Figure 1.
Time courses of single transport studies of piroxicam across PBMEC/C1-2, ECV304 and RBMEC layers.
Comparison between the cleared volume vs. time graphs of internal standards for the transcellular transport route (diazepam, 100 µM), the paracellular transport route (carboxyfluorescein, 5 µM) and the NSAID piroxicam (100 µM) showed clearly that piroxicam permeated between the two markers across all three BBB (A: PBMEC/C1-2; B: ECV304; C: RBMEC) in vitro models. In addition, it was proved that as tighter the model is a wider frame between the two markers diazepam and carboxyfluorescein could be provided to analyze and compare permeabilities of single drugs. (n = 3 for each time point, data are presented as means ± SD).
Table 1.
Single transport studies of NSAIDs across PBMEC/C1-2 and ECV304.
Table 2.
Group transport studies of NSAIDs across PBMEC/C1-2 and ECV304.
Figure 2.
Rankings of the group transport studies with NSAIDs across PBMEC/C1-2 layers.
Permeability coefficient of each substance was normalized to the corresponding permeability coefficient of internal standard diazepam of the same experiment. A): Variant substance compositions - results of the group study with all investigated substances (diazepam, piroxicam, ibuprofen, meloxicam, tenoxicam, diclofenac, celecoxib, carboxyfluorescein = CF) were compared to the study without celecoxib (w/o CC), without celecoxib accomplished in serum-free C6 medium (serum-free), without celecoxib accomplished in PBMEC-Fib medium (GCM = glioma conditioned medium) and without celecoxib and carboxyfluorescein (w/o CF). B): Different transport study conditions - results of the group study with all investigated substances (diazepam, piroxicam, ibuprofen, meloxicam, tenoxicam, diclofenac, celecoxib, carboxyfluorescein = CF) were compared to the study without meloxicam (w/o MEL), without meloxicam and with probenecid (with Probenecid) and without meloxicam and with verapamil (with Verapamil). To calculate the statistical significances between the groups, which differed in the substance compositions, a one-way ANOVA was used, to compare the groups with same substance compositions under different experimental transport conditions (in A: w/o CC, serum-free medium, GCM; in B: w/o MEL, with Probenecid, with Verapamil) a two-way ANOVA was accomplished followed by an all pairwise multiple comparison procedure (Holm-Sidak method) with an overall significance level of 0.05. Statistical significance (p<0.05) for each substance is indicated in the figure by * (all vs. w/o CC, all vs. w/o MEL), by # (w/o CC vs. serum-free or GCM; w/o MEL vs. with Probenecid or with Verapamil), by § (serum-free vs. GCM; with Probenecid vs. with Verapamil) or by $ (w/o CC vs. w/o CF). Data are presented as means ± SD (n = 3).
Figure 3.
Rankings of the group transport studies with NSAIDs across ECV304 layers.
Permeability coefficient of each substance was normalized to the corresponding permeability coefficient of internal standard diazepam of the same experiment. A): Variant substance compositions - results of the group study with all investigated substances (diazepam, piroxicam, ibuprofen, meloxicam, tenoxicam, diclofenac, celecoxib, carboxyfluorescein = CF) were compared to the study without celecoxib (w/o CC), without celecoxib accomplished in serum-free C6 medium (serum-free), without celecoxib accomplished in PBMEC-Fib medium (GCM = glioma conditioned medium) and without celecoxib and carboxyfluorescein (w/o CF). B): Different transport study conditions - results of the group study with all investigated substances (diazepam, piroxicam, ibuprofen, meloxicam, tenoxicam, diclofenac, celecoxib, carboxyfluorescein = CF) were compared to the study without meloxicam (w/o MEL), without meloxicam and with probenecid (with Probenecid) and without meloxicam and with verapamil (with Verapamil). To calculate the statistical significances between the groups, which differed in the substance compositions, a one-way ANOVA was used, to compare the groups with same substance compositions under different experimental transport conditions (in A: w/o CC, serum-free medium, GCM; in B: w/o MEL, with Probenecid, with Verapamil) a two-way ANOVA was accomplished followed by an all pairwise multiple comparison procedure (Holm-Sidak method) with an overall significance level of 0.05. Statistical significance (p<0.05) for each substance is indicated in the figure by * (all vs. w/o CC, all vs. w/o MEL), by # (w/o CC vs. serum-free or GCM; w/o MEL vs. with Probenecid or with Verapamil) or by § (serum-free vs. GCM; with Probenecid vs. with Verapamil). Data are presented as means ± SD (n = 3).
Table 3.
Serum binding [%] of NSAIDs in transport media.
Figure 4.
Characterization of the BBB model based on primary rat brain microvascular endothelial cells (RBMEC) and astrocytes.
RBMECs grow in endothelial cell typical spindle-like morphology proven by light and scanning electron microscopy (SEM). Transmission electron microscopic (TEM) images confirmed that RBMEC grow as a monolayer. The enlarged part of the image shows two RBMECs connected to each other directly over a pore of the Transwell insert membrane (A). mRNA expressions of tight junction proteins ZO-1, occludin, claudin-3, claudin-5 and claudin-12, and of adhesion molecules PECAM-1, VCAM, ICAM-1 and CD44. All data were related to endogenous control GAPDH which was set to 1000 (B). Immunofluorescence images of PECAM-1, ZO-1, occludin, claudin-3 and claudin-5 confirmed the protein’s presence and localization in RBMEC layers (C). Transport studies with paracellular marker APTS-dextran ladder confirmed functionality of the barrier. RBMEC layers were able to differentiate between the different dextran fractions in a molecular size-dependent manner. Comparison of the permeability coefficients for APTS-dextran across PBMEC/C1-2, ECV304 and RBMEC layers is presented in the table on the right side (D).
Table 4.
Group transport study of NSAIDs across RBMEC.