- In vitro modulatory effects of flavonoids on human cytochrome P450 2C8 (CYP2C8)
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The inhibitory effects of five flavonoidswith distinct chemical classes (flavones [luteolin], flavonols [quercetin and quercitrin], and flavanones [hesperetin and hespiridin]) on cDNA-expressed CYP2C8 were investigated. CYP2C8 was co-expressed with NADPH-
- Pang, Chia Yong,Mak, Joon Wah,Ismail, Rusli,Ong, Chin Eng
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- Inhibition of CYP2C9 by selective serotonin reuptake inhibitors: In vitro studies with tolbutamide and (S)-warfarin using human liver microsomes
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Objective: To investigate the in vitro potential of selective serotonin reuptake inhibitors (SSRIs) to inhibit two CYP2C9-catalysed reactions, tolbutamide 4-methylhydroxylation and (S)-warfarin 7-hydroxylation. Methods: The formation of 4-hydroxytolbutami
- Hemeryck,De Vriendt,Belpaire
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- An improved synthesis of N-(butylaminocarbonyl)-4-hydroxymethyl-benzenesulfonamide, one of the metabolites of tolbutamide, and synthesis of its formyl derivative
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An improved synthesis of N-(butylaminocarbonyl)-4-hydroxymethyl-benzenesulfonamide, one of the metabolites of tolbutamide, and its transformation to the formyl derivative are described.
- Makaya,Irie,Shibasaki
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- Gliclazide hydroxylation by rat liver microsomes
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The metabolism of gliclazide to hydroxygliclazide has been investigated in Sprague-Dawley rat liver microsomes. The kinetics of hydroxygliclazide formation are consistent with Michaelis-Menten kinetics (mean (± SD, n = 3) apparent K(m) and V(max) = 256 ±
- Rieutord,Stupans,Shenfield,Gross
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- Electrochemically driven drug metabolism via cytochrome P450 2C9 isozyme microsomes with cytochrome P450 reductase and indium tin oxide nanoparticle composites
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We describe herein an electrochemically driven drug metabolism strategy based on nanocomposites that integrate cyt P450 2C9 (CYP2C9) isozyme microsomes with cyt P450 reductase (CPR), indium tin oxide (ITO) nanoparticles and chitosan (CS). This novel bioel
- Xu, Xuan,Wei, Wei,Huang, Minghe,Yao, Li,Liu, Songqin
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- Identification and Enzymatic Activity Evaluation of a Novel CYP2C9 Allelic Variant Discovered in a Patient
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Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus–insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0?mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.
- Zhou, Xiao-Yang,Lu, Xiang-Ran,Li, Ying-Hui,Ma, Ya-Qing,Zhao, Shi-Wen,Wang, Fang,Xu, Ren-Ai,Hu, Guo-Xin,Cai, Jian-Ping
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- Benchmarking of laboratory evolved unspecific peroxygenases for the synthesis of human drug metabolites
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By mimicking the role of human liver P450 monooxygenases, fungal unspecific peroxygenases (UPOs) can perform a range of highly selective oxyfunctionalization reactions on pharmacological compounds, including O-dealkylations and hydroxylations, thereby simulating drug metabolism. Here we have benchmarked human drug metabolite (HDM) synthesis by several evolved UPO mutants, focusing on dextromethorphan, naproxen and tolbutamide. The HDM from dextromethorphan was prepared at the semi-preparative scale as a proof of production. The structural analysis of mutations involved in the synthesis of HDMs highlights the heme access channel as the main feature on which to focus when designing evolved UPOs. These variants are becoming emergent tools for the cost-effective synthesis of HDMs from next-generation drugs.
- Gomez de Santos, Patricia,Cervantes, Fadia V.,Tieves, Florian,Plou, Francisco J.,Hollmann, Frank,Alcalde, Miguel
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p. 1827 - 1831
(2019/02/24)
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- Heme-thiolate sulfenylation of human cytochrome P450 4A11 functions as a redox switch for catalytic inhibition
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Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear. Here, we found that reducing agents (e.g. dithiothreitol and tris(2-carboxyethyl) phosphine) strongly enhanced the catalytic activity of P450 4A11, but not of 10 other human P450s tested. Conversely, added H2O2 attenuated P450 4A11 catalytic activity. Catalytic roles of five of the potentially eight implicated Cys residues of P450 4A11 were eliminated by site-directed mutagenesis. Using an isotope-coded dimedone/iododimedone-labeling strategy and mass spectrometry of peptides, we demonstrated that the heme-thiolate cysteine (Cys-457) is selectively sulfenylated in an H2O2 concentration-dependent manner. This sulfenylation could be reversed by reducing agents, including dithiothreitol and dithionite. Of note, we observed heme ligand cysteine sulfenylation of P450 4A11 ex vivo in kidneys and livers derived from CYP4A11 transgenic mice. We also detected sulfenylation of murine P450 4a12 and 4b1 heme peptides in kidneys. To our knowledge, reversible oxidation of the heme thiolate has not previously been observed in P450s and may have relevance for 20-HETE-mediated functions.
