Claims
- 1. A substantially pure I-conotoxin peptide having the generic formula I: Xaa1-Xaa2-Xaa3-Xaa4-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Cys-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15-Cys-Cys-Xaa16-Xaa17-Xaa18-Cys-Cys-Xaa19-Xaa20-Gly-Xaa21-Cys-Xaa22-Xaa13-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Cys-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-Xaa41 (SEQ ID NO: 1), wherein Xaa1 is des-Xaa1 or Gly; Xaa2 is des-Xaa2, Pro, hydroxy-Pro (Hyp), Ala, His or Gly; Xaa3 is des-Xaa3, Ser, Val, Pro, Hyp, Thr, g-Ser, g-Thr, g-Hyp or any synthetic hydroxylated amino acid; Xaa4 is des-Xaa4, Gly, Glu, γ-carboxy-Glu (Gla), Phe, Pro, Hyp, Arg, Lys, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa5 is an aliphatic amino acids bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), Ile and Val or non-natural derivatives of the aliphatic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys, any synthetic basic amino acid, Gly, Trp (D or L), neo-Trp, halo-Trp (D or L), or any aromatic synthetic amino acid; Xaa6 is Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys, any synthetic basic amino acid, Ala, an aliphatic amino acids bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), Ile and Val or non-natural derivatives of the aliphatic amino acid, Thr, Ser, g-Thr or g-Ser; Xaa7 is Gly, Asp, Glu, Gla, Asn, Gln or any synthetic acidic amino acid; Xaa8 is Gly, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys, any synthetic basic amino acid, Asp, Glu, Gla, Asn, Gln or any synthetic acidic amino acid; Xaa9 is Ala, Val, Met, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa10 is Ala, His, Ser, Thr, Pro, Hyp, g-Ser, g-Thr, g-Hyp, any synthetic hydroxylated amino acid, Asp, Glu, Gla, Asn, Gln, any synthetic acidic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa11 is Gly, Ser, Thr, g-Ser, g-Thr, Asp, Glu, Gla, any synthetic acidic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa12 is Asn, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, any synthetic aromatic amino acid, Gln or Leu (D or L); Xaa13 is Ser, Thr, g-Ser, g-Thr or His; Xaa14 is Ala, Gla, Ser, Thr, g-Ser, g-Thr, His, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa15 is Asp, Glu, His or Gla; Xaa16 is des-Xaa16, Gly, His, Ser, Pro, Hyp, Thr, g-Ser, g-Thr, g-Hyp, any synthetic hyrdroxylated amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa17 is des-Xaa17, His, Ser, Thr, g-Ser, g-Thr, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys, any synthetic basic amino acid, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid; Xaa18 is Val, Asn, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa19 is des-Xaa19, Leu (D or L), Pro, Hyp, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid; Xaa20 is Gly, Ile, Ser, Thr, g-Ser, g-Thr, His, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys, any synthetic basic amino acid, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid; Xaa21 is Ser, Thr, g-Ser, g-Thr, an aliphatic amino acids bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), Ile and Val or non-natural derivatives of the aliphatic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys, any synthetic basic amino acid, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid; Xaa22 is Ala, Gln, Gla, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa23 is Ser, Pro, Hyp, Thr, g-Ser, g-Thr, g-Hyp, any synthetic hyrdroxylated amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa24 is Gln, Ser, Pro, Hyp, Thr, g-Ser, g-Thr, g-Hyp or any synthetic hyrdroxylated amino acid; Xaa25 is des-Xaa25, Ser, Thr, g-Ser or g-Thr; Xaa26 is des-Xaa26, Asn, Gln, Ser, Thr, g-Asn, g-Ser or g-Thr; Xaa27 is des-Xaa27, Val, Gla, Trp (D or L), neo-Trp, halo-Trp (D or L), any aromatic synthetic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa28 is des-Xaa28, an aliphatic amino acids bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), Ile and Val or non-natural derivatives of the aliphatic amino acid; Xaa29 