WO2018036785A1 - PEPTIDES HAVING TrkA-RECEPTOR-AGONISTIC ACTIVITY AND/OR PEPTIDES HAVING NGF-ANTAGONISTIC ACTIVITY - Google Patents

PEPTIDES HAVING TrkA-RECEPTOR-AGONISTIC ACTIVITY AND/OR PEPTIDES HAVING NGF-ANTAGONISTIC ACTIVITY Download PDF

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Publication number
WO2018036785A1
WO2018036785A1 PCT/EP2017/069705 EP2017069705W WO2018036785A1 WO 2018036785 A1 WO2018036785 A1 WO 2018036785A1 EP 2017069705 W EP2017069705 W EP 2017069705W WO 2018036785 A1 WO2018036785 A1 WO 2018036785A1
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Prior art keywords
cys
asn
gly
ser
thr
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PCT/EP2017/069705
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French (fr)
Inventor
Piercarlo Fantucci
Stefano Govoni
Alessia Angela PASCALE
Nicoletta MARCHESI
Emilio VANOLI
Jacopo VERTEMARA
Alessandro Concetto VITRIOLO
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Neuheart S.R.L.
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Publication of WO2018036785A1 publication Critical patent/WO2018036785A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • C07K14/48—Nerve growth factor [NGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • PEPTIDES HAVING TrkA-RECEPTOR-AGONISTIC ACTIVITY AND/OR PEPTIDES HAVING NGF- ANTAGONISTIC ACTIVITY
  • the invention relates to peptides and peptide fragments and the use thereof.
  • the present invention relates to the identification of a specific amino acid sequence (residues 250-348) within the amino acid structure of the human TrkA receptor which is suitable as a new target and ideal model for the creation of interacting peptides that show a strong bond with the receptor.
  • the invention further relates to peptides and peptide fragments which bind to the aforesaid sequence and have a TrkA-agonistic activity and the use thereof for the treatment of conditions, diseases and disorders deriving from a reduced activation of the TrkA receptor, in particular for the treatment of peripheral neuropathies, multiple sclerosis, Alzheimer's disease, Parkinson's disease, hypoxic cerebral lesions and/or optic nerve atrophy.
  • the present invention also relates to peptides and peptide fragments which bind to the aforesaid sequences and have NGF -antagonistic activity and the use thereof for the treatment of conditions, diseases and disorders linked to high levels of NGF, in particular for the treatment of pain and cardiac arrhythmia.
  • the invention further relates to the use of the peptides and peptide fragments of the invention for the treatment of diseases characterised or aggravated by the loss of innervations, such as, for example, ischaemic heart disease, heart failure and diabetes and the comorbidities thereof, in particular diabetic neuropathies.
  • diseases characterised or aggravated by the loss of innervations such as, for example, ischaemic heart disease, heart failure and diabetes and the comorbidities thereof, in particular diabetic neuropathies.
  • the invention further relates to the use of the peptides and peptide fragments of the invention for the treatment of diseases, conditions and disorders that show a benefit from treatment with NGF, such as, for example, ulcers, neurotrophic keratitis and glaucoma.
  • NGF neurotrophic keratitis
  • Nerve growth factor is a protein that regulates the growth and survival of sympathetic and sensory nerve cells (neurons). NGF thus belongs to a family of growth factors called neurotrophins.
  • NGF binds with great affinity to and activates the tyrosine receptor kinase TrkA.
  • TrkA receptor dimerises and autophosphorylates, thus giving rise to a signalling process that is important for the growth and survival of sympathetic neurons.
  • NGF nerve growth factor
  • NGF neurotrophic factor
  • diseases and disorders include: diabetic polyneuropathy, HIV-associated peripheral neuropathy, Alzheimer's disease, Parkinson's disease, optic glioma, advanced optic nerve atrophy and perinatal hypoxic-ischemic brain damage (Aloe et ⁇ , J. Transl. Med. (2012) 10:
  • NGF neurotrophic keratitis and glaucoma
  • the hyperinnervation of heart tissue has been demonstrated to be linked to cardiac arrhythmia and sudden cardiac death (see, for example, Chen et al Cardiovasc Res (2001) 50, 409-416).
  • NGF neurotrophic factor
  • Tanezumab is effective in reducing pain in both groups of patients compared to the placebo group.
  • the invention relates to the use of the amino acid sequence (residues 250-348) of TrkA as the reference domain for the construction of peptides and peptide fragments which have the ability to bind to it.
  • the peptides and peptide fragments of the invention are peptides and peptide fragments having an amino acid sequence listed above and salts thereof, solvates and solvates of the salts thereof.
  • the salts preferred for the purpose of the invention are physiologically acceptable salts of the peptides and peptide fragments of the invention. Also included are salts which are not themselves suitable for pharmaceutical applications, but can be used, for example, to isolate and purify the peptides and peptide fragments of the invention.
  • solvates for the purpose of the invention refer to the forms of peptides and peptide fragments which in a liquid or solid state form a complex by complexation with one or more molecules of solvent. Hydrates are a specific form of solvates in which complexation takes place with water.
  • the term peptide fragment refers to peptides in which the amino acid sequences of the invention are incorporated into a larger peptide molecule. This can occur by means of a chain extension at the C-terminal domain, the N-terminal domain or both domains.
  • the larger peptide molecule is not longer than a total of 50 amino acids, including the amino acid sequences of the invention.
  • the larger peptide molecule is not longer than 35 amino acids, including the amino acid sequences of the invention.
  • the amino acid sequences of the invention extend by no more than 5 amino acids at the two terminal domains.
  • an amino acid comprises proteinogenic amino acids, but also non-proteinogenic amino acids, such as D-amino acids or amino acids in which the amino group and the carbonyl group are not bonded to the same carbon atom, but are rather separated by at least one additional carbon (e.g. ⁇ - or ⁇ -amino acids).
  • D-amino acids amino acids in which the amino group and the carbonyl group are not bonded to the same carbon atom, but are rather separated by at least one additional carbon (e.g. ⁇ - or ⁇ -amino acids).
  • oc-amino acids may be preferred.
  • L-amino acids may be preferred.
  • oc-L-amino acids may be preferred.
  • the peptides or peptide fragments according to the invention can comprise exclusively L-amino acids or exclusively D-amino acids. In some embodiments the peptides and peptide fragments of the invention consist of a mixture of L- and D-amino acids. In some embodiments the peptides and peptide fragments of the invention may contain at least one L-amino acid. In some embodiments the peptides and peptide fragments of the invention may contain at least one D-amino acid. In some embodiments of the invention the amino acid sequences can be phosphorylated.
  • the peptides of the present invention have an amino acid sequence selected from the group consisting in:
  • the peptides and peptide fragments according to the present invention are suitable for use as a medicament for the treatment of diseases and disorders in humans and in animals.
  • the peptides and peptide fragments are in particular distinguished by their ability to bind to and activate the TrkA receptor.
  • the peptides and peptide fragments according to the present invention are thus useful as agonists of the amino acid sequences of the TrkA receptor selected from the group consisting in
  • the peptides and peptide fragments of the invention are advantageous for the treatment of peripheral neuropathies, multiple sclerosis, Alzheimer's disease, Parkinson's disease, hypoxic brain lesions and/or optic nerve atrophy.
  • the peptides and peptide fragments of the invention are also useful in the treatment of diseases and disorders characterised or aggravated by a loss of innervation, such as ischaemic heart disease, heart failure and diabetes and the comorbidities thereof, in particular diabetic neuropathies.
  • the peptides and peptide fragments of the invention are also useful in the treatment of other diseases and disorders that have shown to derive benefits from a treatment with NGF, such as ulcers, neurotrophic keratitis and/or glaucoma.
  • the peptides and peptide fragments of the invention are distinguished in particular by their ability to bind to the TrkA receptor more strongly than NGF without activating that receptor.
  • NGF antagonists are therefore useful as NGF antagonists in the treatment of diseases and disorders associated with an increase in the level of NGF.
  • the peptides or peptide fragments can be used as NGF antagonists in the bond with the amino acid sequences of the TrkA receptor selected from the group consisting in
  • the peptides and peptide fragments are useful in the treatment and management of pain and in the treatment and prophylaxis of hyperinnervation and conditions associated therewith, such as cardiac arrhythmia and sudden cardiac death.
  • the peptides and peptide fragments according to the present invention are useful in the treatment, prophylaxis and/or management of diseases and disorders, in particular diseases and disorders tied to NGF, more in particular to diseases and disorders tied to an increased level of NGF.
  • the present invention further relates to the peptides and peptide fragments of the invention for use in the treatment and/or management of pain.
  • the present invention further relates to the peptides and peptide fragments of the invention for use in the treatment and/or prophylaxis of hyperinnervation.
  • the present invention further relates to the peptides and peptide fragments of the invention for use in the treatment and/or prophylaxis of cardiac an-hythmias and/or of sudden cardiac death.
  • FIG. 1 shows the peptide having the amino acid sequence GLY-GLN-ASN-TRP-GLN- PRO-SER-ALA-TYR-GLN-ALA-TRP-CYS-THR-THR, which binds to the TrkA receptor by overlapping the NGF binding site
  • FIG. 2 shows the peptide having the amino acid sequence CYS-LYS-GLY-TYR-GLY- GLY-LEU-VAL-ALA-GLN-CYS-THR-ASP-GLY-PHE, which binds to the TrkA receptor by overlapping the NGF binding site
  • FIG. 3 shows the peptide having the amino acid sequence ASN-VAL-GLU-PRO-LEU- THR-CYS-VAL-TYR-GLN-PRO-LYS-CYS-GLY-TRP, which binds to the TrkA receptor by overlapping the NGF binding site
  • FIG. 4 shows the peptide having the amino acid sequence LEU-PRO-SER-PRO-CYS- TRP-GLN-GLN-PHE-ARG-CYS-ASP-GLN-LEU-GLN, which binds to the TrkA receptor by overlapping the NGF binding site
  • Figure 5 shows the peptide having the amino acid sequence CYS-GLY-THR-ASP-CYS- PHE-MET-ASN-GLY-ARG-PRO-GLY-GLY-TYR-TYR, which binds to the TrkA receptor by overlapping the NGF binding site
  • Figure 6 shows the simulation box used for molecular docking
  • Figure 7 shows the structure of 58 amino acids of the TrkA receptor involved in the bond with NGF
  • Figure 7 shows the structure of 58 amino acids of the TrkA receptor involved in the bond with NGF.
  • TrkA The residues and type of interaction that is established between TrkA and NGF are shown in detail in Table 1.
  • Table 1 Residues and type of interaction between TrkA and NGF.
  • the third peptide is the one that most covers the points of contact of the TrkA receptor with NGF.
  • the peptides and peptide fragments according to the present invention are characterised by an original sequence - i.e. one that is not necessarily derived from the original sequence of NGF - and they demonstrate to be capable of competing with NGF in the interaction with the TrkA receptor.
  • This objective was pursued by carrying out simulations of molecular docking against TrkA using, as possible ligands, the peptides of a library of 1 ,000 species, generated with a random sequence using Solypep software.
  • the library of 1,000 peptides was constructed using the Solypep tool; this software makes it possible to generate peptide sequences with a random sequence up to a maximum of 15 residues and to generate a corresponding pdb file containing the peptide in linear form.
  • Solypep enables exclusively the use of L-amino acids. For this reason, every sequence was subsequently processed with Maestro in order to invert all the C a stereocentres so as to generate the D-amino acids. Solypep further makes it possible to control the solubility of the sequences generated by it, so that, among all those generated, the 1,000 most soluble forms were selected. Summing up, each peptide created exhibits the following characteristics: • length 15 residues
  • the entire receptor is maintained rigid with the exception of the OH groups of the THR (292,325,330,352,360), TYR(359) and SER(304,320,326) residues.
  • This choice was dictated by the dimensions of the receptor and of the ligands and aimed to reduce the required computing time and power within acceptable limits.
  • the hydroxyl groups of TrkA adjacent to the binding site for NGF are treated as exempt from constraints in order to maximise the possibility of the receptor establishing H-bonds with the ligand, where present, and partly decrease the rigidity of the system.
  • Figures 1, 2, 3, 4 and 5 represent the TrkA receptor 1 , the NGF dimer 2 and the ligands 3. As may be seen from the figures, all of the peptides bind TrkA by overlapping the NGF binding site, suggesting that they are good inhibitors. Also shown are the number and type of interactions that each ligand develops with the receptor; it may be noted that the first peptide with the highest docking score also has a higher number of interactions with TrkA and consequently a stronger solid interaction with the receptor.
  • the peptides and peptide fragments of the present invention bind to the TrkA receptor and activate it, therefore, by acting as receptor agonists. Accordingly, the peptides and peptide fragments of the present invention are useful in the prophylaxis, treatment and management of the diseases and disorders which benefit from a greater activation of the TrkA receptor.
  • the peptides and peptide fragments of the invention are suitable for treating, preventing and/or managing peripheral neuropathies, multiple sclerosis, Alzheimer's disease, Parkinson's disease, hypoxic cerebral lesions and/or optic nerve atrophy.
  • the peptides and peptide fragments of the invention are useful for the treatment, prophylaxis and/or management of diseases or disorders characterised or aggravated by a loss of innervation, such as ischaemic heart disease, heart failure and diabetes and the comorbidities thereof, in particular diabetic neuropathies.
  • the peptides and peptide fragments of the invention are useful for the treatment, prophylaxis and/or management of other diseases and disorders that have shown to benefit from a treatment with NGF, such as ulcers, neurotrophic keratitis and/or glaucoma.
  • the peptides and peptide fragments of the invention can be used in the treatment and prophylaxis of the above-mentioned conditions not only in humans, but also in animals such as primates, swine, ruminants (cattle, sheep, goats), horses, cats, dogs, poultry (for example, chickens, ducks, geese, quail, pigeons, turkeys or ornamental birds), as well as productive and ornamental fish, reptiles and amphibians.
  • animals such as primates, swine, ruminants (cattle, sheep, goats), horses, cats, dogs, poultry (for example, chickens, ducks, geese, quail, pigeons, turkeys or ornamental birds), as well as productive and ornamental fish, reptiles and amphibians.
  • the present invention further relates to a method for the prophylaxis, treatment and/or management of diseases or disorders which benefit from a greater activation of the TrkA receptor in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
  • the present invention further relates to a method for treating, preventing and/or managing peripheral neuropathies, multiple sclerosis, Alzheimer's disease, Parkinson's disease, hypoxic cerebral lesions and/or optic nerve atrophy in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
  • the present invention further relates to a method for treating, preventing and/or managing diseases or disorders characterised or aggravated by a loss of innervation in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
  • the present invention further relates to a method for treating, preventing and/or managing ischaemic heart disease, heart failure and diabetes and the comorbidities thereof, in particular diabetic neuropathies in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
  • the present invention further relates to a method for treating, preventing and/or managing other diseases and disorders which have shown to benefit from a treatment with NGF, in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
  • the present invention further relates to a method for treating, preventing and/or managing ulcers, neurotrophic keratitis and/or glaucoma in human beings and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
  • the peptides and peptide fragments of the present invention can act systemically and/or locally. To this end, they can be administered in a suitable manner, such as, for example, orally, parenterally, pulmonarily, nasally, sublingually, lingually, buccally, rectally, cutaneously, transdermally, optically, or as an implant or stent or coating thereof. For these routes of administration, the peptides and peptide fragments of the invention can be administered in suitable forms of administration.
  • the forms suitable for oral administration include already existing forms of application that work according to the prior art and release the peptides and peptide fragments of the invention rapidly or in a modified fashion, for example as an sustained-release formulation.
  • These forms of application can contain the peptides and peptide fragments of the invention in a crystalline and/or amorphised and/or dissolved form.
  • Examples of such forms of application include tablets (uncoated tablets or tablets with coatings such as enteric coatings or coatings which modify the release of peptides and peptide fragments of the invention, such as slowly dissolving coatings or insoluble coatings), tablets or films/wafers that disintegrate rapidly in the oral cavity, capsules (for example, hard or soft gelatine capsules), lyophilised powders, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
  • coatings such as enteric coatings or coatings which modify the release of peptides and peptide fragments of the invention, such as slowly dissolving coatings or insoluble coatings
  • tablets or films/wafers that disintegrate rapidly in the oral cavity capsules (for example, hard or soft gelatine capsules), lyophilised powders, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
  • Parenteral administration can take place avoiding an absorption step (for example via the intravenous, intra-arterial, intracardiac, intraspinal or intralumbar route), or else with the inclusion of an absorption step (for example, intramuscular, subcutaneous, percutaneous or intraperitoneal).
  • Parenteral forms of administration include, among others, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.
  • Suitable routes of administration are, for example, pharmaceutical forms for inhalation (for example, powder inhalers or nebulisers) nasal drops, nasal solutions or nasal sprays; tablets, film/wafers or capsules for lingual, sublingual or buccal administration, suppositories, preparations for the eyes or ears, vaginal capsules, aqueous suspensions (lotions, mixtures), lipophilic aqueous suspensions, ointments, creams, transdermal therapeutic systems (such as, for example, patches), milk, pastes, foams, implants or stents.
  • pharmaceutical forms for inhalation for example, powder inhalers or nebulisers
  • nasal drops for example, nasal solutions or nasal sprays
  • tablets film/wafers or capsules for lingual, sublingual or buccal administration, suppositories, preparations for the eyes or ears, vaginal capsules, aqueous suspensions (lotions, mixtures), lipophilic aqueous suspensions, ointments,
  • peptides and peptide fragments of the invention can be incorporated into the listed forms of administration. This can take place in ways that are known per se, by mixing them with inert, nontoxic, pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients include, for example, carriers (for example microcrystalline cellulose, lactose and mannitol), solvents, emulsifiers, dispersants and wetting agents (for example sodium dodecyl sulphate, polyoxysorbitan oleate), binders (for example, polyvinylpyrrolidone), natural and synthetic polymers (for example, albumin), stabilisers (for example, antioxidants such as ascorbic acid), colourants (for example, inorganic pigments such as iron oxides) and taste and/or odour masking agents.
