WO2008149115A1 - Precurseur de la tryptophylline-1 et ses utilisations - Google Patents

Precurseur de la tryptophylline-1 et ses utilisations Download PDF

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Publication number
WO2008149115A1
WO2008149115A1 PCT/GB2008/001961 GB2008001961W WO2008149115A1 WO 2008149115 A1 WO2008149115 A1 WO 2008149115A1 GB 2008001961 W GB2008001961 W GB 2008001961W WO 2008149115 A1 WO2008149115 A1 WO 2008149115A1
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Prior art keywords
pro
peptide
qub
amino acid
hyp
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PCT/GB2008/001961
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English (en)
Inventor
Christopher Shaw
Tianbao Chen
Martin O'rourke
Brian Walker
David Hirst
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The Queen's University Of Belfast
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Publication of WO2008149115A1 publication Critical patent/WO2008149115A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present invention relates to peptides and uses thereof. In particular, it relates to bradykinin antagonist molecules.
  • Bradykinins are found in the skin secretions of many anuran amphibians including representative species of the famtHes, Ranidae, Hylidae and Bombinatoridae (Conlon, J. M. & Aronsson, U. (1997) Peptides 18, 361-365; Yasuhara, T., Ishikawa, O., Nakajima, T., Araki, K. and Tachibana, S. (1979) Chem. Pharm. Bull. (Tokyo) 27, 486-491 ; Anastasi, A., Bertaccini, G. and Erspamer, V. (1966) Br. J. Pharmacol.
  • bradykinin The actions of bradykinin are diverse, including vasodilation with subsequent induced hypotension, increasing vascular permeability, induction of pain and contraction of a variety of smooth muscle types (Regoli, D., Rizzi, A. and Cab, G. (1997) Pharmacol. Res. 35, 513- 515).
  • phyllokinin bradykinyl-IYsulphate from Phyllomedusa sp.
  • vasodilation/hypotension induction Asnastasi, A., Bertaccini, G. and Erspamer, V. (1966) Br. J. Pharmacol. 27, 479-485).
  • the present inventors have studied peptide precursor cDNAs from a South American leaf frog Phyllomedusa sauvagei skin secretion- derived library.
  • One of the sequenced clones encodes the precursor of P. sauvagei tryptophyllin-1 (1) - a peptide first isolated and characterised by Erspamer's group in 1985 (Montecucchi (1985), Peptides 6, 187-195).
  • This peptide, named QUB919 was assessed for arterial smooth muscle relaxant activity. In agreement with Erspamer's group, the peptide was found to be devoid of any relaxant activity in the concentration range tested.
  • the inventors further investigated the effects of this peptide and to their surprise discovered that, although the peptide had no arterial smooth muscle relaxant activity, it was found to be an extraordinarily competitive antagonist of the relaxation effects induced by both PdT-1 and bradykinin.
  • This peptide is thus the prototype of a new class of naturally-occurring peptides, herein referred to as QUB919 peptides that can antagonise this receptor, for example in nanomolar concentrations.
  • the present invention provides a method of antagonising bradykinin receptor activity in a cell, tissue or organism, said method comprising administering a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide to said cell, tissue or organism.
  • a method of constricting vascular smooth muscle comprising administering a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide to said smooth muscle.
  • QUB 919 peptides exhibit significant B2 bradykinin receptor antagonism activity enables the use of such QUB 919 peptides and the polynucleotides encoding said peptides in the treatment of diseases which are associated with or symptoms of which are mediated by B2 bradykinin receptor agonism activity, for example diseases of the cardiovascular system, inflammatory diseases, neurological diseases or degenerative diseases, such as Alzheimer's disease.
  • a method of treating a condition associated with B2 bradykinin receptor activity comprising administering a therapeutically effective amount of a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide to a patient in need thereof.
  • the QUB 919 peptides of the invention have also been found to inhibit angiogenesis.
  • a method of inhibiting angiogenesis comprising administering a therapeutically effective amount of a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide to a patient in need thereof.
  • the invention may thus be used to treat disorders mediated by angiogenesis, such as autoimmune disorders, rheumatoid arthritis etc.
  • disorders mediated by angiogenesis such as autoimmune disorders, rheumatoid arthritis etc.
  • a method of treating a disorder mediated by or associated with angiogenesis comprising administering a therapeutically effective amount of a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide to a patient in need thereof.
  • a fifth aspect of the invention provides a method of treatment of cancer, said method comprising administering a therapeutically effective amount of a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide to a patient in need thereof.
  • a physiological activity of QUB919 which may be exploited therapeutically. Accordingly, in a sixth aspect, there is provided a a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide for use in medicine.
  • a pharmaceutical composition comprising a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide, and a pharmaceutically acceptable carrier or excipient. Further provided by the invention is a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide for use in the treatment of a cardiovascular disease.
  • QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide for use in the treatment of a neural degenerative disease is provided by the invention.
  • a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide for use in the treatment of an inflammatory disease.
  • a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide for use in the treatment of a disease associated with angiogenesis.
  • a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide in the preparation of a medicament for the treatment of cancer.
  • a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide in the preparation of a medicament for the treatment of a cardiovascular disease.
  • a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide in the preparation of a medicament for the treatment of a neural degenerative disease.
  • a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide in the preparation of a medicament for the treatment of an inflammatory disease.
  • a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide in the preparation of a medicament for the treatment of pain relief, such as that associated with chronic inflammatory conditions.
  • a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide in the preparation of a medicament for the treatment of a disease associated with angiogenesis.
  • a QUB 919 peptide or a polynucleotide encoding a QUB 919 peptide in the preparation of a medicament for the treatment of cancer.
  • a QUB 919 peptide refers to an isolated peptide having bradykinin receptor antagonist activity, said peptide comprising the amino acid sequence:
  • Lys-Pro-Pro-Pro-Trp-Val-Pro-Val (SEQ ID NO: 1), a fragment or analogue thereof or a therapeutically acceptable salt thereof.
