Characterization of P-Glycoprotein Humanized Mice Generated by Chromosome Engineering Technology: Its Utility for Prediction of Drug Distribution to the Brain in Humans.

P-glycoprotein (P-gp), encoded by the MDR1 gene in humans and by the Mdr1a/1b genes in rodents, is expressed in numerous tissues and performs as an efflux pump to limit the distribution and absorption of many drugs. Owing to species differences of P-gp between humans and rodents, it is difficult to predict the impact of P-gp on pharmacokinetics and the tissue distribution of P-gp substrates in humans from the results of animal experiments. Therefore, we generated a novel P-gp humanized mouse model by using a mouse artificial chromosome (MAC) vector [designated human MDR1-MAC (hMDR1-MAC) mice]. The results showed that hMDR1 mRNA was expressed in various tissues of hMDR1-MAC mice. Furthermore, the expression of human P-gp was detected in the brain capillary fraction and plasma membrane fraction of intestinal epithelial cells isolated from hMDR1-MAC mice, although the expression levels of intestinal P-gp were extremely low. Thus, we evaluated the function of human P-gp at the blood-brain barrier of hMDR1-MAC mice. The brain-to-plasma ratios of P-gp substrates in hMDR1-MAC mice were much lower than those in Mdr1a/1b-knockout mice, and the brain-to-plasma ratio of paclitaxel was significantly increased by pretreatment with a P-gp inhibitor in hMDR1-MAC mice. These results indicated that the hMDR1-MAC mice are the first P-gp humanized mice expressing functional human P-gp at the blood-brain barrier. This mouse is a promising model with which to evaluate species differences of P-gp between humans and mice in vivo and to estimate the brain distribution of drugs in humans while taking into account species differences of P-gp.

Differentiated HASTR/ci35 cells: A promising in vitro human astrocyte model for facilitating CNS drug development studies.

Astrocytes have shown longstanding promise as therapeutic targets for various central nervous system diseases. To facilitate drug development targeting astrocytes, we have recently developed a new conditionally immortalized human astrocyte cell line, termed HASTR/ci35 cells. In this study, in order to further increase their chances to contribute to various astrocyte studies, we report on the development of a culture method that improves HASTR/ci35 cell differentiation status and provide several proofs related to their astrocyte characteristics. The culture method is based on the simultaneous elimination of serum effects and immortalization signals. The results of qPCR showed that the culture method significantly enhanced several astrocyte marker gene expression levels. Using the differentiated HASTR/ci35, we examined their response profiles to nucleotide treatment and inflammatory stimuli, along with their membrane fatty acid composition. Consequently, we found that they responded to ADP or UTP treatment with a transient increase of intracellular Ca2+ concentration, and that they could show reactive response to interleukin-1ß treatments. Furthermore, the membrane phospholipids of the cells were enriched with polyunsaturated fatty acids. To summarize, as a unique human astrocyte model carrying the capability of a differentiation induction properties, HASTR/ci35 cells are expected to contribute substantially to astrocyte-oriented drug development studies.

Effect of Pregnenolone 16α-Carbonitrile on the Expression of P-Glycoprotein in the Intestine, Brain and Liver of Mice.

P-Glycoprotein (P-gp), encoded by the MDR1 (ABCB1) gene in humans and by Mdr1a and Mdr1b genes in rodents, is a member of the superfamily of ATP-binding cassette transporters. Since P-gp is constitutively expressed in numerous tissues and exhibits a broad specificity in substrate recognition, it can play a crucial role in limiting the absorption and distribution of xenobiotics by decreasing their intracellular accumulation. The expression of P-gp is regulated by various nuclear receptors such as pregnane X receptor (PXR). Although the characterization of P-gp induction by PXR ligands is a crucial goal for predicting pharmacokinetics of drugs, findings regarding the induction of P-gp by PXR ligands in vivo are still controversial. In this study, we examined the effect of pregnenolone 16α-carbonitrile (PCN), a murine PXR ligand, on the expression of Mdr1a/1b mRNA and P-gp protein in the intestine, brain and liver of mice. The results showed that PCN increased the expression of both Mdr1a/1b mRNA and P-gp protein in the intestine and the brain. The present study provided the first evidence that P-gp is inducible by PCN in the large intestine. The results also showed that P-gp protein was induced by PCN in the cortex but not in the whole brain. On the other hand, PCN increased the expression of Mdr1a/1b mRNA in the liver, although no increase was observed in the expression of P-gp protein. These results suggested different effect of PCN on the expression of P-gp protein in the intestine, brain and liver of mice.

