Drug Metabolism and Pharmacokinetics
Online ISSN : 1880-0920
Print ISSN : 1347-4367
ISSN-L : 1347-4367
Volume 24 , Issue 3
Showing 1-12 articles out of 12 articles from the selected issue
Editorial
Regular Articles
  • Takahiro TODA, Kanna OHI, Toshiyuki KUDO, Tomoyuki YOSHIDA, Nobutomo I ...
    2009 Volume 24 Issue 3 Pages 201-208
    Published: 2009
    Released: July 02, 2009
    JOURNALS FREE ACCESS
      Ciprofloxacin (CPX), a new quinolone antibiotic, is reported to reduce CYP3A expression in the liver when administered to rats. The present study investigates whether the reduction in intestinal flora is involved in this reduction of CYP3A. While hepatic Cyp3a11 expression and triazolam metabolic activity were significantly reduced by CPX treatment of SPF mice, no significant changes were seen by CPX treatment of germ-free (GF) mice. Lithocholic acid (LCA)-producing bacteria in the feces as well as hepatic level of taurine conjugate of LCA were significantly reduced in CPX-treated SPF mice. Cyp3a11 expression in GF mice was significantly elevated when treated with LCA, known as an activator of fernesoid X receptor and pregnane X receptor. These results indicate that antibiotics such as CPX, having antimicrobial spectrums against LCA-producing bacteria, possibly cause decrease in LCA in the liver, resulting in lower CYP3A expression. The intestinal flora is reported to be altered also by stress, disease and age etc. The findings of the present study suggest that these changes in intestinal flora may modify CYP expression and contribute to individual differences in pharmacokinetics.
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  • Kazunobu AOYAMA, Kouichi YOSHINARI, Hye-Ji KIM, Kiyoshi NAGATA, Yasush ...
    2009 Volume 24 Issue 3 Pages 209-217
    Published: 2009
    Released: July 02, 2009
    JOURNALS FREE ACCESS
      Human hepatocyte culture is widely used to predict human drug metabolism for new drug development. The limited supply and lot-to-lot (donor-to-donor) variations in enzymatic activity, however, hamper its applicability. In the present study, we explore a new cell system with adenovirus-mediated expression of cytochrome P450s (P450s) as an alternative for hepatocytes. In this system, P450 apoprotein levels and catalytic activity increased depending on the amounts of adenoviruses infected for the individual expression of CYP3A4 or CYP2C19 in HepG2 cells. Similar results were observed in the system with co-expression of CYP3A4 and CYP2C19. When HepG2 cells were infected with adednovirus for CYP3A4 and that for CYP2C19 simultaneously at a ratio of 10:1, the ratio of their apoprotein levels was similar to that observed in human hepatocytes and the metabolic profile of diazepam in the system was almost identical to that observed in hepatocyets. These results indicate that this adenovirus-mediated system makes it possible to reproducibly prepare cells expressing multiple P450s at a desired ratio, suggesting a possible use of this system in preclinical metabolic tests for a drug candidate(s), particularly to assess the influence of inter-individual variation in P450 activity.
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  • Satomi NAGASHIMA, Makiko SHIMIZU, Hiroshi YANO, Norie MURAYAMA, Toshio ...
    2009 Volume 24 Issue 3 Pages 218-225
    Published: 2009
    Released: July 02, 2009
    JOURNALS FREE ACCESS
      Human flavin-containing monooxygenase 3 (FMO3)-mediated microsomal oxygenation activity, levels of FMO3 protein and FMO3 mRNA and modifications were investigated in Japanese livers genotyped for the FMO3 gene. Significant correlations were observed for benzydamine N-oxygenation or methyl p-tolyl sulfide S-oxygenation activity (in the range of ~20- to ~40-fold) and FMO3 levels determined immunochemically in liver microsomes (r2=0.73-0.75, p<0.0001, n=16). Preincubation with the reducing agent ascorbate revealed that FMO3 activity in some liver samples is suppressed. Microsomal FMO3 protein content (~40-fold) was correlated with FMO3 mRNA levels (r2=0.55, p=0.0010, n=16), but FMO3 haplotypes did not affect FMO3 mRNA expression (~100-fold) under the conditions used. FMO3 mRNA levels were multivariately correlated with trans-acting factors, i.e. hepatic nuclear factor 4 (HNF-4) mRNA and nuclear factor Y box-binding protein (NF-Y) mRNA (r2=0.31, p=0.0017, n=37). These results suggest that considerable individual differences in FMO3 levels may exist in Japanese livers. The liver-enriched transcription factor HNF-4 appears to be a determinant of FMO3 expression in livers, as well as the ubiquitous factor NF-Y.
