Drug Metabolism Letters - Volume 3, Issue 4, 2009
Volume 3, Issue 4, 2009
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Editorial [Special Board Members Issue]
More LessThis is the second issue of ‘Drug Metabolism Letters’ reporting recent research in a variety of areas of drug metabolism contribted by the Editorial Board Members of the journal. The journal, publishing its third volume, has recently been accepted by PubMed for coverage!! Another milestone development is the launch of a new online manuscript submission and processing system, Contents Management System (CMS; http://bentham-editorial.org). I am sure you will find papers in this issue quite interesting to read: Yamazaki et al. in ‘Effects of histidine-tag ----systems’ have concluded that histidine-tagged system would be non-applicable for P450 3A5 function. Paper by Al-Kadi et al. discusses Peganum harmala L. (Zygophyllaceae), which a common plant in Middle East and it is still used traditionally to treat several diseases. The effect of P. harmala extract on the expression of different cytochrome P450's (CYP) involved in drug metabolism was examined in human HepG2 cells. In the paper by Liu et al. zebrafish has been modeled for investigating the drug metabolism through sulfation by establishing a complete repertoire of the zebrafish Phase II cytosolic sulfotransferases (SULTs). A reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed developmental stage-dependent expression of SULT3 ST3 during embryonic development and throughout the larval stage onto maturity. ‘Cloning, expression, ------ (Macaca fascicularis)’ authored by Yamazaki et al. suggests that cynomolgus macaques that are frequently used in drug metabolism studies due to their evolutionary closeness to humans, have not been fully explored for the presence of genes encoding drug-metabolizing enzymes The results indicate that cynomolgus CYP3A43 is expressed in liver and encodes a functional drug-metabolizing enzyme and could play a role in hepatic drug metabolism. Paper by Aiming Yu et al. describes desipramine (DMI), a CYP2D6 probe, which was used as a model drug to test whether CYP2D6-humanized (Tg-CYP2D6) and wild-type control mice could be used as preclinical animal models to identify the effects of CYP2D6 genotype/phenotype on drug metabolic profiles. Author identified three metabolites, 2-hydroxyl-, 10-hydroxyl, and N-desmethyl-desipramine. According to Zhou et al. the nuclear receptor (NR) superfamily represents an important group of regulating factors that control the expression of a number of target genes including those encoding important drug metabolizing enzymes and drug transporters. Their results indicate that both SIFT and PolyPhen are useful and efficient tools to predict the functional effects of nsSNPs of human NR genes....
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Effects of Histidine-Tag on Recombinant Human Cytochrome P450 3A5 Catalytic Activity in Reconstitution Systems
Authors: Chie Emoto, Norie Murayama, Sachiko Wakiya and Hiroshi YamazakiCytochrome P450 (P450) plays an important role for oxidation of a variety of chemicals. Human P450 3A5 is recognized as the secondly most important enzymes among P450 3A subfamily, with some differences in substrate specificity, catalytic activity, and regioselectivity of P450 3A4. Because addition of histidine-tag to microsomal P450s is universally used for recombinant protein purifications, purified P450 3A4 protein is commercially available, but P450 3A5 is not at present. In the present study, although catalytic activity of purified P450 3A5 expressed using the original plasmid from bacterial membranes was observed in reconstitution systems containing NADPH-P450 reductase, cytochrome b5, and phospholipids, a histidine-tagged P450 3A5 protein did not show any detectable midazolam hydroxylation activities in the same reconstitution systems. From the docking simulation of midazolam into a P450 3A5 homology model based on the published P450 3A4 structure, it was supported that midazolam could not easily access to the center of the heme of histidine-tagged P450 3A5, with the high interaction energy compared with the original P450 3A5. In conclusion, these results suggest that the histidine-tagged system would be non-applicable for P450 3A5 function.
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Peganum harmala L. Differentially Modulates Cytochrome P450 Gene Expression in Human Hepatoma HepG2 Cells
Authors: Mohamed A.M. El Gendy and Ayman O.S. El-KadiPeganum harmala L. (Zygophyllaceae) is a common plant in Middle East and it is still used traditionally to treat several diseases. The effect of P. harmala extract on the expression of different cytochrome P450's (CYP) involved in drug metabolism was examined in human HepG2 cells. Therefore, HepG2 cells were incubated with increasing concentrations of plant extract and the CYP gene expression was determined by real-time PCR. Our results showed that P. harmala extract significantly increased the expression of CYP1A2, 2C19, and 3A4 whereas; CYP2B6, 2D6 and 2E1 was significantly decreased. We concluded that care should be taken when P. harmala is co-administered with other drugs.
