Simple Search Results

Query String: KETOCONAZOLE

US9180183, Ketoconazole US9394290, KTZ (ketoconazole) US9271963, Ketoconazole US11739089, Compound Ketoconazole US9963439, Ketoconazole BDBM151585 US8987315, Ketoconazole
US9150527, Ketoconazole BDBM60666
Nizoral BDBM31768 Ketoconazole Panfungol CHEMBL295698
24F2-1,25(OH)D3 KTZ Ketoconazole BDBM8610 US9144538, Ketoconozole 1-[4-(4-{[(2R,4S)-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)piperazin-1-yl]ethan-1-one Ketoconazole (k) US9138393, Ketoconozole CHEMBL75
(2R,4R)-ketoconazole 1-acetyl-4-(4-{[(2R,4R)-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)piperazine CHEMBL328863 BDBM50004441
(2S,4S)-ketoconazole CIS-1-ACETYL-4-(4-((2-(2,4-DICHLOROPHENYL)-2-(1H-IMIDAZOL-1-YLMETHYL)-1,3-DIOXOLAN-4-YL)METHOXY)PHENYL)PIPERAZINE BDBM50004442 CHEMBL319160 1-acetyl-4-(4-{[(2S,4S)-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)piperazine

Rotstein, DM; Kertesz, DJ; Walker, KA; Swinney, DC Stereoisomers of ketoconazole: preparation and biological activity. J Med Chem 35: 2818-25 (1992)
Blass, BE; Iyer, P; Abou-Gharbia, M; Childers, WE; Gordon, JC; Ramanjulu, M; Morton, G; Arumugam, P; Boruwa, J; Ellingboe, J; Mitra, S; Nimmareddy, RR; Paliwal, S; Rajasekhar, J; Shivakumar, S; Srivastava, P; Tangirala, RS; Venkataramanaiah, K; Yanamandra, M Design, synthesis, and evaluation of (2S,4R)-Ketoconazole sulfonamide analogs as potential treatments for Metabolic Syndrome. Bioorg Med Chem Lett 26: 5825-5829 (2016)
Wacher, VJ; Silverman, JA; Wong, S; Tran-Tau, P; Chan, AO; Chai, A; Yu, XQ; O'Mahony, D; Ramtoola, Z Sirolimus oral absorption in rats is increased by ketoconazole but is not affected by D-alpha-tocopheryl poly(ethylene glycol 1000) succinate. J Pharmacol Exp Ther 303: 308-13 (2002)
Das, BC; Madhukumar, AV; Anguiano, J; Kim, S; Sinz, M; Zvyaga, TA; Power, EC; Ganellin, CR; Mani, S Synthesis of novel ketoconazole derivatives as inhibitors of the human Pregnane X Receptor (PXR; NR1I2; also termed SXR, PAR). Bioorg Med Chem Lett 18: 3974-7 (2008)

ChEMBL_1667565 (CHEMBL4017361) Inhibition of human CYP3A4 using ketoconazole as substrate
ChEMBL_2171309 (CHEMBL5056443) Inhibition of CYP3A4 (unknown origin) using ketoconazole as substrate
ChEMBL_2152998 (CHEMBL5037545) Inhibition of CYP3A4 in human liver microsomes using ketoconazole as substrate incubated for 5 mins in presence of NADPH by LC-MS/MS analysis
ChEMBL_2322480 Inhibition of CYP3A4 in human liver microsomes using ketoconazole as substrate preincubated for 5 mins followed by NADPH addition and measured after 120 mins by LC-MS/MS analysis
ChEMBL_1910067 (CHEMBL4412513) Inhibition of CYP3A4 in liver microsomes (unknown origin) using ketoconazole as substrate preincubated for 5 mins followed by NADPH addition and measured after 15 mins by LC-MS/MS analysis
CYP3A4 Enzyme Inhibition Assay The inhibition of recombinant human CYP3A4 was measured as the ability to perform a dealkylation of 7-benzyloxy-4-trifluoromethylcoumarin (BFC). Before running the assay, it was verified that the test compound fluorescence was not the same wavelength as the BFC metabolite. Ketoconazole was used as a positive control in the assay. The fluorescence was measured using a LJL Analyst (Molecular Devices Corporation, Sunnyvale, CA).
CYP3A4 Enzyme Inhibition Assay The inhibition of recombinant human CYP3A4 was measured as the ability to perform a debenzylation of 7-benzyloxy-4-trifluoromethylcoumarin (BFC). Before running the assay, it was verified that the test compound fluorescence was not the same wavelength as the BFC metabolite. Ketoconazole was used as a positive control in the assay. The fluorescence was measured using a SpectraMax Gemini XS (Molecular Devices Corporation, Sunnyvale, CA).
Inhibition Assay Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration=0.26%) are incubated with human liver microsomes (0.1 mg/mL) and NADPH (1 mM) in the presence of the probe substrate midazolam (2.5 μM) for 5 min at 37� C. The selective CYP3A4 inhibitor, ketoconazole, is screened alongside the test compounds as a positive control.