Development of a Caco-2 Cell Line Carrying the Human Intestine-Type CES Expression Profile as a Promising Tool for Ester-Containing Drug Permeability Studies.

Carboxylesterase 2 (CES2), which is a member of the serine hydrolase superfamily, is primarily expressed in the human small intestine, where it plays an important role in the metabolism of ester-containing drugs. Therefore, to facilitate continued progress in ester-containing drug development, it is crucial to evaluate how CES2-mediated hydrolysis influences its intestinal permeability characteristics. Human colon carcinoma Caco-2 cells have long been widely used in drug permeability studies as an enterocyte model. However, they are not suitable for ester-containing drug permeability studies due to the fact that Caco-2 cells express CES1 (which is not expressed in human enterocytes) but do not express CES2. To resolve this problem, we created a new Caco-2 cell line carrying the human small intestine-type CES expression profile. We began by introducing short-hairpin RNA for CES1 mRNA knockdown into Caco-2 cells to generate CES1-decifient Caco-2 cells (Caco-2CES1KD cells). Then, we developed Caco-2CES1KD cells that stably express CES2 (CES2/Caco-2CES1KD cells) and their control Mock/Caco-2CES1KD cells. The results of a series of functional expression experiments confirmed that CES2-specific activity, along with CES2 mRNA and protein expression, were clearly detected in our CES2/Caco-2CES1KD cells. Furthermore, we also confirmed that CES2/Caco-2CES1KD cells retained their tight junction formation property as well as their drug efflux transporter functions. Collectively, based on our results clearly showing that CES2/Caco-2CES1KD cells carry the human intestinal-type CES expression profile, while concomitantly retaining their barrier properties, it can be expected that this cell line will provide a promising in vitro model for ester-containing drug permeability studies.

Development of a New Conditionally Immortalized Human Liver Sinusoidal Endothelial Cells.

Liver sinusoidal endothelial cells (LSECs), which are specialized endothelial cells that line liver sinusoids, have been reported to participate in a variety of liver functions, such as blood macromolecule clearance and factor VIII production. In addition, LSECs play crucial roles in liver regeneration following acute liver injury, as well as the development and progression of liver diseases or drug-induced hepatotoxicity. However, the molecular mechanisms underlying their roles remain mostly unknown. Therefore, in order to contribute to the clarification of those mechanisms, herein we report on the development of a new immortalized human LSEC (HLSEC) line. To produce this cell line, two immortalized genes were introduced into the primary HLSECs, which eventually resulted in the establishment of the HLSEC/conditionally immortalized, clone-J (HLSEC/ciJ). Consistent with the two-immortalized gene expression, HLSEC/ciJ showed excellent proliferation activity. Additionally, the results of gene expression analyses showed that several LSEC (as well as pan-endothelial) marker mRNAs and proteins were clearly expressed in HLSEC/ciJ. Furthermore, we found that adherence junction proteins were localized at the cell border in the HLSEC/ciJ monolayer, and that the cells exhibited a tube-like structure formation property. Taken together, the results obtained thus far indicate that we have successfully immortalized HLSECs, resulting in creation of HLSEC/ciJ, a cell line that possesses infinite proliferation ability while retaining possession of at least some HLSEC features. We believe that the HLSEC/ciJ have the potential to provide a valuable and unlimited alternative source of HLSECs for use in liver/LSEC physiology/pathophysiology, pharmacology, and toxicology studies.

