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Background: Alzheimer's disease (AD) is a neurodegenerative disorder recognized as the most common cause of chronic dementia among the ageing population. AD is histopathologically characterized by progressive loss of neurons and deposits of insoluble proteins, primarily composed of amyloid-β pelaques and neurofibrillary tangles (NFTs). Methods: Several molecular processes contribute to the formation of AD cellular hallmarks. Among them, post-translational modifications (PTMs) represent an attractive mechanism underlying the formation of covalent bonds between chemical groups/peptides to target proteins, which ultimately result modified in their function. Most of the proteins related to AD undergo PTMs. Several recent studies show that AD-related proteins like APP, Aβ, tau, BACE1 undergo post-translational modifications. The effect of PTMs contributes to the normal function of cells, although aberrant protein modification, which may depend on many factors, can drive the onset or support the development of AD. Results: Here we will discuss the effect of several PTMs on the functionality of AD-related proteins potentially contributing to the development of AD pathology. Conclusion: We will consider the role of Ubiquitination, Phosphorylation, SUMOylation, Acetylation and Nitrosylation on specific AD-related proteins and, more interestingly, the possible interactions that may occur between such different PTMs.

Growing evidences demonstrate that chemokines and chemokine receptors are up-regulated in resident central nervous system cells during Alzheimer's disease contributing to neuroinflammation and neurodegeneration. Prokineticin 2 belongs to a new family of chemokines which recently emerged as a critical player in immune system and inflammatory diseases. Since pharmacological blockade in vitro of the prokineticin system is able to antagonize Amyloid β-induced neurotoxicity, the aim of the present study was to investigate in vivo effects of prokineticin receptor antagonist PC1 on memory impairment in a rodent model of Alzheimer's disease. Rats were intracerebroventricular infused with Aβ1-42 and behavioral responses as well as the expression profile in hippocampus of prokineticin 2 and its receptors were investigated. Results demonstrated that Aβ1–42-infused rats developed significant memory impairments together with a marked up-regulation of both prokineticin 2 and its receptors in hippocampal neurons and astrocytes. Treatment with PC1 significantly improved learning capability of Aβ1–42-infused rats restoring the balance of prokineticin system. This study pointed to a neuroprotective role of PC1 on Aβ1-42-induced memory deficits that could be ascribed to the ability of PC1 to modulate rat hippocampal prokineticin system and to recover the impaired Aβ1-42-induced neurogenesis. This suggests that prokineticin system antagonism could be considered as a new approach for the study of AD etiopathology.

Bv8/prokineticin 2 (PK2) is a member of a bioactive family of peptides that regulate multiple functions in the CNS including hyperalgesia, neurogenesis, neuronal survival and inflammation. Recent studies have associated PK2 and prokineticin receptors (PKR) with human diseases, but because their role in neuropathology is still debated we examined whether prokineticins exert a protective or deleterious role in models of cerebral ischemia and ischemic tolerance in vitro. In order to mimic cerebral ischemia, we exposed primary murine cortical cell cultures or rat organotypic hippocampal slices to appropriate periods of oxygen-glucose deprivation (OGD), which leads to neuronal damage 24 h later. Ischemic tolerance was induced by exposing hippocampal slices to a preconditioning subtoxic pharmacological stimulus (3 μM NMDA for 1 h) 24 h before the exposure to OGD. Bv8 (10–100 nM) attenuated OGD injury in cortical cultures and hippocampal slices, and the effect was prevented by the PKR antagonist PC7. The development of OGD tolerance was associated with an increase in the expression of PK2, PKR1 and PKR2 mRNA and proteins and was prevented by addition of the antagonist PC7 into the medium during preconditioning. Both Bv8 at protective concentrations and the NMDA preconditioning stimulus promoted the phosphorylation of ERK1/2 and Akt. These findings indicate that the prokineticin system can be up-regulated by a defensive preconditioning subtoxic NMDA stimulus and that PK2 may act as an endogenous neuroprotective factor through the activation of the ERK1/2 and Akt transduction pathways.

The mammalian prokineticins family comprises two conserved proteins, EG-VEGF/PROK1 and Bv8/PROK2, and their two highly related G protein-coupled receptors, PKR1 and PKR2. This signaling system has been linked to several important biological functions, including gastrointestinal tract motility, regulation of circadian rhythms, neurogenesis, angiogenesis and cancer progression, hematopoiesis, and nociception. Mutations in PKR2 or Bv8/PROK2 have been associated with Kallmann syndrome, a developmental disorder characterized by defective olfactory bulb neurogenesis, impaired development of gonadotropin-releasing hormone neurons, and infertility. Also, Bv8/PROK2 is strongly upregulated in neutrophils and other inflammatory cells in response to granulocyte-colony stimulating factor or other myeloid growth factors and functions as a pronociceptive mediator in inflamed tissues as well as a regulator of myeloid cell-dependent tumor angiogenesis. Bv8/PROK2 has been also implicated in neuropathic pain. Anti-Bv8/PROK2 antibodies or small molecule PKR inhibitors ameliorate pain arising from tissue injury and inhibit angiogenesis and inflammation associated with tumors or some autoimmune disorders.

Prokineticin-2 (PK2) is a secreted bioactive peptide that signals through two GPCRs, the prokineticin receptors (PKRs), and regulates a variety of biological processes including angiogenesis, immunity and nociception. The PK2 primary transcript has two alternative splice variants, PK2 and PK2L (a Long form) which is cleaved in an active peptide, named PK2β that preferentially binds to PKR1 receptor. The aim of this study was to characterize the PK2β. Using different Saccharomyces cerevisiae strains, we examined the specificity of PKR1 and PKR2 G-protein coupling following PK2β binding. Data obtained in yeast confirmed that PK2 binds both receptors, inducing a comparable response throughout a promiscuous coupling of G protein subtypes. Conversely, we demonstrated, for the first time, that PK2β preferentially binding to PKR1, activates a signaling cascade that not depends on Gαi/o coupling. The binding specificity of PK2β for PKR1 was evaluated by the analysis of PKR mutant in yeast and GST pull-down experiments, suggesting an important role of PKR1 amino-terminal region. We also evaluated the ability of PK2β to differentially activate PKR1 and/or PKR2 by in vivo nociceptive experiments and we showed that PK2β induces intense sensitization of peripheral nociceptors to painful stimuli through the activation of PKR1. To analyze PK2β-induced signal transduction, we demonstrated the inability of PK2β to induce STAT3 protein phosphorylation in organotypic primary explants from mice Dorsal Root Ganglion (DRG), an important pain station. The control of the concentration ratio between PK2β and PK2 could be one of the keys to allow the specificity of the cell response of prokineticin signaling pathway.

Prokineticin1 and prokineticin2 belong to a new family of chemokines identified in several species including mammals and characterized by the presence of five disulfide bridges. These proteins signal through two G-coupled receptors (prokineticin-receptor1 and prokineticin- receptor2) widely expressed in all tissues and involved in a large spectrum of biological activities, including angiogenesis, hematopoiesis, immune processes, inflammation and nociceptive transmission. Prokineticin2 is overexpressed in inflamed tissues and has a crucial role in neutrophil dependent inflammation and hypernociception. Following tissue inflammation, peripheral nerve injury, cancer, bone metastasis the expression of prokineticin2 and of the prokineticin-receptor2 is increased also within dorsal root ganglia and spinal cord. Prokineticin receptors, highly expressed in nociceptor endings and dorsal root ganglia, exert a tonic activation of TRPV1 and TRPA1 contributing to peripheral sensitization. Prokineticin2-induces activation of the prokineticin receptors in the spinal dorsal horn and in activated astrocytes contributes to central sensitization and maintains chronic and neuropathic pain. Prokineticin2, acting on prokineticin receptors on monocytes, macrophages and dendritic cells, induces chemotaxis and release of inflammatory and pronociceptive cytokines. Hence, the prokineticin system represents a novel therapeutic target in chronic pain conditions. Evaluation of the mechanism of action of prokineticin2 and the potential effectiveness of its inhibition is discussed.