  • the residues in the QUB 919 amino acid sequence which are conserved amongst the tryptophyllin family are Lysine at position 1 , Proline at position 2, Tryptophan at position 5 and Proline at position 7.
  • the QUB919 peptide is an analogue of the peptide having the amino acid sequence shown as Sequence ID No: 1 , wherein the analogue comprises the amino acid sequence as shown as Sequence ID No: 2, or a therapeutically acceptable salt thereof:
  • AA2 is Pro or Hyp
  • AA3 is Pro or Hyp
  • AA4 is any amino acid
  • AA6 is any amino acid
  • X is an NH2 group or AA8, wherein AA8 is any amino acid; wherein where X is an NH2 group, AA4 is Pro or Hyp wherein where AA4 is not Pro or Hyp, X is AA8.
  • X is AA8 and AA4 is Hyp or Pro.
  • AA6 is a hydrophobic amino acid, for example valine or isoleucine.
  • AA8 is a hydrophobic amino acid, for example valine or isoleucine.
  • the QUB919 peptide is an polypeptide comprising the amino acid sequence as shown as Sequence ID No: 3:
  • Lys- AA2- AA3- AA4-Trp-AA6-Pro-X (Sequence ID No: 3); wherein AA2 is Pro or Hyp, AA3 is Pro or Hyp, AA4 is Pro or Hyp, AA6 is a hydrophobic amino acid and X is a hydrophobic amino acid.
  • the QUB 919 peptide consists of the amino acid sequence Lys-Pro-Pro-Pro-Trp-Val-Pro-Val (SEQ ID NO: 1 ) or a therapeutically acceptable salt thereof.
  • QUB 919 peptides include fragments and analogues of the QUB919 peptide having amino acid sequence SEQ ID NO: 1. Fragments and analogues of the invention have QUB919 biological activity i.e. bradykinin receptor antagonist activity and/or anti- angiogenic activity.
  • a “fragment” of a QUB 919 peptide means a stretch of amino acid residues which retains QUB 919 biological activity.
  • a “fragment” of a QUB 919 peptide means a stretch of at least 4 amino acids, for example at least six or at least seven amino acids of the amino acid sequence as shown as Sequence ID No: 1.
  • a QUB 919 analogue of and for use in the invention means a polypeptide modified by varying the amino acid sequence of SEQ ID NO: 1 e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself, wherein said analogue has QUB919 biological activity i.e. bradykinin receptor antagonist activity and/or anti-angiogenic activity.
  • Such analogues may involve substitution or deletion of 5 or fewer amino acids, more preferably of 4 or fewer, even more preferably of 3 or fewer, most preferably of 1 or 2 amino acids only and/or the insertion or addition of one or more, for example, two, three, four or five or more amino acid residues.
  • Analogues of the invention also include multimeric peptides including such QUB919 peptides and prodrugs including such sequences, derivatives of the peptides of the invention, including the peptide linked to a coupling partner, e. g. an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule.
  • a coupling partner e. g. an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule.
  • Analogues of the invention include fusion peptides.
  • analogues may comprise peptides of the invention linked, for example, to antibodies that target the peptides to diseased tissue, for example, heart tissue or tumour tissue.
  • the QUB 919 peptides described herein may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH 1 , CH2, CH3, or any combination thereof) and portions thereof, resulting in chimeric polypeptides.
  • immunoglobulins IgA, IgE, IgG, IgM
  • CH 1 , CH2, CH3, or any combination thereof immunoglobulins
  • Such fusion proteins may be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995).
  • Fusion proteins of and for use in the invention also include QUB 919 peptides fused with albumin, for example recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, , EP Patent 0 413 622, and U.S. Pat. No. 5,766,883).
  • the use of polynucleotides encoding such fusion proteins described herein are also encompassed by the invention.
  • Analogues of and for use in the present invention further include reverse-or retro-analogues of natural QUB 919 peptides or their synthetic derivatives.
  • reverse peptides For details relating to reverse peptides, see, for example, EP 0497 366, U.S. 5,519,115, and Merrifield et al., 1995, PNAS, 92:3449-53 for details relating to reverse peptides, the disclosures of which are herein incorporated by reference.
  • reverse peptides are produced by reversing the amino acid sequence of a naturally occurring or synthetic peptide. Such reverse-peptides retain the same general three-dimensional structure (e.
  • alpha-helix as the parent peptide except for the conformation around internal protease-sensitive sites and the characteristics of the N-and C-termini.
  • Reverse peptides are purported not only to retain the biological activity of the non-reversed "normal” peptide but may possess enhanced properties, including increased biological activity. (See Iwahori et al., 1997, Biol. Pharm. Bull. 20: 267- 70).
  • Analogues of and for use in the present invention may therefore comprise reverse peptides of natural and synthetic QUB 919 peptides.
  • a reverse peptide of the invention includes a peptide having the generic formula:
  • AA7 is Pro or Hyp
  • AA6 is Pro or Hyp
  • AA5 is any amino acid
  • AA3 is any amino acid
  • X is an NH2 group or AA1 , wherein AA1 is any amino acid; wherein where X is an NH2 group, AA5 is Pro or Hyp wherein where AA5 is not Pro or Hyp, X is AA1.
  • X is AA1 and AA5 is Hyp or Pro.
  • AA3 is a hydrophobic amino acid, for example valine or isoleucine.
  • AA1 is a hydrophobic amino acid, for example valine or isoleucine.
  • the QUB919 peptide is an analogue comprising the amino acid sequence as shown as Sequence ID No: 5:
  • AA5 is Pro or Hyp
  • AA6 is Pro or Hyp
  • AA47 is Pro or Hyp
  • AA3 is a hydrophobic amino acid
  • X is a hydrophobic amino acid
  • a particularly preferred reverse peptide of and for use in the present invention has the amino acid sequence shown as SEQ ID NO: 6:
  • Peptides including reverse peptides and fragments of either of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid.
