Neuropeptide FF Receptor Antagonist Research Articles

Abstract P422: Neuropeptide FF and Its Receptor NPFFR2 Decrease Renal Sodium Excretion and Increase Blood Pressure in Mice

The kidney is critical in the overall regulation of fluid and electrolyte balance and blood pressure. Neuropeptide FF (NPFF), a morphine-modulating peptide, regulates cardiovascular function through its interaction with two receptors, NPFFR1 and NPFFR2. We now report that NPFF and its receptors, mainly NPFFR2, are expressed in the kidney. NPFF (9.25 mmol, 0.5 mL/hr, 0.05 nmol/day, 7 days) chronically infused underneath the renal capsule, decreased renal sodium excretion (UNaV) from 0.68±0.07 mEq/day, n=5 to 0.43±0.06 mEq/day (n=6/group) in conscious C57BL/6 mice. This was accompanied by an increase in systolic blood pressure (SBP, 114.5±5.0 mm Hg, n=4); the infusion of vehicle (saline) did not affect the blood pressure (96.4±3.0 mm Hg, n=4). The renal subcapsular injection of a single dose (10 µg in 100 µL) of NPFF also increased SBP within 15-35 min (NPFF: 105.5±3.44 mm Hg, n=6); saline injection did not affect SBP (82.6±5.65 mm Hg, n=4). RF-9 (10 µg in 100 µL), an antagonist of NPFF receptors prevented the NPFF-mediated increase in SBP (NPFF alone: 105.5±3.44 mm Hg, n=6; RF+NPFF: 88.5±5.90 mm Hg, n=5), whereas RF-9, alone, had no effect (Baseline: 97.6±2.70 mmHg; RF-9: 90.9±4.70 mm Hg, n=5). The renal subcapsular injection of scrambled peptide (10 µg in 100 µL) had no effect on SBP (86.1±1.83 mm Hg, n=4). However, SBP was decreased by the renal subcapsular infusion of Npffr2 siRNA (98±4 vs 113±3 mmHg; P < 0.05; n=3-5/group) in C57Bl/6 mice fed with a high salt diet (4% NaCl). Furthermore, the SBP of conventional germline Npffr2 knockout (KO) mice fed a high salt (4% NaCl) diet was lower than the SBP of wild-type (WT) littermates (WT: 105.6±2.9 mm Hg, n=5; KO: 90.5±5.5 mm Hg, n=4, P < 0.05). By contrast, in mice fed normal salt (0.8% NaCl) diet, SBP was not different between Npffr2 KO mice and WT littermates (WT: 96.5±4.5 mm Hg, n=5; KO: 98.7±11.7 mm Hg, n=4, P > 0.05). Taken together, NPFF and its receptor, NPFFR2, in the kidney cause salt-sensitive hypertension in mice. Uncovering the functional relevance of renal NPFF in the dynamic regulation of renal sodium transport will lead to a better understanding of blood pressure homeostasis.

Renal autocrine neuropeptide FF (NPFF) signaling regulates blood pressure

The kidney and brain play critical roles in the regulation of blood pressure. Neuropeptide FF (NPFF), originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the roles of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, are not known. In this study, we found that the transcripts and proteins of NPFF and its receptors, NPFF-R1 and NPFF-R2, were expressed in mouse and human renal proximal tubules (RPTs). In mouse RPT cells (RPTCs), NPFF, but not RF-amide-related peptide-2 (RFRP-2), decreased the forskolin-stimulated cAMP production in a concentration- and time-dependent manner. Furthermore, dopamine D1-like receptors colocalized and co-immunoprecipitated with NPFF-R1 and NPFF-R2 in human RPTCs. The increase in cAMP production in human RPTCs caused by fenoldopam, a D1-like receptor agonist, was attenuated by NPFF, indicating an antagonistic interaction between NPFF and D1-like receptors. The renal subcapsular infusion of NPFF in C57BL/6 mice decreased renal sodium excretion and increased blood pressure. The NPFF-mediated increase in blood pressure was prevented by RF-9, an antagonist of NPFF receptors. Taken together, our findings suggest that autocrine NPFF and its receptors in the kidney regulate blood pressure, but the mechanisms remain to be determined.