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A Novel Hydroxysteroid-Sulfating Cytosolic Sulfotransferase, SULT3 ST3, from Zebrafish: Identification, Characterization, and Ontogenic Study
To establish the zebrafish as a model for investigating the drug metabolism through sulfation, we had embarked on establishing a complete repertoire of the zebrafish Phase II cytosolic sulfotransferases (SULTs). By searching the expressed sequence tag database, a zebrafish cDNA encoding a putative cytosolic sulfotransferase (SULT) was identified. Based on the sequence analysis, this zebrafish SULT was found to belong to the SULT3 gene family. The recombinant protein of the zebrafish SULT, designated SULT3 ST3, was expressed in and purified from BL21 (DE3) Escherichia coli cells transformed with the pGEX-2TK expression vector harboring SULT3 ST3 cDNA. Upon enzymatic characterization, purified SULT3 ST3 displayed sulfating activity toward hydroxysteroids, particularly pregnenolone and dehydroepiandrosterone (DHEA), as well as several drugs among various endogenous and xenobiotic compounds tested as substrates. The pH-dependence and kinetic constants of this enzyme with DHEA were determined. The regulatory effects of various divalent metal cations on the DHEA-sulfating activity of SULT3 ST3 were quantitatively evaluated. A reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed developmental stage-dependent expression of SULT3 ST3 during embryonic development and throughout the larval stage onto maturity. Collectively, these results suggest a possible involvement of the newly discovered SULT3 ST3 in the metabolism of hydroxysteroids and xenobiotics including drugs in zebrafish.
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Cloning, Expression, and Characterization of CYP3A43 in Cynomolgus Macaque (Macaca fascicularis)
Authors: Yasuhiro Uno, Kiyomi Matsuno, Chika Nakamura, Masahiro Utoh and Hiroshi YamazakiCynomolgus macaques are frequently used in drug metabolism studies due to their evolutionary closeness to humans. Despite their importance, genes encoding drug-metabolizing enzymes have not been fully identified in this species. In this study, the cDNA orthologous to human cytochrome P450 3A43 (CYP3A43) was isolated. The deduced amino acid sequence of this cDNA had a high sequence identity (∼95%) to human CYP3A43 cDNA and contained characteristic motifs for CYP3A proteins, a heme-binding region and substrate recognition sites. Of the 10 tissues analyzed, cynomolgus CYP3A43 was expressed in liver, adrenal gland, and lung, with the highest expression being in liver. Cynomolgus CYP3A43 protein heterologously expressed in Escherichia coli catalyzed the 1'-hydroxylation of midazolam. These results indicate that cynomolgus CYP3A43 is expressed in liver and encodes a functional drug-metabolizing enzyme and could play a role in hepatic drug metabolism.
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Difference in Desipramine Metabolic Profile Between Wild-Type and CYP2D6-Humanized Mice
Authors: Hong-Wu Shen and Ai-Ming YuDesipramine (DMI), a CYP2D6 probe, was used as a model drug to test whether CYP2D6- humanized (Tg-CYP2D6) and wild-type control mice could be used as preclinical animal models to identify the effects of CYP2D6 genotype/phenotype on drug metabolic profiles. After the analyses by liquid chromatography coupled with tandem mass spectrometry, DMI biotransformations were compared in Tg- CYP2D6 and wild-type mouse liver microsomes (MLM), and in human CYP2D6 extensive and poor metabolizer liver microsomes. Furthermore, urinary DMI metabolic profiles in Tg-CYP2D6 and wild-type mice were evaluated. Three metabolites, 2-hydroxyl-, 10-hydroxyl, and N-desmethyl-desipramine were identified in the incubations of DMI with both wild-type and Tg-CYP2D6 MLM, as well as in human CYP2D6 extensive metabolizer liver microsomes. Three additional metabolites were found in mouse urine samples, and their chemical structures were elucidated. Although the ratio of individual metabolites produced in Tg-CYP2D6 MLM was closer to that in human CYP2D6 extensive metabolizer liver microsomes, the urinary DMI metabolic profiles did not show much difference between wildtype and Tg-CYP2D6 mice. The results suggest that other mouse enzymes have significant contribution to DMI metabolism.
