| Assay Method Information | |
| | Drug-Drug Interaction (DDI) Assay |
| Description: | The assay was set up and executed using a Biomek FXp robotic liquid handling workstation (Beckman Coulter Corp., Fullerton, Calif.), integrated with a Cytomat shaking incubator set at 37° C. (Thermo Electron Corp., Bellefonte, Pa.). A batch of human liver microsomes from 50 donors, pooled and characterized by BD Gentest, (Cat #457111, lot 01220, 20 mg/mL in 250 mM sucrose) was used. Each substrate was incubated at a protein concentration of 0.1, 0.15 or 0.2 mg/mL in a total incubation volume of 0.16 mL. The incubates were prepared in 100 mM potassium phosphate buffer (pH 7.4) supplemented with 5 mM magnesium chloride and 1 mM EDTA. Quinidine was used as a positive control inhibitor for CYP2D6. Quinidine was prepared as a working solution in organic solvent (primarily methanol, with DMSO and acetonitrile as secondary solvents) and was spiked into the microsomal suspension to yield the desired concentration level. The solution was then serially diluted with additional microsomal suspension to yield eight concentration levels. Final organic content was less than 0.07%. A stock solution of the test compound was prepared at a concentration of 50 mM or higher, if possible, in an adequate organic solvent (DMSO, methanol or acetonitrile), depending on solubility limitations. The stock solution was serially diluted with methanol and subsequently spiked into the microsomal suspension to yield final incubation concentrations of 0, 0.1, 0.3, 1, 3, 10, 30 and 100 μM for Comparator Compound and 0, 0.06, 0.18, 0.6, 1.8, 6, 18 and 60 μM for 4-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethyl)-piperazine-1-carboxylic acid (4-chloro-pyridin-3-yl)-amide. The final organic content was 0.2%. Incubations were performed in triplicate for each probe substrate. The control inhibitor (quinidine) and marker substrate (dextromethorphan or bufuralol) were transferred to the incubation vessels (60 μL aliquots each). After a pre-incubation period at 37° C., the reactions were initiated by the addition of a 40 μL aliquot of NADPH regenerating system (BD Gentest). A 40 μL aliquot (diluted 6:19 with incubation buffer) provided final concentrations of 1.3 mM NADP+, 3.3 mM glucose-6-phosphate and 0.4 U/mL glucose-6-phosphate dehydrogenase. Incubation times were 12 minutes for both dextromethorphan and bufuralol. Reactions were terminated by the direct addition of acetonitrile (160 μL) to the incubation mix followed by transfer to a pooling plate containing additional acetonitrile (400 μL). The incubation reactions were pooled by equal test compound or control inhibitor concentration, transferred to a Phenomenex Strata Impact protein precipitation filter plate containing acetonitrile and internal standards (100 μL of a mixture of the following deuterated compounds ranging in concentration from 0.5 to 2.8 μM: hydroxybufuralol-d9, dextrorphan-d3). The resulting filtrate was evaporated to dryness under a nitrogen flow, then reconstituted in 250 μL mobile phase (1:1 methanol:water, containing 0.1% acetic acid). Samples and standards were analyzed on a Sciex API4000 triple quadrupole mass spectrometer. The data were acquired in Analyst 1.4.1 (Applied Biosystems/MDS Sciex). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |