Lysozyme-dalargin self-organization at the aqueous-air and liquid-liquid interfaces.

An experimental study of protein-peptide binding was performed by means of radiochemical and spectroscopic methods. Lysozyme and dalargin were chosen due to their biological and physiological importance. By means of tensiometry and radiochemical assays, it was found that dalargin possesses rather high surface activity at the aqueous-air and aqueous-p-xylene interfaces to be substituted by protein. Dalargin forms a hydrophobic complex with lysozyme in which the secondary structure of lysozyme is preserved. When lysozyme forms a mixed adsorption layer with dalargin at the aqueous-air surface, the peptide prevents protein from concentrating in the subsurface monolayer. In the presence of p-xylene protein in the interface, reorganization occurs quickly, so there is no lag in the interfacial tension time dependence. The interfacial tension in this case is controlled by protein and/or protein-peptide complexes. An increase in the enzymatic activity of lysozyme in the presence of dalargin was confirmed by a docking model that suggests the formation of hydrogen bonds between dalargin and amino acid residues in the active site.

New Combined Eye Gel with Alpha-2-Beta Interferon.

The results of the development of combined eye gel with interferon alpha-2-beta are presented. Experimental samples of the gel based on different gelling agents were prepared and their biotechnological and technological characteristics (the absence of the cytotoxic effect, aggregation stability, osmotic activity, bioadhesion, and rheological parameters) were evaluated. The composition with hydroxyethyl cellulose, Natrosol 250HHX, in a concentration of 1.5% as a gelling agent showed the best results and the best one-year stability.

[The experimental clinical substantiation of treatment of patients with odontogenic phlegmon of maxillofacial area using Dаlargin in complex therapy.]

The patients with odontogenic phlegmon of maxillofacial area suffer from immune depression at the surgical stage of treatment that can unfavorably affect their rehabilitation. The study analyzes possibility of application of medication Dalargin in complex treatment of patients during post-operation period. The experimental study established components of immune system affected by medication. Thereupon, Dalargin was applied in clinical practice of treatment of patients with course of disease of average severity. The study proved immunomodulatory characteristics of Dalargin effecting various fractions of sub-populations of lymphocytes and manifesting by way of decreasing of initially high indices and increasing of initially low indices that is of great importance during treatment of patients with odontogenic hormones, especially in case of severe course of disease.

Hydrophilic interaction liquid chromatography for dalargin separation from its structural analogues and side products.

Retention behaviour of Dalargin and five peptide analogues of Leu-enkephalin, has been extensively studied by hydrophilic interaction liquid chromatography (HILIC) on a bare silica stationary phase (Atlantis® HILIC silica). The influence of buffer pH, ionic strength, and organic modifier content on peptide retentions was examined. Variation of organic modifier content (70-90% ACN) shows that, as expected, the most polar peptide, Dalargin, is the most retained. Moreover, at acidic pH, the retention mechanism for all the peptides studied seems to rely, mainly, on adsorption phenomenon. By varying the pswH buffer (between 4.4-7.5), we observed that the retention of all the peptides was mainly governed by their total number of charges, whatever the variation (increase or decrease) of their retention factor. At pswH 7.5, an increase of the cationic counter-ion concentration (NH4+) lead to a decrease of the retention factor of Dalargin, suggesting a weak cation exchange for this peptide. For the other peptides, the variation of the retention factors was negligible between 5-15mM. Above 15mM, the retention factors of all the peptides increased, probably due to an increase of the water layer thickness at the surface of the stationary phase. In the second part of the study, qualitative analysis of non-purified dalargin, resulting from solid-phase synthesis, was realized. Optimisation of the separation of the target peptide from its side products has been first performed with UV detection. Then, by coupling the HILIC column with ESI-MS, using the optimal separation conditions, it was possible to identify Dalargin and to propose the amino-acids sequence of its side-products.

[THE INFLUENCE OF DEFICIT OF ENDOGENOUS NEUROPEPTIDES ON THE CLINICAL COURSE OF CORONARY HEART DISEASE].

