|Membrane Permeability and P-gp Assessment||MDCK-MDR1 cells cultured on transwell plates
Two concentrations of test article will be treated with and without the P-gp inhibitor.
Test article concentration in donor and receiver compartments will be measured by LCMS. Apparent permeability (Papp) and P-gp efflux ratio will be calculated.
|PPB (Plasma Protein Binding)||Plasma protein binding will be determined using an equilibrium dialysis (RED) method.
Plasma from four species (rat, dog, monkey, and human) will be fortified with test article
at two concentrations and dialyzed against buffer.
Concentration of test article in buffer will be determined by LCMS and protein binding
(bound and unbound) will be calculated.
|Metabolic Stability – Microsomes||Test article (1 µM) will be incubated with rat, dog, monkey, and human liver microsomes (0.5 mg/mL + 1 mM NADPH).
Remaining parent will be determined at 0, 15, 30, and 60 min following incubation. Percent drug remaining will be determined using LCMS and intrinsic clearance and half-life are calculated.
|Metabolic Stability – Hepatocytes||Test article (1 µM) will be incubated with rat, dog, monkey, and human hepatocytes (0.5 million cells/mL)
Remaining parent will be determined at 0, 1, 2 and 3 hours following incubation.
Percent drug remaining will be determined using LCMS and intrinsic clearance and half-life are calculated.
|Cross-species Metabolite Characterization||Metabolic profile of test article will be determined in hepatocytes from rat, dog, monkey, and human at 10 µM for 120 minutes following incubation at 37ºC.
Qualitative LCMS methods using Qtrap will be used for metabolite characterization.
|CYP Reaction Phenotyping||Determine potential of selected CYP enzymes (3A4, 2D6, 1A2, 2C9, and 2C19) involved
in the metabolism of the test article (one concentration) using human liver microsomes + specific CYP-isoform inhibitors with t=0, 30 and 60 min. Metabolism will be assessed by measurement of drug consumption. Metabolic rates (when possible) for each CYP will be compared.
|CYP Inhibition||Test article will be evaluated as a potential inhibitor of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4 using CYP-selective marker substrates.
Seven concentrations of the test article will be used and inhibitory effects compared to appropriate controls.
IC50 is determined. 7 isoforms with 8 reactions (CYP3A4 with 2 substrates)
|CYP450 TDI Assays||TDI assays to determine the IC50 shifts with 30 min preincubation +/- NADPH, followed by addition of specific CYP450 isoform-selective substrates for 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4; single substrate, LC-MS/MS, in duplicate, 7-pt dose-response. IC50 shift. 7 reactions.
|CYP450 Induction in Human Hepatocytes||Using fresh or platable frozen human hepatocytes + compound treatment for 48 hrs at 3 compound concentrations (e.g. 0, 1, 10 uM); and measure the CYP3A4, 1A2 and 2B6 activity by LC-MS/MS; and mRNA levels by qRT-PCR.|
|PXR Activation||Estimate the potential for CYP3A4 induction by test article with the use of the PXR reporter gene assay.
Test article will be incubated with cells stably expressing a PXR reporter gene construct, +/- a know CYP3A4 inducer, rifampicin, at 2 testing concetrations
Potential for activation estimated by comparison to appropriate controls.
|Report||Study report for all studies will be generated and suitable for IND filing|