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Human Liver Microsomes (human + liver_microsome)
Selected AbstractsP71 Metabolism of delta-3-Carene by human cytochrom 450 enzymesCONTACT DERMATITIS, Issue 3 2004Mike Duisken Occupational exposure to monoterpenes occurs in saw mills, particle-board plants, carpentry shops and other types of wood-treating industries. The bicyclic monoterpene delta-3-Carene, one of the components of turpentine, may irritate the skin and muceous membranes and prolonged exposure may result in allergic contact dermatitis or chronic lung function impairment. The effects of low concentrations of delta-3-Carene on alveolar macrophages in vitro were examined and a dose-dependent relationship between the cell viability and the delta-3-Carene concentration was found. Little is known about the metabolism of delta-3-Carene in mammalians. In order to determine the toxic potential of this monoterpene we studied the human metabolism of delta-3-Carene in vitro. Therefore we used pooled human liver S9 and human liver microsomal cytochrome P450 enzymes. By using GC-MS analysis we found one main metabolite produced at high rates. The structure was identified by its mass spectra. The mass fragmentation indicated hydroxylation in allyl position. After synthesis of the assumed product in a four step reaction, it was characterized as delta-3-Carene-10-ol. There was a clear correlation between the concentration of the metabolite production, incubation time and enzyme concentration, respectively. Kinetic analysis showed that Km and Vmax values for the oxidation of delta-3-Carene by human liver microsomes were 0.39 ,M and 0.2 nmol/min/nmol P450. It is the first time that delta-3-Carene-10-ol is described as human metabolite of delta-3-Carene. [source] Validated capillary electrophoresis assay for the simultaneous enantioselective determination of propafenone and its major metabolites in biological samplesELECTROPHORESIS, Issue 8 2006Minoo Afshar Abstract A robust, inexpensive, and fully validated CE method for the simultaneous determination of the enantiomers of propafenone (PPF), 5-hydroxy-propafenone (5OH-PPF) and N -despropyl-propafenone (NOR-PPF) in serum and in in vitro media is described. It is based upon liquid,liquid extraction at alkaline pH followed by analysis of the reconstituted extract by CE in presence of a pH,2.0 running buffer composed of 100,mM sodium phosphate, 19% methanol, and 0.6% highly sulfated ,-CD. For each compound, the S -enantiomers are shown to migrate ahead of their antipodes, and the overall run time is about 30,min. Enantiomer levels between 25 and 1000,ng/mL provide linear calibration graphs, and the LOD for all enantiomers is between 10 and 12,ng/mL. The assay is shown to be suitable for the determination of the enantiomers of PPF and its metabolites in in vitro incubations comprising human liver microsomes or single CYP450 enzymes (SUPERSOMES). Incubations with CYP2D6 SUPERSOMES revealed, for the first time, the simultaneous formation of the enantiomers of 5OH-PPF and NOR-PPF with that enzyme. CE data can be used for the evaluation of the enzymatic N -dealkylation and hydroxylation rates. [source] Interaction of bisphenol a with human UDP-glucuronosyltransferase 1A6 enzymeENVIRONMENTAL TOXICOLOGY, Issue 3 2008Nobumitsu Hanioka Abstract The effects of bisphenol A (BPA) on UDP-glucuronosyltransferase 1A6 (UGT1A6) activities in microsomes from human livers and yeast cells expressing human UGT1A6 (humUGT1A6) were investigated. Serotonin (5-HT) and 4-methylumbelliferone (4-MU) were used as the substrates for UGT1A6. BPA dose-dependently inhibited 5-HT and 4-MU glucuronidation activities in both enzyme sources. The IC50 values of BPA for 5-HT and 4-MU glucuronidation activities were 156 and 163 ,M for liver microsomes, and 84.6 and 80.3 ,M for yeast cell microsomes expressing humUGT1A6, respectively. The inhibitory pattern of BPA for 5-HT and 4-MU glucuronidation activities in human liver microsomes exhibited a mixture of competitive and noncompetitive components, with Ki values of 84.9 and 72.3 ,M, respectively. In yeast cell microsomes expressing humUGT1A6, 5-HT glucuronidation activities were noncompetitively inhibited by BPA (Ki value, 65.5 ,M), whereas the inhibition of 4-MU glucuronidation activities by BPA exhibited the mixed type (Ki value, 42.5 ,M). These results suggest that BPA interacts with human UGT1A6 enzyme, and that the interaction may contribute to the toxicity, such as hormone disruption and reproductive effects, of BPA. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source] Testosterone 1,-hydroxylation by human cytochrome P450 3A4FEBS JOURNAL, Issue 19 2004Joel A. Krauser Human cytochrome P450 3A4 forms a series of minor testosterone hydroxylation products in addition to 6,-hydroxytestosterone, the major product. One of these, formed at the next highest rate after the 6,- and 2,-hydroxy products, was identified as 1,-hydroxytestosterone. This product was characterized from a mixture of testosterone oxidation products using an HPLC-solid phase extraction-cryoprobe NMR/time-of-flight mass spectrometry system, with an estimated total of ,,6 µg of this product. Mass spectrometry established the formula as C19H29O3 (MH+ 305.2080). The 1-position of the added hydroxyl group was established by correlated spectroscopy and heteronuclear spin quantum correlation experiments, and the ,-stereochemistry of the added hydroxyl group was assigned with a nuclear Overhauser correlated spectroscopy experiment (1,-H). Of several human P450s examined, only P450 3A4 formed this product. The product was also formed in human liver microsomes. [source] Metabolism of methoxymorpholino-doxorubicin in rat, dog and monkey liver microsomes: comparison with human microsomesFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 6 2001Dominique Beulz-Riche The morpholino anthracycline, methoxymorpholino-doxorubicin (MMDx) is a novel anticancer agent. The metabolism of this highly lipophilic doxorubicin analogue is not fully elucidated. MMDx is metabolically activated in vivo, resulting in an 80-fold increase in potency over the parent drug. In this study, MMDx in vitro metabolism was compared in rat, dog, monkey and human liver microsomes. When microsomal fractions were incubated with MMDx, 6,8 metabolites were formed depending on the species and on the substrate concentrations. Among these eight metabolites, three comigrated with authentic standards, namely MMDx-ol, PNU156686 and PNU159682, and the five others are in the process of being characterized. Quantitatively, monkey and human metabolize MMDx with a higher rate than rat and dog. Qualitatively, MMDx metabolic profile in dog microsomes was different from the three other species. MMDx-ol was predominant in dog and only minor in other species. In conclusion, MMDx metabolism was species-different. Rat and monkey liver microsomes may be used as models to study MMDx metabolism in humans. Dog liver microsomes may be a good model for studying the formation of MMDx-ol. [source] Glucuronidation of olanzapine by cDNA-expressed human UDP-glucuronosyltransferases and human liver microsomesHUMAN PSYCHOPHARMACOLOGY: CLINICAL AND EXPERIMENTAL, Issue 5 2002Kristian Linnet Abstract Olanzapine is a widely used, newer antipsychotic agent, which is metabolized by various pathways: hydroxylation and N -demethylation by cytochrome P450, N -oxidation by flavin monooxygenase and direct glucuronidation. In vivo studies have pointed towards the latter pathway as being of major importance. Accordingly, the glucuronidation reaction was studied in vitro using cDNA-expressed human UDP-glucuronosyltransferase (UGT) enzymes and a pooled human liver microsomal preparation (HLM). Glucuronidated olanzapine was determined by HPLC after acid or enzymatic hydrolysis. The following UGT-isoenzymes were screened for their ability to glucuronidate olanzapine: 1A1, 1A3, 1A4, 1A6, 1A9, 2B7 and 2B15. Only UGT1A4 was able to glucuronidate olanzapine obeying saturation kinetics. The Km value was 227,,mol/l (SE 43), i.e. of the same order of magnitude as for other psychotropic drugs, and the Vmax value was 2370,pmol/(min,mg) (SE 170). Glucuronidation was also mediated by the HLM preparation, but a saturation level was not reached. The olanzapine glucuronidation reaction was inhibited by several drugs known as substrates for UGT1A4, e.g. amitriptyline, trifluoperazine and lamotrigine. Thus, competition for glucuronidation by UGT1A4 represents a possibility for drug,drug interactions in subjects receiving several of these psychotropic drugs at the same time. Whether such possible interactions are of any clinical importance may await further studies in patients. Copyright © 2002 John Wiley & Sons, Ltd. [source] Inhibition of fipronil and nonane metabolism in human liver microsomes and human cytochrome P450 isoforms by chlorpyrifosJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2007Hyun Joo Abstract Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:76,80, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20161 [source] CYP3A4 is a Human Microsomal Vitamin D 25-Hydroxylase,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2004Ram P Gupta Abstract The human hepatic microsomal vitamin D 25-hydroxylase protein and gene have not been identified with certainty. Sixteen hepatic recombinant microsomal enzymes were screened for 25-hydroxylase activity; 11 had some 25-hydroxylase activity, but CYP3A4 had the highest activity. In characterized liver microsomes, 25-hydroxylase activity correlated significantly with CYP3A4 testosterone 6,-hydroxylase activity. Activity in pooled liver microsomes was inhibited by known inhibitors of CYP3A4 and by an antibody to CYP3A2. Thus, CYP3A4 is a hepatic microsomal vitamin D 25-hydroxylase. Introduction: Studies were performed to identify human microsomal vitamin D-25 hydroxylase. Materials and Methods: Sixteen major hepatic microsomal recombinant enzymes derived from cytochrome P450 cDNAs expressed in baculovirus-infected insect cells were screened for 25-hydroxylase activity with 1,-hydroxyvitamin D2 [1,(OH)D2], 1,-hydroxyvitamin D3 [1,(OH)D3], vitamin D2, and vitamin D3 as substrates. Activity was correlated with known biological activities of enzymes in a panel of 12 characterized human liver microsomes. The effects of known inhibitors and specific antibodies on activity also were determined. Results: CYP3A4, the most abundant cytochrome P450 enzyme in human liver and intestine, had 7-fold greater activity than that of any of the other enzymes with 1,(OH)D2 as substrate. CYP3A4 25-hydroxylase activity was four times higher with 1,(OH)D2 than with 1,(OH)D3 as substrate, was much less with vitamin D2, and was not detected with vitamin D3. 