The accumulation of β-amyloid (Aβ) is one of the hallmarks of Alzheimer disease (AD). Beyond the inflammatory reactions promoted by Aβ, it has been demonstrated that the prokineticin (PK) system, composed of the chemokine prokineticin 2 (PK2) and its receptors, is involved in Aβ toxicity. In this study we have analyzed how the Aβ chronic treatment affects the glutamatergic transmission on neurons from primary cortical cultures, clearly demonstrating the PK system involvement on its action mechanism. In fact, we have observed a significant increase of the ionic current through the AMPA receptors in primary cortical neurons and an up-regulation of the PK system in cultures chronically treated with Aβ. All effects were nullified by the prokineticin antagonist PC-1. Moreover, we have herein firstly demonstrated that the incubation of primary cortical culture with Bv8, the amphibian homologue of PK2, was able to increase in neurons the AMPA currents at specific doses and exposure times, measured both as evoked and as spontaneous currents. This effect was not due to a modification of the AMPA receptor subunit expression. In contrast, the up-modulation of AMPA currents were blocked by PC-1 and were mediated by the activation of the intracellular protein kinase C (PKC) transduction pathways because Gö6983, the PKC inhibitor added in the medium, nullified the effect. Finally, cellular death induced by kainate was also reduced following treatment with PC1. In conclusion, our results show that the prokineticin system may be a key mediator in the Aβ-induced neuronal damage, suggesting PK antagonists as new therapeutic compounds to ameliorate the AD progression.

Background and Purpose Chemokines are involved in neuroinflammation and contribute to chronic pain processing. The new chemokine prokineticin 2 (PROK2) and its receptors (PKR1 and PKR2) have a role in inflammatory pain and immunomodulation. In the present study, we investigated the involvement of PROK2 and its receptors in neuropathic pain. Experimental Approach Effects of single, intrathecal, perineural and s.c. injections of the PKR antagonist PC1, or of 1 week s.c. treatment, on thermal hyperalgesia and tactile allodynia was evaluated in mice with chronic constriction of the sciatic nerve (CCI). Expression and localization of PROK2 and of its receptors at peripheral and central level was evaluated 10 days after CCI, following treatment for 1 week with saline or PC1. IL-1β and IL-10 levels, along with glia activation, were evaluated. Key Results Subcutaneous, intrathecal and perineural PC1 acutely abolished the CCI-induced hyperalgesia and allodynia. At 10 days after CCI, PROK2 and its receptor PKR2 were up-regulated in nociceptors, in Schwann cells and in activated astrocytes of the spinal cord. Therapeutic treatment with PC1 (s.c., 1 week) alleviated established thermal hyperalgesia and allodynia, reduced the injury-induced overexpression of PROK2, significantly blunted nerve injury-induced microgliosis and astrocyte activation in the spinal cord and restored the physiological levels of proinflammatory and anti-inflammatory cytokines in periphery and in spinal cord. Conclusion and Implications The prokineticin system contributes to pain modulation via neuron–glia interaction. Sustained inhibition of the prokineticin system, at peripheral or central levels, blocked both pain symptoms and some events underlying disease progression.

A new efficient synthetic method to obtain prokineticin receptor antagonists based on the triazinedione scaffold is described. In this procedure the overall yield improves from 13% to about 54%, essentially for two factors: 1) N-(chlorocarbonyl) isocyanate is no more used, it represents the yield limiting step with an average yield not exceeding 30%. 2) The Mitsunobu reaction is not involved in the new synthetic scheme avoiding the use of time and solvent consuming column chromatography. All synthesized triazinediones were preliminary pharmacologically screened in vivo for their ability to reduce the Bv8-induced thermal hyperalgesia. In this assay all compounds displayed EC50 values in the picomolar–subpicomolar range, some triazinediones containing a 4-halogen substituted benzyl group in position 5 showed the best activity. The analogues containing a 4-fluorine atom (PC-7) and a 4-bromobenzyl group (PC-25) resulted 10 times more potent than the reference PC-1 that bears a 4-ethylbenzyl group. While the 4-trifluoromethylbenzyl substituted analog (PC-27) was 100 times more potent as compared to PC1.

Background and Purpose Prokineticin 2 (PK2) has recently been shown to acutely reduce food intake in rodents. We aimed to determine the CNS sites and receptors that mediate the anorectic effects of peripherally administered PK2 and its chronic effects on glucose and energy homeostasis. Experimental Approach We investigated neuronal activation following i.p. administration of PK2 using c-Fos-like immunoreactivity (CFL-IR). The anorectic effect of PK2 was examined in mice with targeted deletion of either prokineticin receptor 1 (PKR1) or prokineticin receptor 2 (PKR2), and in wild-type mice following administration of the PKR1 antagonist, PC1. The effect of IP PK2 administration on glucose homeostasis was investigated. Finally, the effect of long-term administration of PK2 on glucose and energy homeostasis in diet-induced obese (DIO) mice was determined. Key Results I.p. PK2 administration significantly increased CFL-IR in the dorsal motor vagal nucleus of the brainstem. The anorectic effect of PK2 was maintained in mice lacking the PKR2 but abolished in mice lacking PKR1 and in wild-type mice pre-treated with PC1. DIO mice treated chronically with PK2 had no changes in glucose levels but significantly reduced food intake and body weight compared to controls. Conclusions and Implications Together, our data suggest that the anorectic effects of peripherally administered PK2 are mediated via the brainstem and this effect requires PKR1 but not PKR2 signalling. Chronic administration of PK2 reduces food intake and body weight in a mouse model of human obesity, suggesting that PKR1-selective agonists have potential to be novel therapeutics for the treatment of obesity.

Background Injection of the noxious peptide Bv8 has previously been shown to induce a biphasic thermal hyperalgesia in rodents, the first peak presumably due to peripheral sensitization. This hypothesis has never been directly confirmed. We have assessed whether Bv8 can indeed sensitize peripheral nerve fibres in the mouse to heat. Methods We used recordings from single cutaneous fibres, cutaneous calcitonin gene-related peptide (CGRP) release and immunostaining in nerves and plantar skin to evaluate the Bv8 effects on cutaneous nerves. Results Application of Bv8 at nanomolar concentrations (30–310 nmol/L) to skin preparations significantly increased the heat-induced discharge, the heat-induced afterdischarge and reduced threshold temperature of single unmyelinated polymodal fibres. Furthermore, application of Bv8 to hind-paw skin flaps or trigeminal ganglia significantly elevated their heat-induced CGRP release. Capsaicin-induced and to a lesser extent also KCl-induced CGRP releases were also augmented after Bv8 treatment. Immunohistochemistry revealed co-localization of prokineticin 2 (Bv8 ortholog in rodents) and CGRP in both plantar skin and nerve tissues. These results confirm that Bv8 sensitizes cutaneous nerve endings to heat, partly, although not exclusively through TRPV1 activation. Conclusion Our results thus support the hypothesis that the first hyperalgesic phase to follow Bv8 injection to hind paws of intact animals is due to peripheral sensitization of nociceptors. What does this study add? Our data provide mechanistic insights into the effect Bv8 application exerts on afferent nerve endings and into the concomitant development of thermal hyperalgesia.

