A Paranigral VTA Nociceptin Circuit that Constrains Motivation for Reward.

Nociceptin and its receptor are widely distributed throughout the brain in regions associated with reward behavior, yet how and when they act is unknown. Here, we dissected the role of a nociceptin peptide circuit in reward seeking. We generated a prepronociceptin (Pnoc)-Cre mouse line that revealed a unique subpopulation of paranigral ventral tegmental area (pnVTA) neurons enriched in prepronociceptin. Fiber photometry recordings during progressive ratio operant behavior revealed pnVTAPnoc neurons become most active when mice stop seeking natural rewards. Selective pnVTAPnoc neuron ablation, inhibition, and conditional VTA nociceptin receptor (NOPR) deletion increased operant responding, revealing that the pnVTAPnoc nucleus and VTA NOPR signaling are necessary for regulating reward motivation. Additionally, optogenetic and chemogenetic activation of this pnVTAPnoc nucleus caused avoidance and decreased motivation for rewards. These findings provide insight into neuromodulatory circuits that regulate motivated behaviors through identification of a previously unknown neuropeptide-containing pnVTA nucleus that limits motivation for rewards.

Synthesis, characterization, and biological study of new synthetic opioid hemorphin-4 peptides containing sterically restricted nonnatural amino acids.

Some new hemorphin-4 analogs with structures of Xxx-Pro-Trp-Thr-NH2 and Tyr-Yyy-Trp-Thr-NH2, where Xxx is 2-amino-3-(4-hydroxy-2,6-dimethylphenyl)propanoic acid or 2-amino-3-(4-dibenzylamino-2,6-dimethylphenyl)propanoic acid, and Yyy is (2S,4S)-4-amino-pyrrolidine-2-carboxylic acid, were synthesized and characterized by electrochemical and spectral analyses. In vivo anticonvulsant and antinociceptive activities of peptide derivatives were studied after intracerebroventricular injection in mice. The therapeutic effects of the modified peptides on seizures and pain in mice were evaluated to provide valuable insights into the potential applications of the novel compounds. Electrochemical characterization showed that the compounds behave as weak protolytes and that they are in a soluble, stable molecular form at physiological pH values. The antioxidant activity of the peptides was evaluated with voltammetric analyses, which were confirmed by applying the 2,2-Diphenyl-1-picrylhydrazyl method. The compounds showed satisfactory results regarding their structural stability, reaching the desired centers for the manifestation of biological activity without hydrolysis processes at 37°C and physiological pH. Dm-H4 and H4-P1 exhibited 100% and 83% potency to suppress the psychomotor seizures in the 6-Hz test compared to 67% activity of H4. Notably, only the H4-P1 had efficacy in blocking the tonic component in the maximal electroshock test with a potency comparable to H4. All investigated peptides containing unnatural conformationally restricted amino acids showed antinociceptive effects. The analogs Db-H4 and H4-P1 showed the most pronounced and long-lasting effect in both experimental models of pain induced by thermal and chemical stimuli. Dm-H4 produced a dose-dependent thermal antinociception and H4-P2 inhibited only formalin-induced pain behavior.

In Vitro and In Vivo Pharmacological Profiles of LENART01, a Dermorphin-Ranatensin Hybrid Peptide.

Diverse chemical and pharmacological strategies are currently being explored to minimize the unwanted side effects of currently used opioid analgesics while achieving effective pain relief. The use of multitarget ligands with activity at more than one receptor represents a promising therapeutic approach. We recently reported a bifunctional peptide-based hybrid LENART01 combining dermorphin and ranatensin pharmacophores, which displays activity to the mu-opioid receptor (MOR) and dopamine D2 receptor (D2R) in rat brains and spinal cords. In this study, we investigated the in vitro binding and functional activities to the human MOR and the in vivo pharmacology of LENART01 in mice after subcutaneous administration. In vitro binding assays showed LENART01 to bind and be selective to the human MOR over the other opioid receptor subtypes and delta, kappa and nociceptin receptors. In the [35S]GTPγS binding assay, LENART01 acted as a potent and full agonist to the human MOR. In mice, LENART01 produced dose-dependent antinociceptive effects in formalin-induced inflammatory pain, with increased potency than morphine. Antinociceptive effects were reversed by naloxone, indicating MOR activation in vivo. Behavioral studies also demonstrated LENART01's properties to induce less adverse effects without locomotor dysfunction and withdrawal syndrome compared to conventional opioid analgesics, such as morphine. LENART01 is the first peptide-based MOR-D2R ligand known to date and the first dual MOR-dopamine D2R ligand for which in vivo pharmacology is reported with antinociceptive efficacy and reduced opioid-related side effects. Our current findings may pave the way to new pain therapeutics with limited side effects in acute and chronic use.

