Novel Cyclic Endomorphin Analogues with Multiple Modifications and Oligoarginine Vector Exhibit Potent Antinociception with Reduced Opioid-like Side Effects.

Endomorphins (EMs) are potent pharmaceuticals for the treatment of pain. Herein, we investigated several novel EM analogues with multiple modifications and oligoarginine conjugation. Our results showed that analogues 1-6 behaved as potent µ-opioid agonists and enhanced stability and lipophilicity. Analogues 5 and 6 administered centrally and peripherally induced significant and prolonged antinociceptive effects in acute pain. Both analogues also produced long-acting antiallodynic effects against neuropathic and inflammatory pain. Furthermore, they showed a reduced acute antinociceptive tolerance. Analogue 6 decreased the extent of chronic antinociceptive tolerance, and analogue 5 exhibited no tolerance at the supraspinal level. Particularly, they displayed nontolerance-forming antinociception at the peripheral level. In addition, analogues 5 and 6 exhibited reduced or no opioid-like side effects on gastrointestinal transit, conditioned place preference (CPP), and motor impairment. The present investigation established that multiple modifications and oligoarginine-vector conjugation of EMs would be helpful in developing novel analgesics with fewer side effects.

Enhancement of Anti-Tumoral Immunity by ß-Casomorphin-7 Inhibits Cancer Development and Metastasis of Colorectal Cancer.

ß-Casomorphin-7 (BCM) is a degradation product of ß-casein, a milk component, and has been suggested to affect the immune system. However, its effect on mucosal immunity, especially anti-tumor immunity, in cancer-bearing individuals is not clear. We investigated the effects of BCM on lymphocytes using an in vitro system comprising mouse splenocytes, a mouse colorectal carcinogenesis model, and a mouse orthotopic colorectal cancer model. Treatment of mouse splenocytes with BCM in vitro reduced numbers of cluster of differentiation (CD) 20+ B cells, CD4+ T cells, and regulatory T cells (Tregs), and increased CD8+ T cells. Administration of BCM and the CD10 inhibitor thiorphan (TOP) to mice resulted in similar alterations in the lymphocyte subsets in the spleen and intestinal mucosa. BCM was degraded in a concentration- and time-dependent manner by the neutral endopeptidase CD10, and the formed BCM degradation product did not affect the lymphocyte counts. Furthermore, degradation was completely suppressed by TOP. In the azoxymethane mouse colorectal carcinogenesis model, the incidence of aberrant crypt foci, adenoma, and adenocarcinoma was reduced by co-treatment with BCM and TOP. Furthermore, when CT26 mouse colon cancer cells were inoculated into the cecum of syngeneic BALB/c mice and concurrently treated with BCM and TOP, infiltration of CD8+ T cells was promoted, and tumor growth and liver metastasis were suppressed. These results suggest that by suppressing the BCM degradation system, the anti-tumor effect of BCM is enhanced and it can suppress the development and progression of colorectal cancer.

Cyclic derivative of morphiceptin Dmt-cyclo-(D-Lys-Phe-D-Pro-Asp)-NH2(P-317), a mixed agonist of MOP and KOP opioid receptors, exerts anti-inflammatory and anti-tumor activity in colitis and colitis-associated colorectal cancer in mice.

Endogenous opioid system is involved in the maintenance of the intestinal homeostasis. Recently, we proved that stimulation of opioid receptors using P-317, a cyclic morphiceptin analog, resulted in the alleviation of acute colitis in mice. The aim of the current study was to assess the effect of P-317 during colitis and colitis-associated colorectal cancer in mice. Colitis was induced by addition of dextran sodium sulfate (DSS) into drinking water. Colitis-associated colorectal cancer was induced by a single intraperitoneal injection of azoxymethane (AOM) and subsequent addition of DSS into drinking water (week 2, 5, 8). During macroscopic damage evaluation the samples were collected and used for biochemical (MPO activity assay), molecular (qPCR and western blot) and histological studies. In experimental colitis, P-317 induced an anti-inflammatory response as indicated by macroscopic and microscopic scores. In the colitis-associated colorectal cancer model, a significant difference in colorectal tumor development was observed between vehicle- and P-317-treated mice. P-317 decreased the total number of colonic tumors and inhibited MPO activity. Hematoxylin and eosin staining confirmed anti-tumor activity of P-317. The expression of TNF-α was decreased in P-317-treated mice as compared to the vehicle-treated group. P-317 decreased proliferation as well as ß-catenin expression in tumors. P-317, a mixed MOP and KOP receptor agonist, induced an anti-inflammatory response in experimental colitis and decreased tumor development in colitis-associated colorectal cancer in mice.