BACKGROUND AND PURPOSE The amphibian peptide Bv8 induces potent nociceptive sensitization in rodents. Its mammalian homologue, prokineticin 2 (PROK2), is strongly up-regulated in inflamed tissues and is a major determinant in triggering inflammatory pain. Bv8 and PROK2 activate two closely related GPCRs, PK1 and PK2, in a relatively non-selective fashion. To characterize better the roles of the two receptors in hyperalgesia and to obtain ligands whose binding affinity and efficacy differed for the two receptors, we modified the Bv8 molecule in regions essential for receptor recognition and activation. EXPERIMENTAL APPROACH We modified the Bv8 molecule by substituting Trp in position 24 with Ala (A-24) and compared it with Bv8 for binding and activating PK1 and PK2 receptors in cell preparations and in affecting nociceptive thresholds in rodents. KEY RESULTS A-24 preferentially bound to PK2 receptors and activated them with a lower potency (5-fold) than Bv8. When systemically injected, A-24 induced Bv8-like hyperalgesia in rats and in mice, at doses 100 times higher than Bv8. Locally and systemically injected at inactive doses, A-24 antagonized Bv8-induced hyperalgesia. In rat and mouse models of inflammatory and post-surgical pain, A-24 showed potent and long-lasting anti-hyperalgesic activity. Unlike Bv8, A-24 increased β-endorphin levels in mouse brain. CONCLUSIONS AND IMPLICATIONS A-24 induced its anti-hyperalgesic effect in rodents by directly blocking nociceptor PK1 receptors and by activating the central opioid system and the descending pain control pathway through brain PK2 receptors.

An efficient route was developed for the synthesis of the Fmoc-protected dipeptide 4, isostere of Gly-Gly containing an alpha-methylene beta-amino acid; the conformationally restricted analogues of Leu-enkephalin, 3a, and Met-enkephalin, 3b, respectively, were prepared by changing 4 for Gly(2)-Gly(3) in the native compounds 3a and 3b whose biological activities were significantly lower than the parent compounds.

On the basis of a Janssen's patent, we approached a new synthesis of some 1,3,5-triazin-4,6-diones as potential non peptidic prokineticin receptor antagonists, containing the following substitutions: (N(1) and N(5) link a 4-methoxybenzyl and a 4-ethylbenzyl, respectively; C(2) can link an amino-ethyl-guanidine (reference compound 1) or an ethylendiamine (2) or an amino-ethyl-amino-2-imidazoline (3). New compounds were assessed for PKR1 and PKR2 affinity. Antagonist properties were evaluated as inhibition of 1 nM Bv8-induced intracellular Ca2+ mobilization.

The prokineticin Bv8, a small protein secreted by the skin of the Bombina variegata frog, is a potent agonist of both the identified prokineticin receptors, the G-protein-coupled PK-R1 and PK-R2. We found in this study that intraperiaqueductal grey (PAG) Bv8, 100 and 200 pmol per rat, exerted a pronociceptive action and caused opposite effects on the ongoing rostral ventromedial medulla (RVM) On- and Off-cell activities in rats. Bv8 increased and decreased the ongoing activity of RVM On and Off cells, respectively. Bv8 decreased tail flick latency and increased the pause and shortened the onset of the Off-cell pause. Bv8 did not change either the tail flick-induced On-cell burst of activity or the onset of On-cell peak firing. Microdialysis analysis, applied in combination with the plantar test, showed that intra-PAG perfusion with Bv8 (0.25 and 0.5 pm) increased GABA, but not glutamate, extracellular levels, and decreased thermoceptive thresholds. These findings show that stimulation of PAG PK-Rs might worsen pain perception and this effect is consistent with both RVM On- and Off-cell ongoing and tail flick-related activities, as well as with the increase induced by Bv8 in PAG GABA levels.

Peripheral neuropathy is characterized by abnormal pain responses triggered by the release of several mediators and neuronal hyperexcitability at the spinal cord level. Emerging evidence indicates that the enhanced activity of dorsal horn neurons requires communication with glia and microglia, cells that are physiologically involved in neuronal wellbeing. Prokineticins (PKs), which include PK1 and PK2, represent a novel family of chemokines characterized by a unique structural motif comprising five disulfide bonds. They are expressed in the peripheral and central nervous system. PKs bind two G protein coupled receptors, PKR1 and PKR2, and participate in the regulation of several biological processes, including pain sensation. This study aimed to investigate the anti-nociceptive effect of PC1, a non-peptide PKR1-preferring antagonist, in a mouse model of neuropathic pain. To do this, we assessed the activity of spinal cord nociceptive neurons as well as astrocyte and microglia phenotypes after repeated administration of PC1 in vivo. PC1 treatment strongly delayed the development of thermal hyperalgesia and tactile and mechanical allodynia. It also reduced spinal microglial and glial activation 8 days post injury in spared nerve injury (SNI) mice. Neuropathic mice showed an increased level of PK2 protein in the spinal cord, mostly in astrocytes. PC1 treatment completely reversed the increased responsiveness to mechanical stimuli, the decreased threshold of neuronal activation, and the increased spontaneous activity that were observed in nociceptive specific (NS) neurons of SNI mice.

Objective: To investigate the potential role of prokineticin 2 (PK2), a bioactive peptide involved in multiple biological functions including immune modulation, in CNS autoimmune demyelinating disease. Methods: We investigated the expression of PK2 in mice with experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS), and in patients with relapsing-remitting MS. We evaluated the biological effects of PK2 on expression of EAE and on development of T-cell response against myelin by blocking PK2 in vivo with PK2 receptor antagonists. We treated with PK2 immune cells activated against myelin antigen to explore the immune-modulating effects of this peptide in vitro. Results: Pk2 messenger RNA was upregulated in spinal cord and lymph node cells (LNCs) of mice with EAE. PK2 protein was expressed in EAE inflammatory infiltrates and was increased in sera during EAE. In patients with relapsing-remitting MS, transcripts for PK2 were significantly increased in peripheral blood mononuclear cells compared with healthy controls, and PK2 serum concentrations were significantly higher. A PK2 receptor antagonist prevented or attenuated established EAE in chronic and relapsing-remitting models, reduced CNS inflammation and demyelination, and decreased the production of interferon (IFN)-γ and interleukin (IL)-17A cytokines in LNCs while increasing IL-10. PK2 in vitro increased IFN-γ and IL-17A and reduced IL-10 in splenocytes activated against myelin antigen. Conclusion: These data suggest that PK2 is a critical immune regulator in CNS autoimmune demyelination and may represent a new target for therapy.

Bv8/Prokineticin 2 (PROK2) is a bioactive peptide initially discovered as a regulator of gastrointestinal motility. Among multiple biological roles demonstrated for PROK2, it was recently established that PROK2 is an insult-inducible endangering mediator for cerebral damage. Aim of the present study was to evaluate the PROK2 and its receptors’ potential involvement in amyloid beta (Aβ) neurotoxicity, a hallmark of Alzheimer’s disease (AD) and various forms of traumatic brain injury (TBI). Analyzing primary cortical cultures (CNs) and cortex and hippocampus from Aβ treated rats, we found that PROK2 and its receptors PKR1 and PKR2 mRNA are up-regulated by Aβ, suggesting their potential involvement in AD. Hence we evaluated if impairing the prokineticin system activation might have protective effect against neuronal death induced by Aβ. We found that a PKR antagonist concentration-dependently protects CNs against Aβ1–42-induced neurotoxicity, by reducing the Aβ-induced PROK2 neuronal up-regulation. Moreover, the antagonist completely rescued LTP impairment in hippocampal slices from 6 month-old Tg2576 AD mice without affecting basal synaptic transmission and paired pulse-facilitation paradigms. These results indicate that PROK2 plays a role in cerebral amyloidosis and that PROK2 antagonists may represent a new approach for ameliorating the defining pathology of AD.