1,(OH)D2 was the substrate in subsequent experiments. In a panel of characterized human liver microsomes, 25-hydroxylase activity correlated with CYP3A4 testosterone 6,-hydroxylase activity (r = 0.93, p < 0.001) and CYP2C91 diclofenac 4,-hydroxylase activity (r = 0.65, p < 0.05), but not with activity of any of the other enzymes. Activity in recombinant CYP3A4 and pooled liver microsomes was dose-dependently inhibited by ketoconazole, troleandomycin, isoniazid, and ,-naphthoflavone, known inhibitors of CYP3A4. Activity in pooled liver microsomes was inhibited by antibodies to CYP3A2 that are known to inhibit CYP3A4 activity. Conclusion: CYP3A4 is a vitamin D 25-hydroxylase for vitamin D2 in human hepatic microsomes and hydroxylates both 1,(OH)D2 and 1,(OH)D3. [source] Inhibition of human cytochrome P450 isoforms and NADPH-CYP reductase in vitro by 15 herbal medicines, including Epimedii herbaJOURNAL OF CLINICAL PHARMACY & THERAPEUTICS, Issue 1 2006K. H. Liu PhD Summary Objective:, We evaluated the potential of 15 herbal medicines (HMs), commonly used in Korea, to inhibit the catalytic activities of several cytochrome P450 (CYP) isoforms and microsomal NADPH-CYP reductase. Methods:, The abilities of 1,1000 ,g/mL of freeze-dried aqueous extracts of 15 HMs to inhibit phenacetin O -deethylation (CYP1A2), tolbutamide 4-methylhydroxylation (CYP2C9), S -mephenytoin 4,-hydroxylation (CYP2C19), dextromethorphan O -demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), midazolam 1-hydroxylation (CYP3A4) and NADPH-CYP reductase were tested using human liver microsomes. Results:, The HMs Epimedii herba, Glycyrrhizae radix and Leonuri herba inhibited one or more of the CYP isoforms or NADPH-CYP reductase. Of the three HMs, Epimedii herba extracts were the most potent inhibitors of several CYP isoforms (IC50 67·5 ,g/mL for CYP2C19, 104·8 ,g/mL for CYP2E1, 110·9 ,g/mL for CYP2C9, 121·9 ,g/mL for CYP3A4, 157·8 ,g/mL for CYP2D6 and 168·7 ,g/mL for CYP1A2) and NADPH-CYP reductase (IC50 185·9 ,g/mL). Conclusion:, These results suggest that some of the HMs used in Korea have the potential to inhibit CYP isoforms in vitro. Although the plasma concentrations of the active constituents of the HMs were not determined, some herbs could cause clinically significant interactions because the usual doses of those individual herbs are several grams of freeze-dried extracts. Controlled trials to test the significance of these results are necessary. [source] Synthesis of five nevirapine metabolitesJOURNAL OF HETEROCYCLIC CHEMISTRY, Issue 2 2000Karl G. Grozinger Nevirapine (1) is a non-nucleoside reverse transcriptase inhibitor marketed for HIV treatment by Boehringer Ingelheim as Viramune® since 1996. In vitro studies of nevirapine biotransformation using human liver microsomes demonstrated the formation of five major metabolites. This paper describes the syntheses of these metabolites. [source] Synthesis of deuterium-labelled 5,- O -[N -(Salicyl)sulfamoyl]adenosine (Sal-AMS-d4) as an internal standard for quantitation of Sal-AMSJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 2 2008Amol Gupte Abstract 5,- O -[N -(Salicyl)sulfamoyl]adenosine (Sal-AMS, 1) is a potent inhibitor of the bifunctional enzyme salicyl-AMP ligase in Mycobacterium tuberculosis. This inhibitor acts by disrupting the biosynthesis of the mycobactin siderophores that are essential for the process of iron acquisition. To aid with in vitro metabolism and in vivo pharmacokinetic studies of Sal-AMS, a stable deuterium-labelled Sal-AMS analog (Sal-AMS-d4) was synthesized. This deuterium-labelled analog was used as an internal standard to conduct in vitro plasma and microsomal stability studies. Sal-AMS was found to be stable for 24,h in human plasma and 1,h in human liver microsomes at 37°C. Copyright © 2008 John Wiley & Sons, Ltd. [source] An algorithm for thorough background subtraction from high-resolution LC/MS data: application for detection of glutathione-trapped reactive metabolitesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2008Haiying Zhang Abstract A control sample background-subtraction algorithm was developed for thorough subtraction of background and matrix-related signals in high-resolution, accurate mass liquid chromatography/mass spectrometry (LC/MS) data to reveal ions of interest in an analyte sample. This algorithm checked all ions in the control scans within a specified time window around the analyte scan for potential subtraction of ions found in that analyte scan. Applying this method, chromatographic fluctuations between runs were dealt with and background and matrix-related signals in the sample could be thoroughly subtracted. The effectiveness of this algorithm was demonstrated using four test compounds, clozapine, diclofenac, imipramine, and tacrine, to reveal glutathione (GSH)-trapped reactive metabolites after incubation with human liver microsomes supplemented with GSH (30 µM compound, 45-min incubation). Using this algorithm with a ± 1.0 min control scan time window, a ± 5 ppm mass error tolerance, and appropriate control samples, the GSH-trapped metabolites were revealed as the major peaks in the processed LC/MS profiles. Such profiles allowed for comprehensive and reliable identification of these metabolites without the need for any presumptions regarding their behavior or properties with respect to mass spectrometric detection. The algorithm was shown to provide superior results when compared to several commercially available background-subtraction algorithms. Many of the metabolites detected were doubly charged species which would be difficult to detect with traditional GSH adduct screening techniques, and thus, some of the adducts have not previously been reported in the literature. Copyright © 2008 John Wiley & Sons, Ltd. [source] Structure elucidation of