Inhibition Assay Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration=0.275%) are incubated with human liver microsomes (0.5 mg/mL) and NADPH (1 mM) in the presence of the probe substrate testosterone (50 μM) for 5 min at 37� C. The selective CYP3A4 inhibitor, ketoconazole, is screened alongside the test compounds as a positive control.
Inhibition of Cytochrome P450 Oxidase Human liver microsomes containing cytochrome P450 (0.253 mg/mL protein) were incubated with test compounds (0.05-50 μM), CYPs substrates (10 μM paracetamol, 5 μM diclofenac, 30 μM mephenytoin, 5 μM dextromethorphan hydrobromide and 2 μM midazolam) and 1.0 mM NADP at 37� C. for 10 min. Naphthoflavone, sulfaphenazole, N-3-benzylnirvanol, quinidine and ketoconazole were used as reference inhibitors.
null A commercially available P450-GLO Assay kit (Promega Corporation, Madison Wis.) is used to screen various compounds for CYP3A4A inhibition activity. CYP3A4A is thought to be one of the primary CYP isoforms responsible for retinoic acid metabolism in the skin. Three benchmark agents, liarozole, climbazole, and ketoconazole, were assessed for CYP3A4 inhibition to confirm that the inhibition activity (the IC50 for CYP3A4 inhibition) measured by the assay corresponds to the activity reported by the published literature. The results show that the substituted azole compounds having the specific structure set forth herein are CYP inhibitors, and thus function as RAMBAs.
CYP 2C9 inhibition The five isoform-selective probe substrate (in a cocktail manner) was used as a measure of activity for the individual cytochrome P450 (CYPs) in a pool of human liver microsomes, i.e., phenacetin for CYP1A2, diclofenac for CYP2C9, S-Mephenyloin for CYP2C19, dextromethorphan for CYP2D6, midazolam for CYP3A. Test compounds, at 7 concentration levels including zero, were incubated in human liver microsomes (HLM) together with the 5 probe substrate (in a cocktail manner). IC50 was determined by monitoring the reduction of the CYP activity as a function of test compound concentration and quantified by product formation using LC-MS/MS. Ketoconazole for CYP3A was included as quality control. All incubations were performed in singlet.
Inhibition of Cytochrome P450 Oxidase The human liver microsomes (0.253 mg/mL protein) containing cytochrome P450, test compounds (0.05-50 μM), CYPs substrates (10 μM p-acetaminophen, 5 μM diclofenac, 30 μM mephenytoin, 5 μM dextromethorphan hydrobromide, 2 μM midazolam), 1.0 mM NADP were incubated at 37� C. for 10 minutes. Naflavone, sulfafenpyrazole, N-3-benzylnivan, quinidine, and ketoconazole were used as reference inhibitors. The results are shown in Table 3. The IC50 of the test compounds are all greater than 50 μM.
Inhibition Assay In the design of clinically safe and effective metalloenzyme inhibitors, use of the most appropriate metal-binding group for the particular target and clinical indication is critical. If a weakly binding metal-binding group is utilized, potency may be suboptimal. On the other hand, if a very tightly binding metal-binding group is utilized, selectivity for the target enzyme versus related metalloenzymes may be suboptimal. The lack of optimal selectivity can be a cause for clinical toxicity due to unintended inhibition of these off-target metalloenzymes. One example of such clinical toxicity is the unintended inhibition of human drug metabolizing enzymes such as CYP2C9, CYP2C19 and CYP3A4 by the currently-available prostate anticancer agent ketoconazole. It is believed that this off-target inhibition is caused primarily by the indiscriminate binding of the currently utilized 1-imidazole to iron in the active site of CYP2C9, CYP2C19 and CYP3A4.
Inhibitory Assay The cytochrome P450 inhibitory potentials of compounds for human recombinant CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A were determined using Vivid CYP450 blue screening kits (Invitrogen, USA). All of the procedures were performed according the instruction provided by the manufacturer. Briefly, serials of diluted compounds were incubated the vivid CYP450 reaction systems including CYP450 BACULOSOMES with different recombinant human CYP450 isozymes and the appropriate vivid CYP450 substrates, and rabbit NADPH-P450 reductase, and the regeneration system. Ketoconazole was used as reference CYP450 inhibitor. After 30 minutes incubation at room temperature, the fluorescence was measured with an excitation at 400 nm and an emission at 460 nm using Envision 2104 multi-label Reader (Perkin Elmer, USA).
