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The endogenous neuropeptide FF (NPFF) and its two cognate G protein-coupled receptors, Neuropeptide FF Receptors 1 and 2 (NPFFR1 and NPFFR2), represent a relatively new target system for many therapeutic applications including pain regulation, modulation of opioid side effects, drug reward, anxiety, cardiovascular conditions and other peripheral effects. Since the cloning of NPFFR1 and NPFFR2 in 2000, significant progress has been made to understand their pharmacological roles and interactions with other receptor systems, notably the opioid receptors. A variety of NPFFR ligands with different mechanisms of action (agonists or antagonists) have been discovered although with limited subtype selectivities. Differential pharmacological effects have been observed for many of these NPFFR ligands, depending on assays/models employed and routes of administration. In this Perspective, we highlight the therapeutic potentials, current knowledge gaps, and the latest updates of the development of peptidic and small molecule NPFFR ligands as tool compounds and therapeutic candidates.

Neuropeptide FF (NPFF) is a neuropeptide that regulates various biological activities. Currently, the regulation of NPFF on the immune system is an emerging field. However, the influence of NPFF on the transcriptome of primary macrophages has not been fully elucidated. In this study, the effect of NPFF on the transcriptome of mouse bone marrow-derived macrophages (BMDMs) was explored by RNA sequencing, bioinformatics, and molecular simulation. BMDMs were treated with 1 nM NPFF for 18 h, followed by RNA sequencing. Differentially expressed genes (DEGs) were obtained, followed by GO, KEGG, and PPI analysis. A total of eight qPCR-validated DEGs were selected as hub genes. Subsequently, the three-dimensional (3-D) structures of the eight hub proteins were constructed by Modeller and Rosetta. Next, the molecular dynamics (MD)-optimized 3-D structure of hub protein was acquired with Gromacs. Finally, the binding modes between NPFF and hub proteins were studied by Rosetta. A total of 2655 DEGs were obtained (up-regulated 1442 vs. down-regulated 1213), and enrichment analysis showed that NPFF extensively regulates multiple functional pathways mediated by BMDMs. Moreover, the 3-D structure of the hub protein was obtained after MD-optimization. Finally, the docking modes of NPFF-hub proteins were predicted. Besides, NPFFR2 was expressed on the cell membrane of BMDMs, and NPFF 1 nM significantly activated NPFFR2 protein expression. In summary, instead of significantly inhibiting the expression of the immune-related gene transcriptome of RAW 264.7 cells, NPFF simultaneously up-regulated and down-regulated the gene expression profile of a large number of BMDMs, hinting that NPFF may profoundly affect a variety of cellular processes dominated by BMDMs. Our work provides transcriptomics clues for exploring the influence of NPFF on the physiological functions of BMDMs.

Background. Spinal cord injury (SCI) has high incidence globally and is frequently accompanied by subsequent cognitive decline. Accurate early risk-categorization of SCI patients for cognitive decline using biomarkers can enable the timely application of appropriate neuroprotective measures and the development of new agents for the management of SCI-associated cognitive decline. Neuropeptide FF is an endogenous neuropeptide with a multitude of functions and is associated with neuroinflammatory processes. This prospective study investigated the predictive value of serum neuropeptide FF levels measured after acute SCI for subsequent cognitive decline. Methods. 88 patients presenting with acute SCI without preexisting neurological injury, brain trauma, or severe systemic illness and 60 healthy controls were recruited. Serum neuropeptide FF levels, clinical, and routine laboratory variables including low-density lipoprotein, high-density lipoprotein, fasting blood glucose, total triiodothyronine (TT3), total thyroxine (TT4), and thyroid-stimulating hormone (TSH) levels collected from all subjects were assessed. Montreal cognitive assessment (MoCA) was performed 3 months after enrollment. SCI patients were grouped according to quartile of serum neuropeptide FF level and MoCA scores were compared using ANOVA. Additionally, multivariate linear regression with clinical and laboratory variables was performed to predict MoCA scores. Results. SCI patients displayed significantly higher baseline serum neuropeptide FF levels than healthy controls ( 38.5 ± 4.1 versus 23.4 ± 2.0 pg / ml , p < 0.001 ∗ ∗ ). SCI patients in higher quartiles of baseline serum neuropeptide FF displayed significantly lower MoCA scores at 3 months. Linear regression analysis indicated serum neuropeptide FF levels as a significant independent predictor of worse MoCA scores after SCI ( r = 0.331 , p = 0.034 ∗ ). Conclusion. Early serum neuropeptide FF levels significantly and independently predicted cognitive decline after acute SCI among patients without preexisting neurological disorders.

