Peptides with growth inhibitory action

• US 20030175745A1
• Filed: 09/30/2002
• Published: 09/18/2003
• Est. Priority Date: 11/28/2001
• Status: Abandoned Application

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Haaland, Perry D., Heidaran, Mohammad A., Spargo, Catherine A., Dean, Cheryl H.
• Application Number:
  US10/259,816
• Publication Number:
  US 20030175745A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61L 27/227 Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
• CPC Class Codes: A61L 27/34 Macromolecular materials
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/49 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C07K 5/1019 with the first amino acid being basic
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C08L 89/00 Compositions of proteins; Compositions of derivatives thereof foodstuff preparations A23J3/00

Abstract

Peptides and peptide compositions are identified which inhibit the growth of abnormal cells. In one embodiment, the peptides are useful for inhibiting the growth of cells dependent on autocrine activation of the PDGF receptor. Such peptides may be used in the treatment of cell proliferative disorders including cancer, fibrotic disorders, myeloproliferative diseases and blood vessel proliferative (angiogenic) disorders characterized by inappropriate PDGF receptor activity. A biomedical device is further disclosed which has associated with it a peptide or peptide composition according to the present invention.

Claims

1. An isolated peptide or polypeptide of no more than about 50 amino acid residues which, when contacted with cells in which a PDGF-R is activated in an autocrine manner, inhibits the growth of said cells, wherein said peptide or polypeptide comprises one or more amino acid sequences selected from the group consisting of KKKK (SEQ ID NO:

1), DDEEK (SEQ ID NO;

     2), KLMSY (SEQ ID NO;

     3), FFFKK (SEQ ID NO;

     4), FFHPV (SEQ ID NO;

     5), or (i) a combination thereof, (ii) a biologically active variant thereof having the same amino acid composition in a different sequence, (iii) or a biologically active substitution or addition variant.

2. The peptide or polypeptide of claim 1 having no more than about 20 amino acids.

3. The peptide of claim 2 having no more than about 10 amino acids.

4. The peptide of claim 1 which is selected from the group of peptides consisting of KKKK (SEQ ID NO:

1), DDEEK (SEQ ID NO;

     2), KLMSY (SEQ ID NO;

     3), FFFKK (SEQ ID NO;

     4), and FFHPV (SEQ ID NO;

     5).

5. The peptide of claim 4 having five amino acids and the sequence DDEEK (SEQ ID NO:

2).

8. A chemically synthesized peptide multimer comprising the peptide or variant of claim 1, which multimer is selected from the group consisting of:

(a) a multimer having the formula P¹_n wherein

(i) P¹ is any one of KKKK (SEQ ID NO;

     1), DDEEK (SEQ IS NO;

     2), KLMSY (SEQ ID NO;

     3), FFFKK (SEQ ID NO;

     4) or FFHPV (SEQ ID NO;

     5), a shuffled sequence variant thereof having the same amino acid composition in any sequence, or a substitution or addition variant thereof, and

(ii) n=2-8,

(b) a multimer having the formula (P¹-X_m)_n-P², wherein

(i) P¹ and P² are peptides KKKK (SEQ ID NO;

     1), DDEEK (SEQ IS NO;

     2), KLMSY (SEQ ID NO;

     3), FFFKK (SEQ ID NO;

     4) or FFHPV (SEQ ID NO;

     5), shuffled sequence variant thereof, or a substitution or an addition variant thereof,

(ii) P¹ and P² are the same or different peptides;

(iii) X iS C₁-C₅ alkyl, C₁-C₅ alkenyl, C₁-C₅ alkynyl, C₁-C₅ polyether containing up to 4 oxygen atoms;

(iv) m=0 or 1; and

(v) n=1-7,

wherein the peptide multimer has the biological activity of inhibiting cell proliferation mediated by autocrine activation of PDGF-R measured in an standard in vitro or in vivo bioassay of cell growth or proliferation.

9. A recombinantly produced peptide multimer comprising the peptide or variant of claim 2, which multimer has the formula (P¹-Gly_z)_n-P², wherein:

(i) P¹ and P² are peptides KKKK (SEQ ID NO;

     1), DDEEK (SEQ IS NO;

     2), KLMSY (SEQ ID NO;

     3), FFFKK (SEQ ID NO;

     4) or FFHPV (SEQ ID NO;

     5), a shuffled sequence variant thereof, or a substitution or an addition variant thereof,

(ii) P¹ and P² are the same or different;

(iii) z=0-6; and

(iv) n=1-100.

wherein the peptide multimer has the biological activity of inhibiting cell proliferation mediated by autocrine activation of PDGF-R measured in an standard in vitro or in vivo bioassay of cell growth or proliferation.

10. An isolated nucleic acid molecule encoding (a) the polypeptide or peptide of claim 1 or any permuted sequence of SEQ ID NO:

2-SEQ ID NO;

5, or (b) the peptide multimer of claim 9.

