Molecular mechanisms of antioxidant action of dalargin on the liver in experimental cholestasis]

Changes of antioxidant activity of dalargin in the liver after naloxone (100 micrograms/kg) administration were examined in experiment on 144 rats with cholestasis. It was found that dalargin inhibited the activity of xanthine oxidase by 32-37% in different time periods after the injection. Dalargin and naloxone, when used in combination, had no effect on the enzyme activity. Glutathione-S-transferase activity rose by 38.0% and 21.8% on hour 1 and 3 after the injection, respectively, while simultaneous injection of dalargin and naloxone induced no changes in the enzyme activity after 1 hour, though decreased it by 36.8% and 26.4% on hour 3 and 5, respectively. Dalargin inhibited lipid peroxidation by 29-35%, simultaneous injection of dalargin and naloxone raised lipid peroxidation by 109.2%, 80.7% and 25.7% after 1, 3 and 5 hours, respectively. Dalargin injection elucidated a marked tendency to lowering of blood release of the liver-specific enzymes histidase and urokaninase in line with enhancement of their activity in the liver. A combined injection of dalargin and naloxone promoted high release of histidase and urokaninase in blood and did not change histidase activity in the liver in all cases. Urokanidase activity elevated in 5 hours. It was noticed that dalargin raised leu-enkephalin levels in the liver 3.5-fold 1 h after the injection. The reduced dalargin antioxidant effect coupled with naloxone pretreatment demonstrated indirect action of the neuropeptide on the liver via neuron receptors of the liver.     

Effect of dalargin on the processes of memory and learning in the rat

In experiments on rats, the influence was studied of dalargin on the elaboration and preservation of various homogeneous and heterogeneous conditioned reflexes (CRs) elaborated in single and multiple pairings. The effect of dalargin on the processes of learning and memory was compared with the action of the peptide on the activity of hypothalamic neurones. Administration of dalargin delayed the elaboration of maze defensive CRs and practically did not affect the elaboration of two-way avoidance. The preservation of CR also deteriorated under the influence of dalargin. Administration of dalargin 10 min before the CRs testing did not prevent their reproduction. When using CRs elaborated in a single pairing, dalargin disturbed the preservation of the drinking CR and improved that of passive avoidance CR. Dalargin in this dose affected the emotional state of animals in the open field and did not significantly affect their motor activity. Dalargin suppressed impulse activity in 17 out of 22 tested neurones of the lateral hypothalamus, with maximum effect in 20-50 min after its administration. The obtained data show that the character of dalargin action on the elaboration of CR and mainly on its consolidation, depends on the character of the elaborated CR and is probably due to great extent to the effect of the peptide on the brain emotional mechanisms.     

Radioimmunologic and radioreceptor studies of the pharmacokinetics of the enkephalin analog dalargin in man

Pharmacokinetics of dalargin, an opioid hexapeptide, was investigated on 7 males by two approaches. Dalargin radioimmunoassay was performed using a highly specific antiserum reacting only with the whole molecule. In radioreceptor assay lyophilized rat brain membranes containing opiate receptors were used. 2-6 min after intravenous introduction of 1-10 mg dalargin, immunoreactive dalargin blood concentration was lower than 0.5 ng/ml. The results of radioreceptor assay were presented as a biexponential curve with a fast main phase of activity changes (90%, characteristic time 1.5-5.0 min) and a slow "clearance" phase (10% of the substance, characteristic time 85-200 min). Prolonged presence of receptor-active substances in the blood can be attributed to the products of dalargin degeneration, namely its N-terminal penta- and tetrapeptides.     

Functioning of the pituitary-adrenal system during the vestibulo-vegetative syndrome and administration of dalargin and nalorphine

Changes in ACTH, cortisol, beta-endorphin have been investigated during vestibulo-vegetative syndrome (VVS) and injections of dalargin (leu-enkephalin analog) and nalorphine (agonist-antagonist of opioid receptors) in 9 volunteers with low level vestibulo-vegetative stability. Cumulative coriolis acceleration test during rotations on a special chair was used for VVS modelling. Dalargin (1-4 mg), nalorphine (5 mg) and placebo (NaCl solution) were injected intravenously 5-15 min before rotation. A significant increase in ACTH, cortisol and beta-endorphin plasma levels has been observed. Mean positive linear correlation (r greater than +0.6) between ACTH and beta-endorphin and ACTH and cortisol was noted immediately after the test only when dalargin was injected. It is suggested that in VVS there develops a hormonal conflict, i. e. an adequate hormonal release is disturbed.     