The new chemokine Prokineticin 2 (PROK2) and its receptors (PKR1and PKR2) have a role in inflammatory pain and immunomodulation. Here we identified PROK2 as a critical mediator of neuropathic pain in the chronic constriction injury (CCI) of the sciatic nerve in mice and demonstrated that blocking the prokineticin receptors with two PKR1-preferring antagonists (PC1 and PC7) reduces pain and nerve damage. PROK2 mRNA expression was upregulated in the injured nerve since day 3 post injury (dpi) and in the ipsilateral DRG since 6 dpi. PROK2 protein overexpression was evident in Schwann Cells, infiltrating macrophages and axons in the peripheral nerve and in the neuronal bodies and some satellite cells in the DRG. Therapeutic treatment of neuropathic mice with the PKR-antagonist, PC1, impaired the PROK2 upregulation and signalling. This fact, besides alleviating pain, brought down the burden of proinflammatory cytokines in the damaged nerve and prompted an anti-inflammatory repair program. Such a treatment also reduced intraneural oedema and axon degeneration as demonstrated by the physiological skin innervation and thickness conserved in CCI-PC1 mice. These findings suggest that PROK2 plays a crucial role in neuropathic pain and might represent a novel target of treatment for this disease.

The small protein Bv8, isolated from the amphibian skin, belongs to a novel family of secreted proteins linked to several biological effects. We describe the expression of Bv8/prokineticins and their receptors in mouse macrophages, and characterize their proinflammatory activities. The rodent analogue of Bv8, prokineticin-2, is expressed by macrophages, as well as its G-protein-coupled receptor prokineticin receptor (PKR-1 and PKR-2). PKR-1 is expressed more abundantly. Bv8 induces potent chemotaxis of macrophages at concentrations as low as 10−12 M. It stimulates lipopolysaccharide-induced production of the proinflammatory cytokines IL-1 and IL-12, reducing that of the anti-inflammatory cytokine IL-10. The effects are observed starting at the very low concentration of 10−11 M. Effects on chemotaxis and cytokine are not pertussis-toxin sensitive, but are completely prevented by addition of the phospholipase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, suggesting a Gq protein is involved in the Bv8-induced effects. Studies in PKR-1 knockout mice indicate that all the activities exerted by Bv8 on macrophages are mediated by the PKR-1 receptor. In conclusion, Bv8 appears to be able to induce the macrophage to migrate and to acquire a proinflammatory phenotype. Keywords: Bv8, chemotaxis, anti-inflammatory cytokines, macrophages, prokineticins, proinflammatory cytokines Introduction The small 77-amino-acid (aa) protein Bv8, isolated from the amphibian skin (Mollay et al., 1999) belongs to a novel family of secreted proteins whose homologues have been found in snakes (VPRA, MIT1) (Joubert & Strydom, 1980; Schweitz et al., 1999), rodents (mouse Bv8 or prokineticin (PK) 1 and 2) (Melchiorri et al., 2001) and humans (PK 1 or EG-VEGF and PK 2) (Masuda et al., 2002). This peptide family has structural shared motives, such as the 20-aa terminal sequence and a pattern of cysteine sequence that folds the molecules into a globular form. The mRNA of murine Bv8-like protein, PK 1 and 2 (PK-1 and PK-2), has been detected in the brain, spinal cord, dorsal root ganglia, gastrointestinal tract, endocrine glands, spleen and circulating leukocytes of mice, rats and humans (Li et al., 2001; Melchiorri et al., 2001; Masuda et al., 2002; Le Couter et al., 2004). Two receptors for this family of secretory proteins have been identified in humans, rats and mice (Lin et al., 2002; Masuda et al., 2002). These receptors, PKR-1 and PKR-2, belong to the G-protein-coupled receptor and share approximately 85% aa identity. They are distributed in the brain and peripheral organs (Melchiorri et al., 2001; Negri et al., 2002), including the spleen and leukocytes (Le Couter et al., 2004). The list of biological activities associated with Bv8/PK peptides is rapidly growing. They seem to influence complex behaviors such as feeding and drinking (Negri et al., 2004), and circadian rhythms (Cheng et al., 2002), and are involved in neuronal survival (Melchiorri et al., 2001), angiogenesis (Le Couter et al., 2001; Le Couter & Ferrara, 2003), and the reproductive cycle (Wechselberger et al., 1999). Bv8 and EG-VEGF are also related to regulation of hematopoiesis and hematopoietic cell mobilization (Le Couter et al., 2004). Moreover, an hyperalgesic activity of Bv8 has been clearly demonstrated. When injected intravenously or subcutaneously in rats, Bv8 induces intense systemic nociceptive sensitization to mechanical and thermal stimuli applied to the tail and paws (Mollay et al., 1999; Negri et al., 2002). The cytokines interleukin (IL-)1β and tumor necrosis factor alpha (TNF-α) are closely involved in peripheral nerve and neural hyperexcitability, leading to the development of persistent hyperalgesia (Watkins & Maier, 1999; 2002). However, anti-inflammatory cytokines have been reported to limit the hyperalgesic responses induced by inflammatory stimuli and by IL-1β and TNF (Poole et al., 1995). Considering that lymphoid organs, circulating leukocytes and hematopoietic cells all express high levels of Bv8-like protein (Le Couter et al., 2004), we assessed whether Bv8 could influence the physiology of the macrophage, the main cell involved in inflammatory responses. Macrophages play a central role in both innate and adaptive immunity (Schiffmann, 1982; Beutler, 2004). They are fundamental to the innate immune response and their ability to be chemotactically attracted to the site of initial microbial invasion or to an inflammatory focus is crucial for full activation of the immune/inflammatory response that follows (Beutler, 2004). They are the main producers of the proinflammatory cytokines IL-1β and TNF and the major anti-inflammatory cytokine IL-10 (Moore et al., 1993; Beutler, 2004; Hoebe et al., 2004). Macrophages also synthesize and release IL-12, the critical factor driving the development of T helper (Th)-1 cells (Moore et al., 1993; Trincheri, 1995; Mosman & Sad, 1996; Hoebe et al., 2004), linked to cellular immune responses and tissue injury. Therefore, considering the importance of macrophages in all aspects of the inflammatory response, we evaluated the effect of Bv8 in vitro on several macrophage functions. First of all we investigated whether murine peritoneal macrophages expressed PK receptors and PK 1/2. We then analyzed how Bv8 affected macrophage migration and the production of the pro-inflammatory cytokines IL-1β, TNF and IL-12 and the anti-inflammatory cytokine IL-10. In order to identify the type of receptor involved, we designed some experiments with macrophages from PKR-1 knockout (KO) mice, and tried to characterize the type of G-protein signaling in the effects observed.

The small protein Bv8, secreted by the skin of the frog Bombina variegata, belongs to a novel family of secreted proteins whose orthologues have been identified in snakes (MIT) and in mammals (prokineticins (PKs)). A characteristic feature of this protein family is the same N-terminal sequence, AVITGA, and the presence of 10 cysteines with identical spacing in the C-terminal domain. Two closely related G protein-coupled receptors that mediate signal transduction of Bv8/PKs have been cloned (PK-R1 and PK-R2). In mammals, the Bv8/PK protein family is involved in a number of biological activities such as ingestive behaviours, circadian rhythms, angiogenesis and pain sensitization. In an attempt to identify the structural determinants required for the pronociceptive activity of Bv8, we prepared Bv8 derivatives lacking one (des-Ala-Bv8) or two (des-Ala-Val-Bv8) residues from the N-terminus. des-Ala-Bv8 displayed a receptor affinity five times lower than that of Bv8, it was five times less potent in inducing [Ca2+]i transients and in causing p42/p44 MAPK phosphorylation in CHO-cells expressing PK-R1 and PK-R2. Moreover, dA-Bv8 was about 20 times less potent than Bv8 in inducing hyperalgesia in rats. The deletion of the first two amino acids of Bv8 abolished any biological activity both ‘in vitro’ and ‘in vivo’; however, des-AlaVal-Bv8 is able to antagonize the Bv8-induced hyperalgesia, binding the PK-Rs on peripheral and central projections of the primary sensitive neurons. British Journal of Pharmacology (2005) 146, 625–632. doi:10.1038/sj.bjp.0706376