The opioid-induced analgesia is associated with a number of side effects such as addiction, tolerance and respiratory depression. The involvement of neuropeptide FF (NPFF) in modulation of pain perception, opioid-induced tolerance and dependence was well documented in contrast to respiratory depression. Therefore, the aim of the present study was to examine the potency of NPFF to block post-opioid respiratory depression, one of the main adverse effects of opioid therapy. Urethane-chloralose anaesthetized Wistar rats were injected either intravenously (iv) or intracerebroventricularly (icv) with various doses of NPFF prior to iv endomorphin-1 (EM-1) administration. Iv NPFF diminished the number of EM-1-induced apneas without affecting their length and without influence on the EM-1 induced blood pressure decline. Icv pretreatment with NPFF abolished the occurrence of post-EM-1 apneas and reduced also the maximal drop in blood pressure and heart rate. These effects were completely blocked by the NPFF receptor antagonist RF9, which was given as a mixture with NPFF before systemic EM-1 administration. In conclusion, our results showed that centrally administered neuropeptide FF is effective in preventing apnea evoked by stimulation of &mu, opioid receptors and the effect was due to activation of central NPFF receptors. Our finding indicates a potential target for reversal of opioid-induced respiratory depression.

Restoring the control of food intake is the key to obesity management and prevention. The arcuate nucleus (ARC) of the hypothalamus is extensively being studied as a potential anti-obesity target. Animal studies showed that neuropeptide FF (NPFF) reduces food intake by its action in neuropeptide Y (NPY) neurons of the hypothalamic ARC, but the detailed mode of action observed in human neurons is missing, due to the lack of a human-neuron-based model for pharmacology testing. Here, we validated and utilized a human-neural-stem-cell-based (hNSC) model of ARC to test the effects of NPFF on cellular pathways and neuronal activity. We found that in the human neurons, decreased cAMP levels by NPFF resulted in a reduced rate of cytoplasmic calcium oscillations, indicating an inhibition of ARC NPY neurons. This suggests the therapeutic potential of NPFFR2 in obesity. In addition, we demonstrate the use of human-stem-cell-derived neurons in pharmacological applications and the potential of this model to address functional aspects of human hypothalamic neurons.

Opioid peptides and receptors are broadly expressed throughout peripheral and central nervous systems and have been the subject of intense long-term investigations. Such studies indicate that some endogenous neuropeptides, called anti-opioids, participate in a homeostatic system that tends to reduce the effects of endogenous and exogenous opioids. Anti-opioid properties have been attributed to various peptides, including melanocyte inhibiting factor (MIF)-related peptides, cholecystokinin (CCK), nociceptin/orphanin FQ (N/OFQ), and neuropeptide FF (NPFF). These peptides counteract some of the acute effects of opioids, and therefore, they are involved in the development of opioid tolerance and addiction. In this work, the anti-opioid profile of endogenous peptides was described, mainly taking into account their inhibitory influence on opioid-induced effects. However, the anti-opioid peptides demonstrated complex properties and could show opioid-like as well as anti-opioid effects. The aim of this review is to detail the phenomenon of crosstalk taking place between opioid and anti-opioid systems at the in vivo pharmacological level and to propose a cellular and molecular basis for these interactions. A better knowledge of these mechanisms has potential therapeutic interest for the control of opioid functions, notably for alleviating pain and/or for the treatment of opioid abuse.

Pharmacological evidence indicated a functional interaction between neuropeptide FF (NPFF) and cannabinoid systems, and the cannabinoids combined with the NPFF receptor agonist neuropeptide VF (NPVF) produced antinociception without tolerance. In the present study, VF-13, a chimeric peptide containing the pharmacophores of the endogenous cannabinoid peptide VD-hemopressin(α) (VD-Hpα) and NPVF, was synthesized and pharmacologically evaluated. In vitro, VF-13 significantly upregulated the phosphorylated level of extracellular signal-regulated kinase 1/2 (ERK1/2) in CHO cells stably expressing CB1 receptors and inhibited forskolin-induced cAMP accumulation in HEK293 cells stably expressing NPFF1 or NPFF2 receptors. Moreover, VF-13 induced neurite outgrowth in Neuro 2A cells via CB1 and NPFF receptors. These results suggest that VF-13 exhibits multifunctional agonism at CB1, NPFF1 and NPFF2 receptors in vitro. Interestingly, intracerebroventricular VF-13 produced dose-dependent antinociception in mouse models of tail-flick and carrageenan-induced inflammatory pain via the TRPV1 receptor. In contrast, the reference compound (m)VD-Hpα-NH2 induced CB1 receptor-mediated supraspinal antinociception. Additionally, subcutaneous injection of (m)VD-Hpα-NH2 and VF-13 produced significant antinociception in carrageenan-induced inflammatory pain model. In the tetrad assay, our data demonstrated that VF-13 elicited hypothermia, but not catalepsy and hypoactivity after intracerebroventricular injection. Notably, VF-13 produced non-tolerance forming antinociception over 6 days treatment in both acute and inflammatory pain models. Furthermore, VF-13 had no apparent effects on gastrointestinal transit, pentobarbitone-induced sedation, food intake, and motor coordination at the supraspinal level. In summary, VF-13, a novel chimeric peptide of VD-Hpα and NPVF, produced non-tolerance forming antinociception in preclinical pain models with reduced cannabinoid-related side effects.