The opioid peptide ß-endorphin interferes with the pituitary-testis axis in the Mozambique tilapia Oreochromis mossambicus.

Although the involvement of ß-endorphin (ß-ERP) in vertebrate reproduction has been suggested, its role in testicular activity is not clear in fish. We describe the influence of ß-ERP on spermatogenesis in a cichlid fish in the present paper. In comparison to the control group, the administration of ß-ERP (3 µg) caused a significant increase in the number of spermatogonia-A and spermatids. Following treatment with ß-ERP (6 µg), a significant increase in the number of spermatogonia-A was observed, whereas the numbers of all the other germ cells, excluding spermatogonia-B, significantly decreased in comparison to those in the control group. In addition, treatment of fish with 6 µg ß-ERP resulted in a significant reduction in the dimensions of the lumen and seminiferous lobules, the level of immunopositive androgen receptor (AR) expression in Sertoli cells, and the percentage of luteinizing hormone (LH) immunolabeled in the pituitary compared to those in the control group or the group treated with 3 µg ß-ERP. In contrast, the intensity of AR immunoreactivity and the percentage of LH immunolabeling were substantially increased in fish treated with 3 µg ß-ERP compared to those in the control group. These findings reveal for the first time that a low dose of ß-ERP stimulates the recruitment of spermatogonia as well as spermateleosis, whereas a high concentration affects the recruitment of germ cells prior to meiotic division in tilapia. These results suggest that ß-ERP exerts modulatory effects at the testicular and hypophysial levels through alterations in AR expression and LH secretory activity, respectively, in teleosts.

Acute single non-sedative doses of NOP receptor agonists affect acquisition of object location memory but repeated high doses do not induce long-lasting deficits.

The Nociceptin/Orphanin FQ (N/OFQ) system has been shown to modulate various aspects of long-term memory. It is therefore important to study the effects on memory impairment by nociceptin receptor (NOP) agonists under preclinical development. In the present study, we investigated the effect of systemic injection of two small molecule selective NOP agonists, AT-202 and AT-524, in the object location memory task in male and female mice. Since high doses of NOP agonists have been shown to induce sedation, we first determined the sedative doses for the two compounds and found them to be higher in female than in male mice. We then observed that sub-sedative doses of NOP agonists administered before learning, induced memory impairment during a test session performed 24 h later. Again, female mice were less sensitive to the amnesic effects than males. On the contrary, in male mice, NOP agonists did not produce amnesia when they were injected after learning, suggesting that they do not affect the consolidation of object location memory. Finally, repeated administration of high doses of NOP agonists over 7 days did not impair long-term spatial memory. Together, our data show for the first time that NOP receptor agonists impair the acquisition of object location memory with sex-dependent potency but do not affect memory consolidation, and that repeated stimulation of the receptor does not compromise long-term episodic-like spatial memory.

The influence of a synthetic growth hormone-releasing hormone analogue G11 and opioid peptide biphalin on selected fibroblasts parameters relevant to wound healing.

The treatment of hard-to-heal chronic wounds is still a major medical problem and an economic and social burden. In this work, we examine the proregenerative potential of two peptides, G11 (a trypsin-resistant analogue of growth hormone-releasing hormone [GHRH]) and biphalin (opioid peptide), and their combination in vitro on human fibroblasts (BJ). G11, biphalin and their combination exhibited no toxicity against BJ cells. On the contrary, these treatments significantly stimulated proliferation and migration of fibroblasts. Under inflammatory conditions (LPS-induced BJ cells), we noticed that the tested peptides decreased the levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and interleukin 1ß (IL-1ß). This was correlated with diminished phosphorylation levels of p38 kinase, but not those of ERK1/2. We found also that G11, biphalin and their combination activated the ERK1/2 signalling pathway, which has been previously implicated in promigratory activity of some regeneration enhancers, including opioids or GHRH analogues. Potential application of their combination requires further work, in particular in vivo experiments, in which the organism-level relevance of the discussed cell-level effects would be proven and, additionally, analgesic action of the opioid ingredient could be quantified.