  • the peptides of and for use in the present invention can be readily prepared according to well- established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984).
  • QUB 919 peptides of and for use in the invention may be in the form of multimers.
  • multimers of 2, 3 or more individual QUB 919 analogue monomeric units
  • Such multimers may be used to prepare a monomeric peptide by preparing a multimeric peptide that includes the monomeric unit, and a cleavable site (i.e., an enzymatically cleavable site), and then cleaving the multimer to yield a desired monomer.
  • a cleavable site i.e., an enzymatically cleavable site
  • the use of multimers can increase the binding affinity for a receptor.
  • the binding affinity of the peptides of the invention to the bradykinin B2 receptor can often be increased by using multimers of 2-5, preferably 2-3 receptor binding moieties.
  • the multimers can be homomers or heteromers.
  • the term homomer refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO:1 or fragments, variants, splice variants, fusion proteins, or other QUB 919 analogs described herein. These homomers may contain QUB 919 peptides having identical or different amino acid sequences.
  • the multimers can include only QUB 919 peptides having an identical amino acid sequence, or can include different amino acid sequences.
  • the multimer can be a homodimer (e.g., containing QUB 919 peptides having identical or different amino acid sequences), homotrimer or homotetramer.
  • heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., non- QUB 919 peptides) in addition to the QUB 919 peptides described herein.
  • the multimers may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
  • multimers are formed when the QUB 919 peptides described herein contact one another in solution.
  • heteromultimers are formed when QUB 919 and non- QUB 919 peptides contact antibodies to the polypeptides described herein (including antibodies to the heterologous polypeptide sequence in a fusion protein described herein) in solution.
  • multimers described herein may be formed by covalent associations with and/or between the QUB 919 peptides (and optionally non- QUB 919 peptides) described herein.
  • covalent associations can involve one or more amino acid residues contained in a QUB 919 sequence (e.g., that recited in SEQ ID NO:1 ).
  • the covalent associations are the consequence of chemical or recombinant manipulation.
  • such covalent associations can involve one or more amino acid residues contained in the heterologous polypeptide sequence in a QUB 919 fusion protein.
  • covalent associations are between the heterologous sequence contained in a fusion protein described herein (see, e.g., U.S. Pat. No. 5,478,925).
  • the covalent associations are between the heterologous sequence contained in a QUB 919-Fc fusion protein.
  • covalent associations of fusion proteins described herein are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, for example, oseteoprotegerin (see, e.g.WO 98/49305).
  • two or more polypeptides described herein are joined through peptide linkers.
  • peptide linkers include those peptide linkers described in U.S. Pat. No. 5,073,627. Proteins comprising multiple QUB 919 peptides separated by peptide linkers can be produced using conventional recombinant DNA technology.
  • Multimers may also be prepared by fusing the QUB 919 peptides to a leucine zipper or isoleucine zipper polypeptide sequence.
  • leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
  • leucine zipper domains suitable for producing soluble multimeric proteins described herein are those described in PCT application WO 94/10308.
  • Recombinant fusion proteins comprising a polypeptide described herein fused to a polypeptide sequence, that dimerizes or trimerizes in solution, can be expressed in suitable host cells, and the resulting soluble multimeric fusion protein can be recovered from the culture supernatant using techniques known in the art.
  • the multimers may also be generated using chemical techniques known in the art.
  • polypeptides desired to be contained in the multimers described herein may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925).
  • the multimers can be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925).
  • polypeptides described herein may be routinely modified by the 8 001961
  • cysteine or biotin may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925). Additionally, techniques known in the art can be used to prepare liposomes containing two or more QUB 919 peptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925,).
  • those multimers including only naturally-occurring amino acids can be formed using genetic engineering techniques known in the art.
  • those that include post-translational or other modifications can be prepared by a combination of recombinant techniques and chemical modifications.
  • the QUB 919 peptides are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).
  • polynucleotides coding for a homodimer described herein can be generated by ligating a polynucleotide sequence encoding a QUB 919 peptide described herein to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925).
  • Pro-Drugs The peptides described herein are intended, at least in some embodiments, to be administered to a human or other mammal to treat or prevent a disorder mediated by bradykinin release.
  • Peptides are typically administered parenterally, and may be readily metabolized by plasma proteases. Oral administration, which is perhaps the most attractive route of administration, may be even more problematic. In the stomach, acid degrades and enzymes break down the peptides. Those peptides that survive to enter the intestine intact are subjected to additional proteolysis as they are continuously barraged by a variety of enzymes, including gastric and pancreatic enzymes, exo- and endopeptidases, and brush border peptidases. As a result, passage of peptides from the lumen of the intestine into the bloodstream can be severely limited. However, various prodrugs have been developed that enable parenteral and oral administration of therapeutic peptides.
  • Peptides of and for use in the invention can be conjugated to various moieties, such as polymeric moieties, to modify the physiochemical properties of the peptide drugs, for example, to increase resistance to acidic and enzymatic degradation and to enhance penetration of such drugs across mucosal membranes.
  • moieties such as polymeric moieties
  • Abuchowski and Davis have described various methods for derivatizating enzymes to provide water-soluble, non-immunogenic, in vivo stabilized products ("Soluble polymers-Enzyme adducts," Enzymes as Drugs, Eds. Holcenberg and Roberts, J. Wiley and Sons, New York, N.Y. (1981 )).
  • Abuchowski and Davis discuss various ways of conjugating enzymes with polymeric materials, such as dextrans, polyvinyl pyrrolidones, glycopeptides, polyethylene glycol and polyamino acids.
  • the resulting conjugated polypeptides retain their biological activities and solubility in water for parenteral applications.
  • U.S. Patent No. 4,179,337 to Davis, et al. teaches coupling peptides to polyethylene glycol or polypropylene glycol having a molecular weight of 500 to 20,000 Daltons to provide a physiologically active non-immunogenic water soluble polypeptide composition.
