WO2008120263A2 - Antagonistes des récepteurs des prokinéticines, leurs dérivés et leurs utilisations - Google Patents

Antagonistes des récepteurs des prokinéticines, leurs dérivés et leurs utilisations Download PDF

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WO2008120263A2
WO2008120263A2 PCT/IT2008/000216 IT2008000216W WO2008120263A2 WO 2008120263 A2 WO2008120263 A2 WO 2008120263A2 IT 2008000216 W IT2008000216 W IT 2008000216W WO 2008120263 A2 WO2008120263 A2 WO 2008120263A2
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pain
peptide according
ala
paw
induced
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PCT/IT2008/000216
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WO2008120263A3 (fr
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Lucia Negri
Donatella Barra
Pietro Malchiorri
Rossella Miele
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Università degli Studi di Roma 'La Sapienza'
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention concerns derivatives of the protein Bv8 having an agonist activity on the prokineticins receptors and their use in pain treatment.
  • tissue lesions 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.
  • PDRl receptor 1
  • PLR2 receptor 2
  • Bv8 Bombina variegata Bv8 protein mRNA, complete cds. Accession No.
  • 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].
  • MIT-I protein A
  • Bv8 protein isolated from skin secretions of the frog Bombina variegata
  • 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)
  • PRRl Homo sapiens prokineticin receptor 1 (PKRl) mRNA, complete cds. Accession No. AF506287; Mus musculus prokineticin receptor 1 (Prokrl), mRNA. Accession No.
  • 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).
  • 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).
  • TRPVl Transient Receptor Potential Vanilloid 1
  • 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].
  • 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
  • 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.
  • 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.
  • the authors describe the potent anti-hyperalgesic activity of a variant of Bv8, named [Ala 24 ]Bv8, obtained using a recombinant method, in the yeast Pichia pastoris, for instance, wherein the tryptophan at position 24 is substituted with an alanine.
  • 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);
  • 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 [Ala 24 ]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.
  • 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
  • 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.
  • FIG. 1 RP-HPLC purification of [Ala 24 ]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 H 2 O with a retention time of 104 min. In the same conditions, Bv8 elutes at 23.5% of acetonitrile in H 2 O with a retention time of 120 min.
  • FIG. 3 Paw pressure test in the rat.
  • the subcutaneous (SC) administration of 20 ⁇ g/kg of [Ala 24 ]Bv8 blocks the hyperalgesic effect induced by the SC (a), intraplantar (IPL) (b) and intrathecal (IT) (c) administration of Bv8.
  • IT administration of 10 ng of [Ala 24 ]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.
  • FIG. 4 Paw pressure test in the rat.
  • the IT administration of [Ala 24 ]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 [Ala 24 ]Bv8 was administered 4 minutes before the Bv8.
  • FIG. 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 [Ala 24 ]Bv8.
  • AUC area under curve, calculated from integrals by the computer software.
  • FIG. 7 Pressure test in a model of inflammation induced by complete Freund's adjuvant (CFA) in the rat.
  • CFA complete Freund's adjuvant
  • Systemic SC and IV injection of [Ala 24 ]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 ⁇ g/kg of [Ala 24 ]Bv8.
  • a dose of 0.5 ⁇ g/kg has no effect (a).
  • the dose of 0.5 ⁇ g/kg abolishes the hyperalgesia for 2 hours; a dose of 0.1 ⁇ g/kg, IV has no effect (b).
  • FIG. 8 Paw immersion test in the model of CFA-induced inflammation in the mouse.
  • a SC dose of 20 ⁇ g/kg [Ala 24 ]Bv8 abolishes thermal hyperalgesia (paw immersion test at 48 0 C) 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.
  • Bv8 Bombina variegata Bv8 protein mRNA, complete cds. Accession No. AFl 68790
  • Bv8 Bombina variegata Bv8 protein mRNA, complete cds. Accession No. AFl 68790
  • Bv8 Bombina variegata Bv8 protein mRNA, complete cds. Accession No. AFl 68790
  • 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.
  • the cDNA of Bombina variegata coding for Bv8 was cloned in a 9.3 Kb insertion vector, pPIC9K ⁇ lnvitroge ⁇ ), 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.
  • the vector 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 ⁇ -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 B ⁇ mHI and Xhol. The fragment comprising approximately 250 base pairs called PS B ⁇ mHl-Xhol was extracted from the gel and digested with the enzymes B ⁇ mHl and Xhol.
  • the cDNA of Bv8, corresponding to the mature peptide (i.e. lacking its signal sequence) was amplified with the oligonucleotides Bv ⁇ upATz ⁇ l 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.
  • the two fragments obtained was used as a template for a new preparatory PCR conducted with the oligonucleotides pa ⁇ -B ⁇ mHl and Bv8-£coRIdw.
  • the fragment obtained was digested with the restriction enzymes B ⁇ mHl 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.
  • 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.
  • 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 Bv8Ec ⁇ RIdw (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
  • Tm melting temperature
  • 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 mutW ⁇ ) 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 GSi 15 (his4) auxotrophic for histidine and was used to transform the yeast by electroporation.
  • the His + transformants were selected on a minimum MD selective medium.
  • the authors screened the colonies by PCR using specific primers, Pal-Z? ⁇ mHI and Bv8- £coRIdw (Table 1).
  • " Ala24]Bv8 expression in 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.
  • Confluent CHO cells (approximately 20 x 10 6 ) 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).
  • Tactile sensitivity in the plantar region of the rear paws of mice and rats 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 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.
  • Bv8 and [Ala 24 ]Bv8 wore injected subcutaneously (SC), intrathecally (IT) or into the paw (IPL).
  • SC subcutaneously
  • IPL intrathecally
  • IPL paw
  • the drugs were dissolved in 0.9% NaCl and injected into the plantar (20 ⁇ l) and dorsal (20 ⁇ l) regions of the paw using a microsyringe and a 30 gauge needle.
  • An equal volume of saline solution was injected in control rats.
  • 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 ⁇ l of the medium alone or containing the tested compounds in solution, followed by 5 ⁇ l of cerebral spinal fluid.
  • 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
  • the left paw of the mouse or rat was inflamed by injecting Complete Freund's Adjuvant, CFA (20 or 100 ⁇ l), 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.
  • 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. '
  • 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.
  • [Ala 24 ]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).
  • CHO cells stably transfected with the receptors PKRl or PKR2.
  • Trp24 The substitution of Trp24 with Ala induces a 30-fold reduction in the affinity of [Ala 24 ]Bv8 for the receptor PKRl and only an 8-fold reduction for the receptor PKR2 by comparison with Bv8.
  • Anti-nociceptive activity is a 30-fold reduction in the affinity of [Ala 24 ]Bv8 for the receptor PKRl and only an 8-fold reduction for the receptor PKR2 by comparison with Bv8.
  • the Bv8 mutein, [Ala 24 ]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.
  • the SC administration (-15 min) of [Ala 24 ]Bv8 (2 ⁇ g/kg - 20 ⁇ g/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).
  • Intrathecal prior injection (-5 min) of [Ala 24 ]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).
  • [Ala 24 ]Bv8 up to 10 ng IT (-5 min) does not affect the local hyperalgesia induced by Bv8.
  • [Ala 24 ]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, [Ala 24 ]Bv8 abolishes the second phase of hyperalgesia, which depends on the activation of the central sites (Fig 3d).
  • the systemic administration of [Ala 24 ]Bv8 (20 ⁇ g/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 [Ala 24 ]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)
  • 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.
  • non-mammalian prokineticins such as Bv8
  • 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).
  • Bv8 induces chemotaxis of the macrophages and a greater release of lipopolysaccharide-induced cytokines.
  • TRPVl vanilloid receptor
  • 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.
  • 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.
  • 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 [Ala 24 ]Bv8, abolishes the thermal, mechanical and tactile hypersensitivity induced by paw inflammation and incision, supporting the role for PKRs in pain induced by inflammation.

