Dalargin Research Articles

Nanoparticles Based on Quaternary Ammonium Chitosan-methyl-β-cyclodextrin Conjugate for the Neuropeptide Dalargin Delivery to the Central Nervous System: An In Vitro Study.

Peptide oral administration is a hard goal to reach, especially if the brain is the target site. The purpose of the present study was to set up a vehicle apt to promote oral absorption of the neuropeptide dalargin (DAL), allowing it to cross the intestinal mucosal barrier, resist enzymatic degradation, and transport drugs to the brain after crossing the blood–brain barrier. Therefore, a chitosan quaternary ammonium derivative was synthesized and conjugated with methyl-β-cyclodextrin to prepare DAL-medicated nanoparticles (DAL-NP). DAL-NP particle size was 227.7 nm, zeta potential +8.60 mV, encapsulation efficiency 89%. DAL-NP protected DAL from degradation by chymotrypsin or pancreatin and tripled DAL degradation time compared to non-encapsulated DAL. Use of DAL-NP was safe for either Caco-2 or bEnd.3 cells, with the latter selected as a blood–brain barrier model. DAL-NP could also cross either the Caco-2 or bEnd.3 monolayer by the transepithelial route. The results suggest a potential DAL-NP ability to transport to the brain a DAL dose fraction administered orally, although in vivo experiments will be needed to confirm the present data obtained in vitro.

Improvement of Peptide Affinity and Stability by Complexing to Cyclodextrin-Grafted Ammonium Chitosan.

Cyclodextrin-grafted polymers are attractive biomaterials that could bring together the host–guest complexing capability of pristine cyclodextrin and the pharmaceutical features of the polymeric backbone. The present paper is aimed at characterizing the potential application of ammonium–chitosan grafted with 2-methyl-β-cyclodextrin (N+-rCh-MCD) as the functional macromolecular complexing agent for the oral administration of the neuropeptide dalargin (DAL). Specific NMR characterization procedures, along with UV and fluorescence techniques, as well as biological in vitro assessments have been performed. The results indicate that N+-rCh-MCD forms water-soluble complexes with DAL, with a prevalent involvement of Tyr or Phe over Leu and Ala residues. The association constant of DAL with the polymeric derivative is one order of magnitude higher than that with the pristine cyclodextrin (Ka: 2600 M−1 and 120 M−1, respectively). Additionally, N+-rCh-MCD shields DAL from enzymatic degradation in gastrointestinal in vitro models with a three-fold time delay, suggesting a future pharmaceutical exploitation of the polymeric derivative. Therefore, the greater affinity of N+-rCh-MCD for DAL and its protective effect against enzymatic hydrolysis can be attributed to the synergistic cooperation between cyclodextrin and the polymer, which is realized only when the former is covalently linked to the latter.

INFLUENCE OF POLYOXIDONIUM, Poly(I:C), DALARGIN ON THE PROTECTIVE EFFICACY OF YERSINIA PESTIS VACCINE STRAIN EV LINE NIIEG IN EXPERIMENTAL PLAGUE

In this study, the use of immunoadjuvants polyoxidonium (azoximer bromide), Poly (I:C) as a synthetic analog of double-stranded RNA (TLR3 ligand), and synthetic analog of leu-enkephalin dalargin (DA) was experimentally investigated for their potential to minimize ImD50 Yersinia pestis vaccine strain EV line NIIEG co-administrated via invasive (subcutaneous) and noninvasive (intranasal) routes in lethal bubonic and pneumonic models of plague followed by challenge with virulent Y. pestis strains of the main and non-main subspecies from various natural plague foci. The data showed that in all cases immunoadjuvants significantly increased protective efficacy of Y. pestis vaccine strain EV line NIIEG co-administrated to BALB/c inbreed mice in case of lethal challenge with virulent Y. pestis strains in spite of varying magnitude of humoral immune response. Y. pestis vaccine strain EV line NIIEG formulated with polyoxidonium provided more effective protection against lethal challenge with wild-type high virulent strain Y. pestis in pneumonic model of plague. Polyoxidonium introduced into vaccine formula resulted in four-fold rise in total survival in animals with pneumonic plaque. Feasibility of using immunoadjuvants for regimen of specific and urgent plaque prevention is justified.

The Bactericidal Activity of Temporin Analogues Against Methicillin Resistant Staphylococcus aureus.

Staphylococcus aureus is a major infectious agent responsible for a plethora of superficial skin infections and systemic diseases, including endocarditis and septic arthritis. Recent epidemiological data revealed the emergence of resistance to commonly used antibiotics, including increased numbers of both hospital- and community-acquired methicillin-resistant S. aureus (MRSA). Due to their potent antimicrobial functions, low potential to develop resistance, and immunogenicity, antimicrobial peptides (AMPs) are a promising alternative treatment for multidrug-resistant strains. Here, we examined the activity of a lysine-rich derivative of amphibian temporin-1CEb (DK5) conjugated to peptides that exert pro-proliferative and/or cytoprotective activity. Analysis of a library of synthetic peptides to identify those with antibacterial potential revealed that the most potent agent against multidrug-resistant S. aureus was a conjugate of a temporin analogue with the synthetic Leu-enkephalin analogue dalargin (DAL). DAL-PEG-DK5 exerted direct bactericidal effects via bacterial membrane disruption, leading to eradication of both planktonic and biofilm-associated staphylococci. Finally, we showed that accumulation of the peptide in the cytoplasm of human keratinocytes led to a marked clearance of intracellular MRSA, resulting in cytoprotection against invading bacteria. Collectively, the data showed that DAL-PEG-DK5 might be a potent antimicrobial agent for treatment of staphylococcal skin infections.