  • carriers for example microcrystalline cellulose, lactose and mannitol
  • solvents for example, emulsifiers, dispersants and wetting agents (for example sodium dodecy
  • the peptides and peptide fragments of the invention may be useful to administer the peptides and peptide fragments of the invention in forms of administration which modulate their pharmacokinetics and increase their delivery through the blood-brain barrier.
  • forms of administration can be provided by absorbing or using the peptides and peptide fragments of the invention on/in nanoparticulate carriers.
  • the carriers which modulate the pharmacokinetics and increase delivery through the blood-brain barrier include carriers based on biodegradable polymers such as poly(alkyl cyanoacrylates), polyesters, polyanhydrides, polyethers or mixtures or copolymers thereof.
  • Examples of potentially useful nanometer-scale carriers comprise carriers based on poly(butyl cyanoacrylate), polylactic acid, poly (lactic-co-glycolic acid), chitosan, polyethylene glycol or mixtures or copolymers thereof.
  • the nanoparticulate carriers it might be useful for the nanoparticulate carriers to be treated on the surface, for example coated with further compounds to promote delivery through the blood-brain barrier.
  • Such further compounds for the surface treatment of nanoparticulate carriers include surfactants, polyethers, biological molecules and mixtures thereof.
  • the invention further relates to medicaments comprising at least one peptide or peptide fragment of the invention, usually together with at least one inert, nontoxic, pharmaceutically acceptable excipient and the use thereof for the above-mentioned purposes.
  • peptides and peptide fragments of the invention may be preferable to combine with another pharmaceutically active compound, for example another neuroprotective compound.
  • the peptides and peptide fragments of the present invention bind to the TrkA receptor and block it by acting, therefore, as NGF antagonists. Accordingly, the peptides and peptide fragments of the present invention are useful in the prophylaxis, treatment and management of the diseases and disorders correlated to a high level of NGF.
  • the peptides and peptide fragments of the invention are suitable for the treatment and/or management of pain, above all chronic pain, such as osteoarthritis pain, rheumatoid arthritis, chronic lumbar pain, interstitial cystitis, prostatitis, chronic pelvic pain syndrome, fibromyalgia, endometriosis, degenerative disease of the intervertebral disc and cancer pain.
  • the peptides and peptide fragments of the invention are useful for the treatment and/or the prophylaxis of hyperinnervation and conditions correlated thereto, such as cardiac arrhythmias and sudden cardiac death.
  • the peptides and peptide fragments of the invention can be used in the treatment and prophylaxis of the above-mentioned conditions not only in humans, but also in animals such as primates, swine, ruminants (cattle, sheep, goats), horses, cats, dogs, poultry (for example, chickens, ducks, geese, quail, pigeons, turkeys or ornamental birds), as well as productive and ornamental fish, reptiles and amphibians.
  • animals such as primates, swine, ruminants (cattle, sheep, goats), horses, cats, dogs, poultry (for example, chickens, ducks, geese, quail, pigeons, turkeys or ornamental birds), as well as productive and ornamental fish, reptiles and amphibians.
  • the present invention further relates to a method of prophylaxis, treatment and/or management of diseases or disorders tied to NGF, in particular to high levels of NGF in human beings and animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
  • the present invention further relates to a method for the treatment and/or management of pain in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
  • the present invention further relates to a method for treating and/or preventing hyperinnervation in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
  • the present invention further relates to a method for treating and/or preventing cardiac arrhythmias and/or the sudden cardiac death in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
  • the expression pain management relates to all treatment regimens that do not completely eliminate pain from the patient, but reduce pain in order to improve or significantly improve the patient's quality of life.
  • the peptides and peptide fragments of the present invention may act systemically and/or locally: To this end, they can be administered in a suitable manner, such as, for example, orally, parenterally, pulmonarily, nasally, sublingually, lingually, buccally, rectally, cutaneously, transdermally, optically, or as an implant or stent.
  • the peptides and peptide fragments of the invention can be administered in suitable forms of administration
  • the forms suitable for oral administration are forms of application that work according to the prior art and release the peptides and peptide fragments of the invention rapidly or in a modified fashion, for example as an sustained-release formulation.
  • These forms of application can contain the peptides and peptide fragments of the invention in a crystalline and/or amorphised and/or dissolved form.
  • Examples of such forms of application include tablets (uncoated tablets or tablets with coatings such as enteric coatings or coatings which modify the release of peptides and peptide fragments of the invention, such as slowly dissolving coatings or insoluble coatings), tablets or films/wafers that disintegrate rapidly in the oral cavity, capsules (for example, hard or soft gelatine capsules), films, lyophilised powders, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
  • coatings such as enteric coatings or coatings which modify the release of peptides and peptide fragments of the invention, such as slowly dissolving coatings or insoluble coatings
  • tablets or films/wafers that disintegrate rapidly in the oral cavity such as capsules (for example, hard or soft gelatine capsules), films, lyophilised powders, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
  • Parenteral administration can take place avoiding an absorption step (for example via the intravenous, intra-arterial, intracardiac, intraspinal or intralumbar route), or else with the inclusion of an absorption step (for example, intramuscular, subcutaneous, percutaneous or intraperitoneal).
  • Parenteral forms of administration include, among others, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.
  • Suitable routes of administration are, for example, pharmaceutical forms for inhalation (for example, powder inhalers or nebulisers) nasal drops, nasal solutions or nasal sprays; tablets, film/wafers or capsules for lingual, sublingual or buccal administration, suppositories, preparations for the eyes or ears, vaginal capsules, aqueous suspensions (lotions, mixtures), lipophilic aqueous suspensions, ointments, creams, transdermal therapeutic systems (such as, for example, patches), milk, pastes, foams, implants or stents.
  • pharmaceutical forms for inhalation for example, powder inhalers or nebulisers
  • nasal drops for example, nasal solutions or nasal sprays
  • tablets film/wafers or capsules for lingual, sublingual or buccal administration, suppositories, preparations for the eyes or ears, vaginal capsules, aqueous suspensions (lotions, mixtures), lipophilic aqueous suspensions, ointments,
  • peptides and peptide fragments of the invention can be incorporated into the listed forms of administration. This can take place in ways that are known per se, by mixing them with inert, nontoxic, pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients include, for example, carriers (for example microcrystalline cellulose, lactose and mannitol), solvents, emulsifiers, dispersants and wetting agents (for example sodium dodecyl sulphate, polyoxysorbitan oleate), binders (for example, polyvinylpyrrolidone), natural and synthetic polymers (for example, albumin), stabilisers (for example, antioxidants such as ascorbic acid), colourants (for example, inorganic pigments such as iron oxides) and taste and/or odour masking agents.
  • carriers for example microcrystalline cellulose, lactose and mannitol
  • solvents for example, emulsifiers, dispersants and wetting agents (for example sodium dodecy
  • the peptides and peptide fragments of the invention may be useful to administer the peptides and peptide fragments of the invention in forms of administration which modulate their pharmacokinetics and increase their delivery through the blood-brain barrier.
  • forms of administration can be provided by absorbing or using the peptides and peptide fragments of the invention on/in nanoparticulate carriers.
  • the carriers which modulate the pharmacokinetics and increase delivery through the blood-brain barrier include carriers based on biodegradable polymers such as polyialk l cyanoacrylates), polyesters, polyanhydrides, polyethers or mixtures or copolymers thereof.
  • Examples of potentially useful nanometer-scale carriers comprise carriers based on poly(butyl cyanoacrylate), polylactic acid, poly (lactic-co-glycolic acid), chitosan, polyethylene glycol or mixtures or copolymers thereof.
  • nanoparticulate carriers it might be useful for the nanoparticulate carriers to be treated on the surface, for example coated with further compounds to promote delivery through the blood-brain barrier.
  • further compounds for the surface treatment of nanoparticulate carriers include surfactants, polyethers, biological molecules and mixtures thereof.
  • nanoparticulate carriers it may be preferable to treat the nanoparticulate carriers with one or more compounds selected from the group consisting of polysorbate 80, Pluronic, poly(ethylene glycol), poly( vinyl alcohol), human albumin and mixtures thereof.
  • the invention further relates to medicaments comprising at least one peptide or peptide fragment of the invention, usually together with at least one inert, nontoxic, pharmaceutically acceptable excipient and the use thereof for the above-mentioned memeposes.
  • peptides and peptide fragments of the invention may be preferable to combine with other pharmaceutically active compounds, for example other analgesics or compounds useful in the treatment of cardiac arrhythmias and sudden cardiac death such as, for example, beta-blockers.
  • other pharmaceutically active compounds for example other analgesics or compounds useful in the treatment of cardiac arrhythmias and sudden cardiac death such as, for example, beta-blockers.
  • Analgesics suitable for use in combination with the peptides and peptide fragments of the invention include, for example, non-steroidal anti-inflammatory drugs such as paracetamol, ibuprofen, aspirin, diclofenac or naproxen, COX-2 inhibitors, such as rofecoxib, celecoxib and etoricoxib, and opioids, such as codeine, oxycodone, hydrocodone, dihydromorphine or pethidine, as well as other known analgesics.
  • non-steroidal anti-inflammatory drugs such as paracetamol, ibuprofen, aspirin, diclofenac or naproxen
  • COX-2 inhibitors such as rofecoxib, celecoxib and etoricoxib
  • opioids such as codeine, oxycodone, hydrocodone, dihydromorphine or pethidine, as well as other known analgesics.
  • Beta-blockers suitable for use in combination with the peptides and peptide fragments of the invention include, for example, acebutolol, atenolol, betaproxol, bisoprolol, bucindolol, butaxamine, carteolol, carvedilol, celiprolol, esmolol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol and timolol.
  • the invention further relates to medicaments comprising at least one peptide or peptide fragment having TrkA-agonistic activity or NGF-antagonistic activity according to the invention, usually in combination with at least one other pharmaceutically active ingredient and the use thereof for the above-mentioned purposes.
  • the minimum amount of the peptides and peptide fragments of the invention to be administered is a therapeutic amount.
  • therapeutically effective amount means an amount of compound which prevents the onset of or alleviates the symptoms, manages, stops the progression and/or eliminates a disease, disorder or condition by virtue of the altered activation of the TrkA receptor, a disease, disorder or condition characterised or aggravated by a loss of innervation or another disease, disorder or condition that has shown to benefit from a treatment with NGF, or a disease or disorder tied to high levels of NGF.
  • an effective dosing regimen for the peptides and peptide fragments of the invention in adults is about 1 to 1000 ⁇ g kg of the peptides or peptide fragments of the invention, preferably 5 to 500 ⁇ g kga.
  • the effective dose is 5 to 100 ⁇ ig/ kg or 10 to 50 ig I kg.
  • the effective dose is 1 to 20 ng kg or 2 to 10 ⁇ ig/kg.
  • the peptides and peptide fragments of the invention are administered daily.
  • the peptides or peptide fragments of the invention are administered every other day or every 3, 5, 7 or 10 days. In some cases it might be necessary to test the patient in order to find the optimal dose.
  • amino acids mentioned can be either a-L-amino acids or D-amino acids unless specified otherwise.
  • c represents the protein-ligand interaction energy
  • eFlex represents the loss of flexibility of the ligand
  • CHB and FHB are compensatory functions for the non-ideality of the specific bond and are expressed as a sum over all hydrogen-bond sites;
  • AD is a term for the atomic desolvation which describes variations in the solvation field of ligand atoms.
  • TrkA and NGF form a unique complex made up of two molecules of NGF and two molecules of TrkA, forming two homo- dimers.
  • the present inventors mapped the short contact distances between the two TrkA molecules and the two NGF molecules in order to find the smallest example of the TrkA-NGF complex. This study showed that the largest number of short-range interactions occur between the first NGF molecule and second TrkA molecule.
  • NGF 7A between the NGF molecule and the TrkA molecule are to be found in the first 24 amino acids of NGF; in particular, they are to be found in the amino acid residues 2 to 24 of NGF.
  • TrkA receptor a peptide based on the amino acids 2 to 24 of NGF.
  • the corresponding binding domain of the TrkA receptor is represented by residues 250-348, as previously described.
  • amino acid sequence of the starting peptide is given below:
  • the free energy of the starting peptide for binding to TrkA was calculated as -13.02 Kcal/mol. Based on this, the present inventors investigated various mutated forms of the starting peptide showing one, two or three mutations. They then selected those peptides which showed an increase of at least -4 kcal/mol in binding free energy compared to the starting peptide for further investigation. This increase in binding free energy shows an increased ability of the peptide to bind to TrkA and activate it. The increases in the binding free energy of the mutated peptides are given below in Table 6.
  • the peptides described above can be prepared by conventional solid state synthesis.
  • the process of synthesis takes place according to the well-known cycle of a coupling step, a washing step, a step of removing the protective group and a washing step, after which another coupling step follows or, if the peptide is complete, the peptide is cleaved from the solid support.
  • a polystyrene resin can be used as the support.
  • the functionality of the amino acid can be protected, for example, using a tert-butyloxycarbonyl
  • Boc 9-fluorenylmethyloxycarbonyl
  • the coupling reaction is carried out using coupling agents that are usual in the art, such as, for example, a combination of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or ⁇ , ⁇ '- dicyclohexylcarbodiimide (DCC) with 1 -hydroxy-benzotriazole (HOBt) and l-hydroxy-7-aza- benzotriazole (hOAT), where appropriate in the presence of a base, in particular a hindered base such as diisopropylethylamine (DIE A) or tetramethylpiperidine (TMP).
  • coupling agents that are usual in the art, such as, for example, a combination of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or ⁇ , ⁇ '- dicyclohexylcarbodiimide (DCC) with 1 -hydroxy-benzotriazole (HO
  • Suitable solvents for the coupling reaction include dimethylforniamide (DMF), N- methylpyrrolidone (NMP), trichloromethane (TCM) and dichloromethane (DCM) or mixtures thereof.
  • TFE trifluorethanol
  • FFIP hexafluoro-2-propanol
  • DMSO dimethylsulfoxide
  • the removal of the protecting groups is achieved by using suitable deprotecting agents.
  • the Boc protecting group is usually removed under acidic conditions using, for example, hydrochloric acid (HC1) or trifluoroacetic acid (TFA), where appropriate in a suitable solvent such as, for example, methanol or ethanol for HC1 and, for example, DCM or TCM for TFA.
  • the Fmoc protecting group is usually removed in the presence of a base, such as, for example, pyridine, where appropriate in a suitable solvent.
  • the peptide Once the peptide is complete it can be cleaved from the solid support in a suitable manner and purified as necessary. The whole process can be performed on an automated synthesizer.
  • the ability of the peptides and peptide fragments of the invention to act as a TrkA receptor agonist can for example be measured using an enzyme-linked immunosorbent assay (ELISA) for the phosphorylated TrKA receptor.
  • ELISA enzyme-linked immunosorbent assay
  • Such an ELISA kit is commercially available, for example, from Cell Signalling Technologies under the name PathScan® Phospho-TrkA (Tyr674/675) Sandwich ELISA Kit (catalogue number 7212).
  • the assay is used according to the manufacturer's instructions, exposing the PC12 and/or SH- SY5Y cell lines as cellular types expressing TrkA receptors to the peptides and peptide fragments of the invention.
  • the cells grow in a suitable cell culture medium (for example: 60- mm Falcon ⁇ ) at a certain cell density (e.g. 0.5-lxl 0 6 ) in a specific and complete culture medium, as described, for example, in Marchesi et al. (J Cell Physiol. 2014 Nov; 229(1 1):1776- 86) for the SH-SY5Y cell line and Rossi et al. (Bioorg Med Chem. 2011 Nov l ;19(21):6210-24) for the PC 12 cell line.
  • c ⁇ t e a erent ce s g t e o ng:
  • the culture medium when the cell culture is at 80-90% confluence, the culture medium is replaced with a fresh one containing a low percentage of Fetal Bovine Serum (FBS) and for a pre-established period of time.
  • FBS Fetal Bovine Serum
  • the cells are detached from the plate with a scraper and transferred into an appropriate tube stored in ice, where they are then sonicated.
  • the suspension is centrifuged for 10 minutes (at 13- 14000 rpm) at 4°C and the supernatant is transferred into a new tube and stored at -80°C.
  • the percentage of phosphorylated TrkA after exposure to the peptides can be compared with a calibration curve constructed using NGF as a reference.
  • the ability of the peptides and peptide fragments of the invention to act as a TrkA receptor agonist can be measured using a ⁇ -NGF-induced neurite regeneration bioassay.
  • the assay used is based on the method described by Chandler et al. (J. Biol. Chem. (1984) 259, No. 1 1, 6882- 6889).
  • PC 12 cells are grown on tissue culture plates (for example 60 mm Falcon ⁇ ) at an initial cell density of 4-5 x 10 5 cells/plate. During the cell culture, ⁇ -NGF is added on 8 consecutive days. The cells are harvested mechanically with a Pasteur pipette, washed once in serum-containing growth medium and twice in serum-free growth medium and collected by centrifugation (500 g for 3 minutes each time). The cells are resuspended in the serum-free growth medium at 6 x 10 cells/ml and 0.5 ml is dispensed into each well of a 24-well culture plate.
  • tissue culture plates for example 60 mm Falcon ⁇
  • ⁇ -NGF is added on 8 consecutive days.
