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Japan Atomic Energy Research Institute is constructing the International Thermonuclear Experimental Reactor common test facility for the Central Solenoid Model Coil which is around 180 tons, a forced-flow cooled magnet with the maximum pulsed operation of 2 T/s and generates the rated magnetic field of 13 T at 48 kA with stored energy of 668 MJ. The test facility consists of a coil vacuum chamber, a cryogenic system with the 5-kW refrigerator and 500-g/s cryogenic pump, two pairs of 50-kA current leads, two DC power supplies (50kA and 60kA) and two JT-60 pulsed power supplies (50 kA, [+ or -]4.5 kV and [+ or -]40 kA, [+ or -]1.5 kV). The facility will be demonstrating the refrigeration and operation of a fusion pulsed magnet and the design and construction will accumulate experience towards the construction of ITER.

The [Nb.sub.3]Sn cable is being fabricated for the Central Solenoid (CS) Model Coil under the ITER Engineering-Design Activity. The cable consists of about 1000 strands whose diameter is 0.81 mm. The design current is 48 kA at a magnetic field of 13T. The 0.6-GJ CS model coil is operated in a pulse mode (0.5T/s). The first trial fabrication of a 100-m dummy cable and a 20-m superconducting cable was completed successfully. The second trial fabrication of a 1000-m dummy cable was performed to establish the stable manufacturing procedure in January, 1995. We measured the AC losses of the full-sized conductor and could determine the cable coupling time constant. We analyzed the heat generation of the CS model coil and calculated the temperature rise of the cable for the model coil.

To quantitatively understand the events in the human liver, we modeled a hepatic disposition of bosentan and its three known metabolites (Ro 48-5033, Ro 47-8634, and Ro 64-1056) in sandwich-cultured human hepatocytes based on the known metabolic pathway. In addition, the hepatotoxicity of Ro 47-8634 and Ro 64-1056 was investigated because bosentan is well known as a hepatotoxic drug. A model illustrating the hepatic disposition of bosentan and its three metabolites suggested the presence of a novel metabolic pathway(s) from the three metabolites. By performing in vitro metabolism studies on human liver microsomes, a novel metabolite (M4) was identified in Ro 47-8634 metabolism, and its structure was determined. Moreover, by incorporating the metabolic pathway of Ro 47-8634 to M4 into the model, the hepatic disposition of bosentan and its three metabolites was successfully estimated. In hepatocyte toxicity studies, the cell viability of human hepatocytes decreased after exposure to Ro 47-8634, and the observed hepatotoxicity was diminished by pretreatment with tienilic acid (CYP2C9-specific inactivator). Pretreatment with 1-aminobenzotriazole (broad cytochrome P450 inactivator) also tended to maintain the cell viability. Furthermore, Ro 64-1056 showed hepatotoxicity in a concentration-dependent manner. These results suggest that Ro 64-1056 is directly involved in bosentan-induced liver injury partly because CYP2C9 specifically mediates hydroxylation of the t-butyl group of Ro 47-8634. Our findings demonstrate the usefulness of a quantitative modeling of hepatic disposition of drugs and metabolites in sandwich-cultured hepatocytes. In addition, the newly identified metabolic pathway may be an alternative route that can avoid Ro 64-1056-induced liver injury., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Paroxetine, a selective serotonin reuptake inhibitor, is metabolized in the liver and excreted into bile and urine as metabolites, but species differences have been observed in hepatic disposition between rats and humans. A major metabolite in rats is M1-glucuronide, whereas M1-glucuronide and M1-sulfate are found in humans. The primary excretion route of paroxetine-derived radioactivity in rats and humans is bile and urine, respectively. The aim of this study was to examine the usefulness of sandwich-cultured hepatocytes (SCH) to evaluate in vivo species differences of the hepatic disposition of paroxetine between rats and humans. The metabolite profile of [(3)H]paroxetine in SCH was similar to that in hepatocytes in suspension, and the in vitro metabolite profiles were similar to the published in vivo metabolic pathways for both species. Furthermore, the biliary excretion index (BEI) of formed M1-glucuronide in rat SCH (25.8-50.9%) was higher than that in human SCH (15.1-16.7%). The BEI of formed M1-sulfate (16.4-29.1%) was comparable to that of M1-glucuronide in human SCH, whereas the BEIs of paroxetine were negligible in SCH of both species. Moreover, M1-glucuronide was demonstrated to be a multidrug resistance-associated protein 2 substrate in both species, as determined by its uptake into ATP-binding cassette transporter-expressing membrane vesicles. SCH should prove to be useful to evaluate the processes of hepatic uptake and metabolism of parent drugs and the simultaneous examination of the biliary excretion of both parent drug and liver-derived metabolites.