- Albertolle, Matthew E.,Kim, Donghak,Nagy, Leslie D.,Yun, Chul-Ho,Pozzi, Ambra,Savas, üzen,Johnson, Eric F.,Guengerich, F. Peter
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p. 11230 - 11242
(2017/08/08)
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- Acetylshikonin is a novel non-selective cytochrome P450 inhibitor
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Acetylshikonin is a biologically active compound with anti-cancer and anti-inflammatory activity, which is isolated from the roots of Lithospermum erythrorhizoma. An inhibitory effect of acetylshikonin against CYP2J2 activity was discovered recently. Based on this result, this study was expanded to evaluate the inhibitory effects of acetylshikonin against nine different cytochrome P450 (P450) isoforms in human liver microsomes (HLMs) using substrate cocktails incubation assay. Acetylshikonin showed a strong inhibitory effect against all P450s tested with IC50 values of 1.4–4.0 μ m. Pre-incubation of acetylshikonin with HLMs and NADPH did not alter the inhibition potency, indicating that acetylshikonin is not a mechanism-based inhibitor. SKF-525A, a widely used non-specific P450 inhibitor, had no inhibitory activity against CYP1A2, 2A6, 2E1 and 2J2, while it showed an inhibitory effect against CYP2B6, CYP2C19 and 2D6 with IC50 values of 2.5, 3.6 and 0.5 μ m, respectively. Our findings indicate that acetylshikonin may be a novel general P450 inhibitor, which could replace SKF-525A.
- Shon, Jong Cheol,Phuc, Nguyen Minh,Kim, Won Cheol,Heo, Jae Kyung,Wu, Zhexue,Lee, Hyunyoung,Liu, Kwang-Hyeon
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p. 553 - 556
(2017/12/15)
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- Effects of artemisinin antimalarials on Cytochrome P450 enzymes in vitro using recombinant enzymes and human liver microsomes: Potential implications for combination therapies
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1. Cytochrome P450 enzyme system is the most important contributor to oxidative metabolism of drugs. Modification, and more specifically inhibition, of this system is an important determinant of several drug-drug interactions (DDIs). 2. Effects of the antimalarial agent artemisinin and its structural analogues, artemether, artesunate and dihydroartemisinin, on seven of the major human liver CYP isoforms (CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6 and 3A4) were evaluated using recombinant enzymes (fluorometric assay) and human liver microsomes (LC-MS/MS analysis). Inhibitory potency (IC50) and mechanisms of inhibition were evaluated using nonlinear regression analysis. In vitro-in vivo extrapolation using the [I]/Ki ratio was applied to predict the risk of DDI in vivo. 3. All compounds tested inhibited the enzymatic activity of CYPs, mostly through a mixed type of inhibition, with CYP1A2, 2B6, 2C19 and 3A4 being affected. A high risk of interaction in vivo was predicted if artemisinin is coadministrated with CYP1A2 or 2C19 substrates. 4. With respect to CYP1A2 inhibition in vivo by artemisinin compounds, our findings are in line with previously published data. However, reported risks of interaction may be overpredicted and should be interpreted with caution.
- Ericsson, Therese,Sundell, Jesper,Torkelsson, Angelica,Hoffmann, Kurt-Jürgen,Ashton, Michael
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p. 615 - 626
(2014/06/23)
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- Preparation of human drug metabolites using fungal peroxygenases
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The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic organic chemists. Here we report that aromatic peroxygenases (APOs; EC 1.11.2.1) secreted by the agaric fungi Agrocybe aegerita and Coprinellus radians catalyzed the H2O2-dependent selective monooxygenation of diverse drugs, including acetanilide, dextrorphan, ibuprofen, naproxen, phenacetin, sildenafil and tolbutamide. Reactions included the hydroxylation of aromatic rings and aliphatic side chains, as well as O- and N-dealkylations and exhibited different regioselectivities depending on the particular APO used. At best, desired HDMs were obtained in yields greater than 80% and with isomeric purities up to 99%. Oxidations of tolbutamide, acetanilide and carbamazepine in the presence of H218O2 resulted in almost complete incorporation of 18O into the corresponding products, thus establishing that these reactions are peroxygenations. The deethylation of phenacetin-d1 showed an observed intramolecular deuterium isotope effect [(kH/kD) obs] of 3.1 ± 0.2, which is consistent with the existence of a cytochrome P450-like intermediate in the reaction cycle of APOs. Our results indicate that fungal peroxygenases may be useful biocatalytic tools to prepare pharmacologically relevant drug metabolites.
- Poraj-Kobielska, Marzena,Kinne, Matthias,Ullrich, Rene,Scheibner, Katrin,Kayser, Gernot,Hammel, Kenneth E.,Hofrichter, Martin
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experimental part
p. 789 - 796
(2012/07/14)
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- Inhibition of human drug metabolizing cytochrome P450 by buprenorphine
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The effects of buprenorphine, a powerful mixed agonist/antagonist analgesic, on several cytochrome P450 (CYP) isoform specific reactions in human liver microsomes were investigated to predict drug interaction of buprenorphine in vivo from in vitro data. The following eight CYP-catalytic reactions were used in this study: CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4-hydroxylation, CYP2D6-mediated bufuralol 1′-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6β-hydroxylation. Buprenorphine strongly inhibited the CYP3A4- and CYP2D6-catalyzed reactions with Ki values of 14.7 μM and 21.4 μM, respectively. The analgesic also weakly inhibited specific reactions catalyzed by CYP1A1/2 (Ki=132 μM), CYP2B6 (Ki=133 μM), CYP2C19 (Ki=146 μM), CYP2C8/9 (IC50>300 μM), and CYP2E1 (IC 50>300 μM), but not CYP2A6 mediated pathway. In consideration of the Ki values obtained in this study and the therapeutic concentration of buprenorphine in human plasma, buprenorphine would not be predicted to cause clinically significant interactions with other CYP-metabolized drugs.
- Umehara, Ken,Shimokawa, Yoshihiko,Miyamoto, Gohachiro
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p. 682 - 685
(2007/10/03)
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