11. The nucleic acid molecule of claim 10 comprising one or more of SEQ ID NO:

6-SEQ ID NO;

341, inclusive.

12. An expression vector comprising the nucleic acid molecule of claim 10 operatively linked to:

(a) a promoter, and

(b) optionally, additional regulatory sequences that regulate expression of said nucleic acid in a eukaryotic cell,

which vector is capable of expressing said peptide in a host cell.

13. An expression vector comprising the nucleic acid molecule of claim 11 operatively linked to:

(a) a promoter, and

(b) optionally, additional regulatory sequences that regulate expression of said nucleic acid in a eukaryotic cell,

which vector is capable of expressing said peptide in a host cell.

14. The expression vector of claim 12 which is a plasmid.

15. The expression vector of claim 12 which is a viral vector.

16. A solid phase article comprising the polypeptide or peptide of claim 1 in contact with a solid surface.

17. A solid phase article comprising the multimer of claim 8 or 9 associated with or chemically linked to a solid surface.

18. The article of claim 16 wherein said solid surface comprises a synthetic polymer, natural polymer, or a combination thereof.

19. The article of claim 16 further comprising an additional layer of a cell adhesion resistant material between said polypeptide or peptide and said surface.

20. The article of claim 16 wherein said cell adhesion resistant material is material selected from the group consisting of (a) polyethylene glycol, (b) glyme, (c) a glyme derivative, (d) poly-HEMA, (e) polyisopropylacrylamide, (f) hyaluronic acid, (g) alginic acid and (h) a combination of any of (a)-(g).

21. The article of claim 16 wherein said solid surface comprises a synthetic polymer selected from the group consisting of poly(hydroxyethyl methacrylate), poly(ethylene terephthalate), poly(tetrafluoroethylene), fluorinated ethylene, poly(dimethyl siloxane), and a combination thereof.

22. The article of claim 16 wherein said solid surface comprises a natural polymer selected from the group consisting of collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharides, fibrin, gelatin, and a combination thereof.

23. The article of claim 16 wherein said peptide is chemically linked to said surface through a linker molecule.

24. A biomedical device for inhibition of abnormal or undesired cell attachment, cell growth or both attachment and growth, comprising a biocompatible surface having chemically and/or physically associated with said surface a proliferation inhibiting amount of the peptide, polypeptide or combination of claims 1 or a nucleic acid molecule encoding said peptide or polypeptide.

25. A biomedical device for inhibition of abnormal or undesired cell attachment, cell growth or both attachment and growth, comprising a biocompatible surface having chemically and/or physically associated with said surface a proliferation inhibiting amount of a multimer of claim 8 or 9 or a nucleic acid molecule encoding said multimer.

26. The device of claim 24 further comprising an additional layer of a cell adhesion resistant material between said polypeptide or peptide and said surface.

27. The device of claim 24 wherein said peptide or polypeptide is impregnated in or coated on said surface.

28. The device of claim 24 wherein said peptide or polypeptide is present in a controlled release composition.

29. A therapeutic composition that inhibits the undesired growth of cells mediated by abnormal activation or activity of PDGF-R, comprising

(a) the peptide, polypeptide or combination of claim 1;

and.

(b) a therapeutically acceptable carrier or excipient.

30. A therapeutic composition that inhibits the undesired growth of cells mediated by abnormal activation or activity of PDGF-R, comprising

(a) the multimer of claim 8 or 9;

and.

(b) a therapeutically acceptable carrier or excipient.

31. The therapeutic composition of claim 29 wherein said abnormal activation comprises autocrine activation of the PDGF-R.

32. A therapeutic composition that inhibits the undesired growth of cells mediated by abnormal activation or activity of PDGF-R, comprising

(a) the expression vector of claim 12;

and

(b) a therapeutically acceptable carrier or excipient.

wherein expression of said nucleic acid molecule results in production and growth inhibitory action of said peptide.

33. A method of inhibiting cell proliferation comprising contacting cells undergoing undesired proliferation with an effective amount of the peptide, polypeptide or combination of claim 1.

34. The method of claim 33 wherein the cell being inhibited is a tumor or cancer cell.

35. The method of claim 34 wherein the tumor or cancer cell is a carcinoma cell, an osteocarcinoma cell, a sarcoma cell, an osteosarcoma cell, a glioma cell, a melanoma cell, a myxoma cell, an adenoma cell, a neuroblastoma cell, or a rhabdomyoma-derived cell.

36. The method of claim 33 wherein the cell being inhibited is a lung cell, a breast cell, a colon cell, a prostate cell, a kidney cell, an ovary cell, a testicular cell, a skin cell, a pancreatic cell, a thyroid cell, an adrenal cell, a pituitary cell, a brain cell, a muscle cell or a bone cell.