  • the cells are harvested mechanically with a Pasteur pipette, washed once in serum-containing growth medium and twice in serum-free growth medium and collected by centrifugation (500 g for 3 minutes each time). The cells are resuspended in the serum-free growth medium at 6 x 10 cells/
  • the tissue culture plate is pre-treated with 50 ng/ml of poly-L-lysine (Sigma Aldrich) and washed four times with sterile distilled water.
  • the cells are incubated at 37° C for 30 min and 400 ⁇ of modified medium (Dulbecco's modified Eagle's medium) containing bovine serum albumin (BSA) is added to bring the final serum albumin concentration to 1 mg/ml.
  • modified medium Dulbecco's modified Eagle's medium
  • BSA bovine serum albumin
  • the cells are returned to the incubator for 15 min. before 10 pm of ⁇ -NGF or various concentrations of the peptides of the invention (e.g. 0, 1, 2, 5, 10, 20 pm) are added. At least one set of cells is used as a control without the addition of ⁇ -NGF.
  • the cells are incubated for 24 hours at 37 ° C and read out using a phase contrast microscope.
  • the positive cells are the ones which show a minimum neurite growth > 25 microns.
  • the TrkA-agonistic activity of the peptides of the invention is evaluated based on their ability to stimulate the growth of neurons compared to the NGF-induced positive cells.
  • the ability of the peptides and peptide fragments of the invention in the treatment, management or prevention of diseases or disorders which benefit from a greater activation of the TrkA receptor, or of other diseases and disorders which have shown to benefit from a treatment with NGF, can be observed in suitable animal models.
  • peptides of the invention in the treatment or prevention of Alzheimer's disease can be demonstrated, for example, in a rat model using the Morris water maze.
  • the Morris water maze can generally provide information about the activity of the tested compounds in relation to memory, a target of potential interest for aging and dementia-related diseases, additional conditions that might benefit from greater TrkA receptor agonism.
  • peptides of the invention in the treatment of diabetic ulcers can be demonstrated, for example, in a mouse model. Diabetes can be induced in mice with streptozotocin. The diabetic mice are then wounded, according to described methods, using for example a biopsy punch, and treated topically with the peptides and peptide fragments in a suitable solvent, for example saline solution. The closure of the wound is then observed visually or using digital imaging and a comparison is made with control animals treated with a placebo, for example the suitable solvent on its own without the addition of the peptides or peptide fragments of the invention.
  • a suitable solvent for example saline solution
  • the peptide is completely dissolved in a calculated volume of DMSO.
  • the DMSO solution is then suspended in the plasma and the suspension is mixed until a clear solution is obtained.
  • the solution is sterilised by filtration and dispensed into appropriate containers.
  • c represents the protein-ligand interaction energy
  • eFlex represents the loss of flexibility of the ligand
  • CUB and F H B are compensatory functions for the non-ideality of the specific bond and are expressed as a sum over all hydrogen-bond sites
  • AD is a term for the atomic desolvation which describes variations in the solvation field of ligand atoms.
  • NGF 7A between the NGF molecule and the TrkA molecule are to be found in the first 24 amino acids of NGF; in particular, they are to be found in the amino acid residues 2 to 24 of NGF.
  • TrkA receptor a peptide based on the amino acids 2 to 24 of NGF.
  • the corresponding binding domain of the TrkA receptor is represented by residues 250-348, as previously described.
  • amino acid sequence of the starting peptide is given below:
  • the free energy of the starting peptide for binding to TrkA was calculated as -13.02 Kcal/mol. Based on this, the present inventors investigated various mutated forms of the starting peptide showing one, two or three mutations. They then selected those peptides which showed an increase of at least -4 kcal/mol in binding free energy compared to the starting peptide for further investigation. This increase in binding free energy shows an increased ability of the peptide to bind to the TrkA receptor and to displace NGF.
  • the peptides Due to their small size, the peptides lack the ability to dimerise or induce dimerisation in the TrkA receptor. Consequently, these peptides cannot activate the TrkA receptor.
  • the peptides described above can be prepared by conventional solid state synthesis.
  • the process of synthesis takes place according to the well-known cycle of a coupling step, a washing step, a step of removing the protective group and a washing step, after which another coupling step follows or, if the peptide is complete, the peptide is cleaved from the solid support.
  • a polystyrene resin can be used as the support.
  • the functionality of the amino acid can be protected, for example, using a tert-butyloxycarbonyl
  • Boc 9-fluorenylmethyloxycarbonyl
  • the coupling reaction is carried out using coupling agents that are usual in the art, such as, for example, a combination of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or ⁇ , ⁇ '- dicyclohexylcarbodiimide (DCC) with 1-hydroxy-benzotriazole (HOBt) and l-hydroxy-7-aza- benzotriazole (HO At), where appropriate in the presence of a base, in particular a hindered base such as diisopropylethylamine (DIE A) or tetramethylpiperidine (TMP).
  • DIE A diisopropylethylamine
  • TMP tetramethylpiperidine
  • Suitable solvents for the coupling reaction include dimethylformamide (DMF), N- methylpyrrolidone (NMP), trichloromethane (TCM) and dichloromethane (DCM) or mixtures thereof. Furthermore, other solvents such as trifluorethanol (TFE), hexafluoro-2-propanol (FIFIP) or dimethylsulfoxide (DMSO) can be added to suppress peptide aggregation.
  • DMF dimethylformamide
  • NMP N- methylpyrrolidone
  • TCM trichloromethane
  • DCM dichloromethane
  • other solvents such as trifluorethanol (TFE), hexafluoro-2-propanol (FIFIP) or dimethylsulfoxide (DMSO) can be added to suppress peptide aggregation.
  • the washing steps generally use solvents similar to those of the coupling steps.
  • the removal of the protecting groups is achieved by using suitable deprotecting agents.
  • the Boc protecting group is usually removed under acidic conditions using, for example, hydrochloric acid (HCl) or trifluoroacetic acid (TFA), where appropriate in a suitable solvent such as, for example, methanol or ethanol for HCl and, for example, DCM or TCM for TFA.
  • the Fmoc protecting group is usually removed in the presence of a base, such as, for example, pyridine, where appropriate in a suitable solvent.
  • the peptide Once the peptide is complete it can be cleaved from the solid support in a suitable manner and purified as necessary. The whole process can be performed on an automated synthesizer.
  • peptides and peptide fragments of the invention can for example be measured using an enzyme-linked immunosorbent assay (ELISA) for the phosphorylated TrKA receptor.
  • ELISA enzyme-linked immunosorbent assay
  • Such an ELISA kit is commercially available, for example, from Cell Signalling Technologies under the name PathScan® Phospho-TrkA (Tyr674/675) Sandwich ELISA Kit (catalogue number 7212).
  • the assay is used according to the manufacturer's instructions, exposing the PC 12 and/or SH- SY5Y cell lines as cellular types expressing TrkA receptors to the peptides and peptide fragments of the invention.
  • the cells grow in a suitable cell culture medium (for example: 60- mm Falcon ⁇ ) at a certain cell density (e.g. 0.5-lxl0 6 ) in a specific and complete culture medium, as described, for example, in Marchesi et al. (J Cell Physiol. 2014 Nov;229(l l): 1776- 86) for the SH-SY5Y cell line and Rossi et al. (Bioorg Med Chem. 2011 Nov l;19(21):6210-24) for the PC 12 cell line.
  • a suitable cell culture medium for example: 60- mm Falcon ⁇
  • a certain cell density e.g. 0.5-lxl0 6
  • the procedure for the adherent cells is the following:
  • the culture medium when the cell culture is at 80-90% confluence, the culture medium is replaced with a fresh one containing a low percentage of Fetal Bovine Serum (FBS) and for a pre-established period of time.
  • FBS Fetal Bovine Serum
  • the cells are detached from the plate with a scraper and transferred into an appropriate tube stored in ice, where they are then sonicated.
  • the suspension is centrifuged for 10 minutes (at 13- 14000 rpm) at 4°C and the supernatant is transferred into a new tube and stored at -80°C.
  • the percentage of phosphorylated TrkA after exposure to the peptides can be compared with a ... . . 3 ⁇ 4 ⁇ réelle ⁇ , , ⁇ ⁇ calibration curve constructed using NGF as a reierence.
  • the peptides having ainaguinsL auuvity prevent activation by NGF.
  • peptides and peptide fragments of the invention can be measured using a ⁇ -NGF-induced neurite regeneration bioassay.
  • the assay used is based on the method described by Chandler et al. (J. Biol. Chem. (1984) 259, No. 1 1, 6882-6889).
  • PC 12 cells are grown on tissue culture plates (for example 60 mm Falcon ⁇ ) at an initial cell density of 4-5 x 10 5 cells/plate. During the cell culture, ⁇ -NGF is added on 8 consecutive days. The cells are harvested mechanically with a Pasteur pipette, washed once in serum-containing growth medium and twice in serum-free growth medium and collected by centrifugation (500 g for 3 minutes each time). The cells are resuspended in the serum-free growth medium at 6 x 10 cells/ml and 0.5 ml is dispensed into each well of a 24-well culture plate.
  • tissue culture plates for example 60 mm Falcon ⁇
  • ⁇ -NGF is added on 8 consecutive days.
  • the cells are harvested mechanically with a Pasteur pipette, washed once in serum-containing growth medium and twice in serum-free growth medium and collected by centrifugation (500 g for 3 minutes each time). The cells are resuspended in the serum-free growth medium at 6 x 10 cells/
  • the tissue culture plate is pre-treated with 50 ⁇ g/ml of poly-L-lysine (Sigma Aldrich) and washed four times with sterile distilled water.
  • the cells are incubated at 37° C for 30 min and 400 ⁇ of modified medium (Dulbecco's modified Eagle's medium) containing bovine serum albumin (BSA) is added to bring the final serum albumin concentration to 1 mg/ml.
  • modified medium Dulbecco's modified Eagle's medium
  • BSA bovine serum albumin
  • the cells are returned to the incubator for 15 min. before 10 pm of ⁇ -NGF or various concentrations of the peptides of the invention (e.g. 0, 1, 2, 5, 10, 20 pm) are added. At least one set of cells is used as a control without the addition of ⁇ -NGF.
  • the cells are incubated for 24 hours at 37 ° C and read out using a phase contrast microscope.
  • the positive cells are the ones which show a minimum neurite growth > 25 microns.
  • the NGF-antagonistic activity of the peptides of the invention is evaluated based on their ability to reduce the number of NGF-induced positive cells.
  • the ability of the peptides of the invention in the treatment or management of pain can be demonstrated in a mouse or rat model.
  • Arthritis can be induced by the injection of bovine type II collagen into the joints of a test animal. After the arthritis has been induced in the animals, they are treated once or several times with different doses of the peptides and peptide fragments of the invention.
  • the ability of peptides and peptide fragments of the invention in treating pain can be observed through the restoration of mobility in arthritic animals.
  • the peptide is completely dissolved in a calculated volume of DMSO.
  • the DMSO solution is then suspended in plasma and the suspension is mixed until a clear solution is obtained.
  • the solution is sterilized by filtration and dispensed into suitable containers.

Abstract

The present invention relates to novel peptides and peptide fragments and the uses thereof. In particular, the present invention relates to peptides or peptide fragments having TrkA- receptor-agonistic activity useful in the treatment, prevention or control of diseases and disorders such as peripheral neuropathies, multiple sclerosis, Alzheimer's disease, Parkinson's disease, hypoxic cerebral lesions, ischaemic heart disease, heart failure and diabetes and the comorbidities thereof, in particular diabetic neuropathies, optic nerve atrophy, ulcers, keratitis and/or glaucoma. The invention further discloses peptides and peptide fragments having NGF-antagonistic activity useful in the treatment, prevention or control of diseases and disorders such as pain, hyperinnervation, cardiac arrhythmias and sudden cardiac death.

Description

PEPTIDES HAVING TrkA-RECEPTOR-AGONISTIC ACTIVITY AND/OR PEPTIDES HAVING NGF- ANTAGONISTIC ACTIVITY
DESCRIPTION
The invention relates to peptides and peptide fragments and the use thereof.
In particular, the present invention relates to the identification of a specific amino acid sequence (residues 250-348) within the amino acid structure of the human TrkA receptor which is suitable as a new target and ideal model for the creation of interacting peptides that show a strong bond with the receptor.
The invention further relates to peptides and peptide fragments which bind to the aforesaid sequence and have a TrkA-agonistic activity and the use thereof for the treatment of conditions, diseases and disorders deriving from a reduced activation of the TrkA receptor, in particular for the treatment of peripheral neuropathies, multiple sclerosis, Alzheimer's disease, Parkinson's disease, hypoxic cerebral lesions and/or optic nerve atrophy.
In particular, the present invention also relates to peptides and peptide fragments which bind to the aforesaid sequences and have NGF -antagonistic activity and the use thereof for the treatment of conditions, diseases and disorders linked to high levels of NGF, in particular for the treatment of pain and cardiac arrhythmia.
The invention further relates to the use of the peptides and peptide fragments of the invention for the treatment of diseases characterised or aggravated by the loss of innervations, such as, for example, ischaemic heart disease, heart failure and diabetes and the comorbidities thereof, in particular diabetic neuropathies.
The invention further relates to the use of the peptides and peptide fragments of the invention for the treatment of diseases, conditions and disorders that show a benefit from treatment with NGF, such as, for example, ulcers, neurotrophic keratitis and glaucoma. TECHNICAL BACKGROUND
Nerve growth factor (NGF) is a protein that regulates the growth and survival of sympathetic and sensory nerve cells (neurons). NGF thus belongs to a family of growth factors called neurotrophins.
NGF binds with great affinity to and activates the tyrosine receptor kinase TrkA.
Upon activation, the TrkA receptor dimerises and autophosphorylates, thus giving rise to a signalling process that is important for the growth and survival of sympathetic neurons.
The ability of NGF to activate the TrkA receptor and thereby stimulate the survival and growth of neurons has made it of great interest in the field of research aimed at discovering new treatment options for diseases and disorders of the central and peripheral nervous system.
In particular, the bond of NGF to its receptor has been investigated and some of the domains involved in the interaction between this neurotrophic factor and its receptor have been identified.
However, it is worth noting the possibility of identifying new domains of interaction that may be used for the purpose of creating interacting drugs.
The discovery of NGF, its receptor and the signalling mechanisms correlated to it has fostered numerous studies on the role of NGF in physiopathology and on the use ot NGF in the treatment of various diseases and disorders of the central and peripheral nervous system.
These diseases and disorders include: diabetic polyneuropathy, HIV-associated peripheral neuropathy, Alzheimer's disease, Parkinson's disease, optic glioma, advanced optic nerve atrophy and perinatal hypoxic-ischemic brain damage (Aloe et οί, J. Transl. Med. (2012) 10:
239).
Furthermore, other studies indicate that NGF might be suitable for the treatment of other diseases and disorders such as ulcers, neurotrophic keratitis and glaucoma (v.s.).
A large multicentre trial under the name of ADMIRE-HF has recently provided clinical evidence documenting the inverse relationship between cardiac innervation and survival in heart failure. On the other hand, experimental data show that the administration of NGF induces nerve sprouting in the upper thoracic sympathetic ganglia, which is evident also at the cardiac level with an increase of the density of the sympathetic nerve endings.
These data indicate that it might be possible to use NGF to restore innervation when the pathological process compromises its integrity with negative prognostic consequences. Diseases that benefit from such treatment include heart failure and diabetes.
In light of the above, novel compounds having TrkA-receptor-agonistic activity would help to provide new therapeutic options.
Although in many cases the growth of neurons is desirable, their uncontrolled growth can be associated with medical problems.
For example, the hyperinnervation of heart tissue has been demonstrated to be linked to cardiac arrhythmia and sudden cardiac death (see, for example, Chen et al Cardiovasc Res (2001) 50, 409-416).
In recent years NGF has also been identified as an important mediator of pain, in particular chronic pain, and has accordingly become a target in the search for new pain control strategies. One approach in this direction relies on NGF sequestration using anti-NGF antibodies such as, for example, Tanezumab (Pfizer), Fulranumab (Johnson and Johnson), REGN475/SAR164877 (Regeneron together with Sanofi Aventis) or medi578 (AstraZeneca). For an analysis of pain control through the intervention in the NGF-mediated regulation of pain, see for example McKelvey et ai, J. Neu ochm. (2013) 124, 276-289.
Clinical trials performed on patients who suffer from osteoarthritic hip pain (Brown et al, Arthritis & Rheumatism (2013) 65, Vol. 7, 1795-1803) and pain caused by osteoarthritis of the knee (Lane et al, N. Engl. J. Med. (2010), 363, 1521-1531) have demonstrated that Tanezumab is effective in reducing pain in both groups of patients compared to the placebo group.
Trials for the treatment of osteoarthritic knee pain using Tanezumab were suspended by the FDA (US Food and Drug Administration) in June 2010 due to a certain number of patients involved in the trial needing joint replacements because of progressively worsening knee and hip osteoarthritis (Schnitzer et al, Osteoarthritis and Cartilage (201 1) 19, 639-646).
Despite the authorisation of the FDA to resume the clinical trials with Tanezumab in March 2012, it is clear that further compounds with NGF-antagonistic activity and the possibility of different modes of action would be desirable in order to add additional options for controlling pain, in particular chronic pain, as well as for the treatment and prevention of cardiac arrhythmias.
The invention relates to the use of the amino acid sequence (residues 250-348) of TrkA as the reference domain for the construction of peptides and peptide fragments which have the ability to bind to it.