The opioid peptide leucine-enkephalin disrupts seasonal and gonadotropin-induced ovarian recrudescence in the gecko Hemidactylus frenatus.

The enkephalins are known to regulate many physiological functions, including reproduction in vertebrates. However, the role of leucine-enkephalin (L-ENK) in the ovarian recrudescence activity of reptiles is not known. In the present study, we studied the influence of L-ENK on seasonal and FSH-induced ovarian recrudescence during the breeding and non-breeding phases of the cycle in the tropical and subtropical gecko Hemidactylus frenatus. In the first experiment, treatment with 5 and 25 µg L-ENK resulted in a dose-dependent inhibitory effect on the hypothalamic gonadotropin-releasing hormone (GnRH) neurons and ovary, as indicated by a significantly decreased percent area of GnRH-immunoreactive (GnRH-ir) fibres in the median eminence and pars distalis of the pituitary gland, concomitant with complete absence of stage V (late vitellogenic) follicles in the ovary compared to those of experimental controls. In the second experiment, administration of FSH to lizards in the regression phase stimulated the recruitment of stage IV and V (vitellogenic) follicles in contrast to their absence in initial controls or treatment controls. However, similar treatment of FSH in combination with 25 µg L-ENK did not result in the development of stage IV or V follicles. Together, these results suggest for the first time that treatment with 5 and 25 µg L-ENK exerts a dose-dependent inhibitory effect on the hypothalamic GnRH release into the median eminence and pituitary gland, leading to the blockade of ovarian recrudescence. These results also suggest a possible direct inhibitory effect of L-ENK at the level of the ovary in the gecko.

The vital role of arcuate nociceptin/orphanin FQ neurones in mounting an oestradiol-dependent adaptive response to negative energy balance via inhibition of nearby proopiomelanocortin neurones.

We tested the hypothesis that N/OFQ neurones in the arcuate nucleus (N/OFQARC ) inhibit proopiomelanocortin (POMCARC ) neurones in a diet- and hormone-dependent manner to promote a more extensive rebound hyperphagia upon re-feeding following an 18 h fast. We utilized intact male or ovariectomized (OVX) female mice subjected to ad libitum-feeding or fasting conditions. N/OFQARC neurones under negative energy balance conditions displayed heightened sensitivity as evidenced by a decreased rheobase threshold, increased firing frequency, and increased burst duration and frequency compared to ad libitum-feeding conditions. Stimulation of N/OFQARC neurones more robustly inhibited POMCARC neurones under fasting conditions compared to ad libitum-feeding conditions. N/OFQARC inhibition of POMCARC neurones is hormone dependent as chemostimulation of N/OFQARC neurones from fasted males and OVX females produced a sizable outward current in POMCARC neurones. Oestradiol (E2 ) markedly attenuated the N/OFQ-induced POMCARC outward current. Additionally, N/OFQ tonically inhibits POMCARC neurones to a greater degree under fasting conditions than in ad libitum-feeding conditions as evidenced by the abrogation of N/OFQ-nociceptin opioid peptide (NOP) receptor signalling and inhibition of N/OFQ release via chemoinhibition of N/OFQARC neurones. Intra-arcuate nucleus application of N/OFQ further elevated the hyperphagic response and increased meal size during the 6 h re-feed period, and these effects were mimicked by chemostimulation of N/OFQARC neurones in vivo. E2 attenuated the robust N/OFQ-induced rebound hyperphagia seen in vehicle-treated OVX females. These data demonstrate that N/OFQARC neurones play a vital role in mitigating the impact of negative energy balance by inhibiting the excitability of anorexigenic neural substrates, an effect that is diminished by E2 in females. KEY POINTS: Nociceptin/orphanin FQ (N/OFQ) promotes increased energy intake and decreased energy expenditure under conditions of positive energy balance in a sex- and hormone-dependent manner. Here it is shown that under conditions of negative energy balance, i.e. fasting, N/OFQ inhibits anorexigenic proopiomelanocortin (POMC) neurones to a greater degree compared to homeostatic conditions due to fasting-induced hyperexcitability of N/OFQ neurones. Additionally, N/OFQ promotes a sustained increase in rebound hyperphagia and increase in meal size during the re-feed period following a fast. These results promote greater understanding of how energy balance influences the anorexigenic circuitry of the hypothalamus, and aid in understanding the neurophysiological pathways implicated in eating disorders promoting cachexia.