Enzyme Activity Assay Test Example 4: 100 mM PBS buffer was formulated, which was then used to formulate 2.5 mg/ml microsome solution and 5 mM NADPH solution. The 5� concentration of the compound working solution was diluted with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM). The 5� concentration of ketoconazole working solution was diluted with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM). Dextromethorphan working solution was diluted with PBS to a concentration of 50 μM.20 μl of 2.5 mg/ml microsome solution, 20 μl of 50 μM testosterone working solution, 20 μl of MgCl2 solution and 20 μl of the compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM, different reaction systems for each concentration) were taken respectively and mixed well. For the positive control group, the compound was replaced with the same concentration of ketoconazole. The mixture together with 5 mM NADPH solution were pre-incubated at 37� C. for 5 minutes. After 5 minutes, 20 μl of NADPH were added to each well, the reaction was started and incubated for 30 minutes. All the incubated samples were present in duplicate. After 30 minutes, 250 μl of acetonitrile containing internal standard were added to all samples, mixed well, shaken at 800 rpm for 10 minutes, and then centrifuged at 3700 rpm for 10 minutes. 80 μl of the supernatant were taken and analyzed by LC-MS/MS.
CYP450 Inhibition Assay The ability of the R and S enantiomers of (4-fluorophenyl)(4-((5-methyl-1H-pyrazol-3-yl)amino)quinazolin-2-yl)methanol to inhibit the common drug metabolizing isoforms of cytochrome P450 (CYP) was evaluated against the following isoforms: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. The compounds were incubated in duplicate with eight test compound concentrations (final DMSO concentration of 0.20%) with human liver microsomes (0.25 or 0.50 mg/mL) and NADPH (1 mM) in the presence of CYP isoform specific probe substrates (phenacetin, bupropion, taxol, diclofenac, mephenyloin, dextromethorphan, testosterone) at the Km for 10-20 minutes at 37° C. Selective CYP isoform inhibitors (furafulline, ticlopidine, quercetin, sulfaphenazole, ticlopidine, quinidine, ketoconazole) were screened alongside the test compounds as positive controls.
Human Cytochrome P450 Inhibition Assay CYP450 enzymes (final protein 75 pmol/mL for CYP1A2; 12.5 pmol/mL for CYP2C19; and 25 pmol/mL for CYP2C9, 2D6, and 3A4), 0.1 M phosphate buffer pH 7.4, probe and test compound (final concentration 50, 15.8, 5, 1.58, 0.5, and 0.158 μM; diluted from 10 mM stock solution to give a final DMSO concentration of 1%) were pre-incubated at 37� C. for 5 minutes. The reaction was initiated by the addition of 20 μL of 10 mM NADPH in phosphate buffer. The final incubation volume was 200 μL. The following control inhibitors were used for each CYP450 inhibition assay: CYP1A2: α-naphthoflavone; CYP2C9: sulfaphenazole; CYP2C19: tranylcypromine; CYP2D6: quinidine; CYP3A4: ketoconazole.
Inhibition Assay CYP17 activity was assayed according to the following procedure. Solutions of each test compound and isozyme inhibitor (ketoconazole) were separately prepared at concentrations of 2700, 540, 90, 18, 3, 0.6 and 0.1 uM by serial dilution with DMSO:ACN (50:50 v/v). The individual test compound and isozyme inhibitor solutions were then diluted 20-fold with deionized water (50:950 v/v) to concentrations of 135, 27, 4.5, 0.9, 0.15, 0.03 and 0.005 uM. The percent of organic solvent attributable to the test compound or inhibitor mixture in the final reaction mixture is 1%. Pooled rat testicular microsome suspension (20 mg/mL) was diluted with phosphate buffer to obtain a 1.25 mg/mL suspension. A solution of NADPH was prepared in phosphate buffer at a concentration of 2.5x. A stock solution of the substrate was prepared in DMSO:MeCN (50:50 v/v), mixed, and diluted in phosphate buffer to obtain a single solution containing the substrate at 5 uM.
Inhibition Assay Mixtures of isozyme inhibitors (sulfaphenazole, tranylcypromine, and ketoconazole as specific inhibitors of isozymes 2C9, 2C19, and 3A4, respectively) were prepared containing each inhibitor at concentrations of 6000, 2000, 600, 200, 60, 20, 6, and 2 uM by serial dilution with DMSO:ACN (50:50 v/v). The mixed inhibitor solutions were then diluted 20-fold with DMSO:MeCN:deionized water (5:5:180 v/v/v) to concentrations of 300, 100, 30, 10, 3, 1, 0.3, and 0.1 uM. The percent of organic solvent attributable to the test compound or inhibitor mixture in the final reaction mixture was 2% v/v. Pooled human liver microsome suspension (20 mg/mL) was diluted with phosphate buffer to obtain a 5 mg/mL suspension. A solution of NADPH was prepared in phosphate buffer at a concentration of 5 mM. Separate stock solutions of each substrate were prepared in DMSO:MeCN (50:50 v/v), mixed, and diluted in phosphate buffer to obtain a single solution.
