Determination of transport in the Caco-2 cell assay of compounds varying in lipophilicity using LC–MS: enhanced transport of Leu-enkephalin analogues

Abstract

Purpose: To synthesize a number of analogues of Leu-enkephalin with different lipophilicities and to develop an LC–MS method for determining the Caco-2 cell permeability values of these compounds. Methods: A number of sugar and sugar plus lipoamino acid analogues of Leu-enkephalin were synthesized by solid-phase and solution methods. An LC–MS method was developed for analyzing the Caco-2 cell assay samples and validated against the traditional method using radiolabelled compounds. Results: A sensitive and specific LC–MS assay was developed. Standard curves were linear in the range of 0.025–5 μM. Apparent permeability values determined by LC–MS and liquid scintillation counter were identical, for both a hydrophilic drug, cephalexin and a lipophilic Leu-enkaphalin analogue. Caco-2 permeability values for the analogues of Leu-enkephalin were determined and it was found that attachment of sugar or sugar and lipoamino acid to the Leu-enkephalin peptide resulted in an increase in the apparent permeability values compared to the native peptide, which was not transported across the Caco-2 cell monolayers. Conclusions: A rapid, generic LC–MS method for analyzing a range of compounds was developed. Attachment of a sugar or sugar and lipoamino acid to Leu-enkephalin improves the apparent permeability across Caco-2 cell monolayers.