Cancer-Type OATP1B3 mRNA in Extracellular Vesicles as a Promising Candidate for a Serum-Based Colorectal Cancer Biomarker.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) mRNA is a variant isoform of the liver-type OATP1B3. Because Ct-OATP1B3 mRNA shows an excellent cancer-specific expression profile in colorectal cancer (CRC), and that its expression levels are associated with CRC prognosis, it holds the potential to become a useful CRC detection and diagnosis biomarker. While the potential is currently justified only at the tissue level, if existence of Ct-OATP1B3 mRNA in CRC-derived extracellular vesicles (EVs) is validated, the findings could enhance its translational potential as a CRC detection and diagnosis biomarker. Therefore, this study aims at proving that Ct-OATP1B3 mRNA exists in CRC-derived EVs, and can be detected using serum specimens. To examine the possibility of Ct-OATP1B3 mRNA being existed in extracellular milieu, we isolated EVs from the human CRC (HCT116, HT-29, and SW480) cell lines, and prepared their cDNAs. The RT-PCR results showed that Ct-OATP1B3 mRNA was clearly present in EVs derived from the human CRC cell lines. Then, in order to further explore the possibility that Ct-OATP1B3 mRNA in CRC-derived EVs can be detected in serum, we isolated serum EVs derived from human CRC xenograft mice, and then performed RT-PCR. The results showed that Ct-OATP1B3 mRNA could be found in all serum EV and CRC tissue samples of the mice examined. Collectively, our findings, which show that Ct-OATP1B3 mRNA exists in EVs and can be detected in (at least) mouse serum, strengthen the potential use of Ct-OATP1B3 mRNA as a serum-based CRC biomarker.

Indirect activation of pregnane X receptor in the induction of hepatic CYP3A11 by high-dose rifampicin in mice.

Rifampicin (RIF), a typical ligand of human pregnane X receptor (PXR), powerfully induces the expression of cytochrome P450 3A4 (CYP3A4) in humans. Although it is thought that RIF is not a ligand of rodent PXR, treatment with high-dose RIF (e.g. more than 20 mg/kg) increases the expression of CYP3A in the mouse liver. In this study, we investigated whether the induction of CYP3A by high-dose RIF in the mouse liver is mediated via indirect activation of mouse PXR (mPXR). The results showed that high-dose RIF increased the expression of CYP3A11 and other PXR-target genes in the liver of wild-type mice but not PXR-knockout mice. However, the results of reporter gene and ligand-dependent assembly assays showed that RIF does not activate mPXR in a ligand-dependent manner. In addition, high-dose RIF stimulated nuclear accumulation of mPXR in the mouse liver, and geldanamycin and okadaic acid attenuated the induction of Cyp3a11 and other PXR-target genes in primary hepatocytes, suggesting that high-dose RIF triggers nuclear translocation of mPXR. In conclusion, the present study suggests that high-dose RIF stimulates nuclear translocation of mPXR in the liver of mice by indirect activation, resulting in the transactivation of Cyp3a11 and other PXR-target genes.

Establishment and characterization of a new conditionally immortalized human astrocyte cell line.

Astrocytes are the most abundant cell types in mammalian brains, within which they participate in various neuronal activities, partly by utilizing the numerous transporters expressed at their plasma membranes. Accordingly, detailed characterization of astrocytic functions, including transporters, are essential for understanding of mechanistic basis of normal brain functions, as well as the pathogenesis and treatment of various brain diseases. As a part of overall efforts to facilitate such studies, this study reports on the establishment of a new human astrocyte cell line, which is hereafter referred to as human astrocyte/conditionally immortalized, clone 35 (HASTR/ci35). This line, which was developed utilizing a cell immortalization method, showed excellent proliferative ability and expressed various astrocyte markers, including glial fibrillary acidic protein. When co-cultured with neuronal cells, HASTR/ci35 cells could facilitate their dendritic network formation. Furthermore, HASTR/ci35 cells not only possessed significant glutamate and adenosine transporter activities but also exhibited organic ion transporter activities. To summarize, HASTR/ci35 cells possess several key astrocytic characteristics, including various transporter functions, while simultaneously showing infinite proliferation and scalability. Based on these findings, HASTR/ci35 cells can be expected to contribute significantly to various human astrocyte study fields. In vitro astrocyte models are valuable experimental tools in various astrocyte studies. Here, we report the establishment of a new human astrocyte cell line, HASTR/ci35, which show various key astrocyte properties, including astrocytic transporter activities, glycogen storage and facilitation of neuronal cell differentiation. Thus, HASTR/ci35 is expected to significantly contribute to advances toward detailed understanding of human astrocyte functions.