Effects of dalargin on proliferative processes and vertical migration of cells of the corneal epithelium during stress

We studied the effect of synthetic analogue of opioid peptides dalargin on kinetics of cell population of corneal epithelium during stress. It was found out that dalargin introduction before stress decreased the level of pathological mitoses induced by the acute stress action in white rats early in the morning. Dalargin prevented the activation of the proliferative process after the repeated stress activation, made stress milder. The stress induced vertical migration of the cells of the corneal epithelium normalized by dalargin. We believe these results show that dalargin has a good protective effect.     

Effect of the synthetic Leu enkephalin dalargin on the protein and nucleic acid content of the muscles in the rainbow trout

After dalargin treatment of fish eggs (at the stage of swollen egg envelopes) and on juveniles (at the stage of early meiotic oocyte appearance in gonads) DNA content is observed to raise up to 85% and 66%, respectively in yearlings and up to 20% and 23% respectively in second-year fish as compared to the control ones. Dalargin also influences the exchange of muscle RNA in yearlings and second-year rainbow-trout. Dalargin effect is higher when peptide influences at the very onset of organism differentiation.     

Intranasal midazolam in piglets: pharmacodynamics (0.2 vs 0.4 mg/kg) and pharmacokinetics (0.4 mg/kg) with bioavailability determination

Intranasal midazolam was studied in two series of piglets: series 1, n = 20 (18 +/- 3 kg), a randomized double blind pharmacodynamic study to compare doses of 0.2 mg/kg and 0.4 mg/kg; series 2, n = 9 (42 +/- 8 kg), a pharmacokinetic study with a 0.4 mg/kg dose administered either intravenously (i.v.) or intranasally (i.n.) in a cross-over protocol with a one-week wash-out period between each. In series 1, midazolam caused significant anxiolysis and sedation within 3 to 4 min, without a significant difference between 0.2 and 0.4 mg/kg doses for any of the studied parameters. In series 2, after intranasal midazolam administration of 0.4 mg/kg, plasma concentrations attained a maximum (Cmax) of 0.13 +/- 0.04 mg/l at 5 min (median Tmax) and remained higher than 0.04 mg/l until 60 min. The bioavailability factor (F) in this study was F = 0.64 +/- 0.17 by the intranasal route. The terminal half-life (T1/2 lambda z) = 145 +/- 138 min was comparable with the i.v. administration half-life (158 +/- 127 min). In conclusion, optimal intranasal midazolam dose in piglets was 0.2 mg/kg, which procures rapid and reliable sedation, adapted to laboratory piglets.     

The effect of dalargin on stress-induced changes in the 5'-nucleotidase activity of the macrophages and in the level of endogenous blood cortisol in mice]

The action of dalargin, synthetic analogue of leuenkephaline, on stress-induced changes in 5'-nucleotidase activity and endogenous hydrocortisone levels was investigated in mice. It was found that there is a direct relation between the activity of 5'-nucleotidase and the level of hydrocortisone in CBA mice. For C57Bl/6 mice the relation was inverse. Dalargin is able to change the dependence from direct to opposite in CBA mice.     

Effect of dalargin on the activity of cytochrome oxidase and glutamate dehydrogenase in rat spinal cord dissociated cultured neurons

Dissociated cultured neurons from the rat embryo spinal cord were grown for six days in the presence of dalargin, the synthetic analog of leu-enkephalin. Then the activities of two enzymes of energy metabolism, cytochrome oxidase (CO) and glutamate dehydrogenase (GDH), were studied in these neurons using quantitative cytochemical technique. Dalargin, which possesses the properties of nerve growth factor, enhanced the nerve cell growth and increased the activity of the above enzymes, with GDH activity being increased more significantly. According to the classical standpoint, increased GDH activity under conditions of acute energy deflciency favors the invoivement of some amino acids in a citric acid cycle for subsequent reproduction. One can suggest, in this relation, that the increased energy production caused by the enhanced nerve cell growth in the presence of dalargin was partially compensated by the amino acid splitting. The results allow us to suggest that the effect of dalargin (growth factor) on the nerve cells is similar to the effects of the extremal factors, and requires additional energy to be supplied.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 95–99, March–June, 1996.     