We recently characterized a novel bifunctional agonist for opioid and neuropeptide FF receptors, named BN-9, which exhibited potent analgesia in the mouse tail-flick test when given centrally. To further evaluate its potential therapeutic efficacy for translational-medical development, the current work was performed to explore the antinociceptive activities of intraperitoneal (i.p.) administration of BN-9 in mouse models of tail-flick assay, formalin pain, visceral pain and post-operative pain. In the tail-flick test, BN-9 induced a dose-related antinociceptive effect, which was fully blocked by systemic pretreatment with the peripheral acting opioid receptor antagonist naloxone methiodide, but not supraspinal naloxone methiodide, implying the involvement of the peripheral opioid receptors. In addition, the systemic antinociception of BN-9 was antagonized by the selective antagonists of the μ- and κ-opioid receptors, independently of the δ-opioid and neuropeptide FF receptors. Similarly, dose-dependent analgesia was also produced by systemic BN-9 in different pain models via the peripheral opioid receptors, independently of the neuropeptide FF receptors. Furthermore, the side-effects of systemic BN-9 on motor performance, tolerance development and gastrointestinal transit inhibition were also evaluated. Repeated systemic injection of BN-9 produced non-tolerance analgesia over 8 days. Compared with morphine, intraperitoneal administration of BN-9 exerted less inhibition of gastrointestinal transit. These data show that BN-9 induced systemic analgesia with reduced side-effects on tolerance and constipation. This article suggests that systemic injection of BN-9 causes effective antinociception in different preclinical pain models via the peripheral opioid receptors, providing an attractive approach to develop peripherally acting opioid analgesics with multiple targeting properties.

FOXOs belong to the forkhead transcription factor superfamily, several of which are suggested to be involved in the control of food intake. Previously, we proved that the neuropeptide FF (NPFF) peptide was involved in feeding regulation in spotted sea bass. In the present study, seven members of the foxo family were identified in the whole genome of spotted sea bass. The distributions of these genes in different tissues were analyzed by qRT-PCR. Variations in the foxo1a and npff expression profiles during short-term starvation showed similar expression patterns. The colocalization of foxo1a and npff in the telencephalon, hypothalamus, stomach and intestine further provided evidence that foxo1a may act directly to promote the transcription of npff. Thirteen predicted FOXO1 binding sites were found in the 5' upstream region of npff. Luciferase assay results showed that FOXO1A was able to activate npff transcriptional responses by directly binding DNA response elements, and the key regulatory areas and sites of FOXO1A on the npff promoter were confirmed by deletion and site-directed mutagenesis analyses. These findings may help to elucidate the role of FOXO1 in the regulation of feeding processes in teleosts.

Neuropeptide FF (NPFF) and Neuropeptide VF (NPVF) are part of the extended RFamide peptide family characterized by their common arginine (R) and amidated phenylalanine (F)-motif at the carboxyl terminus. Both peptides signal through their respective high affinity G-protein coupled receptors, NPFFR2 and NPFFR1, but also show binding affinity for the other receptor due to their sequence similarity. NPFF and NPVF are highly conserved throughout evolution and can be found across the whole animal kingdom. Both have been implicated in a variety of biological mechanisms, including nociception, locomotion, reproduction, and response to pain and stress. However, more recently a new major functional role in the control of energy homeostasis has been discovered. In this article we will summarise the current knowledge on the distribution of NPFF, NPVF, and their receptors in central and peripheral tissues, as well as how this relates to the regulation of food intake and energy balance, which will help to better understand their role in these processes and thus might help finding treatments for impaired energy homeostasis disorders, such as obesity or anorexia.

The activation of opioid and neuropeptide FF (NPFF) receptors plays important roles to modulate nociceptive signal in inflammatory pain states. Recently, BN-9 (Tyr-D. Ala-Gly-Phe-Gln-Pro-Gln-Arg-Phe-NH2) was pharmacologically characterized as a novel bifunctional agonist at both opioid and NPFF receptors. In the present study, the anti-allodynic activity and site(s) of action of BN-9 were assessed in a mouse model of carrageenan-induced inflammatory pain. In mice, BN-9 induced a dose-dependent anti-allodinic effect through opioid receptor at supraspinal or spinal level, and this effect was augmented by pretreatment with the NPFF receptor antagonist at the same level. In contrast, peripheral administration of BN-9 produced opioid receptor-mediated anti-allodynia, which was insensitive of the NPFF receptor antagonist. In addition, systemic BN-9 produced anti-allodynic effect via opioid receptors, independent of NPFF system. Therefore, these data indicate that central, peripheral or systemic administrations of BN-9 exert potent analgesic activities in inflammatory pain model via opioid receptor, and central effects of BN-9 are associated with NPFF system. Interestingly, systemic anti-allodynia of BN-9 was blocked by intraperitoneal administration of the opioid receptor antagonists, naloxone and naloxone methiodide, but not by intracerebroventricular injection of the peripherally acting opioid antagonist naloxone methiodide. Furthermore, BN-9-induced systemic anti-allodynia was reversed by intraplantar administration of naloxone, but not by peripheral administration of the NPFF receptor antagonist. Taken together, our data further suggest that systemic BN-9-induced anti-allodynic effect is mainly mediated by peripheral opioid receptors, independent of NPFF receptors.

