Endosomal signaling of delta opioid receptors is an endogenous mechanism and therapeutic target for relief from inflammatory pain.

Whether G protein-coupled receptors signal from endosomes to control important pathophysiological processes and are therapeutic targets is uncertain. We report that opioids from the inflamed colon activate δ-opioid receptors (DOPr) in endosomes of nociceptors. Biopsy samples of inflamed colonic mucosa from patients and mice with colitis released opioids that activated DOPr on nociceptors to cause a sustained decrease in excitability. DOPr agonists inhibited mechanically sensitive colonic nociceptors. DOPr endocytosis and endosomal signaling by protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways mediated the sustained inhibitory actions of endogenous opioids and DOPr agonists. DOPr agonists stimulated the recruitment of Gαi/o and ß-arrestin1/2 to endosomes. Analysis of compartmentalized signaling revealed a requirement of DOPr endocytosis for activation of PKC at the plasma membrane and in the cytosol and ERK in the nucleus. We explored a nanoparticle delivery strategy to evaluate whether endosomal DOPr might be a therapeutic target for pain. The DOPr agonist DADLE was coupled to a liposome shell for targeting DOPr-positive nociceptors and incorporated into a mesoporous silica core for release in the acidic and reducing endosomal environment. Nanoparticles activated DOPr at the plasma membrane, were preferentially endocytosed by DOPr-expressing cells, and were delivered to DOPr-positive early endosomes. Nanoparticles caused a long-lasting activation of DOPr in endosomes, which provided sustained inhibition of nociceptor excitability and relief from inflammatory pain. Conversely, nanoparticles containing a DOPr antagonist abolished the sustained inhibitory effects of DADLE. Thus, DOPr in endosomes is an endogenous mechanism and a therapeutic target for relief from chronic inflammatory pain.

[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.

Dose-dependent neuroprotection of delta-opioid peptide [D-Ala(2), D-Leu(5)] enkephalin on spinal cord ischemia-reperfusion injury by regional perfusion into the abdominal aorta in rabbits.

OBJECTIVE: In our prior study, we showed that delta-opioid peptide [D-Ala(2), D-Leu(5)] enkephalin (DADLE), by regional perfusion into the abdominal aorta, could protect the spinal cord against ischemia-reperfusion (I/R) injury caused by aortic occlusion. However, the relative dose-response effects of DADLE still remain unclear. This study investigated whether DADLE has a dose-dependent efficiency on spinal cord I/R injury. METHODS: New Zealand White rabbits were randomly divided into one of six groups: normal saline (NS; n = 8), DADLE (D) groups D0.0005 (n = 8), D0.005 (n = 8), D0.05 (n = 8), and D0.5 mg/kg (n = 8), and a sham group (n = 6). In the NS and DADLE groups, spinal cord ischemia was induced by infrarenal aortic occlusion for 30 minutes. During the occlusion, the same volume of NS or DADLE at the indicated doses was infused continuously through a catheter to the distally clamped abdominal aorta. Heart rate, blood pressure, and core temperature were monitored continuously to evaluate the potential adverse effects of DADLE. Neurologic behavioral function was assessed with the Tarlov scale system at 1, 6, 24, 48, and 72 hours after reperfusion. Neuronal injury evaluation in the ventral horn of the gray matter was evaluated by counting the normal motor neurons at 72 hours after reperfusion. RESULTS: The therapeutic benefits increased at the doses of DADLE from 0.0005 to 0.05 mg/kg and decreased at 0.5 mg/kg, whereas the hemodynamic parameter was suppressed temporarily at the dose of 0.5 mg/kg. CONCLUSIONS: These data revealed that regional administration of DADLE through the abdominal aorta provided dose-dependent protection on spinal cord I/R in rabbits.

[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.

Chitosan nanoparticles are efficient carriers for delivering biodegradable drugs to neuronal cells.

Chitosan nanoparticles (NPs) are biocompatible drug carriers able to cross the blood-brain barrier and represent a promising drug delivery system to the central nervous system. We used chitosan NPs to deliver the D-Ala2-D-Leu5-enkephalin (DADLE) to neuronal cells in vitro. DADLE is a hypometabolising synthetic opioid potentially useful for biomedical applications, but its short plasmatic half-life makes its in vivo administration ineffective. Here, we demonstrate by immunoelectron microscopy that (1) chitosan NPs are capable to deliver the opioid to neuronal cells; (2) DADLE is released from the internalised, opioid-loaded NPs up to 48 h; (3) in the nucleus, DADLE binds the transcription/splicing sites; (4) cells treated with DADLE-loaded NPs undergo a decrease in transcription factor amounts and proliferation rate without damage to cell organelles. In this model, chitosan NPs protected the loaded opioid from degradation, thereby prolonging its intracellular effects. These findings suggest that these NPs are efficient for the systemic and tissue administration of opioids in vivo.

