Epidermal penetration of a therapeutic peptide by lipid conjugation; Stereo-selective peptide availability of a topical diastereomeric lipopeptide

In inflammatory disorders (e.g. psoriasis), local concentrations of human neutrophil elastase (HNE), also known as polymorphonuclear leukocyte elastase (HLE), possibly overwhelm its natural inhibitors leading to extracellular matrix degradation. Elevated levels of HNE have been reported in a variety of inflammatory disorders, including psoriasis. Peptidic HNE inhibitors have a common hydrophobic sequence (Ala-Ala-Pro-Val). This peptide sequence inhibits HNE competitively; however the stratum corneum presents an effective barrier to the delivery of this tetrapeptide across the skin. The current work investigates the delivery of the modified peptide whereby the tetrapeptide was lipidated to enhance its ability to penetrate the stratum The tetrapeptide Was Coupled to a racaemic mixture of a short chain lipoamino acid (LAA) resulting in two diastereomers of the lipoamino acid-modified tetrapeptide. The penetration of the lipopeptide mixture was assessed across human epidermis in vitro. The percentage of applied dose penetrating to the receptor over 8 h following administration was 2.53% for the D-LAA conjugate and 1.47% for the L-diastereomer, compared to 0% for the peptide. The D-diastereomer appears to be relatively stable but the L-diastereomer appears to degrade releasing possibly the tetrapeptide and peptide fragment(s). Therefore the results clearly indicate that coupling the tetrapeptide to a short chain LAA enhances its delivery across human epidermis.

A new principle for tight junction modulation based on occludin peptides

The aim of this study was to investigate whether peptides from the extracellular loops of the tight junction protein occludin could be used as a new principle for tight junction modulation. Peptides of 4 to 47 amino acids in length and covering the two extracellular loops of the tight junction protein occludin were synthesized, and their effect on the tight junction permeability in Caco-2 cells was investigated using [C-14] mannitol as a paracellular marker. Lipopeptide derivatives of one of the active occludin peptides (OPs), synthesized by adding a lipoamino acid containing 14 carbon atoms (C-14-) to the N terminus of the peptide, were also investigated. Peptides corresponding to the N terminus of the first extracellular loop of occludin increased the permeability of the tight junctions without causing short-term toxicity. However, the peptides had an effect only when added to the basolateral side of the cells, which could be partly explained by degradation by apical peptidases and aggregate formation. By contrast, the lipopeptide C-14-OP90-103, which protects the peptide from degradation and aggregation, displayed a rapid apical effect. The L- and D-diastereomers of C-14-OP90-103 had distinctly different effects. The D-isomer, which releases intact OP90-103 from the lipoamino acid, displayed a rapid and transient increase in tight junction permeability. The L- isomer, which releases OP90-103 more rapidly, gave a more sustained increase in tight junction permeability. In conclusion, C-14-OP90-103 represents a prototype of a new class of tight junction modulators that act on the extracellular domains of tight junction proteins.

Development of lipid-core-peptide (LCP) based vaccines for the prevention of the group A streptococcal (GAS) infection

Traditional vaccines consisting of whole attenuated micro-organisms. or microbial components administered with adjuvant, have been demonstrated as one of the most cost-effective and successful public health interventions. Their use in large scale immunisation programs has lead to the eradication of smallpox, reduced morbidity and mortality from many once common diseases, and reduced strain on health services. However, problems associated with these vaccines including risk of infection. adverse effects, and the requirement for refrigerated transport and storage have led to the investigation of alternative vaccine technologies. Peptide vaccines, consisting of either whole proteins or individual peptide epitopes, have attracted much interest, as they may be synthesised to high purity and induce highly specific immune responses. However, problems including difficulties stimulating long lasting immunity. and population MHC diversity necessitating multiepitopic vaccines and/or HLA tissue typing of patients complicate their development. Furthermore, toxic adjuvants are necessary to render them immunogenic. and as such non-toxic human-compatible adjuvants need to be developed. Lipidation has been demonstrated as a human compatible adjuvant for peptide vaccines. The lipid-core-peptide (LCP) system. incorporating lipid adjuvant, carrier, and peptide epitopes, exhibits promise as a lipid-based peptide vaccine adjuvant. The studies reviewed herein investigate the use of the LCP system for developing vaccines to protect against group A streptococcal (GAS) infection. The studies demonstrate that LCP-based GAS vaccines are capable of inducing high-titres of antigen specific IgG antibodies. Furthermore. mice immunised with an LCP-based GAS vaccine were protected against challenge with 8830 strain GAS.