The quantity and activation state of adipose tissue macrophages (ATMs) impact the development of obesity-induced metabolic diseases. Appetite-controlling hormones play key roles in obesity; however, our understanding of their effects on ATMs is limited. Here, we have shown that human and mouse ATMs express NPFFR2, a receptor for the appetite-reducing neuropeptide FF (NPFF), and that NPFFR2 expression is upregulated by IL-4, an M2-polarizing cytokine. Plasma levels of NPFF decreased in obese patients and high-fat diet-fed mice and increased following caloric restriction. NPFF promoted M2 activation and increased the proliferation of murine and human ATMs. Both M2 activation and increased ATM proliferation were abolished in NPFFR2-deficient ATMs. Mechanistically, the effects of NPFF involved the suppression of E3 ubiquitin ligase RNF128 expression, resulting in enhanced stability of phosphorylated STAT6 and increased transcription of the M2 macrophage-associated genes IL-4 receptor α (Il4ra), arginase 1 (Arg1), IL-10 (Il10), and alkylglycerol monooxygenase (Agmo). NPFF induced ATM proliferation concomitantly with the increase in N-Myc downstream-regulated gene 2 (Ndrg2) expression and suppressed the transcription of Ifi200 cell-cycle inhibitor family members and MAF bZIP transcription factor B (Mafb), a negative regulator of macrophage proliferation. NPFF thus plays an important role in supporting healthy adipose tissue via the maintenance of metabolically beneficial ATMs.

Purpose: Gabapentin, a drug for neuropathic pain, exerts its therapeutic effect via inhibition of the a 2 d subunit of N-type Ca 2+ channels. Thus, finding peptides that specifically displace gabapentin from its binding site may lead to the development of new drugs. Methods: Displacement of bound [ 3 H]-gabapentin in membrane preparations of rat cerebral cortex and of human Cav2.2/β 3 /α 2 δ 1 expressed in CHO cell line. Results: Neuropeptide FLFQPQRF-NH2 specifically displaced bound [ 3 H]-gabapentin in membrane preparations from rats and CHO cells. Truncation of the C-terminus of FLFQPQRF-NH2 by three amino acid residues to produce FLFQP-NH 2 improved the displacement of gabapentin. FLFQP-NH 2 displaced bound [ 3 H]-gabapentin with IC 50 and K i values of 2.7 µM and 1.7 µM, respectively. Deletion of two amino acid residues (FQ) in the middle of the FLFQP-NH 2 sequence yielded FLP-NH 2 that displaced bound [ 3 H]-gabapentin with a lower affinity. IC 50 and K i values were 11.9 µM and 7.8 µM, respectively. Neutral binding cooperativity existed when of FLFQP-NH 2 , FLP- NH 2 and gabapentin when incubated together. FLFQPQRF-NH 2 but not FLFQP-NH 2 displaced bound [ 3 H]-gabapentin to membrane preparations of human Cav2.2/b 3 /a 2 d 1 expressed in CHO cells. Conclusion: FLFQPQRF-NH 2 , FLFQP-NH 2 and FLP-NH 2 displace bound gabapentin in membrane preparations of rat cerebral cortex. Binding cooperativity was detected when GBP/FLFQP-NH 2 /FLP-NH 2 were incubated together. These novel binding sites may provide new approaches to modulate L-type Ca 2+ channels.

The great majority of neurons in the superficial dorsal horn of the spinal cord are excitatory interneurons, and these are required for the normal perception of pain and itch. We have previously identified 5 largely non-overlapping populations among these cells, based on the expression of four different neuropeptides (cholecystokinin, neurotensin, neurokinin B and substance P) and of green fluorescent protein driven by the promoter for gastrin-releasing peptide (GRP) in a transgenic mouse line. Another peptide (neuropeptide FF, NPFF) has been identified among the excitatory neurons, and here we have used an antibody against the NPFF precursor (pro-NPFF) and a probe that recognises Npff mRNA to identify and characterise these cells. We show that they are all excitatory interneurons, and are separate from the five populations listed above, accounting for ~ 6% of the excitatory neurons in laminae I-II. By examining phosphorylation of extracellular signal-regulated kinases, we show that the NPFF cells can respond to different types of noxious and pruritic stimulus. Ablation of somatostatin-expressing dorsal horn neurons has been shown to result in a dramatic reduction in mechanical pain sensitivity, while somatostatin released from these neurons is thought to contribute to itch. Since the great majority of the NPFF cells co-expressed somatostatin, these cells may play a role in the perception of pain and itch., Highlights • NPFF is expressed by around 6% of the excitatory interneurons in the superficial dorsal horn of the mouse spinal cord. • NPFF cells differ from those that express substance P, cholecystokinin, neurotensin or neurokinin B. • Although some NPFF cells express gastrin-releasing peptide (GRP), they do not express GFP in a GRP-GFP mouse line. • Some NPFF cells are activated by noxious or pruritic stimuli.

