Igor A. Prudchenko

Delta sleep inducing peptide (DSIP): effect on respiration activity in rat brain mitochondria and stress protective potency under experimental hypoxia

Neuromodulatory delta sleep inducing peptide (DSIP) seems to be implicated in the attenuation of stress-induced pathological metabolic disturbances in various animal species and human beings. Mitochondria, as cell organelles, are considered especially sensitive to stress conditions. In this work, the influence of DSIP and Deltaran((R))-a recently developed product based upon DSIP-on processes of oxidative phosphorylation and ATP production in rat brain mitochondria and rat brain homogenates was studied. A polarographic measurement of oxygen consumption was applied to evaluate the impact of DSIP on maximal rates of mitochondrial respiration and coupling of respiration to ATP production. We provide evidence that DSIP affected the efficiency of oxidative phosphorylation on isolated rat brain mitochondria. This peptide significantly increased the rate of phosphorylated respiration V3, while the rate of uncoupled respiration V(DNP) remaining unchanged. It enhanced the respiratory control ratio RCR and the rate of ADP phosphorylation. DSIP and Deltaran exhibited the same action in rat brain homogenates. We also examined the influence of DSIP under hypoxia when mitochondrial respiratory activity is altered. In rats subjected to hypoxia, we detected a significant stress-mediated reduction of V3 and ADP/t values. Pretreatment of rats with DSIP at the dose of 120 microgram/kg (i.p.) prior to their subjection to hypoxia completely inhibited hypoxia-induced reduction of mitochondrial respiratory activity. The revealed capacity of DSIP to enhance the efficiency of oxidative phosphorylation found in vitro experiments could contribute to understanding pronounced stress protective and antioxidant action of this peptide in vivo.

Effects of delta-sleep inducing peptide (DSIP) and some analogues on the activity of monoamine oxidase type A in rat brain under hypoxia stress

Metabolic effects of delta‐sleep inducing peptide (DSIP) under hypoxia stress were investigated in rats subjected to short‐term hypoxic conditions (about 0.26 Bar). It was found that DSIP partially restricted stress‐induced changes in activity of mitochondrial monoamine oxidase type A (MAO‐A) and serotonin level in rat brain. A number of DSIP analogues was tested and among them there were some compounds with enhanced ability to counteract hypoxia induced changes in MAO‐A activity and serotonin content in comparison with native neuropeptide.

JmjC-domain-containing histone demethylases of the JMJD1B type as putative precursors of endogenous DSIP.

Delta sleep inducing peptide (WAGGDASGE, DSIP) is a well known multifunctional regulatory peptide. Numerous studies have confirmed its stress-protective and adaptive activity which is independent of the origin or nature of the stress or other harmful factors. However, the biosynthetic origin of DSIP remains obscure, since nothing is known of its protein precursor(s) and their encoding gene(s). We have performed a comprehensive analysis of available gene and protein databases for homologous peptide sites within mammalian resources including man. A family of Jumonji C (JmjC)-domain-containing histone demethylases was shown to contain a sequence fragment closely homologous to DSIP. One type of these ubiquitous and phylogenetically ancient proteins encoded by JMJD1B gene includes the WKGGNASGE sequence that differs from DSIP by only 2 amino acid residues in positions 2 and 5. The respective peptide was synthesized and its biological effects were evaluated in a preliminary way in the forced swimming and antitoxic tests. We suggest that the histone demethylases of the JmjC-group containing DSIP-related region can be considered as possible protein precursors of endogenous peptides with DSIP-like activity.

Thrombin receptor agonist peptide entrapped in poly(D,L)-lactide-co-glycolide microparticles: Preparation and characterization

Thrombin receptor agonist peptide (TRAP-6) could advantageously replace thrombin in terms of accelerating wound healing being less expensive and more stable. To promote TRAP-6 pharmacological action as a tissue reconstruction stimulator this study investigated its entrapment within poly(D,L)-lactide-co-glycolide (PLGA) microparticles. Due to its low molecular weight and water solubility, TRAP-6 microencapsulated form is expected to be more useful. This paper reports TRAP-6 microencapsulation by a double (w/o/w) emulsion-evaporation technique. TRAP-6 release kinetics were evaluated by both chemical (HPLC) and biological assays in vitro. The results revealed a high level of TRAP-6 sensitivity to physico-chemical events during the microencapsulation. The surface morphology difference between control microparticles (without TRAP-6) and microparticles with entrapped TRAP-6 during in vitro degradation highlighted a particular role of TRAP-6. The results can allow one to optimize the microencapsulation procedure and to encounter a new promising approach to development of biodegradable polymer drug delivery systems for wound healing.

