Aniko Horvath

Anti‐inflammatory effect of synthetic somatostatin analogues in the rat

Somatostatin (6.11 nmol kg−1 i.p.) inhibited neurogenic plasma extravasation evoked by 1% mustard oil and non‐neurogenic oedema induced by 5% dextran in the rat skin. Cyclic synthetic octapeptide (TT‐248 and TT‐250) and heptapeptide (TT‐232) somatostatin analogues proved to be more effective in reducing neurogenic and non‐neurogenic inflammatory reactions but octreotide had no influence on either neurogenic or non‐neurogenic inflammation. TT‐232 administered i.p. or i.v. (1.06 – 42.40 nmol kg−1) inhibited in a dose‐dependent manner the plasma extravasation evoked by mustard oil in the rats paw. Neither diclofenac (15.78 – 315.60 μmol kg−1) nor the selective COX‐2 inhibitor meloxicam (2.95 – 569.38 μmol kg−1) attenuated the mustard oil‐induced neurogenic plasma extravasation. TT‐232, diclofenac and meloxicam dose‐dependently diminished non‐neurogenic dextran‐oedema of the paw the ED35 values were 1.73 nmol kg−1 for TT‐232 and 34.37 μmol kg−1 for diclofenac. TT‐232 inhibited in the dose range of 1.06 – 21.21 nmol kg−1 the bradykinin‐induced plasma extravasation in the skin of the chronically denervated paw. Mustard oil‐induced cutaneous plasma extravasation was dose‐dependently diminished by s.c. TT‐232 1, 2, 4, 6 or 16 h after the treatment. TT‐232 (2×106, 2×212 and 2×530 nmol kg−1 per day s.c. for 18 days) caused dose‐dependent inhibition of chronic Freund adjuvant‐induced arthritis during the experimental period. TT‐232 (200 and 500 nM) inhibited the release of SP, CGRP and somatostatin from the rat isolated trachea induced by electrical field stimulation (40 V, 0.1 ms, 10 Hz, 120 s) or by capsaicin (10−7 M), but did not influence the basal, non‐stimulated peptide release. It is concluded that somatostatin analogues without endocrine functions as TT‐232 are promising compounds with a novel site of action for inhibition of non‐neurogenic and neurogenic inflammatory processes.

Potential of lipid core peptide technology as a novel self-adjuvanting vaccine delivery system for multiple different synthetic peptide immunogens.

ABSTRACT This study demonstrates the effectiveness of a novel self-adjuvanting vaccine delivery system for multiple different synthetic peptide immunogens by use of lipid core peptide (LCP) technology. An LCP formulation incorporating two different protective epitopes of the surface antiphagocytic M protein of group A streptococci (GAS)—the causative agents of rheumatic fever and subsequent rheumatic heart disease—was tested in a murine parenteral immunization and GAS challenge model. Mice were immunized with the LCP-GAS formulation, which contains an M protein amino-terminal type-specific peptide sequence (8830) in combination with a conserved non-host-cross-reactive carboxy-terminal C-region peptide sequence (J8) of the M protein. Our data demonstrated immunogenicity of the LCP-8830-J8 formulation in B10.BR mice when coadministered in complete Freunds adjuvant and in the absence of a conventional adjuvant. In both cases, immunization led to induction of high-titer GAS peptide-specific serum immunoglobulin G antibody responses and induction of highly opsonic antibodies that did not cross-react with human heart tissue proteins. Moreover, mice were completely protected from GAS infection when immunized with LCP-8830-J8 in the presence or absence of a conventional adjuvant. Mice were not protected, however, following immunization with an LCP formulation containing a control peptide from a Schistosoma sp. These data support the potential of LCP technology in the development of novel self-adjuvanting multi-antigen component vaccines and point to the potential application of this system in the development of human vaccines against infectious diseases.

A lipid core peptide construct containing a conserved region determinant of the group A streptococcal M protein elicits heterologous opsonic antibodies.

