The influence of different drug transport routes in intestinal drug permeability screening assays was studied. Three experimental models were compared: the small-intestine-like 2/4/A1 cell model, which has a leaky paracellular pathway, the Caco-2 cell model, which has a tighter paracellular pathway, and artificial hexadecane membranes (HDMs), which exclusively model the passive transcellular pathway. The models were investigated regarding their ability to divide passively and actively transported compounds into two permeability classes and to rank compounds according to human intestinal absorption. In silico permeability models based on two-dimensional (2D) and three-dimensional (3D) molecular descriptors were also developed and validated using external test sets. The cell-based models classified 80% of the acceptably absorbed compounds (FA >/= 30%) correctly, compared to 60% correct classifications using the HDM model. The best compound ranking was obtained with 2/4/A1 (r(s) = 0.74; r(s) = 0.95 after removing actively transported outliers). The in silico model based on 2/4/A1 permeability gave results of similar quality to those obtained when using experimental permeability, and it was also better than the experimental HDM model at compound ranking (r(s) = 0.85 and 0.47, respectively). We conclude that the paracellular transport pathway present in the cell models plays a significant role in models used for intestinal permeability screening and that 2/4/A1 in vitro and in silico models are promising alternatives for drug discovery permeability screening.
The aim of this study was to investigate whether peptides from the extracellular loops of the tight junction protein occludin could be used as a new principle for tight junction modulation. Peptides of 4 to 47 amino acids in length and covering the two extracellular loops of the tight junction protein occludin were synthesized, and their effect on the tight junction permeability in Caco-2 cells was investigated using [14 C]mannitol as a paracellular marker. Lipopeptide derivatives of one of the active occludin peptides (OPs), synthesized by adding a lipoamino acid containing 14 carbon atoms (C 14 -) to the N terminus of the peptide, were also investigated. Peptides corresponding to the N terminus of the first extracellular loop of occludin increased the permeability of the tight junctions without causing shortterm toxicity. However, the peptides had an effect only when added to the basolateral side of the cells, which could be partly explained by degradation by apical peptidases and aggregate formation. By contrast, the lipopeptide C 14 -OP 90 -103 , which protects the peptide from degradation and aggregation, displayed a rapid apical effect. The L-and D-diastereomers of C 14 -OP 90 -103 had distinctly different effects. The D-isomer, which releases intact OP 90 -103 from the lipoamino acid, displayed a rapid and transient increase in tight junction permeability. The L-isomer, which releases OP 90 -103 more rapidly, gave a more sustained increase in tight junction permeability. In conclusion, C 14 -OP 90 -103 represents a prototype of a new class of tight junction modulators that act on the extracellular domains of tight junction proteins.
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