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Heterotropic activation of the midazolam hydroxylase activity of CYP3A by a positive allosteric modulator of mGlu5: in vitro to in vivo translation and potential impact on clinically relevant drug-drug interactions.
Blobaum AL, Bridges TM, Byers FW, Turlington ML, Mattmann ME, Morrison RD, Mackie C, Lavreysen H, Bartolomé JM, Macdonald GJ, Steckler T, Jones CK, Niswender CM, Conn PJ, Lindsley CW, Stauffer SR, Daniels JS
(2013) Drug Metab Dispos 41: 2066-75
MeSH Terms: Allosteric Regulation, Animals, Cytochrome P-450 CYP3A, Drug Interactions, Hepatocytes, Humans, Intestinal Mucosa, Intestines, Ketoconazole, Kinetics, Liver, Male, Mice, Microsomes, Microsomes, Liver, Midazolam, Mixed Function Oxygenases, Rats, Rats, Sprague-Dawley
Show Abstract · Added February 19, 2015
Allosteric modulation of G protein-coupled receptors has gained considerable attention in the drug discovery arena because it opens avenues to achieve greater selectivity over orthosteric ligands. We recently identified a series of positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu(5)) for the treatment of schizophrenia that exhibited robust heterotropic activation of CYP3A4 enzymatic activity. The prototypical compound from this series, 5-(4-fluorobenzyl)-2-((3-fluorophenoxy)methyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine (VU0448187), was found to activate CYP3A4 to >100% of its baseline intrinsic midazolam (MDZ) hydroxylase activity in vitro; activation was CYP3A substrate specific and mGlu(5) PAM dependent. Additional studies revealed the concentration-dependence of CYP3A activation by VU0448187 in multispecies hepatic and intestinal microsomes and hepatocytes, as well as a diminished effect observed in the presence of ketoconazole. Kinetic analyses of the effect of VU0448187 on MDZ metabolism in recombinant P450 or human liver microsomes resulted in a significant increase in V(max) (minimal change in K(m)) and required the presence of cytochrome b5. The atypical kinetics translated in vivo, as rats receiving an intraperitoneal administration of VU0448187 prior to MDZ treatment demonstrated a significant increase in circulating 1- and 4-hydroxy- midazolam (1-OH-MDZ, 4-OH-MDZ) levels compared with rats administered MDZ alone. The discovery of a potent substrate-selective activator of rodent CYP3A with an in vitro to in vivo translation serves to illuminate the impact of increasing intrinsic enzymatic activity of hepatic and extrahepatic CYP3A in rodents, and presents the basis to build models capable of framing the clinical relevance of substrate-dependent heterotropic activation.
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19 MeSH Terms
High-throughput fluorescence assay of cytochrome P450 3A4.
Cheng Q, Guengerich FP
(2013) Methods Mol Biol 987: 157-62
MeSH Terms: Coumarins, Cytochrome P-450 CYP3A, Cytochrome P-450 CYP3A Inhibitors, Enzyme Assays, Humans, Inhibitory Concentration 50, Ketoconazole, Spectrometry, Fluorescence
Show Abstract · Added March 26, 2014
Microtiter plate-based fluorescence assays allow rapid measurement of the catalytic activities of cytochrome P450 oxygenases (P450s). We describe a high-throughput fluorescence assay of P450 3A4, one of the key enzymes involved in xenobiotic metabolism. The assay involves the oxidative debenzylation of 7-hydroxy-4-trifluoromethyl coumarin, producing an increase in fluorescence.
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8 MeSH Terms
Cimetidine-associated patent ductus arteriosus is mediated via a cytochrome P450 mechanism independent of H2 receptor antagonism.