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Prediction of Deleterious Functional Effects of Non-Synonymous Single Nucleotide Polymorphisms in Human Nuclear Receptor Genes Using a Bioinformatics Approach
Authors: Ya-He Liu, Chun G. Li and Shu-Feng ZhouThe nuclear receptor (NR) superfamily represents an important group of regulating factors that control the expression of a number of target genes including those encoding important drug metabolizing enzymes and drug transporters. Single nucleotide polymorphism (SNP) is the most common mutation in the human genome and a large number of SNPs have been identified to date. It is unlikely to examine the functional impact of all these mutations using an experimental approach. As such, we employed two algorithms, Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen) to predict the impact of non-synonymous SNPs (nsSNPs) on NR activities and disease susceptibility. We identified 442 nsSNPs in a systematic screening of 48 human NR genes. Using SIFT, of 442 amino acid substitutions, 289 (65.38%) were classified as “intolerant“. The PolyPhen program classified 269 (60.86%) of them as “probably damaging” or “possibly damaging”. The results from the two algorithms were in concordance. Among the 442 mutations, 229 of them have been functionally characterized. SIFT predicted 192 of these nsSNPs as “intolerant”, resulting in a correct prediction rate of 83.84%, while PolyPhen gave a prediction rate of 76.86%. For 216 nsSNPs of the androgen receptor gene, 149 nsSNPs have been functionally studied and most (121) of them resulted in a reduction of receptor activity. SIFT sorted 187 out of 216 as “intolerant” (86.57%) and PolyPhen identified 159 out of 216 as “potentially intolerant” (73.61%). These results indicate that both SIFT and PolyPhen are useful and efficient tools to predict the functional effects of nsSNPs of human NR genes.
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Interaction Between Clopidogrel and Proton Pump Inhibitors: Hypothesis to Explain Multifactorial CYP2C19 Inhibition
Authors: Huixia Zhang, Isabelle Ragueneau-Majlessi and Rene H. LevyClopidogrel is an antiplatelet drug that requires bioactivation to its active metabolite to demonstrate its antiplatelet effect. Formation of the active metabolite involves multiple cytochrome P450 enzymes, with CYP2C19 playing an important role. Clopidogrel is often co-administered with proton pump inhibitors (PPIs) to decrease GI-tract bleeding, and decreased antiplatelet effect has been observed in these patients. This observation cannot be explained by the weak inhibitory effect of PPIs on CYP2C19. A hypothesis is proposed to interpret the phenomenon of PPI inhibition based in part on the finding that clopidogrel is itself an inhibitor of CYP2C19.
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The Role of P-Glycoprotein in the Pharmacokinetics and Tissue Distribution of a Hepatitis C Virus Protease Inhibitor
Authors: Kevin X. Chen, Bancha Vibulbhan, Tongtong Liu, Lisa Broske, Genfeng Wang, Cheng Li, F. G. Njoroge, Annette S. Uss and K.-C. ChengS5, a hepatitis C virus protease inhibitor, displays partially saturable efflux in the Caco-2 system. In addition, the efflux can be reversed by cyclosporine, indicating that S5 may be a human Pglycoprotein (P-gp) substrate. S5 can also activate the ATPase activity in vesicle membranes containing mouse P-gp 1a and 1b, suggesting that S5 may be a substrate for mouse P-gp. The pharmacokinetics and tissue distribution of S5 were evaluated after intravenous and oral administration to wild-type and 1a/1b knockout mice. Plasma and kidney levels of this compound in knockout mice were transiently higher than those in wild-type mice only after oral dosing, indicating effective P-gp efflux occurs in wild-type mice. The levels of S5 in brain samples from knockout mice were higher than those from wild-type mice after both intravenous and oral administration, but much more significantly after intravenous administration. The levels in liver were four time higher in knockout mice than in wild-type mice after oral administration, but were not different between knockout and wild-type mice after intravenous administration. These results suggest that P-gp efflux limits exposure to S5 in the brain and liver, and that the effect is dependent on the route of administration.
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Prediction of Human Hepatic Clearance Using an In Vitro Plated Hepatocyte Clearance Model
Authors: Piyun Chao, Jeffrey Barminko, Eric Novik, Yi Han, Timothy Maguire and K.-C. ChengPreviously we have used human hepatocytes in suspension by measuring the parent loss for prediction of metabolic clearance according to a 1st-order kinetic model. In this study, we evaluated a novel integrative approach using plated human hepatocytes to include both uptake processes and metabolism in a single assay. Test articles were added in the medium, and the intrinsic clearance was determined based on the disappearance of the parent compound from the medium. Three different methods: direct, well-stirred, and parallel tube were tested for scaling purpose. With 30 randomly selected compounds with clinical clearance data, the scaled clearance showed reasonable linear correlation with r2 values of 0.67, 0.72, and 0.70 for direct, well-stirred and parallel tube models, respectively. When human serum albumin (HSA) was added to the incubation medium a shift to lower in vitro clearance was observed for most of the compounds, suggesting that protein binding may have an effect on the metabolic clearance. In the presence of 4% of HSA, which is equivalent to the albumin concentration in the human plasma, the in vitro clearance data have the best prediction of human clearance when using the well-stirred method, followed by the parallel tube method and direct method. This study demonstrates the utility of using plated human hepatocyte as an integrated system for the prediction of human metabolic clearance. In addition, evaluation of the protein binding shift in the clearance showed that a significant number of compounds may not follow the equilibrium assumption according to the well-stirred model.
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