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

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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.

Introduction

Peptides and proteins are important potential therapeutic agents for a number of disease states. Hydrolytic instability in the gastrointestinal tract is a major limitation for the preferred oral route of administration of these compounds Lee and Yamamoto, 1990, Banga and Chien, 1988. Attachment of sugar units and lipoamino acids to peptides can increase the stability of a peptide towards degradation and increase the permeability of hydrophilic peptides across the intestinal membrane (Toth et al., 1999). Lipoamino acids are synthetic amino acids with a hydrophobic side chain that increases the lipophilicity of the compound to which it is attached, and have been used to increase the stability to degradation of a number of peptides (Wong and Toth, 2001). Conjugation of sugar units has also been used to increase the stability of peptides to degradation, mainly through protection of the N- or C-termninal end to which it is attached (Wong and Toth, 2001). A library of analogues can be synthesised by attaching these units at various positions in the peptide, usually the N- and C-termini. We have used the Caco-2 assay as an in vitro model to study intestinal epithelial transport of a number of peptide analogues. The human intestinal cell line Caco-2, spontaneously differentiates on microporous filter membranes forming monolayers with tight cellular junctions (Wilson et al., 1990). The permeability of compounds through the Caco-2 cell monolayer can be related to the extent of oral absorption in humans, which provides important information for selecting lead compounds for further study (Artursson and Karlsson, 1991). Detection of the concentration of drug passing through the Caco-2 monolayer is usually determined by following a radiolabelled drug. However, disadvantages of this technique include the cost and hazards of radiolabelled drugs and the problems of detecting degraded or metabolised radiolabelled compounds.

Liquid chromatography–electrospray ionisation mass spectrometry (LC–MS) has been used by a number of researchers to replace the usual quantitation techniques of radiolabelled drugs utilising liquid scintillation counting or liquid chromatography–ultraviolet (LC–UV) detection of unlabelled drugs Wang et al., 2000, Stevenson et al., 1999, Caldwell et al., 1998. LC–MS has shown increased sensitivity and selectivity over these conventional techniques for a number of standard drugs. As part of our work with lipoamino acid modified peptides, we analyse very hydrophobic compounds. In this study, we examined the use of LC–MS with the Caco-2 assay to test some Leu-enkephalin analogues, which vary in lipophilicity and discuss some of the difficulties in running very hydrophobic, poorly water-soluble compounds. Analysis of a water-soluble drug, cephalexin and modified Leu-enkephalin by both radioactivity measurement and LC–MS, confirms the applicability of our LC–MS protocol.

Section snippets

Chemicals

MBHA resin, Chloro Trityl resin, Rink amide resin and protected amino acids were obtained from Novabiochem (Melbourne, Australia). DMF and TFA of peptide synthesis grade were purchased from Auspep (Parkville, Australia). Caco-2 cells were obtained from American Type Culture Collection (Rockville, MD), Transwell polycarbonate insets were from Costar (Cambridge, MA) and cell culture reagents were purchased from Gibco-BRL (Grand Island, NY).

Synthesis

The radiolabelled acetylated cephalexin 1 and acetylated

Comparison of permeability via scintillation counting versus LC–MS

Table 2 shows the apparent permeability values obtained for the readily water soluble radiolabelled acetylated cephalexin 1 as determined by liquid scintillation counter, plus the permeabilities of acetylated cephalexin 2 and cephalexin 3 as quantitated by LC–MS. The permeability values obtained for 1 and 2 via the two different methods are statistically the same, as shown in Table 1. Whereas, acetylated 1 and the non-acetylated 3 are statistically different, most probably due to the increased

Discussion

Difficulties of analysing poorly water-soluble compounds have been overcome by the addition of DMSO (5%) and acidification of the standards and collected receiver solutions. This serves to prevent precipitation of peptides from the aqueous solution and in addition prevents disappearance of the peptides upon standing, which we have observed for solutions without acid and DMSO. This disappearance is attributed to binding of the peptide to the container surfaces, also observed by us for other

Conclusions

A rapid LC–MS method has been developed for determining the apparent permeabilities of compounds varying in lipophilicity in the Caco-2 cell assay (19). Problems associated with poor water solubility of the lipoamino acid-coupled peptides were overcome by acidification with formic acid (10%) and adding DMSO (5%) to the standards and samples collected after the transport experiments. Leu-enkephalin was rapidly degraded after contact with Caco-2 cells, however, sugar and sugar–lipoamino acid

Acknowledgements

This work was supported by a Wellcome Trust equipment grant 057160/Z/99.

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