Novel 14-alkoxy-substituted (e.g. allyloxy, benzyloxy, naphthylmethoxy) morphinan-6-one derivatives were synthesized and biologically evaluated. Compounds 6−9 and 11 displayed affinities in the subnanomolar range to μ opioid receptors which were comparable to 14-O-methyloxymorphone (1) and 14-methoxymetopon (3), and higher than oxymorphone (2). Opioid binding affinity was sensitive to the character and length of the substituent in position 14. In smooth muscle preparations they behaved as potent agonists. Antinociceptive potencies of compounds 6−11 in the hot-plate test after sc administration in mice were considerably greater than the potency of morphine. In the colonic propulsion test, the most potent analgesic compound 7 showed negligible constipating activity at the analgesic dose. These findings provide further evidence that the nature of the substituent at position 14 has a major impact on the abilities of morphinans to interact with opioid receptors. Introduction of a 5-methyl group has no signific...

A fluorescent tripeptide probe derived by coupling fluorescein to H-Dmt-Tic-Glu-NH2 was developed to interact with delta-opioid receptors with high affinity (K-i = 0.035 nM) and selectivity (K-i(mu)/K-i(delta) = 4371). It acts as an irreversible delta-opioid receptor antagonist, and binding to NG108-15 cells is blocked by the standard nonpeptidic delta-opioid receptor antagonist naltrindole. This probe should prove useful in the study of the distribution of delta-opioid receptors in tissues and the internalization of opioid peptides during signal transduction.

1. The small protein Bv8, secreted by the skin of the frog Bombina variegata, belongs to a novel family of secreted proteins whose mammalian orthologues have been identified and named prokineticins (PK-1 and PK-2). 2. Bv8 (from 2.5 to 60 pmol) injected into the lateral ventricles of rat brain suppressed diurnal, nocturnal, deprivation-induced and neuropeptide Y-stimulated feeding and stimulated diurnal drinking. Nocturnal drinking was increased only in fasted rats. 3. PK-2 mRNA is expressed in discrete areas of the rat brain, including the suprachiasmatic nucleus (SCN), medial preoptic area (MPA) and nucleus of the solitary tract (NTS). In the SCN neurons, PK-2 mRNA is highest during the light phase of the circadian cycle and undetectable during the dark phase. 4. The G-protein-coupled receptor prokineticin receptor 2 (PKR-2), which binds Bv8 and PK-2 with high affinity, is mainly expressed in the piriform cortex, paraventricular thalamic nucleus, parataenial nucleus (PT), SCN, hypothalamic paraventricular (PVH) and dorsomedial (DMH) nuclei, arcuate nucleus (ARC) and subfornical organ (SFO) of the rat brain. 5. Bv8 microinjected into the ARC, at doses from 0.02 to 2.0 pmol during night-time or from 0.2 to 5 pmol in 24-h-fasted rats, selectively suppressed feeding without affecting drinking. When injected into the SFO, Bv8 (from 0.2 to 2 pmol) stimulated drinking but did not affect feeding. Bv8 injections into other brain areas left rat ingestive behaviours unchanged. 6. We hypothesize that PK-2-rich projections from SCN neurons to PKR-expressing ARC neurons could transmit the circadian rhythm of feeding, whereas inputs from the PK-2-expressing NTS neurons to the PKR-2-expressing SFO neurons could transmit visceral information on the water-electrolyte balance and osmotic regulation.

The [D-Ala2]deltorphin 1 sequence in which the aspartic acid residue is replaced by the N gamma-OCH3-alpha, gamma-diamino (S) butanoyl residue was synthesized using the Fmoc-chemistry-based solid phase procedure. The resulting deltorphin analogue was chemoselectively glucosylated by reaction with unprotected D-glucose (Glc). The Asn4-, (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-galactopyranosyl)-Asn4- and the (2-acetamido-2-deoxy-D-galactopyranosyl)-Asn4-deltorphin I were also prepared for comparison. The affinity of the new compounds for the delta-opioid receptor was expressed by the inhibition constant (Ki) of the binding of the delta-receptor selective ligand [3H]naltrindole (NTI) to rat brain membrane preparations. The in vitro biological activity of the synthetic peptides was compared with that of the mu-opioid receptor agonist dermorphin in guinea pig ileum (GPI) preparations and with that of the delta-opioid receptor agonist deltorphin I in mouse vas deferens (MVD) preparations. The substitution of Asp4 with Asn failed to affect drastically the Ki and IC50 values for delta-sites, suggesting that an electrostatic interaction does not play an essential role in the binding to delta-opioid sites. The steric hindrance of the side chain of the residue in position 4 affects binding to delta-sites. The increase of the Ki value is smaller when the sugar-peptide linkage involves the gamma-nitrogen of the Dab residue in comparison with the Asn amide side chain.

The small protein Bv8, isolated from amphibian skin, belongs to a novel family of secretory proteins (Bv8-Prokineticin family, SWISS-PROT: Q9PW66) whose orthologues have been conserved throughout evolution, from invertebrates to humans. When injected intravenously or subcutaneously (from 0.06 to 500 pmol kg−1) or intrathecally (from 6 fmol to 250 pmol) in rats, Bv8 produced an intense systemic nociceptive sensitization to mechanical and thermal stimuli applied to the tail and paws. Topically delivered into one rat paw, 50 fmol of Bv8 decreased by 50% the nociceptive threshold to pressure in the injected paw without affecting the threshold in the contralateral paw. The two G-protein coupled prokineticin receptors, PK-R1 and PK-R2, were expressed in rat dorsal root ganglia (DRG) and in dorsal quadrants of spinal cord (DSC) and bound Bv8 and the mammalian orthologue, EG-VEGF, with high affinity. In DSC, PK-R1 was more abundant than PK-R2, whereas both receptors were equally expressed in DRG. IC50 of Bv8 and EG-VEGF to inhibit [125I]-Bv8 binding to rat DRG and DSC were 4.1±0.4 nM Bv8 and 76.4±7.6 nM EG-VEGF, in DRG; 7.3±0.9 nM Bv8 and 330±41 nM EG-VEGF, in DSC. In the small diameter neurons (

A new improved synthetic methodology to form triazinediones as potential antagonists for prokineticin receptors is described.

Alzheimer's disease (AD) is a complex disorder that affects the central nervous system causing a severe neurodegeneration. This pathology affects an increasing number of people worldwide due to the overall aging of the human population. In recent years SUMO protein modification has emerged as a possible cellular mechanism involved in AD. Some of the proteins engaged in the physiopathological process of AD, like BACE1, GSK3-β tau, AβPP, and JNK, are in fact subject to protein SUMO modifications or interactions. Here, we have investigated the SUMO/deSUMOylation balance and SUMO-related proteins during the onset and progression of the pathology in the Tg2576 mouse model of AD. We examined four age-stages (1.5, 3, 6, 17 months old) and observed shows an increase in SUMO-1 protein conjugation at 3 and 6 months in transgenic mice with respect to WT in both cortex and hippocampus. Interestingly this is paralleled by increased expression levels of Ubc9 and SENP1 in both brain regions. At 6 months of age also the SUMO-1 mRNA resulted augmented. SUMO-2-ylation was surprisingly decreased in old transgenic mice and was unaltered in the other time windows. The fact that alterations in SUMO/deSUMOylation equilibrium occur from the early phases of AD suggests that global posttranslational modifications may play an important role in the mechanisms underlying disease pathogenesis, thus providing potential targets for pharmacological interventions.