Blood flow in skin wound tissue and systemic hemodynamics as affected by a collagen-dalargin complex

In has been demonstrated that a collagen-dalargin complex increases blood flow in the granulation tissue (microsphere technique) due to the formation of new capillaries and a decrease in the vascular resistance. Dalargin had no effect on the capillary blood flow in the unaffected tissues. It has been concluded that the wound healing effect of dalargin may be related to its involvement in angiogenesis regulation.     

Effects of dalargin on the proliferative processes of the gastric epithelium under repeated action of different stressors]

Using radiography with H-thymidine method we studied the synthesis of DNA process in pyloric parts of stomach epithelium in white rats, which have been five-fold effected by different kinds of stressors against a background of dalargin injections. In the first hour after animals were stressed, DNA synthesis was depressed. Dalargin injections caused DNA synthesis normalization in the first hour after hypoxia and hyperthermia. Since 24 hours after hyperthermia and immobilization against a background of dalargin injections the normalization of DNA synthesis took place, and after hypoxia the post-stressing IMN activation was growing week. One of the mechanisms of dalargin correction of DNA synthesis breach under the influence of stressors is a stabilization of noradrenaline and histamine concentration in tissue of the stomach.     

Intranasal Drug Delivery of Olanzapine-Loaded Chitosan Nanoparticles

The aim of this study was to investigate olanzapine (OZ) systemic absolute bioavailability after intranasal (i.n.) administration in vivo to conscious rabbits. Furthermore, the study investigated the potential use of chitosan nanoparticles as a delivery system to enhance the systemic bioavailability of olanzapine following intranasal administration. Olanzapine-loaded chitosan nanoparticles were prepared through ionotropic gelation of chitosan with tripolyphosphate anions and studied in terms of their size, drug loading, and in vitro release. The OZ nanoparticles were administered i.n. to rabbits, and OZ plasma concentration at predetermined time points was compared to i.n. administration of OZ in solution. The concentrations of OZ in plasma were analyzed by ultra performance liquid chromatography mass spectroscopy (UPLC/MS). OZ-loaded chitosan nanoparticles significantly (p < 0.05) enhanced systemic absorption with 51 ± 11.2% absolute bioavailability as compared to 28 ± 6.7% after i.n. administration of OZ solution. The results of the present study suggest that intranasal administration of OZ-loaded chitosan nanoparticles formulation could be an attractive modality for delivery of OZ systemically.KEY WORDS: bioavailability, intranasal, nanoparticles, olanzapine, pharmacokinetic     

A placebo-controlled trial of intranasal growth hormone-releasing hormone [GHRH(1-44)-NH2] administration in normal young adults

Growth hormone-releasing hormone, GHRH(1-44), was administered intranasally to 16 healthy young adult male volunteers in a placebo-controlled study using a dose of 1,000 micrograms dissolved in two different solvent vehicles: water alone and water with the surface tension-lowering agent Tween 80 (0.12%). The growth hormone (GH)-releasing effects of intranasal GHRH as well as that of the vehicle were established and compared to the effects of 80 micrograms intravenous GHRH. Plasma GH response was assessed by frequent blood sampling over an 180-min period, using both peak response and area under the curve (AUC). The results show that the GH-release effects of intranasal GHRH are comparable whichever vehicle is used, and are similar, with the dose of 1,000 micrograms, to the response obtained following the administration of 80 micrograms intravenous GHRH. Peak GH responses to GHRH (means +/- SEM) were 25.6 +/- 4.2 ng/ml (1,000 micrograms GHRH with water), 32.9 +/- 9.1 ng/ml (1,000 micrograms with water plus Tween 80) and 36.3 +/- 7.8 ng/ml (80 micrograms i.v. administration) (not significant). There was no significant GH response to placebo. Mean peak GH responses occurred after approximately 30 min in all three active treatments (29.2 +/- 2.7, 33.9 +/- 3.2 and 30.9 +/- 3.9 min, respectively). The AUC values (ng.min.ml-1) were not statistically different: 1,914.4 +/- 386.7 (water), 2,176.2 +/- 599.9 (water plus Tween 80) and 2,419.2 +/- 506.9 (i.v.) (not significant). Intranasal GHRH administration was well tolerated in all subjects. Occasional local reactions consisted of a prickly sensation in the nostrils or sneezing irrespective of the vehicle used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of a stable analog of leu-enkephalin, dalargin, on the cell division of the corneal epithelium in white rats

