WO2009053725A2 - Peptides and uses thereof - Google Patents
Peptides and uses thereof Download PDFInfo
- Publication number
- WO2009053725A2 WO2009053725A2 PCT/GB2008/003637 GB2008003637W WO2009053725A2 WO 2009053725 A2 WO2009053725 A2 WO 2009053725A2 GB 2008003637 W GB2008003637 W GB 2008003637W WO 2009053725 A2 WO2009053725 A2 WO 2009053725A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- peptide
- cgat
- seq
- fragment
- acid sequence
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- 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
- C07K14/575—Hormones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to peptides, compositions and uses thereof.
- it relates to Chromogranin A (CgA) derived peptide fragments and their use in methods of therapy.
- CgA Chromogranin A
- Chromogranin A (CgA), a protein of neuroendocrine cell secretory granules, has long been postulated to represent a precursor of bioactive peptides by nature of the fact that in normal physiological and in many pathological conditions, CgA-derived peptides are abundant in both tissues and the circulation.
- CgA-derived peptides are abundant in both tissues and the circulation.
- the primary structure of Chromogranin A contains multiple paired basic amino acid residue sites consistent with site-directed non-random endogenous prohormone convertase processing.
- CgA-derived peptides Although many biological activities have been associated with CgA-derived peptides, a large number of those CgA- derived peptides used have been generated from intact CgA by inappropriate and often exogenous proteases such as trypsin and endoproteinase Lys-C, with little or unconvincing evidence that they exist in vivo. Additionally, some, such as the archetypal CgA-derived peptide, pancreastatin, are derived from relatively poorly-conserved domains within the intact protein.
- the classical CgA-derived peptides have inhibitory effects such as pancreastatin, which inhibits insulin release from the pancreas; vasostatin, which inhibits vasoconstriction; catestatin and chromostatin, which inhibit catecholamine-release from adrenomedullary cells; and parastatin, which inhibits parathormone release from parathyroid cells.
- pancreastatin which inhibits insulin release from the pancreas
- vasostatin which inhibits vasoconstriction
- catestatin and chromostatin which inhibit catecholamine-release from adrenomedullary cells
- parastatin which inhibits parathormone release from parathyroid cells.
- Two of the present inventors, Shaw and Chen have previously reported the structures of the authentic CgA-derived peptides generated by endogenous prohormone convertases in human neuroendocrine tumours (Proteomics. 2002 Nov; 2(11):1586-600). (See also UniProtKB/
- each of these peptides stimulates release of insulin from human islets of Langerhans cells.
- the present invention enables the novel use of such Chromogranin A tropic (CGAT) peptides, as described herein, as human tropins.
- CGAT Chromogranin A tropic
- Such peptides may be used, for example, in the treatment of diseases which are associated with insulin deregulation, such as diabetes mellitus and related disorders.
- a method of stimulating release of a regulatory substance from a cell comprising administering a Chromogranin A tropic (CGAT) peptide or a polynucleotide encoding a CGAT peptide to said cell.
- CGAT Chromogranin A tropic
- a Chromogranin A tropic (CGAT) peptide is a fragment, or variant thereof, of a full-length Chromogranin A peptide, , which is able to stimulate release of a regulatory substance from a cell in the absence of a stimulatory concentration of glucose, i.e. in the absence of glucose or in the presence of a sub-stimulatory concentration of glucose.
- a sub-stimulatory concentration of glucose is less than 4mM, for example less than 3mM, such as 2mM.
- the stimulation of regulatory substance release or activity by a CGAT peptide is glucose independent.
- a regulatory substance is any naturally secreted substance which has a modulatory effect on another cell.
- Such regulatory substances may include, but are not limited to, hormones, peptides, biogenic amines and neurotransmitters.
- the regulatory substance is a hormone.
- the regulatory substance is insulin.
- the present inventors have shown that the amount of insulin released from a beta cell line is increased significantly by each of AL-11 GV-19, and WE-14 peptides over what would be expected within the time frame of observation in the absence of any extrinsic stimulation.
- the CGAT peptide is derived from the C-terminal region of Chromogranin A.
- the C-terminal region of Chromogranin A consists of the region of Chromogranin A having the amino acid sequence corresponding to amino acid residue 233 through to the amino acid residue 439 of the full length Chromogranin A sequence, as shown in Figure 1.
- a CGAT peptide for use in the invention comprises an amino acid sequence as shown as SEQ ID NO: 1 (AL-11):
- the CGAT peptide consists of a polypeptide having the amino acid sequence as shown as SEQ ID NO:1.
- the CGAT peptide comprises an amino acid sequence as shown as SEQ ID NO: 2 (GV-19):
- the CGAT peptide consists of a polypeptide having the amino acid sequence as shown as SEQ ID NO:2.
- the CGAT peptide is a peptide which comprises an amino acid sequence as shown as SEQ ID NO: 3
- Trp-Ser-X-Met-Asp-Y-Leu-Ala-Lys-Glu-Leu-Thr-Ala-Glu (SEQ ID NO. 3) wherein X represents Lys or Arg; and Y represents GIn or Arg.
- X is Lys and Y is GIn.
- the CGAT peptide peptide consists of a polypeptide having the amino acid sequence as shown as SEQ ID NO: 4 (WE-14):
- the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 5
- the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 6:
- the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 7:
- X represents Lys or Arg
- Y represents GIn or Arg
- X is Lys and Y is GIn.
- the WE14 reverse peptide consists of the amino acid sequence as shown as SEQ ID NO: 8.:
- the CGAT peptide consists of of a polypeptide having theamino acid sequence as shown as SEQ ID NO: 5 or SEQ ID NO: 6, SEQ ID NO: 7or SEQ ID NO: 8.
- a CGAT peptide can be formed of a continuous sequence of at least TO amino acids, for example at least 20 amino acids, such as at least 30 amino acids, at least 40 amino acids, at least 50 amino acids comprising the contiguous amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4,SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.
- the present invention also encompasses the use of derivatives and fragments of CGAT peptides as defined herein.
- Derivatives of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 include peptides having one or more, for example two, three, four or five, amino acid variations, wherein said variations comprise substitutions, insertions, deletions, or a combination thereof, of the contiguous amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.
- the derivatives may have additional amino acids at the N- or C-terminals, N-terminal and/or C- terminal capping, or protease resistant peptide bonding.
- Derivatives for use in the present invention retain the ability to stimulate release of a regulatory substance from a cell in the absence of a stimulatory concentration of glucose. Unless the context demands otherwise, references to CGAT peptides for use in the invention encompass such derivatives and fragments.
- the cell from which release of the regulatory substance is stimulated is an endocrine cell.
- the CGAT Peptide is insulinotropic i.e. capable of stimulating insulin release.
- the cell in which insulin release or activity is stimulated can be any type of insulin secreting cell such as a pancreatic beta cell. In one embodiment the cell is a human pancreatic beta cell.
- Example 1 the inventors have demonstrated enhanced insulin release by the CGAT peptide as described in the present invention at sub-stimulatory concentrations of glucose.
- the stimulation of insulin release or activity by a CGAT peptide is glucose independent.
- a method of stimulating release of insulin from a cell comprising administering a Chromogranin A tropic (CGAT) peptide or a polynucleotide encoding said CGAT peptide to said cell, in the presence of a stimulatory concentration of glucose.
- CGAT Chromogranin A tropic
- the combination of the stimulatory concentration of glucose and the CGAT peptide results in a synergistic effect upon insulin release.
- the CGAT peptide is a GV-19 peptide, fragment or derivative thereof, for example, a peptide which comprises an amino acid sequence as shown as SEQ ID NO: 2 (GV-19), or a or a fragment or derivative thereof, or a reverse peptide thereof, e.g. peptide which comprises an amino acid sequence as shown as SEQ ID NO: 6, or a fragment or derivative thereof.
- synergistic effect is meant, an effect which is greater than the additive effects of administration of the CGAT peptide alone and the glucose alone.
- the synergistic effect provides at least a 2 fold increase, such as at least a 5 fold increase, over the calculated additive effect.
- a stimulatory concentration of glucose is a concentration at which glucose, when administered alone, stimulates release of a regulatory substance in the cells of interest.
- the stimulatory concentration of glucose is greater than 5 mM, for example, greater than 7mM, such as, greater than 1OmM but less than or equal to 2OmM.
- the methods of the invention may be performed in vitro. Alternatively, they may be performed in vivo. Where practised in vivo, the methods of the invention may be used in methods of treatment of animals or humans.
- the invention may be used to treat disorders or conditions associated with decreased activity of a regulatory substance, for example a hormone, for example insulin activity.
- a regulatory substance for example a hormone, for example insulin activity.
- Such conditions may be the result of decreased release of such a regulatory substance or may, alternatively or additionally, be the result of decreased uptake or activity of the substance at a site of action.
- a method of increasing calcium concentration within a cell comprising administering a Chromogranin A tropic (CGAT) peptide or a polynucleotide encoding a CGAT peptide to said cell.
- CGAT Chromogranin A tropic
- a method of treating a condition or disorder associated with an imbalance in a regulatory substance, for example hormone, in a subject comprising administering a therapeutically effective amount of a CGAT peptide or polynucleotide encoding a CGAT peptide, to a subject in need thereof.
- imbalance in a regulatory substance is meant a deficiency in the regulatory substance wherein the secretion or release of the regulatory substance is impaired or inhibited, e.g. a hyposecretory condition.
- the condition is a hyposecretory condition.
- the term “imbalance” should also be understood to refer to conditions in, which secretion is within normal ranges but activity or uptake is reduced; the increase in regulatory substance released as a result of the CGAT peptide may overcome such reduced activity or uptake.
- the regulatory substance imbalance is an imbalance in a hormone, e.g. insulin.