Enzymatic Activity of CYP2C9, CYP2D6, and CYP3A4 I. Experimental materials and instruments1. Human liver microsome (Corning 452117)2. NADPH (Solarbio 705Y021)3. Positive substrates diclofenac (Sigma SLBV3438), dextromethorphan (TRC 3-EDO-175-1), and midazolam (Cerilliant FE01161704)4. Positive inhibitors sulfaphenazole (D. Ehrenstorfer GmbH 109012), quinidine (TCI WEODL-RE), and ketoconazole (Sigma 100M1091V)5. AB Sciex Triple Quad 5500 liquid chromatography-mass spectrometry systemII. Procedures1. Preparation of 100 mM phosphate-buffered saline (PBS): 7.098 g Na2HPO4 was weighed. 500 mL pure water was added. The mixture was dissolved by sonication to give solution A. 3.400 g KH2PO4 was weighed. 250 mL pure water was added. The mixture was dissolved by sonication to give solution B. The solution A was placed in a stirrer, and the solution B was slowly added until the pH reached 7.4, so that the 100 mM PBS buffer was prepared.2. A 10 mM NADPH solution was prepared with a 100 mM PBS buffer. A 10 mM stock solution of the compound of the present application was diluted with DMSO to give a compound working solution at a concentration of 200�(6000, 2000, 600, 200, 60, 20, and 0 μM). The positive inhibitor stock solution was diluted with DMSO to give a positive inhibitor working solution at a concentration of 200�(sulfaphenazole, 1000, 300, 100, 30, 10, 3, and 0 μM; quinidine/ketoconazole, 100, 30, 10, 3, 1, 0.3, and 0 μM). Substrate working solutions (120 μM diclofenac, 400 μM dextromethorphan, and 200 μM midazolam) at a concentration of 200� were prepared with water, acetonitrile, or acetonitrile/methanol.3. 2 μL of 20 mg/mL liver microsome solution, 1 μL of substrate working solution, 1 μL of compound working solution, and 176 μL of PBS buffer were taken, mixed well, and placed in a 37� C. water bath for pre-incubation for 15 min. 1 μL of sulfaphenazole, quinidine, or ketoconazole working solution was added to the positive control group instead of the compound working solution. At the same time, 10 mM NADPH solution was placed together in the 37� C. water bath for pre-incubation for 15 min. After 15 min, 20 μL of NADPH was added to each well to initiate the reaction. The mixture was incubated at 37� C. for 5 min (CYP2C9), 20 min (CYP2D6), or 5 min (CYP3A4). All incubated samples were in duplicate. After incubation for the corresponding period of time, 400 μL of icy methanol containing internal standard was added to all samples to stop the reaction. The mixture was vortexed, mixed well, and centrifuged for 40 min at 4� C. at 3220 g. 100 μL of the supernatant was transferred to a feeding plate after the centrifugation was completed, and 100 μL ultrapure water was added. The mixture was well mixed for LC-MS/MS analysis.
Inhibition Assay Solutions of each test compound were separately prepared at concentrations of 20000, 6000, 2000, 600, 200, and 60 μM by serial dilution with DMSO:MeCN (50:50 v/v). The individual test compound solutions were then diluted 20-fold with DMSO:MeCN:deionized water (5:5:180 v/v/v) to concentrations of 1000, 300, 100, 30, 10, and 3 μM. Mixtures of isozyme inhibitors (sulfaphenazole, tranylcypromine, and ketoconazole as specific inhibitors of isozymes 2C9, 2C19, and 3A4, respectively) were prepared containing each inhibitor at concentrations of 6000, 2000, 600, 200, 60, 20, 6, and 2 μM by serial dilution with DMSO:ACN (50:50 v/v). The mixed inhibitor solutions were then diluted 20-fold with DMSO:MeCN:deionized water (5:5:180 v/v/v) to concentrations of 300, 100, 30, 10, 3, 1, 0.3, and 0.1 μM. The percent of organic solvent attributable to the test compound or inhibitor mixture in the final reaction mixture was 2% v/v.
Inhibition Assay Solutions of each test compound were separately prepared at concentrations of 20000, 6000, 2000, 600, 200, and 60 uM by serial dilution with DMSO:MeCN (50:50 v/v). The individual test compound solutions were then diluted 20-fold with DMSO:MeCN:deionized water (5:5:180 v/v/v) to concentrations of 1000, 300, 100, 30, 10, and 3 uM. Mixtures of isozyme inhibitors (sulfaphenazole, tranylcypromine, and ketoconazole as specific inhibitors of isozymes 2C9, 2C19, and 3A4, respectively) were prepared containing each inhibitor at concentrations of 6000, 2000, 600, 200, 60, 20, 6, and 2 uM by serial dilution with DMSO:ACN (50:50 v/v). The mixed inhibitor solutions were then diluted 20-fold with DMSO:MeCN:deionized water (5:5:180 v/v/v) to concentrations of 300, 100, 30, 10, 3, 1, 0.3, and 0.1 uM. The percent of organic solvent attributable to the test compound or inhibitor mixture in the final reaction mixture was 2% v/v.Pooled human liver microsome suspension (20 mg/mL) was determined.