Biodegradable microparticles loaded with thrombin receptor agonist peptide for gastric ulcer treatment in rats

The aim of the current paper was to elaborate an immobilization method of thrombin receptor agonist peptide (TRAP-6) in biodegradable biocompatible poly(d,l)-lactide-co-glycolide (PLGA) microparticles and to demonstrate the effect of the entrapped peptide for tissue repair, namely for a gastric ulcer treatment in rats. TRAP-6 was entrapped in polymer using w/o/w double emulsion-evaporation technique. The morphology of empty and TRAP-6 loaded microparticles was evaluated by light and scanning electron microscopy (SEM). In vitro release kinetics profile of TRAP-6 from microparticles was studied by HPLC. To investigate gastric mucosal protection effect in vivo, TRAP-6-loaded microparticles were administered in a rat stomach after a previous mucosal injury (a gastric ulcer). Microparticles with entrapped TRAP-6 were found to reduce both an inflammation and proliferation phases of wound healing, and thus accelerated tissue repair in rats.

Delta-sleep inducing peptide entrapment in the charged macroporous matrices

Various biomolecules, for example proteins, peptides etc., entrapped in polymer matrices, impact interactions between matrix and cells, including stimulation of cell adhesion and proliferation. Delta-sleep inducing peptide (DSIP) possesses numerous beneficial properties, including its abilities in burn treatment and neuronal protection. DSIP entrapment in two macroporous polymer matrices based on copolymer of dimethylaminoethyl methacrylate and methylen-bis-acrylamide (Co-DMAEMA-MBAA) and copolymer of acrylic acid and methylen-bis-acrylamide (Co-AA-MBAA) has been studied. Quite 100% of DSIP has been entrapped into positively charged Co-DMAEMA-MBAA matrix, while the quantity of DSIP adsorbed on negatively charged Co-AA-MBAA was only 2-6%. DSIP release from Co-DMAEMA-MBAA was observed in saline solutions (0.9% NaCl and PBS) while there was no DSIP release in water or 25% ethanol, thus ionic strength was a reason of this process.

Entrapment and in vitro release of delta-sleep inducing peptide from polymer hydrogels based on modified polyvinyl alcohol

The aim of this study was to entrap delta-sleep inducing peptide (DSIP) in cross-linked poly(vinyl alcohol)-based hydrogels of different structures and to determine kinetics of the peptide release from these hydrogels using an in vitro model. Isotropic and macroporous hydrogels based on poly(vinyl alcohol) acrylic derivative (Acr-PVA) and also macroporous epoxy groups containing hydrogels synthesized by copolymerization of this macromer and glycidyl methacrylate, have been used in this study. Isotropic hydrogels were prepared at positive temperatures while macroporous ones were obtained by formation in cryo-conditions. The peptide was entrapped into macroporous PVA hydrogels by adding the peptide solution onto preformed matrices, while peptide immobilization on PVA-GMA hydrogels, containing free epoxy groups, was carried out by sorption of peptide from its aqueous solution. In the case of DSIP entrapment into isotropic PVA gel the peptide solution was added into the polymer mixture at hydrogel formation. The kinetics of peptide release from hydrogels was studied by incubating matrices in PBS solution (pH 7.4), in physiological solution (0.9% NaCl) and in water. DSIP concentration in supernatants was determined by reverse-phase HPLC. Incubation of macroporous PVA gels in PBS, 0.9% NaCl, and water for 30 min caused release of 74, 70, and 64% DSIP, respectively, and this processes completed within 3 h. From hydrogel containing epoxy groups the release of neither peptide nor its degradation products was observed even after incubation for 48 h. For freshly prepared isotropic hydrogel the release kinetics was as follows: 27 and 78% DSIP were released within first 30 min and 33 h, relatively. For the lyophilized hydrogel samples the peptide release was 63% after incubation for 30 min, while drying of samples at room temperature for 3 days caused significant peptide loss because of its structure damage.

Biodegradable microparticles with immobilized peptide for wound healing

Thrombin receptor agonist peptide (TRAP-6) may be successfully used instead of thrombin to stimulate regeneration of damaged tissues. Thrombin application is limited by its high price, instability, and proin-flammatory effect at high concentrations. Immobilization of TRAP-6 into a matrix based on lactic and glycolic acid copolymer (PLGA) prevents its destruction by peptidases located in the wound and can also provide controlled release of the peptide. PLGA microparticles with the immobilized peptide were prepared by the double emulgation method. The presence of the immobilized peptide increased the porosity of the microparticle surface detected by scanning electron microscopy. Kinetics of the TRAP-6 release was characterized by a dramatic increase in its concentration in buffer solution (pH 7.5) during the first 2 h after the experiment beginning, and the complete release of the peptide after 20 h. An investigation of TRAP-6 destruction by scanning electron microscopy revealed the increase in the microparticle size and surface porosity already after one day of incubation, and the destroyed microparticles were aggregated by the seventh day of the incubation. Thus, peptide immobilization into PLGA microparticles may be employed for elaboration of a prolonged action preparation with the controlled release of the active agent (peptide).