- the CGAT peptides described in the present invention are secretory granule proteins common to many neuroendocrine cells
- the utility of the CGAT peptides as stimulatory agents as described herein may be used to treat hyposecretory states in many different endocrine cells. Examples of such states include, but are not limited to, hypothyroidism and hypopituitarism and the like.
- site specific delivery can be used to deliver the CGAT peptide to a predetermined and localised area in the body.
- site specific delivery may be achieved by the use of implants comprising the CGAT peptide, for example coupled to an inert carrier polymer, being implanted in the area of interest.
- targetting molecules for example targetting antibodies, may be used to deliver the CGAT peptide to specific cells.
- a CGAT peptide, or polynucleotide encoding a CGAT for use in medicine, for example in the treatment of a condition or disorder associated with deregulation of endocrine release of a regulatory substance.
- a CGAT peptide, or polynucleotide encoding a CGAT for use in the treatment of a condition or disorder associated with deregulation of endocrine release of a regulatory substance.
- a therapeutically effective amount of a CGAT peptide in the preparation of a medicament for the treatment of a condition or disorder associated with deregulation of release of a regulatory substance.
- a therapeutically effective amount of a CGAT peptide and (ii) a stimulatory amount of glucose in the preparation of a medicament for the treatment of a condition or disorder associated with deregulation of endocrine release of a regulatory substance is provided.
- condition or disorder for which the invention may be used is associated with an imbalance or hyposecretion of an endocrine.
- the endocrine is insulin.
- the CGAT peptides of and for use in the invention may be used to treat a condition associated with insulin secretion deficiency or insulin resistance, such as diabetes, e.g. Type I or Type 2 diabetes, borderline diabetes, or a related condition.
- the endocrine is pituitary hormone.
- the CGAT peptides of and for use in the invention may be used to treat a pituitary disorder such as hypopituarism.
- the endocrine is thyroid hormone.
- CGAT peptide of and for use in the invention may be used to treat a thyroid disorder such as hypothyroidism.
- CGAT peptides As described in the Examples, the inventors investigated the effect of CGAT peptides on resistance to apoptosis. Each of the CGAT peptides tested was shown to have no adverse effect on cell survival or indeed a cytoprotective effect. This demonstration of a cytoprotective effect associated with the use of CGAT peptides may be exploited in treatment regimens involving the transplant ion of cells, for example in the transplantation of beta cells for the treatment of diabetes. Beta cell transplants are associated with problems in cell survival; although it is estimated that a patient can lose more than 80% of the islets of the pancreas without the development of diabetes, in transplantation typically much greater than 20% of the typical normal number of islets required before successful restoration of normal blood glucose levels are achieved. The demonstration of cytoprotection using CGAT peptides may thus boost the survival of beta cells following transplantation and enhance the success of transplantation, particularly of pancreatic beta cells.
- a method of enhancing survival of transplanted cells comprising administering a Chromogranin A tropic (CGAT) peptide, or a polynucleotide encoding a CGAT peptide, to a transplant recipient.
- CGAT Chromogranin A tropic
- a CGAT peptide, or polynucleotide encoding a CGAT peptide for use in enhancing the viability of transplanted cells in a transplant recipient.
- the transplanted cells are pancreatic beta cells.
- the transplanted cells are pituitary cells.
- the transplanted cells are thyroid cells.
- the CGAT peptide is GV-19 or a fragment or variant or reverse peptide thereof.
- the CGAT peptide is WE-14 or a fragment, variant or derivative or reverse peptide thereof.
- a pharmaceutical composition comprising a CGAT peptide and a pharmaceutically acceptable carrier or excipient.
- the present invention is based on the demonstration that fragments of Chromogranin A, fragments of which have hitherto all been believed to have either inhibitory or no biological activity, have stimulatory activity.
- the invention thus provides the use of this class of Chromogranin peptide fragment, herein referred to Chromogranin A tropic (CGAT) peptides, in the stimulation of release of regulatory substance(s) from a cell.
- CGAT Chromogranin A tropic
- a CGAT peptide for use in the invention comprise amino acid sequence as shown as SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 , SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or fragments or derivatives thereof.
- a fragment of SEQ ID NO: 1 or SEQ ID NO: 5 can be a peptide having at least 6, for example at least 7, at least 8, at least 9, or at least 10 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 5.
- a fragment of SEQ ID NO: 2 or SEQ ID NO: 6 can be a peptide having at least 6, for example at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 6.
- a fragment of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 8 can be a peptide having at least 6, for example at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, or at least 13 contiguous amino acids of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 8.
- a derivative for use in the invention means a polypeptide modified by varying the amino acid sequence of a CGAT peptide, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself.
- Such derivatives of natural CGAT peptides such as AL-11 , GV-19, or WE- 14 amino acid sequence may involve one, two, three, four or five amino acid variations, wherein said variations comprise substitutions, insertions, deletions, or a combination thereof, while providing a peptide capable of selectively stimulating tropic responses in regulatory substance-producing cells.
- a coupling partner e. g. an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule.
- CGAT analogues may comprise peptides of the invention linked, for example, to antibodies that target the peptides to particular regulatory substance producing cells, such as endocrine cells.
- the antibodies may target the peptides to particular endocrine cells, for example insulin producing cells.
- the CGAT peptides described herein may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1 , CH2, CH3, or any combination thereof or portions thereof), resulting in chimeric polypeptides.
- immunoglobulins IgA, IgE, IgG, IgM
- CH1 , CH2, CH3, or any combination thereof or portions thereof resulting in chimeric polypeptides.
- fusion proteins can facilitate purification and show an increased half-life in vivo.
- 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 which may be used in the invention also include CGAT 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).
- Derivatives for use in the present invention also include reverse-or retro- analogues of natural CGAT peptides or their synthetic derivatives. See, for example, EP 0497 366, U.S. 5,519,115, and Merrifield et al., 1995, PNAS, 92:3449-53, 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 peptides.
- Such reverse-peptides retain the same general three-dimensional structure (e. g., 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). Derivatives of and for use in the present invention may therefore comprise reverse peptides of natural and synthetic CGAT peptides.
- CGAT peptides (including derivatives, 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)).
- CGAT peptides for use in the invention may be in the form of multimers.
- multimers for example of 2, 3 or more individual CGAT peptides monomeric units
- a cleavable site i.e., an enzymatically cleavable site
- 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 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 , SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or fragments, variants, splice variants, fusion proteins, or other CGAT peptides such as AL-11 , GV-19 or WE-14 analogs described herein.
- These homomers may contain CGAT peptides having identical or different amino acid sequences.
- the multimers can include only AL-11 or GV-19 peptides having an identical amino acid sequence, or can include different amino acid sequences.
- the multimer can be a homodimer, homotrimer or homotetramer.
- heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., non-CGAT peptides) in addition to the CGAT 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 CGAT peptides contact one another in solution.
- heteromultimers are formed when for example CGAT and non-CGAT 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 CGAT peptides (and optionally non-CGAT peptides) described herein.
- covalent associations can involve one or more amino acid residues contained in the CGAT sequence (e.g., that recited in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8).
- 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 CGAT 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 CGAT peptide-Fc fusion protein.
- two or more polypeptides described herein are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627. Proteins comprising multiple CGAT peptides separated by peptide linkers can be produced using conventional recombinant DNA technology. Multimers may also be prepared by fusing CGAT peptides to a leucine zipper or isoleucine zipper polypeptide sequence. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
- leucine, zipper domains suitable for producing multimeric proteins described herein are those described in PCT application WO 94/10308.
- Recombinant fusion proteins comprising a peptide described herein fused to a peptide 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.
- 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 addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art 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 CGAT 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.
- CGAT 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 CGAT peptide described herein to 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).
- CGAT peptides that contain a transmembrane domain (or hydrophobic or signal peptide) and that can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925).
- the CGAT 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 associated with regulatory substance e.g. hormone imbalance or deficiency.
- 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 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 polypropropylene 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, MJ. , 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 the invention or pro-drugs from which peptides of the invention (including analogues and fragments) are released or are releaseable 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 1 14 C, 15 N, 18 0, 17 O, and 35 S, respectively.
- peptides of the present invention Use of 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. Peptidomimetics
- the present invention further encompasses the use of mimetic CGAT peptides, which can be used as therapeutic peptides.
- Mimetic CGAT peptides are short peptides which mimic the biological activity of a CGAT peptide.
- 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 CGAT peptides.
- the peptides for use in the method of the present invention may be in the form of a pharmaceutical composition.
- the pharmaceutical composition may comprise, in addition to active ingredients, 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 such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
- 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.
- the peptides for use in the method of the present invention 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.
- the peptides for use in the method of the present invention may be administered alone.
- the peptides for use in the method of the present invention are preferably in the form of a pharmaceutical composition, which will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected dependent on the intended route of administration.
- the peptides for use in the method of the present invention 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 or peptides for use in the method of the present invention.
- Some 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 peptides for use in the method of the present invention 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.
- the peptides for use in the method of the present invention 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 3,773,919; 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 4,485,045 and US 4,544,545. Ordinarily, 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.
- Targeting therapies may be used to deliver the active agent e.g. peptide more specifically, for example, to particular tissue e.g. endocrine tissue such as pancreatic islets, by the use of targeting systems such as antibodies or cell specific ligands.
- the peptides for use in the present invention may be used in the control and/or treatment of a wide variety of clinical conditions in mammals, including humans.
- the peptides for use in the method of the present invention may be used in the treatment of any condition or disorder associated with imbalance of regulatory substances, such as hormone imbalance or deficiency.