Therefore, a series of structures have been generated, which include, in a non-limiting form, the following:
• GLY-GLN-ASN-TRP-GLN-PRO-SER-ALA-TYR-GLN-ALA-TRP-CYS-THR-THR,
• CYS-LYS-GLY-TYR-GLY-GLY-LEU-VAL-ALA-GLN-CYS-THR-ASP-GLY-PHE,
• ASN-VAL-GLU-PRO-LEU-THR-CYS-VAL-TYR-GLN-PRO-LYS-CYS-GLY-1RP,
• LEU-PRO-SER-PRO-CYS-TRP-GLN-GLN-PHE-ARG-CYS-ASP-GLN-LEU-GLN,
• CYS-GLY-THR-ASP-CYS-PHE-MET-ASN-GLY-ARG-PRO-GLY-GLY-TYR-TYR,
• TYR-GLU-LYS-MET-CYS-ASP-THR-GLY-VAL-LEU-ILE-GLN-GLY-LYS-CYS,
• TRP-GLY-THR-ASN-ILE-GLY-CYS-LEU-SER-LYS-CYS-GLU-GLY-CYS-SER,
• ALA-ILE-SER-LYS-GLN-THR-PHE-ASP-THR-LYS-ASP-VAL-TRP-MET-SER,
• THR-VAL-ASP-GLU-PHE-HIS-CYS-MET-LYS-GLY-CYS-CYS-GLU-LYS-ASN,
• THR-GLU-ASN-TRP-GLY-VAL-ARG-CYS-ALA-ASN-TRP-THR-VAL-PHE-VAL,
• PRO-CYS-PHE-ASP-ALA-GLY-GLN-SER-PHE-TRP-THR-PHE-LYS-VAL-CYS,
• ILE-TRP-SER-GLN-CYS-ASP-GLY-ASN-CYS-TRP-PRO-ARG-ALA-ASN-VAL, • MET-GLN-TYR-HIS-CYS-ARG-ASN-THR-GLU-TYR-ASP-GLY-VAL-ASN-TRP,
• CYS-CYS-GLN-TYR-PRO-GLY-GLY-ILE-ASN-ASN-LYS-VAL-VAL-ASP-SER,
• ILE-LEU-PHE-PHE-GLY-TYR-THR-PRO-ASN-GLN-ALA-SER-TRP-CYS-CYS,
• VAL-TYR-CYS-ALA-VAL-ASN-ASN-GLU-PHE-HIS-GLY-ASP-ASN-LYS-TRP,
• GLY-ASN-CYS-VAL-VAL-PRO-HIS-CYS-LYS-ASP-ASP-GLY-ASN-TYR-PHE,
• ALA-CYS-CYS-LYS-PHE-GLY-TYR-MET-MET-ALA-LYS-GLU-CYS-ASP-GLY,
• GLN-LEU-ARG-GLN-CYS-TYR-GLY-GLU-SER-ASN-PHE-THR-ALA-TRP-SER,
• PHE-CYS-TRP-PRO-ASN-SER-PRO-TRP-CYS-THR-PRO-ASN-SER-ALA-SER,
• PRO-LEU-CYS-CYS-PRO-HIS-ILE-CYS-LEU-VAL-ASP-SER-GLN-GLY-ASN,
• VAL-PRO-HIS-ILE-GLN-THR-VAL-ASP-CYS-CYS-SER-ASP-HIS-ASN-PRO,
• ILE-ALA-CYS-ALA-ALA-ASN-ASN-MET-THR-HIS-ASP-GLN-TRP-SER-SER,
• CYS-PHE-GLY-THR-ASN-ALA-GLY-PRO-TRP-THR-CYS-PRO-CYS-ASN-VAL,
• PHE-ASP-GLY-ASN-TYR-GLN-SER-GLY-PRO-SER-CYS-LEU-LYS-CYS-MET,
• SER-THR-THR-PRO-GLY-CYS-LEU-MET-TYR-LEU-MET-SER-TYR-THR-GLN,
• PHE-GLY-TYR-CYS-SER-LEU-GLU-ALA-THR-VAL-TYR-ARG-ASP-LYS-ILE,
• MET-ASN-ASN-CYS-ASN-ALA-MET-TRP-ASP-ARG-SER-ASN-SER-PHE-CYS,
• GLY-TRP-GLU-ASP-LYS-HIS-CYS-MET-GLN-CYS-GLN-GLY-GLN-TRP-CYS,
• GLY-ASP-VAL-LEU-ASN-CYS-THR-GLN-HIS-VAL-THR-CYS-ASN-TYR-ALA,
• CYS-MET-LYSLYS-ASN-CYS-LEU-TYR-ASN-MET-PHE-ASP-ASP-ASN-MET,
• CYS-GLU-THR-HIS-MET-ASN-GLU-ILE-HIS-ASN-TRP-MET-GLY-THR-ASN,
• PHE-GLN-GLY-ILE-VAL-GLU-HIS-GLU-THR-CYS-LYS-ASP-THR-LYS-ALA,
• ILE-GLY-HIS-LEU-SER-GLU-ASN-GLN-THR-VAL-THR-LEU-LEU-GLN-GLN,
• THR-GLN-LYS-ASP-SER-ILE-ARG-PHE-PHE-SER-ALA-CYS-PRO-GLU-VAL,
• THR-VAL-ALA-TYR-CYS-MET-LYS-ASP-ILE-ALA-ALA-ASN-GLN-ASN-GLY,
• GLY-LYS-ASN-CYS-PHE-GLU-CYS-LEU-GLN-SER-TYR-ASN-SER-SER-ALA, • GLY-ASN-LEU-GLY-ARG-GLY-SER-GLY-CYS-CYS-ASP-ARG-CYS-GLU-ILE,
• GLN-ILE-LYS-PHE-SER-GLU-PHE-TYR-ARG-ASP-CYS-GLN-TRP-THR-PHE,
• TYR-TRP-VAL-HIS-GLN-PHE-GLY-TRP-ASN-THR-ASN-PRO-ASN-ASPC-YS,
• LEU-LYS-VAL-ILE-LYS-CYS-GLU-GLU-THR-GLY-SER-ALA-CYS-THR-ALA,
• ALA-ALA-PRO-HIS-ASP-THR-GLU-SER-CYS-PHE-CYS-VAL-ALA-ARG-GLN,
• LEU-ASN-HIS-CYS-SER-ASP-GLY-CYS-LEU-ASP-GLN-CYS-LYS-ASN-ALA,
• SER-ILE-ILE-ALA-TYR-ASN-HIS-SER-VAL-TRP-LEU-ASP-ALA-CYS-CYS,
• LEU-GLY-TYR-LYS-CYS-GLY-CYS-PRO-ASN-GLU-GLY-CYS-ASN-PHE-GLN,
• TRP-THR-LYS-ASP-PRO-TYR-ARG-TYR-PHE-GLN-SER-ASN-ASP-SER-MET,
• GLN-TRP-PRO-CYS-CYS-ASP-GLU-GLY-ALA-ASN-ALA-LYS-LYS-TYR-ASN,
• PHE-GLN-ASP-ILE-SER-PHE-LYS-ALA-GLU-GLY-GLN-CYS-MET-HIS-VAL,
• GLY-GLN-TYR-HIS-ASP-CYS-GLU-LEU-ARG-VAL-PHE-THR-HIS-GLU-CYS,
• THR-GLN-SER-ASP-ASN-ASN-ASP-LYS-ARG-ILE-VAL-GLY-PRO-GLN-ASN,
• TYR-HIS-ASP-GLN-ARG-PHE-SER-TYR-ALA-PHE-LEU-SER-GLU-GLY-ASN, » PRO-GLU-ARG-PHE-THR-ASP-ARG-ILE-ARG-GLU-ASN-SER-LEU-GLY-LEU,
• TRP-GLY -TYR-PRO-L YS-LYS-TRP-GLU-CYS-C YS-SER-GL Y- ASP-GLN- ALA,
• GLN-ARG-THR-ASP-CYS-SER-MET-GLY-LEU-GLN-LYS-GLU-ALA-SER-TYR,
• ALA-THR-SER-ARG-ASP-GLY-ALA-SER-LEU-PRO-ASN-ASP-CYS-HIS-THR,
• PRO-ASP-GLY-GLY-ASN-HIS-MET-ASN-CYS-ILE-TYR-GLY-LEU-CYS-THR,
• ASN-VAL-MET-GLN-THR-PRO-ILE-MET-CYS-TYR-ASP-ALA-HIS-ALA-CYS,
• MET-ASN-ALA-SER-PRO-HIS-TRP-ILE-ASP-SER-MET-SER-ASP-LYS-CYS,
• PRO-TRP-TRP-CYS-GLN-LEU-THR-SER-THR-ASN-GLN-ASP-LYS-CYS-ASN,
• SER-TYR-GLY-PRO-SER-ASN-LYS-LEU-ARG-ASN-ASP-ILE-TYR-ASP-TYR,
• ALA-THR-SER-LEU-PHE-LYS-TRP-TYR-CYS-GLN-MET-SER-ASN-ASP-ASN,
• THR-THR-ILE-PHE-VAL-LYS-GLN-SER-ASN-GLU-GLY-ALA-ILE-ASN-ALA, • GLY-LEU-THR-LYS-ILE-TRP-CYS-ASN-CYS-ASP-SER-TYR-CYS-ASN-SER,
• GLN-ILE-GLY-SER-PRO-ILE-SER-GLU-ARG-ASP-ARG-CYS-ASN-ALA-ALA,
• CYS-THR-GLU-PRO-MET-LEU-PHE-GLY-PHE-SER-THR-ARG-PHE-THR-MET,
• ALA-CYS-PRO-MET-SER-GLU-GLU-CYS-PHE-PRO-LYS-VAL-LYS-GLY-ILE,
• MET-ASN-ASN-PHE-CYS-THR-ASP-PRO-ARG-GLY-TRP-MET-SER-THR-THR,
• PHE-MET-THR-CYS-TYR-GLN-GLY-ARG-ALA-THR-GLU-CYS-TRP-CYS-VAL,
• PHE-VAL-MET-ALA-ASN-PHE-GLU-GLN-LYS-ALA-MET-ASP-MET-HIS-ASN,
• PRO-LEU-CYS-ASP-PHE-GLY-VAL-ASN-SER-LYS-ASP-LYS-ALA-TYR-ALA,
• PHE-LYS-GLU-LEU-TYR-ALA-MET-GLU-VAL-HIS-CYS-CYS-GLY-GLY-TRP,
• GLN-TYR-SER-ASN-CYS-LYS-TYR-CYS-ALA-THR-TYR-GLU-ILE-ASN-GLN,
• PHE-GLU-ARG-ASN-MET-GLN-ILE-LYS-VAL-GLU-THR-PHE-LEU-SER-ASN,
• GLY-MET-CYS-PRO-CYS-VAL-PHE-LEU-GLY-PRO-ASP-TRP-HIS-ASN-GLY,
• CYS-PHE-LYS-SER-CYS-GLU-GLY-VAL-VAL-TYR-GLY-ILE-SER-SER-ILE,
• CYS-SER-LYS-ASN-PHE-PHE-MET-LEU-CYS-TYR-GLU-LYS-ILE-GLU-GLY,
• ALA-THR-ASN-ASN-GLN-CYS-TRP-ARG-THR-GLY-VAL-LEU-GLU-SER-PHE,
• CYS-ASH-CYS-CYS-LEU-MET-SER-LYS-PRO-MET-SER-TYR-GLU-THR-ASN,
• ASN-GLU-CYS-THR-LYS-GLN-PRO-ALA-THR-HIS-GLU-GLN-GLY-TYR-CYS,
• SER-CYS-VAL-PRO-GLU-LEU-ASP-LYS-HIS-ASN-SER-ILE-MET-THR-SER,
• CYS-TRP-PHE-ASP-ASN-MET-SER-PHE-VAL-GLN-THR-LEU-SER-LYS-GLN,
• MET-GLU-SER-HIS-CYS-ALA-GLY-PHE-ILE-LYS-GLU-ALA-ALA-GLN-LEU,
• TYR-TRP-PHE-GLY-CYS-GLN-ARG-GLU-GLN-GLY-TRP-GLY-TRP-TYR-CYS,
• PHE-ASN-CYS-TYR-GLY-LYS-CYS-THR-SER-ASP-PHE-TRP-MET-PHE-THR,
• PHE-LYS-TRP-LEU-ASN-ILE-CYS-THR-ASP-CYS-GLY-GLY-ALA-PHE-SER,
• GLN-ASN-LEU-GLU-ILE-SER-VAL-ALA-TYR-ARG-THR-GLY-TRP-PHE-CYS,
• GLN-TRP-GLN-CYS-THR-VAL-PHE-ALA-TYR-THR-GLN-VAL-SER-SER-GLY, • SER-PHE-THR-TRP-GLY-VAL-LYS-ILE-SER-GLU-GLN-TRP-ALA-CYS-LEU,
• VAL-CYS-LEU-GLN-GLY-ASN-SER-LEU-CYS-ILE-LYS-ALA-SER-GLU-LEU,
• THR-GLU-ARG-SER-THR-ALA-ALA-ASP-LEU-SER-VAL-LYS-TRP-ALA-TYR,
• ALA-THR-ALA-LYS-SER-TRP-ASN-PRO-TRP-ASP-LEU-TRP-MET-SER-GLN,
• CYS-GLU-SER-ALA-LYS-PRO-HIS-ASP-CYS-THR-LEU-SER-MET-LEU-SER,
• ASN-PHE-THR-TRP-PRO-LYS-TYR-ILE-CYS-ILE-THR-GLY-ASP-THR-ALA,
• PHE-VAL-ILE-GLY-ASP-LYS-SER-SER-GLN-CYS-PRO-ASP-PHE-HIS-CYS,
• CYS-GLY-LYS-LYS-VAL-TYR-CYS-THR-LEU-GLU-TYR-PHE-GLN-GLU-GLY,
• TRP-MET-MET-ALA-CYS-GLN-TYR-HIS-CYS-ASP-GLU-HIS-PRO-PRO-TRP,
• CYS-SER-GLY-VAL-PRO-GLU-HIS-LYS-ASP-ASN-ASP-SER-MET-LYS-SER,
• GLN-ARG-MET-CYS-CYS-THR-ASN-GLY-TYR-PRO-THR-SER-ASP-CYS-THR, and
• GLY-ASN-TYR-ARG-GLU-CYS-VAL-MET-GLN-CYS-CYS-LEU-SER-ILE-TRP, and salts thereof, solvates and solvates of the salts thereof.
The peptides and peptide fragments of the invention are peptides and peptide fragments having an amino acid sequence listed above and salts thereof, solvates and solvates of the salts thereof. The salts preferred for the purpose of the invention are physiologically acceptable salts of the peptides and peptide fragments of the invention. Also included are salts which are not themselves suitable for pharmaceutical applications, but can be used, for example, to isolate and purify the peptides and peptide fragments of the invention.
The solvates for the purpose of the invention refer to the forms of peptides and peptide fragments which in a liquid or solid state form a complex by complexation with one or more molecules of solvent. Hydrates are a specific form of solvates in which complexation takes place with water. In the context of the present invention, the term peptide fragment refers to peptides in which the amino acid sequences of the invention are incorporated into a larger peptide molecule. This can occur by means of a chain extension at the C-terminal domain, the N-terminal domain or both domains. Preferably, the larger peptide molecule is not longer than a total of 50 amino acids, including the amino acid sequences of the invention. Even more preferably, the larger peptide molecule is not longer than 35 amino acids, including the amino acid sequences of the invention. In some embodiments it may be preferable that the amino acid sequences of the invention extend by no more than 5 amino acids at the two terminal domains. In some embodiments it may be preferable for the peptide or the peptide fragment of the invention to have an amino acid sequence which does not comprise further amino acids added at both terminal domains. In any case, the amino acid sequences of the invention will always be present in the sequences listed above and not interrupted by other amino acids.
In the context of the invention, the reference to an amino acid comprises proteinogenic amino acids, but also non-proteinogenic amino acids, such as D-amino acids or amino acids in which the amino group and the carbonyl group are not bonded to the same carbon atom, but are rather separated by at least one additional carbon (e.g. β- or γ-amino acids). If an amino acid is mentioned only by name, this includes both L- and D-amino acids and also β-, γ-amino acids and higher order amino acids. In some embodiments oc-amino acids may be preferred. In some embodiments L-amino acids may be preferred. In some embodiments oc-L-amino acids may be preferred. In some embodiments the peptides or peptide fragments according to the invention can comprise exclusively L-amino acids or exclusively D-amino acids. In some embodiments the peptides and peptide fragments of the invention consist of a mixture of L- and D-amino acids. In some embodiments the peptides and peptide fragments of the invention may contain at least one L-amino acid. In some embodiments the peptides and peptide fragments of the invention may contain at least one D-amino acid. In some embodiments of the invention the amino acid sequences can be phosphorylated.
In the context of the invention, the names and abbreviations used have the usual meaning known by the person skilled in the art.
Preferably, the peptides of the present invention have an amino acid sequence selected from the group consisting in:
• GLY-GLN-ASN-TRP-GLN-PRO-SER-ALA-TYR-GLN-ALA-TRP-CYS-THR- THR,
• CYS-LYS-GLY-TYR-GLY-GLY-LEU-VAL-ALA-GLN-CYS-THR-ASP-GLY- PHE,
• ASN-VAL-GLU-PRO-LEU-THR-CYS-VAL-TYR-GLN-PRO-LYS-CYS-GLY- TRP,
• LEU-PRO-SER-PRO-CYS-TRP-GLN-GLN-PHE-ARG-CYS-ASP-GLN-LEU- GLN,
• CYS-GLY-THR-ASP-CYS-PHE-MET-ASN-GLY-ARG-PRO-GLY-GLY-TYR- TYR,
and salts thereof, solvates and solvates of the salts thereof.