The effect of Leu-enkephalin analog--dalargin--on the corneal epithelium proliferation has been studied in white rats. 10 microliter dalargin per 1 kg body weight were administered intraperitoneally at 8 a.m. The mitotic index (MI), DNA synthesis cell index and label intensity (LI) were determined every 4 hours over a 24-hour period. The results obtained demonstrate that dalargin stimulates DNA synthesis in cells throughout the entire period of action. MI increased only 4, 8, 12 hours after dalargin administration. Mean daily DNA synthesis cell index and MI increased 2.1-fold and 3.1-fold, respectively after dalargin administration. It is suggested that dalargin activates the cell division processes by speeding up mitosis, shortening the premitotic period, accelerating the speed of the DNA synthesis and increasing cell proliferation pool.     

Ligands for opioid and sigma-receptors improve cardiac electrical stability in rat models of post-infarction cardiosclerosis and stress.

The effects of the extremely selective mu-opioid receptor agonist, [D-Arg2,Lys4]-dermorphin-(1-4)-amide (DALDA), the mu-opioid receptor agonist morphine, the mu/delta agonist D-Ala2, Leu5, Arg6-enkephalin (dalargin), the kappa-opioid receptor agonist spiradoline, and the sigma1-receptor antagonist DuP 734 on ventricular fibrillation threshold (VFT) was investigated in an experimental post-infarction cardiosclerosis model and an immobilization stress-induced model in rats. Both models produced a significant decrease in VFT. The postinfarction cardiosclerosis-induced decrease in VFT was significantly reversed by intravenous administration of dalargin (0.1 mg/kg), DALDA (0.1 mg/kg), or morphine HCl (1.5 mg/kg). Pretreatment with naloxone (0.2 mg/kg) completely eliminated the increase in cardiac electrical stability produced by DALDA. Both spiradoline (8 mg/kg, i.p.) and DuP 734 (1 mg/kg, i.p.) produced a significant increase in VFT in rats with post-infarction cardiosclerosis. This effect of spiradoline was blocked by nor-binaltorphimine. The immobilization stress-induced decrease in VFT was significantly reversed by administration of either DALDA, spiradoline or DuP 734. In conclusion, activation of either mu- or kappa1-opioid receptors or blockade of sigma1-receptors reversed the decrease in VFT in both cardiac compromised models. Since DALDA and dalargin essentially do not cross blood brain barriers, their effects on VFT may be mediated through peripheral mu-opioid receptors.     

Effect of opioid peptides on the lymphatic drainage of the pancreas in the rat and dog

The effect of dalargin and some other ligands of the opioid receptors on drainage function of the pancreatic lymphatic system was studied in rats and dogs. In rats, dalargin (30-1000 micrograms/kg, subcutaneously) accelerated the elimination of Evans blue from beneath the pancreatic capsule in a dose-related manner. The effect of dalargin was attenuated by naloxone. Specific agonists of mu-, delta- and sigma-opioid receptors had no dalargin-like activity. In dogs, dalargin (60-80 micrograms/kg, subcutaneously) after the administration of Evans blue into the pancreas increased its concentration in the truncus lymphaticus and slowed down its penetration into the blood. Thus, dalargin accelerates the elimination of Evans blue from the pancreas due to the intensification of lymphatic drainage. The effect of dalargin was mediated by subpopulation of opioid receptors with which their certain selective ligands have but slight interaction.     

Intranasal vaccination of recombinant adeno-associated virus encoding receptor-binding domain of severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein induces strong mucosal immune responses and provides long-term protection against SARS-CoV infection