- conditions or disorders for which the invention may be used include, but are not limited to, a condition or disorder selected from the following group: diabetes, e.g. Type I or Type 2 diabetes, borderline diabetes, and for treatment or prevention, as appropriate, of conditions such as diabetic retinopathy, neuropathy or nephropathy impaired glucose intolerance, obesity, hypertension, syndrome X, cognitive disorders, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers, dyslipidaemia, polycystic ovarian syndrome (PCOS), acute pancreatitis, chronic pancreatitis and hemochromatosis, hypothyroidism, cretinism and hypopituitarism.
- diabetes e.g. Type I or Type 2 diabetes, borderline diabetes
- conditions such as diabetic retinopathy, neuropathy or nephropathy impaired glucose intolerance, obesity, hypertension, syndrome X, cognitive disorders, atherosclerosis, myo
- the peptides for use in the methods of the present invention may be encoded by polynucleotides or may be in the form of therapeutically acceptable salts thereof.
- 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.
- FIG 1 shows the primary structure of Chromogranin A
- Figure 2 shows a dose response graph demonstrating the effect of 0.1 ⁇ M, 1 ⁇ M and 10 ⁇ M AL-11 on insulin secretion from MIN6 ⁇ -cell monolayers in the presence of 2mM glucose
- Figure 3 shows a dose response graph demonstrating the effect of 0.1 ⁇ M, 1 ⁇ M and 10 ⁇ M GV-19 on insulin secretion from MIN6 ⁇ -cell monolayers in the presence of 2mM glucose;
- Figure 4 shows a graph representing the effect of 1 ⁇ M AL-11 on insulin secretion over time from MIN6 pseudbislets in the presence of 2mM glucose;
- Figure 5 shows a graph representing the effect of 1 ⁇ M GV-19'on insulin secretion over time from MIN6 cell pseudoislets in the presence of 2mM glucose
- Figure 6 shows a graph representing the effect of 1 ⁇ M AL-11 on insulin secretion over time from MIN6 pseudoislets in the presence of 2OmM glucose
- Figure 7 shows a graph representing the effect of 1 ⁇ M GV-19 on insulin secretion over time from MIN6 cell pseudoislets in the presence of 2OmM glucose
- Figure 8 shows a graph representing the effect of 1 ⁇ M AL-11 on intracellular calcium release, [Ca 2+ ] in Fura 2-loaded MIN6 ⁇ -cells in the presence of 2mM glucose;
- Figure 9 shows a graph representing the effect of 1 ⁇ M and 10 ⁇ M GV-19 on intracellular calcium release, [Ca 2+ ] in Fura 2-loaded MIN6 ⁇ -cells in the presence of 2mM glucose;
- Figure 10 shows a graph representing the effect of 1 ⁇ M AL-11 on insulin secretion over time in isolated human islet of Langerhans cells;
- Figure 11 shows a graph representing the effect of 1 ⁇ M GV-19 on insulin secretion over time in isolated human islet of Langerhans cells
- Figure 12 shows a graph representing the effect of 10 ⁇ M GV-19 on intracellular calcium over time in isolated human islet cells
- Figure 13 shows a graph representing the effect of 1 ⁇ M WE-14 on insulin secretion over time in isolated human islet of Langerhans cells
- Figure 14 shows a graph representing the effect of 1 ⁇ M WE-14 on intracellular calcium over time in Fura 2-loaded MIN6 ⁇ -cells
- Figure 15 shows a graph of caspase activity in mouse islets in the presence and absence of 1 ⁇ M AL-11 and 1 ⁇ M GV-19;
- Figure 16 shows a graph of caspase activity in mouse islets in the presence and absence of 1 ⁇ M WE-14.
- FIGs 2 and 3 demonstrate that at a sub-stimulatory concentration of glucose (2mM), a small increase in insulin secretion from the MIN6 mouse ⁇ -cell line was obtained in response to AL-11 and GV-19 (0.1-10 ⁇ M) respectively. These relatively small effects may be a consequence, to some extent, of the late passage of the MIN6 cells used (MIN6 cells lose responsiveness when maintained in culture for extended periods as monolayers).
- the static incubation experiments shown in Figures 2 and 3 indicated that AL-11 and GV-19 could stimulate increases in insulin secretion at a sub- stimulatory concentration of glucose (2mM).
- MIN6 pseudoislets show improved secretory output compared to monolayer MIN6 cells and they more closely resemble primary islets in their characteristics (Hauge-Evans et al., Diabetes 48: 1402-1408, 1999).
- the pseudoislets were exposed to a buffer supplemented with 2mM glucose for 10 minutes after which the media were supplemented with 1 ⁇ M AL-11 or 1 ⁇ M GV-19. Fractions were collected every 2 minutes and insulin secretion was quantified by radioimmunoassay.
- Figure 6 indicates that treatment of MIN6 pseudoislets with a stimulatory concentration of glucose (2OmM) alone or with AL-11 and 2OmM glucose given together stimulates insulin secretion with similar potency.
- treatment with both GV-19 and 2OmM glucose potentiates glucose-stimulated insulin secretion from MIN6 pseudoislets when compared with 2OmM glucose treatment alone as shown in Figure 7. This synergistic effect is reversible.
- Figures 8 and 9 represent calcium microfluorimetry experiments with Fura 2-loaded MIN6 cells which indicate that both the AL-11 and GV-19 peptide stimulate an increase in intracellular [Ca 2+ ] , at the same concentration (1 ⁇ M) at which they stimulate insulin secretion. Data points represent the mean ⁇ SEM, 84 cells ( Figure 8) and 50 cells ( Figure 9).
- Both AL-11 and GV-19 stimulated rapid, reversible increases in insulin secretion at both 2mM and 2OmM glucose.
- Isolated human islets were dissociated by exposure to trypsin, and cells were plated onto glass coverslips.
- Adherent human islet cells were loaded with 10 ⁇ M Fura 2/AM and exposed to 10 ⁇ M GV-19 (shown by bar) in the presence of 2mM glucose. The results are shown in Figure 12. GV-19 stimulated a reversible increase in Ca 2+ levels. Data are mean ⁇ SEM, 20 separate cells.
- Isolated human islets were perifused with a physiological salt solution at 2mM glucose and supplemented with 1 ⁇ M WE-14 as shown in Figure 13.
- WE-14 stimulated a rapid, reversible increase in insulin secretion at 2mM glucose.
- cytokine induced caspase activity was measured in mouse islets in the presence and absence of each of AL-11 ( Figure 15), GV-19 ( Figure 15) and WE-14 ( Figure 16). Briefly, isolated mouse islets were maintained overnight in standard culture medium (white bars) or in medium supplemented with mixed: cytokines (IL-I , IFN ⁇ , TNF ⁇ ; black bars) in the absence (control) or presence of 1 ⁇ M of AL-11 , GV-19 or WE-14. Islet caspase activity was quantified by a commercial luminescence kit.
- Cytokines significantly (P ⁇ 0.01 ) elevated caspase activity and this was abolished by 1 ⁇ M AL-11 (P ⁇ 0.05 versus control; P>0.2 versus non-cytokine-treated islets) and reduced by 1 ⁇ M WE-14 (P ⁇ 0.05 versus control);. Data are mean ⁇ SEM, n 8 groups of islets.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to fragments of Chromogranin A (CgA) derived peptide fragments and their use in methods of therapy, for example in the treatment of diabetes etc. The peptides, for example AL-11, GV-19 and WE-14, are tropic, i.e stimulate release of regulatory substances and may be used in the absence of a stimulatory concentration of glucose.
Description
PEPTIDES AND USES THEREOF
The present invention relates to peptides, compositions and uses thereof. In particular, it relates to Chromogranin A (CgA) derived peptide fragments and their use in methods of therapy.
Introduction
Chromogranin A (CgA), a protein of neuroendocrine cell secretory granules, has long been postulated to represent a precursor of bioactive peptides by nature of the fact that in normal physiological and in many pathological conditions, CgA-derived peptides are abundant in both tissues and the circulation. In addition, the primary structure of Chromogranin A contains multiple paired basic amino acid residue sites consistent with site-directed non-random endogenous prohormone convertase processing. Although many biological activities have been associated with CgA-derived peptides, a large number of those CgA- derived peptides used have been generated from intact CgA by inappropriate and often exogenous proteases such as trypsin and endoproteinase Lys-C, with little or unconvincing evidence that they exist in vivo. Additionally, some, such as the archetypal CgA-derived peptide, pancreastatin, are derived from relatively poorly-conserved domains within the intact protein. The classical CgA-derived peptides have inhibitory effects such as pancreastatin, which inhibits insulin release from the pancreas; vasostatin, which inhibits vasoconstriction; catestatin and chromostatin, which inhibit catecholamine-release from adrenomedullary cells; and parastatin, which inhibits parathormone release from parathyroid cells.
Two of the present inventors, Shaw and Chen have previously reported the structures of the authentic CgA-derived peptides generated by endogenous prohormone convertases in human neuroendocrine tumours (Proteomics. 2002 Nov; 2(11):1586-600). (See also UniProtKB/Swiss-Prot P10645 (CMGAJHUMAN). Reviewed. P10645; Q96E84; Q96GL7; Q9BQB5. 24 July 2007, entry version 93. Under derived peptides). A series of novel peptides were described for the first time, including AL-11 (CGA 380-390, numbering includes signal peptide) and GV-19 (CGA 393- 411 , numbering includes signal peptide) all of which were consistent and major products of CgA processing in a range of such tumours. In addition, a peptide representing the rat/mouse homologue of AL-11 has been found in extracts of brain tissue from these rodents indicating that its generation is not species-specific or artefactual (Svensson M, Skold K, Svenningsson P and Andren PE. (2003) Peptidomics-based discovery of novel neuropeptides. Journal of Proteome Research, 2, 213-219). Another CgA peptide, WE-14, representing residues 325-338 of human chromogranin A, has been described by Curry et al. 1992 (FEBS Lett. 301 ,319). This peptide was isolated from a human carcinoid tumour and was subsequently found to be present in a wide spectrum of normal and neoplastic human neuroendocrine tissues. However, neither the biological activity nor the physiological role for each of AL-11 , GV-19, or WE-14, each of which are derived from the C-terminal of the full length CgA, have been previously described.