ABSTRACT The study reported here investigated the immunogenicity and protective potential of a lipid core peptide (LCP) construct containing a conserved region determinant of M protein, defined as peptide J8. Parenteral immunization of mice with LCP-J8 led to the induction of high-titer serum immunoglobulin G J8-specific antibodies when the construct was coadministered with complete Freunds adjuvant (CFA) or administered alone. LCP-J8 in CFA had significantly enhanced immunogenicity compared with the monomeric peptide J8 given in CFA. Moreover, LCP-J8/CFA and LCP-J8 antisera opsonized four different group A streptococcal (GAS) strains, and the antisera did not cross-react with human heart tissue proteins. These data indicate the potential of an LCP-based M protein conserved region GAS vaccine in the induction of broadly protective immune responses in the absence of a conventional adjuvant.

Nanoparticles based on PLGA: Poloxamer blends for the delivery of proangiogenic growth factors

New blood vessel formation is a critical requirement for treating many vascular and ischemia related diseases, as well as for many tissue engineering applications. Angiogenesis and vasculogenesis, in fact, represent crucial processes for the functional regeneration of complex tissues through tissue engineering strategies. Several growth factors (GFs) and signaling molecules involved in blood vessels formation have been identified, but their application to the clinical setting is still strongly limited by their extremely short half-life in the body. To overcome these limitations, we have developed a new injectable controlled release device based on polymeric nanoparticles for the delivery of two natural proangiogenic GFs: platelet derived growth factor (PDGF-BB) and fibroblast growth factor (FGF-2). The nanoparticle system was prepared by a modified solvent diffusion technique, encapsulating the GF both in presence and in the absence of two stabilizing agents: bovine serum albumin (BSA) and heparin sodium salt (Hp). The developed nanocarriers were characterized for morphology, size, encapsulation efficiency, release kinetics in vitro and GF activity in cell cultures. The results have indicated that the coencapsulation of stabilizing agents can preserve the GF active structure and, in addition, increase their encapsulation efficiency into nanoparticles. Through this optimization process, we were able to raise the encapsulation efficiency of FGF-2 to 63%, and that of PDGF-BB to 87%. These PLGA:poloxamer blend nanoparticles loaded with GFs were able to release PDGF-BB and FGF-2 in a sustained fashion for more than a month. This work also confirms other positive features of PLGA:poloxamer nanoparticles. Namely, they are able to maintain their stability in simulated biological medium, and they are also nontoxic to cell culture models. Incubation of nanoparticles loaded with FGF-2 or PDGF-BB with endothelial cell culture models has confirmed that GFs are released in a bioactive form. Altogether, these results underline the interest of PLGA:poloxamer nanoparticles for the controlled delivery of GFs and substantiate their potential for the treatment of ischemic diseases and for tissue engineering applications.

Intranasal Administration Is an Effective Mucosal Vaccine Delivery Route for Self-Adjuvanting Lipid Core Peptides Targeting the Group A Streptococcal M Protein

BACKGROUND We investigated the lipid core peptide (LCP) system for mucosal vaccine delivery against infection with group A streptococcus (GAS)--the causative pathogen of rheumatic fever and rheumatic heart disease. METHODS An LCP vaccine formulation containing 2 different peptide epitopes of the antiphagocytic M protein of GAS--a conformational epitope from the carboxyterminal conserved C-repeat region and an aminoterminal serotypic epitope--was intranasally administered to mice with cholera toxin B subunit or without additional adjuvant. RESULTS Our data demonstrate that the LCP vaccine formulation induced the elicitation of antigen-specific systemic immunoglobulin G responses when administered with or without cholera toxin B subunit, whereas cholera toxin B subunit was required for the induction of antigen-specific mucosal immunoglobulin A responses. Immune serum samples from vaccinated mice were capable of opsonization of a homologous GAS strain, as well as opsonization of a heterologous GAS strain. Furthermore, mice were protected from GAS challenge following immunization with the LCP vaccine formulation, even in the absence of additional adjuvant. CONCLUSIONS These data support the potential of the LCP system in the development of a self-adjuvanting, synthetic, peptide-based mucosal GAS vaccine for the prevention of diseases caused by GAS.

Capillary Electrophoretic Analysis of Somatostatin Analog Peptides. Effect of Organic Solvents as Buffer Modifiers

Abstract Capillary electrophoretic (CE) method utilizing triethylammonium phosphate/organic modifier solvents as CE buffer was developed for analysis of new proprietary Somatostatin analog peptides synthetized in our laboratory. Acetonitrile, methanol, ethanol and i-propanol were applied as organic modifiers. Applicability of the systems in the analysis of Somatostatin analog peptides was evaluated and optimum conditions of the separation were determined. The effects of the organic solvents on the migration times of the S-analog peptides, elution order and selectivity in the CE systems were investigated. The migration time, elution order and selectivity can be influenced modifying the composition of the electrophoretic buffer with organic solvents. Applying different organic solvents as modifiers the elution order of the peptides can be changed in different ways. The effect of the alcohols on the CE migration time of the peptides is quite opposite to that of the acetonitrile. The degree of the effect of t...

