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Atsushi Kurihara - One of the best experts on this subject based on the ideXlab platform.
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hepatic microsomal thiol methyltransferase is involved in stereoselective methylation of pharmacologically Active Metabolite of prasugrel
Drug Metabolism and Disposition, 2014Co-Authors: Miho Kazui, Katsunobu Hagihara, Toshihiko Ikeda, Takashi Izumi, Atsushi KuriharaAbstract:Prasugrel, a thienopyridine antiplatelet drug, is converted in animals and humans to the pharmacologically Active Metabolite R-138727 [(2Z)-{1-[(1RS)-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-sulfanylpiperidin-3-ylidene}ethanoic acid], which has two chiral centers, occurring as a mixture of four isomers. The RS and RR isomers are more Active than the SS and SR isomers (RS > RR > > SR = SS). The pharmacologically Active Metabolite is further metabolized to an S-methylated Metabolite that is the major identified inActive Metabolite in humans. In rat, dog, and human liver microsomes supplemented with S-adenosyl methione, the SS and SR isomers of the Active Metabolite were extensively S-methylated while the RS and RR isomers were not. Addition of 2,3-dichloromethyl benzylamine (50 µM) completely inhibited the S-methylation reaction, indicating that the microsomal and cytosolic thiol methyltransferase but not the cytosolic thiopurine S-methyltransferase is involved in the methylation. The hepatic intrinsic clearance values for methylation of the RS, RR, SS, and SR isomers (ml/min/kg) were 0, 0, 40.4, and 37.6, respectively, in rat liver microsomes, 0, 0, 11.6, and 2.5, respectively, in dog liver microsomes, and 0, 0, 17.3, and 17.7, respectively, in human liver microsomes, indicating that the RS and RR isomers are not methylated in vitro and that the methylation of SS and SR isomers is high with rat > human > dog. This finding in vitro agreed well with the in vivo observation in rats and dogs, where the S-methylated SS and SR isomers were the major Metabolites in the plasma whereas negligible amounts of S-methylated RS and RR isomers were detected after intravenous administration of the pharmacologically Active Metabolites.
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biotransformation of prasugrel a novel thienopyridine antiplatelet agent to the pharmacologically Active Metabolite
Drug Metabolism and Disposition, 2010Co-Authors: Katsunobu Hagihara, Miho Kazui, Atsushi Kurihara, Nagy A Farid, Osamu Okazaki, Haruo Iwabuchi, Minoru Ishikawa, Hiroyuki Kobayashi, Naoki Tanaka, Toshihiko IkedaAbstract:Prasugrel, a novel thienopyridine antiplatelet agent, undergoes rapid hydrolysis in vivo to a thiolactone, R-95913, which is further converted to its thiol-containing, pharmacologically Active Metabolite, R-138727, by oxidation via cytochromes P450 (P450). We trapped a sulfenic acid Metabolite as a mixed disulfide with 2-nitro-5-thiobenzoic acid in an incubation mixture containing the thiolactone R-95913, expressed CYP3A4, and NADPH. Further experiments investigated one possible mechanism for the conversion of the sulfenic acid to the Active thiol Metabolite in vitro. A mixed disulfide form of R-138727 with glutathione was found to be a possible precursor of R-138727 in vitro when glutathione was present. The rate constant for the reduction of the glutathione conjugate of R-138727 to R-138727 was increased by addition of human liver cytosol to the human liver microsomes. Thus, one possible mechanism for the ultimate formation of R-138727 in vitro can be through formation of a sulfenic acid mediated by P450s followed possibly by a glutathione conjugation to a mixed disulfide and reduction of the disulfide to the Active Metabolite R-138727.