Trans-chromosomic mice containing a human CYP3A cluster for prediction of xenobiotic metabolism in humans.

Human CYP3A is the most abundant P450 isozyme present in the human liver and small intestine, and metabolizes around 50% of medical drugs on the market. The human CYP3A subfamily comprises four members (CYP3A4, CYP3A5, CYP3A7, CYP3A43) encoded on human chromosome 7. However, transgenic mouse lines carrying the entire human CYP3A cluster have not been constructed because of limitations in conventional cloning techniques. Here, we show that the introduction of a human artificial chromosome (HAC) containing the entire genomic human CYP3A locus recapitulates tissue- and stage-specific expression of human CYP3A genes and xenobiotic metabolism in mice. About 700 kb of the entire CYP3A genomic segment was cloned into a HAC (CYP3A-HAC), and trans-chromosomic (Tc) mice carrying a single copy of germline-transmittable CYP3A-HAC were generated via a chromosome-engineering technique. The tissue- and stage-specific expression profiles of CYP3A genes were consistent with those seen in humans. We further generated mice carrying the CYP3A-HAC in the background homozygous for targeted deletion of most endogenous Cyp3a genes. In this mouse strain with 'fully humanized' CYP3A genes, the kinetics of triazolam metabolism, CYP3A-mediated mechanism-based inactivation effects and formation of fetal-specific metabolites of dehydroepiandrosterone observed in humans were well reproduced. Thus, these mice are likely to be valuable in evaluating novel drugs metabolized by CYP3A enzymes and in studying the regulation of human CYP3A gene expression. Furthermore, this system can also be used for generating Tc mice carrying other human metabolic genes.

Different interaction profiles of direct-acting anti-hepatitis C virus agents with human organic anion transporting polypeptides.

Simeprevir (SMV), asunaprevir (ASV), daclatasvir (DCV), and sofosbuvir (SFV), which are newly developed direct-acting antiviral agents (DAAs) against hepatitis C virus (HCV) infection, are among the key components of anti-HCV regimens. Preclinical studies have identified inhibitory properties for some of these DAAs against organic anion transporting polypeptide 1B (OATP1B) functions. However, their details remain mostly uncharacterized. Because OATP1B1 and OATP1B3 play determinant roles in the pharmacokinetics of various drugs via their uptake into human hepatocytes, it is plausible that the inhibition of these OATP1Bs by a DAA would create a potential risk of drug-drug interaction, which has been an emerging concern in anti-HCV therapy. Accordingly, in the present study, we intended to clarify the inhibitory characteristics of newly developed DAAs toward OATP1B1 and -1B3 functions. The results of our coincubation inhibition assays have shown that all tested DAAs could inhibit OATP1B1 functions and that SMV, ASV, and DCV (to a lesser extent), but not SFV, exhibited long-lasting preincubation inhibitory effects on OATP1B1 functions. It was also found that the preincubation inhibitory effects of SMV and ASV could augment their coincubation inhibition potency. Furthermore, significant, but differential, inhibitory effects of the DAAs on the OATP1B3 function were identified. To summarize, our results clearly show that the newly developed DAAs are newly identified OATP1B1 and OATP1B3 inhibitors with distinctive interaction properties. It is believed that these inhibition profiles will provide essential information to all concerned parties with respect to the clinical significance of DAA-mediated inhibition of OATP1Bs in anti-HCV therapy.