When administered subcutaneously HS-599, a new didehydroderivative of buprenorphine (18,19-dehydrobuprenorphine), produced a long-lasting antinociceptive response in rats. Its potency exceeded twice that of buprenorphine. In the tail-flick test it acted as a full agonist but in the plantar test only as a partial agonist. Whereas the μ-opioid antagonists naloxone and naltrexone antagonized HS-599 antinociception the δ-opioid antagonist naltrindole and the κ-opioid antagonist nor-binaltorphimine did not. Unlike buprenorphine and morphine, HS-599 never induced conditioned place-preference in rats. In radioligand binding assays, compared with buprenorphine HS-599 had 3 fold higher μ-opioid receptor affinity but lower δ- and κ-opioid receptor affinity. In isolated guinea-pig ileum preparations, HS-599 only partially inhibited the electrically-stimulated contraction, acting as a partial opioid agonist. When tested against the μ-opioid receptor agonist dermorphin, it behaved as a non-equilibrium antagonist. Conversely, in mouse vas deferens (rich in δ-opioid receptors) and rabbit vas deferens preparations (rich in κ-opioid receptors) HS-599 acted as a pure equilibrium antagonist, shifting the log-concentration-response curves of the δ-opioid agonist deltorphin I and the κ-opioid agonist U-69593 to the right. In conclusion, HS-599 is a novel buprenorphine derivative with higher affinity, selectivity and potency than the parent compound, for μ-opioid receptors. It produces intense and long-lasting antinociception and does not induce place-preference in rats. British Journal of Pharmacology (2001) 134, 441–447; doi:10.1038/sj.bjp.0704280

To reduce the density of δ-opioid receptor protein, five antisense phosphorothioate oligodeoxynucleotides (aODN), targeting the three exons of rat δ-opioid receptor mRNA (DOR), were injected twice daily for 4 days or continuously infused for 7 days into brain lateral ventricles (i.c.v.) of Sprague-Dawley rats. Rats acting as controls were infused or injected with a mismatch sequence (mODN) of each aODN. The density of opioid receptors in rat brain membranes was measured by saturation binding experiments using selective ligands for δ, μ and κ opioid receptors. aODNs injected twice a day for 4 days left rat brain δ-opioid receptor density unchanged. The ODN targeting the DOR nucleotide sequence 280–299 (aODN280–299, exon 2), decreased brain δ-opioid receptor density significantly more than aODNs targeting exon 1 (aODN239–258), exon 2 (aODN361–380), or exon 3 (aODN741–760) (to 52% vs 79, 72, and 68%). None of the aODNs to the DOR changed the brain density of μ- or k-opioid receptors. When in a novel environment (but not when kept in their home cages), the locomotor activity of aODN280–299 treated rats was significantly lower than that of saline or mODN treated rats. The δ-opioid agonist SNC80 (5 mg kg−1, s.c.) significantly and potently stimulated locomotion and delayed colonic propulsion in saline- and mODN-infused rats, but left motor behaviour and colonic transit of δ-knockdown rats unchanged. The baseline nociceptive threshold and the antinociceptive response to morphine were unchanged in δ-knockdown rats. British Journal of Pharmacology (1999) 128, 1554–1560; doi:10.1038/sj.bjp.0702931

From skin secretions of Bombina variegata and Bombina bombina, we isolated a small protein termed Bv8. The sequence of its 77 amino acids was established by peptide analysis and by cDNA cloning of the Bv8 precursor. Bv8 stimulates the contraction of the guinea-pig ileum at nanomolar concentrations. The contraction is not inhibited by a variety of antagonists. Injection of a few micrograms of Bv8 into the brain of rats elicits, as assessed by the tail-flick test and paw pressure threshold, a marked hyperalgesia which lasts for about 1 h. Bv8 is related to protein A, a component of the venom of the black mamba. After i.c.v. injection, protein A is even more active than Bv8 in inducing hyperalgesia.

1. In order to improve the in vivo stability of the opioid peptide dermorphin we synthesized O-betaglucosylated analogs ([Ser7-O-betaGlc]dermorphin and [Ser7-O-betaGlc(Ac)4]-dermorphin) and C-alphagalactosylated analogs ([Ala7-C-alphaGal]dermorphin and [Ala7-C-alphaGal(Ac)4]-dermorphin). 2. O- and C-glycosylation of dermorphin halved the peptide affinity for brain mu-opioid receptors and the biological potency in guinea-pig ileum assay (GPI). Despite their lower opioid receptor affinity, when administered intracerebroventricularly (i.c.v., 8-40 pmol) and subcutaneously (s.c., 0.5-3 micromol kg(-1)) in rats, glycosylated analogs were two times more potent than dermorphin in reducing the nociceptive response to radiant heat. Acetylation of sugar hydroxyl groups reduces 5-10 times both biological activity on GPI and mu-receptor affinity, whereas the antinociceptive potency was equal to (i.c.v.) or only two-three times lower (s.c.) than dermorphin potency. 3. Blood-Brain Barrier Permeability Index (BBB-PI) of the glycodermorphins was significantly higher than that of dermorphin, indicating a facilitated entry into the brain: O-beta-linked glucoconiugates are expected to enter CNS by the glucose transporter GLUT-1 of the endothelial barrier. However the calculated BBB-PI for the C-alphagalactoside was about two times higher than that of the O-betaglucoside, excluding the implication of GLUT-1 that is known to be selective for O-beta-links and preferring for the exose glucose. 4. The enhanced brain permeability with the subsequent decrease in peripheral dosage of these opioid peptides did not result in lowering constipation.

Changes in respiratory variables, arterial blood pressure and heart rate were studied in awake rats after injection of the opioid peptide [Lys7]dermorphin and its main metabolites, [1-5]dermorphin and [1-4]dermorphin. Fifteen minutes after injection, doses of [Lys7]dermorphin producing antinociception (i.c.v., 36–120 nmol; s.c., 0.12–4.7 μmol kg−1) significantly increased respiratory frequency and minute volume of rats breathing air or hypoxic inspirates. This respiratory stimulation was reversed to depression by the 5-HT receptor antagonist ritanserin (2 mg kg−1, s.c.), was blocked by naloxone (0.1 mg kg−1, s.c.), significantly reduced by the μ1 opioid receptor antagonist naloxonazine (10 mg kg−1, s.c., 24 h before) but unaffected by peripherally acting opioid antagonist naloxone methyl bromide (3 mg kg−1, s.c.). Forty five minutes after injection, doses of the peptide producing catalepsy (s.c., 8.3–14.2 μmol kg−1, i.c.v., 360 nmol) significantly reduced respiratory frequency and volume of rats breathing air and blocked the hypercapnic ventilator response of rats breathing from 4% to 10% CO2. I.c.v. administration of [1-5]dermorphin and [1-4]dermorphin (from 36 to 360 nmol) never stimulated respiration but significantly reduced basal and CO2-stimulated ventilation. Opioid respiratory depression was only antagonized by naloxone. In awake rats, [Lys7]dermorphin (0.1–1 mg kg−1, s.c.) decreased blood pressure. This hypotensive response was abolished by naloxone, reduced by naloxone methyl bromide and unaffected by naloxonazine. In conclusion, the present study indicates that analgesic doses of [Lys7]dermorphin stimulate respiration by activating central μ1 opioid receptors and this respiratory stimulation involves a forebrain 5-hydroxytryptaminergic excitatory pathway.

