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Pmol Mg Protein (pmol + mg_protein)
Selected AbstractsEvaluation of SupermixTM as an in vitro model of human liver microsomal drug metabolismBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 5 2002Karthik Venkatakrishnan Abstract SUPERMIXTM is a commercially available formulation of insect cell-expressed human drug-metabolizing cytochrome P450 (CYP) isoforms, mixed in proportions that are optimized to parallel their relative activities in human liver microsomes. We have evaluated the apparent functional affinity and capacity of individual CYP isoforms in SUPERMIXTM in comparison with microsomes from a panel of 12 human livers, using enzyme kinetic studies of isoform-selective index reactions. In addition, we have measured the concentration of NADPH cytochrome P450 oxidoreductase (OR) in SUPERMIXTM and compared it with the concentrations of this accessory electron transfer protein in human liver microsomes. No important differences were evident in the catalytic activities of CYPs 1A2, 2C8, 2C9, 2C19, 2D6 and 3A4 between SUPERMIXTM and human liver microsomes. However, SUPERMIXTM lacks CYP2B6 activity and did not hydroxylate the antidepressant bupropion, a clinically relevant substrate of this enzyme. In addition, the concentration of OR in SUPERMIXTM (1198 pmol mg protein,1) is 17-fold higher than the mean value in human liver microsomes (70 pmol mg protein,1). In conclusion, SUPERMIXTM lacks CYP2B6 activity and contains supraphysiological concentrations of the accessory electron transfer protein OR. These factors should be considered when this formulation is used as an in vitro model in human liver microsomal drug metabolism studies. Copyright © 2002 John Wiley & Sons, Ltd. [source] Pharmacological effects of carcinine on histaminergic neurons in the brainBRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2004Zhong Chen Carcinine (, -alanyl histamine) is an imidazole dipeptide. The present study was designed to characterize the pharmacological effects of carcinine on histaminergic activity in the brain and on certain neurobehavior. Carcinine was highly selective for the histamine H3 receptor over H1 or H2 receptor (Ki (,M)=0.2939±0.2188 vs 3621.2±583.9 or 365.3±232.8 ,M, respectively). Carcinine at a dose of 20 mg kg,1 slightly increased histidine decarboxylase (HDC) activity in the cortex (from 0.186±0.069 to 0.227±0.009 pmol mg protein,1 min,1). In addition, carcinine (10, 20, and 50 mg kg,1) significantly decreased histamine levels in mice brain. Like thioperamide, a histamine H3 receptor antagonist, carcinine (20, 50 ,M) significantly increased 5-HT release from mice cortex slices, but had no apparent effect on dopamine release. Carcinine (20 mg kg,1) significantly inhibited pentylenetetrazole-induced kindling. This inhibition was completedly reversed by (R)- , -methylhistamine, a representative H3 receptor agonist, and , -fluromethylhistidine, a selective HDC inhibitor. Carcinine (20 mg kg,1) ameliorated the learning deficit induced by scopolamine. This amelioration was reversed by (R)- , -methylhistamine as evaluated by the passive avoidance test in mice. Like thioperamide, carcinine dose-dependently increased mice locomotor activity in the open-field test. The results of this study provide first and direct evidence that carcinine, as a novel histamine H3 receptor antagonist, plays an important role in histaminergic neurons activation and might be useful in the treatment of certain diseases, such as epilepsy, and locomotor or cognitive deficit. British Journal of Pharmacology (2004) 143, 573,580. doi:10.1038/sj.bjp.0705978 [source] Characterisation of the binding of [3H]-SB-674042, a novel nonpeptide antagonist, to the human orexin-1 receptorBRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2004Christopher J Langmead This study characterises the binding of a novel nonpeptide antagonist radioligand, [3H]SB-674042 (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone), to the human orexin-1 (OX1) receptor stably expressed in Chinese hamster ovary (CHO) cells in both a whole cell assay and in a cell membrane-based scintillation proximity assay (SPA) format. Specific binding of [3H]SB-674042 was saturable in both whole cell and membrane formats. Analyses suggested a single high-affinity site, with Kd values of 3.76±0.45 and 5.03±0.31 nM, and corresponding Bmax values of 30.8±1.8 and 34.4±2.0 pmol mg protein,1, in whole cell and membrane formats, respectively. Kinetic studies yielded similar Kd values. Competition studies in whole cells revealed that the native orexin peptides display a low affinity for the OX1 receptor, with orexin-A displaying a ,five-fold higher affinity than orexin-B (Ki values of 318±158 and 1516±597 nM, respectively). SB-334867, SB-408124 (1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) and SB-410220 (1-(5,8-difluoro-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) all displayed high affinity for the OX1 receptor in both whole cell (Ki values 99±18, 57±8.3 and 19±4.5 nM, respectively) and membrane (Ki values 38±3.6, 27±4.1 and 4.5±0.2 nM, respectively) formats. Calcium mobilisation studies showed that SB-334867, SB-408124 and SB-410220 are all functional antagonists of the OX1 receptor, with potencies in line with their affinities, as measured in the radioligand binding assays, and with approximately 50-fold selectivity over the orexin-2 receptor. These studies indicate that [3H]SB-674042 is a specific, high-affinity radioligand for the OX1 receptor. The availability of this radioligand will be a valuable tool with which to investigate the physiological functions of OX1 receptors. British Journal of Pharmacology (2004) 141, 340,346. doi:10.1038/sj.bjp.0705610 [source] Arachidonic acid-mediated cooxidation of all- trans -retinoic acid in microsomal fractions from human liverBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2000Louise Nadin The quantitative importance of prostaglandin H synthase (PGHS)-mediated cooxidation of all- trans -retinoic acid (ATRA) was evaluated in human liver microsomes (n=17) in relation to CYP-dependent ATRA 4-hydroxylation. Observed rates of ATRA cooxidation (4.6,20 pmol mg protein,1 min,1) and 4-hydroxylation (8.7,45 pmol mg protein,1 min,1) were quantitatively similar and exhibited similar individual variation (4 and 5 fold, respectively). From kinetic studies cooxidation was an efficient process in human hepatic microsomes (VmaxKm,1=0.25) compared with NADPH- and NADH-mediated 4-hydroxylation by CYP (VmaxKm,1=0.14 and 0.02, respectively). The capacity of lipid hydroperoxide metabolites of arachidonic acid to mediate ATRA oxidation was established directly, but downstream products (D, E, F and I-series prostaglandins) were inactive. cDNA-expressed CYPs supported ATRA oxidation by lipid hydroperoxides. Whereas CYPs 2C8, 2C9 and 3A4, but not CYPs 1A2 or 2E1, were effective catalysts of the NADPH-mediated reaction, cooxidation supported by 15(S)-hydroperoxyeicosatetraenoic acid was mediated by all five CYPs. The cooxidation reaction in human hepatic microsomes was inhibited by the CYP inhibitor miconazole. These findings indicate that ATRA oxidation is quantitatively significant in human liver. Lipid hydroperoxides generated by intracellular enzymes such as prostaglandin synthase and lipoxygenases are sources of activated oxygen for CYP-mediated deactivation of ATRA to polar products. British Journal of Pharmacology (2000) 131, 851,857; doi:10.1038/sj.bjp.0703579 [source] | ||||||||||