Summary of the Invention
In an effort to define the functional properties of these peptides, the present inventors have utilised the CgA C-terminal fragments AL-11 , GV- 19 and WE-14 in a set of experiments. Surprisingly, the results of these experiments demonstrate that each of these peptides have biological
activity. Moreover, in contrast to all CgA peptides for which biological activity has previously been identified, the activity associated with these peptides is not inhibitory. Indeed the inventors have shown that each of these CgA C-terminal-derived peptides have tropic effects Specifically the inventors have shown that these peptides stimulate insulin release from a mouse beta cell line, MIN6, in an apparently glucose-independent manner. Moreover, as described in the Examples, each of these peptides stimulates release of insulin from human islets of Langerhans cells. Thus, the present invention enables the novel use of such Chromogranin A tropic (CGAT) peptides, as described herein, as human tropins. Such peptides may be used, for example, in the treatment of diseases which are associated with insulin deregulation, such as diabetes mellitus and related disorders.
Thus, according to the present invention, there is provided a method of stimulating release of a regulatory substance from a cell wherein the method comprises administering a Chromogranin A tropic (CGAT) peptide or a polynucleotide encoding a CGAT peptide to said cell.
In the context of the present invention, a Chromogranin A tropic (CGAT) peptide is a fragment, or variant thereof, of a full-length Chromogranin A peptide, , which is able to stimulate release of a regulatory substance from a cell in the absence of a stimulatory concentration of glucose, i.e. in the absence of glucose or in the presence of a sub-stimulatory concentration of glucose.
In an embodiment of the invention, a sub-stimulatory concentration of glucose is less than 4mM, for example less than 3mM, such as 2mM.
Thus, in one embodiment, the stimulation of regulatory substance release or activity by a CGAT peptide is glucose independent.
In the context of the present invention, a regulatory substance is any naturally secreted substance which has a modulatory effect on another cell. Such regulatory substances may include, but are not limited to, hormones, peptides, biogenic amines and neurotransmitters. In one embodiment, the regulatory substance is a hormone. In one particular embodiment, the regulatory substance is insulin.
As described in the Examples, the present inventors have shown that the amount of insulin released from a beta cell line is increased significantly by each of AL-11 GV-19, and WE-14 peptides over what would be expected within the time frame of observation in the absence of any extrinsic stimulation.
In one embodiment, the CGAT peptide is derived from the C-terminal region of Chromogranin A. As herein defined, the C-terminal region of Chromogranin A consists of the region of Chromogranin A having the amino acid sequence corresponding to amino acid residue 233 through to the amino acid residue 439 of the full length Chromogranin A sequence, as shown in Figure 1.
In one embodiment, a CGAT peptide for use in the invention comprises an amino acid sequence as shown as SEQ ID NO: 1 (AL-11):
Ala-Tyr-Gly-Phe-Arg-Gly-Pro-Gly-Pro-Gln-Leu (SEQ ID NO: 1)
or a fragment or derivative thereof.
In a particular embodiment, the CGAT peptide consists of a polypeptide having the amino acid sequence as shown as SEQ ID NO:1.
In an alternative embodiment, the CGAT peptide comprises an amino acid sequence as shown as SEQ ID NO: 2 (GV-19):
Gly-Trp-Arg-Pro-Ser-Ser-Arg-Glu-Asp-Ser-Leu-Glu-Ala-Gly-Leu-Pro-Leu- GIn-VaI (SEQ ID NO: 2)
or a fragment or derivative thereof.
In a particular embodiment, the CGAT peptide consists of a polypeptide having the amino acid sequence as shown as SEQ ID NO:2.
In a further particular embodiment, the CGAT peptide is a peptide which comprises an amino acid sequence as shown as SEQ ID NO: 3
Trp-Ser-X-Met-Asp-Y-Leu-Ala-Lys-Glu-Leu-Thr-Ala-Glu (SEQ ID NO. 3) wherein X represents Lys or Arg; and Y represents GIn or Arg.
In one such embodiment, X is Lys and Y is GIn. In a particular embodiment, the CGAT peptide peptide consists of a polypeptide having the amino acid sequence as shown as SEQ ID NO: 4 (WE-14):
Trp-Ser-Lys-Met-Asp-Gln-Leu-Ala-Lys-Glu-Leu-Thr-Ala-Glu
(SEQ ID NO. 4)
It has been found that reverse peptides i.e. peptides in which the amino acid sequence is reversed from that of a naturally occurring or synthetic peptide, can retain the biological activity of the non-reversed peptide.
Accordingly, in a further embodiment of the present invention the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 5
Leu-Gln-Pro-Gly-Pro-Gly-Arg-Phe-Gly-Tyr-Ala (SEQ ID NO: 5)
or a fragment or derivative thereof.
In a further embodiment of the present invention, the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 6:
Val-Gln-Leu-Pro-Leu-Gly-Ala- Glu-Leu-Ser-Asp-Glu-Arg-Ser-Ser-Pro-Arg- Trp-Gly (SEQ ID NO: 6)
or a fragment or derivative thereof.
In a further embodiment of the present invention, the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 7:
Glu-Ala-Thr-Leu-Glu-Lys-Ala-Leu-Y-Asp-Met-X-Ser-Trp (SEQ ID NO. 7)
where X represents Lys or Arg; and Y represents GIn or Arg.
In one embodiment, X is Lys and Y is GIn. In a particular embodiment, the WE14 reverse peptide consists of the amino acid sequence as shown as SEQ ID NO: 8.:
Glu-Ala-Thr-Leu-Glu-Lys-Ala-Leu-Gln-Asp-Met-Lys-Ser-Trp (SEQ ID NO. 8)
In particular embodiments of the invention the CGAT peptide consists of of a polypeptide having theamino acid sequence as shown as SEQ ID NO: 5 or SEQ ID NO: 6, SEQ ID NO: 7or SEQ ID NO: 8.
In further embodiments, a CGAT peptide can be formed of a continuous sequence of at least TO amino acids, for example at least 20 amino acids, such as at least 30 amino acids, at least 40 amino acids, at least 50 amino acids comprising the contiguous amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4,SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.
The present invention also encompasses the use of derivatives and fragments of CGAT peptides as defined herein.
Derivatives of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 include peptides having one or more, for example two, three, four or five, amino acid variations, wherein said variations comprise substitutions, insertions, deletions, or a combination thereof, of the contiguous amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In embodiments, alternatively or additionally, the derivatives may have additional amino acids at the N- or C-terminals, N-terminal and/or C- terminal capping, or protease resistant peptide bonding.
Derivatives for use in the present invention retain the ability to stimulate release of a regulatory substance from a cell in the absence of a stimulatory concentration of glucose.
Unless the context demands otherwise, references to CGAT peptides for use in the invention encompass such derivatives and fragments.
In one embodiment of the invention, the cell from which release of the regulatory substance is stimulated is an endocrine cell.
In one embodiment, the CGAT Peptide is insulinotropic i.e. capable of stimulating insulin release. The cell in which insulin release or activity is stimulated can be any type of insulin secreting cell such as a pancreatic beta cell. In one embodiment the cell is a human pancreatic beta cell.
As described in Example 1 , the inventors have demonstrated enhanced insulin release by the CGAT peptide as described in the present invention at sub-stimulatory concentrations of glucose.
Thus, in one embodiment, the stimulation of insulin release or activity by a CGAT peptide is glucose independent.
The demonstration that the stimulatory effects of CGAT peptides may be seen at sub-stimulatory concentrations of glucose suggests that such peptides may be used in the treatment of conditions caused by a deficiency of a regulatory substance, e.g. hormone, which is not directly controlled by glucose levels. Indeed, in view of the findings of the present invention and, given that CGAT peptides, such as AL-11 , GV-19 and WE- 14, have been found to be present in tissues unrelated to insulin secretion, a role for such peptides in regulation of release of other regulatory substances appears very likely.
However, as well as demonstrating a role for these CgA tropic peptides, which is glucose independent, the present inventors, in the course of their
characterisation of these peptides, have also demonstrated a significant surprising elevation in the amount of insulin released by pancreatic beta cells when the cells are treated with a composition including the CGAT peptide, GV-19, and a concentration of glucose which would typically provide insulin stimulation in such a cell. The observed elevation in insulin release was more than additive.
Accordingly, in a further aspect of the present invention, there is provided a method of stimulating release of insulin from a cell, said method comprising administering a Chromogranin A tropic (CGAT) peptide or a polynucleotide encoding said CGAT peptide to said cell, in the presence of a stimulatory concentration of glucose. In a particular embodiment of this aspect of the invention, the combination of the stimulatory concentration of glucose and the CGAT peptide results in a synergistic effect upon insulin release. In an embodiment of this aspect of the invention, the CGAT peptide is a GV-19 peptide, fragment or derivative thereof, for example, a peptide which comprises an amino acid sequence as shown as SEQ ID NO: 2 (GV-19), or a or a fragment or derivative thereof, or a reverse peptide thereof, e.g. peptide which comprises an amino acid sequence as shown as SEQ ID NO: 6, or a fragment or derivative thereof.
By "synergistic effect" is meant, an effect which is greater than the additive effects of administration of the CGAT peptide alone and the glucose alone. In one embodiment, the synergistic effect provides at least a 2 fold increase, such as at least a 5 fold increase, over the calculated additive effect.
In the context of the present invention, a stimulatory concentration of glucose is a concentration at which glucose, when administered alone, stimulates release of a regulatory substance in the cells of interest. In one
embodiment, the stimulatory concentration of glucose is greater than 5 mM, for example, greater than 7mM, such as, greater than 1OmM but less than or equal to 2OmM.