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identification of the human cytochrome p450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically Active Metabolite
Drug Metabolism and Disposition, 2010Co-Authors: Miho Kazui, Nagy A Farid, Katsunobu Hagihara, Osamu Okazaki, Toshihiko Ikeda, Yumi Nishiya, Tomoko Ishizuka, Atsushi KuriharaAbstract:The aim of the current study is to identify the human cytochrome P450 (P450) isoforms involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically Active Metabolite. In the in vitro experiments using cDNA-expressed human P450 isoforms, clopidogrel was metabolized to 2-oxo-clopidogrel, the immediate precursor of its pharmacologically Active Metabolite. CYP1A2, CYP2B6, and CYP2C19 catalyzed this reaction. In the same system using 2-oxo-clopidogrel as the substrate, detection of the Active Metabolite of clopidogrel required the addition of glutathione to the system. CYP2B6, CYP2C9, CYP2C19, and CYP3A4 contributed to the production of the Active Metabolite. Secondly, the contribution of each P450 involved in both oxidative steps was estimated by using enzyme kinetic parameters. The contribution of CYP1A2, CYP2B6, and CYP2C19 to the formation of 2-oxo-clopidogrel was 35.8, 19.4, and 44.9%, respectively. The contribution of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 to the formation of the Active Metabolite was 32.9, 6.76, 20.6, and 39.8%, respectively. In the inhibition studies with antibodies and selective chemical inhibitors to P450s, the outcomes obtained by inhibition studies were consistent with the results of P450 contributions in each oxidative step. These studies showed that CYP2C19 contributed substantially to both oxidative steps required in the formation of clopidogrel Active Metabolite and that CYP3A4 contributed substantially to the second oxidative step. These results help explain the role of genetic polymorphism of CYP2C19 and also the effect of potent CYP3A inhibitors on the pharmacokinetics and pharmacodynamics of clopidogrel in humans and on clinical outcomes.
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a possible mechanism for the differences in efficiency and variability of Active Metabolite formation from thienopyridine antiplatelet agents prasugrel and clopidogrel
Drug Metabolism and Disposition, 2009Co-Authors: Katsunobu Hagihara, Miho Kazui, Atsushi Kurihara, Nagy A Farid, Michiharu Yoshiike, Kokichi Honda, Osamu Okazaki, Toshihiko IkedaAbstract:The efficiency and interindividual variability in bioactivation of prasugrel and clopidogrel were quantitatively compared and the mechanisms involved were elucidated using 20 individual human liver microsomes. Prasugrel and clopidogrel are converted to their thiol-containing Active Metabolites through corresponding thiolactone Metabolites. The formation rate of clopidogrel Active Metabolite was much lower and more variable [0.164 ± 0.196 μl/min/mg protein, coefficient of variation (CV) = 120%] compared with the formation of prasugrel Active Metabolite (8.68 ± 6.64 μl/min/mg protein, CV = 76%). This result was most likely attributable to the less efficient and less consistent formation of clopidogrel thiolactone Metabolite (2.24 ± 1.00 μl/min/mg protein, CV = 45%) compared with the formation of prasugrel thiolactone Metabolite (55.2 ± 15.4 μl/min/mg protein, CV = 28%). These differences may be attributed to the following factors. Clopidogrel was largely hydrolyzed to an inActive acid Metabolite (approximately 90% of total Metabolites analyzed), and the clopidogrel concentrations consumed were correlated to human carboxylesterase 1 activity in each source of liver microsomes. In addition, 48% of the clopidogrel thiolactone Metabolite formed was converted to an inActive thiolactone acid Metabolite. The oxidation of clopidogrel to its thiolactone Metabolite correlated with variable activities of CYP1A2, CYP2B6, and CYP2C19. In conclusion, the Active Metabolite of clopidogrel was formed with less efficiency and higher variability than that of prasugrel. This difference in thiolactone formation was attributed to hydrolysis of clopidogrel and its thiolactone Metabolite to inActive acid Metabolites and to variability in cytochrome P450-mediated oxidation of clopidogrel to its thiolactone Metabolite, which may contribute to the poorer and more variable Active Metabolite formation for clopidogrel than prasugrel.
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quantitative determination of clopidogrel Active Metabolite in human plasma by lc ms ms
Journal of Pharmaceutical and Biomedical Analysis, 2008Co-Authors: Makoto Takahashi, Nagy A Farid, Henrianna Pang, Kiyoshi Kawabata, Atsushi KuriharaAbstract:A quantitative method for the determination of clopidogrel Active Metabolite (AM) in human plasma was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Clopidogrel AM contains a thiol group, thus requiring stabilization in biological samples. The alkylating reagent 2-bromo-3'-methoxyacetophenone was used to stabilize clopidogrel AM in blood. An analog of the derivatized clopidogrel AM was used as the internal standard (IS). The derivatized samples were subjected to solid-phase extraction with a C2 disk plate and the overall procedure exhibited good reaction (more than 90%) and recovery efficiencies (from 85% to 105%). The derivative of clopidogrel AM (MP-AM) and IS were separated on an ODS column and quantified by tandem mass spectrometry with electrospray ionization. No significant endogenous peaks corresponding to MP-AM or IS were detected in blank human plasma samples, and no significant matrix effect was observed for MP-AM and IS in human plasma samples (from 102% to 121%). The calibration curve ranged from 0.5 to 250 ng/mL with good linearity, and extended by validation of a 50-fold dilution. In the intra- and inter-assay reproducibility tests, the accuracy and precision were within 12% relative error and 6% coefficient of variation, respectively. The derivatized MP-AM was stable in human plasma for 4 months at -80 degrees C. The validated method was successfully used to analyze clinical samples and determine the pharmacokinetics of clopidogrel AM.