  • the polyethylene glycol or polypropylene glycol protects the polypeptide from loss of activity and the composition can be injected into the mammalian circulatory system with substantially no immunogenic response.
  • U.S. Patent Nos. 5,681 ,811 , 5,438,040 and 5,359,030 teach stabilized, conjugated polypeptide complexes including a therapeutic agent coupled to an oligomer that includes lipophilic and hydrophilic moieties.
  • Garmen, et al. describe a protein-PEG prodrug (Garman, AJ. , and Kalindjian, S. B., FEBS Lett, 1987, 223, 361 -365).
  • a prodrug can be prepared using this chemistry, by first preparing a maleic anhydride reagent from polydispersed MPEG5000 and then conjugating this reagent to the peptides disclosed herein. The reaction of amino acids with maleic anhydrides is well known.
  • the hydrolysis of the maleyl- amide bond to reform the amine-containing drug is aided by the presence of the neighbouring free carboxyl group and the geometry of attack set up by the double bond.
  • the peptides can be released (by hydrolysis of the prodrugs) under physiological conditions.
  • the peptides can also be coupled to polymers, such as polydispersed PEG, via a degradable linkage, for example, the degradable linkage shown (with respect to pegylated interferon ⁇ -2b) in Roberts, M.J., et al., Adv. Drug Delivery Rev., 2002, 54, 459-476.
  • the peptides can also be linked to polymers such as PEG using 1 ,6 or 1 ,4 benzyl elimination (BE) strategies (see, for example, Lee, S., et al., Bioconjugate Chem., (2001), 12, 163-169; Greenwald, R.B., et al., U.S. Patent No. 6,180,095, 2001 ; Greenwald, R.B., et al., J. Med. Chem., 1999, 42, 3657-3667.); the use of trimethyl lock lactonization (TML) (Greenwald, R.B., et al., J. Med.
  • TML trimethyl lock lactonization
  • the peptides have free amino, amido, hydroxy and/or carboxylic groups and these functional groups can be used to convert the peptides into prodrugs.
  • Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues which are covalently joined through peptide bonds to free amino, hydroxy or carboxylic acid groups of various polymers, for example, polyalkylene glycols such as polyethylene glycol.
  • Prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters which are covalently bonded to the above peptides through the C-terminal carboxylic acids.
  • Some approaches involve using enzyme inhibitors to slow the rate of degradation of proteins and peptides in the gastrointestinal tract; manipulating pH to inactivate local digestive enzymes; using permeation enhancers to improve the absorption of peptides by increasing their paracellular and transcellular transports; using nanoparticles as particulate carriers to facilitate intact absorption by the intestinal epithelium, especially, Peyer's patches, and to increase resistance to enzyme degradation; liquid emulsions to protect the drug from chemical and enzymatic breakdown in the intestinal lumen; and micelle formulations for poorly water-solubulized drugs.
  • the peptides can be provided in a suitable capsule or tablet with an enteric coating, so that the peptide is not released in the stomach.
  • the peptide can be provided as a prodrug.
  • the peptides are present in these drug delivery devices as prodrugs.
  • Prodrugs comprising the peptides of and for use in the invention or pro-drugs from which peptides of and for use in the invention (including analogues and fragments) are released or are releasable are considered to be analogues of the invention.
  • Isotopically-labelled peptides or peptide prodrugs and their use in methods of the invention are also encompassed by the invention.
  • Such peptides or peptide prodrugs are identical to the peptides or peptide prodrugs of the invention, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 0, 17 O, and 35 S, respectively.
  • Peptides of the present invention prodrugs thereof, and/or the prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention.
  • Certain isotopically-labeled compounds of the present invention for example those into which radioactive isotopes such as 3 H and 14 C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability.
  • Isotopically-labelled peptides and prodrugs thereof can generally be prepared by carrying out readily known procedures, including substituting a readily available isotopically-labelled reagent for a non-isotopically-labelled reagent, e.g., a labelled amino acid.
  • the present invention further encompasses the use of mimetic peptides of QUB 919 which can be used as therapeutic peptides.
  • Mimetic QUB 919 peptides are short peptides which mimic the biological activity of QUB 919 by binding to the bradykinin B 2 receptor and functioning as an antagonist at that receptor.
  • Such mimetic peptides can be obtained from methods known in the art such as, but not limited to, phage display or combinatorial chemistry. For example, the method disclosed by Wrighton, et al., Science 273:458-463 (1996) can be used to generate mimetic QUB 919 peptides.
  • the present invention may utilise nucleic acids in an expression system.
  • nucleic acids which may be used in the invention include any isolated polynucleotide encoding a QUB 919 peptide (which may be a reverse peptide QUB 919).
  • the polynucleotide comprises the nucleic acid sequence:
  • nucleic acid according to and for use in the present invention is provided as an isolate, in isolated and/or purified form, or free or substantially free of material with which it is naturally associated, such as free or substantially free of nucleic acid flanking the gene in the frog genome, except possibly one or more regulatory sequence (s) for expression.
  • Nucleic acid may be wholly or partially synthetic and may include genomic DNA, cDNA or RNA.
  • Nucleic acid sequences encoding a peptide in accordance with or for use in the present invention can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art.
  • Modifications to the sequences can be made, e. g. using site directed mutagenesis, to lead to the expression of modified peptide or to take account of codon preference in the host cells used to express the nucleic acid.
  • the sequences can be incorporated in a vector having one or more control sequences operably linked to the nucleic acid to control its expression.
  • the vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the peptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell.
  • Peptides can then be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the peptide is produced and recovering the peptide from the host cells or the surrounding medium.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e. g. phage, or phagemid, as appropriate.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems.
  • the nucleic acid of and for use in the invention may be integrated into the genome (e. g. chromosome) of the host cell.