Inhibition Assay Solutions of each test compound were separately prepared at concentrations of 20000, 6000, 2000, 600, 200, and 60 uM by serial dilution with DMSO:MeCN (50:50 v/v). The individual test compound solutions were then diluted 20-fold with DMSO:MeCN:deionized water (5:5:180 v/v/v) to concentrations of 1000, 300, 100, 30, 10, and 3 M. Mixtures of isozyme inhibitors (sulfaphenazole, tranylcypromine, and ketoconazole as specific inhibitors of isozymes 2C9, 2C19, and 3A4, respectively) were prepared containing each inhibitor at concentrations of 6000, 2000, 600, 200, 60, 20, 6, and 2 uM by serial dilution with DMSO:CH3CN (50:50 v/v). The mixed inhibitor solutions were then diluted 20-fold with DMSO: CH3CN:deionized water (5:5:180 v/v/v) to concentrations of 300, 100, 30, 10, 3, 1, 0.3, and 0.1 uM. The percent of organic solvent attributable to the test compound or inhibitor mixture in the final reaction mixture was 2% v/v. Pooled human liver microsome suspension (20 mg/mL) was diluted with phosphate buffer to obtain a 5 mg/mL suspension.
Cell-Based Inhibitor Assay CYP26A1 or CYP26B1 stably transfected HeLa cells were maintained in Minimum Essential Medium (MEM) containing 10% fetal bovine serum (FBS) and 100 μg/mL hygromycin. Exponentially growing cells were harvested by incubation in trypsin. Cells were then collected and re-plated in 48-well culture plates at 5�105 cells in 0.2 mL of culture medium containing 0.05 μCi [3H]-RA in the presence or absence of increasing concentrations of a test compound. The test compounds were diluted in dimethyl sulfoxide (DMSO) and then added in duplicate to wells at 0.01, 0.1, 1, 10 and 100 μM final concentration. As a positive control for inhibition of RA metabolism, cells were also incubated with ketoconazole at the same concentrations as above. Cells were incubated for 3 hours at 37� C. Retinoids were then extracted using the procedure of Bligh, et al., (1959), Canadian Journal of Biochemistry 37, 911-917, which was modified by using dichloromethane instead of chloroform. Each sample's water soluble radioactivity level was quantified using a β-scintillation counter (using Ecolume scintillation fluid, available from MP Biomedicals of Solon, Ohio, USA). IC50 values represented the concentration of inhibitor required to inhibit all trans-RA metabolism by 50%, and were derived from log transformed data.
Enzymatic Activity Assay I. Test Materials and Instruments1. Human liver microsomes (Corning 452117)2. NADPH (Solarbio 705Y021)3. Positive substrates diclofenac (Sigma SLBV3438), dextromethorphan (TRC 3-EDO-175-1) and midazolam (Cerilliant FE01161704)4. Positive inhibitors sulfaphenazole (D. Ehrenstorfer GmbH 109012), quinidine (TCI WEODL-RE) and ketoconazole (Sigma 100M1091V)5. AB Sciex Triple Quad 5500 liquid chromatography-mass spectrometryII. Test Steps: 1. Preparation of 100 mM phosphate buffered saline (PBS): 7.098 g of Na2HPO4 was weighed, 500 mL of pure water was added and subjected to ultrasonic dissolution to obtain solution A. 3.400 g of KH2PO4 was weighed, 250 mL of pure water was added and subjected to ultrasonic dissolution to obtain solution B. Solution A was placed on a stirrer and solution B was slowly added until pH reaches 7.4 to prepare 100 mM PBS buffer. 2. Preparation of 10 mM NADPH solution with 100 mM PBS buffer. 10 mM stock solution of compounds of the invention was diluted with DMSO to obtain a 200� concentration of compound working solution (6000, 2000, 600, 200, 60, 20, 0 �M). The stock solution of positive inhibitors was diluted with DMSO to obtain a 200� concentration of positive inhibitor working solution (sulfaphenazole, 1000, 300, 100, 30, 10, 3, 0 �M; quinidine/ketoconazole, 100, 30, 10, 3, 1, 0.3, 0 �M). A 200 � concentration of substrate working solution (120 �M of diclofenac, 400 �M of dextromethorphan, and 200 �M of midazolam) was prepared in water, acetonitrile, or acetonitrile/methanol. 3. 2 �l of 20 mg/ml liver microsome solution, 1 �l of substrate working solution, 1 �l of compound working solution and 176 �l of PBS buffer were taken, mixed uniformly, and pre-incubated in a 37� C. water bath for 15 minutes. To the positive control group was added 1 �l of diclofenac, dextromethorphan or midazolam working solution to replace the compound working solution. Simultaneously, 10 mM of NADPH solution was pre-incubated together in a 37� C. water bath for 15 minutes. After 15 minutes, 20 �l of NADPH was added to each well to initiate the reaction, and the reaction was incubated at 37� C. for 5 minutes (CYP2C9) or 20 minutes (CYP2D6). Double samples were set for all incubation samples. After incubation for corresponding time, the reaction was terminated by adding 400 ul of ice methanol containing an internal standard to all samples. The mixture was mixed uniformly by vortex and centrifuged at 3220 g at 4� C. for 40 minutes. After centrifugation, 100 �L of the supernatant was transferred to a loading plate, and 100 �L of ultrapure water was added and mixed uniformly for LC-MS/MS analysis.
