Determination of transport in the Caco-2 cell assay of compounds varying in lipophilicity using LC-MS: enhanced transport of Leu-enkephalin analogues.
OBJECTIVE
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 microM. 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.
School of Pharmacy, The University of Queensland, 4072, St. Lucia, Australia
Allan K
AK
Wong
Benjamin P
BP
Ross
Yiu Ngok
YN
Chan
Per
P
Artursson
Lucia
L
Lazorova
Alun
A
Jones
Istvan
I
Toth
D000042:Absorption; D001692:Biological Transport / Q000502:physiology; D018938:Caco-2 Cells / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D006801:Humans; D008055:Lipids / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods
D008055:Lipids; D004743:Enkephalin, Leucine