In vivo antinociception studies demonstrate that deltorphins are opioid peptides with an unusually high blood–brain barrier penetration rate. In vitro , isolated bovine brain microvessels can take up deltorphins through a saturable nonconcentrative permeation system, which is apparently distinct from previously described systems involved in the transport of neutral amino acids or of enkephalins. Removing Na + ions from the incubation medium decreases the carrier affinity for deltorphins (−25%), but does not affect the V max value of the transport. The nonselective opiate antagonist naloxone inhibits deltorphin uptake by brain microvessels, but neither the selective δ-opioid antagonist naltrindole nor a number of opioid peptides with different affinities for δ- or μ-opioid receptors compete with deltorphins for the transport. Binding studies demonstrate that μ-, δ-, and κ-opioid receptors are undetectable in the microvessel preparation. Preloading of the microvessels with l -glutamine results in a transient stimulation of deltorphin uptake. Glutamine-accelerated deltorphin uptake correlates to the rate of glutamine efflux from the microvessels and is abolished by naloxone.

The density and affinity of binding sites for the δ-selective opioid ligands [3H]-[d-Ala2, Asp4]deltorphin (DELT-I), [3H]-[d-Ala2Glu4]-deltorphin (DELT-II), [3H]-[d-Pen2,d-Pen5]enkephalin (DPDPE), and [3H]-naltrindole (NTI) were determined in whole brain from 10, 15, 25 and 60 day-old C57BL mice. At all ages, the analyses of the homologous displacement curves, gave best fits to single rather than to multiple site models. The binding capacity (Bmax) labelled by [3H]-NTI was about one half that labelled by [3H]-DELT-I, [3H]-DELT-II and [3H]-DPDPE. In 25 and 60 day-old mouse brain the DPDPE Bmax was 25% less than the deltorphin-II Bmax. In saturation experiments, specific binding of [3H]-DELT-I on adult mouse brain homogenates was best fitted by a two-site model (34% high affinity site, Kd=1.08 nm and 66% low affinity sites, Kd=39.9 nm). DPDPE produced a biphasic inhibition of specific [3H]-DELTI-I binding, from 15 days of age onwards. The relative percentage of high and low affinity sites was 72% and 28% in 15 day-, 65% and 35% in 25 day- and 30% and 70% in 60 day-old mice. In adult mouse brain labelled with [3H]-DELT-I, DELT-II recognized 71% of high-affinity and 29% of low-affinity sites. DELT-I and DPDPE produced monophasic inhibition of specific [3H]-DELT-II binding to brain homogenates of adult mice. These data suggest that a sub-population of δ-sites (probably the δ2-subtype), recognized by DELT-I, with high affinity for DELT-II and low affinity for DPDPE develops from 25 days onward. In electrically stimulated mouse vas deferens (MVD) the rank order of potency of the three δ-agonists was: DELT-I> DELT-II> DPDPE in 10 day-old mice, and: DELT-I= DELT-II> DPDPE, from 25 days onward. During this time, the potency of DELT-II increased about 15 fold whereas the potency of DELT-I and DPDPE increased only 5 times. The higher efficacy of DELT-II could depend on receptor maturation towards the δ2-subtype. British Journal of Pharmacology (1997) 120, 989–994; doi:10.1038/sj.bjp.0700990

The Bv8/Prokineticins (PKs) are a new family of peptides identified in frog, fish, reptiles and mammals that signal through two highly homologous G-protein coupled receptors, PKR1 and PKR2. Over the past few years, several biological functions of Bv8/PK proteins have been elucidated. The high expression level of human Bv8/PK2 in bone marrow, lymphoid organs and leukocytes suggested an involvement of these peptides in hematopoiesis and in inflammatory and immunomodulatory processes. Neutrophils are the main endogenous source of Bv8/PK2, while G-CSF is the the only cytokine able to activate PK2 expression in neutrophiles. Moreover G-CSF derived pain seems to be stronlgly related to blood levels of Bv8/PK. According to these results, we started a clinical experimental trial in order to explore role and expression of Bv8/PK proteins in patients treated with chemotherapy for early breast cancer and receiving G-CSF (filgrastim vs peg-filgrastim), investigating also the relationship with G-CSF related pain. Methods: 20 BC pts were submitted to adjuvant FEC100. G-CSF was given as primary prophylaxis. 12 pts received Lenograstim (L: 5 doses, beginning 96 hours after CT) while 8 pts received Pegfilgrastim (P: 1 dose 24 hours after CT). Real-Time PCR and Elisa test were used to analyze Bv8/PK2 expression both in pts receiving L and P. Blood samples for Bv8/PK2 analysis were taken in different steps and days, in order to evaluate Bv8/PK2 expression according to CT time and G-CSF administration. Incidence of neutropenia (N), febrile-N and BP (NRS) were evaluated in all CT cycles. 6 healthy women were recruited as controls to evaluate their Bv8/PK2 basal level. Results: An impressive increase of Bv8/PK2 expression was observed both in L and P group. N occurred in 41.6% of pts treated with L (G3: 8.3%; G4: 16.7%) and in 75% of pts with P (G3: 12.5%; G4: 62.5%; 1 FN). In pts receiving L an incidence of BP occurred in 59.3% (NRS 7-8: 41%) with a mean duration of 6 days; in P group incidence was 37.5% (NRS 7-8: 37.5%), BP lasting 4 days. In all pts BP started within 24-48 hours after G-CSF administration, concurrently with Bv8/PK2 overexpression. Conclusions: Our preliminary results suggest an emerging role of Bv8/prokineticin system in occurrence of G-CSF related BP in primary prevention of CT induced N. Therefore, inhibition of Bv8/PK2 activity should constitute a promising therapeutic strategy to prevent inflammatory pain and perhaps other cancer characteristics. Citation Format: Luigi Rossi, Anselmo Papa, Federica Tomao, Lucia Negri, Silverio Tomao. Relationship of G-CSF related bone pain (BP) with Bv8/PK2 expression in breast cancer (BC) patients (pts) treated with FEC100 adjuvant chemotherapy (CT). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1437.

Background Opioid analgesics are the cornerstone drugs for the treatment of moderate-to-severe pain. Morphine and other analgesics like fentanyl, oxycodone and oxymorphone activate the μ opioid (MOP) receptor, the main type targeted for pharmacotherapy of pain. These drugs share the same pharmacological profiles including severe adverse effects such as respiratory depression, constipation, tolerance and physical dependence. Chemical approaches towards the identification of novel MOP analgesics with reduced side effects include structural modifications of morphinan-6-ones in key positions that are important for binding, selectivity, potency and efficacy at opioid receptors. A representative example is the development of the 14-O-methyl-substituted derivative of the clinically used MOP analgesic oxymorphone, namely 14-O-methyloxymorphone, and its 5-methyl-substituted analogue, 14-methoxymetopon. The focus of the present work is on structure-activity relationship (SAR) studies and in vitro and in vivo pharmacological investigations on a series of opioid ligands differently substituted in positions 5 and 14 of the morphinan skeleton.

Bv8 is a pronociceptive peptide that binds to two G-protein coupled prokineticin receptors, PK-R1 and PK-R2. These receptors are localized in the dorsal horn of the spinal cord and dorsal root ganglia (DRG) of nociceptive neurons in rodents. Systemic administration of Bv8 elicits a biphasic reduction in nociceptive thresholds to thermal and mechanical stimuli. Here, the possibility that Bv8 might directly modulate the expression and release of excitatory transmitters within the early and late phases of hyperalgesia was evaluated. Administration of Bv8 to mouse lumbar spinal cord sections produced a direct, significant and concentration-related release of CGRP. Bv8- or capsaicin-stimulated CGRP release was markedly enhanced in tissues taken from Bv8-pretreated mice during the late, but not the early, phase of hyperalgesia. Pretreatment of rats with protein synthesis inhibitors blocked the expression of the late, but not early, phase of Bv8-induced hyperalgesia. Finally, during the late-phase of hyperalgesia, there was an upregulation of CGRP and substance P immunoreactivity in the rat lumbar dorsal horn and DRG. Such upregulation was prevented by pretreatment with protein synthesis inhibitors. These data suggest that Bv8 induces hyperalgesia by direct release of excitatory transmitters in the spinal cord, consistent with the first phase of hyperalgesia. Additionally, Bv8 elicits a subsequent, protein-synthesis dependent increase in expression and release of excitatory transmitters that may underlie the long-lasting second phase of hyperalgesia. Activation of prokineticin receptors may therefore contribute to persistent hyperalgesia occurring as a consequence of tissue injury further suggesting that these receptors are attractive targets for development of therapeutics for pain treatment.