ORM‐10103, a novel specific inhibitor of the Na+/Ca2+ exchanger, decreases early and delayed afterdepolarizations in the canine heart

At present there are no small molecule inhibitors that show strong selectivity for the Na+/Ca2+ exchanger (NCX). Hence, we studied the electrophysiological effects of acute administration of ORM‐10103, a new NCX inhibitor, on the NCX and L‐type Ca2+ currents and on the formation of early and delayed afterdepolarizations.

Comparison of high-performance liquid chromatography and capillary electrophoresis in the analysis of somatostatin analogue peptides

HPLC and CE methods were developed for analysis of somatostatin analogue (S-analogue) peptides utilizing triethylammonium phosphate-organic solvent modifier solvents as the CE buffer and HPLC eluent. Acetonitrile, methanol, ethanol and 2-propanol were applied as organic modifiers. The applicability of HPLC and CE systems was evaluated and compared. Optimum conditions for the separation were determined for both methods. Retention (migration) time, elution order and selectivity can be influenced by modifying the composition of the eluent (buffer) with organic solvents not only in HPLC but also in CE. Although the HPLC system reacted to changes in the organic solvent concentration in a much more sensitive way than the CE system did (from the point of view of retention time), CE proved to be a more suitable method for separating the peptides investigated. Baseline separation could be achieved within 6-9 min by CE, a result which was impossible to achieve with HPLC working in the isocratic mode. In CE the effect of the alcohols on migration times proved to be opposite to that of acetonitrile. Whereas ACN decreased, the alcohols increased the migration times in a concentration-dependent way. The results suggest that CE can be applied very advantageously in peptide analysis. Its performance regarding selectivity, resolution, theoretical plate number, duration and cost is comparable or sometimes superior to that of HPLC.

PLGA:poloxamer blend micro- and nanoparticles as controlled release systems for synthetic proangiogenic factors.

Tissue engineering is one of the most promising research areas in bioregenerative medicine. However, the restoration of biological functionalities by implanting bioartificially engineered tissues is still highly limited because of their lack of vascular networks. The use of proangiogenic molecules delivered from a controlled release device is a promising strategy to induce tissue vascularization. Indeed, the controlled release system can enhance the therapeutic effect in vivo of many short half-life drugs, while circumventing the need for repeated administrations. In this work, PLGA:poloxamer blend based micro- and nanoparticles have been developed for the sustained delivery of a recently developed synthetic proangiogenic compound: SHA-2-22. Drug-loaded PLGA:poloxamer blend microparticles were prepared by an oil-in-oil solvent extraction/evaporation technique. Drug-loaded PLGA:poloxamer nanoparticles were prepared by a modified solvent diffusion technique. These drug carriers were characterized with regard to their physicochemical properties, morphology, drug encapsulation efficiency and release kinetics in vitro. The results show that by adjusting the formulation conditions, it is possible to obtain PLGA:poloxamer micro- and nanoparticles with very high drug loadings, and with the capacity to release the active compound in a controlled way for up to one month. In vitro cell assays performed in an endothelial cell model confirmed the bioactivity of SHA-22-2 encapsulated in PLGA:poloxamer microparticles.

A lipophilic adjuvant carrier system for antigenic peptides

A lipoamino acid based synthetic peptide, (Lipid Core Peptide, LCP) derived from the conserved region of group A streptococci (GAS) was evaluated as potential candidate in a vaccine to prevent GAS-associated diseases, including rheumatic heart disease and post-streptococcal acute glomerulonephritis. Multiple copies of a peptide sequence from the bacterial surface M protein were incorporated into a lipid core and it was used to immunize mice with and without the application of adjuvant. The LCP construct had significantly enhanced immunogenicity compared with the monomeric peptide epitope. Furthermore, the peptides incorporated into the LCP system generated antibodies without the use of any conventional adjuvant.