The peptides and peptide fragments according to the present invention are suitable for use as a medicament for the treatment of diseases and disorders in humans and in animals.
In one aspect of the invention, the peptides and peptide fragments are in particular distinguished by their ability to bind to and activate the TrkA receptor.
The peptides and peptide fragments according to the present invention are thus useful as agonists of the amino acid sequences of the TrkA receptor selected from the group consisting in
PHE-PRO-ALA-SER-VAL-GLN-LEU-HIS-THR-ALA-VAL-GLU-MET-HIS-HIS-
TRP-CYS-ILE-PRO-PHE-SER-VAL-ASP-GLY-GLN-PRO-ALA-PRO-SER-LEU-
ARG-TRP-LEU-PHE-ASN-GLY-SER-VAL-LEU-ASN-GLU-THR-SER-PHE-ILE-
PHE-THR-GLU-PHE-LEU-GLU-PRO-ALA-ALA-ASN-GLU-THR-VAL-ARG-HIS-
GLY-CYS-LEU-ARG-LEU-ASN-GLN-PRO-THR-HIS-VAL-ASN-ASN-GLY-ASN- TYR-THR-LEU-LEU-ALA-ALA-ASN-PRO-PHE-GLY-GLN-ALA-SER-ALA-SER-
ILE-MET-ALA-ALA-PHE-MET-ASP-ASN-PRO.
They can therefore be used in the treatment of diseases and disorders which benefit from an increased activation of the TrkA receptor.
In particular, the peptides and peptide fragments of the invention are advantageous for the treatment of peripheral neuropathies, multiple sclerosis, Alzheimer's disease, Parkinson's disease, hypoxic brain lesions and/or optic nerve atrophy.
The peptides and peptide fragments of the invention are also useful in the treatment of diseases and disorders characterised or aggravated by a loss of innervation, such as ischaemic heart disease, heart failure and diabetes and the comorbidities thereof, in particular diabetic neuropathies. The peptides and peptide fragments of the invention are also useful in the treatment of other diseases and disorders that have shown to derive benefits from a treatment with NGF, such as ulcers, neurotrophic keratitis and/or glaucoma.
In another aspect of the present invention, the peptides and peptide fragments of the invention are distinguished in particular by their ability to bind to the TrkA receptor more strongly than NGF without activating that receptor.
They are therefore useful as NGF antagonists in the treatment of diseases and disorders associated with an increase in the level of NGF.
In particular, the peptides or peptide fragments can be used as NGF antagonists in the bond with the amino acid sequences of the TrkA receptor selected from the group consisting in
PHE-PRO-ALA-SER-VAL-GLN-LEU-HIS-THR-ALA-VAL-GLU-MET-HIS-HIS-
TRP-CYS-ILE-PRO-PHE-SER-VAL-ASP-GLY-GLN-PRO-ALA-PRO-SER-LEU-
ARG-TRP-LEU-PHE-ASN-GLY-SER-VAL-LEU-ASN-GLU-THR-SER-PHE-ILE-
PHE-THR-GLU-PHE-LEU-GLU-PRO-ALA-ALA-ASN-GLU-THR-VAL-ARG-HIS-
GLY-CYS-LEU-ARG-LEU-ASN-GLN-PRO-THR-HIS-VAL-ASN-ASN-GLY-ASN- TYR-THR-LEU-LEU-ALA-ALA-ASN-PRO-PHE-GLY-GLN-ALA-SER-ALA-SER-
ILE-MET-ALA-ALA-PHE-MET-ASP-ASN-PRO.
Preferably, the peptides and peptide fragments are useful in the treatment and management of pain and in the treatment and prophylaxis of hyperinnervation and conditions associated therewith, such as cardiac arrhythmia and sudden cardiac death.
The peptides and peptide fragments according to the present invention are useful in the treatment, prophylaxis and/or management of diseases and disorders, in particular diseases and disorders tied to NGF, more in particular to diseases and disorders tied to an increased level of NGF.
The present invention further relates to the peptides and peptide fragments of the invention for use in the treatment and/or management of pain.
The present invention further relates to the peptides and peptide fragments of the invention for use in the treatment and/or prophylaxis of hyperinnervation.
The present invention further relates to the peptides and peptide fragments of the invention for use in the treatment and/or prophylaxis of cardiac an-hythmias and/or of sudden cardiac death. The features and advantages of the invention will become more apparent from the detailed description that follows of the tests and results that have led to the definition thereof, provided together with the reference drawings, in which:
- Figure 1 shows the peptide having the amino acid sequence GLY-GLN-ASN-TRP-GLN- PRO-SER-ALA-TYR-GLN-ALA-TRP-CYS-THR-THR, which binds to the TrkA receptor by overlapping the NGF binding site
- Figure 2 shows the peptide having the amino acid sequence CYS-LYS-GLY-TYR-GLY- GLY-LEU-VAL-ALA-GLN-CYS-THR-ASP-GLY-PHE, which binds to the TrkA receptor by overlapping the NGF binding site
- Figure 3 shows the peptide having the amino acid sequence ASN-VAL-GLU-PRO-LEU- THR-CYS-VAL-TYR-GLN-PRO-LYS-CYS-GLY-TRP, which binds to the TrkA receptor by overlapping the NGF binding site
- Figure 4 shows the peptide having the amino acid sequence LEU-PRO-SER-PRO-CYS- TRP-GLN-GLN-PHE-ARG-CYS-ASP-GLN-LEU-GLN, which binds to the TrkA receptor by overlapping the NGF binding site
Figure 5 shows the peptide having the amino acid sequence CYS-GLY-THR-ASP-CYS- PHE-MET-ASN-GLY-ARG-PRO-GLY-GLY-TYR-TYR, which binds to the TrkA receptor by overlapping the NGF binding site
Figure 6 shows the simulation box used for molecular docking
Figure 7 shows the structure of 58 amino acids of the TrkA receptor involved in the bond with NGF
INTERACTIONS BETWEEN TrkA and NGF
Figure 7 shows the structure of 58 amino acids of the TrkA receptor involved in the bond with NGF.
The residues and type of interaction that is established between TrkA and NGF are shown in detail in Table 1.
Table 1 : Residues and type of interaction between TrkA and NGF.
Figure imgf000014_0001
Table 2 shows which TrkA residues involved in the bond with NGF are intercepted by the first five peptides proposed in our study:
Table 2: TrkA residues involved in the bond with NGF residues
GLU295 O O O 0
SER304
HIS297 O O 0
PHE327 O
GLU334 O
ARG347 O O
ASN349 O O
GLN350 O O O 0
HIS353
As can be noted from table 2, the third peptide (POSA3) is the one that most covers the points of contact of the TrkA receptor with NGF.
OPTIMISATION OF MOLECULES AND BIOMOLECULES THAT ARE NGF ANTAGONISTS INNGF-TRKA INTERACTION USING MOLECULAR MODELLING TECHNIQUES The peptides and peptide fragments according to the present invention are characterised by an original sequence - i.e. one that is not necessarily derived from the original sequence of NGF - and they demonstrate to be capable of competing with NGF in the interaction with the TrkA receptor. This objective was pursued by carrying out simulations of molecular docking against TrkA using, as possible ligands, the peptides of a library of 1 ,000 species, generated with a random sequence using Solypep software. CONSTRUCTION OF THE PEPTIDE LIBRARY
The library of 1,000 peptides was constructed using the Solypep tool; this software makes it possible to generate peptide sequences with a random sequence up to a maximum of 15 residues and to generate a corresponding pdb file containing the peptide in linear form. Solypep enables exclusively the use of L-amino acids. For this reason, every sequence was subsequently processed with Maestro in order to invert all the Ca stereocentres so as to generate the D-amino acids. Solypep further makes it possible to control the solubility of the sequences generated by it, so that, among all those generated, the 1,000 most soluble forms were selected. Summing up, each peptide created exhibits the following characteristics: • length 15 residues
• high solubility
• made up of D-amino acids
SIMULATION OF MOLECULAR DOCKING
For the docking simulations, use was made of the Glide program, which is part of the Maestro suite. As regards the identification of the section of TrkA assumed as the receptor, the procedure was as follows. The entire structure of TrkA, taken from the PDB site (structure with the code 2IFG), was treated with PRIME in order to define the bond orders and disulphide bridges, protonate polar and non-polar residues at pH 7.0 and, finally, reconstruct any missing loops or unresolved residues. The simulation box (cubic) used for docking has a size of 36 A, sufficiently large to contain the whole portion of TrkA involved in the binding with NGF. The simulation box is illustrated in Figure 6.
During the docking simulations, the entire receptor is maintained rigid with the exception of the OH groups of the THR (292,325,330,352,360), TYR(359) and SER(304,320,326) residues. This choice was dictated by the dimensions of the receptor and of the ligands and aimed to reduce the required computing time and power within acceptable limits. As seen above, the hydroxyl groups of TrkA adjacent to the binding site for NGF are treated as exempt from constraints in order to maximise the possibility of the receptor establishing H-bonds with the ligand, where present, and partly decrease the rigidity of the system.
As regards the 1,000 peptides of the library, they were not subjected to any type of constraint. For each peptide, Glide explored all the possible conformations and selected those that best interact with TrkA, if present. The force field used for the simulations was OPLS2005 and the degree of accuracy imposed for the docking was SP-peptide (method implemented in Glide for docking with small peptides). All the poses found were ordered from the best to the worst according to the docking score. The score function used is an empirical function representative of the binding free energy expressed in kcal/mol. Table 3 shows the first 100 peptides capable of interacting with TrkAs ordered according to their docking scores value.
Table 3 - Sequences of peptides of the invention identified with a one-letter code
SEQUENCE DOCKING SCORE
G& fWQP A YQiWCTT -1 1 ,070
CKGYGGL VAQCTDGF -10,959
NVEPL TCVYQPKCGW -10,830
LPSPCWQQFRCDOLQ -10,628
CGTDCFMNGRPGGYY -10,510
YE KM CD TG VLIQGKC -10,410
WGTNIGCLSKCEGCS -10,374
AISKQTFDTKD VWMS -10,372
TVDEFHCMKGCCEKN -10,349
TENWG VRCANWTVFV -10,328
PCFDA GQSFWTFKVC -10,319
IWSQCDGNCWPRANV -10,254
MQYHCRNTEYDGVNW -10,202
CCQ YPGGINNKVVDS -10,180
ILFFGYTPNQASWCC -10,143
VYCA VNNEFHGDNKW -10,1 14
GNCVVPHCKDDGNYF -10,099
A CCKFG YMMAKECDG -10,038
QLRQCYGESNFTA WS -9,853
FCWPNSPWCTPNSAS -9,735
PLCCPHICL VDSQGN -9,732
VPHIQTVDCCSDHNP -9,732
lACAANNMTHDQWSS -9,730
CFGTNAGPWTCPCNV -9,705
FDGNYQSGPSCLKCM -9,628
S TTPGCLMYLMS YTQ -9,614
FGYCSLEA TVYRDKI -9,614
MNNCNAMWDRSNSFC -9,598
GWEDKHCMQCQGQWC -9,590
GO VLNCTQHVTCNYA -9,559
CMKKNCL YNMFDDNM -9,536
CETHMNEIHNWMGTN -9,522
FQGIVEHETCKD TKA -9,518
IGHLSENQTVTLLQQ -9,513
TQKDSIRFFSA CPEV -9,506
TVA YCMKDIAANQNG -9,504
GKNCFECL QS YNSSA -9,485
GNLGRGSGCCDRCEI -9,457
QIKFSEFYRDCQ WTF -9,446
YWVHOFGWNTNPNDC -9,443
LKVIKCEETGSACTA -9,425
AAPHDTESCFCVARQ -9,402
LNHCSD GCLDQCKNA -9,390
SUA YNHSVWLDACC -9,363
LGYKCGCPNEGCNFQ -9,352 QWPCCDEGANAKKYN
FQDISFKAEGQCMHV
GQYHDCELR VFTIIEC
TQSDNNDKRIVGPQN
YHDQRFSYA FLSEGN
PERFTDRIRENSL GL
WG YPKKWECCSGDQA
QR TDCSMGLQKEASY
A TSRDGASLPNDCHT
PD GGNIIMNCI YGL CT
NVMQTPIMCYDAHAC
MNASPH WIDSMSDKC
PWWCQL TSTNQDKCN
S YGPSNKLRNDI YD Y
A TSLFK WYCQMSNDN
TTIFVKOSNEGAINA
GL TKIWCNCDSYCNS
QIGSPISERDR CNAA
CTEPMLFGFSTRFTM
A CPMSEECFPKVKGI
MNNFCTDPRGWMSTT
FMTCYQGRA TECWCV
FVMANFEQKAMDMHN
PLCDFG VNSKDKA YA
FKEL YA ME VII CCGG W
Q YSNCKYCA TYEINQ
FERNMQIK VETFLSN
GMCPCVFL GPD WHNG
CFKSCEGVVYGISSI
CSKNFFMLCYEKIEG
A TNNQCWR TG VLESF
CNCCLMSKPMSYETN
NECTKQPA THEQGYC
SCVPELDKHNSIMTS
CWFDNMSFVQ TLSKQ
MESHCA GF1KEAAQL
YWFGCQREQGWGWYC
FNCYGKCTSDFWMFT
FKWLNICTDCGGAFS
QNLEISVA YRTGWFC
QWQCTVFA YTQVSSG
SFTWGVKISEQWACL
VCL QGNSL CIKASEL
TERSTAADLSVKWA Y
A TAKSWNPWDL WMSQ
CESAKPHDCTLSMLS
NFTWPKYICITGDTA
FVIGDKSSQCPDFHC
CGKKVYCTLEYFQEG
WMMACQYHCDEHPPW
CSGVPEIIKDNDSMKS
Figure imgf000018_0001
OiWO 2018/036785 rSDCr -8,606
GNYRECVMQCCLSIW -8,588
ICYR VCNEP VDA RMA -5.118
Figures 1, 2, 3, 4 and 5 represent the TrkA receptor 1 , the NGF dimer 2 and the ligands 3. As may be seen from the figures, all of the peptides bind TrkA by overlapping the NGF binding site, suggesting that they are good inhibitors. Also shown are the number and type of interactions that each ligand develops with the receptor; it may be noted that the first peptide with the highest docking score also has a higher number of interactions with TrkA and consequently a stronger solid interaction with the receptor.
In Table 4, other information in addition to that regarding the binding energy has been added - for the first ten peptides - which improves the description of the peptide-TrkA interaction. In particular, reference is made to the data in the last column which represents the degree of similarity (coverage) of the peptide in question with the internal reference sequence of NGF. A high coverage is thus indicative of a great similarity between the peptide and NGF and thus the interaction of the peptide with TrkA should be more representative of what actually happens in nature.
Table 4: Description of the Peptide-TrkA Interaction
Figure imgf000020_0001
PEPTIDES HA VING TrkA-RECEPTOR-AGONIST ACTIVITY
The peptides and peptide fragments of the present invention bind to the TrkA receptor and activate it, therefore, by acting as receptor agonists. Accordingly, the peptides and peptide fragments of the present invention are useful in the prophylaxis, treatment and management of the diseases and disorders which benefit from a greater activation of the TrkA receptor. In particular, the peptides and peptide fragments of the invention are suitable for treating, preventing and/or managing peripheral neuropathies, multiple sclerosis, Alzheimer's disease, Parkinson's disease, hypoxic cerebral lesions and/or optic nerve atrophy.
Furthermore, the peptides and peptide fragments of the invention are useful for the treatment, prophylaxis and/or management of diseases or disorders characterised or aggravated by a loss of innervation, such as ischaemic heart disease, heart failure and diabetes and the comorbidities thereof, in particular diabetic neuropathies.
Furthermore, the peptides and peptide fragments of the invention are useful for the treatment, prophylaxis and/or management of other diseases and disorders that have shown to benefit from a treatment with NGF, such as ulcers, neurotrophic keratitis and/or glaucoma.
The peptides and peptide fragments of the invention can be used in the treatment and prophylaxis of the above-mentioned conditions not only in humans, but also in animals such as primates, swine, ruminants (cattle, sheep, goats), horses, cats, dogs, poultry (for example, chickens, ducks, geese, quail, pigeons, turkeys or ornamental birds), as well as productive and ornamental fish, reptiles and amphibians.
The present invention further relates to a method for the prophylaxis, treatment and/or management of diseases or disorders which benefit from a greater activation of the TrkA receptor in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
The present invention further relates to a method for treating, preventing and/or managing peripheral neuropathies, multiple sclerosis, Alzheimer's disease, Parkinson's disease, hypoxic cerebral lesions and/or optic nerve atrophy in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
The present invention further relates to a method for treating, preventing and/or managing diseases or disorders characterised or aggravated by a loss of innervation in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
The present invention further relates to a method for treating, preventing and/or managing ischaemic heart disease, heart failure and diabetes and the comorbidities thereof, in particular diabetic neuropathies in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
The present invention further relates to a method for treating, preventing and/or managing other diseases and disorders which have shown to benefit from a treatment with NGF, in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
The present invention further relates to a method for treating, preventing and/or managing ulcers, neurotrophic keratitis and/or glaucoma in human beings and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
The peptides and peptide fragments of the present invention can act systemically and/or locally. To this end, they can be administered in a suitable manner, such as, for example, orally, parenterally, pulmonarily, nasally, sublingually, lingually, buccally, rectally, cutaneously, transdermally, optically, or as an implant or stent or coating thereof. For these routes of administration, the peptides and peptide fragments of the invention can be administered in suitable forms of administration.