  • the nucleic acid may be on an extra-chromosomal vector within the cell, or otherwise identifiably heterologous or foreign to the cell.
  • the introduction which may (particularly for in vitro introduction) be generally referred to without limitation as "transformation", may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome- mediated transfection and transduction using retrovirus or other virus, e. g. vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
  • direct injection of the nucleic acid could be employed. The introduction may be followed by causing or allowing expression from the nucleic acid, e. g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded polypeptide (or peptide) is produced.
  • polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium.
  • a polypeptide or peptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e. g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e. g. see below).
  • QUB919 peptides of and for use in the present invention have bradykinin receptor antagonist activity.
  • an "antagonist” is a substance that inhibits its binding partner, typically a receptor. Inhibition is defined in the context of the particular assay, or may be apparent in the literature from a discussion herein that makes a comparison to a factor or substance that is accepted as an "agonist” or an “antagonist” of the particular binding partner under substantially similar circumstances as appreciated by ' those of skill in the art. Inhibition may be defined with respect to a decrease in a particular effect or function that is induced by interaction of the antagonist with a binding partner, and can include allosteric effects.
  • Bradykinin receptor antagonist activity may be assessed by any means known in the art or as described herein.
  • a peptide or salt thereof is considered to possess bradykinin receptor antagonist activity if it is capable of inhibiting bradykinin induced vasodilation and/or is capable of inhibiting angiogenesis.
  • a peptide or salt thereof is considered to be biologically active i.e. have bradykinin receptor antagonist activity if it is capable of inhibiting bradykinin induced vasodilation and is capable of inhibiting angiogenesis.
  • the QUB919 peptide of and for use in the present invention antagonises arterial smooth muscle dilation with a pA2 of at least 8.0, for example at least 9.0.
  • pA2 may be calculated using conventional means known in the art, for example using Schild's plot analysis.
  • the pA2 may be determined using any suitable preparation, for example a human umbilical artery preparation, a sheep femoral artery preparation or a rat tail artery preparation.
  • the QUB919 peptide of and for use in the present invention inhibits angiogenesis, for example as assessed in a tubule formation assay, with an EC50 which is less than 10 '6 M, for example less than 10 "7 M, such as less than 10 '8 M, such as less than 10 "9 M.
  • the QUB919 peptide of and for use in the present invention antagonises arterial smooth muscle dilation with a pA2 of a value greater than the pA2 of HOE140 in an equivalent tissue preparation and/or inhibits angiogenesis, with an EC50 which is less than that for HOE140 in an equivalent preparation.
  • the peptides or salts thereof or for use in the invention are devoid of direct smooth muscle effects i.e. devoid of vasoactive effects.
  • the biological activity of the peptides may be assayed by various methods.
  • various immunoassays known in the art can be used, including but not limited to, competitive and non- competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and Immunoelectrophoresis assay
  • binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al., Microbiol. Rev. 59:94-123 (1995).
  • bradykinin antagonism of peptides of and for use in the present application may be assessed using any suitable assay known in the art.
  • activity may be assessed by determining its effect on the relaxation induced by a known B2 receptor agonist such as Pdt1 on a preconstricted (for example using phenylephrine) smooth muscle preparation for example a tail artery preparation, a femoral artery preparation or an umbilical artery preparation.
  • Anti-angiogenesis activity may be assayed using any standard assays, known in the art.
  • Many in vitro and in vivo assays are known in the art. These include Matrigel plug and corneal neovascularization assays, the in vivo/in vitro chick chorioallantoic membrane (CAM) assay, and the in vitro cellular (proliferation, migration, tube formation) and organotypic (aortic ring) assays, the chick aortic arch and the Matrigel sponge assays. Further details of such assays may be found, for example, in Auerbach et al, Clinical Chemistry 49: 32-40, 2003; 10.1373/49.1.32. Further details are also provided in the Examples such as the Matrigel assay and the assays used in the Examples.
  • compositions comprising a QUB 919 peptide (or nucleic acid).
  • Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be, for example, oral, intravenous, or topical.
  • the formulation may be a liquid, for example, a physiologic salt solution containing non-phosphate buffer at pH 6.8-7.6, or a lyophilised powder.
  • compositions are preferably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time- course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and at the discretion of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.
  • Peptides of and for use in the present invention may be administered alone but will preferably be administered as a pharmaceutical composition, which will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected dependent on the intended route of administration.
  • the peptides may be administered to a patient in need of treatment via any suitable route.
  • the precise dose will depend upon a number of factors, including the precise nature of the peptide.
  • suitable routes of administration include (but are not limited to) oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may comprise a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood.
  • sustained release carriers include semipermeable polymer matrices in the form of shared articles, e.g. suppositories or microcapsules.
  • Implantable or microcapsular sustained release matrices include polylactides (US Patent No.
  • EP-A- 0058481 copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers 22(1 ): 547-556, 1985), poly (2- hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15: 167-277, 1981 , and Langer, Chem. Tech. 12:98-105, 1982).
  • Liposomes containing the polypeptides are prepared by well-known methods: DE 3,218, 121 A; Epstein et al, PNAS USA, 82: 3688-3692, 1985; Hwang et al, PNAS USA, 77: 4030- 4034, 1980; EP-A-0052522; E-A-0036676; EP-A-0088046; EP-A- 0143949; EP-A-0142541 ; JP-A-83-11808; US Patent Nos 4,485,045 and 4,544,545.
  • the liposomes are of the small (about 200- 800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal rate of the polypeptide leakage.
  • the peptides and nucleic acids for use in the invention may be used in the control and/or treatment of a wide variety of clinical conditions in mammals, including humans.
  • the peptides and methods of the invention may be used in the treatment of any condition or disorder for which bradykinin antagonists and/or vasoconstrictors may be useful.
  • Treatment includes any regime that can benefit a human or non-human animal.