We investigated the metabolism of pranlukast, a selective leukotriene agonist, and the potential for drug-drug interactions. Although cytochrome P450 (CYP) 3A4 appeared to be the major cytochrome P450 isoform involved in the metabolism of pranlukast, the results suggested that pranlukast metabolism was inhibited less than 50% by ketoconazole, a reversible CYP3A4 inhibitor, or by anti-CYP3A4 antibodies. Irreversible macrolide CYP3A4 inhibitors, clarithromycin, erythromycin and roxithromycin, exhibited little effect on pranlukast metabolism. On the other hand, pranlukast reversibly inhibited CYP2C8 and/or 2C9, and CYP3A4, with K(i) values of 3.9 and 4.1 micromol/l, respectively. The [I](in,max,u)/K(i) ratios were 0.004 and 0.003, respectively. The K(i) values were about 300-fold greater than the [I](in,max,u), therefore it is suggested that, at clinical doses, pranlukast will not affect the pharmacokinetics of concomitantly administered drugs that are primarily metabolized by CYP2C8 and/or 2C9 or CYP3A4.

Human multidrug resistance protein 1 (MRP1) has a total of 17 transmembrane (TM) helices arranged in three membrane-spanning domains, MSD0, MSD1, and MSD2, with a 5 + 6 + 6 TM configuration. Photolabeling studies indicate that TMs 10 and 11 in MSD1 and 16 and 17 in MSD2 contribute to the substrate binding pocket of the protein. Previous mutational analyses of charged and polar amino acids in predicted TM helices 11, 16, and 17 support this suggestion. Mutation of Trp(553) in TM10 also affects substrate specificity. To extend this analysis, we mutated six additional polar residues within TM10 and the remaining uncharacterized polar residue in TM16, Asn(1208). Although mutation of Asn(1208) was without effect, two of six mutations in TM10, T550A and T556A, modulated the drug resistance profile of MRP1 without affecting transport of leukotriene C4, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), and glutathione. Mutation T550A increased vincristine resistance but decreased doxorubicin resistance, whereas mutation T556A decreased resistance to etoposide (VP-16) and doxorubicin. Although conservative mutation of Tyr(568) in TM10 to Phe or Trp had no apparent effect on substrate specificity, substitution with Ala decreased the affinity of MRP1 for E(2)17betaG without affecting drug resistance or the transport of other substrates tested. These analyses confirm that several amino acids in TM10 selectively alter the substrate specificity of MRP1, suggesting that they interact directly with certain substrates. The location of these and other functionally important residues in TM helices 11, 16, and 17 is discussed in the context of an energy-minimized model of the membrane-spanning domains of MRP1.

Human multidrug resistance protein 1 (MRP1) is an ATP binding cassette (ABC) transporter that confers resistance to many natural product chemotherapeutic agents and can transport structurally diverse conjugated organic anions. MRP1 has three polytopic transmembrane domains (TMDs) and a total of 17 TM helices. Photolabeling and mutagenesis studies of MRP1 indicate that TM11, the last helix in the second TMD, may form part of the protein's substrate binding pocket. We have demonstrated that certain polar residues within a number of TM helices, including Arg(593) in TM11, are determinants of MRP1 substrate specificity or overall activity. We have now extended these analyses to assess the functional consequences of mutating the remaining seven polar residues within and near TM11. Mutations Q580A, T581A, and S585A in the predicted outer leaflet region of the helix had no detectable effect on function, while mutation of three residues close to the membrane/cytoplasm interface altered substrate specificity. Two of these mutations affected only drug resistance. N597A increased and decreased resistance to vincristine and VP-16, respectively, while S605A decreased resistance to vincristine, VP-16 and doxorubicin. The third, S604A, selectively increased 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. In contrast, elimination of the polar character of the residue at position 590 (Asn in the wild-type protein) uniformly impaired the ability of MRP1 to transport potential physiological substrates and to confer resistance to three different classes of natural product drugs. Kinetic and photolabeling studies revealed that mutation N590A not only decreased the affinity of MRP1 for cysteinyl leukotriene 4 (LTC(4)) but also substantially reduced the binding of ATP to nucleotide binding domain 1 (NBD1). Thus, polar interactions involving residues in TM11 influence not only the substrate specificity of MRP1 but also an early step in the proposed catalytic cycle of the protein.