Changes in feeding behavior, locomotor activity, and metabolism in rats upon modulation of opioid receptors in the gastrointestinal tract.

We studied the role of µ-, δ-, and κ-opioid receptors of the stomach in the regulation of natural feeding behavior, metabolism, and locomotor activity of rats. Locomotor activity (number of crossed squares), food and water intake, oxygen consumption, and carbon dioxide release in animals were estimated in the standard home cage using a Phenomaster device (TSE) for 24 h at 40-min intervals. Administration of a µ-opioid receptor agonist DAMGO suppressed feeding behavior of animals in the light phase, but had little effect on locomotor activity and metabolism. Treatment with a δ-opioid receptor agonist DADLE was followed by the increase in metabolism over 24 h. These changes were accompanied by a decrease in locomotor activity during the light phase and activation of feeding behavior in the transition period. Intragastric administration of a κ-opioid receptor agonist ICI-204,448 inhibited feeding behavior, metabolism, and locomotor activity of rats only in the nighttime. These data suggest that opioid peptides produced in the stomach during food digestion play an important role in the regulation of food motivation and metabolism in rats. Various subtypes of opioid receptors probably regulate feeding behavior and metabolism of animals in different phases of vital activity.

[DADLE suppresses the proliferation of human liver cancer HepG2 cells by activation of PKC pathway and elevates the sensitivity to cis-diammine dichloridoplatium].

OBJECTIVE: To investigate the effect of DADLE, a δ-opioid receptor agonist, on the proliferation of human liver cancer HepG2 cells and explore the mechanism involving PKC pathway. METHODS: HepG2 cells were treated with DADLE at different doses (0.01, 0.1, 1.0 and 10 µmol/L). Cell viability was determined using methyl thiazolyl terazolium (MTT) assay. The expression of PKC mRNA and p-PKC protein were examined by RT-PCR and Western blot assay. After treated separately with DADLE plusing NAL or PMA, the cell cycle of HepG2 cells was analyzed by flow cytometer. MTT was used to detect their proliferation capacity and Western blot was used to examine the p-PKC expression. The growth inhibitory rate of HepG2 cells treated with DADLE and cis-diammine dichloridoplatinum (CDDP) was analyzed. RESULTS: DADLE at different concentrations showed an inhibitory effect on the proliferation of HepG2 cells though inhibiting the expression of PKC mRNA and p-PKC protein. The results of flow cytometry showed that compared with the control group, the percentage of S + G(2)/M cells in DADLE-treated group was lowered by 3.94% (P < 0.01). Meanwhile, after treated with NAL and PMA, the percentage was elevated by 3.22% and 3.63%, respectively (P < 0.01). The MTT and Western blot assays showed that compared with the control group, the values of A570 and p-PKC protein levels in the HepG2 cells of DADLE-treated group were significantly decreased (P < 0.01). After treatment with NAL and PMA, the values of A570 and p-PKC protein levels were elevated significantly (P < 0.01). The growth inhibitory rate of DADLE + CDDP group was 79.9%, significantly lower than 25.2% and 43.2% of the DADLE and CDDP groups, respectively. CONCLUSIONS: Activation of δ-opioid receptor by DADLE inhibits the apoptosis of human liver cancer HepG2 cells. The underlying mechanism may be correlated with PKC pathway. DADLE can enhance the chemosensitivity of HepG2 cells to CDDP.

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.

[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.

[Regulatory peptides influence on hypoxia induced tooth germ pathology of new born albino rats].

Intrauterine hypoxia decreased the nucleolar organizator quantity in enameloblasts of newborn albino rats tooth germ. Influence the leu-encepgalin synthetic analog dalargin and non-opiate synthetic leu-encephalin on newborn rats from 2 to 6 day abolished the intrauterine hypoxia affect. The main mechanism of peptide antioxidant effect may be NO system stimulation.

A strategy for delivering peptides into the central nervous system by sequential metabolism.

Most peptides do not enter the central nervous system because of their hydrophilic character and the presence of peptidolytic enzymes in the lipoidal blood-brain barrier. To achieve brain delivery of a peptide conjugate, an opioid peptide (enkephalin) was placed in a molecular environment that disguises its peptide nature and provides biolabile, lipophilic functions to penetrate the blood-brain barrier by passive transport. The strategy also incorporates a 1,4-dihydrotrigonellinate targetor that undergoes an enzymatically mediated oxidation to a hydrophilic, membrane-impermeable trigonellinate salt. The polar targetorpeptide conjugate that is trapped behind the lipoidal blood-brain barrier is deposited in the central nervous system. Analgesia was observed with "packaged" enkephalin but not with the unmodified peptide or lipophilic peptide precursors.