The methods of the invention may be performed in vitro. Alternatively, they may be performed in vivo. Where practised in vivo, the methods of the invention may be used in methods of treatment of animals or humans.
In one embodiment, the invention may be used to treat disorders or conditions associated with decreased activity of a regulatory substance, for example a hormone, for example insulin activity. Such conditions may be the result of decreased release of such a regulatory substance or may, alternatively or additionally, be the result of decreased uptake or activity of the substance at a site of action.
As described in the examples, the inventors have demonstrated that the CGAT peptides tested, when administered to pancreatic beta cells - caused an increase in intracellular calcium concentration, [Ca2+] L
Accordingly, in a further aspect of the invention, there is provided a method of increasing calcium concentration within a cell, wherein the method comprises administering a Chromogranin A tropic (CGAT) peptide or a polynucleotide encoding a CGAT peptide to said cell. In a further aspect of the present invention, there is provided a method of treating a condition or disorder associated with an imbalance in a regulatory substance, for example hormone, in a subject, said method comprising administering a therapeutically effective amount of a CGAT peptide or polynucleotide encoding a CGAT peptide, to a subject in need thereof.
By the term " imbalance" in a regulatory substance is meant a deficiency in the regulatory substance wherein the secretion or release of the regulatory substance is impaired or inhibited, e.g. a hyposecretory condition. In one embodiment, the condition is a hyposecretory condition. However, in an alternative embodiment, the term "imbalance" should also be understood to refer to conditions in, which secretion is within normal ranges but activity or uptake is reduced; the increase in regulatory substance released as a result of the CGAT peptide may overcome such reduced activity or uptake.
In one embodiment, the regulatory substance imbalance is an imbalance in a hormone, e.g. insulin.
However, as the CGAT peptides described in the present invention are secretory granule proteins common to many neuroendocrine cells, the utility of the CGAT peptides as stimulatory agents as described herein, may be used to treat hyposecretory states in many different endocrine cells. Examples of such states include, but are not limited to, hypothyroidism and hypopituitarism and the like.
In one embodiment, site specific delivery can be used to deliver the CGAT peptide to a predetermined and localised area in the body. For example, site specific delivery may be achieved by the use of implants comprising the CGAT peptide, for example coupled to an inert carrier polymer, being implanted in the area of interest. In a particular embodiment, targetting molecules, for example targetting antibodies, may be used to deliver the CGAT peptide to specific cells.
In a fourth aspect of the invention, there is provided a CGAT peptide, or polynucleotide encoding a CGAT, for use in medicine, for example in the
treatment of a condition or disorder associated with deregulation of endocrine release of a regulatory substance.
In a fifth aspect of the invention, there is provided a CGAT peptide, or polynucleotide encoding a CGAT, for use in the treatment of a condition or disorder associated with deregulation of endocrine release of a regulatory substance.
According to a sixth aspect of the present invention, there is provided the use of a therapeutically effective amount of a CGAT peptide in the preparation of a medicament for the treatment of a condition or disorder associated with deregulation of release of a regulatory substance.
In a seventh aspect of the present invention, there is provided (i) a therapeutically effective amount of a CGAT peptide and (ii) a stimulatory amount of glucose in the preparation of a medicament for the treatment of a condition or disorder associated with deregulation of endocrine release of a regulatory substance.
In a particular embodiment of the invention, the condition or disorder for which the invention may be used is associated with an imbalance or hyposecretion of an endocrine.
In one embodiment the endocrine is insulin. Thus, in one embodiment of the invention, the CGAT peptides of and for use in the invention may be used to treat a condition associated with insulin secretion deficiency or insulin resistance, such as diabetes, e.g. Type I or Type 2 diabetes, borderline diabetes, or a related condition.
In another embodiment, the endocrine is pituitary hormone. Thus, in one embodiment of the invention, the CGAT peptides of and for use in the invention may be used to treat a pituitary disorder such as hypopituarism.
In a further embodiment, the endocrine is thyroid hormone. Thus, in one embodiment of the invention, CGAT peptide of and for use in the invention may be used to treat a thyroid disorder such as hypothyroidism.
As described in the Examples, the inventors investigated the effect of CGAT peptides on resistance to apoptosis. Each of the CGAT peptides tested was shown to have no adverse effect on cell survival or indeed a cytoprotective effect. This demonstration of a cytoprotective effect associated with the use of CGAT peptides may be exploited in treatment regimens involving the transplant ion of cells, for example in the transplantation of beta cells for the treatment of diabetes. Beta cell transplants are associated with problems in cell survival; although it is estimated that a patient can lose more than 80% of the islets of the pancreas without the development of diabetes, in transplantation typically much greater than 20% of the typical normal number of islets required before successful restoration of normal blood glucose levels are achieved. The demonstration of cytoprotection using CGAT peptides may thus boost the survival of beta cells following transplantation and enhance the success of transplantation, particularly of pancreatic beta cells.
Accordingly, in an eighth aspect of the present invention, there is provided a method of enhancing survival of transplanted cells, said method comprising administering a Chromogranin A tropic (CGAT) peptide, or a polynucleotide encoding a CGAT peptide, to a transplant recipient.
In a ninth aspect of the invention, there is provided a CGAT peptide, or polynucleotide encoding a CGAT peptide, for use in enhancing the viability of transplanted cells in a transplant recipient.
In one embodiment of the eighth or ninth aspect of the present invention, the transplanted cells are pancreatic beta cells.
In another embodiment of the eighth or ninth aspects of the present invention, the transplanted cells are pituitary cells.
In a further embodiment of the eighth or ninth aspects of the present invention, the transplanted cells are thyroid cells.
In one embodiment of the eighth or ninth aspects of the invention, the CGAT peptide is GV-19 or a fragment or variant or reverse peptide thereof.
In another embodiment of the eighth or ninth aspects of the invention, the CGAT peptide is WE-14 or a fragment, variant or derivative or reverse peptide thereof.
According to an tenth aspect of the present invention, there is provided a pharmaceutical composition comprising a CGAT peptide and a pharmaceutically acceptable carrier or excipient.
Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis.
Detailed Description
As described herein, the present invention is based on the demonstration that fragments of Chromogranin A, fragments of which have hitherto all been believed to have either inhibitory or no biological activity, have stimulatory activity. The invention thus provides the use of this class of Chromogranin peptide fragment, herein referred to Chromogranin A tropic (CGAT) peptides, in the stimulation of release of regulatory substance(s) from a cell.
In particular embodiments, a CGAT peptide for use in the invention comprise amino acid sequence as shown as SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 , SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or fragments or derivatives thereof.
In the context of the present invention, a fragment of SEQ ID NO: 1 or SEQ ID NO: 5 can be a peptide having at least 6, for example at least 7, at least 8, at least 9, or at least 10 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 5. In the context of the present invention, a fragment of SEQ ID NO: 2 or SEQ ID NO: 6 can be a peptide having at least 6, for example at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 6. In the context of the present invention, a fragment of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 8 can be a peptide having at least 6, for example at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, or at least 13 contiguous amino acids of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 8.
A derivative for use in the invention means a polypeptide modified by varying the amino acid sequence of a CGAT peptide, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself.
Such derivatives of natural CGAT peptides such as AL-11 , GV-19, or WE- 14 amino acid sequence may involve one, two, three, four or five amino acid variations, wherein said variations comprise substitutions, insertions, deletions, or a combination thereof, while providing a peptide capable of selectively stimulating tropic responses in regulatory substance-producing cells.
Other derivatives of CGAT peptides which may be used in the present invention include multimeric or fusion peptides including such CGAT peptides, prodrugs including such sequences, CGAT peptides linked to a coupling partner, e. g. an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule. Techniques for coupling CGAT peptides of the invention to both peptidyl and non-peptidyl coupling partners are well known in the art.
CGAT analogues may comprise peptides of the invention linked, for example, to antibodies that target the peptides to particular regulatory substance producing cells, such as endocrine cells. In particular embodiments, the antibodies may target the peptides to particular endocrine cells, for example insulin producing cells.
The CGAT peptides described herein may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1 , CH2, CH3, or any combination thereof or portions thereof), resulting in chimeric polypeptides. These fusion proteins can facilitate purification and show an increased half-life in vivo. 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 which may be used in the invention also include CGAT 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 is also encompassed by the invention.
Derivatives for use in the present invention also include reverse-or retro- analogues of natural CGAT peptides or their synthetic derivatives. See, for example, EP 0497 366, U.S. 5,519,115, and Merrifield et al., 1995, PNAS, 92:3449-53, the disclosures of which are herein incorporated by reference. As described in EP 0497 366, reverse peptides are produced by reversing the amino acid sequence of a naturally occurring or synthetic peptides. Such reverse-peptides retain the same general three-dimensional structure (e. g., 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). Derivatives of and for use in the present invention may therefore comprise reverse peptides of natural and synthetic CGAT peptides.
CGAT peptides (including derivatives, 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. For example, 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)).
Multimeric Peptides
As described above, CGAT peptides for use in the invention may be in the form of multimers. Thus multimers (for example of 2, 3 or more individual CGAT peptides monomeric units) and their use are within the scope of the invention. Moreover, 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.
The multimers can be homomers or heteromers. As used herein, the term homomer refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 , SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or fragments, variants, splice variants, fusion proteins, or other CGAT peptides such as AL-11 , GV-19 or WE-14 analogs described herein. These homomers may contain CGAT peptides having identical or different amino acid sequences. For example, the multimers can include only AL-11 or GV-19 peptides having an identical amino acid sequence, or can include different amino acid sequences. The multimer can be a homodimer, homotrimer or homotetramer.