Nagy A Farid - One of the best experts on this subject based on the ideXlab platform.
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biotransformation of prasugrel a novel thienopyridine antiplatelet agent to the pharmacologically Active Metabolite
Drug Metabolism and Disposition, 2010Co-Authors: Katsunobu Hagihara, Miho Kazui, Atsushi Kurihara, Nagy A Farid, Osamu Okazaki, Haruo Iwabuchi, Minoru Ishikawa, Hiroyuki Kobayashi, Naoki Tanaka, Toshihiko IkedaAbstract:Prasugrel, a novel thienopyridine antiplatelet agent, undergoes rapid hydrolysis in vivo to a thiolactone, R-95913, which is further converted to its thiol-containing, pharmacologically Active Metabolite, R-138727, by oxidation via cytochromes P450 (P450). We trapped a sulfenic acid Metabolite as a mixed disulfide with 2-nitro-5-thiobenzoic acid in an incubation mixture containing the thiolactone R-95913, expressed CYP3A4, and NADPH. Further experiments investigated one possible mechanism for the conversion of the sulfenic acid to the Active thiol Metabolite in vitro. A mixed disulfide form of R-138727 with glutathione was found to be a possible precursor of R-138727 in vitro when glutathione was present. The rate constant for the reduction of the glutathione conjugate of R-138727 to R-138727 was increased by addition of human liver cytosol to the human liver microsomes. Thus, one possible mechanism for the ultimate formation of R-138727 in vitro can be through formation of a sulfenic acid mediated by P450s followed possibly by a glutathione conjugation to a mixed disulfide and reduction of the disulfide to the Active Metabolite R-138727.
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identification of the human cytochrome p450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically Active Metabolite
Drug Metabolism and Disposition, 2010Co-Authors: Miho Kazui, Nagy A Farid, Katsunobu Hagihara, Osamu Okazaki, Toshihiko Ikeda, Yumi Nishiya, Tomoko Ishizuka, Atsushi KuriharaAbstract:The aim of the current study is to identify the human cytochrome P450 (P450) isoforms involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically Active Metabolite. In the in vitro experiments using cDNA-expressed human P450 isoforms, clopidogrel was metabolized to 2-oxo-clopidogrel, the immediate precursor of its pharmacologically Active Metabolite. CYP1A2, CYP2B6, and CYP2C19 catalyzed this reaction. In the same system using 2-oxo-clopidogrel as the substrate, detection of the Active Metabolite of clopidogrel required the addition of glutathione to the system. CYP2B6, CYP2C9, CYP2C19, and CYP3A4 contributed to the production of the Active Metabolite. Secondly, the contribution of each P450 involved in both oxidative steps was estimated by using enzyme kinetic parameters. The contribution of CYP1A2, CYP2B6, and CYP2C19 to the formation of 2-oxo-clopidogrel was 35.8, 19.4, and 44.9%, respectively. The contribution of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 to the formation of the Active Metabolite was 32.9, 6.76, 20.6, and 39.8%, respectively. In the inhibition studies with antibodies and selective chemical inhibitors to P450s, the outcomes obtained by inhibition studies were consistent with the results of P450 contributions in each oxidative step. These studies showed that CYP2C19 contributed substantially to both oxidative steps required in the formation of clopidogrel Active Metabolite and that CYP3A4 contributed substantially to the second oxidative step. These results help explain the role of genetic polymorphism of CYP2C19 and also the effect of potent CYP3A inhibitors on the pharmacokinetics and pharmacodynamics of clopidogrel in humans and on clinical outcomes.