The ATP-binding cassette transporter multidrug resistance protein 1 (MRP1) confers resistance to a number of clinically important chemotherapeutic agents. The proximal promoter region of MRP1 is GC-rich and contains binding sites for members of the Sp1 family of trans-acting factors that seem to be important for basal expression. As an approach to searching for other elements that may contribute to expression, we have sequenced and functionally compared the promoters of the murine and rat mrp1 genes with that of the human gene. All three promoters are GC-rich, TATA-less, and CAAT-less. Conservation of sequence between rodent and human promoters is limited to a proximal region of 100 nucleotides containing binding sites for members of the Sp1 family and a putative activator protein-1 element. The 5'-untranslated region (UTR) of human MRP1 contains an insertion of approximately 160 nucleotides comprising a GCC-triplet repeat and a GC-rich tandem repeat that is absent from the rodent sequences. Transient transfection analyses demonstrated that the conserved GC-boxes of all three genes are the major determinants of basal activity. Based on electrophoretic mobility shift assays, each GC-box can be bound by Sp1 or Sp3. Unlike the rodent genes, the human MRP1 5'UTR also binds Sp1 but not Sp3, and the human promoter retains substantial activity even in the absence of the conserved GC-boxes. Finally, we show that the tumor suppressor protein p53 can repress the human and rodent promoters by a mechanism that is independent of the Sp1 elements.

Multidrug resistance protein 1 (MRP1) transports a wide range of structurally diverse conjugated and nonconjugated organic anions and some peptides, including oxidized and reduced glutathione (GSH). The protein confers resistance to certain heavy metal oxyanions and a variety of natural product-type chemotherapeutic agents. Elevated levels of MRP1 have been detected in many human tumors, and the protein is a candidate therapeutic target for drug resistance reversing agents. Previously, we have shown that human MRP1 (hMRP1) and murine MRP1 (mMRP1) differ in their substrate specificity despite a high degree of structural conservation. Since rat models are widely used in the drug discovery and development stage, we have cloned and functionally characterized rat MRP1 (rMRP1). Like mMRP1 and in contrast to hMRP1, rMRP1 confers no, or very low, resistance to anthracyclines and transports the two estrogen conjugates, 17beta-estradiol-17-(beta-d-glucuronide) (E217betaG) and estrone 3-sulfate, relatively poorly. Mutational studies combined with vesicle transport assays identified several amino acids conserved between rat and mouse, but not hMRP1, that make major contributions to these differences in substrate specificity. Despite the fact that the rodent proteins transport E217betaG poorly and the GSH-stimulated transport of estrone 3-sulfate is low compared with hMRP1, site-directed mutagenesis studies indicate that different nonconserved amino acids are involved in the low efficiency with which each of the two estrogen conjugates is transported. Our studies also suggest that although rMRP1 and mMRP1 are 95% identical in primary structure, their substrate specificities may be influenced by amino acids that are not conserved between the two rodent proteins.

Human dihydrodiol dehydrogenase (DD) 4/aldo-keto reductase (AKR) 1C4 is a major isoform of hepatic DD that oxidizes trans-dihydrodiols of polycyclic aromatic hydrocarbons to reactive and redox-active o-quinones and that reduces several ketone-containing drugs. To investigate the mechanism of transcriptional regulation of the human DD4 gene, the 5'-flanking region of the gene was fused to the luciferase gene. The results of luciferase assays using HepG2 cells and of 1,10-phenanthroline-copper footprinting indicated that two positive regulatory regions were located in regions from -701 to -684 and from -682 to -666. The former region contained a putative hepatocyte nuclear factor (HNF)-4 binding motif, and the latter region contained an HNF-1 consensus binding sequence. DNA fragments of the HNF-4 or HNF-1 motif gave a shifted band in a gel-shift assay with nuclear extracts from HepG2 cells. The formation of the DNA-protein complex was inhibited by the HNF-4 or HNF-1 motif of the alpha(1)-antitrypsin gene. A supershift assay using antibodies to human HNF-4alpha, HNF-4gamma and HNF-1alpha showed that HNF-4alpha and HNF-4gamma bound to the HNF-4 motif, and that HNF-1alpha interacted with the HNF-1 motif. Introduction of mutations into the HNF-4 or HNF-1 motif lowered the luciferase activity to 10 or 8% respectively of that seen with the intact human DD4 gene. These results indicate that HNF-4alpha, HNF-4gamma and HNF-1alpha regulate co-operatively the transcription of the human DD4 gene in HepG2 cells.