Cotton RB, Shah LP, Poole SD, Ehinger NJ, Brown N, Shelton EL, Slaughter JC, Baldwin HS, Paria BC, Reese J
(2013) J Mol Cell Cardiol 59: 86-94
MeSH Terms: Cimetidine, Cytochrome P-450 Enzyme System, Ductus Arteriosus, Patent, Histamine H2 Antagonists, Humans, Immunohistochemistry, Infant, Newborn, Ketoconazole, Polymerase Chain Reaction, Randomized Controlled Trials as Topic, Ranitidine, Receptors, Histamine, Retrospective Studies
Show Abstract · Added April 9, 2015
Persistent patency of the ductus arteriosus (PDA) is a common problem in preterm infants. The antacid cimetidine is a potent antagonist of the H2 histamine receptor but it also inhibits certain cytochrome P450 enzymes (CYPs), which may affect DA patency. We examined whether cimetidine contributes to PDA and is mediated by CYP inhibition rather than H2 blockade. Analysis of a clinical trial to prevent lung injury in premature infants revealed a significant association between cimetidine treatment and PDA. Cimetidine and ranitidine, both CYP inhibitors as well as H2 blockers, caused relaxation of the term and preterm mouse DA. CYP enzymes that are inhibited by cimetidine were expressed in DA subendothelial smooth muscle. The selective CYP3A inhibitor ketoconazole induced greater DA relaxation than cimetidine, whereas famotidine and other H2 antagonists with less CYP inhibitory effects caused less dilation. Histamine receptors were developmentally regulated and localized in DA smooth muscle. However, cimetidine caused DA relaxation in histamine-deficient mice, consistent with CYP inhibition, not H2 antagonism, as the mechanism for PDA. Oxygen-induced DA constriction was inhibited by both cimetidine and famotidine. These studies show that antacids and other compounds with CYP inhibitory properties pose a significant and previously unrecognized risk for PDA in critically ill newborn infants.
Copyright © 2013 Elsevier Ltd. All rights reserved.
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13 MeSH Terms
High-throughput fluorescence assay of cytochrome P450 3A4.
Cheng Q, Sohl CD, Guengerich FP
(2009) Nat Protoc 4: 1258-61
MeSH Terms: Coumarins, Cytochrome P-450 CYP3A, Humans, Inhibitory Concentration 50, Ketoconazole, Oxidation-Reduction, Software, Spectrometry, Fluorescence
Show Abstract · Added March 26, 2014
Cytochrome P450 mono-oxygenases (P450s) are the principal enzymes involved in the oxidative metabolism of drugs and other xenobiotics. In this protocol, we describe a fluorescence-based, high-throughput assay for measuring the activity of P450 3A4, one of the key enzymes involved in drug metabolism. The assay involves the oxidative debenzylation of a substituted coumarin, yielding an increase in fluorescence on reaction. The entire procedure can be accomplished in 1 h or less.
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8 MeSH Terms
The arachidonic acid epoxygenase is a component of the signaling mechanisms responsible for VEGF-stimulated angiogenesis.
Yang S, Wei S, Pozzi A, Capdevila JH
(2009) Arch Biochem Biophys 489: 82-91
MeSH Terms: Animals, Antifungal Agents, Cell Proliferation, Cells, Cultured, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System, Cytochrome P450 Family 2, Endothelial Cells, Enzyme Activation, Ketoconazole, Lung, MAP Kinase Signaling System, Mice, Mice, Transgenic, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Neovascularization, Physiologic, Phosphorylation, Proto-Oncogene Proteins c-akt, RNA, Small Interfering, Vascular Endothelial Growth Factor A
Show Abstract · Added February 24, 2014
Cultured lung endothelial cells (LEC) respond to VEGF or arachidonic acid with increases in cell proliferation, the formation of tube-like structures, and the activation of Akt and ERK1/2 mediated growth pathways. LECs express a VEGF inducible Cyp2c44 epoxygenase and its 11,12- and 14,15-EET metabolites increase cell proliferation, tubulogenic activity, and the phosphorylation states of the ERK1/2 and Akt kinases. Ketoconazole, an epoxygenase inhibitor, blocks the cellular responses to VEGF. LECs expressing a Cyp2c44 epoxygenase small interference RNA show reductions in Cyp2c44 mRNA levels, and in their VEGF-stimulated proliferative and tubulogenic capacities; effects that are associated with decreases in VEGF-induced phosphorylation of the ERK1/2 and Akt kinases. We conclude that the Cyp2c44 arachidonic acid epoxygenase is a component of the signaling pathways associated with VEGF-stimulated angiogenesis, and suggest a role for EETs in the growth factor-induced changes in the activation states of the ERK1/2 and Akt kinase pathways.