The study is aimed at elucidating the relationship between the blood b-endorphin level in patients with coronary heart disease (CHD) with metabolic syndrome (MS) and cardiovascular risk factors and evaluating the possibility to correct them by dalargin therapy. The study included 123 patients (61 men and 62 women) at the mean age 57.6±5,2 years randomized into 2 groups. The patients of group 1 (n=63) were given the standard treatment, those of group 2 (n=60) additionally received 2 mg/day of dalargin for 10 days (3 courses during 3 months). The group of comparison (n=84) contained 84 CHD patients without MS. Biochemical and immunological characteristics were measured by immuno enzyme and immunochemiluminescent assays before and 3 months after treatment. The study revealed inverse correlation between b-endorphin levels and those of leptin, insulin, cortisol, TNF-a, IL-6, oxidized LDLP, triglycerides (TG), and HDLP cholesterol. Standard therapy resulted in a 6.5% reduction of insulin level, 9,4% , 6,1%, and 17,4% reduction of TNF-a , IL-6, TG levels respectively; it increased the HDLP cholesterol level by 10,3% (p<0,05 for all values) but did not change other parameters of interest. Dalargin therapy caused a 32,6% and 17,4%, rise in the b-endorphin and HDLP cholesterol levels but decreased leptin, insulin, cortisol, TNF-a, IL-6, LDLP, and tG levels by 36,1%, 22,4%, 23,9%, 55%, 56,3%, 14% and 27,2% respectively (p<0,001). It is concluded that the decrease of the blood b-endorphin level in the patients with coronary heart disease and metabolic syndrome is associated with enhanced blood atherogenicity, hyperinsulinemia, hypercortisolemia, activation of pro-inflammatory cytokines and lipid peroxidation. Supplementation of conventional therapy with dalargin results in the increased b-endorphin level, enhanced anti-atherogenic effect, reduced activity of pro-inflammatory cytokines and lipid peroxidation, reduction of leptin, insulin and cortisol levels.

Correction of Negative Effect of Antenatal Hypoxia on Liver Tissue Homeostasis in Newborn Albino Rats with Opioid Peptides.

We studied the possibility of correction of the negative effects of antenatal hypoxia on the liver tissue homeostasis in 7-day-old albino rats by administration of opioid peptides in a dose of 100 µg/kg on postnatal days 2-6. Administration of mixed µ/δ-opioid receptor agonist Dalargin neutralized deviations of gravimetric indicators, parameters of proliferative activity, and activity of the nucleolar apparatus of hepatocytes. Administration of the non-opiate Leu-enkephalin analogue did not normalize gravimetric parameters and nucleolar apparatus parameters, however, it significantly increased the pool of proliferating hepatocytes. Both peptides significantly reduced the intensity of free radical oxidation, improved antioxidant antiradical defense and resistance to peroxidation in the liver tissue of animals subjected to antenatal hypoxia.

[ЕXPERIMENTAL ESTIMATION OF THE LOCAL APPLICATION EFFICACY OF BIOLOGICAL STIMULATOR FOR THE SOFT TISSUES REPARATION IN TREATMENT OF CHRONIC GASTRIC ULCERS].

Results of experimental investigation for studying of the local application possibility for the reparative processes stimulators, including autologic purified lipoaspirate, dalargin and a platelet­rich plasma (PRP) for treatment of chronic gastric ulcers, were analyzed. The advantages of PRP application, as the growth factors donator, independently and in combination with dalargin, were proved. The PRP capacity to stimulate the fibroblasts activity and creation of vessels in young connective tissue was proved, what have leaded to the ulcer tissues oxygenation improvement and cellular proliferation promotion, acceleration of the connective tissue maturation, and the ulcer healing.

[THE ANTIOXIDANT EFFECT OF DALARGIN IN PATIENTS WITH CORONARY HEART DISEASE AND METABOLIC SYNDROME].