As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., non-CGAT peptides) in addition to the CGAT 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. Thus, in one embodiment, multimers are formed when CGAT peptides contact one another in solution. In another embodiment, heteromultimers are formed when for example CGAT and non-CGAT peptides contact antibodies to the polypeptides described herein (including antibodies to the heterologous polypeptide sequence in a fusion protein described herein) in solution. In other embodiments, multimers described herein may be formed by covalent associations with and/or between the CGAT peptides (and optionally non-CGAT peptides) described herein.
Such covalent associations can involve one or more amino acid residues contained in the CGAT sequence (e.g., that recited in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8). In one embodiment, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations can involve one or more amino acid residues contained in the heterologous polypeptide sequence in a CGAT fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein described herein (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a CGAT peptide-Fc fusion protein. In another embodiment, two or more polypeptides described herein are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627. Proteins comprising multiple CGAT peptides separated by peptide linkers can be produced using conventional recombinant DNA technology.
Multimers may also be prepared by fusing CGAT peptides to a leucine zipper or isoleucine zipper polypeptide sequence. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine, zipper domains suitable for producing multimeric proteins described herein are those described in PCT application WO 94/10308. Recombinant fusion proteins comprising a peptide described herein fused to a peptide 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.
Multimers may also be generated using chemical techniques known in the art. For example, 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). Additionally, 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). Further, polypeptides described herein may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art 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 CGAT peptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925,).
Alternatively, those multimers including only naturally-occurring amino acids can be formed using genetic engineering techniques known in the
art. Alternatively, those that include post-translational or other modifications can be prepared by a combination of recombinant techniques and chemical modifications. In one embodiment, CGAT 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). For example, polynucleotides coding for a homodimer described herein can be generated by ligating a polynucleotide sequence encoding a CGAT peptide described herein to 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). The recombinant techniques described herein or otherwise known in the art can be applied to generate recombinant CGAT peptides that contain a transmembrane domain (or hydrophobic or signal peptide) and that can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925).
Pro-Drugs
The CGAT 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 associated with regulatory substance e.g. hormone imbalance or deficiency. 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 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. For example, 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 polypropropylene 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 α-2b) in Roberts, MJ. , 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. Chem., 2000, 43, 475-487); the coupling of PEG carboxylic acid to a hydroxy-terminated carboxylic acid linker (Roberts, MJ., J. Pharm. Sci., 1998, 87(11), 1440-1445), and PEG prodrugs involving families of MPEG phenyl ethers and MPEG benzamides linked to an amine-containing drug via an aryl carbamate (Roberts, MJ., et al., Adv. Drug Delivery Rev., 2002, 54, 459-476), including a prodrug structure involving a meta relationship between the carbamate and the PEG amide or ether (U.S. Patent No. 6,413,507 to Bently, et al.); and prodrugs
involving a reduction mechanism as opposed to a hydrolysis mechanism (Zalipsky, S., et al., Bioconjugate Chem., 1999, 10(5), 703-707).
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.
In some cases, 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. Alternatively, or additionally, the peptide can be provided as a prodrug. In one embodiment, the peptides are present in these drug delivery devices as prodrugs.
Prodrugs comprising the peptides of the invention or pro-drugs from which peptides of the invention (including analogues and fragments) are released or are releaseable 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. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine and chlorine, such as 2H, 3H, 13C1 14C, 15N, 180, 17O, and 35S, respectively. Use of 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 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. 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.
Peptidomimetics
The present invention further encompasses the use of mimetic CGAT peptides, which can be used as therapeutic peptides. Mimetic CGAT peptides are short peptides which mimic the biological activity of a CGAT peptide. 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 CGAT peptides.
Pharmaceutical Compositions
The peptides for use in the method of the present invention may be in the form of a pharmaceutical composition. The pharmaceutical composition may comprise, in addition to active ingredients, 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.
Dose
The peptides for use in the method of the present invention 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.
Administration
The peptides for use in the method of the present invention may be administered alone. However, the peptides for use in the method of the present invention are preferably in the form of a pharmaceutical composition, which will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected dependent on the intended route of administration.
The peptides for use in the method of the present invention 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 or peptides for use in the method of the present invention.
Some 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.
For intravenous injection, or injection at the site of affliction, the peptides for use in the method of the present invention will be in the form of a
parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, 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.
Pharmaceutical 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.
The peptides for use in the method of the present invention may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood. Suitable examples of 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 3,773,919; 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 4,485,045
and US 4,544,545. Ordinarily, 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.
Examples of the techniques and protocols mentioned above and other techniques and protocols which may be used in accordance with the invention can be found in Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro AR, et al, eds., Lippincott Williams & Wilkins, 2005.
Targeting therapies may be used to deliver the active agent e.g. peptide more specifically, for example, to particular tissue e.g. endocrine tissue such as pancreatic islets, by the use of targeting systems such as antibodies or cell specific ligands.
Therapeutic Uses
The peptides for use in the present invention may be used in the control and/or treatment of a wide variety of clinical conditions in mammals, including humans. The peptides for use in the method of the present invention may be used in the treatment of any condition or disorder associated with imbalance of regulatory substances, such as hormone imbalance or deficiency.
Examples of conditions or disorders for which the invention may be used include, but are not limited to, a condition or disorder selected from the following group: diabetes, e.g. Type I or Type 2 diabetes, borderline diabetes, and for treatment or prevention, as appropriate, of conditions such as diabetic retinopathy, neuropathy or nephropathy impaired glucose
intolerance, obesity, hypertension, syndrome X, cognitive disorders, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers, dyslipidaemia, polycystic ovarian syndrome (PCOS), acute pancreatitis, chronic pancreatitis and hemochromatosis, hypothyroidism, cretinism and hypopituitarism.
In one embodiment, the peptides for use in the methods of the present invention may be encoded by polynucleotides or may be in the form of therapeutically acceptable salts thereof.
"Treatment" or "therapy" 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.
Throughout the specification, unless the context demands otherwise, the terms 'comprise' or 'include', or variations such as 'comprises' or 'comprising1, 'includes' or 'including' will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.
The present invention will now be described, by way of example only, with reference to the accompanying drawings, wherein;
Figure 1 shows the primary structure of Chromogranin A;
Figure 2 shows a dose response graph demonstrating the effect of 0.1 μM, 1μM and 10 μM AL-11 on insulin secretion from MIN6 β-cell monolayers in the presence of 2mM glucose;
Figure 3 shows a dose response graph demonstrating the effect of 0.1 μM, 1μM and 10 μM GV-19 on insulin secretion from MIN6 β-cell monolayers in the presence of 2mM glucose;
Figure 4 shows a graph representing the effect of 1 μM AL-11 on insulin secretion over time from MIN6 pseudbislets in the presence of 2mM glucose;
Figure 5 shows a graph representing the effect of 1 μM GV-19'on insulin secretion over time from MIN6 cell pseudoislets in the presence of 2mM glucose;
Figure 6 shows a graph representing the effect of 1 μM AL-11 on insulin secretion over time from MIN6 pseudoislets in the presence of 2OmM glucose;
Figure 7 shows a graph representing the effect of 1 μM GV-19 on insulin secretion over time from MIN6 cell pseudoislets in the presence of 2OmM glucose;
Figure 8 shows a graph representing the effect of 1 μM AL-11 on intracellular calcium release, [Ca2+] in Fura 2-loaded MIN6 β-cells in the presence of 2mM glucose; and
Figure 9 shows a graph representing the effect of 1 μM and 10μM GV-19 on intracellular calcium release, [Ca2+] in Fura 2-loaded MIN6 β-cells in the presence of 2mM glucose;
Figure 10 shows a graph representing the effect of 1 μM AL-11 on insulin secretion over time in isolated human islet of Langerhans cells;
Figure 11 shows a graph representing the effect of 1 μM GV-19 on insulin secretion over time in isolated human islet of Langerhans cells;
Figure 12 shows a graph representing the effect of 10 μM GV-19 on intracellular calcium over time in isolated human islet cells;
Figure 13 shows a graph representing the effect of 1 μM WE-14 on insulin secretion over time in isolated human islet of Langerhans cells;
Figure 14 shows a graph representing the effect of 1 μM WE-14 on intracellular calcium over time in Fura 2-loaded MIN6 β-cells;
Figure 15 shows a graph of caspase activity in mouse islets in the presence and absence of 1μM AL-11 and 1μM GV-19; and
Figure 16 shows a graph of caspase activity in mouse islets in the presence and absence of 1μM WE-14.
Example 1
Figures 2 and 3 demonstrate that at a sub-stimulatory concentration of glucose (2mM), a small increase in insulin secretion from the MIN6 mouse β-cell line was obtained in response to AL-11 and GV-19 (0.1-10μM) respectively. These relatively small effects may be a consequence, to some extent, of the late passage of the MIN6 cells used (MIN6 cells lose responsiveness when maintained in culture for extended periods as monolayers).
The static incubation experiments shown in Figures 2 and 3 indicated that AL-11 and GV-19 could stimulate increases in insulin secretion at a sub- stimulatory concentration of glucose (2mM). Hence further experiments were performed to examine the time-course and reversibility of insulin secretion in response to the peptide from MIN6 β-cells configured as 3D clusters (pseudoislets). MIN6 pseudoislets show improved secretory output compared to monolayer MIN6 cells and they more closely resemble primary islets in their characteristics (Hauge-Evans et al., Diabetes 48: 1402-1408, 1999). In these experiments the pseudoislets were exposed to a buffer supplemented with 2mM glucose for 10 minutes after which the media were supplemented with 1μM AL-11 or 1μM GV-19. Fractions were collected every 2 minutes and insulin secretion was quantified by radioimmunoassay.
The perfusion data presented in Figure 4 and Figure 5 indicate that 1 μM of AL-11 or GV-19 initiate insulin secretion at 2mM glucose, with a rapid increase in secretion upon exposure to each peptide.