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21 MeSH Terms
Characteristics of the heterologously expressed human lanosterol 14alpha-demethylase (other names: P45014DM, CYP51, P45051) and inhibition of the purified human and Candida albicans CYP51 with azole antifungal agents.
Lamb DC, Kelly DE, Waterman MR, Stromstedt M, Rozman D, Kelly SL
(1999) Yeast 15: 755-63
MeSH Terms: Antifungal Agents, Binding Sites, Candida albicans, Carbon Monoxide, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System, Formates, Humans, Inhibitory Concentration 50, Itraconazole, Ketoconazole, Kinetics, Ligands, Oxidoreductases, Recombinant Proteins, Spectrum Analysis, Sterol 14-Demethylase, Titrimetry
Show Abstract · Added February 12, 2015
Human and Candida albicans CYP51 were purified to homogeneity after GAL10-based heterologous expression in yeast in order to resolve the basis for the selective inhibition of the fungal enzyme over the human orthologue by the azole drugs ketoconazole and itraconazole, used in the treatment of systemic fungal infection. The purified proteins have similar spectral characteristics, both giving a maximum at 448 nm in reduced carbon monoxide difference spectra. Substrate affinity constants of 20.8 and 29.4 microM and Vmax of 0. 15 and 0.47 nmol/min/nmol were observed for C. albicans and human enzymes, respectively, in reconstituted enzymatic assays, using an intermediate of the demethylation reaction [32-3H]-3beta-hydroxylanost-7-en-32-ol as the substrate. Both enzymes gave similar type II spectra on titration with drugs, but a reduced affinity was observed for human CYP51 using the ability of carbon monoxide to displace the drug as a ligand and by calculation of IC50. However, although the results indicate higher affinity of the drugs for their target CYP51 in the major fungal pathogen C. albicans, when compared directly to CYP51 from humans, the difference was less than 10-fold. This difference is an order of magnitude lower than previously reported data based on measurements using unpurified human CYP51 enzyme preparations. Consequently, increased azole doses to combat resistant candidaemia may well inhibit endogenous human CYP51 and the potential consequences are discussed.
Copyright 1999 John Wiley & Sons, Ltd.
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18 MeSH Terms
Different contributions of cytochrome P450 2C19 and 3A4 in the oxidation of omeprazole by human liver microsomes: effects of contents of these two forms in individual human samples.
Yamazaki H, Inoue K, Shaw PM, Checovich WJ, Guengerich FP, Shimada T
(1997) J Pharmacol Exp Ther 283: 434-42
MeSH Terms: Animals, Anti-Ulcer Agents, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 CYP2C19, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System, Fluvoxamine, Humans, Hydroxylation, Ketoconazole, Microsomes, Liver, Mixed Function Oxygenases, Omeprazole, Rabbits, Recombinant Proteins, Sulfaphenazole
Show Abstract · Added May 26, 2014
Omeprazole 5-hydroxylation and sulfoxidation activities were determined in liver microsomes of different humans whose levels of individual forms of cytochrome P450 (P450 or CYP) varied. Correlation coefficients between omeprazole 5-hydroxylation activities (when determined at a substrate concentration of 10 microM) and S-mephenytoin 4'-hydroxylation and testosterone 6beta-hydroxylation activities were found to be 0.64 and 0.67, respectively, in liver microsomes of 84 human samples examined. Omeprazole sulfoxidation activities in these human samples were correlated with testosterone 6beta-hydroxylation activities (r = 0. 86). Omeprazole 5-hydroxylation by liver microsomes of a human sample that contained relatively high levels of CYP3A4 and low levels of CYP2C19 were inhibited very significantly by ketoconazole and anti-CYP3A4 antibodies, although a human sample having high in CYP2C19 and low in CYP3A4 was found to be sensitive toward fluvoxamine and anti-CYP2C9 antibodies. Sulfaphenazole (at 100 microM) did not affect the omeprazole 5-hydroxylation and sulfoxidation catalyzed by human liver microsomes. Both recombinant human CYP2C19 and CYP3A4 enzymes had activities for omeprazole 5-hydroxylation, with low Km and high Vmax values for the former enzyme and high Km and low Vmax values for the CYP3A4. These results suggest that contributions of CYP2C19 and CYP3A4 in the omeprazole 5-hydroxylation depend upon the ratio of these two P450 levels in human liver microsomes. Omeprazole 5-hydroxylation activities of different human samples were found to be related to predicted values calculated from the kinetic parameters of recombinant enzymes and the levels of liver microsomal CYP2C19 and CYP3A4 enzymes. Finally, when recombinant human CYP2C19 and CYP3A4 were mixed at levels found in different human samples, relatively similar profiles of omeprazole oxidation by the recombinant and microsomal enzyme systems were determined by analysis of high-performance liquid chromatography. These results suggest that both CYP2C19 and CYP3A4 are involved in the 5-oxidation of omeprazole (at a substrate concentration of 10 microM) in human liver microsomes and that contributions of these P450 enzymes depend on the compositions of CYP2C19 and CYP3A4 in liver.