The aim of this study was to evaluate the effect of dalargin on the state of lipid peroxidation (LPO) and antioxidant system in patients with coronary heart disease (CHD) and on the background of metabolic syndrome (MS) in a group of 123 patients with stable coronary artery disease and MS (mean age 56.7 ± 5.1 years). For this purpose, the blood redox potential (EP), total antioxidant activity (TAA), level of oxidized low density lipoproteins (LDL), and activity of superoxide dismutase (SOD) were compared between the group receiving a standard medical therapy (ST) for coronary heart disease (group 1, n = 63) and that with supplementary dalargin administration (ST + D) in a dose of 1 mg intranasally twice a day for 10 days (group 2, n = 60), using the same dose for 10 days in the next two months (total 3 courses over 3 months). It was found that patients with CHD + MS upon 3-month ST showed no statistically significant changes in parameters characterizing the oxidative potential of blood (EP) and antioxidant protection of blood (oxidized LDL level, SOD activity). The inclusion of dalargin into therapy (ST + D) led to a significant decrease in the oxidative stress parameters (blood EP by 10.5%, oxidized LDL level by 14%, p < 0.001) and increase in the blood antioxidant properties (SOD activity by 36.1%, TAA by 25.3%, p < 0.001).

[Comparison of pharmacological renal preconditioning with dalargin and lithium ions in the model of gentamycin-induced acute renal failure].

PURPOSE: To examine the efficacy of renal preconditioning effect of dalargin and lithium ions by observing the model of gentamycin-induced acute renalfailure. MATERIALS AND METHODS: The experiments were performed on white rats, male. The influence of dalargin and lithium ions on the development of gentamycin-induced acute renalfailure was studied in vivo. On the first 24 hours after dalargin injections were terminated, the rats were euthanized humanly. After this we took the blood for a biochemistry study and a renal culture for biochemical test and also for the test of gsk-3ß activity. Concentrations of creatinine and urea were studied in serum. The culture samples of renal tubular epithelium before insertion of gentamycin were incubated in dalargin or lithium ions in different concentrations. After that the substratum was immediately changed to gentamycin in different concentrations also and the incubated for 24 hours. After all the standards MTT-test was performed (based on the ability of living cells to reduce the unpainted form by 3-4,5-dimethylthiazol-2-yl-2,5-difenilterarazola to blue crystalline farmazan). RESULTS: Lithium precondition leads to the 250% increase of gsk-3ß concentration (p = 0.035). The same results were observed after injection of dalargin in 50 mcg/kg concentration. Concentration of creatinine was 44% lower in the dalargin group than in the control group (p = 0.022). Concentration of creatinine was 32% lower in the lithium group than in the control group (p = 0.030). Concentration of urea was 27% lower in the lithium group than in the control group (p = 0.049). Morphological inflammatory changes in the control group were more significant also. In vitro studies showed the maximum efficacy in the lithium group. The most effective dalargin concentration was 5 mg/ml. CONCLUSION: Lithium and dalargine preconditioning lowers the signs of gentamycine induced acute renal failure and damage rate of renal parenchyma in vivo and in vitro.

Use of electrochemical oxidation and model peptides to study nucleophilic biological targets of reactive metabolites: the case of rimonabant.

Electrochemical oxidation of drug molecules is a useful tool to generate several different types of metabolites. In the present study we developed a model system involving electrochemical oxidation followed by characterization of the oxidation products and their propensity to modify peptides. The CB1 antagonist rimonabant was chosen as the model drug. Rimonabant has previously been shown to give high covalent binding to proteins in human liver microsomes and hepatocytes and the iminium ion and/or the corresponding aminoaldehyde formed via P450 mediated α-carbon oxidation of rimonabant was proposed to be a likely contributor. This proposal was based on the observation that levels of covalent binding were significantly reduced when iminium species were trapped as cyanide adducts but also following addition of methoxylamine expected to trap aldehydes. Incubation of electrochemically oxidized rimonabant with peptides resulted in peptide adducts to the N-terminal amine with a mass increment of 64 Da. The adducts were shown to contain an addition of C5H4 originating from the aminopiperidine moiety of rimonabant. Formation of the peptide adducts required further oxidation of the iminium ion to short-lived intermediates, such as dihydropyridinium species. In addition, the metabolites and peptide adducts generated in human liver microsomes were compared with those generated by electrochemistry. Interestingly, the same peptide modification was found when rimonabant was coincubated with one of the model peptides in microsomes. This clearly indicated that reactive metabolite(s) of rimonabant identical to electrochemically generated species are also present in the microsomal incubations. In summary, electrochemical oxidation combined with peptide trapping of reactive metabolites identified a previously unobserved bioactivation pathway of rimonabant that was not captured by traditional trapping agents and that may contribute to the in vitro covalent binding.

