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

The transepithelial transport of a synthetic peptide combinatorial library containing 375000 individual peptides was assessed using Caco-2 cell monola yers in order to screen for permeability and deliverability. A series of 15 0 pools, each containing 2500 tripeptide sequences, were applied to the api cal side of Caco-2 monolayers. Basolateral side samples were collected afte r 4 h and screened by capillary high-pressure liquid chromatography. The ma jority of pools showed no permeable species, due to low solubility, limited permeability and extensive metabolism. Several pools contained permeable s tructures, and transport proved reproducible with passage number and time. Permeable structures were identified by liquid chromatography/mass spectrom etry/mass spectrometry (LC/MS/MS). To discriminate between isobaric structu res, several tripeptides were resynthesized and tested as discrete compound s. For example, 1-2% D-Phe-D-Ala-D-Ser-OH was transported across the Caco-2 cell monolayer with a P-app value of 0.35-0.69 x 10(-6) cm/s, which is com parable with the permeability of amino acids (Leu, P-app = 0.30 x 10(6) cm/ s) and dipeptides (L-Val-L-Val, P-app = 0.18 x 10(-6) cm/s) (Lennernas, H., Palm, K., Fagerholm, U., Artursson, P., 1996. Comparison between active an d passive drug transport in human intestinal epithelial (Caco-2) cells in v itro and human jejunum in vivo. Int. J. Pharm. 127, 103-107; Tamura, K., Bh atnagar, 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 li braries for permeability across Caco-2 cells and structurally identify the resulting compounds. Such methodology can be of importance in the achieveme nt of structure-permeability relationships, useful in the design of pharmac euticallly bioavailable drugs. (C) 1999 Elsevier Science B.V. All rights re served.