Moderate protective effect of Kyotorphin against the late consequences of intracerebroventricular streptozotocin model of Alzheimer's disease.

The established decrease in the level of endogenous kyotorphin (KTP) into the cerebrospinal fluid of patients with an advanced stage of Alzheimer's disease (AD) and the found neuroprotective activity of KTP suggested its participation in the pathophysiology of the disease. We aimed to study the effects of subchronic intracerebroventricular (ICV) treatment (14 days) with KTP on the behavioral, biochemical and histological changes in rats with streptozotocin (STZ-ICV)-induced model of sporadic AD (sAD). Three months after the administration of STZ-ICV, rats developed increased locomotor activity, decreased level of anxiety, impaired spatial and working memory. Histological data from the STZ-ICV group demonstrated decreased number of neurons in the CA1 and CA3 subfields of the hippocampus. The STZ-ICV group was characterized with a decrease of total protein content in the hippocampus and the prefrontal cortex as well as increased levels of the carbonylated proteins in the hippocampus. KTP treatment of STZ-ICV rats normalized anxiety level and regained object recognition memory. KTP abolished the protein loss in prefrontal cortex and decrease the neuronal loss in the CA3 subfield of the hippocampus. STZ-ICV rats, treated with KTP, did not show significant changes in the levels of the carbonylated proteins in specific brain structures or in motor activity and spatial memory compared to the saline-treated STZ-ICV group. Our data show a moderate and selective protective effect of a subchronic ICV administration of the dipeptide KTP on the pathological changes induced by an experimental model of sAD in rats.

The protective effect of beta-casomorphin-7 via promoting Foxo1 activity and nuclear translocation in human lens epithelial cells.

PURPOSE: To investigate the protective effect of beta-casomorphin-7 (ß-CM-7) in oxidative stressed human lens epithelial cells (HLECs) and to explore the possible mechanism for oxidative stress in HLECs induced by high glucose. METHODS: We used HLECs to determine the effect of different concentrations of ß-CM-7 on cell viability by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolimol/L bromide (MTT) assay. We used flow cytometry to determine the content of reactive oxygen species (ROS) induced by oxidative stress and a bioassay kit to determine the oxidant malondialdehyde (MDA) and antioxidant enzyme superoxide dismutase (SOD) levels. We used Western blotting and an immunofluorescence assay to determine the expression of Forkhead box o1 (Foxo1), SP1, and the related protein glutathione peroxidase (GSH-px) at the molecular biology level as well as their intracellular localization. RESULTS: The expression of Foxo1 and SP1 was weakly expressed when the glucose concentration was 40 mM/L, but was highly expressed when cells were pre-treated with an appropriate concentration of ß-CM-7. After pre-treatment with ß-CM-7, the cells treated with 40 mM/L glucose for 48 h showed Foxo1 was transferred to the nucleus, and the expression of SP1 was increased. The content of ROS and MDA in the HLECs that were pre-treated with ß-CM-7 was lower than in those that was not pre-treated (p <0.05). Accordingly, SOD was elevated in the cells pre-treated with ß-CM-7. The relative expression of GSH-px increased with increases of Foxo1 and SP1. CONCLUSION: ß-CM-7 protects HLECs from oxidative damage by upregulating the relative expression of Foxo1 and promoting Foxo1 nuclear translocation.

Por qué la infusión preventiva de una dosis baja de sulfato de magnesio influye en la percepción del dolor postoperatorio y el nivel sérico de beta-endorfinas en las histerectomías abdominales totales / Whether preventive low dose magnesium sulphate infusion has an influence on postoperative pain perception and the level of serum beta-endorphin throughout the total abdominal hysterectomy