The Therapeutic Potential of Naturally Occurring Peptides in Counteracting SH-SY5Y Cells Injury.

Peptides have revealed a large range of biological activities with high selectivity and efficiency for the development of new drugs, including neuroprotective agents. Therefore, this work investigates the neuroprotective properties of naturally occurring peptides, endomorphin-1 (EM-1), endomorphin-2 (EM-2), rubiscolin-5 (R-5), and rubiscolin-6 (R-6). We aimed at answering the question of whether well-known opioid peptides can counteract cell injury in a common in vitro model of Parkinson's disease (PD). Antioxidant activity of these four peptides was evaluated by the 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activity, oxygen radical absorbance capacity (ORAC), and ferric-reducing antioxidant power (FRAP) assays, while neuroprotective effects were assessed in a neurotoxic model induced by 6-hydroxydopamine (6-OHDA) in a human neuroblastoma cell line (SH-SY5Y). The mechanisms associated with neuroprotection were investigated by the determination of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, and Caspase-3 activity. Among the tested peptides, endomorphins significantly prevented neuronal death induced by 6-OHDA treatment, decreasing MMP (EM-1) or Caspase-3 activity (EM-2). Meanwhile, R-6 showed antioxidant potential by FRAP assay and exhibited the highest capacity to recover the neurotoxicity induced by 6-OHDA via attenuation of ROS levels and mitochondrial dysfunction. Generally, we hypothesize that peptides' ability to suppress the toxic effect induced by 6-OHDA may be mediated by different cellular mechanisms. The protective effect caused by endomorphins results in an antiapoptotic effect (mitochondrial protection and decrease in Caspase-3 activity), while R-6 potency to increase a cell's viability seems to be mediated by reducing oxidative stress. Our results may provide new insight into neurodegeneration and support the short peptides as a potent drug candidate to treat PD. However, further studies should be conducted on the detailed mechanisms of how tested peptides could suppress neuronal injuries.

Glutamate Buffering Capacity and Blood-Brain Barrier Protection of Opioid Receptor Agonists Biphalin and Nociceptin.

Opioids play crucial roles in the regulation of many important brain functions including pain, memory, and neurogenesis. Activation of opioid receptors is reported to have neuroprotective effects after ischemic reperfusion injury. The objective of this study was to understand the role of biphalin and nociceptin, opioid receptor agonists, on blood-brain barrier (BBB) integrity during ischemic stroke. In this study, we aimed to measure the effect of biphalin and nociceptin on astrocytic glutamate uptake and on expression of excitatory amino acid transporter to study the indirect role of astrocytes on opioid receptor-mediated BBB protection during in vitro stroke conditions. We used mouse brain endothelial cells (bEnd.3) and primary astrocytes as an in vitro BBB model. Restrictive BBB properties were evaluated by measuring [14C] sucrose paracellular permeability and the redistribution of the tight junction proteins. The protective effect of biphalin and nociceptin on BBB integrity was assessed after exposing cells to oxygen glucose deprivation (OGD) and glutamate. It was observed that combined stress (2 mM glutamate and 2 hours of OGD) significantly reduced glutamate uptake by astrocytes; however, biphalin and nociceptin treatment increased glutamate uptake in primary astrocytes. This suggests a role of increased astrocytic buffering capacity in opioid-meditated protection of the BBB during ischemic stroke. It was also found that the combined stress significantly increased [14C] sucrose paracellular permeability in an in vitro BBB model. Biphalin and nociceptin treatment attenuated the effect of the combined stress, which was reversed by the opioid receptor antagonists, suggesting the role of opioid receptors in biphalin and nociception's BBB modulatory activity. SIGNIFICANT STATEMENT: There is an unmet need for discovering new efficacious therapeutic agents to offset the deleterious effects of ischemic stroke. Given the confirmed roles of opioid receptors in the regulation of central nervous system functions, opioid receptor agonists have been studied as potential neuroprotective options in ischemic conditions. This study adds to the knowledge about the cerebrovascular protective effects of opioid receptor agonists and provides insight about the mechanism of action of these agents.

Regulation of Opioid Receptors by Their Endogenous Opioid Peptides.