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  • Miki KATOH, Yuko YOSHIOKA, Nao NAKAGAWA, Tsuyoshi YOKOI
    2009 Volume 24 Issue 3 Pages 226-234
    Published: 2009
    Released: July 02, 2009
    JOURNALS FREE ACCESS
      Kampo is a traditional Japanese herbal medicine and widely used in clinical practice in Japan. Little is known about interactions between Kampo and other medicines. Kampo contains many aglycones, which can be conjugated by UDP-glucuronosyltransferase (UGT). Therefore, in the present study, the effects of Kampo on human UGT1A1 activity were investigated in vitro. Substrates of human UGT1A1, β-estradiol or 7-ethyl-10-hydroxycamptothecin (SN-38), were incubated with human liver microsomes in the presence of 51 Kampos, 14 medicinal herbs and their components. β-Estradiol 3-glucuronidation was strongly inhibited by some Kampos such as Bofu-tsusho-san, Mashinin-gan and Otsuji-to. Medicinal herbs such as Daio (Rhei Rhizoma), Kanzo (Glycyrrhizae Radix), Keihi (Cinnamomi Cortex) and Ogon (Scutellariae Radix) exhibited potent inhibition on that activity. On β-estradiol 3-glucuronidation, the major component of Keihi (cinnamaldehyde) and Ogon (wogonin) exhibited mixed-type inhibition of Ki with values of 0.7 μM and 2.8 μM, respectively. On SN-38 glucuronidation, the inhibitory potencies of Kampos, medicinal herbs and their components tended to be similar to those on β-estradiol 3-glucuronidation. In the present study, Kampo was clarified to inhibit β-estradiol and SN-38 glucuronidation mainly catalyzed by UGT1A1.
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  • Ryoichi FUJIWARA, Miki NAKAJIMA, Tetsunori YAMAMOTO, Hidemi NAGAO, Tsu ...
    2009 Volume 24 Issue 3 Pages 235-244
    Published: 2009
    Released: July 02, 2009
    JOURNALS FREE ACCESS
      UDP-Glucuronosyltransferases (UGTs) are predominant drug metabolizing enzymes in the liver and extrahepatic tissues. Human UGT1A9 is uniquely stable against heat treatment. To understand the unique properties of UGT1A9, the three-dimensional structure was constructed by homology modeling using a crystal structure of TDP-epi-vancosaminyltransferase as template. Sequence alignment analysis revealed that 13 amino acid residues (Arg42, Lys91, Ala92, Tyr106, Gly111, Tyr113, Asp115, Asn152, Leu173, Leu219, His221, Arg222, and Glu241) are unique to UGT1A9 as compared with UGT1A7, UGT1A8 and UGT1A10. To examine the roles of these residues in the conformational stability of UGT1A9, molecular dynamics simulation of the structures was carried out at 310 K and 360 K in aqueous solution for 3.0 nanoseconds. Root mean square deviation analyses revealed that Arg42, Leu173, Leu219, His221 and Arg222 were responsible for the thermal stability. Root mean square fluctuation analyses and a dynamical cross correlation map revealed that Lys91, Ala92, Tyr106, Gly111, Tyr113, Asp115, Leu219, His221, Arg222 and Glu241 were responsible for the thermal stability. In vitro study using mutants of these residues demonstrated that all these amino acids may be collectively involved in the thermal stability of UGT1A9. The results presented here provide a molecular basis for the thermal stability of human UGT1A9.
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  • Kazuko INOUE, Yoshihiro SHIBATA, Hiroyuki TAKAHASHI, Tomoyuki OHE, Mas ...
    2009 Volume 24 Issue 3 Pages 245-254
    Published: 2009
    Released: July 02, 2009
    JOURNALS FREE ACCESS
      A trapping approach for semi-quantitative assessment of bioactivation potential has been established for new chemical entities by using [35S]cysteine and [14C]sodium cyanide as trapping reagents. Reactive metabolites were trapped as radioactive adducts with the trapping reagents to be analyzed by radio-LC(/MS). As a reference, hepatotoxic drugs (clozapine, diclofenac, R-(+)-pulegone and troglitazone) were tested in the [35S]cysteine trapping assay and the proposed structures of the cysteine adducts were consistent with glutathione adducts previously reported. The accuracy of this methodology to predict bioactivation potential of structurally diverse non-radiolabeled test compounds was evaluated by comparing the radiochromatographic peak area obtained in this assays with the extent of covalent binding to protein assessed by the conventional method using radiolabeled test compounds. The value obtained from the [35S]cysteine trapping assay in human liver microsomes predicted potential for covalent binding of the test compounds to proteins with reasonable accuracy. A combination of trapping reagents ([35S]cysteine and [14C]cyanide) improved the accuracy for prediction of bioactivation potential by simultaneously trapping both types of electrophilic reactive metabolites. This method is expected to be a useful to prioritize compounds for further development based on the bioactivation liability, especially at the lead optimization stage.
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  • Saadi Abdul VAHAB, Supratim SEN, Nivedita RAVINDRAN, Sridevi MONY, Ani ...
