Use of Caco-2 cells and LC/MS/MS to screen a peptide combinatorial library for permeable structures

The transepithelial transport of a synthetic peptide combinatorial library containing 375,000 individual peptides was assessed using Caco-2 cell monolayers in order to screen for permeability and deliverability. A series of 150 pools, each containing 2500 tripeptide sequences, were applied to the apical side of Caco-2 monolayers. Basolateral side samples were collected after 4 h and screened by capillary high-pressure liquid chromatography. The majority of pools showed no permeable species, due to low solubility, limited permeability and extensive metabolism. Several pools contained permeable structure, and transport proved reproducible with passage number and time. Permeable structures were identified by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). To discriminate between isobaric structures, several tripeptides were resynthesized and tested as discrete compounds. For example, 1-2% D-Phe-D-Ala-D-Ser-OH was transported across the Caco-2 cell monolayer with a Papp value of 0.35-0.69 x 10(-6) cm/s, which is comparable with the permeability of amino acids (Leu, Papp = 0.30 x 10(-6) cm/s) and dipeptides (L-Val-L-Val, Papp = 0.18 x 10(-6) cm/s) (Lennernas, H., Palm, K., Fagerholm, U., Artursson, P., 1996. Comparison between active and passive drug transport in human intestinal epithelial (Caco-2) cells in vitro and human jejunum in vivo. Int. J. Pharm. 127, 103-107; Tamura, K., Bhatnagar, P.K., Takata, J.S., Lee, C.P., Smith, P.L., Borchardt, R.T., 1996. Metabolism, uptake, and transepithelial transport of the diastereomers of Val-Val in the human intestinal cell line Caco-2. Pharm. Res. 13, 1213-1218). These studies demonstrate the techniques used to screen combinatorial libraries for permeability across Caco-2 cells and structurally identify the resulting compounds. Such methodology can be of importance in the achievement of structure-permeability relationships, useful in the design of pharmaceutically bioavailable drugs.