The forms suitable for oral administration include already existing forms of application that work according to the prior art and release the peptides and peptide fragments of the invention rapidly or in a modified fashion, for example as an sustained-release formulation. These forms of application can contain the peptides and peptide fragments of the invention in a crystalline and/or amorphised and/or dissolved form. Examples of such forms of application include tablets (uncoated tablets or tablets with coatings such as enteric coatings or coatings which modify the release of peptides and peptide fragments of the invention, such as slowly dissolving coatings or insoluble coatings), tablets or films/wafers that disintegrate rapidly in the oral cavity, capsules (for example, hard or soft gelatine capsules), lyophilised powders, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
Parenteral administration can take place avoiding an absorption step (for example via the intravenous, intra-arterial, intracardiac, intraspinal or intralumbar route), or else with the inclusion of an absorption step (for example, intramuscular, subcutaneous, percutaneous or intraperitoneal). Parenteral forms of administration include, among others, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.
Other suitable routes of administration are, for example, pharmaceutical forms for inhalation (for example, powder inhalers or nebulisers) nasal drops, nasal solutions or nasal sprays; tablets, film/wafers or capsules for lingual, sublingual or buccal administration, suppositories, preparations for the eyes or ears, vaginal capsules, aqueous suspensions (lotions, mixtures), lipophilic aqueous suspensions, ointments, creams, transdermal therapeutic systems (such as, for example, patches), milk, pastes, foams, implants or stents.
The peptides and peptide fragments of the invention can be incorporated into the listed forms of administration. This can take place in ways that are known per se, by mixing them with inert, nontoxic, pharmaceutically acceptable excipients. Such pharmaceutically acceptable excipients include, for example, carriers (for example microcrystalline cellulose, lactose and mannitol), solvents, emulsifiers, dispersants and wetting agents (for example sodium dodecyl sulphate, polyoxysorbitan oleate), binders (for example, polyvinylpyrrolidone), natural and synthetic polymers (for example, albumin), stabilisers (for example, antioxidants such as ascorbic acid), colourants (for example, inorganic pigments such as iron oxides) and taste and/or odour masking agents.
In some aspects of the invention, it may be useful to administer the peptides and peptide fragments of the invention in forms of administration which modulate their pharmacokinetics and increase their delivery through the blood-brain barrier. Such forms of administration can be provided by absorbing or using the peptides and peptide fragments of the invention on/in nanoparticulate carriers. The carriers which modulate the pharmacokinetics and increase delivery through the blood-brain barrier include carriers based on biodegradable polymers such as poly(alkyl cyanoacrylates), polyesters, polyanhydrides, polyethers or mixtures or copolymers thereof. Examples of potentially useful nanometer-scale carriers comprise carriers based on poly(butyl cyanoacrylate), polylactic acid, poly (lactic-co-glycolic acid), chitosan, polyethylene glycol or mixtures or copolymers thereof.
In some aspects of the invention, it might be useful for the nanoparticulate carriers to be treated on the surface, for example coated with further compounds to promote delivery through the blood-brain barrier. Such further compounds for the surface treatment of nanoparticulate carriers include surfactants, polyethers, biological molecules and mixtures thereof. In some cases it may be preferable to treat the nanoparticulate carriers with one or more compounds selected from the group consisting of polysorbate 80, Pluronic, poly(ethylene glycol), poly(vinyl alcohol), human albumin and mixtures thereof. The invention further relates to medicaments comprising at least one peptide or peptide fragment of the invention, usually together with at least one inert, nontoxic, pharmaceutically acceptable excipient and the use thereof for the above-mentioned purposes.
In some embodiments, it may be preferable to combine the peptides and peptide fragments of the invention with another pharmaceutically active compound, for example another neuroprotective compound.
PEPTIDES HA VING NGF- ANT A GONISTIC A CTIVITY.
The peptides and peptide fragments of the present invention bind to the TrkA receptor and block it by acting, therefore, as NGF antagonists. Accordingly, the peptides and peptide fragments of the present invention are useful in the prophylaxis, treatment and management of the diseases and disorders correlated to a high level of NGF. In particular, the peptides and peptide fragments of the invention are suitable for the treatment and/or management of pain, above all chronic pain, such as osteoarthritis pain, rheumatoid arthritis, chronic lumbar pain, interstitial cystitis, prostatitis, chronic pelvic pain syndrome, fibromyalgia, endometriosis, degenerative disease of the intervertebral disc and cancer pain.
Furthermore, the peptides and peptide fragments of the invention are useful for the treatment and/or the prophylaxis of hyperinnervation and conditions correlated thereto, such as cardiac arrhythmias and sudden cardiac death.
The peptides and peptide fragments of the invention can be used in the treatment and prophylaxis of the above-mentioned conditions not only in humans, but also in animals such as primates, swine, ruminants (cattle, sheep, goats), horses, cats, dogs, poultry (for example, chickens, ducks, geese, quail, pigeons, turkeys or ornamental birds), as well as productive and ornamental fish, reptiles and amphibians.
The present invention further relates to a method of prophylaxis, treatment and/or management of diseases or disorders tied to NGF, in particular to high levels of NGF in human beings and animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
The present invention further relates to a method for the treatment and/or management of pain in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
The present invention further relates to a method for treating and/or preventing hyperinnervation in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
The present invention further relates to a method for treating and/or preventing cardiac arrhythmias and/or the sudden cardiac death in humans and in animals, comprising the administration of a therapeutically effective amount of peptides or peptide fragments of the invention.
The expression pain management relates to all treatment regimens that do not completely eliminate pain from the patient, but reduce pain in order to improve or significantly improve the patient's quality of life.
The peptides and peptide fragments of the present invention may act systemically and/or locally: To this end, they can be administered in a suitable manner, such as, for example, orally, parenterally, pulmonarily, nasally, sublingually, lingually, buccally, rectally, cutaneously, transdermally, optically, or as an implant or stent.
For these routes of administration, the peptides and peptide fragments of the invention can be administered in suitable forms of administration
The forms suitable for oral administration are forms of application that work according to the prior art and release the peptides and peptide fragments of the invention rapidly or in a modified fashion, for example as an sustained-release formulation. These forms of application can contain the peptides and peptide fragments of the invention in a crystalline and/or amorphised and/or dissolved form. Examples of such forms of application include tablets (uncoated tablets or tablets with coatings such as enteric coatings or coatings which modify the release of peptides and peptide fragments of the invention, such as slowly dissolving coatings or insoluble coatings), tablets or films/wafers that disintegrate rapidly in the oral cavity, capsules (for example, hard or soft gelatine capsules), films, lyophilised powders, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
Parenteral administration can take place avoiding an absorption step (for example via the intravenous, intra-arterial, intracardiac, intraspinal or intralumbar route), or else with the inclusion of an absorption step (for example, intramuscular, subcutaneous, percutaneous or intraperitoneal). Parenteral forms of administration include, among others, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.
Other suitable routes of administration are, for example, pharmaceutical forms for inhalation (for example, powder inhalers or nebulisers) nasal drops, nasal solutions or nasal sprays; tablets, film/wafers or capsules for lingual, sublingual or buccal administration, suppositories, preparations for the eyes or ears, vaginal capsules, aqueous suspensions (lotions, mixtures), lipophilic aqueous suspensions, ointments, creams, transdermal therapeutic systems (such as, for example, patches), milk, pastes, foams, implants or stents.
The peptides and peptide fragments of the invention can be incorporated into the listed forms of administration. This can take place in ways that are known per se, by mixing them with inert, nontoxic, pharmaceutically acceptable excipients. Such pharmaceutically acceptable excipients include, for example, carriers (for example microcrystalline cellulose, lactose and mannitol), solvents, emulsifiers, dispersants and wetting agents (for example sodium dodecyl sulphate, polyoxysorbitan oleate), binders (for example, polyvinylpyrrolidone), natural and synthetic polymers (for example, albumin), stabilisers (for example, antioxidants such as ascorbic acid), colourants (for example, inorganic pigments such as iron oxides) and taste and/or odour masking agents.
In some aspects of the invention it may be useful to administer the peptides and peptide fragments of the invention in forms of administration which modulate their pharmacokinetics and increase their delivery through the blood-brain barrier. Such forms of administration can be provided by absorbing or using the peptides and peptide fragments of the invention on/in nanoparticulate carriers. The carriers which modulate the pharmacokinetics and increase delivery through the blood-brain barrier include carriers based on biodegradable polymers such as polyialk l cyanoacrylates), polyesters, polyanhydrides, polyethers or mixtures or copolymers thereof. Examples of potentially useful nanometer-scale carriers comprise carriers based on poly(butyl cyanoacrylate), polylactic acid, poly (lactic-co-glycolic acid), chitosan, polyethylene glycol or mixtures or copolymers thereof.
In some aspects of the invention it might be useful for the nanoparticulate carriers to be treated on the surface, for example coated with further compounds to promote delivery through the blood-brain barrier. Such further compounds for the surface treatment of nanoparticulate carriers include surfactants, polyethers, biological molecules and mixtures thereof. In some cases it may be preferable to treat the nanoparticulate carriers with one or more compounds selected from the group consisting of polysorbate 80, Pluronic, poly(ethylene glycol), poly( vinyl alcohol), human albumin and mixtures thereof.
The invention further relates to medicaments comprising at least one peptide or peptide fragment of the invention, usually together with at least one inert, nontoxic, pharmaceutically acceptable excipient and the use thereof for the above-mentioned puiposes.
In some embodiments, it may be preferable to combine the peptides and peptide fragments of the invention with other pharmaceutically active compounds, for example other analgesics or compounds useful in the treatment of cardiac arrhythmias and sudden cardiac death such as, for example, beta-blockers.
Analgesics suitable for use in combination with the peptides and peptide fragments of the invention include, for example, non-steroidal anti-inflammatory drugs such as paracetamol, ibuprofen, aspirin, diclofenac or naproxen, COX-2 inhibitors, such as rofecoxib, celecoxib and etoricoxib, and opioids, such as codeine, oxycodone, hydrocodone, dihydromorphine or pethidine, as well as other known analgesics.
Beta-blockers suitable for use in combination with the peptides and peptide fragments of the invention include, for example, acebutolol, atenolol, betaproxol, bisoprolol, bucindolol, butaxamine, carteolol, carvedilol, celiprolol, esmolol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol and timolol.
The invention further relates to medicaments comprising at least one peptide or peptide fragment having TrkA-agonistic activity or NGF-antagonistic activity according to the invention, usually in combination with at least one other pharmaceutically active ingredient and the use thereof for the above-mentioned purposes.
The minimum amount of the peptides and peptide fragments of the invention to be administered is a therapeutic amount. The term "therapeutically effective amount" means an amount of compound which prevents the onset of or alleviates the symptoms, manages, stops the progression and/or eliminates a disease, disorder or condition by virtue of the altered activation of the TrkA receptor, a disease, disorder or condition characterised or aggravated by a loss of innervation or another disease, disorder or condition that has shown to benefit from a treatment with NGF, or a disease or disorder tied to high levels of NGF.
Typically, an effective dosing regimen for the peptides and peptide fragments of the invention in adults is about 1 to 1000 μg kg of the peptides or peptide fragments of the invention, preferably 5 to 500 μg kga. In some embodiments the effective dose is 5 to 100 ^ig/ kg or 10 to 50 ig I kg. In other embodiments the effective dose is 1 to 20 ng kg or 2 to 10 ^ig/kg. In some embodiments the peptides and peptide fragments of the invention are administered daily. In other embodiments the peptides or peptide fragments of the invention are administered every other day or every 3, 5, 7 or 10 days. In some cases it might be necessary to test the patient in order to find the optimal dose.
It may however be necessary to deviate from the above amounts if required by the circumstances. Such deviations might be due to body weight, the route of administration, the individual response to the active ingredient, the severity of the condition, disease or disorder, the type of preparation and the time or interval over which the administration takes place. Therefore, in some cases it might be sufficient to use doses that are smaller than the above-mentioned minimum amount, whereas in other cases the upper limit mentioned above must be exceeded. In the event of the administration of large amounts of the peptides and peptide fragments of the invention, it may be advisable to distribute them in a plurality of single doses over the day.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT RELATED TO PEPTIDES
HAVING TrkA- RECEPTOR AGONISTIC ACTIVITY
The present invention will now be illustrated in greater detail based on selected but non-limiting examples. In the following examples all the amino acids mentioned can be either a-L-amino acids or D-amino acids unless specified otherwise.
A.) Binding considerations
In the examples that follow, all calculations were performed using the "Molecular Operating Environment" (MOE) software available from the Chemical Computing Group. All values are based on the "London dG" scoring function.
The relevant term for the calculations was
in which
c represents the protein-ligand interaction energy;
eFlex represents the loss of flexibility of the ligand;
CHB and FHB are compensatory functions for the non-ideality of the specific bond and are expressed as a sum over all hydrogen-bond sites; AD, is a term for the atomic desolvation which describes variations in the solvation field of ligand atoms.
It is known that during the action of NGF on the TrkA receptor, TrkA and NGF form a unique complex made up of two molecules of NGF and two molecules of TrkA, forming two homo- dimers. The present inventors mapped the short contact distances between the two TrkA molecules and the two NGF molecules in order to find the smallest example of the TrkA-NGF complex. This study showed that the largest number of short-range interactions occur between the first NGF molecule and second TrkA molecule.
The specific distances between the closest ct-carbon atoms in the first NGF molecule and the second TrkA molecule are given in Table 5
Table 5: Distances between the between the a-carbon atoms of NGF(l) and TrkA(2)
Figure imgf000033_0001
Based on the calculations, it becomes clear that all α-carbon atoms with a distance of less than
7A between the NGF molecule and the TrkA molecule are to be found in the first 24 amino acids of NGF; in particular, they are to be found in the amino acid residues 2 to 24 of NGF.
On the basis of this information the present inventors have defined a peptide based on the amino acids 2 to 24 of NGF. The corresponding binding domain of the TrkA receptor is represented by residues 250-348, as previously described.
The amino acid sequence of the starting peptide is given below:
SE -SER-HIS-PRO-ILE-PHE-HIS-ARG-GLY-GLU-PHE-SER-VAL-CYS-ASP-SER-VAL- SER-VAL-TRP-VAL-GLY-ASP
The free energy of the starting peptide for binding to TrkA was calculated as -13.02 Kcal/mol. Based on this, the present inventors investigated various mutated forms of the starting peptide showing one, two or three mutations. They then selected those peptides which showed an increase of at least -4 kcal/mol in binding free energy compared to the starting peptide for further investigation. This increase in binding free energy shows an increased ability of the peptide to bind to TrkA and activate it. The increases in the binding free energy of the mutated peptides are given below in Table 6.
Table 6 - Increase in binding free energy of mutated peptides compared to starting peptide
Figure imgf000034_0001
2SER=>LYS
-4.509VAL=>LEU 2SER=>LYS
-4.702GLY => CYS 2SER=>LYS
-4.552GLY => GLU SER=>LYS
-6.931PHE=>PR0 PRO => VAL
-6.722GLY => HIS PRO => VAL
-4.109VAL=>ILE ILE => TRP
-4.102GLY => HIS 8SER=>GLU
-4.520TRP => PHE 2SER=>LYS 8SER=>HIS -5.29 SER => THR 2SER->LYS 8SER=>HIS -4.40 ILE => VAL 2SER=>LYS
-4.218SER=>HIS 10GLU => ASP
12SER => LYS
18SER => HIS -5.06
20TRP => ILE
B.) Peptide synthesis
The peptides described above can be prepared by conventional solid state synthesis.
The process of synthesis takes place according to the well-known cycle of a coupling step, a washing step, a step of removing the protective group and a washing step, after which another coupling step follows or, if the peptide is complete, the peptide is cleaved from the solid support.
In such a synthesis, for example, a polystyrene resin can be used as the support.
The functionality of the amino acid can be protected, for example, using a tert-butyloxycarbonyl
(Boc) protecting group. Other suitable protecting groups can be used for any reactive side-chain functionality that may be present. In the event that orthogonal protection is necessary, the Boc group can be replaced by a 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group.
The coupling reaction is carried out using coupling agents that are usual in the art, such as, for example, a combination of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or Ν,Ν'- dicyclohexylcarbodiimide (DCC) with 1 -hydroxy-benzotriazole (HOBt) and l-hydroxy-7-aza- benzotriazole (hOAT), where appropriate in the presence of a base, in particular a hindered base such as diisopropylethylamine (DIE A) or tetramethylpiperidine (TMP).
Suitable solvents for the coupling reaction include dimethylforniamide (DMF), N- methylpyrrolidone (NMP), trichloromethane (TCM) and dichloromethane (DCM) or mixtures thereof.
Furthermore, other solvents such as trifluorethanol (TFE), hexafluoro-2-propanol (FIFIP) or dimethylsulfoxide (DMSO) can be added to suppress peptide aggregation. The washing steps generally use solvents similar to those of the coupling steps.
The removal of the protecting groups is achieved by using suitable deprotecting agents. The Boc protecting group is usually removed under acidic conditions using, for example, hydrochloric acid (HC1) or trifluoroacetic acid (TFA), where appropriate in a suitable solvent such as, for example, methanol or ethanol for HC1 and, for example, DCM or TCM for TFA.
The Fmoc protecting group is usually removed in the presence of a base, such as, for example, pyridine, where appropriate in a suitable solvent.
Once the peptide is complete it can be cleaved from the solid support in a suitable manner and purified as necessary. The whole process can be performed on an automated synthesizer.