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a possible mechanism for the differences in efficiency and variability of Active Metabolite formation from thienopyridine antiplatelet agents prasugrel and clopidogrel
Drug Metabolism and Disposition, 2009Co-Authors: Katsunobu Hagihara, Miho Kazui, Atsushi Kurihara, Nagy A Farid, Michiharu Yoshiike, Kokichi Honda, Osamu Okazaki, Toshihiko IkedaAbstract:The efficiency and interindividual variability in bioactivation of prasugrel and clopidogrel were quantitatively compared and the mechanisms involved were elucidated using 20 individual human liver microsomes. Prasugrel and clopidogrel are converted to their thiol-containing Active Metabolites through corresponding thiolactone Metabolites. The formation rate of clopidogrel Active Metabolite was much lower and more variable [0.164 ± 0.196 μl/min/mg protein, coefficient of variation (CV) = 120%] compared with the formation of prasugrel Active Metabolite (8.68 ± 6.64 μl/min/mg protein, CV = 76%). This result was most likely attributable to the less efficient and less consistent formation of clopidogrel thiolactone Metabolite (2.24 ± 1.00 μl/min/mg protein, CV = 45%) compared with the formation of prasugrel thiolactone Metabolite (55.2 ± 15.4 μl/min/mg protein, CV = 28%). These differences may be attributed to the following factors. Clopidogrel was largely hydrolyzed to an inActive acid Metabolite (approximately 90% of total Metabolites analyzed), and the clopidogrel concentrations consumed were correlated to human carboxylesterase 1 activity in each source of liver microsomes. In addition, 48% of the clopidogrel thiolactone Metabolite formed was converted to an inActive thiolactone acid Metabolite. The oxidation of clopidogrel to its thiolactone Metabolite correlated with variable activities of CYP1A2, CYP2B6, and CYP2C19. In conclusion, the Active Metabolite of clopidogrel was formed with less efficiency and higher variability than that of prasugrel. This difference in thiolactone formation was attributed to hydrolysis of clopidogrel and its thiolactone Metabolite to inActive acid Metabolites and to variability in cytochrome P450-mediated oxidation of clopidogrel to its thiolactone Metabolite, which may contribute to the poorer and more variable Active Metabolite formation for clopidogrel than prasugrel.
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quantitative determination of clopidogrel Active Metabolite in human plasma by lc ms ms
Journal of Pharmaceutical and Biomedical Analysis, 2008Co-Authors: Makoto Takahashi, Nagy A Farid, Henrianna Pang, Kiyoshi Kawabata, Atsushi KuriharaAbstract:A quantitative method for the determination of clopidogrel Active Metabolite (AM) in human plasma was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Clopidogrel AM contains a thiol group, thus requiring stabilization in biological samples. The alkylating reagent 2-bromo-3'-methoxyacetophenone was used to stabilize clopidogrel AM in blood. An analog of the derivatized clopidogrel AM was used as the internal standard (IS). The derivatized samples were subjected to solid-phase extraction with a C2 disk plate and the overall procedure exhibited good reaction (more than 90%) and recovery efficiencies (from 85% to 105%). The derivative of clopidogrel AM (MP-AM) and IS were separated on an ODS column and quantified by tandem mass spectrometry with electrospray ionization. No significant endogenous peaks corresponding to MP-AM or IS were detected in blank human plasma samples, and no significant matrix effect was observed for MP-AM and IS in human plasma samples (from 102% to 121%). The calibration curve ranged from 0.5 to 250 ng/mL with good linearity, and extended by validation of a 50-fold dilution. In the intra- and inter-assay reproducibility tests, the accuracy and precision were within 12% relative error and 6% coefficient of variation, respectively. The derivatized MP-AM was stable in human plasma for 4 months at -80 degrees C. The validated method was successfully used to analyze clinical samples and determine the pharmacokinetics of clopidogrel AM.