Knockout of mouse Cyp3a gene enhances synthesis of cholesterol and bile acid in the liver.

Here, we studied the effects of cytochrome P450 (CYP)3A deficiency on the mRNA expression of genes encoding regulators of hepatic cholesterol levels using Cyp3a-knockout (Cyp3a(-/-)) mice. The mRNA expression levels of genes encoding enzymes involved in cholesterol biosynthesis in the livers of Cyp3a(-/-) mice were higher than those of wild-type (WT) mice. Nuclear levels of sterol regulatory element-binding protein-2 (SREBP-2), which enhances cholesterol biosynthesis, were also higher in the livers of Cyp3a(-/-) mice. Binding of SREBP-2 to the Hmgcs1 gene promoter was more abundant in the livers of Cyp3a(-/-) mice. These results suggest that deficiency of CYP3A enzymes enhances transcription of genes encoding enzymes involved in cholesterol biosynthesis via activation of SREBP-2. On the other hand, hepatic cholesterol levels in Cyp3a(-/-) mice were 20% lower than those in WT mice. The mRNA expression levels of genes encoding enzymes involved in bile acid synthesis, plasma levels of 7α-hydroxy-4-cholesten-3-one and hepatic levels of total bile acid were significantly higher in Cyp3a(-/-) mice than in WT mice. These findings suggest that reduction of hepatic total cholesterol in Cyp3a(-/-) mice would be the consequence of enhanced bile acid synthesis. Therefore, CYP3A enzymes appear to play roles in the synthesis of cholesterol and bile acid in vivo.

Acrolein stimulates the synthesis of IL-6 and C-reactive protein (CRP) in thrombosis model mice and cultured cells.

Measurements of protein-conjugated acrolein (PC-Acro), IL-6, and C-reactive protein (CRP) in plasma were useful for identifying silent brain infarction with high sensitivity and specificity. The aim of this study was to determine whether acrolein causes increased production of IL-6 and CRP in thrombosis model mice and cultured cells. In mice with photochemically induced thrombosis, acrolein produced at the locus of infarction increased the level of IL-6 and then CRP in plasma. This was confirmed in cell culture systems - acrolein stimulated the production of IL-6 in mouse neuroblastoma Neuro-2a cells, mouse macrophage-like J774.1 cells, and human umbilical vein endothelial cells (HUVEC), and IL-6 in turn stimulated the production of CRP in human hepatocarcinoma cells. The level of IL-6 mRNA was increased by acrolein through an increase in phosphorylation of the transcription factors, c-Jun, and NF-κB p65. Furthermore, CRP stimulated IL-6 production in mouse macrophage-like J774.1 cells and HUVEC. IL-6 functioned as a protective factor against acrolein toxicity in Neuro-2a cells and HUVEC. These results show that acrolein stimulates the synthesis of IL-6 and CRP, which function as protecting factors against acrolein toxicity, and that the combined measurement of PC-Acro, IL-6, and CRP is effective for identification of silent brain infarction. The combined measurements of protein-conjugated acrolein (PC-Acro), IL-6, and C-reactive protein (CRP) in plasma were useful for identifying silent brain infarction. The aim of this study was to determine whether acrolein causes increased production of IL-6 and CRP, and indeed acrolein increased IL-6 synthesis and IL-6 in turn increased CRP synthesis. Furthermore, IL-6 decreased acrolein toxicity in several cell lines.

Transplantation of side population cells restores the function of damaged exocrine glands through clusterin.

