WO2008120263A2

WO2008120263A2 - Prokineticins receptors antagonists, derivatives and uses thereof

Info

Publication number: WO2008120263A2
Application number: PCT/IT2008/000216
Authority: WO
Inventors: Lucia Negri; Donatella Barra; Pietro Malchiorri; Rossella Miele
Original assignee: Università degli Studi di Roma 'La Sapienza'
Priority date: 2007-04-03
Filing date: 2008-04-01
Publication date: 2008-10-09
Also published as: WO2008120263A3; ITRM20070182A1

Abstract

The present invention relates to peptides derived from the protein Bv8 having an antagonist activity for the prokineticins receptors PKRl and PKR2, with no hyperalgesic activity, and their use in the treatment of pain.

Description

Prokineticins receptors antagonists, derivatives and uses thereof

TECHNICAL FIELD OF THE INVENTION

The present invention concerns derivatives of the protein Bv8 having an agonist activity on the prokineticins receptors and their use in pain treatment.

STATE OF THE ART

An important consequence of tissue lesions is nociceptors activation and sensitization.

Nociceptors sensitization favours sensitivity to both harmful stimuli and those normally not harmful, producing an immediate and persistent pain. Nociceptors sensitization is clinically relevant in the stabilization of a lesion because many states of acute and chronic pain respond to drugs (e.g. non-steroidal anti-inflammatory drugs) that block the activity of the substances producing nociceptors sensitization (e.g. prostanoids). Nociceptors sensitization is an acute process that occurs soon after a tissue lesion and that depends on extracellular mediators that modulate the function of sensory transducers on the nociceptors. However, it also includes a long-term process, that involves the transcription and trafficking of sensory molecules, resulting in a prolonged sensitization of the nociceptors and a consequent persistence of the pain [McMahon et al., 2006]. Prokineticins receptors have recently been discovered and named as receptor 1 (PKRl) and receptor 2 (PKR2) [Lin et al., 2002; Masuda et al., 2002; Soga et al., 2002]. Their activation by peptides belonging to the Bv8-EG-VEGF-prokineticins (PK) family (Bv8: Bombina variegata Bv8 protein mRNA, complete cds. Accession No. AF 168790, EG- VEGF: Homo sapiens prokineticin 1 (PROKl), mRNA. Accession No. NM_032414, PKl : Homo sapiens prokineticin 1 precursor (PROKl) mRNA, complete cds. Accession No. AF333024, Human Bv8: Homo sapiens Bv8 protein (BV8) mRNA, partial cds. Accession No. AFl 82069, PK2: Rattus norvegicus prokineticin 2 precursor, mRNA, complete cds. Accession No. AY089984) constitutes a new mechanism responsible for peripheral nociceptor sensitization following a lesion. Mammalian prokineticins (e.g. PKl and PK2) [Li et al., 2001; LeCouter et al., 2001] are homologous sharing a partial sequence identity with a protein originally isolated from black mamba snake venom named protein A (MIT-I) [Schweitz et al., 1999; Joubert et al., 1980] and to a protein isolated from skin secretions of the frog Bombina variegata, named Bv8 [Mollay et al., 1999; Kaser et al., 2003]. Bv8 (8-1 1 kDa) is a valid pharmacological "tool" that has enabled the prediction and characterization of the physiological role of the endogenous agonists, the prokineticins, in animals. The systemic, spinal and intraplantar administration of Bv8 reduces the nociceptive threshold in relation to a broad spectrum of physical and chemical stimuli [Mollay et al, 1999; Negri et al, 2002, 2006]. Prokineticin 1 (PKl) and prokineticin 2 (PK2) are present in the spinal bone marrow and the dorsal root ganglion cells (DRG), suggesting their role in the transmission of harmful stimuli. PK2, but not PKl, is also expressed in the skin, probably in the granulocytes, dendritic cells and macrophages, and its increased expression in inflammatory processes [LeCouter et al., 2004; Dorsch et al., 2005] supports its exacerbatory role in mediating inflammatory pain [Martucci et al.,2006]. Both PKl and PK2 non-selectively activate the PK receptors (PKRl and PKR2) (PKRl : Homo sapiens prokineticin receptor 1 (PKRl) mRNA, complete cds. Accession No. AF506287; Mus musculus prokineticin receptor 1 (Prokrl), mRNA. Accession No. NM_021381, PKR2: Mus musculus prokineticin receptor 2 (Pkr2) mRNA, complete cds. Accession No. AF487279; Homo sapiens prokineticin receptor 2 (PKR2) mRNA, complete cds. Accession No. AF506288).