is des-Xaa29, an aliphatic amino acids bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), Ile and Val or non-natural derivatives of the aliphatic amino acid; Xaa30 is des-Xaa30, Ile, Ser, Pro, Hyp, Thr, g-Ser, g-Thr, g-Hyp, any synthetic hyrdroxylated amino acid, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid; Xaa31 is des-Xaa31 or Gly; Xaa32 is Ser, Thr, g-Ser, g-Thr, Trp (D or L), neo-Trp, halo-Trp (D or L), any aromatic synthetic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa33 is Val, Ser, Thr, g-Ser, g-Thr, Tip (D or L), neo-Trp, halo-Trp (D or L) or any aromatic synthetic amino acid; Xaa34 is Gly, Ile, Asp, Glu, Gla, Asn, Ser, Thr, g-Asn, g-Ser or g-Thr; Xaa35 is des-Xaa35, Val, Met, Gln, Pro, Hyp, Ser, Thr, g-Ser, g-Thr, g-Hyp or any synthetic hydroxylated amino acid; Xaa36 is des-Xaa36, Val, Thr, Ser, g-Thr, g-Ser, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid; Xaa37 is des-Xaa37, Gln, Asn, Thr, Ser, g-Ser, g-Ser, g-Asn, Met, Leu, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, any synthetic aromatic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa38 is des-Xaa38, Leu, Ser, Thr, g-Ser, g-Thr, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa39 is des-Xaa39, Ile, Ala, Thr, Ser, g-Ser, g-Thr, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; Xaa40 is des-Xaa40, Asp, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N′-dimethyl-Lys, N,N′,N″-trimethyl-Lys or any synthetic basic amino acid; and Xaa41 is des-Xaa41, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid.Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid, with the proviso that the peptide is not J029 (SEQ ID NO:2).
- 2. A substantially pure I-conotoxin peptide selected from the group consisting of the mature toxins set forth in Table 1 with the proviso that the peptide is not J029 (SEQ ID NO:2).
- 3. The substantially pure I-conotoxin peptide of claim 1, which is modified to contain an O-glycan, an S-glycan or an N-glycan.
- 4. The substantially pure I-conotoxin peptide of claim 2 which is modified to contain an O-glycan, an S-glycan or an N-glycan.
- 5. A substantially pure I-conotoxin peptide derivative comprising a derivative of the conotoxin peptide of claim 2.
- 6. The substantially pure I-conotoxin peptide derivative of claim 5, wherein the Arg residues may be substituted by Lys, ornithine, homoargine, nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any synthetic basic amino acid; the Lys residues may be substituted by Arg, ornithine, homoargine, nor-Lys, or any synthetic basic amino acid; the Tyr residues may be substituted with any synthetic hydroxy containing amino acid; the Ser residues may be substituted with Thr or any synthetic hydroxylated amino acid; the Thr residues may be substituted with Ser or any synthetic hydroxylated amino acid; the Phe and Trp residues may be substituted with any synthetic aromatic amino acid; and the Asn, Ser, Thr or Hyp residues may be glycosylated; the Cys residues may be in D or L configuration; the Cys residues may be substituted with homocysteine (D or L); the Tyr residues may also be substituted with the 3-hydroxyl or 2-hydroxyl isomers (meta-Tyr or ortho-Tyr, respectively) and corresponding O-sulpho- and O-phospho-derivatives; the acidic amino acid residues may be substituted with any synthetic acidic amino acid, e.g., tetrazolyl derivatives of Gly and Ala; pairs of Cys residues may be replaced pairwise with isoteric lactam or ester-thioether replacements, such as Ser/(Glu or Asp), Lys/(Glu or Asp), Cys(Glu or Asp) or Cys/Ala combinations; the aliphatic amino acids may be substituted by synthetic derivatives bearing non-natural aliphatic branched or linear side chains CnH2n+2 up to and including n=8; the Leu residues may be substituted with Leu (D); the Glu residues may be substituted with Gla; the Gla residues may be substituted with Glu; the N-terminal Gln residues may be substituted with pyroGlu; and the Met residues may be substituted with norleucine.