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Abstract

La présente invention concerne des peptides dérivés de la protéine Bv8 ayant une activité antagoniste des récepteurs des prokinéticines PKR1 et PKR2, sans aucune activité hyperalgésique. L'invention concerne également leur utilisation dans le traitement de la douleur.
PCT/IT2008/000216 2007-04-03 2008-04-01 Antagonistes des récepteurs des prokinéticines, leurs dérivés et leurs utilisations WO2008120263A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009039337A2 (fr) * 2007-09-21 2009-03-26 Genentech, Inc. Inhibition de l'angiogenèse
WO2012006003A1 (fr) * 2010-06-28 2012-01-12 Janssen Pharmaceutica Nv Antagonistes du récepteur-1 de la prokinéticine pour le traitement de la douleur
WO2012006004A1 (fr) * 2010-06-28 2012-01-12 Janssen Pharmaceutica Nv Antagonistes du récepteur-1 de la prokinéticine pour le traitement de la douleur
JP2016502520A (ja) * 2012-11-13 2016-01-28 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． 抗プロキネチシン受容体（ｐｒｏｋｒ）抗体およびその使用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004087054A2 (fr) * 2003-03-25 2004-10-14 The Regents Of The University Of California Procedes de modulation de la secretion gastrique mettant en oeuvre des antagonistes du recepteur de la prokineticine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004087054A2 (fr) * 2003-03-25 2004-10-14 The Regents Of The University Of California Procedes de modulation de la secretion gastrique mettant en oeuvre des antagonistes du recepteur de la prokineticine