Objetivo. Debido al conocido papel preventivo que juegan las bajas dosis de sulfato de magnesio en el tratamiento del dolor postoperatorio, en este estudio aleatorizado a doble ciego y controlado con placebo tratamos de investigar la posible relación entre la infusión intraoperatoria de sulfato de magnesio, la analgesia postoperatoria y el nivel de beta-endorfinas séricas en las histerectomías abdominales totales realizadas bajo anestesia general. Métodos. Se distribuyó aleatoriamente a 40 mujeres sometidas a histerectomía abdominal total en 2 grupos (20 en cada uno de ellos). Quince minutos antes de la inducción de anestesia, al grupo de estudio se le administró una infusión intravenosa de sulfato de magnesio (15mg/kg/h), y al grupo control con placebo se le administró el mismo volumen de solución salina isotónica. Las puntuaciones del dolor se evaluaron a las 0, 6, 12 y 24h posteriores a la intervención, utilizando la escala de calificación numérica verbal. Se registró de manera precisa el consumo de petidina. Se determinó el nivel sérico de beta-endorfinas 15min antes de la inducción y al finalizar las intervenciones, utilizando el método ELISA. Resultados. A las 6 y 12h posteriores a las intervenciones, el valor de la escala de calificación numérica verbal en el grupo de estudio fue considerablemente menor que en el grupo control con placebo (p=0,0001). A las 24h de la intervención, el consumo de petidina fue significativamente inferior en el grupo de estudio en comparación con el grupo control (p=0,0001). En el grupo de estudio, el nivel sérico de beta-endorfinas descendió considerablemente al final de las intervenciones, en comparación con el momento anterior a la inducción (p=0,04). Conclusión. Demostramos que la baja dosis preventiva e intraoperatoria de sulfato de magnesio reduce el dolor postoperatorio, tiene un efecto opioide moderado y disminuye la concentración sérica de beta-endorfinas en las histerectomías abdominales totales (AU)

Objective. Due to the known role of preventive low dose magnesium sulphate on postoperative pain management, in this randomized, double-blinded, placebo-controlled study, we tried to investigate the possible relationship between low dose intra-operative magnesium sulphate infusion, postoperative analgesia and the level of serum beta-endorphin during total abdominal hysterectomy under general anesthesia. Methods. Forty women undergoing total abdominal hysterectomy were randomly allocated into 2 groups (20 in each arm). Fifteen minutes before induction of anaesthesia, the case group received a continuous intravenous infusion of magnesium sulphate (15mg/kg/h) and placebo control group received the same volume of isotonic saline. Pain scores were assessed at 0, 6, 12, and 24h after operations using Verbal Numeric Rating Scale. Pethidine consumption was recorded precisely. Serum level of beta-endorphin just 15min before the induction and at the end of the operations was determined by ELISA technique. Results. At 6 and 12h after the operations, Verbal Numeric Rating Scale in the case group was significantly lower than that of placebo control group (P=.0001). Over 24h after the operations, pethidine consumption was significantly lower in the case group compared with control group (P=.0001). In the case group, serum level of beta-endorphin was significantly decreased at the end of the operations compared with before the induction (P=.04). Conclusion. We illustrated that preventive low dose intra-operative magnesium sulphate infusion reduces postoperative pain, has opioid sparing effect and declines serum beta-endorphin concentration during total abdominal hysterectomy (AU)

Synthesis and activity of opioid peptidomimetics with ß2- and ß3-amino acids.

Morphiceptin (Tyr-Pro-Phe-Pro-NH2) is a selective ligand of the mu opioid receptor, an important target in pain regulation. In this study, morphiceptin was modified at positions 2 or 3 by introduction of ß2- or ß3-amino acids and additionally in position 1 by replacing Tyr by Dmt (2',6'-dimethyltyrosine), which resulted in obtaining enzymatically stable analogs with mixed opioid receptor affinity profiles. An analog of the sequence Dmt-d-Ala-(R)-ß2-1-Nal-Pro-NH2 [Nal=3-(1-naphthyl)-alanine] showed very high activity at the mu and delta receptors in the calcium mobilization functional test but did not cross the artificial membrane imitating the blood-brain barrier. In the in vivo test this analog induced strong antinociceptive effect in the writhing test in mice after intraperitioneal but also oral administration and inhibited diarrhea similarly to loperamide. Therefore, it may become an interesting lead compound in the development of peripherally restricted drugs for the treatment of gastrointestinal disorders.

Orally administered novel cyclic pentapeptide P-317 alleviates symptoms of diarrhoea-predominant irritable bowel syndrome.