- 7. The substantially pure I-conotoxin peptide derivative of claim 5 which is modified to contain an O-glycan, an S-glycan or an N-glycan.
- 8. The substantially pure I-conotoxin peptide derivative of claim 6 which is modified to contain an O-glycan, an S-glycan or an N-glycan.
- 9. An isolated nucleic acid comprising a nucleic acid coding for a I-conotoxin precursor comprising an amino acid sequence selected from the group of amino acid sequences set forth in Table 1.
- 10. The nucleic acid of claim 9 wherein the nucleic acid comprises a nucleotide sequence selected from the group of nucleotide sequences set forth in Table 1 or their complements.
- 11. A substantially pure I-conotoxin protein precursor comprising an amino acid sequence selected from the group of amino acid sequences set forth in Table 1.
- 12. A pharmaceutical composition comprising a I-conotoxin peptide of claim 1 or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.
- 13. The pharmaceutical composition of claim 12, wherein said I-conotoxin peptide is modified to contain an O-glycan, a n S-glycan or an N-glycan.
- 14. A pharmaceutical composition comprising a I-conotoxin peptide of claim 2 or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.
- 15. The pharmaceutical composition of claim 14, wherein the Arg residues may be substituted by Lys, ornithine, homoargine, nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any synthetic basic amino acid; the Lys residues may be substituted by Arg, ornithine, homoargine, nor-Lys, or any synthetic basic amino acid; the Tyr residues may be substituted with any synthetic hydroxy containing amino acid; the Ser residues may be substituted with Thr or any synthetic hydroxylated amino acid; the Thr residues may be substituted with Ser or any synthetic hydroxylated amino acid; the Phe and Trp residues may be substituted with any synthetic aromatic amino acid; and the Asn, Ser, Thr or Hyp residues may be glycosylated; the Cys residues may be in D or L configuration; the Cys residues may be substituted with homocysteine (D or L); the Tyr residues may also be substituted with the 3-hydroxyl or 2-hydroxyl isomers (meta-Tyr or ortho-Tyr, respectively) and corresponding O-sulpho- and O-phospho-derivatives; the acidic amino acid residues may be substituted with any synthetic acidic amino acid, e.g., tetrazolyl derivatives of Gly and Ala; pairs of Cys residues may be replaced pairwise with isoteric lactam or ester-thioether replacements, such as Ser/(Glu or Asp), Lys/(Glu or Asp), Cys/(Glu or Asp) or Cys/Ala combinations; the aliphatic amino acids may be substituted by synthetic derivatives bearing non-natural aliphatic branched or linear side chains CnH2n+2 up to and including n=8; the Leu residues may be substituted with Leu (D); the Glu residues may be substituted with Gla; the Gla residues may be substituted with Glu; the N-terminal Gln residues may be substituted with pyroGlu; and the Met residues may be substitued with norleucine.
- 16. The pharmaceutical composition of claim 14 wherein said I-conotoxin peptide is modified to contain an O-glycan, an S-glycan or an N-glycan.
- 17. The pharmaceutical composition of claim 15 said I-conotoxin peptide is modified to contain an O-glycan, an S-glycan or an N-glycan.
- 18. A method for regulating the flow of potassium through potassium channels in an individual in need thereof which comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 12.
- 19. The method of claim 18, wherein said individual in need thereof suffers from a disorder selected from the group consisting of multiple sclerosis, other demyelinating diseases (such as acute dissenmiated encephalomyelitis, optic neuromyelitis, adrenoleukodystrophy, acute transverse myelitis, progressive multifocal leukoencephalopathy), sub-acute sclerosing panencephalomyelitis (SSPE), metachromatic leukodystrophy, Pelizaeus-Merzbacher disease, spinal cord injury, botulinum toxin poisoning, Huntington's chorea, compression and entrapment neuropathies (such as carpal tunnel syndrome, ulnar nerve palsy), cardiovascular disorders (such as cardiac arrhythmias, congestive heart failure), reactive gliosis, hyperglycemia, immunosuppression, cocaine addiction, cancer, cognitive dysfunction, disorders resulting from defects in neurotransmitter release (such as Eaton-Lambert syndrome), and reversal of the actions of curare and other neuromuscular blocking drugs.