aplidine metabolites formed in vitro by human liver microsomes using triple quadrupole mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2005Esther F. A. Brandon Abstract The cyclic depsipeptide aplidine is a new anti-cancer drug of marine origin. Four metabolites of this compound were found after incubation with pooled human microsomes using gradient high-performance liquid chromatography with ultraviolet detection. After chromatographic isolation, the metabolites have been identified using nano-electrospray triple quadrupole mass spectrometry. A highly specific sodium-ion interaction with the cyclic structure opens the depsipeptide ring, and cleavage of the amino acid residues gives sequence information when activated by collision-induced dissociation in the second quadrupole. The aplidine molecule could undergo the following metabolic reactions: hydroxylation at the isopropyl group (metabolites apli-h 1 and apli-h 2); C-dealkylation at the N(Me)-leucine group (metabolite apli-da); hydroxylation at the isopropyl group and C-dealkylation at the N(Me)-leucine group (metabolite apli-da/h), and C-demethylation at the threonine group (metabolite apli-dm). The identification of these metabolites formed in vitro may greatly aid the elucidation of the metabolic pathways of aplidine in humans. Copyright © 2005 John Wiley & Sons, Ltd. [source] Allosteric kinetics of human carboxylesterase 1: Species differences and interindividual variability,JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 12 2008Shiori Takahashi Abstract Esterified drugs such as imidapril, derapril, and oxybutynin hydrolyzed by carboxylesterase 1 (CES1) are extensively used in clinical practice. The kinetics using the CES1 substrates have not fully clarified, especially concerning species and tissue differences. In the present study, we performed the kinetic analyses in humans and rats in order to clarify these differences. The imidaprilat formation from imidapril exhibited sigmoidal kinetics in human liver microsomes (HLM) and cytosol (HLC) but Michaelis-Menten kinetics in rat liver microsomes and cytosol. The 2-cyclohexyl-2-phenylglycolic acid (CPGA) formation from oxybutynin were not detected in enzyme sources from rats, although HLM showed high activity. The kinetics were clarified to be different among species, tissues, and preparations. In individual HLM and HLC, there was large interindividual variability in imidaprilat (31- and 24-fold) and CPGA formations (15- and 9-fold). Imidaprilat formations exhibited Michaelis-Menten kinetics in HLM and HLC with high activity but sigmoidal kinetics in those with low activity. CPGA formations showed sigmoidal kinetics in high activity HLM but Michaelis-Menten kinetics in HLM with low activity. We revealed that the kinetics were different between individuals. These results could be useful for understanding interindividual variability and for the development of oral prodrugs. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5434,5445, 2008 [source] Characterization of in vitro and in vivo metabolic pathways of the investigational anticancer agent, 2-methoxyestradiolJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 7 2007Nehal J. Lakhani Abstract The aim of this study was to characterize the metabolic pathways of 2-methoxyestradiol (2ME2), an investigational anticancer drug. In vitro metabolism studies were performed by incubation of 2ME2 with human liver microsomes under various conditions and metabolite identification was performed using liquid chromatography-tandem mass spectrometry. In microsomal mixtures, four major oxidative metabolites and two glucuronic acid conjugates were observed originating from 2ME2. Human liver S9 protein fraction was used to screen for in vitro sulfation but no prominent conjugates were observed. The total hepatic clearance as estimated using the well-stirred model was approximately 712 mL/min. In vivo metabolism, assessed using 24-h collections of urine from cancer patients treated with 2ME2 revealed that <0.01% of the total administered dose of 2ME2 is excreted unchanged in urine and about 1% excreted as glucuronides. Collectively, this suggests that glucuronidation and subsequent urinary excretion are elimination pathways for 2ME2. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 1821,1831, 2007 [source] Stereoselective disposition of talinolol in manJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2002Michael Zschiesche Abstract The disposition of the ,-blocking drug talinolol is controlled by P-glycoprotein in man. Because talinolol is marketed as a racemate, we reevaluated the serum-concentration time profiles of talinolol of a previously published study with single intravenous (30 mg) and repeated oral talinolol (100 mg for 14 days) before and after comedication of rifampicin (600 mg per day for 9 days) in eight male healthy volunteers (age 22,26 years, body weight 67,84 kg) with respect to differences in the kinetic profiles of the two enantiomers S(,) talinolol and R(+) talinolol. Additionally, the metabolism of talinolol in human liver microsomes was examined. After oral administration, S(,) talinolol was slightly less absorbed and faster eliminated than R(+) talinolol. The absolute bioavailabilty of the R(+) enantiomer of talinolol was slightly but significantly higher than of its S(,) enantiomer. Coadministration of rifampicin further intensified this difference in the disposition of R(+) and S(,) talinolol (p,<,0.05). Formation of 4-trans hydroxytalinolol was the major metabolic pathway in human liver microsomes. All Clint values of S(,) were higher than of R(+) talinolol; 0.1 ,M ketoconazole inhibited the formation of all metabolites. In conclusion, the stereoselectivity of talinolol disposition is of minor importance, and most likely caused by presystemic biotransformation via CYP3A4. The less active R(+) talinolol might be suitable for phenotyping P-glycoprotein expression in man. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:303,311, 2002 [source] Ethanol inhibits in-vitro metabolism of nifedipine, triazolam and testosterone in human liver microsomesJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 8 2004Kiran C. Patki Although extended exposure to ethanol induces CYP3A metabolism in-vivo, the acute effects of ethanol on CYP3A metabolism have not been fully evaluated in-vitro. We assessed the effect of ethanol on CYP3A-mediated biotransformation using human liver microsomes in-vitro with three prototypic CYP3A-mediated reactions: nifedipine to oxidized nifedipine, triazolam to its 1-hydroxy (1-OH TRZ) and 4-hydroxy (4-OH TRZ) metabolites, and testosterone to 6,-hydroxytestosterone (6,-OH TST). Ethanol inhibited metabolism of nifedipine (oxidized nifedipine IC50 3 mg dL,1, where the IC50 value is the inhibitor concentration corresponding to a 50% reduction in metabolite formation velocity), triazolam (1-OH TRZ IC50 1.1 mg dL,1, 4-OH TRZ IC50 2.7 mg dL,1) and testosterone (6,-OH TST IC50 2.4 mg dL,1). The inhibitory potency of ethanol was similar for the three substrates representing the three hypothetical CYP3A substrate categories. The IC50 values obtained were lower than clinically relevant blood alcohol concentrations. In conclusion, ethanol is an inhibitor of human CYP3A metabolism and may contribute to clinically important interactions. [source] Inhibition of CYP3A-mediated oxidation in human hepatic microsomes by the dietary derived complex phenol, gallic acidJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 2 2002Ieva Stupans Plant polyphenols, such as gallic acid, have been reported to have a range of biological activities including antimutagenic effects. Previously, we reported that gallic acid (3,4,5-trihydroxy-benzoic acid), an agent found in wine and tea, inhibits androstenedione 6,-hydroxylase activity (Ki 70 ,M), a cytochrome P450 (CYP3A) marker in human liver microsomes. The preincubation of gallic acid (100 ,M) with human liver microsomes in the absence of NADPH, as compared with the presence of NADPH, before assay of androstenedione 6,-hydroxylase activity significantly increased the inhibitory effects of the gallic acid (0.03 ± 0.03 nmol (mg microsomal protein),1 min,1 compared with 0.20 ± 0.06 nmol (mg microsomal protein),1 min,1 (P< 0.05)). The antioxidant ascorbic acid and the radical scavenger glutathione prevented this observed increase in inhibition. Removal of gallic acid-derived products from the incubation completely restored CYP3A activity. In contrast, the activities of CYP1A and CYP2E, and non-CYP mediated reductive microsomal 17,-hydroxysteroid dehydrogenase activity were refractory to inhibition by gallic acid. [source] Metabolism of the major Echinacea alkylamide N -isobutyldodeca-2E,4E,8Z,10Z -tetraenamide by human recombinant cytochrome P450 enzymes and human liver microsomesPHYTOTHERAPY RESEARCH, Issue 8 2010F. Toselli Abstract Echinacea preparations are used for the treatment and prevention of upper respiratory tract infections. The phytochemicals believed responsible for the immunomodulatory properties are the alkylamides found in ethanolic extracts, with one of the most abundant being the N -isobutyldodeca-2E,4E,8Z,10Z -tetraenamide (1). In this study, we evaluated the human cytochrome P450 enzymes involved in the metabolism of this alkylamide using recombinant P450s, human liver microsomes and pure synthetic compound. Epoxidation, N -dealkylation and hydroxylation products were detected, with different relative amounts produced by recombinant P450s and microsomes. The major forms showing activity toward the metabolism of 1 were CYP1A1, CYP1A2 (both producing the same epoxide and N -dealkylation product), CYP2A13 (producing two epoxides), and CYP2D6 (producing two epoxides and an hydroxylated metabolite). Several other forms showed less activity. In incubations with human liver microsomes and selective inhibitors, CYP2E1 was found to be principally responsible for producing the dominant, hydroxylation product, whereas CYP2C9 was the principal source of the epoxides and CYP1A2 was responsible for the dealkylation product. In summary, in this study the relative impacts of the main human xenobiotic-metabolizing cytochrome P450s on the metabolism of a major Echinacea alkylamide have been established and the metabolites formed have been identified. Copyright © 2010 John Wiley & Sons, Ltd. [source] Toxicological determination and in vitro metabolism of the designer drug methylenedioxypyrovalerone (MPDV) by gas chromatography/mass spectrometry and liquid chromatography/quadrupole time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2010Sabina Strano-Rossi A method for the toxicological screening of the new designer drug methylenedioxypyrovalerone (MDPV) is described; with an emphasis on its application for anti-doping analysis. The metabolism of MDPV was evaluated in vitro using human liver microsomes and S9 cellular fractions for CYP450 phase I and uridine 5,-diphosphoglucuronosyltransferase (UGT) and sulfotransferase (SULT) phase II metabolism studies. The resulting metabolites