We have previously reported that a subunit protein vaccine based on the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein and a recombinant adeno-associated virus (rAAV)-based RBD (RBD-rAAV) vaccine could induce highly potent neutralizing Ab responses in immunized animals. In this study, systemic, mucosal, and cellular immune responses and long-term protective immunity induced by RBD-rAAV were further characterized in a BALB/c mouse model, with comparison of the i.m. and intranasal (i.n.) routes of administration. Our results demonstrated that: 1) the i.n. vaccination induced a systemic humoral immune response of comparable strength and shorter duration than the i.m. vaccination, but the local humoral immune response was much stronger; 2) the i.n. vaccination elicited stronger systemic and local specific cytotoxic T cell responses than the i.m. vaccination, as evidenced by higher prevalence of IL-2 and/or IFN-gamma-producing CD3+/CD8+ T cells in both lungs and spleen; 3) the i.n. vaccination induced similar protection as the i.m. vaccination against SARS-CoV challenge in mice; 4) higher titers of mucosal IgA and serum-neutralizing Ab were associated with lower viral load and less pulmonary pathological damage, while no Ab-mediated disease enhancement effect was observed; and 5) the vaccination could provide long-term protection against SARS-CoV infection. Taken together, our findings suggest that RBD-rAAV can be further developed into a vaccine candidate for prevention of SARS and that i.n. vaccination may be the preferred route of administration due to its ability to induce SARS-CoV-specific systemic and mucosal immune responses and its better safety profile.     

Proof of stimulating effect of dalargin of the process of cell division through opiate receptors

The role of opiate receptors on cell division in corneal epithelium during administration of dalargin was analysed. Naloxone injection/200 micrograms/kg/decreased MI two times, DNA-synthesis 1.4 times over 24 hours. Naloxone prevented dalargin effect on cell proliferation. Another testimonies of dalargin opiate-binding mitogenic effect were the results of the study with dalargin analogues. They are agonists of opiate receptors too. These drugs, as well as dalargin, in a dose 10 micrograms/kg increased DNA-synthesis 1.5 times, MI and MIK 2.2 times. It turned out, that the administration of another two analogues of dalargin, which are not ligands of opiate receptors, probably do not cause an adequate increase of DNA-synthesis and mitotic index.     

Antidepressant-like effects exerted by the intranasal administration of a glucagon-like peptide-2 derivative containing cell-penetrating peptides and a penetration-accelerating sequence in mice

The intracerebroventicular (i.c.v.) administration of glucagon-like peptide-2 (GLP-2) to rodents was shown to have antidepressant-like effects in imipramine-resistant depression-model mice. In order to utilize GLP-2 as a clinical treatment tool for depression, we herein focused on the intranasal delivery that is non-invasive approach, because the i.c.v. administration is invasive and impractical. In the present study, we prepared a GLP-2 derivative containing cell penetrating peptides (CPPs) and a penetration accelerating sequence (PAS) (PAS-CPPs-GLP-2) for the intranasal (i.n.) administration. PAS-CPPs-GLP-2 (i.n.) exhibited antidepressant-like effects in the forced-swim test (FST) and tail suspension test (TST) in naïve mice as well as adrenocorticotropic hormone (ACTH) treated-mice. However, PAS-CPPs-GLP-2 (i.v.) and the GLP-2 derivative containing CPPs without a PAS (CPPs-GLP-2) (i.n.) did not affect the immobility time in the mouse FST. Moreover, fluorescein isothiocyanate (FITC)-labeled PAS-CPPs-GLP-2 (i.n.), but not FITC-labeled CPPs-GLP-2 (i.n.) was distributed through the mouse brain after the FST session. These results suggest that PAS-CPPs-GLP-2 is effective for i.n. delivery to the brain, and may be useful in the clinical treatment of major depression.     

Pharmacokinetics of bestatin and oral activity for treatment of experimental metastases

Summary Bestatin is a low molecular weight aminopeptidase inhibitor originally isolated from culture filtrates of Streptomyces olivoreticuli. The serum pharmacokinetics in mice are dependent on route of administration, with a short t1/2 (1.69 min t1/2 and 12.8 min t1/2), but a high initial serum level following i.v. administration. When administered via the i.p., s.c., i.m., or p.o. routes of administration, bestatin had serum t1/2s of 8.56, 16.91, 19.25, or 15.4 min, respectively. The maximum area under the curve (concentration×time) occurred following i.v. and i.m. administration, with a lower level following p.o. or i.p. administration. Bestatin had therapeutic activity for experimental metastases, not only following i.v., i.p., and i.m. routes of administration but also following oral administration. Because of its brief serum t1/2, bestatin's therapeutic activity depends on aggressive (either daily or twice daily injection, especially following p.o. administration) and high-dose administration. Thus, the rate-limiting aspect of bestatin's therapeutic activity appears to be associated with its pharmacokinetics.This research was supported by the National Cancer Institute, DHHS, under contract No. N01-23910 with Program Resources, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government