Permeability

CaCo-2 cells were grown in Transwell permeable supports, and confluency was assessed by transepithelial electrical resistance measurements (>270 Ω/cm²) and by unidirectional permeability (apical-basolateral) of the low- and high-permeability reference compounds, ¹⁴C-mannitol and ³H-propranolol. NDD-713.HCl and -825.HCl (20 µM in HBSS that contained 20 mM HEPES, pH 7.4) were transferred to the donor compartment of Transwell plates and the acceptor compartments were filled with blank HBSS. Permeability was determined over 90 min by sampling both compartments. Quantitation of compound concentrations in donor and acceptor samples was by liquid chromatography–mass spectrometry (LC-MS; lower limit of quantification, 0.5–1.5 ng/ml), and for ¹⁴C-mannitol and ³H-propranolol, by liquid scintillation counting. Apparent permeability coefficients (P_app) were calculated as follows: P_app (cm/s) = dQ/dT × 1/(C₀ × A), where dQ/dt = apparent steady-state transport rate (μmol/s); C₀ = initial concentration in the chamber (μmol/cm³); and A = surface area of CaCo-2 monolayer (0.3cm²). Efflux ratios were calculated as the ratio of mean P_app values in B→A and A→B directions. Studies were carried out by CDCO.