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16 MeSH Terms
Induction of renal arachidonate cytochrome P-450 epoxygenase after uninephrectomy: counterregulation of hyperfiltration.
Takahashi K, Harris RC, Capdevila JH, Karara A, Makita K, Jacobson HR, Munger KA, Badr KF
(1993) J Am Soc Nephrol 3: 1496-500
MeSH Terms: Alkaline Phosphatase, Animals, Arachidonic Acid, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System, Glomerular Filtration Rate, Ketoconazole, Kidney, Male, Microsomes, Nephrectomy, Oxygenases, Rats, Rats, Wistar, Renal Circulation, Sodium-Potassium-Exchanging ATPase
Show Abstract · Added August 19, 2013
After unilateral nephrectomy (UNx) in the rat, cytochrome P-450 (cP-450)-linked arachidonate enzymatic activity was markedly and specifically induced in microsomal fractions from the remaining kidney. The enzymatic activity reached 200% at 1 wk and 285% at 2 wk post-UNx as compared with non-UNx controls. Mean baseline values for GFR and RPF rate in the remaining kidney 2 wk after UNx were 1.56 +/- 0.10 and 6.47 +/- 0.35 mL/min, respectively. In these rats, the administration of ketoconazole, a cP-450 inhibitor, led to 75% inhibition of renal cP-450 arachidonate metabolism and was associated with acute augmentations in both GFR and RPF to 1.82 +/- 0.18 (P < 0.05 versus baseline) and 7.54 +/- 0.37 mL/min (P < 0.05 versus baseline), respectively. Because vasoconstrictor arachidonate epoxygenase products are endogenously generated in the rat kidney, these findings suggest that the stimulation of renal cP-450-mediated oxygenation of arachidonic acid may subserve an important counterregulatory function in mitigating the renal hyperperfusion and hyperfiltration that follow reductions in renal mass.
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16 MeSH Terms
Progesterone 16 alpha-hydroxylase activity is catalyzed by human cytochrome P450 17 alpha-hydroxylase.