CYP3A4 Inhibitory Activity Assay Test compounds, DMSO (negative control), and ketoconazole (positive control) were diluted to 4� final concentrations in water. The standard final Compound concentrations were 37, 111, 333, 1000, and 3000 nM. 12.5 μL of the Compound dilutions were transferred to a white 96-well plate. 1450 μL (enough for a whole plate) of 4� assay buffer (400 mM potassium phosphate buffer (10 mL 1M potassium phosphate buffer: 8.02 mL 1M K2HPO4 +1.98 mL 1M KH2PO4 (1.4 g K2HPO4 +0.27 g KH2PO4 in 10 mL H2O), 32 μM Luciferin-IPA (Promega V9002)) 580 μl of 1 M K3PO4 buffer, 870 μL H2O, 14 μL of 3 mM Luciferin-IPA, and 18 μL of human liver microsome (Sigma M0317-1VL) was made. 12.5 μL of 4� assay buffer was added to each well. For the well of blank control, 12.5 μL of 4� assay buffer without liver microsome was added. The plate was incubated at room temperature for 15 minutes. 2.75 mL NADPH buffer was made as follows: 2.42 mL H2O, 275 μL solution A and 55 μL solution B (NADPH regeneration system, Promega V9510). 25 μL of the buffer was added to each well. The plates were incubated at 37� C. for 11 minutes. 50 μL of luciferin detection reagent (Promega V9002) was added and the plates were incubated at room temperature for 5 minutes. The plate was read with a luminometer.
Inhibition Assay The potential of the test compound to act as a competitive inhibitor of CYP3A4 was evaluated in in vitro assays, using human liver microsomes and the reference substrate midazolam. The test compound was solved in acetonitrile. Human liver microsomal preparation (pool of HLM) was applied for the assay. A stock solution of the test compound was added to phosphate buffer containing EDTA, NADP, glucose 6-phosphate, and glucose 6-phosphate dehydrogenase. This mixture was sequentially diluted on a Genesis Workstation (Tecan, Crailsheim, FRG). After pre-warming, reaction was initiated by addition of a mixture of probe substrate (midazolam). Finally, the incubation mixtures contained human liver microsomes at protein concentration of 60 ug/mL, NADPH-regenerating system (1 mM NADP, 5.0 mM glucose 6-phosphate, glucose 6-phosphate dehydrogenase (1.5 U/mL), 1.0 mM EDTA, the test compound at 6 different concentrations, 2.5 uM midazolam as probe substrate, and phosphate buffer (50 mM, pH 7.4) in a total volume of 200 uL. Incubations were performed on a Genesis Workstation (Tecan, Crailsheim, FRG) in 96-well plates (Microtiter plate, 96-well plate) at 37° C. Stock solution of probe substrate was prepared in water (midazolam 10 mM). Ketoconazole was used as positive control of a direct-acting inhibitor. The reference samples (substrate, but no inhibitor) were incubated in parallel in sextuple and contained the same amount of solvent as the test incubations. Reactions were stopped by addition of 100 uL acetonitrile containing the internal standard. Precipitated proteins were removed by centrifugation of the well plate, supernatants were analyzed by LC-MS/MS.
Luciferase-Based P450-Glo Assay Five CYP isoforms (0.5 pmol) were tested, namely 1A2, 2C9, 2C19, 2D6 and 3A4 (each isoform was assayed in a separate assay plate). Each assay plate contained several compounds at 2 concentrations (10 uM and 1 uM), with 2 replicates at each concentration or a small number of compounds per plate in dose response by duplicate (50, 16.5, 5.4, 1.8, 0.6, 0.2, 0.066, 0.022, 0.007 uM). In addition, each assay plate contained 8 different concentrations of an isoform-specific inhibitor (Furafylline, Sulfaphenazole, N-3-benzylnirvanol, Quinidine and Ketoconazole as inhibitors of CYP 1A2, 2C9, 2C19, 2D6 and 3A4, respectively), with two replicates at each concentration. The test compounds and the reference inhibitors were tested at a final DMSO concentration of 0.5%. The assay plate included also 8 replicates a vehicle control containing 0.5% DMSO/H2O. The membranes containing the CYPs, test compound and the probe substrate were pre-incubated 10 min at 37°C. in the absence of NADPH, NADPH was then added following incubation for 60 minutes at 37°C., the reaction was terminated by the addition of Luciferin detection reagent. After 20 min incubation at 37°C., the assay plate was read in the Envision 2104 Multilable reader. Values were normalized against the control activity included for each CYP. These values were plotted against the inhibitor concentration and were fitted to a sigmoid dose-response curve by using the model sigmoidal Four-Parameter Logistc inplement for Activity base software.