The PKRl and PKR2 receptors bind to the proteins Gq/11, Gqi and Gs, inducing an increase in intracellular calcium [Lin et al., 2002; Negri et al., 2002; Vellani et al 2006]. Both PKRl and PKR2 are present in the DRG and in the dorsal horn of the spinal bone marrow. In the smaller-diameter DRG cells, PKRl is colocalized with the vanilloid receptor TRPVl (Transient Receptor Potential Vanilloid 1), thought to be responsible for the perception of painful thermal and tactile stimuli (and known as the capsaicin receptor). This means that PKRl is found on primary sensory fibers responsible for pain perception (nociceptors). In contrast, PKR2 is found mainly in the medium-large diameter cells and is apparently not significantly colocalized with TRPVl. The endogenous agonists of these receptors, the prokineticins (PK), are abundantly expressed in the leukocytes that infiltrate inflammatory tissues, suggesting that the PKs released at sites of inflammation can activate the PKRs on the nerve endings, thus having a significant role in sensitizing the nociceptors [Negri et al., 2002, 2006; Vellani et al 2006]. In vitro studies on cultures of neurons isolated from DRG have demonstrated, in approximately 90% of the neurons examined, that the functional PKRl receptors (that respond to Bv8) are colocalized with the functional TRPVl receptors (that respond to capsaicin), providing the anatomical grounds for PKRl/TRPVl interactions. The authors of the present invention have demonstrated that genetic PKRl deletion in the mouse prevents or considerably reduces nociceptor activation by capsaicin, inflammation or protons. By contrast, the genetic deletion of the TRPVl channel in the mouse prevents nociceptor activation by Bv8. These findings suggest a positive mutual interaction between the receptor PKRl coupled to G protein and the TRPVl channel in the perception of painful thermal, mechanical and chemical stimuli [Negri et al., 2006; Vellani et al., 2006]. Many studies support the therapeutic potential of TRPVl channel antagonists [Honore et al., 2005, Ghilardi et al., 2005]. Inactivation of the prokineticins receptors (PKRs) could prove therapeutically useful for two main reasons. First, PKR antagonists prevent the sensitizing action of endogenous prokineticins. Second, PKR antagonists may prevent the activation of the TRPVl channel by endogenous channel activators. They would therefore certainly be useful in all painful processes involving TRPVl channel activation, i.e. in pain with a significant inflammatory component, including postoperative and deep muscle pain, as well as other types of pain (e.g. due to bone cancer).

Thus by blocking PKRl, and possibly PKR2, it is possible to effectively attenuate pain due to tissue lesions. Furthermore, it is possible to accelerate the return to a normal sensitivity after a tissue lesion. The development of Bv8 analogues as potential antagonists Nuclear magnetic resonance (NMR) methods [Boisbouvier et al. (1998)] have shown that the characteristic structure of disulphide bridges, already determined for colipase [Van Tilbeurgh et al., 1992], can also be found in the protein isolated from black mamba snake venom. This is probably also true for the other proteins in the AVIT family (i.e. Bv8, EG- VEGF, PKs, which all have the same AVIT amino acid sequence at the N-terminal end of the molecule [Kaser et al., 2003]). The venom protein has a compact structure, stabilized by five disulphide bridges, with the N- and C- terminal fragments present on the surface (PDB, No. HTM). Many charged amino acid residues are hidden in the molecule, while some hydrophobic residues, such as Trp24, are exposed to the solvent. The steric conformation of the protein is ellipsoid: one pole has a distinctly positive charge, while the opposite pole is hydrophobic.

Patent applications WO2005/042717 and WO2005/091925 concern a method for selecting

PKR2 antagonists, also for use in pain modulation.

Patent application WO2004/081229 concerns the use of human Bv8 and EG-VEGF to promote hematopoiesis, while patent application WO03/020892 concerns the use of human Bv8 polypeptides to induce the proliferation and stimulate the growth of endothelial cells. Structure-activity relationship studies have demonstrated that the N-terminal portion of the molecule is essential for binding and activating the PKRs. However the C-terminal portion contributes significantly to the hyperalgesic effect of PKRl and PKR2 activation on the nociceptors [Bulloc et al 2004; Negri et al., 2005]. An antagonist of the prokineticins receptors (PKRs) has recently been developed and tested by the authors of the present invention. This is a Bv8 analogue lacking the first two amino acids (dAV-Bv8): this molecule has no biological activity, neither in vitro nor in vivo, but it retains the capacity to bind the PKRs with an affinity 200 times higher than Bv8. In vivo, administering this antagonist peptide results in a rightward shift of the dose-effect curve relative to the mechanical hyperalgesia induced by Bv8. However, the antagonist effect of dAV-Bv8 is short-lasting of approximately 2 hours [Negri et al., 2005]. No prior art documents indicate the role and use of Bv8 derivatives in which certain mutations are able to produce variants acting as antagonists of the receptors PKRl and PKR2 with a prolonged effect.

SUMMARY OF THE INVENTION

In the present invention, the authors describe the potent anti-hyperalgesic activity of a variant of Bv8, named [Ala²⁴]Bv8, obtained using a recombinant method, in the yeast Pichia pastoris, for instance, wherein the tryptophan at position 24 is substituted with an alanine.

Using the recently resolved three-dimensional structure of "Mamba Intestinal Toxin 1" (MIT) as a template, the authors obtained a homologue model for Bv8. The degree of each residue's evolutionary conservation was thus evaluated by analyzing multiple alignments of Bv8 homologue protein sequences and the results obtained were mapped on the structure modeled to enable the identification of important functional residues and variable regions. The results suggest that each modification in any of the amino acid positions from 6 to 40 of the primary structure of Bv8 produces molecules capable of behaving as PKR antagonists.

