Oral absorption enhancement of dipeptide L-Glu-L-Trp-OH by lipid and glycosyl conjugation.

In recent years, the conjugation of sugar moieties and lipoamino acids has been extensively investigated as a mean to enhance the stability towards enzymatic degradation and the permeability across biological membranes of poorly orally available drugs, including peptides. In this prospect, a library of novel derivatives of the dipeptide L-Glu-L-Trp, a naturally occurring thymic immunomodulator with high hydrophilic character and low membrane permeability, was designed and synthesised by conjugating 2-amino-dodecanoic acid (C(12)) and/or 1-amino-beta-D-glucuronic acid (GlcAN), beta-D-glucuronic acid (GlcA) and N-beta-D-glucopyranosylamine succinamic acid (GlsNS) residues to the Glu-Trp scaffold, using an Fmoc solid-phase peptide synthesis strategy on trichlorotrityl resin. A cellobiose derivative was also prepared in solution. The synthesized peptides showed no sign of toxicity to red blood cells at 200 microM (haemolysis assay) and their resistance against enzymatic hydrolysis, assessed in Caco-2 homogenates, was usually significantly increased, particularly for the C-terminal conjugates. Several derivatives also saw their apparent permeability values greatly enhanced and one of the conjugates tested proved to be able to release the initial dipeptide after penetrating Caco-2 monolayers. An initial in vivo experiment was then carried out in male Wistar rats to examine the effect of conjugation on the absorption rate and bioavailability.

Lipidation and glycosylation of a T cell antigen receptor (TCR) transmembrane hydrophobic peptide dramatically enhances in vitro and in vivo function.

A T cell antigen receptor (TCR) transmembrane sequence derived peptide (CP) has been shown to inhibit T cell activation both in vitro and in vivo at the membrane level of the receptor signal transduction. To examine the effect of sugar or lipid conjugations on CP function, we linked CP to 1-aminoglucosesuccinate (GS), N-myristate (MYR), mono-di-tripalmitate (LP1, LP2, or LP3), and a lipoamino acid (LA) and examined the effects of these compounds on T cell activation in vitro and by using a rat model of adjuvant-induced arthritis, in vivo. In vitro, antigen presentation results demonstrated that lipid conjugation enhanced CP's ability to lower IL-2 production from 56.99%+/-15.69 S.D. observed with CP, to 12.08%+/-3.34 S.D. observed with LA. The sugar conjugate GS resulted in only a mild loss of in vitro activity compared to CP (82.95%+/-14.96 S.D.). In vivo, lipid conjugation retarded the progression of adjuvant-induced arthritis by approximately 50%, whereas the sugar conjugated CP, GS, almost completely inhibited the progression of arthritis. This study demonstrates that hydrophobic peptide activity is markedly enhanced in vitro and in vivo by conjugation to lipids or sugars. This may have practical applications in drug delivery and bioavailability of hydrophobic peptides.

Functional implications of modifying RyR-activating peptides for membrane permeability.

1. Our aim was to determine whether lipoamino acid conjugation of peptides that are high-affinity activators of ryanodine receptor (RyR) channels would (a) render the peptides membrane permeable, (b) alter their structure or (a) reduce their activity. The peptides correspond to the A region of the II-III loop of the skeletal dihydropyridine receptor. 2. The lipoamino acid conjugation increased the apparent permeability of the peptide across the Caco-2 cell monolayer by up to approximately 20-fold. 3. Nuclear magnetic resonance showed that the alpha-helical structure of critical basic residues, required for optimal activation of RyRs, was retained after conjugation. 4. The conjugated peptides were more effective in enhancing resting Ca2+ release, Ca2+-induced Ca2+ release and caffeine-induced Ca2+ release from isolated sarcoplasmic reticulum (SR) than their unconjugated counterparts, and significantly enhanced caffeine-induced Ca2+ release from mechanically skinned extensor digitorum longus (EDL) fibres. 5. The effect of both conjugated and unconjugated peptides on Ca2+ release from skeletal SR was 30-fold greater than their effect on either cardiac Ca2+ release or on the Ca2+ Mg2+ ATPase. 6. A small and very low affinity effect of the peptide in slowing Ca2+ uptake by the Ca2+, Mg2+ ATPase was exacerbated by lipoamino acid conjugation in both isolated SR and in skinned EDL fibres. 7. The results show that lipoamino acid conjugation of A region peptides increases their membrane permeability without impairing their structure or efficacy in activating skeletal and cardiac RyRs.

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

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 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.