The development of multitarget opioid drugs has emerged as an attractive therapeutic strategy to eliminate opioid-related side effects. Our previous study developed a series of opioid and neuropeptide FF pharmacophore-containing chimeric peptides, including DN-9 (Tyr-D.Ala-Gly-NMe.Phe-Gly-Pro-Gln-Arg-Phe-NH2), which produced potent nontolerance forming analgesia at the supraspinal level. In the present study, the antinociceptive effects of DN-9 in a series of preclinical pain models and the potential side-effects were investigated at the spinal level in mice. In the tail-flick test, intrathecal injection of DN-9 produced potent analgesia with an ED50 value at 1.33 pmol, and the spinal antinociception of DN-9 was mainly mediated by μ- and κ-opioid receptors. In addition, DN-9-induced spinal antinociception was augmented by the neuropeptide FF receptors antagonist. Furthermore, DN-9 could decrease both the frequency and amplitude of sEPSCs in lamina IIo neurons of the spinal cord, which were mediated by opioid receptors. In contrast to morphine, chronic intrathecal treatments with DN-9 did not induce analgesic tolerance, c-Fos expression or microglial activation. Intrathecal injection of DN-9 showed potent analgesia with antinociceptive ED50 values between .66 and 55.04 pmol in different pain models, including the formalin test, acetic acid-induced writhing test, carrageenan-induced inflammatory pain and neuropathic pain. Moreover, DN-9 did not show side effects in locomotor function and coordination, gastrointestinal transit inhibition, the cardiovascular system, and body temperature regulation at antinociceptive doses. Taken together, the present study showed DN-9 produced effective, nontolerance forming analgesia with reduced side effects at the spinal level. DN-9 might be a promising compound for developing multifunctional opioid analgesics with limited adverse effects. PERSPECTIVE: This article presents the potent and nontolerance forming analgesia effects of DN-9 in a series of preclinical pain models with less opioid related adverse effects at the spinal level in mice. This study also demonstrates that DN-9 has translational potential into an intrathecal analgesic.

Research has shown that opioids are involved in the rewarding effects of ethanol. Neuropeptide FF (NPFF) has been described as an anti-opioid peptide because, in many cases, it inhibits opioid and ethanol effects in rodents. Kissorphin (KSO) is a new peptide derived from kisspeptin-10 with structural similarities to NPFF. This peptide possesses NPFF-like biological activity in vitro. The aim of the current study was to investigate whether KSO (Tyr-Asn-Trp-Asn-Ser-Phe-NH2) influences the acquisition, expression, and reinstatement of ethanol-induced conditioned place preference (ethanol-CPP) in rats. The ethanol-CPP was established (conditioning for 5 days) by intraperitoneal (i.p.) administration of ethanol (1 g/kg, 20%, w/v) using an unbiased procedure. After that, one group of rats was used in final post-conditioning testing (expression of CPP) and the other group received a priming injection of ethanol after 10 days of extinction (reinstatement of CPP). Our experiments showed that KSO, given intravenously (i.v.) at the doses of 1, 3, and 10 nmol before every ethanol administration, inhibited the acquisition and, given acutely before the post-conditioning test or before the priming dose of ethanol, inhibited the expression and reinstatement of ethanol-CPP, respectively, in a dose-dependent manner. KSO given by itself neither induced place preference nor aversion and did not alter locomotor activity and coordination of rats. These results suggest that KSO can alter rewarding/motivational effects of ethanol. These data suggest this peptide possesses an anti-opioid character.

Neuropeptide FF (NPFF) is a neuropeptide that modulates various physiological processes. The regulatory role of NPFF in the immune and inflammatory response is currently being revealed. However, the effect of NPFF at the transcriptome level in macrophages has not been fully elucidated. Here, the impact of NPFF on gene expression at the transcriptome level of RAW 264.7 cells was investigated by RNA-seq. RAW 264.7 macrophages were treated with NPFF (1 nM) for 18 h, followed by RNA-seq examination. Differentially expressed genes (DEGs) were acquired, followed by GO, KEGG, and PPI analysis. A total of eight qPCR-verified DEGs were obtained. Next, three-dimensional models of the eight hub proteins were constructed by using homology modeling with Modeller (9v23). Finally, molecular dynamics simulation (300 ns) was performed with GROMACS 2018.2 to investigate the structural characteristics of these hub proteins. NPFF had no detectable effect on the morphology of RAW264.7 cells. A total of 211 DEGs were acquired, and an enrichment study demonstrated that the immune response-related pathway was significantly inhibited by NPFF. Moreover, the molecular dynamics optimized-protein models of the hub proteins were obtained. Collectively, NPFF inhibited the expression of immune-related genes in RAW 264.7 cells at the transcriptome level, which suggested a negative relationship between NPFF and this set of immune-related genes in RAW 264.7 macrophages. Therefore, our data may provide direct evidence of the role of NPFF in peripheral or central inflammatory diseases.