Activation of µ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses. The three major endogenous opioid peptides, ß-endorphin, enkephalins, and dynorphins result from the processing of three main precursors: proopiomelanocortin, proenkephalin, and prodynorphin. Using a knockout approach, we sought to determine whether the absence of endogenous opioid peptides would affect the expression or activity of opioid receptors in mice lacking either proenkephalin, ß-endorphin, or both. Since gene knockout can lead to changes in the levels of peptides generated from related precursors by compensatory mechanisms, we directly measured the levels of Leu-enkephalin and dynorphin-derived peptides in the brain of animals lacking proenkephalin, ß-endorphin, or both. We find that whereas the levels of dynorphin-derived peptides were relatively unaltered, the levels of Leu-enkephalin were substantially decreased compared to wild-type mice suggesting that preproenkephalin is the major source of Leu-enkephalin. This data also suggests that the lack of ß-endorphin and/or proenkephalin does not lead to a compensatory change in prodynorphin processing. Next, we examined the effect of loss of the endogenous peptides on the regulation of opioid receptor levels and activity in specific regions of the brain. We also compared the receptor levels and activity in males and females and show that the lack of ß-endorphin and/or proenkephalin leads to differential modulation of the three opioid receptors in a region- and gender-specific manner. These results suggest that endogenous opioid peptides are important modulators of the expression and activity of opioid receptors in the brain.

Involvement of the nociceptin opioid peptide receptor in morphine-induced antinociception, tolerance and physical dependence in female mice.

Nociceptin opioid peptide (NOP) receptor modulates pain transmission and is considered a prospective target for pain management. Under acute pain conditions in rodents, however, no definitive conclusions about effects of systemically intervening NOP receptors on nociception, classical opioid-induced antinociception, tolerance and physical dependence have been drawn. Given that opioid analgesia has sex differences, and females experience greater pain and consume more opioids, clarifying these issues in females will help develop novel analgesics. To clarify the role of NOP receptors on the pharmacological profiles of µ-opioid receptor agonists, in this study, a selective agonist (SCH221510) and antagonist (SB612111) of the NOP receptor were subcutaneously administered in female mice in multiple animal models. In hot-plate test, neither SCH221510 (3 and 10 mg/kg, sc) nor SB612111 (10 mg/kg, sc) produced significant antinociception. SCH221510 (3 mg/kg, sc) attenuated but SB612111 (10 mg/kg, sc) enhanced morphine-induced antinociception, with rightward and leftward shift of morphine dose-response curves, respectively. SCH221510 (3 mg/kg, sc) combined with morphine (10 mg/kg, sc) accelerated the development of morphine antinociceptive tolerance. Conversely, SB612111 (10 mg/kg, sc) delayed morphine tolerance development. Neither SCH221510 (3 mg/kg, sc) nor SB612111 (10 mg/kg, sc) statistically significantly altered the development of morphine-induced physical dependence. Therefore, systemic activation of NOP receptors attenuated morphine antinociception to acute thermal stimuli, facilitated morphine-induced antinociceptive tolerance but did not robustly alter physical dependence in female mice. Systemic blockade of NOP receptors produced opposite actions. These findings demonstrate that N/OFQ-NOP receptor system plays diverse roles in modulating pharmacological profiles of µ-opioid receptor agonists.

Non-Opioid Peptides Targeting Opioid Effects.

Opioids are the most potent widely used analgesics, primarily, but not exclusively, in palliative care. However, they are associated with numerous side effects, such as tolerance, addiction, respiratory depression, and cardiovascular events. This, in turn, can result in their overuse in cases of addiction, the need for dose escalation in cases of developing tolerance, and the emergence of dose-related opioid toxicity, resulting in respiratory depression or cardiovascular problems that can even lead to unintentional death. Therefore, a very important challenge for researchers is to look for ways to counteract the side effects of opioids. The use of peptides and their related compounds, which have been shown to modulate the effects of opioids, may provide such an opportunity. This short review is a compendium of knowledge about the most important and recent findings regarding selected peptides and their modulatory effects on various opioid actions, including cardiovascular and respiratory responses. In addition to the peptides more commonly reported in the literature in the context of their pro- and/or anti-opioid activity-such as neuropeptide FF (NPFF), cholecystokinin (CCK), and melanocyte inhibiting factor (MIF)-we also included in the review nociceptin/orphanin (N/OFQ), ghrelin, oxytocin, endothelin, and venom peptides.