Use of halogenated precursors to define a transcription time window after treatment with hypometabolizing molecules.

A new method is proposed which combines the high spatial resolution of transmission electron microscopy with information on the dynamics of transcription. Incorporation of two different RNA precursors was used to define a time transcription window on cultured cells treated with hypometabolizing peptides which are known to modulate transcription. This procedure allows detecting a single fibril of newly synthesized RNA in the time range in which it is transcribed.

Co-localization of hypocretin-1 and leucine-enkephalin in hypothalamic neurons projecting to the nucleus of the solitary tract and their effect on arterial pressure.

Experiments were done to investigate whether hypothalamic hypocretin-1 (hcrt-1; orexin-A) neurons that sent axonal projections to cardiovascular responsive sites in the nucleus of the solitary tract (NTS) co-expressed leucine-enkephalin (L-Enk), and to determine the effects of co-administration of hcrt-1 and D-Ala2,D-Leu5-Enkephalin (DADL) into NTS on mean arterial pressure (MAP) and heart rate. In the first series, in the Wistar rat the retrograde tract-tracer fluorogold (FG) was microinjected (50nl) into caudal NTS sites at which L-glutamate (0.25 M; 10 nl) elicited decreases in MAP and where fibers hcrt-1 immunoreactive fibers were observed that also contained L-Enk immunoreactivity. Of the number of hypothalamic hcrt-1 immunoreactive neurons identified ipsilateral to the NTS injection site (1207 ± 78), 32.3 ± 2.3% co-expressed L-Enk immunoreactivity and of these, 2.6 ± 1.1% were retrogradely labeled with FG. Hcrt-1/L-Enk neurons projecting to NTS were found mainly within the perifornical region. In the second series, the region of caudal NTS found to contain axons that co-expressed hcrt-1 and L-Enk immunoreactivity was microinjected with a combination of hcrt-1 and DADL in α-chloralose anesthetized Wistar rats. Microinjection of DADL into NTS elicited depressor and bradycardia responses similar to those elicited by microinjection of hcrt-1. An hcrt-1 injection immediately after the DADL injection elicited an almost twofold increase in the magnitude of the depressor and bradycardia responses compared to those elicited by hcrt-1 alone. Prior injections of the non-specific opioid receptor antagonist naloxone or the specific opioid δ-receptor antagonist ICI 154,129 significantly attenuated the cardiovascular responses to the combined hcrt-1-DADL injections. Taken together, these data suggest that activation of hypothalamic-opioidergic neuronal systems contribute to the NTS hcrt-1 induced cardiovascular responses, and that this descending hypothalamo-medullary pathway may represent the anatomical substrate by which hcrt-1/L-Enk neurons function in the coordination of autonomic-cardiovascular responses during different behavioral states.

Nephroprotective effect of GSK-3ß inhibition by lithium ions and δ-opioid receptor agonist dalargin on gentamicin-induced nephrotoxicity.

Nephrotoxicity and ototoxicity are the most considerable side effects of aminoglycoside antibiotics, such as gentamicin that seriously limits its application in medicine. The major mechanism of negative effect of gentamicin on kidney cells involves damage of mitochondria and induction of an oxidative stress that causes cell death resulting in kidney dysfunction. In this work we compared effects of the lithium ions and δ-opioid receptors agonist, dalargin on gentamicin-induced kidney injury. It was revealed that LiCl and dalargin treatment reduced renal tubular cell death and diminished kidney injury caused by gentamicin. Both LiCl and dalargin were found to enhance phosphorylation of glycogen synthase kinase 3ß in the kidney which points to induction of nephroprotective signaling pathways. Thus, we conclude that lithium ions and dalargin might be considered as novel promising agents for future use to prevent negative consequences of therapy with aminoglycoside antibiotics.