  • the treatment may be in respect of an existing condition or may be prophylactic (preventative treatment). Treatment may include curative, alleviation or prophylactic effects.
  • the peptides are used for the treatment of cardiovascular disorders such as hypotension.
  • Other conditions for which the peptides and methods of the invention may be useful include inflammation, either chronic or acute e.g. as an anti-inflammatory agent.
  • cardiovascular disorders such as hypotension, pain, common cold, allergies and immunology/allergy disorders, asthma, pancreatitis, burns and other skin disorders, viral infection and other infectious diseases such as sepsis, multiple trauma and the like.
  • Specific conditions include, but are not limited to, respiratory diseases, diuresis, natriuresis, calciuresis, COPD (chronic obstructive pulmonary disease), itching, cystitis, pruritis, rheumatoid arthritis, osteoarthritis, migraine, neuropathic pain, post-traumatic and post ischemic cerebral eczema, liver cirrhosis and other liver/kidney diseases, rhinitis, hepatorenal failure, diabetes and other metabolic diseases, metastasis, pancreatitis, neovascularization, corneal haze, glaucoma, ocular pain, ocular hypertension and other eye diseases, angio edema and the like in mammals, especially humans.
  • respiratory diseases include, but are not limited to, respiratory diseases, diuresis, natriuresis, calciuresis, COPD (chronic obstructive pulmonary disease), itching, cystitis, pruritis, rheumato
  • bradykinin B2 receptor activation is believed to be associated with some of the symptoms of Alzheimer's disease.
  • the invention further encompasses the use of the QUB 919 peptides or nucleic acids encoding such peptides as anti plaque forming agents in neurological or other degenerative disease.
  • disorders include, but are not limited to, Huntington's disease, Parkinson's disease and other central nervous system disorders such as amyotrophic lateral sclerosis, multiple sclerosis, stroke, head trauma, post-surgical brain edema, brain edema (general), cytotoxic brain edema (such as that associated with brain tumours, stroke, head trauma, etc.), brain edema associated with metabolic diseases (renal failure, pediatric metabolic diseases, etc.), brain tumour and other cancers, pseudotumor cerebri, hydrocephalus, spinal cord trauma, spinal cord edema, neurodegenerative diseases, post-traumatic brain injury, other head injuries, and the like.
  • Huntington's disease Parkinson's disease and other central nervous system disorders
  • central nervous system disorders such as amyotrophic lateral sclerosis, multiple sclerosis, stroke, head trauma, post-surgical brain edema, brain edema (general), cytotoxic brain edema (such as that associated with brain tumours, stroke, head trauma, etc.), brain edem
  • the peptides and methods of the invention may be used to inhibit angiogenesis.
  • Angiogenesis can be inhibited by administering an effective amount of a QUB 919 peptide or a nucleic acid encoding said peptide to a patient in need of such treatment.
  • the methods can be used to treat tumours, various autoimmune disorders, hereditary disorders, ocular disorders and other angiogenesis-mediated disorders.
  • the therapeutic and diagnostic methods described herein typically involve administering an effective amount of the peptides, nucleic acids or compositions including the peptides or nucleic acids to a patient.
  • the exact dose to be administered will vary according to the use of the compositions and on the age, sex and condition of the patient, and can readily be determined by the treating physician.
  • the compositions may be administered as a single dose or in a continuous manner over a period of time. Doses may be repeated as appropriate.
  • compositions and methods can be used to treat angiogenesis- mediated disorders including hemangioma, solid tumors, leukemia, metastasis, telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis, Crohn's disease, plaque neovascularization, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, diabetic neovascularization, macular degeneration, wound healing, peptic ulcer, Helicobacter related diseases, fractures, keloids, and vasculogenesis.
  • angiogenesis- mediated disorders including hemangioma, solid tumors, leukemia, metastasis, telangiectasia, psoriasis, scleroderma,
  • Carcinomas that may be treated using the peptides, compounds, compositions and methods of the invention include colorectal carcinoma, gastric carcinoma, signet ring type, esophageal carcinoma, intestinal type, mucinous type, pancreatic carcinoma, lung carcinoma, breast carcinoma, renal carcinoma, bladder carcinoma, prostate carcinoma, testicular carcinoma, ovarian carcinoma, endometrial carcinoma, thyroid carcinoma, liver carcinoma, larynx carcinoma, mesothelioma, neuroendocrine carcinomas, neuroectodermal tumors, melanoma, gliomas, neuroblastomas, sarcomas, leiomyosarcoma, MFII, fibrosarcoma, liposarcoma, MPNT, chondrosarcoma, and lymphomas.
  • ocular disorders are mediated by angiogenesis, and may be treated using the compounds, compositions and methods described herein.
  • a disease mediated by angiogenesis is ocular neovascular disease, which is characterized by invasion of new blood vessels into the structures of the eye and is the most common cause of blindness.
  • the associated visual problems are caused by an ingrowth of chorioidal capillaries through defects in Bruch's membrane with proliferation of fibrovascular tissue beneath the retinal pigment epithelium.
  • Angiogenic damage is also associated with diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma and retrolental fibroplasia.
  • corneal neovascularization diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, periphigoid radial keratotomy, and corneal graph rejection.
  • Diseases associated with retinal/choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, presumed myopia, optic pits, chronic retinal detachment, hyperviscosity syndromes, trauma and post-laser complications.
  • Other diseases include, but are not limited to, diseases associated with rubeosis (neovascularization of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy.
  • the peptides and methods of the invention may also be used to treat angiogenesis-mediated disorders such as inflammation, including various forms of arthritis, such as rheumatoid arthritis and osteoarthritis.
  • angiogenesis-mediated disorders such as inflammation, including various forms of arthritis, such as rheumatoid arthritis and osteoarthritis.
  • treatment with combinations of the compounds described herein with other agents useful for treating the disorders such as cyclooxygenase-2 (COX-2) inhibitors, which are well known to those of skill in the art.