Stem cell-based therapy has been proposed as a promising strategy for regenerating tissues lost through incurable diseases. Side population (SP) cells have been identified as putative stem cells in various organs. To examine therapeutic potential of SP cells in hypofunction of exocrine glands, SP cells isolated from mouse exocrine glands, namely, lacrimal and salivary glands, were transplanted into mice with irradiation-induced hypofunction of the respective glands. The secretions from both glands in the recipient mice were restored within 2 months of transplantation, although the transplanted cells were only sparsely distributed and produced no outgrowths. Consistent with this, most SP cells were shown to be CD31-positive endothelial-like cells. In addition, we clarified that endothelial cell-derived clusterin, a secretory protein, was an essential factor for SP cell-mediated recovery of the hypofunctioning glands because SP cells isolated from salivary glands of clusterin-deficient mice had no therapeutic potential, whereas lentiviral transduction of clusterin restored the hypofunction. In vitro and in vivo studies showed that clusterin had an ability to directly inhibit oxidative stress and oxidative stress-induced cell damage. Thus, endothelial cell-derived clusterin possibly inhibit oxidative stress-induced hypofunction of these glands.

ENT1, a ribavirin transporter, plays a pivotal role in antiviral efficacy of ribavirin in a hepatitis C virus replication cell system.

We previously showed that equilibrative nucleoside transporter 1 (ENT1) is a primary ribavirin transporter in human hepatocytes. However, because the role of this transporter in the antiviral mechanism of the drug remains unclear, the present study aimed to elucidate the role of ENT1 in ribavirin antiviral action. OR6 cells, a hepatitis C virus (HCV) replication system, were used to evaluate both ribavirin uptake and efficacy. The ribavirin transporter in OR6 cells was identified by mRNA expression analyses and transport assays. Nitrobenzylmercaptopurine riboside (NBMPR) and micro-RNA targeted to ENT1 mRNA (miR-ENT1) were used to reduce the ribavirin uptake level in OR6 cells. Our results showed that ribavirin antiviral activity was associated with its accumulation in OR6 cells, which was also closely associated with the uptake of the drug. It was found that the primary ribavirin transporter in OR6 cells was ENT1 and that inhibition of ENT1-mediated ribavirin uptake by NBMPR significantly attenuated the antiviral activity of the drug as well as its accumulation in OR6 cells. The results also showed that even a small reduction in the ENT1-mediated ribavirin uptake, achieved in this case using miR-ENT1, caused a significant decrease in its antiviral activity, thus indicating that the ENT1-mediated ribavirin uptake level determined its antiviral activity level in OR6 cells. In conclusion, our results show that by facilitating its uptake and accumulation in OR6 cells, ENT1 plays a pivotal role in the antiviral effectiveness of ribavirin and therefore provides an important insight into the efficacy of the drug in anti-HCV therapy.

Identification of a new organic anion transporting polypeptide 1B3 mRNA isoform primarily expressed in human cancerous tissues and cells.

Organic anion transporting polypeptide 1B3 (OATP1B3) is a hepatocyte plasma membrane protein that transports various endogenous and xenobiotic compounds. Although it is exclusively expressed in the human liver under normal conditions, OATP1B3 can be also expressed in various human cancer tissues that have been associated with prognosis and clinical outcomes. However, despite the potential significance of the latter finding, no experimental evidence addressing the molecular entity of cancer-associated OATP1B3 has been provided to date. In this paper, we report the identification of a new OATP1B3 mRNA isoform expressed in human colon and lung cancer tissues, which we named cancer-type OATP1B3 (Ct-OATP1B3). Our results also make known a previously unidentified transcription start site and an alternative promoter region, localized at intron 2, from which Ct-OATP1B3 mRNA is generated. Isoform specific mRNA quantification showed that the Ct-OATP1B3 mRNA level was strikingly higher than that of Lt-OATP1B3 mRNA in human cancer tissues. In addition, the results showed that the translation occurred at three out of four open reading frames. To summarize, our results clearly demonstrate that the newly-identified Ct-OATP1B3 (but not Lt-OATP1B3) is the primary mRNA isoform, at least in the human cancerous samples we have examined. In line with the possibility that its translation products play important biological roles in cancer cells, we strongly believe that the existence of Ct-OATP1B3 should be taken into account during future studies of OATP1B3 associated with cancer prognosis and clinical outcomes.

Contribution of ribavirin transporter gene polymorphism to treatment response in peginterferon plus ribavirin therapy for HCV genotype 1b patients.