C.) Assessment of physiological activity
In vitro
ELISA
The ability of the peptides and peptide fragments of the invention to act as a TrkA receptor agonist can for example be measured using an enzyme-linked immunosorbent assay (ELISA) for the phosphorylated TrKA receptor. Such an ELISA kit is commercially available, for example, from Cell Signalling Technologies under the name PathScan® Phospho-TrkA (Tyr674/675) Sandwich ELISA Kit (catalogue number 7212).
The assay is used according to the manufacturer's instructions, exposing the PC12 and/or SH- SY5Y cell lines as cellular types expressing TrkA receptors to the peptides and peptide fragments of the invention. The cells grow in a suitable cell culture medium (for example: 60- mm Falcon ©) at a certain cell density (e.g. 0.5-lxl 06) in a specific and complete culture medium, as described, for example, in Marchesi et al. (J Cell Physiol. 2014 Nov; 229(1 1):1776- 86) for the SH-SY5Y cell line and Rossi et al. (Bioorg Med Chem. 2011 Nov l ;19(21):6210-24) for the PC 12 cell line. c ^ t e a erent ce s g t e o ow ng:
when the cell culture is at 80-90% confluence, the culture medium is replaced with a fresh one containing a low percentage of Fetal Bovine Serum (FBS) and for a pre-established period of time. After washing with a phosphate saline buffer, 0.4/0.5 ml of cold IX Cell Lysis Buffer (specific name of the buffer) containing protease inhibitors is added to each plate, in contact with the cells, for 5 minutes in ice.
The cells are detached from the plate with a scraper and transferred into an appropriate tube stored in ice, where they are then sonicated. The suspension is centrifuged for 10 minutes (at 13- 14000 rpm) at 4°C and the supernatant is transferred into a new tube and stored at -80°C.
The percentage of phosphorylated TrkA after exposure to the peptides can be compared with a calibration curve constructed using NGF as a reference.
Neurite Regeneration Bioassay
The ability of the peptides and peptide fragments of the invention to act as a TrkA receptor agonist can be measured using a β-NGF-induced neurite regeneration bioassay. The assay used is based on the method described by Chandler et al. (J. Biol. Chem. (1984) 259, No. 1 1, 6882- 6889).
PC 12 cells are grown on tissue culture plates (for example 60 mm Falcon ©) at an initial cell density of 4-5 x 105 cells/plate. During the cell culture, β-NGF is added on 8 consecutive days. The cells are harvested mechanically with a Pasteur pipette, washed once in serum-containing growth medium and twice in serum-free growth medium and collected by centrifugation (500 g for 3 minutes each time). The cells are resuspended in the serum-free growth medium at 6 x 10 cells/ml and 0.5 ml is dispensed into each well of a 24-well culture plate. The tissue culture plate is pre-treated with 50 ng/ml of poly-L-lysine (Sigma Aldrich) and washed four times with sterile distilled water. The cells are incubated at 37° C for 30 min and 400 μΐ of modified medium (Dulbecco's modified Eagle's medium) containing bovine serum albumin (BSA) is added to bring the final serum albumin concentration to 1 mg/ml. The cells are returned to the incubator for 15 min. before 10 pm of β-NGF or various concentrations of the peptides of the invention (e.g. 0, 1, 2, 5, 10, 20 pm) are added. At least one set of cells is used as a control without the addition of β-NGF. The cells are incubated for 24 hours at 37 ° C and read out using a phase contrast microscope. The positive cells are the ones which show a minimum neurite growth > 25 microns. The TrkA-agonistic activity of the peptides of the invention is evaluated based on their ability to stimulate the growth of neurons compared to the NGF-induced positive cells.
In vivo
The ability of the peptides and peptide fragments of the invention in the treatment, management or prevention of diseases or disorders which benefit from a greater activation of the TrkA receptor, or of other diseases and disorders which have shown to benefit from a treatment with NGF, can be observed in suitable animal models.
The ability of peptides of the invention in the treatment or prevention of Alzheimer's disease can be demonstrated, for example, in a rat model using the Morris water maze. For an overview of rat models for Alzheimer's disease, see for example Do Carmo and Cuello, Neurodegeneration (2013) 8: 37. Furthermore, the Morris water maze can generally provide information about the activity of the tested compounds in relation to memory, a target of potential interest for aging and dementia-related diseases, additional conditions that might benefit from greater TrkA receptor agonism.
The ability of peptides of the invention in the treatment of diabetic ulcers can be demonstrated, for example, in a mouse model. Diabetes can be induced in mice with streptozotocin. The diabetic mice are then wounded, according to described methods, using for example a biopsy punch, and treated topically with the peptides and peptide fragments in a suitable solvent, for example saline solution. The closure of the wound is then observed visually or using digital imaging and a comparison is made with control animals treated with a placebo, for example the suitable solvent on its own without the addition of the peptides or peptide fragments of the invention.
D.) Example of a pharmaceutical composition
1% DMS0 / 99% Plasma
The peptide is completely dissolved in a calculated volume of DMSO. The DMSO solution is then suspended in the plasma and the suspension is mixed until a clear solution is obtained. The solution is sterilised by filtration and dispensed into appropriate containers.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT IN RELATION TO NGF- ANTA GONISTIC A CTIVITY
The present invention will now be illustrated in greater detail based on selected but non-limiting examples. In the following examples all the amino acids mentioned are a-L or D-amino acids unless specified otherwise
A.) Binding considerations
In the examples that follow, all calculations were performed using the "Molecular Operating Environment" (MOE) software available from the Chemical Computing Group. All values are based on the "London dG" scoring function.
The relevant term for the calculations was in which,
c represents the protein-ligand interaction energy; eFlex represents the loss of flexibility of the ligand; CUB and FHB are compensatory functions for the non-ideality of the specific bond and are expressed as a sum over all hydrogen-bond sites; and AD, is a term for the atomic desolvation which describes variations in the solvation field of ligand atoms. It is known that during the action of NGF on the TrkA receptor, TrkA and NGF form a unique complex from two molecules of NGF and two molecules of TrkA, forming two homo-dimers. The present inventors mapped the short contact distances between the two TrkA molecules and the two NGF molecules in order to find the smallest example of the TrkA-NGF complex. This study showed that the largest number of short-range interactions occur between the first NGF molecule and second TrkA molecule.
The specific distances between the closest a-carbon atoms in the first molecule NGF and in the second TrkA molecule are given in the above-mentioned Table 5.
Based on the calculations, it becomes clear that all α-carbon atoms with a distance of less than
7A between the NGF molecule and the TrkA molecule are to be found in the first 24 amino acids of NGF; in particular, they are to be found in the amino acid residues 2 to 24 of NGF.
On the basis of this information the present inventors have defined a peptide based on the amino acids 2 to 24 of NGF. The corresponding binding domain of the TrkA receptor is represented by residues 250-348, as previously described.
The amino acid sequence of the starting peptide is given below:
SER-SER-HIS-PRO-ILE-PHE-HIS-ARG-GLY-GLU-PHE-SER-VAL-CYS-ASP-SER-VAL- SER-VAL-TRP-VAL-GLY-ASP
The free energy of the starting peptide for binding to TrkA was calculated as -13.02 Kcal/mol. Based on this, the present inventors investigated various mutated forms of the starting peptide showing one, two or three mutations. They then selected those peptides which showed an increase of at least -4 kcal/mol in binding free energy compared to the starting peptide for further investigation. This increase in binding free energy shows an increased ability of the peptide to bind to the TrkA receptor and to displace NGF.
Due to their small size, the peptides lack the ability to dimerise or induce dimerisation in the TrkA receptor. Consequently, these peptides cannot activate the TrkA receptor.
Thanks to their increased binding free energy, these peptides will displace NGF and consequently act as NGF antagonists. The increases in the binding free energy of the mutated peptides are given below in Table 7:
7 Increase in binding free energy of mutated peptides compared to the starting peptide
Figure imgf000042_0001
2SER=>LYS
-4.509VAL=>LEU 2SER=>LYS
-4.702GLY => CYS 2SER=>LYS
-4.552GLY => GLU SER=>LYS
-6.931PHE=>PR0 PRO => VAL
-6.722GLY => HIS PRO => VAL
-4.109VAL=>ILE ILE => TRP
-4.102GLY => HIS 8SER=>GLU
-4.520TRP => PHE 2SER=>LYS 8SER=>HIS -5.29 SER => THR 2SER=>LYS 8SER=>HIS -4.40 ILE => VAL 2SER=>LYS
-4.218SER=>HIS 10GLU => ASP
12SER => LYS
18SER => HIS -5.06
20TRP => ILE
B.) Peptide synthesis
The peptides described above can be prepared by conventional solid state synthesis.
The process of synthesis takes place according to the well-known cycle of a coupling step, a washing step, a step of removing the protective group and a washing step, after which another coupling step follows or, if the peptide is complete, the peptide is cleaved from the solid support.
In such a synthesis, for example, a polystyrene resin can be used as the support.
The functionality of the amino acid can be protected, for example, using a tert-butyloxycarbonyl
(Boc) protecting group. Other suitable protecting groups can be used for any reactive side-chain functionality that may be present. In the event that orthogonal protection is necessary, the Boc group can be replaced by a 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group.
The coupling reaction is carried out using coupling agents that are usual in the art, such as, for example, a combination of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or Ν,Ν'- dicyclohexylcarbodiimide (DCC) with 1-hydroxy-benzotriazole (HOBt) and l-hydroxy-7-aza- benzotriazole (HO At), where appropriate in the presence of a base, in particular a hindered base such as diisopropylethylamine (DIE A) or tetramethylpiperidine (TMP).
Suitable solvents for the coupling reaction include dimethylformamide (DMF), N- methylpyrrolidone (NMP), trichloromethane (TCM) and dichloromethane (DCM) or mixtures thereof. Furthermore, other solvents such as trifluorethanol (TFE), hexafluoro-2-propanol (FIFIP) or dimethylsulfoxide (DMSO) can be added to suppress peptide aggregation.
The washing steps generally use solvents similar to those of the coupling steps. The removal of the protecting groups is achieved by using suitable deprotecting agents. The Boc protecting group is usually removed under acidic conditions using, for example, hydrochloric acid (HCl) or trifluoroacetic acid (TFA), where appropriate in a suitable solvent such as, for example, methanol or ethanol for HCl and, for example, DCM or TCM for TFA.
The Fmoc protecting group is usually removed in the presence of a base, such as, for example, pyridine, where appropriate in a suitable solvent.
Once the peptide is complete it can be cleaved from the solid support in a suitable manner and purified as necessary. The whole process can be performed on an automated synthesizer.
C.) Assessment of physiological activity
In vitro
ELISA
The ability of peptides and peptide fragments of the invention to act as a TrkA receptor antagonist can for example be measured using an enzyme-linked immunosorbent assay (ELISA) for the phosphorylated TrKA receptor. Such an ELISA kit is commercially available, for example, from Cell Signalling Technologies under the name PathScan® Phospho-TrkA (Tyr674/675) Sandwich ELISA Kit (catalogue number 7212).
The assay is used according to the manufacturer's instructions, exposing the PC 12 and/or SH- SY5Y cell lines as cellular types expressing TrkA receptors to the peptides and peptide fragments of the invention. The cells grow in a suitable cell culture medium (for example: 60- mm Falcon ©) at a certain cell density (e.g. 0.5-lxl06) in a specific and complete culture medium, as described, for example, in Marchesi et al. (J Cell Physiol. 2014 Nov;229(l l): 1776- 86) for the SH-SY5Y cell line and Rossi et al. (Bioorg Med Chem. 2011 Nov l;19(21):6210-24) for the PC 12 cell line.
The procedure for the adherent cells is the following:
when the cell culture is at 80-90% confluence, the culture medium is replaced with a fresh one containing a low percentage of Fetal Bovine Serum (FBS) and for a pre-established period of time. After washing with a phosphate saline buffer, 0.4/0.5 ml of cold IX Cell Lysis Buffer (specific name of the buffer) containing protease inhibitors is added to each plate, in contact with the cells, for 5 minutes in ice.
The cells are detached from the plate with a scraper and transferred into an appropriate tube stored in ice, where they are then sonicated. The suspension is centrifuged for 10 minutes (at 13- 14000 rpm) at 4°C and the supernatant is transferred into a new tube and stored at -80°C. The percentage of phosphorylated TrkA after exposure to the peptides can be compared with a ... . . ¾ τ^„ · , , · · calibration curve constructed using NGF as a reierence. The peptides having ainaguinsL auuvity prevent activation by NGF.
Neurite Regeneration Bioassay
The ability of peptides and peptide fragments of the invention to act as NGF antagonists can be measured using a β-NGF-induced neurite regeneration bioassay. The assay used is based on the method described by Chandler et al. (J. Biol. Chem. (1984) 259, No. 1 1, 6882-6889).
PC 12 cells are grown on tissue culture plates (for example 60 mm Falcon ©) at an initial cell density of 4-5 x 105 cells/plate. During the cell culture, β-NGF is added on 8 consecutive days. The cells are harvested mechanically with a Pasteur pipette, washed once in serum-containing growth medium and twice in serum-free growth medium and collected by centrifugation (500 g for 3 minutes each time). The cells are resuspended in the serum-free growth medium at 6 x 10 cells/ml and 0.5 ml is dispensed into each well of a 24-well culture plate. The tissue culture plate is pre-treated with 50 μg/ml of poly-L-lysine (Sigma Aldrich) and washed four times with sterile distilled water. The cells are incubated at 37° C for 30 min and 400 μΐ of modified medium (Dulbecco's modified Eagle's medium) containing bovine serum albumin (BSA) is added to bring the final serum albumin concentration to 1 mg/ml. The cells are returned to the incubator for 15 min. before 10 pm of β-NGF or various concentrations of the peptides of the invention (e.g. 0, 1, 2, 5, 10, 20 pm) are added. At least one set of cells is used as a control without the addition of β-NGF. The cells are incubated for 24 hours at 37 ° C and read out using a phase contrast microscope. The positive cells are the ones which show a minimum neurite growth > 25 microns. The NGF-antagonistic activity of the peptides of the invention is evaluated based on their ability to reduce the number of NGF-induced positive cells.
In vivo
The ability of the peptides of the invention in the treatment or management of pain can be demonstrated in a mouse or rat model. Arthritis can be induced by the injection of bovine type II collagen into the joints of a test animal. After the arthritis has been induced in the animals, they are treated once or several times with different doses of the peptides and peptide fragments of the invention. The ability of peptides and peptide fragments of the invention in treating pain can be observed through the restoration of mobility in arthritic animals.
D.) Example of a pharmaceutical composition
1% DMSO / 99% Plasma
The peptide is completely dissolved in a calculated volume of DMSO. The DMSO solution is then suspended in plasma and the suspension is mixed until a clear solution is obtained. The solution is sterilized by filtration and dispensed into suitable containers.