Multitarget μ-Opioid Receptor Agonists─Neuropeptide FF Receptor Antagonists Induce Potent Antinociception with Reduced Adverse Side Effects.

The design of bifunctional compounds is a promising approach toward the development of strong analgesics with reduced side effects. We here report the optimization of the previously published lead peptide KGFF09, which contains opioid receptor agonist and neuropeptide FF receptor antagonist pharmacophores and is shown to induce potent antinociception and reduced side effects. We evaluated the novel hybrid peptides for their in vitro activity at MOP, NPFFR1, and NPFFR2 and selected four of them (DP08/14/32/50) for assessment of their acute antinociceptive activity in mice. We further selected DP32 and DP50 and observed that their antinociceptive activity is mostly peripherally mediated; they produced no respiratory depression, no hyperalgesia, significantly less tolerance, and strongly attenuated withdrawal syndrome, as compared to morphine and the recently FDA-approved TRV130. Overall, these data suggest that MOP agonist/NPFF receptor antagonist hybrids might represent an interesting strategy to develop novel analgesics with reduced side effects.

Bifunctional Peptidomimetic G Protein-Biased Mu-Opioid Receptor Agonist and Neuropeptide FF Receptor Antagonist KGFF09 Shows Efficacy in Visceral Pain without Rewarding Effects after Subcutaneous Administration in Mice.

There is still an unmet clinical need to develop new pharmaceuticals for effective and safe pain management. Current pharmacotherapy offers unsatisfactory solutions due to serious side effects related to the chronic use of opioid drugs. Prescription opioids produce analgesia through activation of the mu-opioid receptor (MOR) and are major contributors to the current opioid crisis. Multifunctional ligands possessing activity at more than one receptor represent a prominent therapeutic approach for the treatment of pain with fewer adverse effects. We recently reported on the design of a bifunctional MOR agonist/neuropeptide FF receptor (NPFFR) antagonist peptididomimetic, KGFF09 (H-Dmt-DArg-Aba-βAla-Bpa-Phe-NH2), and its antinociceptive effects after subcutaneous (s.c.) administration in acute and persistent pain in mice with reduced propensity for unwanted side effects. In this study, we further investigated the antinociceptive properties of KGFF09 in a mouse model of visceral pain after s.c. administration and the potential for opioid-related liabilities of rewarding and sedation/locomotor dysfunction following chronic treatment. KGFF09 produced a significant dose-dependent inhibition of the writhing behavior in the acetic acid-induced writhing assay with increased potency when compared to morphine. We also demonstrated the absence of harmful effects caused by typical MOR agonists, i.e., rewarding effects (conditioned-place preference test) and sedation/locomotor impairment (open-field test), at a dose shown to be highly effective in inhibiting pain behavior. Consequently, KGFF09 displayed a favorable benefit/side effect ratio regarding these opioid-related side effects compared to conventional opioid analgesics, such as morphine, underlining the development of dual MOR agonists/NPFFR antagonists as improved treatments for various pain conditions.

Intracerebroventricular Neuropeptide FF Diminishes the Number of Apneas and Cardiovascular Effects Produced by Opioid Receptors’ Activation

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 μ-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.

Spinal administration of the multi-functional opioid/neuropeptide FF agonist BN-9 produced potent antinociception without development of tolerance and opioid-induced hyperalgesia

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.

Central and peripheral modulation of gastrointestinal transit in mice by DN-9, a multifunctional opioid/NPFF receptor agonist.