    2009 Volume 24 Issue 3 Pages 255-260
    Published: 2009
    Released: July 02, 2009
    JOURNALS FREE ACCESS
      The transmembrane P-glycoprotein that functions as a drug-efflux transporter coded by ATP-binding cassette, subfamily B, member 1/Multidrug Resistance 1 (ABCB1/MDR1) gene is considered relevant to drug absorption and elimination, with access to the central nervous system. Effects of three ABCB1 single nucleotide polymorphisms (SNPs) in genotypic and haplotypic combination have been evaluated in a south Indian population for risk of pediatric medically refractory epilepsy. The study included age and sex matched medically refractory (N=113) cases and drug responsive epilepsy patients (N=129) as controls, belonging to the same ethnic population recruited from a tertiary referral centre, of Karnataka, Southern India. The genotype frequencies of SNPs c.1236C>T, c.2677G>T/A, and c.3435C>T were determined from genomic DNA of the cases and controls by PCR- RFLP and confirmatory DNA sequencing. 256 normal population samples of the same ethnicity were genotyped for the three loci to check for population stratification. Results indicate that there was no statistically significant difference between allele and genotype frequencies of refractory and drug responsive epilepsy patients. The predicted haplotype frequencies of the three polymorphisms did not show significant difference between cases and controls. The results confirm earlier observations on absence of association of ABCB1 polymorphisms with medically refractory epilepsy.
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  • Hege CHRISTENSEN, Liv MATHIESEN, Lillian W. POSTVOLL, Bjørn WIN ...
    2009 Volume 24 Issue 3 Pages 261-268
    Published: 2009
    Released: July 02, 2009
    JOURNALS FREE ACCESS
      In vitro drug metabolism techniques with human CYP c-DNA expressed systems are frequently used to predict human drug metabolism in vivo. The aim of this study was to compare midazolam enzyme kinetics in recombinant expressed CYP3A4 microsomes from human and insect cells. The amounts of 1′- hydroxymidazolam and 4-hydroxymidazolam formed in CYP3A4 microsomes from transfected human liver epithelial cells (T5-3A4 microsomes) and baculovirus-infected insect cells (with and without coexpressed cytochrome b5) were analysed by LC-MS. Enzyme kinetic parameters were estimated by nonlinear regression. Mean Km for the formation of 1′-hydroxymidazolam was 3- and 4-fold higher in T5-3A4 microsomes than in insect microsomes (p<0.05), with and without coexpressed cytochrome b5, respectively. Only minor differences in Vmax were observed and the higher Km in T5-3A4 microsomes was reflected by significantly lower Clint compared to insect microsomes (p<0.001). For formation of 1′-hydroxymidazolam, human microsomes displayed Michaelis-Menten kinetics, while insect microsomes showed substrate inhibition kinetics. The different enzyme kinetics of midazolam observed in recombinant CYP3A4 microsomes from human and insect sources, especially the substantially higher Km obtained in human microsomes compared to insect microsomes, should be further evaluated since it may have implications for correlations to in vivo situation.
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Note
  • Atsushi MIYAJIMA, Tomomi FURIHATA, Kan CHIBA
    2009 Volume 24 Issue 3 Pages 269-276
    Published: 2009
    Released: July 02, 2009
    JOURNALS FREE ACCESS
      Although there is a putative GC box in the cytochrome P450 1A2 (CYP1A2) promoter, its function has remained undetermined. To understand the molecular mechanisms controlling CYP1A2 gene expression in the liver, we studied the roles of the GC box in promoter activity and the effects of its CpG methylation on CYP1A2 gene expression. The results of luciferase assays showed that promoter activity was significantly dependent on the presence of the intact GC box. The results of bisulfate sequencing showed that the CpG methylation status of the GC box was strongly associated with CYP1A2 mRNA expression in human cell lines and tissues, suggesting that CpG methylation is involved in the tissue-specific regulation of CYP1A2 gene expression. However, effects of in vitro CpG methylation of the GC box on the promoter activity were not so dramatic in the luciferase assay, suggesting that the major function of the methylated-CpG is not to inhibit transcription factors in binding to the GC box. Taken together, our results show that the GC box is a critical element for the CYP1A2 promoter and its epigenetic regulation mediated by CpG methylation may play important roles in the tissue-specific CYP1A2 gene expression. Genome-based approaches may be necessary for understanding this tissue-specific epigenetic mechanism.
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SNP Communication
  • Su-Ryang KIM, Yoshiro SAITO, Masaya ITODA, Keiko MAEKAWA, Manabu KAWAM ...
    2009 Volume 24 Issue 3 Pages 277-281
    Published: 2009
    Released: July 02, 2009
    JOURNALS FREE ACCESS
      The bile salt export pump (BSEP) encoded by ABCB11 is located in the canalicular membrane of hepatocytes and mediates the secretion of numerous conjugated bile salts into the bile canaliculus. In this study, 28 ABCB11 exons (including non-coding exon 1) and their flanking introns were comprehensively screened for genetic variations in 120 Japanese subjects. Fifty-nine genetic variations, including 19 novel ones, were found: 14 in the coding exons (6 nonsynonymous and 8 synonymous variations), 4 in the 3′-UTR, and 41 in the introns. Three novel nonsynonymous variations, 361C>A (Gln121Lys), 667C>T (Arg223Cys), and 1460G>T (Arg487Leu), were found as heterozygotes and at 0.004 allele frequencies. These data provide fundamental and useful information for genotyping ABCB11 in the Japanese and probably other Asian populations.
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Letter to the Editor
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