Mounting evidences indicate the functional interactions between neuropeptide FF (NPFF) and opioids, including the endogenous opioids. In the present work, EN-9, a chimeric peptide containing the functional domains of the endogenous opioid endomorphin-2 (EM-2) and NPFF, was synthesized and pharmacologically characterized. In vitro cAMP assay demonstrated that EN-9 was a multifunctional agonist of κ-opioid, NPFF1 and NPFF2 receptors. In the mouse tail-flick test, intracerebroventricularly (i.c.v.) administration of EN-9 produced significant antinociception with an ED50 value of 13.44 nmol, which lasted longer than that of EM-2. In addition, EN-9 induced potent antinociception after both intravenous (i.v.) and subcutaneous (s.c.) injection. Furthermore, the experiments using the antagonists of opioid and NPFF receptors indicated that the central antinociception of EN-9 was mainly mediated by κ-opioid receptor, independently on NPFF receptors. Notably, the central antinociception of EN-9 was not reduced over a period of 6 days repeated i.c.v. injection. Repeated i.c.v. administration of EN-9 with the NPFF1 and NPFF2 receptors antagonist RF9 resulted in a progressive loss of analgesic potency, consistent with the development of tolerance. Moreover, central administration of EN-9 induced the place conditioning aversion only at a high dose of 60 nmol, but not at low doses. At supraspinal level, only high dose of EN-9 (60 nmol, i.c.v.) inhibited gastrointestinal transit via NPFF receptors. Similarly, systemic administration of EN-9 also inhibited gastrointestinal transit at high doses (10 and 30 mg/kg, i.v.). Taken together, the multifunctional agonist of κ-opioid and NPFF receptors EN-9 produced a potent, non-tolerance forming antinociception with limited side effects.

Methionine-enkephalin-Arg-Phe is an endogenous amphiactive analgesic peptide. Neuropeptide FF, on the other hand, is reported for its role in opioid modulation and tolerance development. Based on these reports, in the present study we designed a chimeric peptide NPYFa (YGGFMKKKPQRFamide), having the Met-enkephalin (opioid) and PQRFamide sequence of neuropeptide FF, which can then target both the opioid and neuropeptide FF receptors. We hypothesized that the chimeric peptide so designed would have both analgesic properties and further aid in understanding of the role of neuropeptide FF in the development of opiate tolerance. Our studies indicated that NPYFa induced an early onset, potent, dose-dependent and prolonged antinociception. Additionally, antagonists (MOR, KOR, and DOR) pretreatment studies determined a KOR-mediated antinociception activity of the ligand. Further, in vitro binding studies using the Eu-GTP-γS binding assay on cell lines expressing opioid and NPFF receptors showed binding to both the opioid and neuropeptide FF receptors suggesting a multiple receptor binding character of NPYFa. Moreover, chronic (6 days) treatment with NPYFa exhibited an absence of tolerance development subsequent to its analgesia. The current study proposes NPYFa as a potent, long-acting antinociceptor lacking tolerance development as well as a probe to study opioid analgesia and the associated complex mechanisms of tolerance development.

Neuropeptide FF (NPFF) system has recently been reported to modulate cannabinoid-induced antinociception. The aim of the present study was to further investigate the roles of NPFF system in the antinociceptive effects induced by intracerebroventricular (i.c.v.) administration of mouse VD-hemopressin(α), a novel endogenous agonist of cannabinoid CB1 receptor, in naive and VD-hemopressin(α)-tolerant mice. The effects of NPFF system on the antinociception induced by VD-hemopressin(α) were investigated in the radiant heat tail-flick test in naive mice and VD-hemopressin(α)-tolerant mice. The cannabinoid-tolerant mice were produced by given daily injections of VD-hemopressin(α) (20 nmol, i.c.v.) for 5 days and the antinociception was measured on day 6. In naive mice, intracerebroventricular injection of NPFF dose-dependently attenuated central analgesia of VD-hemopressin(α). In contrast, neuropeptide VF (NPVF) and D.NP(N-Me)AFLFQPQRF-NH2 (dNPA), two highly selective agonists for Neuropeptide FF1 and Neuropeptide FF2 receptors, enhanced VD-hemopressin(α)-induced antinociception in a dose-dependent manner. In addition, the VD-hemopressin(α)-modulating activities of NPFF and related peptides were antagonized by the Neuropeptide FF receptors selective antagonist 1-adamantanecarbonyl-RF-NH2 (RF9). In VD-hemopressin(α)-tolerant mice, NPFF failed to modify VD-hemopressin(α)-induced antinociception. However, both neuropeptide VF and dNPA dose-dependently potentiated the antinociception of VD-hemopressin(α) and these cannabinoid-potentiating effects were reduced by RF9. The present works support the cannabinoid-modulating character of NPFF system in naive and cannabinoid-tolerant mice. In addition, the data suggest that a chronic cannabinoid treatment modifies the pharmacological profiles of NPFF, but not the cannabinoid-potentiating effects of neuropeptide VF and dNPA.

Chronic opioids treatment is impeded by the development of analgesic tolerance and opioid-induced hyperalgesia. Recent studies have shown that multi-functional opioid compounds produce analgesic activities with limited side effects. We developed a novel multi-functional peptide targeting opioid and neuropeptide FF receptors named BN-9, which produced potent and non-tolerance forming antinociceptive effect after supraspinal and systemic administrations. In the present study, the analgesic properties and potential side effects of intrathecal BN-9 were investigated in a range of preclinical rodent models. In complete Freund's adjuvant-induced inflammatory pain model, intrathecal BN-9 dose-dependently produced analgesic effect via opioid receptors, and the spinal antinociceptive effect was augmented by the neuropeptide FF receptor antagonist RF9. In contrast, in plantar incision-induced postoperative pain model, BN-9 exhibited potent anti-allodynic effect via opioid receptors and, at least partially, neuropeptide FF receptors. In mouse models of acetic acid-induced visceral pain and formalin pain, BN-9-induced spinal antinociception was mainly mediated by opioid receptors, independent of neuropeptide FF receptors. Furthermore, at the spinal level, chronic treatments with BN-9 did not lead to analgesic tolerance and cross-tolerance to morphine. Moreover, opioid-induced hyperalgesia was observed after repeated administration of morphine, but not BN-9. Taken together, our present study suggests that intrathecal BN-9 produces potent and non-tolerance forming antinociception, and does not cause opioid-induced hyperalgesia. Thus, BN-9 might serve as a promising lead compound in the development of multi-functional opioid analgesics with minimized side effects.