Conjugate of Enkephalin and Temporin Peptides as a Novel Therapeutic Agent for Sepsis.

Antimicrobial peptides (AMPs) exhibit a wide spectrum of actions, ranging from a direct bactericidal effect to multifunctional activities as immune effector molecules. The aim of this study was to examine the anti-inflammatory properties of a DAL-PEG-DK5 conjugate composed of a lysine-rich derivative of amphibian temporin-1CEb (DK5) and dalargin (DAL), the synthetic Leu-enkephalin analogue. Detailed study of the endotoxin-neutralizing activity of the peptide revealed that DAL-PEG-DK5 interacts with LPS and the LPS binding protein (LBP). Moreover, DAL-PEG-DK5 prevented dimerization of TLR4 at the macrophage surface upon LPS stimulation. This inhibited activation of the NF-κB signaling pathway and markedly reduced pro-inflammatory cytokine production. Finally, we showed that aggregation of DAL-PEG-DK5 into amyloid-like structures induced by LPS neutralized the endotoxin proinflammatory activity. Consequently, DAL-PEG-DK5 reduced morbidity and mortality in vivo, in a mouse model of endotoxin-induced septic shock. Collectively, the data suggest that DAL-PEG-DK5 is a promising therapeutic compound for sepsis.

The impact of Immunomodulators on Reactivity the immune System cells in Model Vaccinal process against Tularemia

Investigated is the effect of azoximer bromide (polioxidonium - PO) and dalargin (DA) on the immune system cell reactivity in the anti-tularemia vaccine prosess modeling in Balb/c mice. The animals were immunized subcutaneously with a dose of 10 4 live microbial cells of vaccine strain Francisella tularensis 15 NIIEG. PO or DA subcutaneously administered 60 min before vaccination, respectively, in doses of 4 mg and 2 mg. On 3rd and 21st days of immunogenesis the peripheral organs of the immune system and the contents of the abdominal cavity were investigated. Cell response to vaccination and investigated immunomodulators evaluated by flow cytometry (CyAn ADP) for two light scatter parameters and for changing the proliferating and apoptotic cell number. Additionally, for the characterization of changes in the functional status of lymphocytes nuclei the light microscopy was used and titers of specific antibodies were determined. Installed is the different response of intact mice lymphocytes and phagocytes to PO and DA introduction. As distinct from PO, DA stimulated the lymphocete proliferation and macrophage apoptosis by acting on their cytoplasmic granules. PO activated the migration of phagocytes with high cytoplasmic granules from mice abdominal cavity in the spleen. Only when using the PO as an immunomodulator in anti-tularemia vaccine process modeling recorded a significant increase in specific antibody titers against the background of a long-term functional splenocyte activation and macrophages reduced macrophage damage intensity in the spleen and abdomen.

Effects of dalargin on excitation induced by L-glutamate agonists in the frog vestibular organs.

We have used an electrophysiological approach to investigate the action of a synthetic analog of leu-enkephalin dalargin (DAL) on chemically induced afferent activity in the frog vestibular organs. Administration of 5.0 microM kainic acid (KA), 5.0 microM (AMPA) and 50 microM NMDA produced an increase in the frequency of the resting discharge. Firing evoked by KA, AMPA or NMDA could be depressed by administration of 1 nM Dal by 55.5 +/- 9.9% (n = 10, p < 0.05), 64.5 +/- 11.2% (n = 13, p < 0.05) and 21.3 +/- 11.1% (n = 14, p = 0.051), respectively. Thus, the frequency decrease under NMDA was statistically non-significant. These results show that non-NMDA, but not NMDA subtypes of receptors are mostly involved in opioid action at the vestibular organs of the frog.

Opioid peptides may modulate afferent synaptic transmission in the frog semicircular canal

With the aid of external perfusion techniques we tested the action of a synthetic analog of leu-enkephalin dalargin (DAL) and the specific opiate antagonist naloxone (NAL) at the afferent synapses of the semicircular canals in the frog. DAL at concentrations of 0.1- 100 nM depresses the resting discharge frequency. NAL (1-100 μ M) antagonizes responses induced by DAL, which suggests a specific DAL action. DAL inhibits the excitatory action of L-glutamate (L-GLU). The data obtained speak in favor of the presence of opiate receptors and that the neurotransmitter L-GLU is involved in opiate action at the peripheral vestibular system of the frog. The results appear to demonstrate that opioid peptides may act as neuromodulators in this system.

Actions of dalargin upon single unit activity in the ampullae of Lorenzini of the skate Raja clavata.

In the present study we have shown by single afferent unit recording in electroreceptors of skates (the ampullae of Lorenzini) that the synthetic analogue of leu-enkephalin, dalargin (DAL) at concentrations between 10(-6)-10(-10) M cause a concentration-dependent decrease in the resting discharge frequency as well as a decrease in stimulus evoked responses. The specific opiate antagonist naloxone (NAL, 10(-6) M) antagonizes responses induced by DAL. DAL depresses the excitatory action of L-glutamate (L-GLU). The data obtained speak in favour of the presence of opiate receptors at the synaptic membrane of the ampullae of Lorenzini.