OBJECTIVE: The aim of our study was to characterize the effect of P-317, a novel cyclic derivative of morphiceptin, on gastrointestinal (GI) motility and abdominal pain in mouse models mimicking symptoms of diarrhoea-predominant irritable bowel syndrome (IBS-D). METHODS: The effect of P-317 on mouse intestinal motility was characterized in vitro and in vivo in physiological and pathophysiological conditions. The antinociceptive action of P-317 was characterized in the mustard oil-induced abdominal pain model and the writhing test. Locomotor activity and grip-strength tests were used to evaluate the effect of P-317 in the central nervous system (CNS). To translate our study to clinical conditions, the semi-quantitative expression of µ-opioid receptors (MOP) and κ-opioid receptors (KOP) messenger RNA (mRNA) in human colonic samples from IBS-D patients was quantified. KEY FINDINGS: In vitro, P-317 (10(-10) -10(-6) M) inhibited colonic and ileal smooth muscle contractions in a concentration-dependent, ß-funaltrexamine and nor-binaltorphimine-reversible manner. In vivo, P-317 (0.1 mg/kg, i.p. and 1 mg/kg, p.o.) inhibited GI transit, displayed a potent antinociceptive action in abdominal pain tests and did not influence the CNS. CONCLUSION: P-317 produced a potent analgesic and antidiarrhoeal action in the mouse GI tract after oral administration. Given lower expression of MOP and KOP mRNA in IBS-D patients, P-317 is a promising peptide-based drug candidate for IBS-D therapy.

Development and characterization of functionalized niosomes for brain targeting of dynorphin-B.

A niosomal formulation, functionalized with N-palmitoylglucosamine, was developed as potential brain targeted delivery system of dynorphin-B. In fact, this endogenous neuropeptide, selective agonist of k opioid receptors, is endowed with relevant pharmacological activities on the central nervous system, including a marked antinociceptive effect, but is unable to cross the blood brain barrier (BBB), thus requiring intracerebroventricular administration. Statistical design of experiments was utilized for a systematic evaluation of the influence of variations of the relative amounts of the components of the vesicle membrane (Span 60, cholesterol and SolulanC24) on vesicle mean diameter, polydispersity index and drug entrapment efficiency, chosen as the responses to optimize. A Scheffé simplex-centroid design was used to obtain the coefficients of the postulated mathematical model. The study of the response surface plots revealed that variations of the considered factors had different effects on the selected responses. The desirability function enabled for finding the optimal mixture composition, which represented the best compromise to simultaneously optimize all the three responses. The experimental values obtained with the optimized formulation were very similar to the predicted ones, proving the validity of the proposed regression model. The optimized niosomal formulation of dynorphin-B administered intravenously to mice (100mg/kg) showed a pronounced antinociceptive effect, significantly higher (P<0.05) than that given by i.v. administration of the simple solution of the peptide at the same concentration, proving its effectiveness in enabling the peptide brain delivery. These positive results suggest that the proposed approach could be successfully extended to other neuro-active peptides exerting a strong central action, even at low doses, but unable to cross the BBB.

Protective effect of ß-casomorphin-7 on cardiomyopathy of streptozotocin-induced diabetic rats via inhibition of hyperglycemia and oxidative stress.

ß-Casomorphin-7 (ß-CM-7) is regarded as the most representative milk-derived bioactive peptide. The present work studies the efficacy of ß-CM-7 against myocardial injury in streptozotocin-induced diabetic rats, focusing on the following assays: (1) the level of blood glucose and advanced glycosylation end product (AGE), the activity of lactate dehydrogenase (LDH) in serum; (2) the level of hydrogen peroxide (H2O2), the activity of Na(+)K(+)-ATPase, Ca(2+)Mg(2+)-ATPase and enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) in myocardial tissue; (3) the protein expression of glucose transporter-4 (GLUT-4) in myocardial tissue. It showed that with the influence of ß-CM-7, the levels of blood glucose of ß-CM-7 treatment group decreased markedly compared with model group (P<0.01) accompanied with their alleviated symptoms of diabetes. In the antioxidant and oxidant levels, ß-CM-7 treatment group signified a remarkable increase in the activity of GSH-Px, SOD and CAT of the anti-oxidation system and meanwhile demonstrated a considerable reduction in H2O2 content (all P<0.05) in comparison with model group. We also found both the content of AGE and the activity of LDH of ß-CM-7 treated group considerably reduced while the content of GLUT-4 and the activity of Na(+)K(+)-ATPase and Ca(2+)Mg(2+)-ATPase of ß-CM-7 treated group increased obviously (P<0.05). Meanwhile the cardiac indexes were significantly lessened. Thus our assay validates that the remedy employing ß-CM-7 may treat diabetic cardiomyopathy with high efficacy predominantly associated with the mechanism that ß-CM-7 ameliorates myocardial energy metabolism and abates free-radical-mediated oxidative stress in blood and myocardium.