Encapsulation of lipopeptides within liposomes: Effect of number of lipid chains, chain length and method of liposome preparation

The purpose of this study was to systematically investigate the effect of lipid chain length and number of lipid chains present on lipopeptides on their ability to be incorporated within liposomes. The peptide KAVYNFATM was synthesized and conjugated to lipoamino acids having acyl chain lengths of C-8, C-12 and C-16. The C-12 construct was also prepared in the monomeric, dimeric and trimeric form. Liposomes were prepared by two techniques: hydration of dried lipid films (Bangham method) and hydration of freeze-dried monophase systems. Encapsulation of lipopeptide within liposomes prepared by hydration of dried lipid films was incomplete in all cases ranging from an entrapment efficiency of 70% for monomeric lipoamino acids at a 5% (w/w) loading to less than 20% for di- and trimeric forms at loadings of 20% (w/w). The incomplete entrapment of lipopeptides within liposomes appeared to be a result of the different solubilities of the lipopeptide and the phospholipids in the solvent used for the preparation of the lipid film. In contrast, encapsulation of lipopeptide within liposomes prepared by hydration of freeze-dried monophase systems was high, even up to a loading of 20% (w/w) and was much less affected by the acyl chain length and number than when liposomes were prepared by hydration of dried lipid films. Freeze drying of monophase systems is better at maintaining a molecular dispersion of the lipopeptide within the solid phospholipid matrix compared to preparation of lipid film by evaporation, particularly if the solubility of the lipopeptide in solvents is markedly different from that of the polar lipids used for liposome preparation. Consequently, upon hydration, the lipopeptide is more efficiently intercalated within the phospholipid bilayers. (C) 2005 Elsevier B.V. All rights reserved.

Synthesis and immunological evaluation of M protein targeted tetra-valent and tri-valent group A streptococcal vaccine candidates based on the lipid-core peptide system

Group A streptococcus (GAS) is responsible for causing many clinical complications including the relatively benign streptococcal pharyngitis and impetigo. However. if left untreated. these conditions may lead to more severe diseases such as rheumatic fever (RF) and rheumatic heart disease (RHD). These diseases exhibit high morbidity and mortality, Particularly in developing countries and in indigenous populations of affluent countries. Only ever occur following GAS infection, a vaccine offers Promise for their Prevention. As stich, we have investigated the Use of the lipid-core peptide (LCP) system for the development of multi-valent Prophylactic GAS vaccines. The current study has investigated the capacity of this system to adjuvant LIP to four different GAS peptide epitopes. Presented are the synthesis and immunological assessment of tetra-valent and tri-valent GAS LCP systems. We demonstrated their capacity to elicit systemic IgG antibody responses in B10.BR mice to all GAS peptide epitopes. The data also showed that the LCP systems Were self-adjuvanting. These findings are particularly encouraging for the development of multi-valent LCP-based GAS vaccines.

Method for the synthesis of highly pure vaccines using the lipid core peptide system

Traditional vaccines consisting of whole attenuated microorganisms, killed microorganisms, or microbial components, administered with an adjuvant (e.g. alum), have been proved to be extremely successful. However, to develop new vaccines, or to improve upon current vaccines, new vaccine development techniques are required. Peptide vaccines offer the capacity to administer only the minimal microbial components necessary to elicit appropriate immune responses, minimizing the risk of vaccination associated adverse effects, and focusing the immune response toward important antigens. Peptide vaccines, however, are generally poorly immunogenic, necessitating administration with powerful, and potentially toxic adjuvants. The attachment of lipids to peptide antigens has been demonstrated as a potentially safe method for adjuvanting peptide epitopes. The lipid core peptide (LCP) system, which incorporates a lipidic adjuvant, carrier, and peptide epitopes into a single molecular entity, has been demonstrated to boost immunogenicity of attached peptide epitopes without the need for additional adjuvants. The synthesis of LCP systems normally yields a product that cannot be purified to homogeneity. The current study describes the development of methods for the synthesis of highly pure LCP analogs using native chemical ligation. Because of the highly lipophilic nature of the LCP lipid adjuvant, difficulties (e.g. poor solubility) were experienced with the ligation reactions. The addition of organic solvents to the ligation buffer solubilized lipidic species, but did not result in successful ligation reactions. In comparison, the addition of approximately 1% (w/v) sodium dodecyl sulfate (SDS) proved successful, enabling the synthesis of two highly pure, tri-epitopic Streptococcus pyogenes LCP analogs. Subcutaneous immunization of B10.BR (H-2(k)) mice with one of these vaccines, without the addition of any adjuvant, elicited high levels of systemic IgG antibodies against each of the incorporated peptides. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.