  • COX-2 cyclooxygenase-2
  • the blood vessels in the synovial lining of the joints can undergo angiogenesis.
  • the endothelial cells form new vascular networks and release factors and reactive oxygen species that lead to pannus growth and cartilage destruction. These factors are believed to actively contribute to rheumatoid arthritis and also to osteoarthritis. Chondrocyte activation by angiogenic-related factors contributes to joint destruction, and also promotes new bone formation. The methods described herein can be used as a therapeutic intervention to prevent bone destruction and new bone formation.
  • Pathological angiogenesis is also believed to be involved with chronic inflammation.
  • disorders that can be treated using the compounds, compositions and methods described herein include ulcerative colitis, Crohn's disease, bartonellosis, and atherosclerosis.
  • the peptides or nucleic acids may be combined with various existing therapeutic agents used for that disease.
  • the QUB 919 peptides may advantageously be employed in combination with one or more other therapeutic agents, including an antibiotic, anti-fungal, anti-viral, non-steroidal anti-inflammatory drug or disease modifying anti-rheumatic drug.
  • the QUB 919 peptides may be combined with agents such as TNF-alpha inhibitors, for example, anti- TNF monoclonal antibodies (such as Remicade, CDP-870 and D 2 E 7 ) and TNF receptor immunoglobulin molecules (such as Enbrel®), COX- 2 inhibitors (such as meloxicam, celecoxib, rofecoxib, valdecoxib and etoricoxib) low dose methotrexate, lefiunomide, hydroxychloroquine, d- penicillamine, auranofin or parenteral or oral gold.
  • TNF-alpha inhibitors for example, anti- TNF monoclonal antibodies (such as Remicade, CDP-870 and D 2 E 7 ) and TNF receptor immunoglobulin molecules (such as Enbrel®), COX- 2 inhibitors (such as meloxicam, celecoxib, rofecoxib, valdecoxib and etoricoxib) low dose
  • the QUB 919 peptides may also be used in combination with existing therapeutic agents for the treatment of osteoarthritis.
  • Suitable agents to be used in combination include standard non-steroidal anti- inflammatory agents (hereinafter NTHE's) such as piroxicam, diclofenac, propionic acids such as naproxen, flubiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates such as mefenamic acid, indomethacin, sulindac, apazone, pyrazolones such as phenylbutazone, salicylates such as aspirin, COX-2 inhibitors such as celecoxib, valdecoxib, rofecoxib and etoricoxib, analgesics and intraarticular therapies such as corticosteroids and hyaluronic acids such as hyalgan and synvisc.
  • NTHE's such as piroxicam, diclofenac
  • the QUB 919 peptides may also be used in combination with anticancer agents such as endostatin and angiostatin or cytotoxic drugs such as adriamycin, daunomycin, cis-platinum, etoposide, taxol, taxotere and alkaloids, such as vincristine, farnesyl transferase inhibitors, VEGF inhibitors, and antimetabolites such as methotrexate.
  • anticancer agents such as endostatin and angiostatin or cytotoxic drugs such as adriamycin, daunomycin, cis-platinum, etoposide, taxol, taxotere and alkaloids, such as vincristine, farnesyl transferase inhibitors, VEGF inhibitors, and antimetabolites such as methotrexate.
  • the QUB 919 peptides may also be used in combination with antiviral agents such as Viracept, AZT, aciclovir and famciclovir, and antisepsis compounds such as Zovant, tifacogin, NOX-100 and 13R270773.
  • antiviral agents such as Viracept, AZT, aciclovir and famciclovir
  • antisepsis compounds such as Zovant, tifacogin, NOX-100 and 13R270773.
  • the QUB 919 peptides may also be used in combination with cardiovascular agents such as calcium channel blockers, lipid lowering agents such as statins, fibrates, beta-blockers, Ace inhibitors, Angiotensin-2 receptor antagonists and platelet aggregation inhibitors.
  • cardiovascular agents such as calcium channel blockers, lipid lowering agents such as statins, fibrates, beta-blockers, Ace inhibitors, Angiotensin-2 receptor antagonists and platelet aggregation inhibitors.
  • the QUB 919 peptides may also be used in combination with CNS agents such as antidepressants (such as sertraline), anti-Parkinsonian drugs (such as deprenyl, L-dopa, Requip, Mirapex, MAOB inhibitors such as selegine and rasagiline, comP inhibitors such as Tasmar, A-2 inhibitors, dopamine reuptake inhibitors, NMDA antagonists, nicotine agonists, Dopamine agonists and inhibitors of neuronal nitric oxide synthase), and anti-Alzheimer's drugs such as donepezil, tacrine, COX-2 inhibitors, propentofylline or metryfonate.
  • CNS agents such as antidepressants (such as sertraline), anti-Parkinsonian drugs (such as deprenyl, L-dopa, Requip, Mirapex, MAOB inhibitors such as selegine and rasagiline, comP inhibitors such as Tasmar, A-2 inhibitor
  • the QUB 919 peptides may also be used in combination with osteoporosis agents such as raloxifene, droloxifene, lasofoxifene or fosomax and immunosuppressant agents such as FK-506 and rapamycin.
  • osteoporosis agents such as raloxifene, droloxifene, lasofoxifene or fosomax
  • immunosuppressant agents such as FK-506 and rapamycin.
  • the QUB 919 peptides may be combined with one or more of the following: (a) leukotriene biosynthesis inhibitors: 5-lipoxygenase (5-LO) inhibitors and 5-lipoxygenase activating protein (FLAP) antagonists selected from the group consisting of zileuton; ABT-761 ; fenleuton; tepoxalin; Abbott-79175; Abbott-85761 ; N-(5-substituted)-thiophene- 2alkylsulfonamides of Formula (5.2.8); 2,6-di-tert-butylphenol hydrazones of Formula (5.2.10); the class of methoxytetrahydropyrans which includes Zeneca ZD-2138 of Formula (5.2.11); the compound SB-210661 of Formula (5.2.12) and the class to which it belongs; the class of pyridinyl-substituted 2-cyanonaphthalene compounds to which L-739,010 belongs; the class of 2-cyan
  • Targeting therapies may be used to deliver the active agent e.g. peptide more specifically to arterial smooth muscle, by using targeting systems such as antibody or cell specific ligands. These targeting systems can be covalently linked to the peptide sequence, or to a drug delivery vehicle (such as a liposome, microsphere, microparticle, microcapsule and the like).