Side-effects of analgesic kyotorphin derivatives: advantages over clinical opioid drugs.

The adverse side-effects associated with opioid administration restrain their use as analgesic drugs and call for new solutions to treat pain. Two kyotorphin derivatives, kyotorphin-amide (KTP-NH2) and ibuprofen-KTP-NH2 (IbKTP-NH2) are promising alternatives to opioids: they trigger analgesia via an indirect opioid mechanism and are highly effective in several pain models following systemic delivery. In vivo side-effects of KTP-NH2 and IbKTP-NH2 are, however, unknown and were evaluated in the present study using male adult Wistar rats. For comparison purposes, morphine and tramadol, two clinically relevant opioids, were also studied. Results showed that KTP-derivatives do not cause constipation after systemic administration, in contrast to morphine. Also, no alterations were observed in blood pressure or in food and water intake, which were only affected by tramadol. A reduction in micturition was detected after KTP-NH2 or tramadol administrations. A moderate locomotion decline was detected after IbKTP-NH2-treatment. The side-effect profile of KTP-NH2 and IbKTP-NH2 support the existence of opioid-based mechanisms in their analgesic actions. The conjugation of a strong analgesic activity with the absence of the major side-effects associated to opioids highlights the potential of both KTP-NH2 and IbKTP-NH2 as advantageous alternatives over current opioids.

ß-Casomorphin-7 attenuates the development of nephropathy in type I diabetes via inhibition of epithelial-mesenchymal transition of renal tubular epithelial cells.

This study was designed to investigate the putative protective effect of ß-casomorphin-7 on diabetic nephropathy in a rat model, and to explore the possible mechanism of this effect. SD rats were randomly divided into the following three groups: control group, diabetes group and ß-casomorphin-7-treatment group. All rats were euthanized after 30 days with or without ß-casomorphin-7 treatment. Biochemical parameters including blood glucose and renal function were quantified. The concentration of plasma TGF-ß1 was measured by ELISA. Histopathological changes to the kidney were studied by Masson and Sirius red staining. Expressions of α-smooth muscle actin (α-SMA), E-cadherin, vimentin, cytokeratin19 and TGF-ß1 mRNA in rat renal cortices were analyzed by real-time PCR. Changes in α-SMA and E-cadherin protein expression in rat renal cortices were quantified by Western blot. ß-Casomorphin-7 treatment of diabetic rats reduced urinary glucose, urinary protein, serum creatinine, blood urinary nitrogen, plasma TGF-ß1 and the ratio of kidney: body weight. Masson and Sirius red staining showed that ß-casomorphin-7 treatment attenuated renal interstitial fibrosis in diabetic rats. Compared to the control rats, diabetic rats had elevated expressions of α-SMA, vimentin and TGF-ß1 mRNA and α -SMA protein and decreased expression of E-cadherin and cytokeratin19 mRNA, and E-cadherin protein. ß-Casomorphin-7 treatment of diabetic rats partially normalized these changes. Our results suggest that administration of ß-casomorphin-7 attenuates renal interstitial fibrosis caused by diabetes. This protective effect may be associated, in part, with down regulation of epithelial-mesenchymal transition of renal tubular epithelial cells.

Chemical conjugation of the neuropeptide kyotorphin and ibuprofen enhances brain targeting and analgesia.