Expression of drug-metabolizing enzymes including cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) in various tissues of Suncus murinus (Suncus) were examined. Northern blot analysis showed that mRNAs hybridizable with cDNAs for rat CYP1A2, human CYP2A6, rat CYP2B1, human CYP2C8, human CYP2D6, rat CYP2E1, human CYP3A4 and rat CYP4A1 were expressed in various tissues from Suncus. The mRNA level of CYP2A in the Suncus lung was very high. Furthermore, it was found that the level of CYP2A mRNA in the Suncus lung was higher compared to the Suncus liver. The expression level of mRNA hybridizable with cDNA for human CYP3A4 was very low. The presence of CYP3A gene in Suncus was proven by the induction of the CYP with dexamethasone. Very low expression levels of mRNAs hybridizable with cDNAs for rat FMO1, rat FMO2, rat FMO3 and rat FMO5 were also seen in Suncus liver. No apparent hybridization band appeared when human FMO4 cDNA was used as a probe. The hepatic expression of mRNAs hybridizable with cDNAs for UDP-glucuronosyltransferase 1*6, aryl sulfotransferase, glutathione S-transferase 1, carboxyesterase and microsomal epoxide hydrolase in the Suncus were observed. These results indicate that the Suncus is a unique animal species in that mRNAs for CYP3A and FMO are expressed at very low levels.

(+)-Cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) is a newly developed drug as a platelet-activating factor receptor antagonist. The disposition of SM-12502 was investigated in plasma from 28 healthy Japanese volunteers after a single i.v. administration of SM-12502. Three of 28 subjects were phenotyped as poor metabolizers (PMs). Genomic DNAs from three extensive metabolizers or three PMs of SM-12502 were analyzed by Southern blot analysis with CYP2A6 cDNA as a probe. DNAs from three PMs digested with SacI and SphI showed novel restriction fragment length polymorphisms (RFLPs); one type without 4.5- and 2.6-kb fragments and a weak density of a 6.4-kb fragment (E-type), and the other type without 7.1- and 5.5-kb restriction fragments (C'-type) as compared with three extensive metabolizers, respectively. The deletional restriction fragments specific to three PMs in SacI- and SphI-RFLPs were identified as CYP2A6. Using polymerase chain reaction-RFLP analyses of the gene from the three PMs, we found that the exon 1, exon 8, and exon 9 in CYP2A6 were absent. A new RFLP characterized by SacI and SphI was found to be due to the entire gene deletion of the three exons and was associated with the decreased metabolism of SM-12502. This study demonstrates a new deletional allele in the human CYP2A6 gene responsible for the poor metabolic phenotype of SM-12502.

The S-oxidation of (4)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) and the 7-hydroxylation of coumarin are primarily catalyzed by cytochrome P450 2A6 (CYP2A6). The activities of SM-12502 S-oxidase and coumarin 7-hydroxylase were investigated with liver microsomes from 20 human individuals. Liver microsomes from individual H16 showed the lowest activities of both enzymes. The expression of CYP2A6 protein was not detectable in liver microsomes from individuals H4, H5, H7, H8, H12 and H16. CYP2A6 mRNA was hardly detectable in the liver of the individual H16. A new SacI-restriction fragment length polymorphism showing the lack of a 2.6 kb fragment was found in two of forty genomic DNA preparations from individuals H16 and No. 594, using CYP2A6 cDNA as a probe. This deletional 2.6 kb fragment was isolated from a genomic library prepared from one individuals showing normal coumarin 7-hydroxylase activity and was sequenced. This fragment contained a CYP2A6 gene region from 319 bp upstream of a putative exon 6 to a SacI site in exon 9, indicating that this region was deleted in the two individuals in this study. We also demonstrated by polymerase chain reaction analysis that the exon 8 of CYP2A6 gene was deleted in individuals H16 and No. 594. These results indicate that the reduced activity of SM-12502 S-oxidase and no activity of coumarin 7-hydroxylase are caused by the lack of CYP2A6 mRNA and CYP2A6 protein caused by the CYP2A6 gene deletion.

Nicotine is primarily metabolized to cotinine in humans. In this study, human cytochrome P450 (CYP) isoform involved in cotinine formation was identified. The formation of cotinine in 16 human liver microsomes was determined with a 50 microM nicotine concentration and with a cytosol preparation as a source of aldehyde oxidase. Cotinine formation in human liver microsomes significantly correlated with immunochemically determined CYP2A6 levels (r = 0.663, p < 0.05), coumarin 7-hydroxylase activities (r = 0.831, p < 0.01), and cotinine 3'-hydroxylase activities (r = 0.735, p < 0.01) that are responsible for CYP2A6. In inhibition studies, cotinine formation in human liver microsomes was inhibited by coumarin and rabbit anti-rat CYP2A1 antibody specifically. When the capability of microsomes of B-lymphoblastoid cells expressing human CYPs to perform biotransformation of nicotine to cotinine was determined, cDNA-expressed CYP2A6 exhibited the highest cotinine formation. The KMapp values from microsome expressing CYP2A6 cDNA were similar to the value obtained from human liver microsomes. The large interindividual variabilities in cotinine formation and immunochemically determined CYP2A6 levels were observed in human liver microsomes, suggesting genetic polymorphism of CYP2A6. Nicotine is a new in vivo probe for phenotyping of CYP2A6 in humans.