The 2 structurally related peptides, neuropeptide FF (NPFF) and neuropeptide AF (NPAF), are encoded by the NPFF gene and have been identified as neuromodulators that regulate nociception and opiate-mediated analgesia via NPFF receptor (NPFFR2) in mammals. However, little is known about these 2 peptides in birds. In this study, we examined the structure, tissue expression profile, and functionality of NPAF and NPFF in chickens. Our results showed that: 1) unlike mammalian NPFF, NPFF from chicken and other avian species is predicted to produce a single bioactive NPAF peptide, whereas the putative avian NPFF peptide likely lacks activity due to the absence of functional RFamide motif at its C-terminus; 2) synthetic chicken (c-) NPAF can potently activate cNPFFR2 (and not cNPFFR1) expressed in HEK293 cells, as monitored by 3 cell-based luciferase reporter systems, indicating that cNPAF is a potent ligand for cNPFFR2, which activation could decrease intracellular cAMP levels and stimulate the MAPK/ERK signaling cascade; interestingly, gonadotropin-inhibitory hormone, a peptide sharing high structural similarity to NPAF, could specifically activate cNPFFR1 (but not cNPFFR2); 3) Quantitative real-time PCR revealed that cNPFF mRNA is widely expressed in chicken tissues with the highest level detected in the hypothalamus, whereas cNPFFR2 is expressed in all tissues examined with the highest level noted in the hypothalamus and anterior pituitary. Taken together, our data reveal that avian NPFF encodes a single bioactive NPAF peptide, which preferentially activates NPFFR2, and provides insights into potential structural and functional changes of NPFF-derived peptides during vertebrate evolution.

Neuropeptide FF (NPFF) has been implicated in many physiological processes. Previously, we have reported that NPFF modulates the viability and nitric oxide (NO) production of RAW264.7 macrophages. In this study, we investigated the influence of NPFF on lipopolysaccharide (LPS)-mediated osteoclast formation of RAW264.7 cells. Our results suggest that, NPFF dose-dependently (1 nM, 10 nM and 100 nM) inhibited osteoclast formation, TRAP enzyme activity and bone resorption in osteoclasts induced by LPS respectively. Moreover, LPS-provoked NO release was also inhibited by NPFF treatment, indicating a NO-dependent pathway is mainly involved. Furthermore, the alterations of osteoclast marker genes were also assessed including TRAP, Cathepsin K, MMP-9, NFATc1 and Runx2. NPFF downregulated LPS-caused gene augmentations of TRAP, Cathepsin K and MMP-9, whereas showed no influences on NFATc1 and Runx2. In addition, NPFF receptor 2 (NPFFR2) mRNA expression was also augmented in response to NPFF treatment, hinting the involvement of NPFFR2 pathway. It should be mentioned that RF9 (1 µ M), a reported pharmacological inhibitor for NPFF receptors, exerted NPFF-like agonist properties as to attenuate osteoclastogenesis. Collectively, our findings provide new evidence for the in vitro activity of NPFF on osteoclasts, which may be helpful to extend the scope of NPFF functions.

Objective Neuropeptide FF (NPFF) has been implicated in many physiological processes but not osteoclastogenesis. We previously demonstrated that NPFF modulates the viability and nitric oxide (NO) production of RAW264.7 macrophages. This study was designed to investigate the effect of NPFF on receptor activator of nuclear factor κB ligand (RANKL)-mediated differentiation of RAW264.7 cells into osteoclast-like cells. Design RAW264.7 cells were cultured in 96-stripwell plates or in Corning Osteo Assay Surface 96-well plates in the presence of various concentrations of NPFF with or without RANKL for 3 or 6 d. The differentiation of osteoclast-like cells was analyzed by tartrate-resistant acid phosphatase (TRAP) stain, TRAP activity and bone resorption capacity, respectively. The mRNA expression of NPFF2 receptor (NPFFR2) and osteoclast genes was evaluated by using real-time quantitative PCR which includes TRAP, RANK (receptor activator of NF-κB), Cathepsin K, MMP-9 (matrix metallopeptidase 9), Intβ3 (integrin β3) and NFATc1 (nuclear factor of activated T cells cytoplasmic 1). In addition, the influence of NPFF on the cell viability and NO release of RAW264.7 cells was measured by MTT assay and Griess method, respectively. Results NPFF dose-dependently inhibited RANKL-induced osteoclast-like cells differentiation including TRAP-positive cell formation, TRAP activity and bone resorption capacity. Moreover, NO release and osteoclast gene expression of osteoclast-like cells were downregulated by NPFF. In addition, NPFFR2 gene expression in osteoclast-like cells was augmented in response to NPFF treatment. Conclusion Our findings showed that NPFF could attenuate osteoclast-like cells differentiation in an in vitro osteoclastogenesis model.