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BERGER V ET AL: "Transport mechanisms of the large neutral amino acid L-phenylalanine in the human intestinal epithelial caco-2 cell line." THE JOURNAL OF NUTRITION NOV 2000, vol. 130, no. 11, November 2000 (2000-11), pages 2780-2788, XP002497333 ISSN: 0022-3166 *
BULLOCK C M ET AL: "STRUCTURAL DETERMINANTS REQUIRED FOR THE BIOACTIVITIES OF PROKINETICINS AND IDENTIFICATION OF PROKINETICIN RECEPTOR ANTAGONISTS" MOLECULAR PHARMACOLOGY, BALTIMORE, MD, US, vol. 65, no. 3, 1 March 2004 (2004-03-01), pages 582-588, XP008028638 ISSN: 0026-895X *
LAI REN ET AL: "Two novel Bv8-like peptides from skin secretions of the toad Bombina maxima." COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY. PART B, BIOCHEMISTRY & MOLECULAR BIOLOGY MAR 2003, vol. 134, no. 3, March 2003 (2003-03), pages 509-514, XP002497330 ISSN: 1096-4959 *
MOLLAY ET AL: "Bv8, a small protein from frog skin and its homologue from snake venom induce hyperalgesia in rats" EUROPEAN JOURNAL OF PHARMACOLOGY, ELSEVIER BV, NL, vol. 374, no. 2, 18 June 1999 (1999-06-18), pages 189-196, XP002123181 ISSN: 0014-2999 *
NEGRI L ET AL: "Nociceptive sensitization by the secretory protein Bv8" BRITISH JOURNAL OF PHARMACOLOGY, BASINGSTOKE, HANTS, vol. 137, no. 8, 1 December 2002 (2002-12-01), pages 1147-1154, XP002371986 ISSN: 0007-1188 *
NEGRI LUCIA ET AL: "Biological activities of Bv8 analogues." BRITISH JOURNAL OF PHARMACOLOGY NOV 2005, vol. 146, no. 5, November 2005 (2005-11), pages 625-632, XP002497331 ISSN: 0007-1188 *
NEGRI LUCIA ET AL: "Modulators of pain: BV8 and prokineticins" CURRENT NEUROPHARMACOLOGY, vol. 4, no. 3, July 2006 (2006-07), pages 207-215, XP002497332 ISSN: 1570-159X *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009039337A2 (fr) * 2007-09-21 2009-03-26 Genentech, Inc. Inhibition de l'angiogenèse
WO2009039337A3 (fr) * 2007-09-21 2009-05-14 Genentech Inc Inhibition de l'angiogenèse
US9284369B2 (en) 2007-09-21 2016-03-15 Genentech, Inc. Inhibition of angiogenesis
WO2012006003A1 (fr) * 2010-06-28 2012-01-12 Janssen Pharmaceutica Nv Antagonistes du récepteur-1 de la prokinéticine pour le traitement de la douleur
WO2012006004A1 (fr) * 2010-06-28 2012-01-12 Janssen Pharmaceutica Nv Antagonistes du récepteur-1 de la prokinéticine pour le traitement de la douleur
JP2013530231A (ja) * 2010-06-28 2013-07-25 ヤンセン ファーマシューティカ エヌ．ベー． 鎮痛処置用プロキネチシン１受容体アンタゴニスト
JP2016502520A (ja) * 2012-11-13 2016-01-28 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． 抗プロキネチシン受容体（ｐｒｏｋｒ）抗体およびその使用
US9951132B2 (en) 2012-11-13 2018-04-24 Regeneron Pharmaceuticals, Inc. Anti-prokineticin receptor (PROKR) antibodies and uses thereof

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