BACKGROUND: Standard-dose ribavirin is crucial for the standard-of-care treatment of chronic hepatitis C virus (HCV) infection. Equilibrative nucleoside transporter 1 (ENT1), encoded by SLC29A1 gene, is the main transporter that imports ribavirin into human hepatocytes. AIMS: To determine whether single nucleotide polymorphisms (SNPs) at the SLC29A1 gene could influence the probability of treatment response compared with other baseline and host genetic factors. METHODS: A total of 526 East Asian patients monoinfected with HCV genotype 1b who had received pegylated interferon alpha plus ribavirin therapy were enrolled in this study. They were assigned randomly to the derivation and confirmatory groups. SNPs related to the IL28B, ITPA and SLC29A1 genes were genotyped using real-time detection polymerase chain reaction. Factors associated with sustained virological response (SVR) were analysed using multiple logistic regression analysis. RESULTS: Multivariate analysis for the derivation group identified six baseline variables significantly and independently associated with SVR: age [P = 0.023, odds ratio (OR) = 0.97], gender (P = 0.0047, OR = 2.25), platelet count (P = 0.00017, OR = 1.11), viral load (P = 0.00026, OR = 0.54), IL28B SNP rs12979860 (P = 1.09 × 10(-7) , OR = 8.68) and SLC29A1 SNP rs6932345 (P = 0.030, OR = 1.85). Using the model constructed by these independent variables, positive and negative predictive values and predictive accuracy were 73.3, 70.1 and 71.9% respectively. For the confirmatory group, they were 71.4, 84.6 and 75.3% respectively. The SLC29A1 and IL28B SNPs were also significantly associated with rapid virological response. CONCLUSIONS: The SNP at the major ribavirin transporter ENT1 gene SLC29A1 was one of significantly independent factors influencing treatment response, although the impact on the prediction was small.

Dexamethasone-mediated transcriptional regulation of rat carboxylesterase 2 gene.

Rat carboxylesterase 2 (rCES2), which was previously identified as a methylprednisolone 21-hemisuccinate hydrolase, is highly inducible by dexamethasone in the liver. In the present study, we investigated the molecular mechanisms by which this induction occurs. Injection of dexamethasone (1 mg/kg weight) into rats resulted in increases in the expression of rCES2 mRNA in a time-dependent manner with a peak at 12 h after injection. In primary rat hepatocytes, the expression level of rCES2 mRNA was increased by treatment with 100 nM dexamethasone, and the increase was completely blocked in the presence of 10 µM mifepristone (RU-486), a potent inhibitor of glucocorticoid receptor (GR), or 10 µg/mL cycloheximide, a translation inhibitor. Luciferase assays revealed that 100 nM dexamethasone increased rCES2 promoter activities, although the effect of dexamethasone on the promoter activity was smaller than that on rCES2 mRNA expression. The increased activities were completely inhibited by treatment of the hepatocytes with 10 µM RU-486. Based on these results, it is concluded that dexamethasone enhances transcription of the rCES2 gene via GR in the rat liver and that the dexamethasone-mediated induction of rCES2 mRNA may be dependent on de novo protein synthesis. Our results provide clues to understanding what compounds induce rCES2.

Identification of CYP isozymes involved in benzbromarone metabolism in human liver microsomes.