- 20. The method of claim 18, wherein said disorder is a demyelinating disease.
- 21. A method for treating disorders associated with radical depolarization of excitable membranes by activating a KATP channel which comprises administering to an individual in need thereof an effective amount of the pharmaceutical composition of claim 12.
- 22. The method of claim 21, wherein said disorder is cardiac ischemia.
- 23. The method of claim 21, wherein said disorder is cerebral ischemia.
- 24. The method of claim 21, wherein said disorder is asthma.
- 25. The method of claim 21, wherein said disorder is ocular ischemia.
- 26. A method for treating or preventing disorders associated with voltage gated ion channel disorders in which comprises administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 12.
- 27. The method of claim 26, wherein said disorder is a neurologic disorder.
- 28. The method of claim 27, wherein said neurologic disorder is a neurotoxic injury associated with conditions of hypoxia, anoxia or ischemia.
- 29. The method of claim 28, wherein said neurotoxic injury is associated with stroke, cerebrovascular accident, brain or spinal cord trauma, myocardial infarct, physical trauma, drownings, suffocation, perinatal asphyxia, or hypoglycemic events.
- 30. The method of claim 27, wherein said neurologic disorder is a seizure.
- 31. The method of claim 30, wherein said seizure is associated with epilepsy.
- 32. The method of claim 26, wherein said disorder is pain.
- 33. The method of claim 32, wherein said pain is migraine, acute pain, persistent pain, neuropathic pain or nociceptive pain.
- 34. The method of claim 26, wherein said disorder is a neuromuscular disorder.
- 35. The method of claim 34, wherein said neuromuscular disorder is myofacial pain syndrome, chronic muscle spasm, dystonias or spasticity.
- 36. A method for providing musculoskeletal relaxation in a patient undergoing a surgical procedure requiring anesthesia which comprises administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 12.
- 37. A method of alleviating pain which comprises administering to a mammal that is either exhibiting pain or is about to be subjected to a pain-causing event a pain-alleviating amount of the pharmaceutical composition of claim 12.
- 38. A method of identifying compounds that mimic the therapeutic activity of a I-conotoxin, comprising the steps of: (a) conducting a biological assay on a test compound to determine the therapeutic activity; and (b) comparing the results obtained from the biological assay of the test compound to the results obtained from the biological assay of a I-conotoxin.
- 39. A substantially pure I-conotoxin peptide derivative comprising a permutant of the peptide of claim 2.
- 40. A substantially pure I-conotoxin peptide derivative comprising a permutant of the peptide of claim 4.
- 41. A substantially pure I-conotoxin peptide derivative comprising a permutant of the peptide of claim 6.
- 42. A substantially pure I-conotoxin peptide derivative comprising a permutant of the peptide of claim 8.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims benefit under 35 USC §119(e) to U.S. provisional patent applications Ser. No. 60/______ filed Jun. 30, 2000 (attorney docket no. 2314-186), Ser. No. 60/243,410 filed Oct. 27, 2000, Ser. No. 60/246,581 filed Nov. 8, 2000, No. 60/247,714 filed Nov. 14, 2000 and No. 60/264,256 filed Jan. 29, 2001, each incorporated herein by reference.
Government Interests
[0002] This invention was made with Government support under Grant No. PO1 GM48677 awarded by the National Institute of General Medical Sciences, National Institutes of Health, Bethesda, Md. The United States Government has certain rights in the invention.
Provisional Applications (5)
|
Number |
Date |
Country |
|
60243410 |
Oct 2000 |
US |
|
60246581 |
Nov 2000 |
US |
|
60247714 |
Nov 2000 |
US |
|
60264256 |
Jan 2001 |
US |
|
60304166 |
Jun 2000 |
US |