Thus, the object of the present invention is a peptide derived from the protein Bv8 characterized in that: - it comprises an amino acid substitution in at least one position from positions from 6 to 40 of the primary sequence of Bv8 (SEQ ID No. 2);

- it is a functional antagonist of the prokineticins receptors PKRl and PKR2;

- it has no hyperalgesic activity. The substitution is preferably a substitution of the tryptophan at position 24 of the SEQ ID No. 2. It is even more preferable for the tryptophan at position 24 of the SEQ ID No. 2 to be substituted with a neutral amino acid. Even more preferably, the peptide is [Ala²⁴]Bv8. The object of the invention is the above-described peptide for medical use. Another object of the invention is the above-described peptide for use in the treatment and/or prevention of pain.

A further object of the invention is the use of the above-described peptide in the preparation of medicament for the treatment and/or prevention of pain. The pain is preferably acute or chronic. Even more preferably, the chronic pain is selected from the group of: chronic inflammatory pain, caused by pancreatitis, kidney stones, headache, dysmenorrhea, musculoskeletal pain, sprains, abdominal pain, ovarian cysts, prostatitis, cystitis, interstitial cystitis, postoperative pain, migraine, trigeminal neuralgia, pain caused by burns and/or injuries, pain associated with trauma, neuropathic pain, pain associated with musculoskeletal diseases, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, periarticular conditions, oncological pain, pain due to bone metastases, HIV- related pain.

Another object of the invention is a pharmaceutical composition comprising the above- described peptide in effective amounts together with adjuvant ingredients and/or excipients and/or diluents. The peptide may be administered subcutaneously or intravenously. An expert in the field can extrapolate the dose to administer from results obtained in animal models. The dosage is preferably from 0.01 to 0.02 mg/kg approximately (about 1 mg for a man weighing 70 kg) twice a day.

The present invention is described below in non-limiting examples, with particular reference to the following figures: Figure 1. cDNA coding sequence for Bv8 from mature Bombina variegata.

Figure 2. RP-HPLC purification of [Ala²⁴]Bv8. After binding, the material is eluted from a Vydac 208TP52 inverse-phase chromatographic column with a linear gradient of acetonitrile/TFA 0.2% in water/TFA 0.2%. The mutant protein is eluted at 22.5% of acetonitrile in H₂O with a retention time of 104 min. In the same conditions, Bv8 elutes at 23.5% of acetonitrile in H₂O with a retention time of 120 min.

Figure 3. Paw pressure test in the rat. The subcutaneous (SC) administration of 20 μg/kg of [Ala²⁴]Bv8 blocks the hyperalgesic effect induced by the SC (a), intraplantar (IPL) (b) and intrathecal (IT) (c) administration of Bv8. (d) IT administration of 10 ng of [Ala²⁴]Bv8 (a dose that blocks both the first and the second phases of hyperalgesia induced by administering IT Bv8) blocks the second phase of hyperalgesia induced by administering SC Bv8, which depends on the activation of the central PKRs receptors. Mechanical nociception was measured with the paw pressure test in the rat. The percentage variation in the nociceptive threshold measured was calculated in respect to the nociceptive baseline threshold, i.e. before the treatment.

Figure 4. Paw pressure test in the rat. The IT administration of [Ala²⁴]Bv8 blocks the hyperalgesic effect induced by administering IT Bv8 (a), but has no effect on the local peripheral hyperalgesia caused by administering IPL Bv8 (b). The percentage variation in the measured nociceptive threshold was calculated in respect to the baseline nociceptive threshold recorded before the treatment. The [Ala²⁴]Bv8 was administered 4 minutes before the Bv8.

Figure 5. Paw pressure test in the rat. The dose-response curve for the hyperalgesic effect induced by the IT injection of Bv8 (expressed as the area under the curve [AUC] of hyperalgesia) is shifted one order of magnitude to the right from the IT pre-administration of 5 ng of [Ala²⁴]Bv8. AUC = area under curve, calculated from integrals by the computer software.

Figure 6. Anti-hyperalgesic effect of [Ala²⁴]Bv8 in the mouse. Systemic administration of [Ala²⁴]Bv8 (20 μg/kg, SC) in the mouse abolishes thermal hyperalgesia (paw immersion test) (a) and significantly reduces tactile allodynia (von Frey) (b) induced by local administration of Bv8 (0.5 ng = 50 fmol, IPL). [Ala²⁴]Bv8 (20 μg/kg, SC) reduces the "licking" induced by capsaicin (5 nmol, IPL) (c).

Figure 7. Pressure test in a model of inflammation induced by complete Freund's adjuvant (CFA) in the rat. Systemic SC and IV injection of [Ala²⁴]Bv8 in the rat abolishes the hyperalgesia caused by CFA-induced inflammation in a dose-dependent manner (paw pressure test). This anti-hyperalgesic effect lasts 8 hours, 4 hours and 3 hours, respectively, after the SC injection of 20, 5, and 2 μg/kg of [Ala²⁴]Bv8. A dose of 0.5 μg/kg has no effect (a). After IV administration, the dose of 0.5 μg/kg abolishes the hyperalgesia for 2 hours; a dose of 0.1 μg/kg, IV has no effect (b).

