGT1(A1*1))Ugt1(-/-) mice]. Adult Tg(UGT1(A1*28))Ugt1(-/-) mice expressed elevated levels of total bilirubin (TB) compared with Tg(UGT1(A1*1))Ugt1(-/-) mice, confirming that the promoter polymorphism associated with the UGT1A1*28 allele contributes to hyperbilirubinemia in mice. However, TB accumulated to near toxic levels during neonatal development, a finding that is independent of the Gilbert's UGT1A1*28 promoter polymorphism. Whereas serum TB levels eventually returned to adult levels, TB clearance in neonatal mice was not associated with hepatic UGT1A1 expression. In approximately 10% of the humanized UGT1 mice, peak TB levels culminated in seizures followed by death. UCB deposition in brain tissue and the ensuing seizures were associated with developmental milestones and can be prevented by enhancing regulation of the UGT1A1 gene in neonatal mice.

[Pharmacy School and Pharm.D. Program in the United States of America].

Pharmacy schools in Japan started to offer the 6-year undergraduate program in 2006. Currently, students are eligible for the national licensing examination to become a pharmacist only after completing the six-year program and receiving a bachelor's degree. Meanwhile, pharmacy schools offer the four-year graduate program in the United States of America (U.S.A.). In addition to the length of the program, there are several significant differences between pharmacy schools in Japan and the U.S.A. In the U.S., students receive a professional doctoral degree in pharmacy, doctor of pharmacy (Pharm.D.), after completion of the program. Fourth-year pharmacy students in the U.S. spend 1600 h in their clinical rotations, which is considerably longer than those in Japanese programs. It is also unique that pharmacists and pharmacy students are authorized to administer vaccines in the U.S. upon completion of immunization training. This symposium review aims to introduce the pharmaceutical education and the Pharm.D. program offered in the U.S.