were subsequently liquid/liquid extracted and analyzed using gas chromatography/mass spectrometry (GC/MS) as trimethylsilyl (TMS) derivatives. The structures of the metabolites were further confirmed by accurate mass measurement using a liquid chromatography/quadrupole time-of-flight (LC/QTOF) mass spectrometer. The studies demonstrated that the main metabolites of MDPV are catechol and methyl catechol pyrovalerone, which are in turn sulfated and glucuronated. The method for the determination of MDPV in urine has been fully validated by assessing the limits of detection and quantification, linearity, repeatability, and accuracy. This validation demonstrates the suitability for screening of this stimulant substance for anti-doping and forensic toxicology purposes. Copyright © 2010 John Wiley & Sons, Ltd. [source] A liquid chromatography/tandem mass spectrometry method for detecting UGT-mediated bioactivation of drugs as their N -acetylcysteine adducts in human liver microsomesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2009Hiroshi Harada The detection of the reactive metabolites of drugs has recently been gaining increasing importance. In vitro trapping studies using trapping agents such as glutathione are usually conducted for the detection of reactive metabolites, especially those of cytochrome P450-mediated metabolism. In order to detect the UDP-glucuronosyltransferase (UGT)-mediated bioactivation of drugs, an invitro trapping method using N -acetylcysteine (NAC) as a trapping agent followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed in this study. After the test compounds (diclofenac and ketoprofen) had been incubated in human liver microsomes with uridine diphosphoglucuronic acid (UDPGA) and NAC, the NAC adducts formed through their acyl glucuronides were analyzed using LC/MS/MS with electrospray ionization (ESI). The NAC adduct showed a mass shift of 145 units as compared to its parent, and the characteristic ion fragmentations reflected the parent. This is a concise and high-throughput method for evaluating reactive metabolites by UGT-mediated bioactivation. Copyright © 2009 John Wiley & Sons, Ltd. [source] Metabolism of a sulfur-containing heteroarotionoid antitumor agent, SHetA2, using liquid chromatography/tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 21 2008Zhongfa Liu SHetA2 {[(4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl)amino]methanethione], NSC 726189}, a sulfur-containing heteroarotinoid, selectively inhibits cancer cell growth and induces apoptosis without activation of nuclear retinoic acid receptors (RARs). The objective of this study was to investigate its in vitro metabolism in rat and human liver microsomes and in vivo metabolism in the mouse and rat using liquid chromatography-ultraviolet/multi-stage mass spectrometry (LC-UV/MSn) on an ion-trap mass spectrometer coupled with a photo-diode array (PDA) detector. In vitro, in the absence of glutathione (GSH), oxidation of the four aliphatic methyl groups of SHetA2 yielded one mono-, two di-, and one tri-hydroxylated SHetA2 metabolites, which were identified based on their UV and multi-stage mass spectra. In the presence of GSH, in addition to these primary oxidative metabolites, four GSH adducts of SHetA2 and a novel rare form thioether GSH adduct was detected and characterized. In vivo, the monohydroxylated SHetA2 metabolites were also detected in mouse and rat plasma and two GSH adducts were detected in rat liver following intravenous (i.v.) bolus administration of SHetA2 at 40,mg/kg. Copyright © 2008 John Wiley & Sons, Ltd. [source] Metabolite identification of small interfering RNA duplex by high-resolution accurate mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 12 2008Yan Zou On-line liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) using an LTQ-Orbitrap mass spectrometer was employed to investigate the metabolite profiles of a model siRNA duplex designated HBV263. The HBV263 duplex was incubated in rat and human serum and liver microsomes in vitro. The siRNA drug and its metabolites were then extracted using a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE), and analyzed by LC/ESI-MS. High-resolution accurate mass data enabled differentiation between two possible metabolite sequences with a monoisotopic molecular mass difference of less than 1,Da. ProMass deconvolution software was used to provide semi-automated data processing. In vitro serum and liver microsome incubation samples afforded different metabolite patterns: the antisense strand of the duplex was degraded preferentially in rat and human serum, while the sense strand of the duplex was less stable in rat and human liver microsomes. Copyright © 2008 John Wiley & Sons, Ltd. [source] Atmospheric pressure desorption/ionization on silicon ion trap mass spectrometry applied to the quantitation of midazolam in rat plasma and determination of midazolam 1,-hydroxylation kinetics in human liver microsomesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 24 2006Rick C. Steenwyk The application of atmospheric pressure desorption/ionization on silicon (AP-DIOS) coupled with ion trap mass spectrometry (ITMS) was investigated for the quantification of midazolam in rat plasma, and determination of midazolam 1,-hydroxylation kinetics in pooled human liver microsomes. Results indicate good sensitivity with absolute detection limits for midazolam in rat plasma of approximately 300 femtograms. A linear dynamic range from approximately 10,5000,ng/mL was obtained in rat plasma with analysis times of 1,min per sample. Kinetic constants for midazolam 1,-hydroxylation in human liver microsomes yielded an apparent Km of 10.0,µM and Vmax of 6.4,nmol/min/mg. Studies investigating the inhibition of 1,-hydroxymidazolam formation by the cytochrome P450 3A4 model inhibitor ketoconazole yielded an IC50 of 0.03,µM. Quantitative precision for replicate analysis of rat plasma and human liver microsomal samples was variable with relative standard deviation (RSD) values ranging from a low of approximately 3% to over 50%, with the highest variability observed in data from human liver microsomal incubations. While preliminary studies investigating the application of AP-DIOS-ITMS suggested feasibility of this technique to typical pharmacokinetic applications, further work is required to understand the underlying causes for the high variability observed in these investigations. Copyright © 2006 John Wiley & Sons, Ltd. [source] The Role of Human CYP2C8 and CYP2C9 Variants in Pioglitazone Metabolism In VitroBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 6 2009Eugen Muschler The present study was conducted to further clarify the role of individual CYPs and of the CYP2C8/9 polymorphisms in the primary metabolism of pioglitazone in vitro. Pioglitazone (2,400 ,M) was incubated with isolated cytochrome P450 enzymes or human liver microsomes, some of them carrying either the CYP2C8*3/*3 genotype (and also the CYP2C9*2/*2 genotype) or the CYP2C8*1/*1 genotype (five samples each). The formation of the primary pioglitazone metabolite M-IV was monitored by HPLC. Enzyme kinetics were estimated assuming a single binding site. Mean intrinsic clearance of pioglitazone to the metabolite M-IV was highest for CYP2C8 and CYP1A2 with 58 pmol M-IV/min/nmol CYP P450/,M pioglitazone each, 53 for CYP2D6*1, 40 for CYP2C19*1, and 34 for CYP2C9*2, respectively. CYP2A6, CYP2B6, CYP2C9*1, CYP2C9*3, CYP2E1, CYP3A4 and CYP3A5 did not form quantifiable amounts of M-IV. CYP2C8*1/*1 microsomes (25 ± 4 pmol M-IV/min/mg protein/,M pioglitazone) showed lower intrinsic clearance of pioglitazone than CYP2C8*3/*3 microsomes (35 ± 9, p = 0.04). In all samples, metabolite formation showed substrate inhibition, while pioglitazone did not inhibit CYP2C8-mediated paclitaxel metabolism. CYP2C8, CYP1A2 and CYP2D6 are major CYPs forming M-IV in vitro. The higher activity of CYP2C8*3/CYP2C9*2 microsomes may result from a contribution of CYP2C9*2, or from differences in CYP2C8 expression. The evidence for substrate-specific inhibitory effects of pioglitazone on CYP2C-mediated metabolism needs to be tested in further studies. [source] Substrate-Dependent Modulation of UDP-Glucuronosyltransferase 1A1 (UGT1A1) by Propofol in Recombinant Human UGT1A1 and Human Liver MicrosomesBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 3 2007Yuji Mano In the present study, we investigated the mechanism of activation, and whether the stimulatory effect occurs when another substrate is used with human liver microsomes. The glucuronidation of 4-MU followed Michaelis-Menten kinetics with a Km value of 101 µM in the absence of propofol. In the presence of 200 µM propofol, a concentration that causes heterotopic activation of 4-MU glucuronidation (4-MUG), the Vmax value increased to 1.5-fold, while the Km value decreased to 0.53-fold. In order to assess whether propofol activates UGT1A1 activity for a substrate other than 4-MU, the effect of propofol on oestradiol 3,-glucuronidation by recombinant UGT1A1 and in human liver microsomes was evaluated. In contrast to 4-MUG activity, propofol inhibited UGT1A1-catalysed oestradiol 3,-glucuronidation in recombinant UGT1A1 as well as in human liver microsomes with IC50 values of 59 and 228 µM, respectively. In addition, a known UGT1A1 modulator, 17,-ethynyloestradiol, stimulated oestradiol 3,-glucuronidation slightly at a concentration of 5 µM, while it inhibited 4-MUG in recombinant UGT1A1 at all concentrations tested (5,100 µM). These findings indicate that the modulation of UGT1A1 by propofol is substrate-dependent, and thus care should be taken when extrapolating the stimulatory effects of drugs for one glucuronidation substrate. [source] Metabolism of Repaglinide by CYP2C8 and CYP3A4 in vitro: Effect of Fibrates and RifampicinBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 4 2005Lauri I. Kajosaari To clarify the mechanisms of observed repaglinide drug interactions, we determined the contribution of the two enzymes to repaglinide metabolism at different substrate concentrations, and examined the effect of fibrates and rifampicin on CYP2C8, CYP3A4 and repaglinide metabolism in vitro. We studied repaglinide metabolism using pooled human liver microsomes, recombinant CYP2C8 and recombinant CYP3A4 enzymes. The effect of quercetin and itraconazole on repaglinide metabolism, and of gemfibrozil, bezafibrate, fenofibrate and rifampicin on CYP2C8 (paclitaxel 6,-hydroxylation) and CYP3A4 (midazolam 1-hydroxylation) activities and repaglinide metabolism were studied using human liver microsomes. At therapeutic repaglinide concentrations (<0.4 ,M), CYP2C8 and CYP3A4 metabolised repaglinide at similar rates. Quercetin (25 ,M) and itraconazole (3 ,M) inhibited the metabolism of 0.2 ,M repaglinide by 58% and 71%, and that of 2 ,M repaglinide by 56% and 59%, respectively. The three fibrates inhibited CYP2C8 (Ki: bezafibrate 9.7 ,M, gemfibrozil 30.4 ,M and fenofibrate 92.6 ,M) and repaglinide metabolism (IC50: bezafibrate 37.7 ,M, gemfibrozil 111 ,M and fenofibrate 