Figure 8. Paw immersion test in the model of CFA-induced inflammation in the mouse. In the rat, a SC dose of 20 μg/kg [Ala²⁴]Bv8 abolishes thermal hyperalgesia (paw immersion test at 48⁰C) for more than 6 hours. Measurements were obtained in 5 control animals (treated with saline solution) and 5 animals treated with Ala24-Bv8: means and two-way ANOVA. Latency was measured in seconds, as explained in the methods. Figure 9. Hypersensitivity to heat (hot plate) (a) and to mechanical stress caused by an intraplantar incision (b) became stable 1 day after the incision. Systemic administration of the PKRs antagonist [Ala²⁴]Bv8 (20 μg/kg SC) completely cancels (A) the hypersensitivity to heat (measured in seconds of latency before the paw is withdrawn from the thermal stimulus) and (B) the hypersensitivity to mechanical stress (measured as the weight in grams at which the animal withdraws its paw from the pressure stimulus). Figure 10. Gel electrophoresis of the amplification products, using RT-PCR, of mRNA for PK2 in the skin of the healthy paw (A), and the skin of the inflamed paw 6, 12 and 24 h, and 30 days after the injection of CFA (B); in the DRG of a healthy rat (C) and in the DRG of a rat with an inflamed paw, 24 h after the injection of CFA (D). Figure 11. Time course of hyperalgesia and PK2 expression in rat paw skin after CFA injection. The onset and duration of hyperalgesia caused by the CFA-induced inflammatory process correlates with the increase in prokineticin 2 expression in the skin of the inflamed paw.

MATERIALS AND METHODS

Design of the |^~Ala²⁴]Bv8 construct and cloning in the pPIC9K expression vector

Bv8 [Bv8: Bombina variegata Bv8 protein mRNA, complete cds. Accession No. AFl 68790] is composed of 77 amino acid residues, with 10 cysteines forming 5 disulphide bridges. It is well known that a large number of disulphide bridges in the polypeptide chain interferes with the proper production of recombinant proteins in Escherichia coli, with the frequent formation of insoluble inclusion bodies. This was also the case in the production of proteins in the AVIT family, such as Bv8. A yeast, Pichia pastor is, was consequently chosen as a system for the recombinant expression of the protein. For this purpose, the cDNA of Bombina variegata coding for Bv8 was cloned in a 9.3 Kb insertion vector, pPIC9K {lnvitrogeή), specific for P. pastoris. pPIC9K can act as a shuttle between bacterium and yeast, since there are two bacteria selection elements, i.e. ampicillin and kanamycin resistance. The presence of the HIS 4 gene enables stable P. pastoris transformants to be generated through homologue recombination between sequences shared by the vector and the host genome. These integrated sequences reveal a strong stability in the absence of selective pressure even when they occur as multiple copies. Upstream from the multiple cloning site, the vector contains the alcohol oxidase 1 (AOXl) gene promoter frequently used to control the expression of heterologous genes. This promoter is strongly repressed in cells grown in the presence of glucose, glycerol, and other sources of carbon, while it is strongly induced by methanol. The vector also contains the sequence coding for the Saccharomyces cerevisiae α-factor signaling peptide, which enables the recombinant protein to be addressed outside the cell. This signal peptide is composed of a pre-sequence of 19 amino acid residues and a pro-sequence of 66 residues. The product's maturation process consists of three phases: 1) removal of the pre-sequence in the endoplasmic reticulum; 2) shearing at a specific site inside the pro-sequence by Kex2; and 3) release of the mature protein by Stel3. Since it is impossible to insert the cDNA directly in the multiple cloning sites, the sequence coding the signal peptide on the vector had to be amplified by means of a preparatory PCR, conducted with the oligonucleotides palBamHl and pa2Xho\, that respectively contain the sites recognized by the enzymes BαmHI and Xhol. The fragment comprising approximately 250 base pairs called PS BαmHl-Xhol was extracted from the gel and digested with the enzymes BαmHl and Xhol.

The cDNA of Bv8, corresponding to the mature peptide (i.e. lacking its signal sequence) was amplified with the oligonucleotides BvδupATzøl and Bv8£coRIdw, enabling the introduction of a site recognized by EcoRl. The fragment of approximately 300 base pairs called Bv8 EcoRl-Xhol was extracted from the gel and digested with the restriction enzymes EcoKl and Xhol.

After binding, the two fragments obtained was used as a template for a new preparatory PCR conducted with the oligonucleotides pa\-BαmHl and Bv8-£coRIdw. The fragment obtained was digested with the restriction enzymes BαmHl and EcoRI and then inserted in the vector pPIC9K, previously shorn with the same enzymes to eliminate the part coding for the signal sequence. The plasmid DNA was extracted from one of the positive colonies, PS-Bv8 #13, and its nucleotide sequence was determined.

