Determination of GNF6702 permeability in Caco-2 assay

A 96-Multiwell Insert System (BD Biosciences) was used for the Caco-2 cell culture and permeability assay as described previously⁴². Caco-2 cells were seeded onto insert wells at a density of 1.48 × 10⁵ cells per ml and allowed to grow for 19–23 days before assays. To measure both absorptive (apical to basolateral [A-B]) and secretory (basolateral to apical [B-A]) compound transport, a solution of GNF6702 at 10 μM concentration in 0.5% DMSO were added to donor wells. The plate was incubated at 37°C for 2 hours, with samples taken at the beginning and end of the incubation from both donor and acceptor wells. The concentration of GNF6702 was determined by LC-MS/MS.

Apparent drug permeability (Papp) was calculated using the following equation:

where dQ/dt is the total amount of a test compound transported to the acceptor chamber per unit of time (nmol/s), A is the surface area of the transport membrane (0.0804 cm²), Cin is the initial compound concentration in the donor chamber (10 μM), and Papp is expressed as cm/s).