164 ,M), but had no effect on CYP3A4. Rifampicin inhibited CYP2C8 (Ki 30.2 ,M), CYP3A4 (Ki 18.5 ,M) and repaglinide metabolism (IC50 13.7 ,M). In conclusion, both CYP2C8 and CYP3A4 are important in the metabolism of therapeutic concentrations of repaglinide in vitro, but their predicted contributions in vivo are highly dependent on the scaling factor used. Gemfibrozil is only a moderate inhibitor of CYP2C8 and does not inhibit CYP3A4; inhibition of CYP-enzymes by parent gemfibrozil alone does not explain its interaction with repaglinide in vivo. Rifampicin competitively inhibits both CYP2C8 and CYP3A4, which can counteract its inducing effect in humans. [source] In vitro Metabolism of Genistein and Tangeretin by Human and Murine Cytochrome P450sBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 1 2003Vibeke M. Breinholt Analysis of the metabolic profile from incubations with genistein and human liver microsomes revealed the production of five different metabolites, of which three were obtained in sufficient amounts to allow a more detailed elucidation of the structure. One of these metabolites was identified as orobol, the 3,-hydroxylated metabolite of genistein. The remaining two metabolites were also hydroxylated metabolites as evidenced by LC/MS. Orobol was the only metabolite formed after incubation with CYP1A2. The two major product peaks after incubation of tangeretin with human microsomes were identical with 4,-hydroxy-5,6,7,8-tetramethoxyflavone and 5,6-dihydroxy-4,,7,8-trimethoxyflavone, previously identified in rat urine in our laboratory. By comparison with UV spectra and LC/MS fragmentation patterns of previously obtained standards, the remaining metabolites eluting after 14, 17 and 20 min. were found to be demethylated at the 4,,7-, 4,,6-positions or hydroxylated at the 3,- and demethylated at the 4,-positions, respectively. Metabolism of tangeretin by recombinant CYP1A2, 3A4, 2D6 and 2C9 resulted in metabolic profiles that qualitatively were identical to those observed in the human microsomes. Inclusion of the CYP1A2 inhibitor fluvoxamine in the incubation mixture with human liver microsomes resulted in potent inhibition of tangeretin and genistein metabolism. Other isozymes-selective CYP inhibitors had only minor effects on tangeretin or genistein metabolism. Overall the presented observations suggest major involvement of CYP1A2 in the hepatic metabolism of these two flavonoids. [source] In vitro metabolism of a new H+/K+ ATPase inhibitor DBM-819 in liver microsomes using HPLC and electrospray mass spectrometryBIOMEDICAL CHROMATOGRAPHY, Issue 8 2001Sung Jin Choi The metabolism of 1-(2-methyl-4-methoxyphenyl)-4-[(3-hydroxypropyl)amino]-6-methyl-2,3-dihydropyrrolo[3,2c]quinoline (DBM-819), a new H+/K+ ATPase inhibitor, has been studied by HPLC with spectrometric detection and on-line LC-electrospray mass spectrometry. In vitro incubation of DBM-819 with rat liver microsomes in the presence of NADPH resulted in the production of four metabolites (M1-4), whereas DBM-819 was oxidized to two metabolites, M2 and M4, by human liver microsomes. M2, M3 and M4 were identified as O-demethyl-DBM-819, 8-hydroxy-DBM-819 and N-dehydroxypropyl-DBM-819, respectively, based on LC/MS/MS analysis with authentic standards. M1 was tentatively identified as 1-(hydroxy-2-methyl-4-methoxyphenyl)-4-[(3-hydroxypropyl)amino]-6-methyl-2,3-dihydropyrrolo[3,2c]quinoline. Rat liver CYP1A1/2 catalyzed the oxidation of DBM-819 to 8-hydroxy-DBM-819 and N-dehydroxypropyl-DBM-819. Human CYP3A4 was a major isozyme for the formation of O-demethyl-DBM-819 as well as N-dehydroxypropyl-DBM-819. Copyright © 2001 John Wiley & Sons, Ltd. [source] Contributions of human cytochrome P450 enzymes to glyburide metabolismBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 4 2010Lin Zhou Abstract Glyburide (GLB) is a widely used oral sulfonylurea for the treatment of gestational diabetes. The therapeutic use of GLB is often complicated by a substantial inter-individual variability in the pharmacokinetics and pharmacodynamics of the drug in human populations, which might be caused by inter-individual variations in factors such as GLB metabolism. Therefore, there has been a continued interest in identifying human cytochrome P450 (CYP) isoforms that play a major role in the metabolism of GLB. However, contrasting data are available in the present literature in this regard. The present study systematically investigated the contributions of various human CYP isoforms (CYP3A4, CYP3A5, CYP2C8, CYP2C9 and CYP2C19) to in vitro metabolism of GLB. GLB depletion and metabolite formation in human liver microsomes were most significantly inhibited by the CYP3A inhibitor ketoconazole compared with the inhibitors of other CYP isoforms. Furthermore, multiple correlation analysis between GLB depletion and individual CYP activities was performed, demonstrating a significant correlation between GLB depletion and the CYP3A probe activity in 16 individual human liver microsomal preparations, but not between GLB depletion and the CYP2C19, CYP2C8 or CYP2C9 probe activity. By using recombinant supersomes overexpressing individual human CYP isoforms, it was found that GLB could be depleted by all the enzymes tested; however, the intrinsic clearance (Vmax/Km) of CYP3A4 for GLB depletion was 4,17 times greater than that of other CYP isoforms. These results confirm that human CYP3A4 is the major enzyme involved in the in vitro metabolism of GLB. Copyright © 2010 John Wiley & Sons, Ltd. [source] | ||||||||||||||||