CYP Inhibition Assay Human liver microsomes (pooled, >30 male and female donors) were incubated with individual CYP isoform-selective standard probes (phenacetin for CYP1A2, amodiquine for CYP2C8, diclofenac for CYP2C9, dextromethorphan for CYP2D6 and midazolam for CYP3A4) in the absence and presence of increasing concentrations of the test compound in order to compare the extent of formation of the respective metabolite. In addition, a set of incubation in the absence of test compound was used as a negative control. Furthermore, the inhibitory potency of standard inhibitors was included as positive controls (fluvoxamine for CYP1A2, montelukast for CYP2C8, sulfaphenazole for CYP2C9, fluoxetine for CYP2D6, ketoconazole for CYP3A4 and mibefradil for CYP3A4-preincubation). Incubation conditions (protein and probe substrate concentration, incubation time) were optimised with regard to linearity and metabolite turnover. Incubation medium consisted of 50 mM potassium phosphate buffer (pH 7.4) containing 1 mM EDTA, NADPH regenerating system (1 mM NADP, 5 mM glucose 6-phosphate, glucose 6-phosphate dehydrogenase (1.5 U/mL). Sequential dilutions and incubations were performed on a Genesis Workstation (Tecan, Crailsheim, FRG) in 96-well plates at 37� C. A final incubation volume of 200 μL was used. Reactions were stopped by addition of 100 μL acetonitrile containing the respective internal standard. Precipitated proteins were removed by centrifugation of the well plate, supernatants were combined and analyses were performed by LC-MS/MS. The LC-MS/MS quantification of the metabolites paracetamol (CYP1A2), desethylamodiaquine (CYP2C8), 4-hydroxydiclofenac (CYP2C9), dextrorphan (CYP2D6), and 1-hydroxymidazolam (CYP3A4) was performed with a PE SCIEX API 3000 LC/MS/MS system (Applied Biosystems, MDS Sciex, Concord, Ontario, Canada).
Inhibition Assay The CYP3A4 inhibition assays were conducted using Human Liver Microsomes purchased from Invitrogen and designed to screen potential inhibitors of Cytochrome P450 in physiological condition. Initially the following reagents/mixtures were prepared: (i) Assay buffer: 0.1 M Phosphate buffer pH 7.4 (ii) Cofactor: 15 mM stock was prepared in assay buffer. Final concentration in assay 1.5 mM (iii) Substrate 50 mM DMSO stock was prepared for testosterone. From this a 10 mM sub-stock was prepared in MeCN. Further, a working stock solution of 700 μM was prepared in assay buffer. Final concentration in assay 70 μM (iv) Enzyme: 20 mg/mL stock was provided by manufacturer. Final concentration in assay was 0.5 mg/mL. At the start of the experiment, various concentrations of compound (7 different concs.) or positive control (Ketoconazole at a single concentration) were prepared in assay buffer. For 100 μL of final reaction system, 2.5 μL of HLM (20 mg/ml), 50 μL of test compound/reference compound from each concentration was added. Subsequently, 10 μL of substrate (testosterone 700 μM) and 10 μL of Cofactor (NADPH; 15 mM) were added. The volume was increased to 100 μL by adding assay buffer. DMSO concentration was kept as 0.5% uniform across all the reactions. The reaction was then allowed to incubate for 45 min at 37� C. After completion of the incubation period, the reaction was terminated by addition of 200 μL of chilled MeCN containing internal standard (Dexamethasone). The samples were than centrifuged and supernatants were analyzed using LCMS/MS.
Recombinant CYP Inhibition Assay Recombinant CYP Inhibition Assay: In a drug discovery program, a rapid screening for cyctochrome P450 (CYP450) inhibitors is a part of the existing standard for avoiding the development of drugs likely to give clinical pharmacokinetic drug-drug interactions and associated toxicities. A microtiter plate-based, direct fluorometric assay for the activities of the principal human drug-metabolizing enzymes, CYP2D6 and CYP3A4 can be used and these assays are rapid and compatible with existing high-throughput assay instrumentation. Fluorometric Enzyme Inhibition Assays: Test compounds were dissolved in 100% organic solvent (CH3CN or DMSO) to make 30 mM stock solutions. Quinidine (CYP2D6 assay, Sigma Aldrich) and ketoconazole (CYP3A4 assay, Sigma Aldrich) ran as positive controls and were dissolved in 100% acetonitrile to make 1 mM stock solutions. A 100 mM potassium phosphate buffer was prepared and adjusted to pH 7.4. The 30 mM stock solution of test and control compounds (1 mM) were further diluted in phosphate buffer (100 mM, pH 7.4) to ensure the final organic solvent content was <0.2% in the reaction. In a separate falcon tube, a 2� enzyme/substrate (E/S) solution was prepared in phosphate buffer. The final concentration of CYP2D6 (Corning) and AMMC was 10 nM and 4 μM, and CYP3A4 (Corning) and BFC was 20 nM and 40 μM, respectively. In a separate falcon tube, a 2� NADPH regenerating system (NRS) was prepared in phosphate buffer. The final concentration for each component in the assay was as follows:CYP2D6 assay=0.008 mM NADPH, 3.3 mM glucose 6-phosphate, 0.4 U of glucose-6-phosphate dehydrogenase/mL CYP3A4 assay=2.45 mM NADPH, 24.7 mM glucose 6-phosphate, 1.25 U of glucose-6-phosphate dehydrogenase/mL.