The pharmaceutical potential of natural analgesic peptides is mainly hampered by their inability to cross the blood-brain barrier, BBB. Increasing peptide-cell membrane affinity through drug design is a promising strategy to overcome this limitation. To address this challenge, we grafted ibuprofen (IBP), a nonsteroidal anti-inflammatory drug, to kyotorphin (l-Tyr-l-Arg, KTP), an analgesic neuropeptide unable to cross BBB. Two new KTP derivatives, IBP-KTP (IbKTP-OH) and IBP-KTP-amide (IbKTP-NH(2)), were synthesized and characterized for membrane interaction, analgesic activity and mechanism of action. Ibuprofen enhanced peptide-membrane interaction, endowing a specificity for anionic fluid bilayers. A direct correlation between anionic lipid affinity and analgesic effect was established, IbKTP-NH(2) being the most potent analgesic (from 25 µmol · kg(-1)). In vitro, IbKTP-NH(2) caused the biggest shift in the membrane surface charge of BBB endothelial cells, as quantified using zeta-potential dynamic light scattering. Our results suggest that IbKTP-NH(2) crosses the BBB and acts by activating both opioid dependent and independent pathways.

Inhibition of nociceptive responses after systemic administration of amidated kyotorphin.

BACKGROUND AND PURPOSE: Kyotorphin (KTP; L-Tyr-L-Arg), an endogenous neuropeptide, is potently analgesic when delivered directly to the central nervous system. Its weak analgesic effects after systemic administration have been explained by inability to cross the blood-brain barrier (BBB) and detract from the possible clinical use of KTP as an analgesic. In this study, we aimed to increase the lipophilicity of KTP by amidation and to evaluate the analgesic efficacy of a new KTP derivative (KTP-amide - KTP-NH(2) ). EXPERIMENTAL APPROACH: We synthesized KTP-NH(2) . This peptide was given systemically to assess its ability to cross the BBB. A wide range of pain models, including acute, sustained and chronic inflammatory and neuropathic pain, were used to characterize analgesic efficacies of KTP-NH(2) . Binding to opioid receptors and toxicity were also measured. KEY RESULTS: KTP-NH(2) , unlike its precursor KTP, was lipophilic and highly analgesic following systemic administration in several acute and chronic pain models, without inducing toxic effects or affecting motor responses and blood pressure. Binding to opioid receptors was minimal. KTP-NH(2) inhibited nociceptive responses of spinal neurons. Its analgesic effects were prevented by intrathecal or i.p. administration of naloxone. CONCLUSIONS AND IMPLICATIONS: Amidation allowed KTP to show good analgesic ability after systemic delivery in acute and chronic pain models. The indirect opioid-mediated actions of KTP-NH(2) may explain why this compound retained its analgesic effects although the usual side effects of opioids were absent, which is a desired feature in next-generation pain medications.

Protective effect of beta-casomorphin-7 on type 1 diabetes rats induced with streptozotocin.

The present study was aimed to investigate the possible effects of beta-casomorphin-7, against hyperglycemia and free radical-mediated oxidative stress in streptozotocin-induced diabetic rats by assaying the blood glucose level and the activity of plasma enzymatic antioxidants, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px). A significant increase in the levels of both blood glucose and oxidative stress with a predominant decrease in antioxidant status was observed in the diabetic rats when compared to control rats. After 15 days oral administration of beta-casomorphin-7 (7.5 x 10(-8) mol/day), the elevated blood glucose level was reduced. Oral administration of beta-CM-7 to diabetic rats showed an increase in the level of plasma insulin, the elevated plasma glucagon level was markedly reduced by the oral administration of beta-CM-7. Oral administration of beta-CM-7 to the diabetic group of rats also showed a significant elevation in the activity of SOD and catalase. Thus, the results of the present study suggest that beta-casomorphin-7 can protect rats from hyperglycemia and free radical-mediated oxidative stress in diabetic rats.

Synthesis and biological evaluation of cyclic endomorphin-2 analogs.

In our previous paper we reported synthesis and biological activity of two cyclic analogs of endomorphin-2 (EM-2): Tyr-c(Lys-Phe-Phe-Asp)-NH(2) and Tyr-c(Asp-Phe-Phe-Lys)-NH(2), achieved by making an amid bond between Lys and Asp side-chains. The first analog did not bind to the mu-opioid receptor, the affinity of the second one was very low. In the present study, we describe the synthesis of four novel cyclic analogs of similar structure, but with d-amino acids in position 2 (D-Lys or D-Asp). All new analogs displayed high affinity for the mu-opioid receptor, were much more stable than EM-2 in rat brain homogenate and showed remarkable antinociceptive activity after intracerebroventricular (i.c.v.) administration. Analgesic effect of the most potent cyclic analog, Tyr-c(D-Lys-Phe-Phe-Asp)NH(2) was much stronger and longer lasting than that of EM-2. This analog elicited analgesia also after peripheral administration and this effect was reversed by concomitant i.c.v. injection of the mu-opioid antagonist, beta-funaltrexamine, which indicated that antinociception was mediated by the mu-opioid receptor in the brain. Central action of the cyclic analog gives evidence that it was able to cross the blood-brain barrier, most likely due to the increased lipophilicity. Our results demonstrate that cyclization might be a promising strategy to enhance bioavailability of peptides and may serve a role in the development of novel endomorphin analogs with increased therapeutic potential.