[Cardiovascular effects of D-ALA2, LEU5, ARG6-enkephalin (dalargin) are mediated by peripheral mu-opioid receptor activation].

It has been established that intravenous administration of dalargin at a dose of 0.1 mg/kg induced hypotensive, positive inotropic, and positive chronotropic response in patients with myocardial infarction, while not producing the "steal syndrome." The positive chronotropic effect of dalargin was mediated by peripheral mu-opioid receptors. Increase in the heart rate induced by dalargin had a reflex nature. Direct action of dalargin on the heart was accompanied by a decrease in the heart rate and a delay in the cardiac conductance in the atrioventricular junction region.

Dose-dependent neuroprotection of delta opioid peptide [D-Ala2, D-Leu5] enkephalin in neuronal death and retarded behavior induced by forebrain ischemia in rats.

Cerebral ischemic insult, mainly induced by cardiovascular disease, is one of the most severe neurological diseases in clinical. There's mounting evidence showing that delta opioid agonist [D-Ala2, D-Leu5] enkephalin (DADLE) has a tissue-protective effect. However, whether this property is effective to prevent neuronal death induced by forebrain ischemia is not clear. This study was aimed to investigate whether intracerebroventricular (ICV) administration of DADLE has a neuroprotective effect against forebrain ischemia in rats. We found in our study that administration of DADLE 45 min before forebrain ischemia had significant protective effect against CA1 neuronal lose. Further more, we found that DADLE had a dose-dependent protection for improving behavioral retardation revealed by Morris water maze and motor score test, while naltrindole, the antagonist of delta opioid receptor, partially abolished neuroprotective effect of DADLE, which implicated that both opioid and non-opioid systems are involved in ischemic insults and neuroprotection.

[Endocrine homeostasis and its correction with stress protectors in children with severe burn injury].

The use of stress protection with dalargin, clofelin, and pentamine in the treatment of children with severe burn injury was ascertained to result in a reduction in the magnitude of abnormal changes in the endocrine system and in the prevention of depletion of its compensatory capacities. This treatment will assist in reducing the severity of endotoxicosis, as well as the frequency and severity of toxic and pyoseptic complications.

Protective effect of delta opioid receptor agonist (D-Ala2, D-Leu5) enkephalin on permanent focal cerebral ischemia in rats.

To investigate the effect of delta opioid receptor agonist (D-Ala, D-Leu) enkephalin (DADLE) on the permanent focal cerebral ischemia in rats. Thirty four male Sprague-Dawley rats were assigned randomly into three groups: sham group (group Sham, n=10), artificial cerebrospinal fluid group (group ACSF, n=12), and DADLE group (group DADLE, n=12). Permanent middle cerebral artery occlusion was performed to induce permanent focal cerebral ischemia in rats. Then, the animals in group DADLE and group ACSF were treated with DADLE or ACSF by an intracerebroventricular injection at 45 min after ischemia. Neurologic deficit scores were assessed according to the Garcia criterion at 24 h after ischemia. Infarct volume was determined using the 2,3,5-triphenyltetrazolium chloride staining method. The histological analysis was used to evaluate the extent of cerebral injury. Compared with the control group, the Garcia scores were significantly higher (P=0.000) and the infarct volumes (P=0.018) were significantly smaller in the DADLE treatment group at 24 h after ischemia. These neurologic changes were closely correlated with the outcome of the infarct volumes. In addition, the histological examination showed more intact neurons in rats treated with DADLE than those treated with ACSF at 24 h after ischemia (P=0.000). DADLE by intracerebroventricular administration at 45 min after ischemia can improve neurologic outcome and mitigate cortical neuronal injury induced by permanent focal cerebral ischemia in rats.

Double-coated poly (butylcynanoacrylate) nanoparticulate delivery systems for brain targeting of dalargin via oral administration.