Inhibition of Cytochrome P450 Enzyme Solutions of each test compound were separately prepared at concentrations of 20000, 6000, 2000, 600, 200, and 60 μM by serial dilution with DMSO:MeCN (50:50 v/v). The individual test compound solutions were then diluted 20-fold with DMSO:MeCN:deionized water (5:5:180 v/v/v) to concentrations of 1000, 300, 100, 30, 10, and 3 μM. Mixtures of isozyme inhibitors (sulfaphenazole, tranylcypromine, and ketoconazole as specific inhibitors of isozymes 2C9, 2C19, and 3A4, respectively) were prepared containing each inhibitor at concentrations of 6000, 2000, 600, 200, 60, 20, 6, and 2 μM by serial dilution with DMSO:CH3CN (50:50 v/v). The mixed inhibitor solutions were then diluted 20-fold with DMSO: CH3CN:deionized water (5:5:180 v/v/v) to concentrations of 300, 100, 30, 10, 3, 1, 0.3, and 0.1 μM. The percent of organic solvent attributable to the test compound or inhibitor mixture in the final reaction mixture was 2% v/v.Pooled human liver microsome suspension (20 mg/mL) was diluted with phosphate buffer to obtain a 5 mg/mL suspension. A solution of NADPH was prepared in phosphate buffer at a concentration of 5 mM. Separate stock solutions of each substrate were prepared in DMSO:MeCN (50:50 v/v), mixed, and diluted in phosphate buffer to obtain a single solution containing each substrate at five times its experimentally determined Km concentration. The percent of organic solvent attributable to substrate mixture in the final reaction mixture was 1% v/v.Substrate solution and microsome suspension were combined in a 1:1 volume ratio, mixed, and distributed to reaction wells of a PCR plate. Individual test compound or combined inhibitor solutions at each concentration were added to the wells and mixed by repetitive aspirate-dispense cycles. For active controls, blank phosphate buffer solution was added in place of test compound solution. Reaction mixtures were allowed to equilibrate at 37� C. for approximately two minutes before adding NADPH solution to initiate reaction, followed by pipette mixing of reaction mixture. Ten minutes after addition of NADPH, the reaction mixtures were quenched with cold acetonitrile.
CYP Inhibition and Pre-Incubation CYP Inhibition Assays Use of in vitro assays to evaluate the inhibition potential of new drug candidates towards CYP-mediated metabolism has been shown to be effective as part of a strategy to minimise the chances of drug interactions with co-administered drugs. The inhibitory potency of the test compound towards 5 human cytochrome P450 isoforms (CYP1A2, 2C8, 2C9, 2D6, and 3A4) was determined. More preferred examples of the present invention have CYP inhibition IC50≥10 μM.For CYP3A4 time dependent inhibitory potential was also tested by applying a 30 min pre-incubation time of the test compound in metabolically active incubation system. If a time-dependent inhibition of CYP3A4 is observed, this is a hint of an irreversible mechanism-based inhibition of the CYP3A4 activity by the test compound. More preferred examples of the present invention have pre-incubation CYP inhibition IC50≥20 μM.Method Description CYP Inhibition AssayHuman liver microsomes (pooled, >30 male and female donors) were incubated with individual CYP isoform-selective standard probes (phenacetin for CYP1A2, amodiquine for CYP2C8, diclofenac for CYP2C9, dextromethorphan for CYP2D6 and midazolam for CYP3A4) in the absence and presence of increasing concentrations of the test compound in order to compare the extent of formation of the respective metabolite. In addition, a set of incubation in the absence of test compound was used as a negative control. Furthermore, the inhibitory potency of standard inhibitors was included as positive controls (fluvoxamine for CYP1A2, montelukast for CYP2C8, sulfaphenazole for CYP2C9, fluoxetine for CYP2D6, ketoconazole for CYP3A4 and mibefradil for CYP3A4-preincubation). Incubation conditions (protein and probe substrate concentration, incubation time) were optimised with regard to linearity and metabolite turnover. Incubation medium consisted of 50 mM potassium phosphate buffer (pH 7.4) containing 1 mM EDTA, NADPH regenerating system (1 mM NADP, 5 mM glucose 6-phosphate, glucose 6-phosphate dehydrogenase (1.5 U/mL). Sequential dilutions and incubations were performed on a Genesis Workstation (Tecan, Crailsheim, FRG) in 96-well plates at 37� C. A final incubation volume of 200 μL was used. Reactions were stopped by addition of 100 μL acetonitrile containing the respective internal standard. Precipitated proteins were removed by centrifugation of the well plate, supernatants were combined and analyses were performed by LC-MS/MS. The LC-MS/MS quantification of the metabolites paracetamol (CYP1A2), desethylamodiaquine (CYP2C8), 4-hydroxydiclofenac (CYP2C9), dextrorphan (CYP2D6), and 1-hydroxyidazolam (CYP3A4) was performed with a PE SCIEX API 3000 LC/MS/MS system (Applied Biosystems, MDS Sciex, Concord, Ontario, Canada).