Vascular effects, angiotensin I-converting enzyme (ACE)-inhibitory activity, and antihypertensive properties of peptides derived from egg white.

In this study, we have identified novel antihypertensive peptides derived from egg-white proteins. The sequences YRGGLEPINF and ESIINF produced an acute blood-pressure-lowering effect in spontaneously hypertensive rats upon a single oral administration. Our results suggest that the antihypertensive action could be attributed to a vascular-relaxing mechanism that would occur in vivo independently of angiotensin I-converting enzyme (ACE) inhibition, because neither these peptides nor their main digestion fragments, except for the dipeptide YR, acted as ACE inhibitors in vitro. The vasodilator and antihypertensive activity of the sequences ESI and NF would explain the blood-pressure-lowering effect of ESIINF. With regard to YRGGLEPINF, in addition to NF, YR appeared as the main fragment responsible for its activity. The dipeptide YR, named kyotorphin and previously identified as an endogenous analgesic neuropeptide in the central nervous system, showed strong vasodilator and antihypertensive properties. The structure-activity features of the vasodilator peptides are discussed.

Gene therapy and chronic pain.

Chronic, unremitting pain is perhaps the most common reason that patients seek medical care. In general, conservative techniques, such as medical management, are implemented as first-line therapy. Local anesthesia and lytic procedures, followed by interventional techniques, such as dorsal column stimulation and intrathecal drug delivery systems, are second-line therapies. However, for refractory and severe pain, which is not adequately controlled by other modes of therapy, new emerging options, including molecular or gene therapy, may become more widely utilized as experimental results are translated into clinical options.

Osteogenic growth peptide effects on primary human osteoblast cultures: potential relevance for the treatment of glucocorticoid-induced osteoporosis.

The osteogenic growth peptide (OGP) is a naturally occurring tetradecapeptide that has attracted considerable clinical interest as a bone anabolic agent and hematopoietic stimulator. In vivo studies on animals have demonstrated that the synthetic peptide OGP (10-14), reproducing the OGP C-terminal active portion [H-Tyr-Gly-Phe-Gly-Gly-OH] increases bone formation, trabecular bone density and fracture healing. In vitro studies performed on cellular systems based on osteoblastic-like cell lines or mouse stromal cells, have demonstrated that OGP (10-14) increases osteoblast proliferation, alkaline phosphatase (ALKP) activity and matrix synthesis and mineralization. In view of a potential application of OGP (10-14) in clinical therapy, we have tested different concentrations of OGP (10-14) on primary human osteoblast (hOB) cultures. We have observed significant increases of hOB proliferation (+35%), ALKP activity (+60%), osteocalcin secretion (+50%), and mineralized nodules formation (+49%). Our experimental model based on mature hOBs was used to investigate if OGP (10-14) could prevent the effects on bone loss induced by sustained glucocorticoid (GC) treatments. A strong decrease in bone formation has been attributed to the effects of GCs on osteoblastogenesis and osteocyte apoptosis, while an increase in bone resorption was due to a transient osteoblastic stimulation, mediated by the OPG/RANKL/RANK system, of osteoclasts recruitment and activation. Moreover, GCs act on hOBs decreasing the release of osteoprotegerin (OPG) a regulator of the RANKL/RANK interaction. Here, we provide evidences that OGP (10-14) inhibits hOB apoptosis induced by an excess of dexamethasone (-48% of apoptotic cells). Furthermore, we show that OGP (10-14) can increase OPG secretion (+20%) and can restore the altered expression of OPG induced by GCs to physiological levels. Our results support the employment of OGP (10-14) in clinical trials addressed to the treatment of different bone remodeling alterations including the GC-induced osteoporosis.