Benzbromarone (BBR) is metabolized to 1'-hydroxy BBR and 6-hydroxy BBR in the liver. 6-Hydroxy BBR is further metabolized to 5,6-dihydroxy BBR. The aim of this study was to identify the CYP isozymes involved in the metabolism of BBR to 1'-hydroxy BBR and 6-hydroxy BBR and in the metabolism of 6-hydroxy BBR to 5,6-dihydroxy BBR in human liver microsomes. Among 11 recombinant P450 isozymes examined, CYP3A4 showed the highest formation rate of 1'-hydroxy BBR. The formation rate of 1'-hydroxy BBR significantly correlated with testosterone 6ß-hydroxylation activity in a panel of 12 human liver microsomes. The formation of 1'-hydroxy BBR was completely inhibited by ketoconazole in pooled human liver microsomes. On the other hand, the highest formation rate of 6-hydroxy BBR was found in recombinant CYP2C9. The highest correlation was observed between the formation rate of 6-hydroxy BBR and diclofenac 4'-hydroxylation activity in 12 human liver microsomes. The formation of 6-hydroxy BBR was inhibited by tienilic acid in pooled human liver microsomes. The formation of 5,6-dihydroxy BBR from 6-hydroxy BBR was catalysed by recombinant CYP2C9 and CYP1A2. The formation rate of 5,6-dihydroxy BBR was significantly correlated with diclofenac 4'-hydroxylation activity and phenacetin O-deethylation activity in 12 human liver microsomes. The formation of 5,6-dihydroxy BBR was inhibited with either tienilic acid or α-naphthoflavone in human liver microsomes. These results suggest that (i) the formation of 1'-hydroxy BBR and 6-hydroxy BBR is mainly catalysed by CYP3A4 and CYP2C9, respectively, and (ii) the formation of 5,6-dihydroxy BBR is catalysed by CYP2C9 and CYP1A2 in human liver microsomes.

[Genetic marker of statin-induced rhabdomyolysis].

This review summarizes genetic factors predisposed to statin-induced rhabdomyolysis. The first genetic risk factor of statin myopathy uncovered by genome-wide analysis of single nucleotide polymorphisms was the common variant of SLCO1B1 gene. Analysis of 30000 genetic markers in 85 patients with myopathy induced by high-dose simvastatin showed a strong association with 521T>C polymorphism of SLCO1B1. Another study also showed that this variant of SLCO1B1 has a significant association with myopathy in patients taking pravastatin or atorvastatin although the number of patients analyzed was limited. In addition to SLCO1B1, recent studies suggested that variants of genes encoding transporters (ABCG2 and ABCB1) and metabolic enzymes (CYP2C8 and UGT1A3) involved in the disposition of statins, and those involved in the metabolic muscle disease (glycogen storage disorders, carnitine palmitoyl-2 deficiency and myoadenylate deaminase deficiency) are also risk factors of statin-induced myopathy. These genetic factors may provide predisposition testing for statin-induced rhabdomyolysis.

Characterization of ribavirin uptake systems in human hepatocytes.

BACKGROUND & AIMS: The purpose of this study was to identify the major ribavirin uptake transporter(s) in human hepatocytes and to determine if these previously unidentified transporters are involved in hepatic ribavirin uptake. Furthermore, we aimed to address what causes the difference in uptake levels among human hepatocytes. METHODS: Profiles of ribavirin uptake and nucleoside transporter mRNA expression in Caucasian hepatocytes (HH268, HH283 and HH291) were characterized by transport assay and reverse transcription-polymerase chain reaction (RT-PCR). The 5'-side of the SLC29A1 gene structure was characterized by determination of transcription start sites and by RT-PCR. RESULTS: Equilibrative nucleoside transporter 1 (ENT1)-mediated uptake was exclusively involved in ribavirin uptake in HH268 and HH283 and was responsible for the largest ribavirin uptake fraction in HH291. The level of ENT1-mediated uptake in HH291 was higher than that in HH268 and HH283. Characterization of the SLC29A1 gene structure revealed the existence of several ENT1 mRNA isoforms in the human liver, and the levels of four ENT1 mRNA isoforms in HH291 were higher than those in HH268 or HH283. No ENT2-mediated uptake was observed in any hepatocyte lines. Na(+)-dependent uptake was detected only in HH291; however, mRNA levels of concentrative nucleoside transporters (CNTs) were at trace levels in all hepatocyte lines. CONCLUSIONS: ENT1, but not ENT2 or CNTs, is a major ribavirin uptake transporter in human hepatocytes. The different ENT1-mediated ribavirin uptake levels in different hepatocyte lines are associated with different expression levels of specific isoforms of ENT1 mRNAs. Furthermore, an unidentified Na(+)-dependent ribavirin transport system might exist in human hepatocytes.