The nonapeptide DN-9 functions as a multifunctional agonist to opioid and neuropeptide FF (NPFF) receptors and exhibits antinociceptive effects at the central and peripheral levels. The effects of DN-9 on small and colonic intestinal transit were evaluated using the upper gastrointestinal (GI) transit test and colonic bead expulsion assay, respectively. Opioid and NPFF receptor antagonists were used to investigate the mechanisms of DN-9-induced GI inhibition. Furthermore, the agonism of the DN-9 analog [Phg9 ]-DN-9 to opioid and NPFF receptors was tested by the cAMP assay. Intracerebroventricular administration of DN-9 dose-dependently slowed upper GI transit and colonic expulsion via mu- and kappa-opioid receptors in the brain, independent of the delta-opioid receptor. Similarly, intraperitoneal injection of DN-9 dose-dependently inhibited GI propulsion via the peripheral opioid receptors. DN-9-induced GI transit inhibitions were significantly aggravated by the NPFF receptor antagonist RF9. Moreover, the DN-9 analog [Phg9 ]-DN-9, an agonist at mu-, delta-, and kappa-opioid receptors but not NPFF receptors, inhibited GI more potently than DN-9. In addition, intracerebroventricular NPFF significantly attenuated the central inhibitory effects induced by [Phg9 ]-DN-9 and morphine. However, central and peripheral injections of NPFF or RF9 almost had no significant effects on GI transit by itself. Intracerebroventricular and intraperitoneal administrations of DN-9 inhibit GI transit via opioid receptors in mice by central and peripheral mechanisms, respectively. In addition, the NPFF agonism of DN-9 possesses antiopioid effects on GI transit, which might explain the reduced constipation at the antinociceptive doses.

Bioanalytical method development and validation of MES207, a neuropeptide FF receptor antagonist, and its application in preclinical pharmacokinetics

The nonpeptide small molecule, MES207, exhibits 17-fold preferential binding to the neuropeptide FF receptor 1 (NPFFR1) over NPFFR2 and shows antagonist functionality at NPFF receptors. In order to further the development of MES207 as a NPFFR1 probe, an UPLC-MS/MS bioanalytical method was developed and validated to quantify MES207 in rat plasma for a linearity range of 3–200 ng/mL. The method was applied in the analysis of the plasma, brain, and urine samples collected during pharmacokinetic studies in healthy male and female Sprague Dawley rats. The animals were dosed through oral gavage (50 mg/kg) and intravenously (2.5 mg/kg). Test samples were analyzed using the validated bioanalytical method to generate plasma concentration-time profiles. The results were further subjected to non-compartmental analysis using Phoenix 6.4®. MES207 exhibits a large volume of distribution (1.2 ± 0.6 L), high clearance (0.8 ± 0.1 L/h), and a poor oral bioavailability (1.7 ± 0.4%). The compound also showed a multiple peak phenomenon with a very short absorption phase. It appears that gender does not significantly influence the differences in pharmacokinetic parameters of this NPFF probe.

Spinal DN-9, a Peptidic Multifunctional Opioid/Neuropeptide FF Agonist Produced Potent Nontolerance Forming Analgesia With Limited Side Effects

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. PerspectiveThis 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.

Discovery of two novel branched peptidomimetics containing endomorphin-2 and RF9 pharmacophores: Synthesis and neuropharmacological evaluation.

It is well known that opioid analgesics produce side effects including tolerance and constipation. Since neuropeptide FF (NPFF) receptor antagonists reversed opioid-induced hyperalgesia and analgesic tolerance, the present work was performed to synthetize two branched peptidomimetics, EKR and RKE, containing the opioid peptide endomorphin-2 (EM-2) and the NPFF receptor antagonist RF9. Our data obtained from the in vitro cyclic adenosine monophosphate experiment demonstrated that EKR functioned as a mixed mu-, delta-opioid receptors agonist and NPFF1 receptor antagonist/NPFF2 receptor partial agonist, whereas RKE acted as a multi-functional peptidomimetic with the mu-opioid agonism and the NPFF1 antagonism/NPFF2 partial agonism. Furthermore, EKR and RKE completely blocked the NPFF2 receptor-mediated neurite outgrowth of Neuro 2A cells. In vivo antinociception studies found that supraspinal administration of EKR and RKE dose-dependently produced potent antinociception via the mu-opioid receptor in the tail-flick test. In carrageenan inflammatory pain model, spinal administration of EKR and RKE induced dose-related analgesia, which was significantly reduced by the opioid antagonist naloxone and the NPFF antagonist RF9. Notably, compared with morphine, intracerebroventricular repeated administration of EKR and RKE maintained prolonged antinociceptive effectiveness. In addition, at the antinociceptive doses, these two branched peptidomimetics did not significantly inhibit gastrointestinal transit. Taken together, the present work suggest that EKR and RKE behave as multi-functional ligands with the opioid agonism and the NPFF1 antagonism/NPFF2 partial agonism, and produce prolonged antinociception with limited side effects. Moreover, our results imply that EKR and RKE might be interesting pharmacological tools for further investigating the biological function of the NPFF and opioid systems.

