Antipyrine

HUVEC, ST, and ST/HUVEC barrier models were prepared. After washing cells, the assay buffer was added to each insert (200 μL), and buffer containing 100 μg/mL antipyrine (Fujifilm Wako chemicals), 200 μM caffeine (Fujifilm Wako chemicals), or 200 μM glyphosate (Fujifilm Wako chemicals) was added to each well (1300 μL). After 30 min, the buffers were collected for HPLC analysis. For time course experiments, cells were exposed to 100 μg/mL antipyrine for 2, 30, 60, 120, 180 min.
The apparent permeability coefficient (Papp) was calculated as follows: Papp (cm/s) = (dQ/dt)/(A × C₀), where dQ/dt is the rate of the substrate appearance in the column insert (nmol/s), A is the surface area of the basement collagen membrane (cm²), and C₀ is the initial concentration of the substrate in the well (μM)^{19 (link)}.

Sodium nitroprusside dihydrate (SNP) and fluorescein isothiocyanate-dextran 4,000 (FD-4) were obtained from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). Carboxy-PTIO (c-PTIO) was obtained from Dojindo Laboratories (Kumamoto, Japan). Griseofulvin, antipyrine, theophylline, and sodium ferrocyanide decahydrate (SFC) were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Flurbiprofen was obtained from LKT Laboratories (St. Paul, MN, USA). Propranolol was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All other reagents were of analytical reagent grade.