The neuropeptide FF (NPFF) system has been implicated in a number of physiological processes including modulating the pharmacological activity of opioid analgesics and several other classes of drugs of abuse. In this study, we report the discovery of a novel proline scaffold with antagonistic activity at the NPFF receptors through a high throughput screening campaign using a functional calcium mobilization assay. Focused structure-activity relationship studies on the initial hit 1 have resulted in several analogs with calcium mobilization potencies in the submicromolar range and modest selectivity for the NPFF1 receptor. Affinities and potencies of these compounds were confirmed in radioligand binding and functional cAMP assays. Two compounds, 16 and 33, had good solubility and blood-brain barrier permeability that fall within the range of CNS permeant candidates without the liability of being a P-glycoprotein substrate. Finally, both compounds reversed fentanyl-induced hyperalgesia in rats when administered intraperitoneally. Together, these results point to the potential of these proline analogs as promising NPFF receptor antagonists.

Although opiates represent the most effective analgesics, their use in chronic treatments is associated with numerous side effects including the development of pain hypersensitivity and analgesic tolerance. We recently identified a novel orally active neuropeptide FF (NPFF) receptor antagonist, RF313, which efficiently prevents the development of fentanyl-induced hyperalgesia in rats. In this study, we investigated the properties of this compound into more details. We show that RF313 exhibited a pronounced selectivity for NPFF receptors, antagonist activity at NPFF1 receptor (NPFF1R) subtype both in vitro and in vivo and no major side effects when administered in mice up to 30 mg/kg. When co-administered with opiates in rats and mice, it improved their analgesic efficacy and prevented the development of long lasting opioid-induced hyperalgesia. Moreover, and in marked contrast with the dipeptidic NPFF receptor antagonist RF9, RF313 displayed negligible affinity and no agonist activity (up to 100 muM) toward the kisspeptin receptor. Finally, in male hamster, RF313 had no effect when administered alone but fully blocked the increase in LH induced by RFRP-3, while RF9 per se induced a significant increase in LH levels which is consistent with its ability to activate kisspeptin receptors. Altogether, our data indicate that RF313 represents an interesting compound for the development of therapeutic tools aiming at improving analgesic action of opiates and reducing adverse side effects associated with their chronic administration. Moreover, its lack of agonist activity at the kisspeptin receptor indicates that RF313 might be considered a better pharmacological tool, when compared to RF9, to examine the regulatory roles of RF-amide-related peptides and NPFF1R in reproduction.

A new radioiodinated photoaffinity compound, [ 125 I]YE(Bpa)WSLAAPQRFNH 2 , derived from a peptide present in the rat neuropeptide FF (NPFF) precursor was synthesized, and its binding characteristics were investigated on a neuroblastoma clone, SH-SY5Y, stably expressing rat NPFF 2 receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction ( K D = 0.24 nM) with a single class of binding sites. It was also able to affinity label NPFF 2 receptor in a specific and efficient manner given that 38% of the bound radioligand at saturating concentration formed a wash-resistant binding after ultraviolet (UV) irradiation. Photoaffinity labeling with [ 125 I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF 2 receptor with apparent molecular weights of 140 and 95 kDa in a 2:1 ratio. The comparison of the results between photoaffinity labeling and Western blot analysis suggests that all receptor forms bind the probe irreversibly with the same efficiency. On membranes of mouse olfactory bulb, only the high molecular weight form of NPFF 2 receptor is observed. [ 125 I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF 2 receptors in vitro.

Neuropeptide FF (NPFF) interacts with specific receptors to regulate diverse biological processes. Its modulatory effect in the immune field, however, has not been fully explored yet. Here, we report that NPFF2 receptors may be functionally expressed in two immune cell models, the primary peritoneal macrophage and RAW 264.7 macrophage. Firstly, the mRNA levels of NPFF2 receptor were up-regulated in macrophages when treated with LPS for 24 to 72 h. Subsequently, our data hinted that NPFF regulates the viability of both kinds of macrophages. After treatment with RF9, a reported antagonist for both NPFF receptors, delayed or inhibited the NPFF-induced macrophages viability augmentation, suggesting the involvement of NPFF2 receptor. Furthermore, down-regulation of nitric oxide (NO) synthases (NOSs) partially significantly inhibited the viability augmentation of macrophages induced by NPFF, implying a nitric oxide synthases- dependent pathway is involved. However, the NOSs are not the only route by which NPFF affects the viability of macrophages. Pharmacological inhibitors of NF-κB signal pathway also blocked the NPFF-induced macrophages growth, suggesting the involvement of the NF-κB signal pathway. The regulation activity of NPFF for macrophages suggests that NPFF could act as a potential hormone in the control of immune system. Collectively, our data provide new evidence about the immune modulatory effect of NPFF, which will be helpful in extending the scope of NPFF functions.