A subtype-specific neuropeptide FF receptor antagonist attenuates morphine and nicotine withdrawal syndrome in the rat

Considerable evidence suggests the Neuropeptide FF (NPFF) and related peptides exert pro-nociceptive and anti-opiate actions, particularly at the supra-spinal level, which may contribute to opiate dependence. The FF1 receptor subtype appears to be primarily responsible for anti-opiate effects. In contrast, stimulation of the FF2 receptor primarily induces pro-opiate effects. AC-262620 is a small molecule, systemically active, selective FF1 receptor antagonist. An initial experiment showed that 10 mg/kg i.p. AC-262620 significantly reduced subsequent naloxone-precipitated somatically expressed withdrawal signs in rats infused s.c. for seven days with 0.3 mg/kg/hr morphine sulfate. A second experiment showed that the same dose of AC-262620 significantly reduced subsequent spontaneous withdrawal signs 23.75 h after termination of seven days s.c. infusion of 0.6 mg/kg/hr morphine sulfate.Chronic nicotine intake may contribute to dependence by overstimulating opiate receptors through release of opiate peptides. By analogy to opiate dependence, it was hypothesized that FF1 receptor activation contributes to nicotine dependence and withdrawal syndrome. AC-262620 significantly reduced somatically expressed withdrawal signs precipitated by the nicotinic antagonist mecamylamine in rats infused for seven days with nicotine bitartrate. Taken together, these findings suggest that NPFF or related neuropeptides contribute to opiate, as well as nicotine, dependence and withdrawal syndrome through the FF1 receptor.

A bifunctional-biased mu-opioid agonist-neuropeptide FF receptor antagonist as analgesic with improved acute and chronic side effects.

Opioid analgesics, such as morphine, oxycodone, and fentanyl, are the cornerstones for treating moderate to severe pain. However, on chronic administration, their efficiency is limited by prominent side effects such as analgesic tolerance and dependence liability. Neuropeptide FF (NPFF) and its receptors (NPFF1R and NPFF2R) are recognized as an important pronociceptive system involved in opioid-induced hyperalgesia and analgesic tolerance. In this article, we report the design of multitarget peptidomimetic compounds that show high-affinity binding to the mu-opioid receptor (MOPr) and NPFFRs. In vitro characterization of these compounds led to identification of KGFF03 and KGFF09 as G-protein-biased MOPr agonists with full agonist or antagonist activity at NPFFRs, respectively. In agreement with their biased MOPr agonism, KGFF03/09 showed reduced respiratory depression in mice, as compared to the unbiased parent opioid agonist KGOP01. Chronic subcutaneous administration of KGOP01 and KGFF03 in mice rapidly induced hyperalgesia and analgesic tolerance, effects that were not observed on chronic treatment with KGFF09. This favorable profile was further confirmed in a model of persistent inflammatory pain. In addition, we showed that KGFF09 induced less physical dependence compared with KGOP01 and KGFF03. Altogether, our data establish that combining, within a single molecule, the G-protein-biased MOPr agonism and NPFFR antagonism have beneficial effects on both acute and chronic side effects of conventional opioid analgesics. This strategy can lead to the development of novel and potent antinociceptive drugs with limited side effects on acute and chronic administration.

The new kisspeptin derivative - kissorphin (KSO) - attenuates acute hyperlocomotion and sensitization induced by ethanol and morphine in mice.

