Caco-2 permeability assay

Caco-2 cells (purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) at passage 30 to 33 were used in this study. Cells were routinely cultured as previously described with minor modifications [49]. Cells were seeded onto Transwell inserts in 12-well dishes at a density of 5 × 10⁵ cells/well (#3401; 0.4 µm pore size, growth area of 1.13 cm², Corning, NY, USA), and the medium was replaced with D-Hanks solution every other day except for the treatment days [50]. No sign of Caco-2 cell death was observed at OCT concentrations of 1, 10, and 50 µM as determined by the LDH-cytotoxicity assay (data not shown).

Cell monolayer integrity was evaluated through the transepithelial electrical resistance (TEER) using a Millicell ERS resistance system (Millipore, Bedford, MA, USA) at 37 °C. Each TEER value was corrected for background resistance determined on an extracellular matrix-coated cell culture insert without cells. Monolayers that exhibited a TEER value > 350 Ω·cm² on the 21st day of cell culture were used for the transport study.

Caco-2 transport study was performed as previously described [50]. Briefly, apical to basolateral (A to B) or basolateral to apical (B to A) transport assay started after gentle removal of the HBSS buffer in the donor compartment and addition of 1.4 mL (1) OCT (10 µM); (2) OCT (10 µM) plus the specific P-gp inhibitor rhodamine 123 (1 mM; Sigma-Aldrich, St. Louis, MO); or (3) OCT (10 µM) plus probenecid (1 mM). A to B assay was performed to determine the extent of OCT absorption, while B to A assay was performed to measure OCT secretion. As regard A to B assay, the apical (upper) chamber was filled with 1.6 mL test drug solution in HBSS, while the receiver basolateral (lower) compartment was filled with 0.6 mL HBSS alone. Conversely, in B to A assay the upper and lower chambers were filled with 0.6 mL HBSS and 1.6 mL test drug, respectively. A small aliquot (0.05 mL) of HBSS was collected to determine the OCT loading concentration C₀. The TEER value was determined at different incubation times to verify the integrity of the monolayer after buffer change. A total amount of 100 µL was collected from the receiver compartment at 5, 15, 30, 60, and 90 min to determine the OCT concentration by LC–MS/MS. The concentration kinetic of OCT uptake was measured at pH 6.0 over a concentration range of 1 to 50 µM at 37 °C, as described above. The apparent permeability coefficient (P_app, cm/s) of OCT across the Caco-2 monolayer was calculated using the following equation:

where ΔA_R is the amount of drug accumulated in the receiver compartment during the interval time (Δt) and Area is the surface area of the filter/membrane of the Transwell (cm²). The permeability direction rate (PDR) was calculated by the ratio of P_app in the B to A direction versus that in the A to B direction, as follows:

Microsomes from the small intestine of healthy rats and PH rats were obtained as previously described [51], and their protein concentration (0.53 ± 0.05 mg/mL and 0.52 ± 0.06 mg/mL total protein, respectively) was measured using the BCA method (Beijing Solarbio Science & Technology Co., Ltd). The reaction mixture (500 µL) containing 0.4 mg intestinal microsome protein, 100 mM potassium phosphate buffer (PPB; pH 7.4), a NADPH-generating system (3.3 mM MgCl₂, 1.3 mM β-NADP⁺, 3.3 mM glucose 6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase), and OCT (1000 ng/mL), was incubated at 37 °C for 30 min with or without ketoconazole (10 µM, Sigma-Aldrich, St. Louis, MO). CYP3A activity was measured using a fluorescent quantitative detection kit (Genmed Scientifics Inc., U.S.A.) with the NADPH regenerating system after pre-incubation at 37 °C for 0, 2, 10, 20 or 30 min. In addition, the effect of OCT pre-incubation on CYP3A inactivation was determined by pre-incubating OCT (1000 ng/mL) with microsomes, prepared in PPB, for 0, 2, 10, 20 or 30 min at 37 °C in the presence of a NADPH regenerating system. The organic phase was evaporated to dryness and the OCT content in the residue was measured by LC–MS/MS.

Microsomes prepared from healthy rats were equally divided into 2 groups: (1) OCT (Group N + OCT) and (2) OCT + ketoconazole (Group N + OCT + K). Similarly, microsomes prepared from PH rats were divided into two groups: (1) OCT (Group PH + OCT) and (2) OCT + ketoconazole (Group PH + OCT + K) (n = 6 in each group). The control group (Group OCT; n = 6) contained OCT without microsomes. Each sample was tested 3 times.

All incubations were performed at 37 °C in a water bath. Recombinant human cDNA-expressing P450 isozyme 3A4 (BD Gentest Supersomes) was carefully thawed on ice prior to each experiment. The reaction mixture (200 µL) contained 10 µL human recombinant CYP3A4, 2 µL OCT (at different concentrations; see below), 168 µl PPB (100 mM), and 20 µL NADPH (1 mM). After a 5-min pre-incubation at 37 °C, the reaction was started by the addition of NADPH and ended 30 min later by the addition of 500 µL methanol. Control samples without NADPH and samples without OCT were also included. At the end of each reaction, samples were centrifuged at 12,000g for 5 min at 4 °C to precipitate the proteins, and the supernatant was subjected to LC–MS/MS analysis to determine the OCT content. Each sample was tested 4 times. CYP3A4 activity with and without OCT incubation was assessed using a fluorescent quantitative detection kit.

To determine the optimal experimental conditions, different incubation times such as 5, 10, 15, 20, and 30 min were used together with a CYP3A4 concentration of 2.5 mg/mL with an OCT concentration of 100 mM to determine the most effective incubation time within a linear range. To determine the optimal protein concentration of recombinant CYP3A4 within a linear range, different recombinant CYP3A4 concentrations were used, such as 1.25, 2.5, 5, 10, and 20 mg/mL using an incubation time of 10 min and an OCT concentration of 100 mM. Based on the optimized CYP3A4 protein concentration at different reaction times, different OCT concentrations (20, 50, 100, 200, and 400 mM) were added to the reaction mixture and the experiment was then performed as described above.