37. The method of claim 33 wherein said contacting is in vivo in a subject.

38. The method of claim 33 wherein said contacting is in vitro.

39. The method of claim 33 which further comprises, administering to the subject a therapeutically effective amount of one or more agents or drugs selected from the group consisting of cisplatin, cyclophosphamide, VP-16, enoxaprin, angiopeptin, endostatin, paclitaxel, 5-fluorouracil, vinblastine, vincristine, an epothilone, angiostatin, hirudin, acetylsalicylic acid, and a thymidine kinase inhibitors.

40. A method of treating a subject suffering from a cell proliferative disorder, comprising contacting cells of said subject which are characterized by inappropriate PDGF receptor activity with an effective amount of a peptide, polypeptide or combination according to claim 1 or with a nucleic acid molecule encoding said peptide or polypeptide, which nucleic acid is expressible in said cells.

41. The method of claim 40 wherein said peptide or polypeptide is associated or chemically linked to a biomedical implant.

42. The method of claim 41 wherein said biomedical implant comprises at least one of a natural polymer or a synthetic polymer.

43. The method of claim 40, further comprising administering to the patient a therapeutically effective amount of a composition comprising an agent selected from the group consisting of cisplatin, cyclophosphamide, VP-16, enoxaprin, angiopeptin, endostatin, paclitaxel, 5-fluorouracil, vinblastine, vincristine, epothilones, angiostatin, hirudin, acetylsalicylic acid, thymidine kinase inhibitors, and combinations thereof.

44. A method of treating a subject who has a solid tumor, the cells of which are characterized by inappropriate PDGF receptor activity, the method comprising contacting tumor cells and/or cells surrounding tumor cells of said subject with an effective amount of a peptide, polypeptide or combination according to claim 1 or a with a nucleic acid molecule encoding said peptide or polypeptide which nucleic acid is expressible in said tumor or surrounding cells.

45. The method of claim 44 wherein said peptide or polypeptide is associated with or chemically linked to a biomedical implant.

46. The method of claim 45 wherein said biomedical implant comprises at least one of a natural or synthetic polymer.

47. The method of claim 44 further comprising administering to the subject a therapeutically effective amount of a composition comprising an agent selected from the group consisting of cisplatin, cyclophosphamide, VP-16, enoxaprin, angiopeptin, endostatin, paclitaxel, 5-fluorouracil, vinblastine, vincristine, epothilones, angiostatin, hirudin, acetylsalicylic acid, thymidine kinase inhibitors, and combinations thereof.

48. The method of claim 44, further comprising prior to said contacting step, the steps of

(i) surgically removing or debulking said solid tumor;

and

(ii) implanting a biomedical device proximal to the site of the surgery,

said device having associated therewith and available for interaction with cells surrounding said site, a synthetic or recombinant peptide or polypeptide of no more than about 50 amino acid residues which, when contacted with cells in which a PDGF-R is activated in an autocrine manner, inhibits the growth of said,wherein said peptide or polypeptide comprises an amino acid sequence selected from the group consisting of KKKK (SEQ ID NO;

     1), DDEEK (SEQ ID NO;

     2), KLMSY (SEQ ID NO;

     3), FFFKK (SEQ ID NO;

     4), FFHPV (SEQ ID NO;

     5), and combinations thereof, or with a nucleic acid molecule encoding said peptide or polypeptide.

49. The method of claim 48, wherein said peptide or polypeptide associated with said device has no more than about 20 amino acids.

50. The method of claim 49 wherein said peptide or polypeptide associated with said device has no more than about 10 amino acids.

51. The method of claim 50 wherein said peptide associated with said device is a pentapeptide selected from the group consisting of KKKK (SEQ ID NO:

1), DDEEK (SEQ ID NO;

     2), KLMSY (SEQ ID NO;

     3), FFFKK (SEQ ID NO;

     4), and FFHPV (SEQ ID NO;

     5).

52. The method of any of claims 48 further comprising administering to the subject a therapeutically effective amount of a composition comprising an agent selected from the group consisting of cisplatin, cyclophosphamide, VP-16, enoxaprin, angiopeptin, endostatin, paclitaxel, 5-fluorouracil, vinblastine, vincristine, epothilones, angiostatin, hirudin, acetylsalicylic acid, thymidine kinase inhibitors, and combinations thereof.

53. A method of inhibiting proliferation of a cell of mesenchymal origin in vivo, the method comprising administering to a subject in which said cells are present a proliferation-inhibiting effective amount of a peptide, polypeptide or combination of claim 1, or a nucleic acid molecule encoding said peptide or polypeptide.

6. A pentapeptide that falls within a parameter space defined by at least two physicochemical parameters of peptides SEQ ID NO:

2-SEQ ID NO;

5, that has the following properties;

(a) inhibits the growth of cells that expressing a PDGF-R that is activated for growth in an autocrine manner;

(b) total charge of between −

4 and +2; and

(c) MlogP value between about −

8.5 and −

2.

7. The pentapeptide of claim 6 having a total charge between −

4 and −

2, and a MlogP value between about −

7 and −

3.5.