Using this construct as a template, the authors used PCR to produce a DNA coding for a variant of Bv8, in which the tryptophan at position 24 is substituted with an alanine. For this purpose, two different fragments were produced by amplification: the first fragment was obtained using the oligonucleotides pal -BamHl and Mut-W-dw (Table 1), capable of annealing on the region upstream from tryptophan 24 and of introducing the restriction site Nhel obtained with a 'silent substitution of a single base. The second fragment was obtained using the oligonucleotides Bv8-W-up and Bv8EcøRIdw (Table 1). The first is annealed upstream from the tryptophan 24 region and contains a mutation of three bases that enables both the substitution of W24A and the insertion of the restriction site Nhel. Table 1. Oligonucleotides used to amplify the DNA by PCR

In addition to the sequence, it also shows the melting temperature (Tm). The mutated bases are shown in bold type.

Oligonucleotide Sequence Tm

PalBamHI 5'-GCG-GAT-CCA-AAC-GAT-GAG- 62.7⁰C

ATT-TCC-3' SEQ ID No. 3

Pa2XhoI 5 ' -GCC-TCG-AGA-GAT-ACC-CCT- 64.2°C

TCT-TC-3' SEQ ID No. 4

Bv8EcoRIdw 5'-ATA-GAA-TTC-AGA-ACA-CTT- 62.6°C

AAA-TTT-TTC-TCC-3' SEQ ID No. 5

Bv8kex2XhoI 5 ' -TTA-TCT-CGA-GAA-AAG-AGC- 71.6°C

TGT-TAT-CAC-TGG-CGC-CTG-TG-3 '

SEQ ID No. 6

Mut-W-up 5 ' -GCT-GCT-AGC-GCT-GCT-TCA- 69.5°C

CGT-AAC-ATC-AG-3' SEQ ID No. 7 Mut-W-up 5 ' -CGC-GCT-AGC-AGC-GCA-GCA- 67.6°C

GG-3 SEQ ID No. 8

The resulting two fragments were digested with Nhel and then bound. The product was used as a template for a new PCR amplification using the oligonucleotides pal-BamHl and Bv8-£coRIdw (Table 1). The new fragment was then digested again with BamHl and EcoRl and inserted in the vector pPIC9K, previously digested with the same enzymes, in order to shear the region coding for the signal sequence. This plasmid (Bv8 mutWΣ) was used to transform E. coli ToplOF-competent cells, and sequenced. The plasmid Bv8 mut#2 was linearized with Sail to facilitate integration in the host strain P. pastoris GSi₁₅ (his4) auxotrophic for histidine and was used to transform the yeast by electroporation.

The His⁺ transformants were selected on a minimum MD selective medium. To confirm the presence of Bv8 cassette expression in the His⁺ transformants of P. pastoris, the authors screened the colonies by PCR using specific primers, Pal-Z?αmHI and Bv8- £coRIdw (Table 1). |^"Ala24]Bv8 expression in P. pastoris Recombinant P. pastoris was grown on a culture medium with glycerol as a source of carbon and energy to obtain a high cell density. The cells were separated by centrifugation and resuspended in a medium containing methanol to induce overexpression of the recombinant protein due to the AOXl promoter.

Ten recombinant clones of P. pastoris were analyzed for their small-scale expression levels and the yield of recombinant protein was determined by inverse-phase HPLC. Using this approach, the authors selected the P. pastoris clone capable of producing high levels of Bv8 for subsequent large-scale expression. The optimal conditions were determined by monitoring the Bv8 content in the cultures with different methanol concentrations, culture times and temperatures. The peak expression of recombinant Bv8 was seen after 120 hours of induction with 1% methanol at 30°C. [AIa²⁴I Bv8 purification

The supernatants from the expression cultures were concentrated and, as a first purification step, they were loaded in a CM-Sephadex column. At a pH of 7, Bv8 with a pi of 8.46 binds to the resin and is eluted with a BES buffer, pH 7.0/0.2 M NaCl. Fractions of recombinant Bv8 were pooled and dialyzed against a Tris-HCl buffer, 20 mM, pH 7.0, then the protein was purified by inverse-phase HPLC in a Cl 8 column (Fig. 3). In these expression conditions, the yield of [Ala²⁴]Bv8 is approximately 1 mg per liter of culture. Binding assays

Confluent CHO cells (approximately 20 x 10⁶) were washed with PBS/EDTA, detached from the culture plates and collected for centrifugation. The resulting precipitate was homogenized in 10 ml of cold homogenization buffer (50 mM Tris-HCl, pH 7.4) using a Polytron homogenizer (PT3000, Kinematica) at 16,000 rpm for 2 minutes. The homogenate was centrifuged at low speed (700 g for 15 min at 4°C) and the resulting supernatant was centrifuged at 100,000 g for 60 minutes at 4°C. The resulting precipitate was resuspended in 10 ml 5OmM Tris-HCl, pH 7.4, and stored at -80°C until use. The protein concentration was determined using the BCA Protein Assay Kit (Pierce, Rockfort, IL, USA). The membranes (20 μg of proteins for PKRl and 40 μg of proteins for PKR2) were incubated with 4 pM [¹²⁵I]MIT (Perkin Elmer, ¹²⁵I-MIT: Kd PKRl = 0.72 nM; Kd PKR2 = 0.29 nM), establishing variable concentrations of Bv8 and [Ala²⁴]Bv8 in a final volume of 1 ml at 37⁰C for 90 min. Each concentration was assayed in duplicate. After incubation, the samples were cooled and the membranes collected on Whatman GF/B filters previously steeped in 0.5% polyethylenimine (Sigma-Aldrich, Milan, Italy), then washed 9 times with 2ml of cold 5OmM Tris-HCl, pH 7.4, and transferred to phials for counting. Radioactivity was measured in a gamma counter (Packard, Cobra II auto- gamma). The nonspecific ligand was determined in the presence of 0.1 mM of Bv8. The shift in the curves and the IC₅₀ values were calculated using PRISM software (GraphPad Software, San Diego, CA, USA). Nociception measurements Tactile sensitivity