Dysregulation of Nociceptin/Orphanin FQ and Dynorphin Systems in the Extended Amygdala of Alcohol Preferring Marchigian Sardinian (msP) Rats.

Previous studies have shown that genetically selected Marchigian Sardinian alcohol-preferring (msP) rats consume excessive amounts of ethanol to self-medicate from negative moods and to relieve innate hypersensitivity to stress. This phenotype resembling a subset of alcohol use disorder (AUD) patients, appears to be linked to a dysregulation of the equilibrium between stress and antistress mechanisms in the extended amygdala. Here, comparing water and alcohol exposed msP and Wistar rats we evaluate the transcript expression of the anti-stress opioid-like peptide nociceptin/orphanin FQ (N/OFQ) and its receptor NOP as well as of dynorphin (DYN) and its cognate κ-opioid receptor (KOP). In addition, we measured the transcript levels of corticotropin-releasing factor (CRF), CRF receptor 1 (CRF1R), brain-derived neurotrophic factor (BDNF) and of the tropomyosin receptor kinase B receptor (Trk-B). Results showed an innately up-regulation of the CRFergic system, mediating negative mood and stress responses, as well as an inherent up-regulation of the anti-stress N/OFQ system, both in the amygdala (AMY) and bed nucleus of the stria terminalis (BNST) of msP rats. The up-regulation of this latter system may reflect an attempt to buffer the negative condition elicited by the hyperactivity of pro-stress mechanisms since results showed that voluntary alcohol consumption dampened N/OFQ. Alcohol exposure also reduced the expression of dynorphin and CRF transmissions in the AMY of msP rats. In the BNST, alcohol intake led to a more complex reorganization of these systems increasing receptor transcripts in msP rats, along with an increase of CRF and a decrease of N/OFQ transcripts, respectively. Moreover, mimicking the effects of alcohol in the AMY we observed that the activation of NOP receptor by intracerebroventricular administration of N/OFQ in msP rats caused an increase of BDNF and a decrease of CRF transcripts. Our study indicates that both stress and anti-stress mechanisms are dysregulated in the extended AMY of msP rats. The voluntary alcohol drinking, as well as NOP agonism, have a significant impact on neuropeptidergic systems arrangement, bringing the systems back to normalization.

In Vitro Analyses of Spinach-Derived Opioid Peptides, Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and ß-Arrestin-Mediated Pathways.

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the ß-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKeyTM assay, cADDis® cAMP assay, and PathHunter® ß-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKeyTM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis® cAMP and PathHunter® ß-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.

The analgesic hybrid of dermorphin/substance P and analog of enkephalin improve wound healing in streptozotocin-induced diabetic rats.

The purpose of this study was to investigate the effect of the peptide analgesic hybrid compounds: AWL3106 analog of dermorphin and substance P (7-11), and biphalin enkephalin analog on wound healing in streptozotocin-induced diabetic rats. The diabetes was induced in 6-7 week-old male Wistar rats by intraperitoneal injection of streptozotocin. After 70 days, the wounds were created on the back of the rats and then, once a day for 21 days, the dressing containing lanolin ointment, 10% of keratin scaffolds, and 1 mM of AWL3106 or biphalin was applied. The wounds histology were analyzed by hematoxylin and eosin staining. The orientation and organization of collagen was analyzed by Masson's trichome staining. The number of macrophages, blood vessels, and fibroblasts were visualized by CD68, CD34, and vimentin immunoreactivity, respectively. Our results demonstrated that the wound area of AWL3106- and biphalin-treated groups was greatly reduced (up to 47% on the 7 day) in comparison with untreated diabetic groups. The immunohistochemical staining of macrophages demonstrated that AWL3106 and biphalin accelerated inflammatory progression and subsequently decreased persistent inflammation. The histological analysis showed that the structure of tissue in the groups under the study was very similar to the one of wound tissue in N-DM group. The H&E and Masson's trichome staining demonstrated that the orientation and organization of collagen as well as the number and shape of blood vessels were better in 3106- and BIF-treated group than in DM group. In conclusion, the obtained data suggested that our hybrid peptides enhanced wound healing, particularly by accelerating the inflammatory phase and promoted the wound closure.

Antinociceptive and Cytotoxic Activity of Opioid Peptides with Hydrazone and Hydrazide Moieties at the C-Terminus.