Mammalian Bv8 (also called prokineticin 2) is a secreted protein that regulates diverse biological processes including pain perception. It belongs to a new family of chemokines, which activate two G-protein linked receptors (prokineticin receptor 1 and 2, PKR1 and PKR2) expressed in regions of the nervous system associated with pain and in cells participating to immuno-inflammatory responses. Primary sensitive neurons co-express PKRs and the transient potential receptor vanilloid 1, cooperating in nociceptor sensitization. Bv8, strongly upregulated in neutrophils and other inflammatory cells, is a main pronociceptive mediator in inflamed tissues, where it sensitizes peripheral nociceptors, stimulates neutrophil chemotaxis and modulates the release of inflammatory and pronociceptive cytokines. Availability of a nonpeptide PKR antagonist, leading to blockade of the Bv8/PKR system, ameliorates pain arising from tissue injury and reduces the time required for recovery from injury.

A novel 17 amino acid peptide, having a d -leucine in position 2 of its sequence, has been isolated from methanol extracts of the skin of the Brazilian frog, Phyllomedusa burmeisteri . The sequence of the peptide is: Tyr- d -Leu-Phe-Ala-Asp-Val-Ala-Ser-Thr-Ile-Gly-Asp-Phe-Phe-His-Ser-Ile-NH 2 . It displays a poor affinity for δ-opioid binding sites, both in the periphery and in the central nervous system. However, the shorter synthetic amidated analogue (1–10) possesses both on the central and peripheral δ binding sites an agonistic potency equalling in affinity and exceeding in selectivity that of the enkephalins. The shorter amidated analogue (1–7) is virtually inactive on opioid binding sites in the periphery, but displays a clear-cut affinity for both δ and μ binding sites on rat brain membranes. To date six different d -amino acid residues have been found, always in position 2 of the sequence, in as many as 11 natural peptide molecules of animal origin.

Bv8 is a small protein secreted by frog skin. Mammalian homologues of Bv8, the prokineticins PK1 and PK2, and their G-protein coupled receptors PKR1 and PKR2 have been identified and linked to several biological effects as gut motility, circadian rhythms, neurogenesis, angiogenesis and cancer progression, haematopoiesis and nociception. In rodents, administration of amphibian Bv8 lowers nociceptor thresholds to a broad spectrum of physical and chemical stimuli. The prokineticin receptors are present in regions of the nervous system associated with pain; primary sensitive neurons expressing PKRs also express the vanilloid receptor TRPV1, providing an anatomical basis for PKR1/TRPV1 cooperative interaction in nociceptor sensitization. Bv8/PK2, strongly up-regulated in neutrophils and other inflammatory cells, is a main pronociceptive mediator in inflamed tissues. Indeed Bv8/PK2 produced by inflammatory cells is released at the site of inflammation where it sensitizes peripheral nociceptors, stimulates chemotaxis and modulates the release of inflammatory and pronociceptive cytokines. Availability of a non-peptide PKR antagonist, leading to blockade the PK/PKR system, ameliorates pain arising from tissue injury and, additionally, reduces the time required for recovery from injury.

Bv8 is an amphibian peptide belonging to the widely distributed AVIT protein family. The mammalian orthologues of Bv8 were named prokineticin 1 and prokineticin 2. Two G-protein-coupled receptors for Bv8–prokineticins have been identified. The biological activities of Bv8/PK proteins range from angiogenesis and involvement in reproduction and cancer, to neuronal survival and neurogenesis, hypothalamic hormone secretion, circadian rhythm control and immunomodulatory processes. Identifying the structural determinants required for receptor binding of Bv8–PKs is mandatory for the design of PKR antagonists, which may be useful in the treatment and prevention of various disease states. Here we describe a procedure for the production in Pichia pastoris of Bv8 and 3 mutants: W24A-Bv8, in which the tryptophan in position 24 is substituted by alanine, the double mutant M1-W24A-Bv8, that contains an additional methionine at the N-terminus and Bv8-TyrTyr that includes two additional tyrosines at the C-terminus. The results evidence a relevant role of tryptophan 24 in Bv8–PKRs interaction.

Three naturally occurring dermorphin-like peptides from the skin of the frog Phyllomedusa bicolor, the related carboxyl-terminal amides, and some substituted analogs were synthesized, their binding profiles to opioid receptors were determined, and their biological activities were studied in isolated organ preparations and intact animals. The opioid binding profile revealed a very high selectivity of these peptides for mu sites and suggested the existence of two receptor subtypes, of high and low affinity. The peptides tested acted as potent mu opioid agonists on isolated organ preparations. They were several times more active in inhibiting electrically evoked contractions in guinea pig ileum than in mouse vas deferens. When injected into the lateral brain ventricle or peritoneum of rats, the high-affinity-site-preferring ligand, [Lys7-NH2]dermorphin, behaved as a potent analgesic agent. By contrast, the low-affinity-site-preferring ligand, [Trp4,Asn7-NH2]dermorphin, produced a weak antinociception but an intense catalepsy.

In experiments to evaluate responses to the activation of cerebral delta-opioid receptors, repeated daily injection of the selective delta-opioid agonist Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 ([D-Ala2]deltorphin II) into rat brain resulted in the development of tolerance, whereas repeated daily injection or continuous infusion of morphine resulted in sensitization to the behavioral activating effects of the delta-opioid agonist. Although the rats did not modify their spontaneous locomotor activity after morphine withdrawal, they became markedly hyperresponsive to the locomotor and stereotypy-producing effects of a challenge dose of the delta-opioid agonist. Sensitization to activation of delta-opioid receptors persisted for at least 60 days after discontinuing morphine treatment. These results show that the development of tolerance and long-term sensitization to opioids involves delta-opioid as well as mu-opioid receptors.

Bv8 is a small protein secreted by frog skin. Mammalian homologues of Bv8, the prokineticins PK1 and PK2, and their G-protein coupled receptors prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2) have been identified and linked to several biological effects as gut motility, neurogenesis, angiogenesis, circadian rhythms, hematopoiesis, and nociception. Emerging evidences indicated that prokineticins are also associated with pathologies of the reproductive and nervous system, myocardial infarction, and tumorigenesis. Bv8 elicits a dose-dependent reduction in nociceptive threshold to thermal, mechanical, and chemical stimuli. The prokineticin receptors are present in a fraction of C- and Adelta-fiber neurons also expressing the vanilloid receptors, TRPV1 and TRPA1. Mice lacking PKR genes exhibit impaired Bv8-induced hyperalgesia, develop deficient responses to noxious heat, capsaicin, and protons and show reduced thermal and mechanical hypersensitivity to paw inflammation, indicating a requirement for PKR signaling in activation and sensitization of primary afferent fibers. Bv8/PK2 is highly expressed by neutrophils and other inflammatory cells and must be considered as new pronociceptive mediators in inflamed tissues. Bv8-like hyperalgesic activity was demonstrated in extracts of rat inflammatory granulocytes. Bv8 stimulates macrophage and T lymphocyte to differentiate towards an inflammatory and Th1 profile indicating that Bv8/PK2 plays a role in immunoinflammatory responses. Blockade of PKRs may represent a novel therapeutic strategy in acute and inflammatory pain conditions.