Tactile sensitivity in the plantar region of the rear paws of mice and rats (accustomed to a metal mesh floor) was tested with flexible filaments calibrated so as to exert variable pressures of 0.41, 0.70, 1.20, 2.00, 3.63, 5.50, 8.5 and 15.1 g (von Frey test). The pressure stimulus was alternately increased and reduced (up-down method) to obtain a variety of positive and negative responses ⁴³'⁴⁴. The 50% paw withdrawal threshold was determined as follows: (10^[xf+kd])/10,000 [χ_f = last von Frey filament used, k = Dixon value and d = interval between stimuli (difference in grams between the various filaments)]. Thermal hypersensitivity The rats were placed on a preheated clear glass plate and allowed to become accustomed to the environment for approximately 30 minutes. The rear paws were then exposed to infrared thermal stimuli and the latency between application of the stimulus and paw withdrawal was measured (with a timer). A maximum time of 32 seconds was used as a "cut-off to prevent tissue damage (Plantar Test, Ugo Basile, Comerio, Italy). A paw immersion test was used, placing the mouse's rear paw in hot water (48⁰C) and measuring the latency time to paw withdrawal. Randall-Selitto mechanical sensitivity

The threshold for harmful stimuli of a mechanical type was measured with an analgesia meter (Ugo Basile, Comerio, Italy). The test was conducted using a gradually increasing pressure (on a linear scale) on the rat's rear paw up until the animal withdrew its paw. A "cut-off of 400 g was used to avoid the risk of tissue damage. Drug administration

Bv8 and [Ala²⁴]Bv8 wore injected subcutaneously (SC), intrathecally (IT) or into the paw (IPL). For the IPL injections, the drugs were dissolved in 0.9% NaCl and injected into the plantar (20 μl) and dorsal (20 μl) regions of the paw using a microsyringe and a 30 gauge needle. An equal volume of saline solution was injected in control rats. For the IT applications, chronic lumbar IT catheters were inserted in rats anesthetized with ketamine and xylazine (60 mg/kg + 10mg/kg, intraperitoneal^) as described elsewhere [Negri et al., 2002]. The medium used to carry the IT dose is an artificial cerebral spinal fluid and each rat received 5 μl of the medium alone or containing the tested compounds in solution, followed by 5 μl of cerebral spinal fluid. For systemic administration, the compounds were dissolved in saline solution and injected in a volume of 2 ml/kg SC. Controls were injected with an equal volume of saline solution. Animal pain models

CFA-induced paw inflammation

The left paw of the mouse or rat was inflamed by injecting Complete Freund's Adjuvant, CFA (20 or 100 μl), while the right paw was injected with saline solution for control purposes. CFA-induced paw edema was assessed by measuring the paw's volume with a plethysmometer 7140 (Ugo Basile). Thermal, tactile and mechanical hypersensitivity developed within 6 hours of the CFA injection, peaked after 12-24 hours and returned to basal values within 4 days of the injection.

The nociceptive threshold for tactile (von Frey), thermal (radiant heat) and mechanical (Randall Selitto) stimulation of the inflamed paw and control paw were evaluated as described above. Two, 6 and 12 hours, and 1, 2, 3 and 4 days after the CFA injection, separate groups of rats were tested for thermal, tactile and mechanical hypersensitivity, before injecting the antagonist, then again 15, 30, 60, 90, 120 and 180 minutes after injecting the antagonist to determine the time trend of the drug's activity. For each dose, separate groups of animals were tested to determine the dose-response curve for the PKR antagonist.

Postoperative pain model (plantar incision)

The rats were anesthetized with isoflurane by inhalation. The plantar region of the left paw was prepared and a 1 cm longitudinal incision was made through the strip of skin and muscle of the plantar surface. The skin was sutured with two 5-0 nylon stitches and the injury covered with antibiotic cream before the animal was woken. Stitches were removed 2 days later. The rats used as controls were anesthetized and prepared for surgery but the incision was not made. '

Thermal, tactile and mechanical hypersensitivity developed within 2 hours of the incision and began to diminish 4 days after the operation, returning to the values prior to the incision (baseline) within 7 days.

The nociceptive threshold for tactile, thermal and mechanical stimuli in the inflamed paw and control paw were assessed as explained above. Two hours and 1 and 4 days after the incision, separate groups of rats were tested for thermal, tactile and mechanical hypersensitivity before injecting the antagonist, then again 15, 30, 60, 90, 120 and 180 minutes after injecting the antagonist to determine the time course of the drug's activity. For each dose, separate groups of animals were tested to determine the dose-response curve for the PKR antagonist.