Nanofiber-based delivery of therapeutic peptides to the brain.

The delivery of therapeutic peptides and proteins to the central nervous system is the biggest challenge when developing effective neuropharmaceuticals. The central issue is that the blood-brain barrier is impermeable to most molecules. Here we demonstrate the concept of employing an amphiphilic derivative of a peptide to deliver the peptide into the brain. The key to success is that the amphiphilic peptide should by design self-assemble into nanofibers wherein the active peptide epitope is tightly wrapped around the nanofiber core. The nanofiber form appears to protect the amphiphilic peptide from degradation while in the plasma, and the amphiphilic nature of the peptide promotes its transport across the blood-brain barrier. Therapeutic brain levels of the amphiphilic peptide are achieved with this strategy, compared with the absence of detectable peptide in the brain and the consequent lack of a therapeutic response when the underivatized peptide is administered.

Intensity of proliferative processes and degree of oxidative stress in the mucosa of the ileum in Crohn's disease.

The intensity of proliferative processes (estimated from Ki-67 expression) and degree oxidative stress (chemiluminescence assay) in biopsy specimens from the terminal portion of the ileal mucosa were studied in patients with Crohn's disease. Crohn's disease is characterized by hyper-regenerative processes in the ileal mucosa. The labeling index (Ki-67 expression) in biopsy specimens from the intact ileal mucosa in patients with the irritable bowel syndrome (reference group) was 10.64±0.62%. The corresponding values in patients receiving monotherapy with mesalazine (group 1) and combination therapy with mesalazine and dalargin (group 2) were 24.05±1.17 and 22.90±0.92%, respectively. Analysis of free radical oxidation showed that this state is accompanied oxidative stress. Spontaneous and H(2)O(2)-induced luminol-dependent chemiluminescence in biopsy specimens from the ileal mucosa was 1.8-2.3-fold higher compared to the reference group. After therapy, the labeling index in groups 1 and 2 decreased to 18.60±1.18 and 14.38±0.81%, respectively. Histologically, normalization of the disease symptoms was more pronounced after combination therapy. The decrease in free radical oxidation in this group of patients was more pronounced than after mesalazine monotherapy. Our results suggest that oxidative stress plays a role in the hyper-regenerative reaction.

Factors that restrict the cell permeation of cyclic prodrugs of an opioid peptide, part 3: Synthesis of analogs designed to have improved stability to oxidative metabolism.

Previously, our laboratory reported that cyclic peptide prodrugs of the opioid peptide H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE) are metabolized by cytochrome P450 (CYP450) enzymes, which limits their systemic exposure after oral dosing to animals. In an attempt to design more metabolically stable cyclic prodrugs of DADLE, we synthesized analogs of DADLE cyclized with a coumarinic acid linker (CA; CA-DADLE), which contained modifications in the amino acid residues known to be susceptible to CYP450 oxidation. Metabolic stability and metabolite identification studies of CA-DADLE and its analogs were then compared using rat liver microsomes (RLM), guinea pig liver microsomes (GPLM), and human liver microsomes (HLM), as well as recombinant human recombinant cytochrome P450 3A4 (hCYP3A4). Similar to the results observed for CA-DADLE, incubation of its analogs with RLM, GPLM, and HLM resulted in monohydroxylation of an amino acid side chain on these cyclic prodrugs. When CA-DADLE was incubated with hCYP3A4, similar oxidative metabolism of the peptide was observed. In contrast, incubation of the CA-DADLE analogs with hCYP3A4 showed that these amino-acid-modified analogs are not substrates for this CYP450 isozyme. These results suggest that the amino-acid-modified analogs of CA-DADLE prepared in this study could be stable to metabolic oxidation by CYP3A4 expressed in human intestinal mucosal cells.

Factors that restrict the cell permeation of cyclic prodrugs of an opioid peptide, part 4: Characterization of the biopharmaceutical and physicochemical properties of two new cyclic prodrugs designed to be stable to oxidative metabolism by cytochrome P-450 enzymes in the intestinal mucosa.