Swart P, Swart AC, Waterman MR, Estabrook RW, Mason JI
(1993) J Clin Endocrinol Metab 77: 98-102
MeSH Terms: 17-alpha-Hydroxyprogesterone, Adrenal Glands, Animals, Aryl Hydrocarbon Hydroxylases, Cell Line, Transformed, Chlorocebus aethiops, Cytochrome P-450 CYP2C8, Cytochrome P-450 CYP2C9, DNA, Humans, Hydroxyprogesterones, Ketoconazole, Kidney, Kinetics, Male, Microsomes, NADP, Pregnenolone, Progesterone, Steroid 16-alpha-Hydroxylase, Steroid 17-alpha-Hydroxylase, Steroid Hydroxylases, Testis, Transfection
Show Abstract · Added February 12, 2015
Progesterone and pregnenolone are metabolized to 17 alpha-hydroxysteroids by a cytochrome P450-dependent 17 alpha-hydroxylase (P450c17). The same enzyme can also catalyze the removal of the side-chain of these 17 alpha-hydroxylated steroids to yield androstenedione and dehydroepiandrosterone, respectively. We investigated the metabolism of progesterone by monkey kidney tumor (COS 1) cells transfected with a plasmid vector containing the cDNA encoding the complete amino acid sequence for human cytochrome P450c17. Transfected COS 1 cells converted progesterone to 17 alpha-hydroxyprogesterone as well as 16 alpha-hydroxyprogesterone, but no detectable androstenedione was produced. However, pregnenolone was converted to 17 alpha-hydroxypregnenolone and, ultimately, dehydroepiandrosterone. No 16 alpha-hydroxypregnenolone was produced. The kinetics of progesterone metabolism by the enzyme expressed in COS 1 cells indicated that both 17 alpha- and 16 alpha-hydroxylated products were products were produced from a common active site. Microsomes prepared from fetal adrenal and adult testis converted progesterone to 17 alpha-hydroxyprogesterone as well as 16 alpha-hydroxyprogesterone. No detectable androstenedione was produced by these preparations. Antibodies raised against porcine cytochrome P450c17 inhibited the 17 alpha- and 16 alpha-hydroxylation of progesterone to the same extent when using fetal adrenal microsomes, whereas no inhibition of 21-hydroxylation of progesterone was observed. Similar results were obtained with the imidazole antimycotic agent ketoconazole, which is a preferential cytochrome P450c17 inhibitor. From these results we conclude that human cytochrome P450c17 exhibits marked progesterone 16 alpha-hydroxylase activity in addition to its 17 alpha-hydroxylase function when expressed not only in a heterologous cell expression system but also, importantly, in human steroidogenic cells. Furthermore, the human enzyme has extremely low C-17,20-lyase activity toward progesterone, 17 alpha-hydroxyprogesterone, and 16 alpha-hydroxyprogesterone and fails to convert these to corresponding C19 steroids.
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24 MeSH Terms
Perilla ketone increases endothelial cell monolayer permeability in vitro.
Waters CM, Alexander JS, Harris TR, Haselton FR
(1993) J Appl Physiol (1985) 74: 2493-501
MeSH Terms: Actins, Animals, Aorta, Thoracic, Blotting, Western, Cattle, Cell Membrane Permeability, Cytochrome P-450 Enzyme Inhibitors, Cytoskeleton, Electrophoresis, Polyacrylamide Gel, Endothelium, Vascular, Female, Ketoconazole, Microscopy, Fluorescence, Monoterpenes, Pregnancy, Sheep, Sodium Fluoride, Staining and Labeling, Terpenes
Show Abstract · Added May 27, 2014
Perilla ketone (PK) is a potent lung toxin that causes increased microvascular permeability pulmonary edema in grazing animals. Because the mechanism of action of PK is not know, we investigated whether PK directly affects endothelial cells. Bovine aortic endothelial cells were grown to confluence on Cytodex-3 microcarrier beads and placed in a chromatographic cell column. Monolayer permeability was evaluated from the elution profiles of three optical tracers: blue dextran (2 x 10(6) mol wt), sodium fluorescein (NaF, 342 mol wt), and cyanocobalamin (B12, 1,355 mol wt). Perfusion with 1.2 mM PK increased permeability within 15 min to NaF and B12 by 51 +/- 6 and 54 +/- 11%, respectively. Permeability returned to baseline after PK removal. These in vitro results suggest that PK produces a rapid and reversible increase in endothelial permeability directly. Staining of fixed cells with rhodamine-phalloidin revealed a major disruption of actin microfilaments after PK treatment. Because previous reports suggested that PK may be activated via cytochrome P-450, we attempted to block this using the cytochrome P-450 inhibitor ketoconazole. Ketoconazole alone did not significantly affect permeability, and the combination of PK and ketoconazole resulted in permeability increases similar to those measured for PK alone. This suggests that PK may not require cytochrome P-450 to increase vascular permeability.
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19 MeSH Terms