Figure 6 indicates that treatment of MIN6 pseudoislets with a stimulatory concentration of glucose (2OmM) alone or with AL-11 and 2OmM glucose given together stimulates insulin secretion with similar potency. Surprisingly, treatment with both GV-19 and 2OmM glucose potentiates glucose-stimulated insulin secretion from MIN6 pseudoislets when compared with 2OmM glucose treatment alone as shown in Figure 7. This synergistic effect is reversible.
Since [Ca2+]s plays a key role in stimulus-response coupling in β-cells, experiments to examine whether increases in [Ca2+] might be a possible mode of action of these peptides in β-cells were performed. Figures 8 and
9 represent calcium microfluorimetry experiments with Fura 2-loaded MIN6 cells which indicate that both the AL-11 and GV-19 peptide stimulate an increase in intracellular [Ca2+] , at the same concentration (1μM) at which they stimulate insulin secretion. Data points represent the mean ± SEM, 84 cells (Figure 8) and 50 cells (Figure 9). The increase in [Ca2+] in response to 1μM AL-11 was delayed in onset by ~120s (Figure 8) and a similar delay in the response to 10μM GV-19 is evident from Figure 9, although in this experiment the cells showed a rapid increase in [Ca2+]j in response to 1μM GV-19.
Example 2 Effect of AL-11 and GV-19 on human islet of Langerhans cells
The inventors investigated the effect of AL-11 and GV-19 in human islet cells. Isolated human islets were perifused with a physiological salt solution at 2mM glucose (t=0-50min) or 2OmM glucose (t=51-90min) and supplemented with 1μM AL-11 (Figure 10) or 1μM GV-19 (Figure 11) as shown. Both AL-11 and GV-19 stimulated rapid, reversible increases in insulin secretion at both 2mM and 2OmM glucose. Data are mean±SEM, n=3 groups of islets per experiment; 2 separate experiments.
Example 3 Effect of GV-19 on intracellular calcium in human islet of Langerhans cells
As with the mouse cells, the inventors investigated whether or not the effects of these peptides in the human islet cells may be mediated by an increase in [Ca2+]. Isolated human islets were dissociated by exposure to trypsin, and cells were plated onto glass coverslips. Adherent human islet cells were loaded with 10μM Fura 2/AM and exposed to 10μM GV-19 (shown by bar) in the presence of 2mM glucose. The results are shown in
Figure 12. GV-19 stimulated a reversible increase in Ca2+ levels. Data are mean±SEM, 20 separate cells.
Example 4: Effect of WE-14 on insulin secretion over time in isolated human islet of Langerhans cells
The inventors investigated the effect of the CgA peptide WE-14 on insulin secretion from human islets of Langerhans. Isolated human islets were perifused with a physiological salt solution at 2mM glucose and supplemented with 1μM WE-14 as shown in Figure 13. As can be seen from that Figure, as with AL-11 and GV-19, WE-14 stimulated a rapid, reversible increase in insulin secretion at 2mM glucose. Data are mean±SEM, n=3 groups of islets per experiment; 2 separate experiments.
Example 5: Effect of WE-14 on intracellular calcium in human islet of Langerhans cells
To investigate whether or not the effect of WE-14 on insulin secretion from islets may be via a mechanism of action involving an increase in [Ca2+], the inventors measured intracellular calcium in β-cells in response to WE- 14. MIN6 mouse β-cells were plated onto glass coverslips and loaded with 10μM Fura 2/AM when adherent. As shown in Figure 14, exposure of MIN6 cells to 1μM and 10μM WE-14 at 2mM glucose led to rapid, reversible elevations in intracellular Ca2+ levels. Data are expressed as fluorescence ratio (340/380nm) data, mean±SEM, 60 separate cells.
Example 6 CGAT peptides are not cytotoxic
In conditions such as Type 1 and Type 2 diabetes, the islet cells are under threat and undergo increased apoptosis. To investigate the effect of the
CGAT peptides on such cells, cytokine induced caspase activity was measured in mouse islets in the presence and absence of each of AL-11 (Figure 15), GV-19 (Figure 15) and WE-14 (Figure 16). Briefly, isolated mouse islets were maintained overnight in standard culture medium (white bars) or in medium supplemented with mixed: cytokines (IL-I , IFNγ, TNFα; black bars) in the absence (control) or presence of 1μM of AL-11 , GV-19 or WE-14. Islet caspase activity was quantified by a commercial luminescence kit. Cytokines significantly (P<0.01 ) elevated caspase activity and this was abolished by 1μM AL-11 (P<0.05 versus control; P>0.2 versus non-cytokine-treated islets) and reduced by 1μM WE-14 (P<0.05 versus control);. Data are mean±SEM, n=8 groups of islets.
These results show that none of the CGAT peptides were cytotoxic, with caspase induced apoptosis not significantly increased by their presence. Significantly, AL-11 and WE-14 were found to be cytoprotective, suggesting that they may find particular utility in transplantation of β cells to protect the transplanted cells and to decrease rejection of transplanted cell and thus improve the success of such treatments.
All documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.
Claims
1. A method of stimulating release of a< regulatory substance from a cell wherein the method comprises administering a Chromogranin A tropic (CGAT) peptide or a polynucleotide encoding a CGAT peptide to said cell, wherein said peptide is a fragment of a full-length Chromogranin A peptide, or variant thereof, which is able to stimulate release of a regulatory substance from a cell in the absence of a stimulatory concentration of glucose.
2. A method of treatment of a condition or disorder mediated by or associated with imbalance in a regulatory substance , said method comprising administering a therapeutically effective amount of a CGAT peptide or polynucleotide to a patient in need thereof.
3. The method according to claim 2 wherein the condition or disorder is selected from the group comprising diabetes, e.g. Type I or Type 2 diabetes, borderline diabetes, diabetic retinopathy, neuropathy or nephropathy impaired glucose intolerance, obesity, hypertension, syndrome X, cognitive disorders, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers, dyslipidaemia, polycystic ovarian syndrome (PCOS), acute pancreatitis, chronic pancreatitis and hemochromatosis, hypothyroidism, cretinism and hypopituitarism.
4. The method according to any one of claims 1 to 3, wherein the CGAT peptide is a Chromogranin A insulinotropic (CGAT) peptide derived from the C-terminal region of Chromogranin A.
5. The method according to any one of claims 1 to 4,, wherein the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 1 :
Ala-Tyr-Gly-Phe-Arg-Gly-Pro-Gly-Pro-Gln-Leu (SEQ ID NO: 1 )
or a fragment or derivative thereof.
6. The method according to any one of claims 1 to 4 wherein the CGAT peptide comprises an amino acid sequence as shown as SEQ ID NO: 2:
Gly-Trp-Arg-Pro-Ser-Ser-Arg-Glu-Asp-Ser-Leu-Glu-Ala-Gly-Leu-Pro-Leu- GIn-VaI (SEQ ID NO: 2)
or a fragment or derivative thereof.
7. The method according: to any one of claims 1 to 4 wherein the CGAT peptide comprises an amino acid sequence:
Trp-Ser-X-Met-Asp-Y-Leu-Ala-Lys-Glu-Leu-Thr-Ala-Glu (SEQ ID NO. 3)
wherein X represents Lys or Arg; and Y represents GIn or Arg.
8. The method according to claim 7, wherein X is Lys and Y is GIn.
9. The method according to any one of claims 1 to 4 wherein the CGAT peptide comprises an acid sequence as shown in SEQ ID NO: 5
Leu-Gln-Pro-Gly-Pro-Gly-Arg-Phe-Gly-Tyr-Ala (SEQ ID NO:5)
or a fragment or derivative thereof.
10. The method according to any one of claims 1 to 4 wherein the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 6:
Val-Gln-Leu-Pro-Leu-Gly-Ala-Glu-Leu-Ser-Asp-Glu-Arg-Ser-Ser-Pro-Arg- Trp-Gly (SEQ ID NO: 6)
or a fragment or derivative thereof.
11. The method according to any one of claims 1 to 4, wherein the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 7:
Glu-Ala-Thr-Leu-Glu-Lys-Ala-Leu-Y-Asp-Met-X-Ser-Trp
(SEQ ID NO. 7)
where X represents Lys or Arg; and Y represents GIn or Arg.
12. The method according to any one of the preceding claims wherein the cell is a pancreatic beta cell.
13. The method according to any one of the preceding claims wherein the regulatory substance is insulin.
14. A Chromogranin A tropic (CGAT) peptide or a polynucleotide encoding a CGAT peptide for use in medicine, wherein said peptide is a fragment of a full-length Chromogranin A peptide, or variant thereof, which is able to stimulate release of a regulatory substance from a cell in the absence of a stimulatory concentration of glucose.
15. A Chromogranin A tropic (CGAT) peptide or a polynucleotide encoding a CGAT peptide for use in the treatment of a condition or disorder associated with imbalance in release of a regulatory substance, wherein said peptide is a fragment of a full-length Chromogranin A peptide, or variant thereof, which is able to stimulate release of a regulatory substance from a cell in the absence of a stimulatory concentration of glucose.
16. Use of a therapeutically effective amount of a CGAT peptide in the preparation of a medicament for the treatment of a condition or disorder associated with imbalance in release of a regulatory substance, wherein said peptide is a fragment of a full-length Chromogranin A peptide, or variant thereof, which is able to stimulate release of a regulatory substance from a cell in the absence of a stimulatory concentration of glucose.
17. The peptide or polynucleotide according to claim 15 or the use according to claim 16, wherein the condition or disorder is selected from the group comprising diabetes, e.g. Type I or Type 2 diabetes, borderline diabetes, and for treatment or prevention, as appropriate, of conditions such as diabetic retinopathy, neuropathy or nephropathy impaired glucose intolerance, obesity, hypertension, syndrome X, cognitive disorders, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers, dyslipidaemia, polycystic ovarian syndrome (PCOS), acute pancreatitis, chronic pancreatitis and hemochromatosis, hypothyroidism, cretinism and hypopituitarism.