Claims

1. A peptide or peptide fragment having an amino acid sequence selected from the group consisting of:
GLY-GLN-ASN-TRP-GLN-PRO-SER-ALA-TYR-GLN-ALA-TPvP-CYS-THR-THR,
CYS-LYS-GLY-TYR-GLY-GLY-LEU-VAL-ALA-GLN-CYS-THR-ASP-GLY-PHE,
ASN-VAL-GLU-PRO-LEU-THR-CYS-VAL-TYR-GLN-PRO-LYS-CYS-GLY-TRP,
LEU-PRO-SER-PRO-CYS-TRP-GLN-GLN-PHE-ARG-CYS-ASP-GLN-LEU-GLN,
CYS-GLY-THR-ASP-CYS-PHE-MET-ASN-GLY-ARG-PRO-GLY-GLY-TYR-TYR,
TYR-GLU-LYS-MET-CYS-ASP-THR-GLY-VAL-LEU-ILE-GLN-GLY-LYS-CYS,
TRP-GLY-THR-ASN-ILE-GLY-CYS-LEU-SER-LYS-CYS-GLU-GLY-CYS-SER,
ALA-ILE-SER-LYS-GLN-THR-PHE-ASP-THR-LYS-ASP-VAL-TRP-MET-SER,
THR-VAL-ASP-GLU-PHE-HIS-CYS-MET-LYS-GLY-CYS-CYS-GLU-LYS-ASN,
THR-GLU-ASN-TRP-GLY-VAL-ARG-CYS-ALA-ASN-TRP-THR-VAL-PHE-VAL,
PRO-CYS-PHE-ASP-ALA-GLY-GLN-SER-PHE-TRP-THR-PHE-LYS-VAL-CYS,
ILE-TRP-SER-GLN-CYS-ASP-GLY-ASN-CYS-TRP-PRO-ARG-ALA-ASN-VAL,
MET-GLN-TYR-HIS-CYS-ARG-ASN-THR-GLU-TYR-ASP-GLY-VAL-ASN-TRP,
CYS-CYS-GLN-TYR-PRO-GLY-GLY-ILE-ASN-ASN-LYS-VAL-VAL-ASP-SER,
ILE-LEU-PHE-PHE-GLY-TYR-THR-PRO-ASN-GLN-ALA-SER-TRP-CYS-CYS,
VAL-TYR-CYS-ALA-VAL-ASN-ASN-GLU-PHE-HIS-GLY-ASP-ASN-LYS-TRP,
GLY-ASN-CYS-VAL-VAL-PRO-HIS-CYS-LYS-ASP-ASP-GLY-ASN-TYR-PHE,
ALA-CYS-CYS-LYS-PHE-GLY-TYR-MET-MET-ALA-LYS-GLU-CYS-ASP-GLY,
GLN-LEU-ARG-GLN-CYS-TYR-GLY-GLU-SER-ASN-PHE-THR-ALA-TRP-SER,
PHE-CYS-TRP-PRO-ASN-SER-PRO-TRP-CYS-THR-PRO-ASN-SER-ALA-SER,
PRO-LEU-CYS-CYS-PRO-HIS-ILE-CYS-LEU-VAL-ASP-SER-GLN-GLY-ASN,
VAL-PRO-HIS-ILE-GLN-THR-VAL-ASP-CYS-CYS-SER-ASP-HIS-ASN-PRO, ILE-ALA-CYS-ALA-ALA-ASN-ASN-MET-THR-HIS-ASP-GLN-TRP-SER-SER,
CYS-PHE-GLY-THR-ASN-ALA-GLY-PRO-TRP-THR-CYS-PRO-CYS-ASN-VAL,
PHE-ASP-GLY-ASN-TYR-GLN-SER-GLY-PRO-SER-CYS-LEU-LYS-CYS-MET,
SER-THR-THR-PRO-GLY-CYS-LEU-MET-TYR-LEU-MET-SER-TYR-THR-GLN,
PHE-GLY-TYR-CYS-SER-LEU-GLU-ALA-THR-VAL-TYR-ARG-ASP-LYS-ILE,
MET-ASN-ASN-CYS-ASN-ALA-MET-TRP-ASP-ARG-SER-ASN-SER-PHE-CYS,
GLY-TRP-GLU-ASP-LYS-HIS-CYS-MET-GLN-CYS-GLN-GLY-GLN-TRP-CYS,
GLY-ASP-VAL-LEU-ASN-CYS-THR-GLN-HIS-VAL-THR-CYS-ASN-TYR-ALA,
CYS-MET-LYSLYS-ASN-CYS-LEU-TYR-ASN-MET-PHE-ASP-ASP-ASN-MET,
CYS-GLU-THR-HIS-MET-ASN-GLU-ILE-HIS-ASN-TRP-MET-GLY-THR-ASN,
PHE-GLN-GLY-ILE-VAL-GLU-HIS-GLU-THR-CYS-LYS-ASP-THR-LYS-ALA,
ILE-GLY-HIS-LEU-SER-GLU-ASN-GLN-THR-VAL-THR-LEU-LEU-GLN-GLN,
THR-GLN-LYS-ASP-SER-ILE-ARG-PHE-PHE-SER-ALA-CYS-PRO-GLU-VAL,
THR-VAL-ALA-TYR-CYS-MET-LYS-ASP-ILE- ALA-ALA- ASN-GLN-ASN-GLY,
GLY-LYS-ASN-CYS-PHE-GLU-CYS-LEU-GLN-SER-TYR-ASN-SER-SER-ALA,
GLY-ASN-LEU-GLY-ARG-GLY-SER-GLY-CYS-CYS-ASP-ARG-CYS-GLU-ILE,
GLN-ILE-LYS-PHE-SER-GLU-PHE-TYR-ARG-ASP-CYS-GLN-TRP-THR-PHE,
TYR-TRP-VAL-HIS-GLN-PHE-GLY-TRP-ASN-THR-ASN-PRO-ASN-ASPC-YS,
LEU-LYS-VAL-ILE-LYS-CYS-GLU-GLU-THR-GLY-SER-ALA-CYS-THR-ALA,
ALA-ALA-PRO-HIS-ASP-THR-GLU-SER-CYS-PHE-CYS-VAL-ALA-ARG-GLN,
LEU-ASN-HIS-CYS-SER-ASP-GLY-CYS-LEU-ASP-GLN-CYS-LYS-ASN-ALA,
SER-ILE-ILE-ALA-TYR-ASN-HIS-SER-VAL-TRP-LEU-ASP-ALA-CYS-CYS,
LEU-GLY-TYR-LYS-CYS-GLY-CYS-PRO-ASN-GLU-GLY-CYS-ASN-PHE-GLN,
TRP-THR-LYS-ASP-PRO-TYR-ARG-TYR-PHE-GLN-SER-ASN-ASP-SER-MET,
GLN-TRP-PRO-CYS-CYS-ASP-GLU-GLY-ALA-ASN-ALA-LYS-LYS-TYR-ASN, PHE-GLN-ASP-ILE-SER-PHE-LYS-ALA-GLU-GLY-GLN-CYS-MET-HIS-VAL,
GLY-GLN-TYR-HIS-ASP-CYS-GLU-LEU-ARG-VAL-PHE-THR-HIS-GLU-CYS,
THR-GLN-SER-ASP-ASN-ASN-ASP-LYS-ARG-ILE-VAL-GLY-PRO-GLN-ASN,
TYR-HIS-ASP-GLN-ARG-PHE-SER-TYR-ALA-PHE-LEU-SER-GLU-GLY-ASN,
PRO-GLU-ARG-PHE-THR-ASP-ARG-ILE-ARG-GLU-ASN-SER-LEU-GLY-LEU,
TRP-GLY-TYR-PRO-LYS-LYS-TRP-GLU-CYS-CYS-SER-GLY-ASP-GLN-ALA,
GLN-ARG-THR-ASP-CYS-SER-MET-GLY-LEU-GLN-LYS-GLU-ALA-SER-TYR,
ALA-THR-SER-ARG-ASP-GLY-ALA-SER-LEU-PRO-ASN-ASP-CYS-HIS-THR,
PRO-ASP-GLY-GLY-ASN-HIS-MET-ASN-CYS-ILE-TYR-GLY-LEU-CYS-THR,
ASN-VAL-MET-GLN-THR-PRO-ILE-MET-CYS-TYR-ASP-ALA-HIS-ALA-CYS,
MET-ASN-ALA-SER-PRO-HIS-TRP-ILE-ASP-SER-MET-SER-ASP-LYS-CYS,
PRO-TRP-TRP-CYS-GLN-LEU-THR-SER-THR-ASN-GLN-ASP-LYS-CYS-ASN,
SER-TYR-GLY-PRO-SER-ASN-LYS-LEU-ARG-ASN-ASP-ILE-TYR-ASP-TYR,
ALA-THR-SER-LEU-PHE-LYS-TRP-TYR-CYS-GLN-MET-SER-ASN-ASP-ASN,
THR-THR-ILE-PHE-VAL-LYS-GLN-SER-ASN-GLU-GLY-ALA-ILE-ASN-ALA,
GLY-LEU-THR-LYS-ILE-TRP-CYS-ASN-CYS-ASP-SER-TYR-CYS-ASN-SER,
GLN-ILE-GLY-SER-PRO-ILE-SER-GLU-ARG-ASP-ARG-CYS-ASN-ALA-ALA,
CYS-THR-GLU-PRO-MET-LEU-PHE-GLY-PHE-SER-THR-ARG-PHE-THR-MET,
ALA-CYS-PRO-MET-SER-GLU-GLU-CYS-PHE-PRO-LYS-VAL-LYS-GLY-ILE,
MET-ASN-ASN-PHE-CYS-THR-ASP-PRO-ARG-GLY-TRP-MET-SER-THR-THR,
PHE-MET-THR-CYS-TYR-GLN-GLY-ARG-ALA-THR-GLU-CYS-TRP-CYS-VAL,
PHE-VAL-MET-ALA-ASN-PHE-GLU-GLN-LYS-ALA-MET-ASP-MET-HIS-ASN,
PRO-LEU-CYS-ASP-PHE-GLY-VAL-ASN-SER-LYS-ASP-LYS-ALA-TYR-ALA,
PHE-LYS-GLU-LEU-TYR-ALA-MET-GLU-VAL-HIS-CYS-CYS-GLY-GLY-TRP,
GLN-TYR-SER-ASN-CYS-LYS-TYR-CYS-ALA-THR-TYR-GLU-ILE-ASN-GLN, PHE-GLU-ARG-ASN-MET-GLN-ILE-LYS-VAL-GLU-THR-PHE-LEU-SER-ASN,
GLY-MET-CYS-PRO-CYS-VAL-PHE-LEU-GLY-PRO-ASP-TRP-HIS-ASN-GLY,
CYS-PHE-LYS-SER-CYS-GLU-GLY-VAL-VAL-TYR-GLY-ILE-SER-SER-ILE,
CYS-SER-LYS-ASN-PHE-PHE-MET-LEU-CYS-TYR-GLU-LYS-ILE-GLU-GLY,
ALA-THR-ASN-ASN-GLN-CYS-TRP-ARG-THR-GLY-VAL-LEU-GLU-SER-PHE,
CYS-ASN-CYS-CYS-LEU-MET-SER-LYS-PRO-MET-SER-TYR-GLU-THR-ASN,
ASN-GLU-CYS-THR-LYS-GLN-PRO-ALA-THR-HIS-GLU-GLN-GLY-TYR-CYS,
SER-CYS-VAL-PRO-GLU-LEU-ASP-LYS-HIS-ASN-SER-ILE-MET-THR-SER,
CYS-TRP-PHE-ASP-ASN-MET-SER-PHE-VAL-GLN-THR-LEU-SER-LYS-GLN,
MET-GLU-SER-HIS-CYS-ALA-GLY-PHE-ILE-LYS-GLU-ALA-ALA-GLN-LEU,
TYR-TRP-PHE-GLY-CYS-GLN-ARG-GLU-GLN-GLY-TRP-GLY-TRP-TYR-CYS,
PHE-ASN-CYS-TYR-GLY-LYS-CYS-THR-SER-ASP-PHE-TRP-MET-PHE-THR,
PHE-LYS-TRP-LEU-ASN-ILE-CYS-THR-ASP-CYS-GLY-GLY-ALA-PHE-SER,
GLN-ASN-LEU-GLU-ILE-SER-VAL-ALA-TYR-ARG-THR-GLY-TRP-PHE-CYS,
GLN-TRP-GLN-CYS-THR-VAL-PHE-ALA-TYR-THR-GLN-VAL-SER-SER-GLY,
SER-PHE-THR-TRP-GLY-VAL-LYS-TLE-SEP-GLU-GLN-TRP-ALA-CYS-LEU,
VAL-CYS-LEU-GLN-GLY-ASN-SER-LEU-CYS-ILE-LYS-ALA-SER-GLU-LEU,
THR-GLU-ARG-SER-THR-ALA-ALA-ASP-LEU-SER-VAL-LYS-TRP-ALA-TYR,
ALA-THR-ALA-LYS-SER-TRP-ASN-PRO-TRP-ASP-LEU-TRP-MET-SER-GLN,
CYS-GLU-SER-ALA-LYS-PRO-HIS-ASP-CYS-THR-LEU-SER-MET-LEU-SER,
ASN-PHE-THR-TRP-PRO-LYS-TYR-ILE-CYS-ILE-THR-GLY-ASP-THR-ALA,
PHE-VAL-ILE-GLY-ASP-LYS-SER-SER-GLN-CYS-PRO-ASP-PHE-HIS-CYS,
CYS-GLY-LYS-LYS-VAL-TYR-CYS-THR-LEU-GLU-TYR-PHE-GLN-GLU-GLY,
TRP-MET-MET-ALA-CYS-GLN-TYR-HIS-CYS-ASP-GLU-HIS-PRO-PRO-TRP,
CYS-SER-GLY-VAL-PRO-GLU-HIS-LYS-ASP-ASN-ASP-SER-MET-LYS-SER, GLN-ARG-MET-CYS-CYS-THR-ASN-GLY-TYR-PRO-THR-SER-ASP-CYS-THR, and GLY-ASN-TYR-ARG-GLU-CYS-VAL-MET-GLN-CYS-CYS-LEU-SER-ILE-TRP, and salts thereof, solvates and solvates of the salts thereof.
2. The peptide or peptide fragment according to claim 1, characterised in that the amino acid sequence is selected from the group consisting of:
GLY-GLN-ASN-TRP-GLN-PRO-SER-ALA-TYR-GLN-ALA-TRP-CYS-THR-THR, CYS-LYS-GLY-TYR-GLY-GLY-LEU-VAL-ALA-GLN-CYS-THR-ASP-GLY-PHE, ASN-VAL-GLU-PRO-LEU-THR-CYS-VAL-TYR-GLN-PRO-LYS-CYS-GLY-TRP, LEU-PRO-SER-PRO-CYS-TRP-GLN-GLN-PHE-ARG-CYS-ASP-GLN-LEU-GLN, and CYS-GLY-THR-ASP-CYS-PHE-MET-ASN-GLY-ARG-PRO-GLY-GLY-TYR-TYR, and salts thereof, solvates and solvates of the salts thereof.
3. The peptide or peptide fragment according to either of the preceding claims for use as a medicament.
4. The peptide or peptide fragment according to any one of the preceding claims for use as an agonist of amino acid sequences of the TrkA receptor selected from the group consisting of: ■ .
PHE-PRO-ALA-SER-VAL-GLN-LEU-HIS-THR-ALA-VAL-GLU-MET-HIS-HIS-TRP- CYS-ILE-PRO-PHE-SER-VAL-ASP-GLY-GLN-PRO-ALA-PRO-SER-LEU-ARG-TRP- LEU-PHE-ASN-GLY-SER-VAL-LEU-ASN-GLU-THR-SER-PHE-ILE-PHE-THR-GLU- PHE-LEU-GLU-PRO-ALA-ALA-ASN-GLU-THR-VAL-ARG-HIS-GLY-CYS-LEU- ARG-LEU-ASN-GLN-PRO-THR-HIS-VAL-ASN-ASN-GLY-ASN-TYR-THR-LEU- LEU- ALA-ALA- ASN-PRO-PHE-GLY-GLN-ALA-SER-ALA-SER-ILE-MET-ALA- ALA-PHE-MET-ASP-ASN-PRO.
5. The peptide or peptide fragment according to any of claims 1 to 3 for use as an antagonist of amino acid sequences of the TrkA receptor selected from the group consisting of PHE-PRO-ALA-SER-VAL-GLN-LEU-HIS-THR-ALA-VAL-GLU-MET-HIS-HIS-TRP-
CYS-ILE-PRO-PHE-SER-VAL-ASP-GLY-GLN-PRO-ALA-PRO-SER-LEU-ARG-TRP-
LEU-PHE-ASN-GLY-SER-VAL-LEU-ASN-GLU-THR-SER-PHE-ILE-PHE-THR-GLU-
PHE-LEU-GLU-PRO-ALA-ALA-ASN-GLU-THR-VAL-ARG-HIS-GLY-CYS-LEU-
ARG-LEU-ASN-GLN-PRO-THR-HIS-VAL-ASN-ASN-GLY-ASN-TYR-THR-LEU-
LEU-ALA-ALA-ASN-PRO-PHE-GLY-GLN-ALA-SER-ALA-SER-ILE-MET-ALA-
ALA-PHE-MET-ASP-ASN-PRO.
The peptide or peptide fragment according to either of claims 1 and 2 for use in the treatment, prophylaxis and/or management of diseases or disorders, particularly diseases or disorders sensitive to a greater activation of the TrkA receptor, particularly for use in the treatment, prophylaxis and/or management of peripheral neuropathies, multiple sclerosis, Alzheimer's disease, Parkinson's disease, hypoxic cerebral lesions and/or optic nerve atrophy.
The peptide or peptide fragment according to either of claims 1 and 2 for use in the treatment, prophylaxis and/or management of diseases and disorders characterised by a loss of innervation, particularly for use in the treatment, prophylaxis and/or management of ischaemic heart disease, heart failure and diabetes and the comorbidities thereof, particularly diabetic neuropathies.
The peptide or peptide fragment according to either of claims 1 and 2 for use in the treatment, prophylaxis and/or management of diseases and disorders that obtain a benefit from a treatment with NGF, particularly for use in the treatment, prophylaxis and/or management of ulcers, neurotrophic keratitis and/or glaucoma.
The peptide or peptide fragment according to either of claims 1 and 2 for use in the treatment, prophylaxis and/or management of diseases and disorders, particularly diseases or disorders tied to NGF, more particularly diseases or disorders tied to high levels of NGF and especially for use in the treatment and/or management of pain and/or the treatment and/or prophylaxis of cardiac arrhythmia and/or sudden cardiac death.
10. A medicament comprising a peptide or a peptide fragment according to either of claims 1 and 2 in combination with at least one inert, nontoxic pharmaceutically acceptable excipient.
1 1. A medicament comprising a peptide or a peptide fragment according to either of claims 1 and 2 in combination with at least one further active ingredient.
12. The medicament according to claim 10 or 11 for use in the treatment, prophylaxis and/or management of diseases or disorders, particularly diseases or disorders sensitive to a greater activation of the TrkA receptor, particularly for use in the treatment, prophylaxis and/or management of peripheral neuropathies, multiple sclerosis, Alzheimer's disease, Parkinson's disease, hypoxic cerebral lesions and/or optic nerve atrophy.
13. The medicament according to claim 10 or 11 for use in the treatment, prophylaxis and/or management of diseases or disorders, characterised by a loss of innervation, particularly for use in the treatment, prophylaxis and/or management of ischaemic heart disease, heart failure and diabetes and the comorbidities thereof, particularly diabetic neuropathies.
14. The medicament according to claim 10 or 11 for use in the treatment, prophylaxis and/or management of diseases or disorders that obtain a benefit from a treatment with NGF, particularly for use in the treatment, prophylaxis and/or management of ulcers, neurotrophic keratitis and/or glaucoma.
15. The medicament according to claim 10 or 11 for use in the treatment, prophylaxis and/or management of diseases or disorders, particularly diseases or disorders tied to NGF, more particularly diseases or disorders tied to high levels of NGF and especially for use in the treatment and/or management of pain and/or the treatment and/or prophylaxis of cardiac arrhythmia and/or sudden cardiac death.
PCT/EP2017/069705 2016-08-23 2017-08-03 PEPTIDES HAVING TrkA-RECEPTOR-AGONISTIC ACTIVITY AND/OR PEPTIDES HAVING NGF-ANTAGONISTIC ACTIVITY WO2018036785A1 (en)

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