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TEER reduction at acidic and slightly basic pH of different CTS grades compared to MTT test results and effect of washing step * Cellular death is accompanied by detachment of cells which leads to a proportional TEER reduction, therefore the comparison of both methods reveals the influence of CTS on tight junctions.

Table 8 TEER reduction at acidic and slightly basic pH of different CTS grades compared to MTT test results and effect of washing step * Cellular death is accompanied by detachment of cells which leads to a proportional TEER reduction, therefore the comparison of both methods reveals the influence of CTS on tight junctions.

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* The acid dissociation constants were determined with a pH-potentiometric method in 0.1 M hydrochloric acid at 25 °C. Approximately 3.8 x 10-4 N of drug was titrated in the presence of excess acid with 0.1 M potassium hydroxide. > The partition equilibria between n-octanol and water, buffered at pH 1, 2, 5, 7, 9, were determined by the flask shaking method. Each test set consiste of three different volume ratios of the two phases, after equilibration they were separated by centrifugation (5000 rpm, 30 min) and subsequently analyze by HPLC. Effect of pH in 0.05 M buffered solutions on ionic state of dissolved FXai and its resulting log P and aqueous solubility Table |
* The acid dissociation constants were determined with a pH-potentiometric method in 0.1 M hydrochloric acid at 25 °C. Approximately 3.8 x 10-4 N of drug was titrated in the presence of excess acid with 0.1 M potassium hydroxide. > The partition equilibria between n-octanol and water, buffered at pH 1, 2, 5, 7, 9, were determined by the flask shaking method. Each test set consiste of three different volume ratios of the two phases, after equilibration they were separated by centrifugation (5000 rpm, 30 min) and subsequently analyze by HPLC. Effect of pH in 0.05 M buffered solutions on ionic state of dissolved FXai and its resulting log P and aqueous solubility Table |
Fig. 1. Structures of FXai parent drug and its prodrugs along with their respective molecular weight and partition coefficient in octanol/water, the pro- moieties have been highlighted with a dotted line.
Fig. 1. Structures of FXai parent drug and its prodrugs along with their respective molecular weight and partition coefficient in octanol/water, the pro- moieties have been highlighted with a dotted line.
Effect of prodrug formation on acid dissociation constants (pKa) and partition coefficients (log P) * Acronyms were formed according to the pro-moiety: B-butyl, E-ethyl, OH-hydroxyl, 1x-mono-prodrug, 2x-double-prodrug. > log P was determined with the method described in Table 1 in 0.05 M PBS at pH 7.0.
Effect of prodrug formation on acid dissociation constants (pKa) and partition coefficients (log P) * Acronyms were formed according to the pro-moiety: B-butyl, E-ethyl, OH-hydroxyl, 1x-mono-prodrug, 2x-double-prodrug. > log P was determined with the method described in Table 1 in 0.05 M PBS at pH 7.0.
Fig. 2. Cumulative mass permeation of FXai O and its prodrugs B2x O, E2x A, OH1x @, B1x A and Elx Macross the Caco-2 monolayer over time (mean sum of prodrug and corresponding ester cleaved metabolite + SD, n= 3).
Fig. 2. Cumulative mass permeation of FXai O and its prodrugs B2x O, E2x A, OH1x @, B1x A and Elx Macross the Caco-2 monolayer over time (mean sum of prodrug and corresponding ester cleaved metabolite + SD, n= 3).
Data represent means + SD of three experimental determinations. * are significantly different (p = 0.05) from FXai value (unpaired Welch‘s t-te: assumed Gaussian distribution). Apparent permeability of drug and prodrugs and their metabolite in apical to basolateral directior Table 3
Data represent means + SD of three experimental determinations. * are significantly different (p = 0.05) from FXai value (unpaired Welch‘s t-te: assumed Gaussian distribution). Apparent permeability of drug and prodrugs and their metabolite in apical to basolateral directior Table 3
Metabolite (OH1x) distribution after basolateral application of double- prodrugs after 120 min Even distribution of the metabolite in both compartments. In contrast to results in Table 3, basolateral application has circumvented the strong esterase activity in the apical region of the polarized cell, leading to the expected distribution of the metabolite.
Metabolite (OH1x) distribution after basolateral application of double- prodrugs after 120 min Even distribution of the metabolite in both compartments. In contrast to results in Table 3, basolateral application has circumvented the strong esterase activity in the apical region of the polarized cell, leading to the expected distribution of the metabolite.
Metabolite (OH1x) distribution after apical application of double- prodrugs after 120 min Intestinal ester cleavage was expected to occur intracellularly, leading to a 50/50 distribution of the metabolite by passive diffusion, since OH1x was found to be not actively transported. The uneven distribution suggests a stronger esterase activity in the apical membrane. This would represent a new absorptive barrier for the double-prodrugs as their metabolite OH1x is much less permeable. All experiments were done in triplicate.
Metabolite (OH1x) distribution after apical application of double- prodrugs after 120 min Intestinal ester cleavage was expected to occur intracellularly, leading to a 50/50 distribution of the metabolite by passive diffusion, since OH1x was found to be not actively transported. The uneven distribution suggests a stronger esterase activity in the apical membrane. This would represent a new absorptive barrier for the double-prodrugs as their metabolite OH1x is much less permeable. All experiments were done in triplicate.
Fig. 3. Apical to basolateral mass permeation of B2x and its metabolite (64M) after 120 min (n=3) without and with three different efflux inhibitors (20 4M each) in absolute numbers (light columns) and as percentage relative to transport buffer (dark columns).
Fig. 3. Apical to basolateral mass permeation of B2x and its metabolite (64M) after 120 min (n=3) without and with three different efflux inhibitors (20 4M each) in absolute numbers (light columns) and as percentage relative to transport buffer (dark columns).
* Calculated according to Eq. (2). For E2x, P. calculation purposes. ‘app (A — B) was set to 1 for Apparent permeability of FXai and its double-prodrugs in the basolateral to apical direction and the resulting efflux ratio
* Calculated according to Eq. (2). For E2x, P. calculation purposes. ‘app (A — B) was set to 1 for Apparent permeability of FXai and its double-prodrugs in the basolateral to apical direction and the resulting efflux ratio
Data represent means + SD of three experimental determinations. * value is significantly different (py = 0.05) from FXai value (unpaired Welch‘s t-test, assumed Gaussian distribution). Effect of 0.1% Imwitor 742 on permeation of parent drug FXai across Caco-2 monolayers
Data represent means + SD of three experimental determinations. * value is significantly different (py = 0.05) from FXai value (unpaired Welch‘s t-test, assumed Gaussian distribution). Effect of 0.1% Imwitor 742 on permeation of parent drug FXai across Caco-2 monolayers
Data represent means + SD of three experimental determinations. * values are significantly different (p = 0.05) from corresponding FXai control (unpaired Welch’s t-test, assumed Gaussian distribution). n.d., not determined. Comparison of FXai permeation across Caco-2 monolayers in combination with different types of CTS at acidic and slightly basic pHs
Data represent means + SD of three experimental determinations. * values are significantly different (p = 0.05) from corresponding FXai control (unpaired Welch’s t-test, assumed Gaussian distribution). n.d., not determined. Comparison of FXai permeation across Caco-2 monolayers in combination with different types of CTS at acidic and slightly basic pHs
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