18. The peptide or polynucleotide according to claim 14, 15 or 17, or the use according to claim 16 or 17, wherein the CGAT peptide is a Chromogranin A insulinotropic (CGAT) peptide derived from the C-terminal region of Chromogranin A.
19. The peptide, polynucleotide or use according to claim 18, wherein the CGAT peptide comprises an amino acid sequence as shown in SEQ
ID NO: 1 , or a fragment or derivative thereof.
20. The peptide, polynucleotide or use according to claim 18, wherein the CGAT peptide comprises an amino acid sequence as shown as SEQ ID NO: 2, or a fragment or derivative thereof.
21. The peptide, polynucleotide or use according to claim 18, wherein the CGAT peptide comprises an amino acid sequence, as shown s SEQ ID NO: 3, or a fragment or variant thereof.
22. The peptide, polynucleotide or use according to claim 21 , wherein X is Lys and Y is GIn.
23. The peptide, polynucleotide or use according to claim 18, wherein the CGAT peptide comprises an acid sequence as shown in SEQ ID NO: 5, or a fragment or derivative thereof.
24. The peptide, polynucleotide or use according to claim 18 wherein the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 6, or a fragment or derivative thereof.
25. The peptide, polynucleotide or use according to claim 18, wherein the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 7, or a fragment or variant thereof
26. A pharmaceutical composition comprising a CGAT peptide and a pharmaceutically acceptable carrier or excipient.
27. The composition, according to claim 26, wherein the CGAT peptide is a Chromogranin A insulinotropic (CGAT) peptide derived from the C-terminal region of Chromogranin A.
28. The composition according to claim 26 or claim 27, wherein the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 1 , or a fragment or derivative thereof.
29. The composition according to claim 26 or claim 27, wherein the CGAT peptide comprises an amino acid sequence as shown as SEQ ID NO: 2, or a fragment or derivative thereof.
30. The composition according to claim 26 or claim 27, wherein the CGAT peptide comprises an amino acid sequence, as shown s SEQ ID NO: 3, or a fragment or variant thereof.
31. The composition according to claim 26 or claim 27, wherein X is Lys and Y is GIn.
32. The composition according to claim 26 or claim 27,wherein the CGAT peptide comprises an acid sequence as shown in SEQ ID NO: 5, or a fragment or derivative thereof.
33. The composition according to claim 26 or claim 27, wherein the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 6, or a fragment or derivative thereof.
34. The composition according to claim 26 or claim 27, wherein the CGAT peptide comprises an amino acid sequence as shown in SEQ ID NO: 7, or a fragment or variant thereof.
35. A method of treating a condition or disorder associated with deregulation of release of a regulatory substance in a subject, said method comprising administering (i) a therapeutically effective amount of a CGAT peptide, or a polynucleotide encoding said CGAT peptide and (ii) a stimulatory amount of glucose.
36. A method of enhancing survival of transplanted cells which produce a regulatory substance, for example a hormone, said method comprising administering a Chromogranin A tropic (CGAT) peptide, or a polynucleotide encoding a CGAT peptide, to a transplant recipient.
37. The method according to claim 36, wherein said transplanted cells are pancreatic beta cells.
38. A method of increasing calcium concentration within a cell, wherein the method comprises administering a Chromogranin A tropic (CGAT) peptide or a polynucleotide encoding a CGAT peptide to said cell.
39. The method according to any one of claims 36 to 38, wherein said CGAT peptide is as defined in any one of claims 4 to 11.
40. The peptide or polynucleotide according to claim 15 or the use according to claim 16, wherein said peptide or polynucleotide is for use in enhancing survival of transplanted cells which produce said regulatory substance.
41. A Chromogranin A tropic (CGAT) peptide or a polynucleotide encoding a CGAT peptide for use in enhancing survival of transplanted cells in a transplant recipient.
42. Use of a therapeutically effective amount of a CGAT peptide in the preparation of a medicament for the prevention of transplant cell death in a transplant recipient.
43. The peptide or polynucleotide according to claim 41 or the use according to claim 42, wherein said transplanted cells produce a regulatory substance.
44. The peptide, polynucleotide, or use according to claim 43 wherein said transplanted cells are pancreatic beta cells and said regulatory substance is insulin.
45. The peptide or polynucleotide according to any one of claims 40, 41 , 43 or 44 or the use according to any one of claims 40, 42, 43 or 44, wherein said CGAT peptide is as defined in any one of claims 4 to 11.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB0721131.1 | 2007-10-27 | ||
GBGB0721131.1A GB0721131D0 (en) | 2007-10-27 | 2007-10-27 | "Peptide and users thereof" |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2009053725A2 true WO2009053725A2 (en) | 2009-04-30 |
WO2009053725A3 WO2009053725A3 (en) | 2009-07-30 |
Family
ID=38830077
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB2008/003637 WO2009053725A2 (en) | 2007-10-27 | 2008-10-27 | Peptides and uses thereof |
Country Status (2)
Country | Link |
---|---|
GB (1) | GB0721131D0 (en) |
WO (1) | WO2009053725A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ITMI20091125A1 (en) * | 2009-06-25 | 2010-12-26 | San Raffaele Centro Fond | USE OF CROMOGRANIN A AND ITS PEPTIDIC DERIVATIVES IN THE TREATMENT OF CHANNEL INFLAMMATIONS |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003083039A2 (en) * | 2001-07-05 | 2003-10-09 | Curagen Corporation | Therapeutic polypeptides, nucleic acids encoding same, and methods of use |
WO2006086769A2 (en) * | 2005-02-11 | 2006-08-17 | Amylin Pharmaceuticals, Inc. | Gip analog and hybrid polypeptides with selectable properties |
WO2007106799A2 (en) * | 2006-03-15 | 2007-09-20 | Allergan, Inc. | Multivalent clostridial toxins |
-
2007
- 2007-10-27 GB GBGB0721131.1A patent/GB0721131D0/en not_active Ceased
-
2008
- 2008-10-27 WO PCT/GB2008/003637 patent/WO2009053725A2/en active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003083039A2 (en) * | 2001-07-05 | 2003-10-09 | Curagen Corporation | Therapeutic polypeptides, nucleic acids encoding same, and methods of use |
WO2006086769A2 (en) * | 2005-02-11 | 2006-08-17 | Amylin Pharmaceuticals, Inc. | Gip analog and hybrid polypeptides with selectable properties |
WO2007106799A2 (en) * | 2006-03-15 | 2007-09-20 | Allergan, Inc. | Multivalent clostridial toxins |
Non-Patent Citations (3)
Title |
---|
BOONEN ET AL: "Neuropeptides of the islets of Langerhans: A peptidomics study" GENERAL AND COMPARATIVE ENDOCRINOLOGY, ACADEMIC PRESS, US, vol. 152, no. 2-3, 1 June 2007 (2007-06-01), pages 231-241, XP022124635 ISSN: 0016-6480 * |
KENNEDY HELEN J ET AL: "Glucose enhances insulin promoter activity in MIN6 beta-cells independently of changes in intracellular Ca2+ concentration and insulin secretion" BIOCHEMICAL JOURNAL, vol. 342, no. 2, 1 September 1999 (1999-09-01), pages 275-280, XP002526291 ISSN: 0264-6021 * |
ORR DAVID F ET AL: "The spectrum of endogenous human chromogranin A-derived peptides identified using a modified proteomic strategy." PROTEOMICS, vol. 2, no. 11, November 2002 (2002-11), pages 1586-1600, XP002526290 ISSN: 1615-9853 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ITMI20091125A1 (en) * | 2009-06-25 | 2010-12-26 | San Raffaele Centro Fond | USE OF CROMOGRANIN A AND ITS PEPTIDIC DERIVATIVES IN THE TREATMENT OF CHANNEL INFLAMMATIONS |
WO2010150082A3 (en) * | 2009-06-25 | 2011-05-19 | Universita' Degli Studi Di Milano | Use of chromogranin a and peptidic derivatives thereof in the treatment of inflammation |
Also Published As
Publication number | Publication date |
---|---|
GB0721131D0 (en) | 2007-12-05 |
WO2009053725A3 (en) | 2009-07-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20240067695A1 (en) | Glucagon-like peptide-2 compositions and methods of making and using same | |
JP6223377B2 (en) | Compositions for long acting peptide analogs | |
TWI710570B (en) | Compositions and methods of use for treating metabolic disorders | |
JP2022023029A (en) | Glucagon analogues | |
JP2019085420A (en) | Therapeutic agent comprising elastin-like peptide | |
TW201619191A (en) | GIP agonist compounds and methods | |
US10968266B2 (en) | GIP peptide analogues | |
WO2022129305A1 (en) | Pharmaceutical composition of glp-1/glp-2 dual agonists | |
WO2009053725A2 (en) | Peptides and uses thereof | |
WO2022241167A1 (en) | Delivery constructs derived from bacterial toxins and uses thereof | |
WO2021156504A1 (en) | Treatment of panx1 associates diseases | |
EP2507266B1 (en) | TREATMENT OF IgE-MEDIATED DISEASE | |
WO2004069857A2 (en) | “peptides, compositions and uses thereof” | |
WO2022177890A1 (en) | Solid oral compositions with zinc | |
KR20200080331A (en) | Glucagon analogues | |
NZ622174B2 (en) | Glucagon-like peptide-2 compositions and methods of making and using same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 08841553 Country of ref document: EP Kind code of ref document: A2 |
|
NENP | Non-entry into the national phase in: |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 08841553 Country of ref document: EP Kind code of ref document: A2 |