Tomomi Furihata

Genomic Structure and Transcriptional Regulation of the Rat, Mouse, and Human Carboxylesterase Genes

The mammalian carboxylesterases (CESs) comprise a multigene family which gene products play important roles in biotransformation of ester- or amide-type prodrugs. Since expression level of CESs may affect the pharmacokinetic behavior of prodrugs in vivo, it is important to understand the transcriptional regulation mechanism of the CES genes. However, little is known about the gene structure and transcriptional regulation of the mammalian CES genes. In the present study, to investigate the transcriptional regulation of the promoter region of the CES1 and CES2 genes were isolated from mouse, rat and human genomic DNA by PCR amplification. A TATA box was not found the transcriptional start site of all CES promoter. These CES promoters share several common binding sites for transcription factors among the same CES families, suggesting that the orthologous CES genes have evolutionally conserved transcriptional regulatory mechanisms. The result of present study suggested that the mammalian CES promoters were at least partly conserved among the same CES families, and some of the transcription factors may play similar roles in transcriptional regulation of the human and murine CES genes.

Identification of Human Cytochrome P450 Isozymes Involved in Diphenhydramine N-Demethylation

Diphenhydramine is widely used as an over-the-counter antihistamine. However, the specific human cytochrome P450 (P450) isozymes that mediate the metabolism of diphenhydramine in the range of clinically relevant concentrations (0.14–0.77 μM) remain unclear. Therefore, P450 isozymes involved in N-demethylation, a main metabolic pathway of diphenhydramine, were identified by a liquid chromatography-mass spectrometry method developed in our laboratory. Among 14 recombinant P450 isozymes, CYP2D6 showed the highest activity of diphenhydramine N-demethylation (0.69 pmol/min/pmol P450) at 0.5 μM. CYP2D6 catalyzed diphenhydramine N-demethylation as a high-affinity P450 isozyme, the Km value of which was 1.12 ± 0.21 μM. In addition, CYP1A2, CYP2C9, and CYP2C19 were identified as low-affinity components. In human liver microsomes, involvement of CYP2D6, CYP1A2, CYP2C9, and CYP2C19 in diphenhydramine N-demethylation was confirmed by using P450 isozyme-specific inhibitors. In addition, contributions of these P450 isozymes estimated by the relative activity factor were in good agreement with the results of inhibition studies. Although an inhibitory effect of diphenhydramine on the metabolic activity of CYP2D6 has been reported previously, the results of the present study suggest that it is not only a potent inhibitor but also a high-affinity substrate of CYP2D6. Therefore, it is worth mentioning that the sedative effect of diphenhydramine might be caused by coadministration of CYP2D6 substrate(s)/inhibitor(s). In addition, large differences in the metabolic activities of CYP2D6 and those of CYP1A2, CYP2C9, and CYP2C19 could cause the individual differences in anti-allergic efficacy and the sedative effect of diphenhydramine.

Purification, molecular cloning, and functional expression of inducible liver acylcarnitine hydrolase in C57BL/6 mouse, belonging to the carboxylesterase multigene family

To identify the peroxisome proliferator-inducible acylcarnitine hydrolase in C57BL/6 mice, acylcarnitine hydrolase was purified to homogeneity using column chromatography. The purified enzyme, named ACH M1, had a subunit molecular weight of 60kDa. ACH M1 could hydrolyze classical carboxylesterase (CES) substrates as well as palmitoyl-dl-carnitine and these activities were inhibited by anti-rat CES antibodies. The peptide fragments of ACH M1 were identical to those of the deduced amino acid sequence of mouse CES2 isozyme. These findings suggested that ACH M1 was a member of the CES2 family. The mouse CES2 cDNA, designated mCES2, was cloned from mouse liver. The recombinant mCES2 expressing in Sf9 cells showed high level of catalytic activity toward acylcarnitines. Furthermore, the biological characteristics of the expressed protein were identical with those of ACH M1 in many cases, suggesting that mCES2 encodes mouse liver ACH M1.

INVOLVEMENT OF HEPATOCYTE NUCLEAR FACTOR 4α IN THE DIFFERENT EXPRESSION LEVEL BETWEEN CYP2C9 AND CYP2C19 IN THE HUMAN LIVER

CYP2C9 and CYP2C19 are clinically important drug-metabolizing enzymes. The expression level of CYP2C9 is much higher than that of CYP2C19, although the factor(s) responsible for the difference between the expression levels of these genes is still unclear. It has been reported that hepatocyte nuclear factor 4α (HNF4α) plays an important role in regulation of the expression of liver-enriched genes, including P450 genes. Thus, we hypothesized that HNF4α contributes to the difference between the expression levels of these genes. Two direct repeat 1 (DR1) elements were located in both the CYP2C9 and CYP2C19 promoters. The upstream and downstream elements in these promoters had the same sequences, and HNF4α could bind to both elements in vitro. The transactivation levels of constructs containing two DR1 elements of the CYP2C9 promoter were increased by HNF4α, whereas those of the CYP2C19 promoter were not increased. The introduction of mutations into either the upstream or downstream element in the CYP2C9 gene abolished the responsiveness to HNF4α. We also examined whether HNF4α could bind to the promoter regions of the CYP2C9 and the CYP2C19 genes in vivo. The results of chromatin immunoprecipitation assays showed that HNF4α could bind to the promoter region of the CYP2C9 gene but not to that of the CYP2C19 promoter in the human liver. Taken together, our results suggest that HNF4α is a factor responsible for the difference between the expression levels of CYP2C9 and CYP2C19 in the human liver.