Kissorphin (KSO) is a new peptide derived from kisspeptin-10. This peptide possesses neuropeptide FF (NPFF)-like biological activity invitro; NPFF, in many cases, inhibits opioid and ethanol effects in rodents. Therefore, the current study explored the influence of KSO on acute ethanol- and morphine-induced hyperactivity, and on the development and expression of locomotor sensitization induced by these drugs. In the present study, sensitization to locomotor effects was induced by repeated exposure to ethanol (2.4g/kg, intraperitoneally [i.p.], 1×4 days) or morphine (10mg/kg, subcutaneously [s.c.], 1×7 days). We found that KSO (1-10nmol/300μL, intravenously [i.v.]) did not have an impact on locomotor activity of naïve mice. However, it reduced both acute ethanol- (10nmol/300μL) and morphine-induced hyperactivity (3 and 10nmol/300μL). Pretreatment of animals with KSO (10nmol/300μL), before every ethanol or morphine injection during development of sensitization or before the ethanol or morphine challenge, attenuated the development, as well as the expression of locomotor sensitization to both substances. Moreover, prior administration of the NPFF receptor antagonist RF9 (10nmol/300μL, i.v.) inhibited the ability of KSO (10nmol/300μL) to reduce the expression of ethanol and morphine sensitization. KSO given alone, at all used doses, did not influence the motor coordination measured via the rotarod test. The results from this study show that KSO effectively attenuated acute and repeated effects of ethanol and morphine. Thus, KSO possesses NPFF-like anti-opioid activity in these behavioral studies.

NPFFR2 Activates the HPA Axis and Induces Anxiogenic Effects in Rodents.

Neuropeptide FF (NPFF) belongs to the RFamide family and is known as a morphine-modulating peptide. NPFF regulates various hypothalamic functions through two receptors, NPFFR1 and NPFFR2. The hypothalamic-pituitary-adrenal (HPA) axis participates in physiological stress response by increasing circulating glucocorticoid levels and modulating emotional responses. Other RFamide peptides, including neuropeptide AF, neuropeptide SF and RFamide related peptide also target NPFFR1 or NPFFR2, and have been reported to activate the HPA axis and induce anxiety- or depression-like behaviors. However, little is known about the action of NPFF on HPA axis activity and anxiety-like behaviors, and the role of the individual receptors remains unclear. In this study, NPFFR2 agonists were used to examine the role of NPFFR2 in activating the HPA axis in rodents. Administration of NPFFR2 agonists, dNPA (intracerebroventricular, ICV) and AC-263093 (intraperitoneal, IP), time-dependently (in rats) and dose-dependently (in mice) increased serum corticosteroid levels and the effects were counteracted by the NPFF receptor antagonist, RF9 (ICV), as well as corticotropin-releasing factor (CRF) antagonist, α-helical CRF(9-41) (intravenous, IV). Treatment with NPFFR2 agonist (AC-263093, IP) increased c-Fos protein expression in the hypothalamic paraventricular nucleus and induced an anxiogenic effect, which was evaluated in mice using an elevated plus maze. These findings reveal, for the first time, that the direct action of hypothalamic NPFFR2 stimulates the HPA axis and triggers anxiety-like behaviors.

Discovery of Novel Proline-Based Neuropeptide FF Receptor Antagonists.

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.

Peripheral and central sites of action for anti-allodynic activity induced by the bifunctional opioid/NPFF receptors agonist BN-9 in inflammatory pain model

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.

RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes effects of RF-amide-related peptide-3 and opioid-induced hyperalgesia in rodents

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 μM) 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.

BN‐9, a chimeric peptide with mixed opioid and neuropeptide FF receptor agonistic properties, produces nontolerance‐forming antinociception in mice

Neuropeptide FF (NPFF) behaves as an endogenous opioid-modulating peptide. In the present study, the opioid and NPFF pharmacophore-containing chimeric peptide BN-9 was synthesized and pharmacologically characterized. Agonist activities of BN-9 at opioid and NPFF receptors were characterized in in vitro cAMP assays. Antinociceptive activities of BN-9 were evaluated in the mouse tail-flick and formalin tests. Furthermore, its side effects were investigated in rotarod, antinociceptive tolerance, reward and gastrointestinal transit tests. BN-9 acted as a novel multifunctional agonist at μ, δ, κ, NPFF1 and NPFF2 receptors in cAMP assays. In the tail-flick test, BN-9 produced dose-related antinociception and was approximately equipotent to morphine; this antinociception was blocked by μ and κ receptor antagonists, but not by the δ receptor antagonist. In the formalin test, supraspinal administration of BN-9 produced significant analgesia. Notably, repeated administration of BN-9 produced analgesia without loss of potency over 8days. In contrast, repeated i.c.v. co-administration of BN-9 with the NPFF receptor antagonist RF9 produced significant antinociceptive tolerance. Furthermore, i.c.v. BN-9 induced conditioned place preference. When given by the same routes, BN-9 had a more than eightfold higher ED50 value for gastrointestinal transit inhibition compared with the ED50 values for antinociception. BN-9 produced a robust, nontolerance-forming analgesia with limited inhibition of gastrointestinal transit. As BN-9 is able to activate both opioid and NPFF systems, this provides an interesting approach for the development of novel analgesics with minimal side effects.