RESULTS

Receptor affinity

[Ala²⁴]Bv8 binds both PKRl and PKR2; its PKRs affinity was determined as the concentration needed to shift 50% of bound 125I-MIT to membrane preparations of CHO cell transfected with PKRl or PKR2 (Table 2).

Table 2: Bv8 and [Ala²⁴]Bv8 protein affinity for prokineticins receptors expressed as the concentration that inhibits [¹²⁵I]MIT-I (in nM) binding to membrane preparations from

CHO cells stably transfected with the receptors PKRl or PKR2.

The substitution of Trp24 with Ala induces a 30-fold reduction in the affinity of [Ala²⁴]Bv8 for the receptor PKRl and only an 8-fold reduction for the receptor PKR2 by comparison with Bv8. Anti-nociceptive activity

Models of hyperalgesia induced by Bv8

The authors have already demonstrated in rats and mice that the injection SC (200 ng/kg) or IT (0.5 ng/rat) of Bv8 produces a characteristic biphasic hyperalgesia to thermal, mechanical and tactile stimuli, and that local injections (IPL) of Bv8 (0.5 ng) elicit a strong, localized thermal and mechanical hypersensitivity similar to that of the initial stage of hyperalgesia induced by the systemic administration of Bv8, but without the second hypersensitive phase. The biphasic hyperalgesia to systemic Bv8 administration consequently seems to be mediated by prokineticin activity at the periphery (in the first phase) and at central sites (in the first and second phases).

At doses up to 50 times higher than the hyperalgesic doses of Bv8, the Bv8 mutein, [Ala²⁴]Bv8, has no hyperalgesic effect, but if it is administered prior (5-15 min previously), it is capable of blocking the thermal, mechanical and tactile hypersensitivity induced by subsequent Bv8 administration. In rats, the SC administration (-15 min) of [Ala²⁴]Bv8 (2 μg/kg - 20 μg/kg) abolishes the first and second phases of mechanical hypersensitivity induced by the SC (200 ng /kg) and IT (0.5 ng/rat) administration of Bv8 (Figs 3a, 3c). A significantly lower dose (0.1 μg/kg, SC, -15 min) suffices to eliminate the local hyperalgesia induced by the IPL injection of 0.5 ng of Bv8 (Fig 3b). These results indicate that the systemic administration of the PKR antagonist blocks both peripheral and spinal PKRs.

Intrathecal prior injection (-5 min) of [Ala²⁴]Bv8 at a dose of 2 ng reduces, while a dose of 5 ng abolishes the first and second phases of hyperalgesia induced by Bv8 (from 0.5 to 3 ng/rat of Bv8, ITH) (Figs 4a, 5). [Ala²⁴]Bv8 up to 10 ng IT (-5 min) does not affect the local hyperalgesia induced by Bv8. [Ala²⁴]Bv8 fails to block the hyperalgesia induced by the IPL injection of Bv8 (Fig 4b) or the first phase of hyperalgesia induced by the SC injection of Bv8 (Fig 3d), By contrast, [Ala²⁴]Bv8 abolishes the second phase of hyperalgesia, which depends on the activation of the central sites (Fig 3d). In mice, the systemic administration of [Ala²⁴]Bv8 (20 μg/kg, SC) abolishes the thermal hyperalgesia and significantly reduces the tactile allodynia induced by the local administration of Bv8 (0.5 ng, IPL) indicating a role of the peripheral PKRs in thermal and tactile hypersensitivity (Figs 6a, b). The mutein [Ala²⁴]Bv8 also reduces the "flinching" induced by capsaicin (Fig 6c), coinciding with a reduced sensitivity to capsaicin of the knockout mice lacking the receptor PKRl. These data confirm the already identified interaction between PKR and the vanilloid receptor TRPVl . Models of inflammatory pain (CFA-induced paw inflammation)

[Ala²⁴]Bv8 (20 μg/kg, SC) abolishes the mechanical and tactile hypersensitivity induced by CFA. It is worth noting that the systemic administration of this dose of [Ala²⁴]Bv8 does not alter body temperature over the 8 hours after its administration: body temperature varied from 37.5 ± .2⁰C to 38 ± 0.03⁰C in rats treated with saline solution and from 37.5 ± 0.2⁰C to 38.1 ± 0.03⁰C in rats treated with [Ala²⁴]Bv8 (p< 0.05, paired t-test, n=6). In the rat, the systemic SC and IV injection of [Ala²⁴]Bv8 abolishes the mechanical hyperalgesia induced by CFA (paw pressure test) in a dose-dependent manner. This anti- hyperalgesic effect lasts for 8, 4 and 3 hours, respectively, after the SC injection of 20, 5 and 2 μg/kg, (Fig. 7). The dose of 0.5 μg/kg has no effect when injected SC, but abolishes hyperalgesia for 2 hours when injected IV. The IV dose of 0.1 μg/kg has no effect. In the mouse, [Ala²⁴]Bv8 at a dose of 20 μg/kg abolishes thermal hyperalgesia (tested immersing the paw at 48°C) for more than 6 hours (Fig 8). Model of postoperative pain (plantar incision)