The aim of this study is to evaluate oral administration of poly (butylcyanoacrylate) nanoparticulate delivery systems (PBCA-NDSs), double-coated with Tween 80 and poly (ethylene) glycol (PEG) 20000 for brain delivery of hexapeptide dalargin, an anti-nociceptive peptide that does not cross blood-brain barrier (BBB) by itself. Studies have proven the brain uptake of Tween 80 overcoated nanoparticles after intravenous administration, but studies for brain delivery of nanoparticles after oral administration had been limited due to reduced bioavailability of nanoparticles and extensive degradation of the peptide and/or nanoparticles by gastrointestinal enzymes. To address this problem, dalargin-loaded PBCA-NDS were successively double-coated with Tween 80 and PEG 20000 in varied concentrations of up to 2% each. Measurement of in vivo central anti-nociceptive effect of dalargin along with a dose response curve was obtained by the tail flick test following the oral administration of PBCA-NDSs to mice. Results from the tail flick test indicated that significant dalargin-induced analgesia was observed from PBCA-NDSs with double-coating of Tween and PEG in comparison with single-coating of either Tween or PEG. Hence, it could be concluded that surface coated PBCA-NDS can be used successfully for brain targeting of dalargin or other peptides administered orally. However, further studies are required to elucidate the exact transport mechanism of PBCA-NDSs from gastrointestinal tract to brain.

[Transcranial electrostimulation combined with dalargin ultraphonophoresis in the treatment of patients with chronic idiopathic eczema].

General condition and local status were analysed in patients with chronic idiopathic eczema before the conventional treatment and combined therapy including sessions of transcranial electrostimulation and dalargin phonophoresis and after these treatments. The latter combination improved the patients' quality of life and promoted their faster recovery.

Protective effect of delta opioid agonist [D-Ala2, D-Leu5] enkephalin on spinal cord ischemia reperfusion injury by regional perfusion into abdominal aorta in rabbits.

[D-Ala(2), D-Leu(5)] enkephalin (DADLE) has been reported to exhibit protective effects against hypoxic or ischemic induced brain insult. However its efficacy on the spinal cord ischemia-reperfusion injury remains unclear. Here we investigate whether DADLE could attenuate ischemia and reperfusion induced neural injury in the rabbit spinal cord. New Zealand white rabbits were subjected to spinal cord ischemia by infrarenal aortic occlusion for 30 min. In the period of spinal cord ischemia, DADLE 0.5 mg/kg or NS were infused continuously into the distal clamped abdominal aorta. The heart rate, blood pressure, and core temperature were monitored continuously during the whole experimental procedure. Then the neurological behavioral function was assessed with Tarlov scale system at 1h, 6h, 24h, 48 h after reperfusion, and neuronal injury evaluation in the ventral horn of gray matter was measured by counting the normal motor neurons at 48 h after reperfusion. Comparing with the control group, the Tarlov scores were significantly higher and the incidences of paraplegia were significantly lower in the DADLE group at four time-point recorded. In addition, the normal neurons numbers in the DADLE group were significant more than those in the control group at 48 h after reperfusion. These results suggested that DADLE infused into the abdominal aorta during ischemia period could attenuate behavioral retardation and the loss of normal motor neuron induced by ischemia-reperfusion in rabbits.

Mu but not delta and kappa opioid receptor involvement in ventrolateral orbital cortex opioid-evoked antinociception in formalin test rats.

The present study was designed to investigate the roles of different subtypes of opioid receptors in ventrolateral orbital cortex (VLO) opioid-evoked antinociception in formalin test by using an automatic detection system for recording the nociceptive behavior (agitation) and a manual method for detecting the duration of licking the injected paw in the conscious rat. Formalin (5%, 50 microl) s.c. injected into the hindpaw produced a biphasic agitation response or lengthening duration of licking. Morphine (5 microg) microinjected unilaterally into VLO significantly inhibited the agitation response and the licking time, and these effects were blocked by pre-administration of the non-selective opioid receptor antagonist naloxone (1.0 microg) into the same site. Microinjection of endomorphin-1 (5 microg), a selective micro-receptor agonist, and [D-Ala2, D-Leu5]-enkephalin (DADLE, 10 microg), a delta-/micro-receptor agonist also inhibited the nociceptive behaviors, and both the effects were blocked by selective mu-receptor antagonist beta-funaltrexamine hydrochloride (beta-FNA; 3.75 microg), but the DADLE-evoked inhibition was not influenced by the selective delta-receptor antagonist naltrindole (5 microg). Microinjection of selective kappa-receptor agonist (+/-)-trans-U-50488 methanesulfonate salt (1.5 microg) failed to alter the nociceptive behaviors induced by formalin injection. The beta-FNA and naloxone applied into VLO and morphine into the adjacent regions ventral and dorsal to VLO had no effect on the formalin-evoked nociceptive behaviors. These results suggest that mu- but not delta- or kappa-opioid receptor is involved in the VLO opioid-evoked antinociception in formalin test rat.