In the present contribution, we analyze the influence that C-terminal extension of short opioid peptide sequences by organic fragments has on receptor affinity, in vivo analgesic activity, and antimelanoma properties. The considered fragments were based on either N-acylhydrazone (NAH) or N'-acylhydrazide motifs combined with the 3,5-bis(trifluoromethyl)phenyl moiety. Eleven novel compounds were synthesized and subject to biological evaluation. The analyzed compounds exhibit a diversified range of affinities for the µ opioid receptor (MOR), rather low δ opioid receptor (DOR) affinities, and no appreciable neurokinin-1 receptor binding. In three out of four pairs, N-acylhydrazone-based derivatives bind MOR better than their N'-acylhydrazide counterparts. The best of the novel derivatives have similar low nanomolar MOR binding affinity as the reference opioids, such as morphine and biphalin. The obtained order of MOR affinities was compared to the results of molecular docking. In vivo, four tested compounds turned out to be relatively strong analgesics. Finally, the NAH-based analogues reduce the number of melanoma cells in cell culture, while their N'-acylhydrazide counterparts do not. The antimelanoma properties are roughly correlated to the lipophilicity of the compounds.

Nociceptin impairs acquisition of novel object recognition memory in perirhinal cortex.

Nociceptin/Orphanin FQ (N/OFQ) plays an important role in the regulation of spatial, fear and recognition memories. N/OFQ receptors are highly distributed in the perirhinal cortex, which is a key brain area involved in modulating novel object recognition (NOR) memory. However, the role of N/OFQ in NOR memory in the perirhinal cortex was still unknown. Moreover, the effects of N/OFQ on different stages of NOR memory were still unclear. In NOR task, we found that pre-training intracerebroventricular (icv) injection of N/OFQ (0.3 and 1 nmol) impaired long-term memory in a dose-dependent manner. However, icv infusion of N/OFQ immediately after training did not affect NOR memory consolidation even at a high dose of 3 nmol. Pre-test icv injection of N/OFQ (1 nmol) also did not influence NOR memory retrieval. These data indicate that N/OFQ negatively modulates long-term NOR memory during the acquisition phase. Furthermore, the amnesia effect of N/OFQ (1 nmol, icv) could be antagonist by pre-treatment with the selective N/OFQ receptor antagonist [Nphe1]N/OFQ(1-13)NH2 (10 nmol, icv), indicating pharmacological specificity. Then, we found that pre-training infusion of N/OFQ (0.1 and 0.3 nmol/side) into the bilateral perirhinal cortex impaired long-term NOR memory, suggesting the perirhinal cortex is a critical brain structure in mediating the amnesic effect of N/OFQ in NOR task. In conclusion, our data, for the first time, indicate that N/OFQ in the perirhinal cortex impairs NOR memory acquisition through the NOP receptors.

The History of N/OFQ and the NOP Receptor.

The discovery of nociceptin/orphanin FQ (N/OFQ) marks the genuine start of the reverse pharmacology era, when systematic hunting for ligands of orphan receptors began. The choice of this particular target was no coincidence as the orphan receptor ORL-1 displayed high similarity to known opioid receptors, and thus its elusive ligand held promise to find more than a ligand but a missing opioid peptide. N/OFQ indeed turned out to belong to the opioid peptide family, but with significant pharmacological and functional distinctions. The quest for understanding N/OFQ's physiological functions has produced some novel insights into stress regulation and many other body functions but is still ongoing almost 25 years after its discovery. This chapter highlights the early steps of orphan receptor research and some of the protagonists who helped to advance the field.

Regulation of the Genes Encoding the ppN/OFQ and NOP Receptor.

Over the years, the ability of N/OFQ-NOP receptor system in modulating several physiological functions, including the release of neurotransmitters, anxiety-like behavior responses, modulation of the reward circuitry, inflammatory signaling, nociception, and motor function, has been examined in several brain regions and at spinal level. This chapter collects information related to the genes encoding the ppN/OFQ and NOP receptor, their regulation, and relative transcriptional control mechanisms. Furthermore, genetic manipulations, polymorphisms, and epigenetic alterations associated with different pathological conditions are discussed. The evidence here collected indicates that the study of ppN/OFQ and NOP receptor gene expression may offer novel opportunities in the field of personalized therapies and highlights this system as a good "druggable target" for different pathological conditions.