The systemic administration of [Ala²⁴]Bv8 abolishes the thermal and tactile hypersensitivity induced by the incision. Administering [Ala²⁴]Bv8 (20 μg/kg SC) 1 day after the incision restores the thermal and mechanical nociceptive threshold to the baseline values (i.e. prior to the incision), comparable with the thermal and mechanical nociceptive threshold of the control (sham) rats within 30 minutes of its administration (Figs 9a, 9b respectively), indicating that acute administration of the PKR antagonist [Ala ]Bv8 abolishes the thermal and mechanical hypersensitivity induced by the incision. This anti- hyperalgesic effect is persistent, and diminishes 7 hours after the injection. PK expression in tissues

RT-PCR studies indicate that CFA-induced inflammation in the rat paw produces a marked increase in PK2 expression, which peaks after 12- 24 h, in the skin of the inflamed paw (Fig 10a), but also produces a significant increase in PK2 expression in the DRG L4-5 receiving the nociceptive afferent fibers from the inflamed paw (Fig 10b). The CFA- induced increase in PK2 mRNA is time-dependent, already significant within the first hour and peaking (increasing 1000-fold) 9 hours after the CFA injection. The levels of PK2 mRNA decline 24 hours after the CFA injection, but remain significantly higher than the baseline values even 30 days afterwards. The onset and duration of hyperalgesia correlates with this marked increase in PK2 expression (Fig 11). Twenty- four hours after CFA administration in the paw, the levels of PK2 mRNA are also increased in the DRG.

It has been demonstrated that administering non-mammalian prokineticins, such as Bv8, lowers the threshold of nociceptors for a broad spectrum of physical and chemical stimuli by activating the PKRs on primary sensory neurons. The characteristic time course of the hypersensitivity induced by Bv8 is consistent with a prokineticin activity at the periphery (in the first phase) and at the central sites (in both the first and the second phases). CFA injection in the rat paw produced a dramatic upregulation of PK2mRNA, both in the inflamed skin and in the DRG (L4-5). In vitro, Bv8 induces chemotaxis of the macrophages and a greater release of lipopolysaccharide-induced cytokines. Moreover, the chemotaxis induced by Bv8 and the increased release of cytokines induced by lipopolysaccharides in the presence of Bv8 were not observed in the macrophages of PKRl knockout mice, indicating that these pro-inflammatory responses are mediated specifically by the receptor PKRl [Martucci et al., 2006]. These, data point to a role of PKs and PKRs in inflammatory pain and suggest that blocking neuronal PKR activity could reduce pain sensitization. Moreover, blocking the leukocyte PKRs might prevent immune cell infiltration in the area of the lesion. It has been demonstrated that the receptor PKRl is essential for the perception of the painful signals known to be mediated by the vanilloid receptor TRPVl . The TRPVl antagonists studied to date are effective as anti-hyperalgesic agents in pathological processes that give rise to tissue acidosis, as in the case of inflammation, injuries and carcinogenesis. However, the TRPVl receptors are present in the central nervous system, and preclinical studies have consistently demonstrated that TRPVl antagonists cause a significant unwanted rise in body temperature. This increase in body temperature seems to be a class effect, not restricted to a distinct chemical structure. An ideal drug should be capable of limiting the activity of the TRPVl channels in painful conditions without the side-effects of the TRPVl antagonists. A potential target is represented by the prokineticins receptors, which are essential to TRPV 1 receptor activation. Accordingly, the present invention has demonstrated that blocking the prokineticins receptors with non-selective antagonists, such as [Ala²⁴]Bv8, abolishes the thermal, mechanical and tactile hypersensitivity induced by paw inflammation and incision, supporting the role for PKRs in pain induced by inflammation. REFERENCES

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Claims

1. A peptide derived from the protein Bv8 characterized in that:

- it comprises an amino acid substitution in at least one position from 6 to 40 of the primary sequence of Bv8 (SEQ ID No. 2); - it is a functional antagonist for the prokineticins receptors PKRl and PKR2;

- it has no hyperalgesic activity.

2. The peptide according to claim 1, wherein the substitution is a substitution of the tryptophan at position 24 of the SEQ ID No. 2.

3. The peptide according to claim 2, wherein the tryptophan at position 24 of the SEQ ID No. 2 is substituted with a neutral amino acid.

4. The peptide according to claim 3 being [Ala²⁴]Bv8.

5. The peptide according to any of the previous claims that is for medical purposes.

6. The peptide according to claim 1-4 for use in the treatment and/or prevention of pain.

7. Use of the peptide according to claim 1-4 for the preparation of a medicament for the treatment and/or prevention of pain.

8. Use according to claim 7, wherein the pain is acute or chronic.

9. Use according to claim 8, wherein the chronic pain is selected from the group of: chronic inflammatory pain, caused by pancreatitis, kidney stones, headache, dysmenorrhea, musculoskeletal pain, sprains, abdominal pain, ovarian cysts, prostatitis, cystitis, interstitial cystitis, postoperative pain, migraine, trigeminal neuralgia, pain caused by burns and/or injuries, pain associated with trauma, neuropathic pain, pain associated with musculoskeletal diseases, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, periarticular conditions, oncological pain, pain due to bone metastases, HIV-related pain.

10. A pharmaceutical composition comprising effective amount of the peptide according to claims 1-4 and adjuvants and/or excipients, and/or diluents.