Neuropeptide FF Promotes Recovery of Corneal Nerve Injury Associated With Hyperglycemia.

To investigate how the neuropeptide FF (NPFF) promotes the recovery of corneal nerve injury associated with hyperglycemia. Gene expression was analyzed using neurotrophin and receptor RT2 profiler polymerase chain reaction arrays in trigeminal (TG) sensory neurons. The role of NPFF in the regeneration of diabetic TG nerves was investigated in vitro by using cultured TG neurons from diabetic BKS.Cg-m+/+Leprdb/J (db/db) mice and in vivo by following corneal injury healing responses. RF9, a selective NPFF receptor (NPFF2R) antagonist, was used to prevent the interactions between NPFF and NPFF2R. Using a mRNA real-time PCR array, NPFF was found to be significantly lower in diabetic TG sensory neurons. Hyperglycemia induced the deficiency of ocular properties in db/db mice. The application of NPFF enhanced neurite elongation in diabetic TG neurons. Through subconjunctival injection, NPFF promoted corneal nerve injury recovery and epithelial wound healing in db/db mice. Furthermore, the application of NPFF rescued the activation of SIRT1 and PPAR-gamma, and downregulated the expression of PTEN and Rb in diabetic TG neurons. The promotion of NPFF on diabetic corneal epithelial healing and corneal innervations was completely abolished by RF9. Moreover, subconjunctivally injected NPFF accelerated the reinnervation of corneal nerves via the ERK1/2 pathway. These results indicate that NPFF signaling through NPFFR2 contributes to diabetic corneal nerve injury recovery and epithelial wound healing. Neuropeptide FF is a potential neuroregenerative factor for diabetic sensory nerve injury. Chinese Abstract.

Neuropeptide VF Enhances Cannabinoid Agonist WIN55,212-2-Induced Antinociception in Mice.

Cannabinoids produce analgesia in several pain models, but the undesirable side effects from high doses of cannabinoid drugs limit their clinic use. Our recent results indicate that cannabinoid-induced antinociception was enhanced by neuropeptide VF (NPVF). Here, we investigate whether low-dose cannabinoid agonists combined with NPVF can produce effective antinociception with limited side effects. The in vivo properties of (R)-(-1)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN55,212-2) given alone and its combination with NPVF were evaluated in nociceptive modulation, locomotor activity, gastrointestinal transit, and tolerance development assays after intracerebroventricular administration in mice. In the radiant tail-flick test, the antinociception of combination of WIN55,212-2 and NPVF was more potent than that of cannabinoid agonist given alone, with an ED50 shift from 3.51 to 0.69 nmol; 9 nmol WIN55,212-2 alone and 3 nmol WIN55,212-2 combined with NPVF induced equivalent antinociception after supraspinal administration. The cannabinoid-potentiating effects of NPVF were reduced by both the cannabinoid receptor type 1 and the neuropeptide FF receptor antagonists. In the formalin assay, WIN55,212-2 combined with NPVF also significantly reduced pain-related behaviors. However, the combination of WIN55,212-2 with NPVF exerted significant hypoactivity in a manner similar to high doses of WIN55,212-2. It was important to note that the combination of WIN55,212-2 with NPVF produced nontolerance-forming antinociception and weaker inhibition of gastrointestinal transit compared with high dose of WIN55,212-2. These data suggest that the cannabinoid agonist combined with NPVF produces effective antinociception-lacking tolerance via both cannabinoid receptor type 1 and neuropeptide FF receptors in the brain.