  • the peptides can also be targeted to ischemic heart tissue or to growing tumor beds (both of which are associated with attached capillary beds) by incorporating the peptides into microparticles or other drug delivery vehicles that are suitably sized so that they pass through the veins but lodge in capillary beds. When lodged in the beds, the peptides can be locally released (rather than systemically released) at a location where they are most useful.
  • the present invention further extends to methods of gene therapy using nucleotides encoding QUB 919 peptides.
  • Figure 1 illustrates the effect of QUB 919 on phenylephrine preconstricted rat tail arteries
  • Figure 3 illustrates the effect of QUB 9,19 on PdT-1 tryptophyllin (10 " 6 M) induced dilation of phenylephrine preconstricted rat tail arteries;
  • Figure 4 illustrates the effect of QUB 919 on bradykinin induced relaxation in the isolated rat-tail artery model
  • Figure 5 illustrates the nucleotide sequence of cDNA encoding the open-reading frame of the precursor of QUB 919, the putative signal peptide (single underlined) and the mature peptide (double underlined);
  • Figure 6 illustrates the effect of QUB 919 on HMEC-1 Migration in in vitro wound scrape
  • Figure 7 illustrates the effect of QUB 919 on the ability of HMEC-1 cells to form tubule structures on Matrigel matrices; and Figure 8 illustrates the effect of QUB 919 on the proliferation of Human Microvascular Endothelial Cells after 24 hours exposure;
  • Figure 9(a) illustrates QUB919 peptides of and for use in the invention; in the table the amino acid residue number is listed on the top row; AA2 is Pro or Hyp, AA3 is Pro or Hyp, AA4 is any amino acid, AA6 is any amino acid, AA8 is any amino acid X is an NH2 group or AA8
  • Figure 9(b) illustrates further QUB919 peptides of and for use in the invention; in the table the amino acid residue number is listed on the top row; AA1 is any amino acid, AA3 is any amino acid, AA5 is any amino acid, AA6 is Pro or Hyp, AA7 is Pro or Hyp X is an NH2 group or AA 1
  • bradykinin B 2 receptor The EC 5O of bradykinin was 2.18 x 10 "7 M and the EC 50 of bradykinin + QUB 919 was 3.43 x 10 "5 M. These effects that have been shown to be mediated via the bradykinin B 2 receptor.
  • QUB 919 is thus the prototype of a new class of naturally- occuring peptides that can antagonise this receptor in nanomolar concentrations.
  • Figure 5 shows the nucleotide sequence of cDNA encoding the open- reading frame of the precursor of tryptophyllin from the skin secretion of P. sauvagei.
  • the amino acid residues constituting the putative signal peptide are single-underlined and those encoding the mature peptide present in skin secretion, are double-underlined.
  • the stop codon is indicated by an arrow.
  • QUB 919 was assessed for potential anti-angiogenic properties properties.
  • the first of such bioassays assessed the effects of the peptide on the ability of human microvessel endothelial cells to migrate into an induced wound on a confluent monolayer of cells. Pro- angiogenics accelerate this process and anti-angiogenics delay wound closure.
  • Figure 6 shows that QUB 919 inhibits wound closure when compared to time matched controls at an optimum concentration of 1 x 10 "7 M.
  • the EC 50 of was 8.7 x 10- 10 M.
  • Matrigel is a complex cell culture medium containing growth factors in which cultured human microvascular endothelial cells form pre- angiogenic tubules through an ordered series of taxic responses and morphological changes. Anti-angiogenic agents, when added to such a medium can inhibit this process.
  • QUB 919 dose-dependently inhibits tubule formation by microvessel endothelial cells in this medium with an EC 50 of 7.76x10 "10 M and an optimum concentration of 10 '7 M.
  • the final assay that is normally employed examines the proliferative effects of agents on the microvessel endothelial cells by use of the MTT assay.
  • Figure 8 clearly shows that peptide QUB 919 does not affect proliferation of such cells when compared with time - matched controls.
  • a tryptophyllin from the skin secretion of the South American leaf frog Phyllomedusa sauvagei thus represents a prototype peptide with potent and highly-specific antagonist properties at the mammalian bradykinin B 2 receptor.
  • the peptide also displays potent anti- angiogenic properties in the bioassays employed.

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Abstract

L'invention porte sur le peptide QUB919, présentant la séquence Lys-Pro-Pro-Pro-Trp- Val-Pro-Val (SEQ ID NO: 1), et sur ses fragments et analogues dont ses peptides inverses, et sur leur utilisation en tant qu'antagonistes du récepteur de la bradykinine ou qu'agents anti-angiogènes.
PCT/GB2008/001961 2007-06-06 2008-06-06 Precurseur de la tryptophylline-1 et ses utilisations WO2008149115A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004074312A2 (fr) * 2003-02-05 2004-09-02 University Of Ulster Peptides de tryptophylline et utilisations correspondantes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004074312A2 (fr) * 2003-02-05 2004-09-02 University Of Ulster Peptides de tryptophylline et utilisations correspondantes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEN TIANBAO ET AL: "Pachymedusa dacnicolor tryptophyllin-1: Structural characterization, pharmacological activity and cloning of precursor cDNA", REGULATORY PEPTIDES, ELSEVIER SCIENCE BV, NL, vol. 117, no. 1, 15 January 2004 (2004-01-15), pages 25 - 32, XP002303677, ISSN: 0167-0115 *

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