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

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:

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.

Mechanism of the decrease in catalytic activity of human cytochrome P450 2C9 polymorphic variants investigated by computational analysis

Cytochrome P450 (CYP) is deeply involved in the metabolism of chemicals including pharmaceuticals. Therefore, polymorphisms of this enzyme have been widely studied to avoid unfavorable side effects of drugs in chemotherapy. In this work, we performed computational analysis of the mechanism of the decrease in enzymatic activity for three typical polymorphisms in CYP 2C9 species: *2, *3, and *5. Based on the equilibrated structure obtained by molecular dynamics simulation, the volume of the binding pocket and the fluctuation of amino residues responsible for substrate holding were compared between the wild type and the three variants. Further docking simulation was carried out to evaluate the appropriateness of the binding pocket to accommodate substrate chemicals. Every polymorphic variant was suggested to be inferior to the wild type in enzymatic ability from the structural viewpoint. F‐G helices were obviously displaced outward in CYP2C9*2. Expansion of the binding pocket, especially the space near F′ helix, was remarkable in CYP2C9*3. Disappearance of the hydrogen bond between K helix and β4 loop was observed in CYP2C9*5. The reduction of catalytic activity of those variants can be explained from the deformation of the binding pocket and the consequent change in binding mode of substrate chemicals. The computational approach is effective for predicting the enzymatic activity of polymorphic variants of CYP. This prediction will be helpful for advanced drug design because calculations forecast unexpected change in drug efficacy for individuals.

Functional analysis of a mutation in the SLCO1B1 gene (c.1628T>G) identified in a Japanese patient with pravastatin-induced myopathy

In the present study, we analyzed the function of a novel mutation (c.1628T>G, p.Leu543Trp) in the solute carrier organic anion transporter (SLCO) 1B1 gene, encoding organic anion transporting polypeptide (OATP) 1B1, which was identified in a patient with pravastatin-induced myopathy. OATP1B1 variants carrying the mutation (OATP1B1*1a+c.1628T>G or *1b+c.1628T>G) showed a reduced transporting activity toward typical substrates and pravastatin compared with the activity of the references (OATP1B1*1a or *1b). This was due to reduction in Vmax values of the variants, not due to change in their Km values. OATP1B1*1b+c.1628T>G was normally expressed on the plasma membrane of HEK293 cells at the same level as that of OATP1B1*1b. Taken together, our results suggest that the mutation c.1628T>G (p.Leu543Trp) reduced the function of OATP1B1 probably due to decrease in turnover rate of one OATP1B1 molecule rather than impairment of protein sorting to the plasma membrane.

Synergistic role of specificity proteins and upstream stimulatory factor 1 in transactivation of the mouse carboxylesterase 2/microsomal acylcarnitine hydrolase gene promoter.

Mouse carboxylesterase 2 (mCES2), a microsomal acylcarnitine hydrolase, is thought to play some important roles in fatty acid (ester) metabolism, and it is therefore thought that the level of transcription of the mCES2 gene is under tight control. Examination of the tissue expression profiles revealed that mCES2 is expressed in the liver, kidney, small intestine, brain, thymus, lung, adipose tissue and testis. When the mCES2 promoter was cloned and characterized, it was revealed that Sp1 (specificity protein 1) and Sp3 could bind to a GC box, that USF (upstream stimulatory factor) 1 could bind to an E (enhancer) box, and that Sp1 could bind to an NFkappaB (nuclear factor kappaB) element in the mCES2 promoter. Co-transfection assays showed that all of these transcription factors contributed synergistically to transactivation of the mCES2 promoter. Taken together, our results indicate that Sp1, Sp3 and USF1 are indispensable factors for transactivation of the mCES2 gene promoter. To our knowledge, this is the first study in which transcription factors that interact with a CES2 family gene have been identified. The results of the present study have provided some clues for understanding the molecular mechanisms regulating mCES2 gene expression, and should be useful for studies aimed at elucidation of physiological functions of mCES2.

Establishment of a new conditionally immortalized cell line from human brain microvascular endothelial cells: A promising tool for human blood–brain barrier studies

The blood-brain barrier (BBB) is formed by brain microvascular endothelial cells (BMEC) working together with astrocytes and pericytes, in which tight junctions and various transporters strictly regulate the penetration of diverse compounds into the brain. Clarification of the molecular machinery that provides such regulation using in vitro BBB models has provided important insights into the roles of the BBB in central nervous system (CNS) disorders and CNS drug development. In this study, we succeeded in establishing a new cell line, hereinafter referred to as human BMEC/conditionally immortalized, clone β (HBMEC/ciβ), as part of our ongoing efforts to develop an in vitro human BBB model. Our results showed that HBMEC/ciβ proliferated well. Furthermore, we found that HBMEC/ciβ exhibited the barrier property of restricting small molecule intercellular penetration and possessed effective efflux transporter functions, both of which are essential to a functioning BBB. Because higher temperatures are known to terminate immortalization signals, we specifically examined the effects of higher temperatures on the HBMEC/ciβ differentiation status. The results showed that higher temperatures stimulated HBMEC/ciβ differentiation, marked by morphological alteration and increases in several mRNA levels. To summarize, our data indicates that the newly established HBMEC/ciβ offers a promising tool for use in the development of a practical in vitro human BBB model that could make significant contributions toward understanding the molecular biology of CNS disorders, as well as to CNS drug development. It is also believed that the development of a specific culture method for HBMEC/ciβ will add significant value to the HBMEC/ciβ-based BBB model.