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increased Active Metabolite formation explains the greater platelet inhibition with prasugrel compared to high dose clopidogrel
Journal of Cardiovascular Pharmacology, 2007Co-Authors: Christopher D Payne, Nagy A Farid, David S Small, John T Brandt, Steven C Ernest, Joseph A Jakubowski, Daniel E Salazar, Kenneth J WintersAbstract:Prasugrel pharmacodynamics and pharmacokinetics after a 60-mg loading dose (LD) and daily 10-mg maintenance doses (MD) were compared in a 3-way crossover study to clopidogrel 600-mg/75-mg and 300-mg/75-mg LD/MD in 41 healthy, aspirin-free subjects. Each LD was followed by 7 days of daily MD and a 14-day washout period. Inhibition of platelet aggregation (IPA) was assessed by turbidometric aggregometry (20 and 5 microM ADP). Prasugrel 60-mg achieved higher mean IPA (54%) 30 minutes post-LD than clopidogrel 300-mg (3%) or 600-mg (6%) (P < 0.001) and greater IPA by 1 hour (82%) and 2 hours (91%) than the 6-hour IPA for clopidogrel 300-mg (51%) or 600-mg (69%) (P < 0.01). During MD, IPA for prasugrel 10-mg (78%) exceeded that of clopidogrel (300-mg/75-mg, 56%; 600-mg/75-mg, 52%; P < 0.001). Active Metabolite area under the concentration-time curve (AUC0-tlast) after prasugrel 60-mg (594 ng.hr/mL) was 2.2 times that after clopidogrel 600-mg. Prasugrel Active Metabolite AUC0-tlast was consistent with dose-proportionality from 10-mg to 60-mg, while clopidogrel Active Metabolite AUC0-tlast exhibited saturable absorption and/or metabolism. In conclusion, greater exposure to prasugrel's Active Metabolite results in faster onset, higher levels, and less variability of platelet inhibition compared with high-dose clopidogrel in healthy subjects.
D M Burger - One of the best experts on this subject based on the ideXlab platform.
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development and validation of a bioanalytical method to quantitate enzalutamide and its Active Metabolite n desmethylenzalutamide in human plasma application to clinical management of patients with metastatic castration resistant prostate cancer
Therapeutic Drug Monitoring, 2018Co-Authors: Guillemette E Benoist, Eric Van Der Meulen, Inge M Van Oort, Jan H Beumer, Diederik M Somford, Jack A Schalken, D M BurgerAbstract:Background:Enzalutamide is a potent androgen-signaling receptor inhibitor and is licensed for the treatment of metastatic castration–resistant prostate cancer. N-desmethylenzalutamide is the Active Metabolite of enzalutamide. A method to quantitate enzalutamide and its Active Metabolite was develope
Randa A Abdelsalam - One of the best experts on this subject based on the ideXlab platform.
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high performance liquid chromatographic determination of 3 methylflavone 8 carboxylic acid the main Active Metabolite of flavoxate hydrochloride in human urine
Journal of Pharmaceutical and Biomedical Analysis, 2007Co-Authors: Alaa Elgindy, Shehab A Sallam, Randa A AbdelsalamAbstract:High performance liquid chromatographic (HPLC) method was presented for the determination of 3-methylflavone-8-carboxylic acid as the main Active Metabolite of flavoxate hydrochloride (FX) in human urine. The proposed method was based on using CN column with mobile phase consisting of acetonitrile-12 mM ammonium acetate (40:60, v/v) and adjusted to apparent pH 4.0 with flow rate of 1.5 ml min(-1). Quantitation was achieved with UV detection at 220 nm. The proposed method was utilized to the determination of dissolution rate for tablets containing flavoxate hydrochloride. The urinary excretion pattern has been calculated using the proposed method.
Ljuba M Mandic - One of the best experts on this subject based on the ideXlab platform.
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simultaneous lc determination of tiazofurin its acetyl and benzoyl esters and their Active Metabolite thiazole 4 carboxamide adenine dinucleotide in biological samples
Journal of Pharmaceutical and Biomedical Analysis, 2002Co-Authors: Jelena Tasic, Slavica Ristic, Vesna Piperski, Mirjana Dacevic, Vjera Pejanovic, Milan Jokanovic, Ljuba M MandicAbstract:A rapid and sensitive HPLC-RP method for simultaneous determination of tiazofurin, its 5'-O acetyl and benzoyl esters and their Active Metabolite thiazole-4-carboxamide adenine dinucleotide was developed and validated. The method allowed determination and quantification of nanomolar quantities of these substances in cell extracts of treated cells, and was also used in kinetic studies of cellular uptake of tiazofurin and its esters from the cultivation medium. Separation of the analyzed substances from unidentified peaks from both biological materials was achieved by gradient elution, thus reducing the possibility of interference. The mobile phase consisted of a 0.1 M sodium-hydrogen phosphate, pH 5.1 and methanol. Run time was 22 min, with 5 min equilibration time.