The biopharmaceutical and physicochemical properties of two new cyclic prodrugs (CA-[cychexalanine (Cha(4)), D-Leu(5) ]-Enkephalin (Enk) and coumarinic acid (CA)-[Cha(4), D-Ala(5)]-Enk) of opioid peptides that were designed to be stable to oxidative metabolism by cytochrome P-450 enzymes in the intestinal mucosa are described in this paper. Two-dimensional nuclear magnetic resonance studies and molecular dynamics simulations showed that these cyclic prodrugs exhibit unique solution conformations (i.e., type I ß-turns), which are favorable for transcellular permeation. The calculated molecular surface areas and cLog P values confirmed that these new cyclic prodrugs are more lipophilic than linear opioid peptides and, thus, they should exhibit better transcellular permeation characteristics. However, Caco-2 cell permeation studies showed that the cyclic prodrugs were substrates for apically polarized efflux transporters (e.g., P-glycoprotein, which significantly limited their transcellular permeation). Permeability studies using an in situ rat intestinal perfusion model confirmed the poor intestinal permeation characteristics of CA-[Cha(4), D-Leu(5) ]-Enk and CA-[Cha(4), D-Ala(5)]-Enk as well as the stability of these two new cyclic prodrugs of opioid peptides to oxidative metabolism. In conclusion, these data clearly show that oral absorption of cyclic prodrugs of opioid peptides can only be achieved by designing molecules devoid of substrate activity for both cytochrome P-450 enzymes and efflux transporters in the intestinal mucosa.

Development and in vivo characterization of a novel peptide drug delivery system providing extended plasma half life.

It was the aim of this study to develop a sustained parenteral peptide (DALCE) delivery system by the immobilization of DALCE to thiolated carboxymethyl dextran-cysteine (CMD-Cys) via disulfide bond formation. The resulting CMD-Cys-DALCE conjugate displayed a 22.6±7.9% (m/m) of DALCE (mean±S.D.; n=3). The conjugation of DALCE with CMD-Cys was confirmed by FTIR-ATR spectroscopy. In vitro release studies of conjugate CMD-Cys-DALCE in the presence of 2 µM/ml reduced glutathione (GSH) being also available in the plasma showed a sustained peptide release over a time period of 8 h, because of thiol/disulfide exchange reactions. For in vivo pharmacokinetic study, DALCE and CMD-Cys-DALCE were administered intravenously to male Sprague-Dawley rats at a dose of 1mg/kg. The AUC(0-8) (ng.min/ml) was determined to be 268848±924 and 40019±495 for CMD-Cys-DALCE and DALCE, respectively. The mean residence time (MRT) was determined to be 256±8 and 53.1±9.5 min for CMD-Cys-DALCE and for DALCE, respectively. CMD-Cys-DALCE showed a more than 5-fold increased elimination half-life (p<0.01), 3-fold decreased volume of distribution (p<0.01) and a 6.7-fold decreased plasma clearance rate (p<0.01) compared to DALCE. According to these findings, CMD-Cys-DALCE seems to act as prodrug by improving half-life and decreasing plasma clearance.

[A new approach to the evaluation of the state of the microcirculatory system in patients presenting with chronic atrophic pharyngitis and treated with a synthetic neuropeptide].

This paper is devoted to the mechanisms of development of chronic atrophic pharyngitis. A method is proposed for studying microcirculation in the mucous membrane at the posterior pharyngeal wall of the patients with this condition using laser Doppler flowmetry. The role of chronic somatic pathology in the development of pharyngeal dystrophy is demonstrated. It is shown that therapy with the synthetic neuropeptide is highly efficacious for the treatment of chronic atrophic pharyngitis.

Effects of dalargin on free radical processes in the blood of rats exposed to moderate hypothermia.

Moderate hypothermia stimulates LPO processes in rat plasma and erythrocytes and simultaneously increases antioxidant activity in the plasma and SOD activity erythrocyte. Dalargin prevents stimulation of LPO in the blood in hypothermia by reducing the generation of active oxygen species and maintaining the level of low-molecular antioxidants.