Compositions comprising conditioned cell culture media and uses thereof

• US 20040142861A1
• Filed: 06/07/2002
• Published: 07/22/2004
• Est. Priority Date: 06/07/2001
• Status: Active Grant

• Current Assignee: Allergan, Inc. (Canada) (Abbvie Incorporated)
• Original Assignee: Skinmedica Incorporated (Abbvie Incorporated)
• Inventors: Mansbridge, Jonathan N.
• Granted Patent: US 7,160,726 B2
• Time in Patent Office: Days
• US Class Current: 514/12
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 2800/522 Antioxidants; Radical scavengers
• CPC Class Codes: A61K 2800/86 Products or compounds obtained by genetic engineering
• 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: A61K 38/1825 Fibroblast growth factor [FGF]
• CPC Class Codes: A61K 38/1866 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: A61K 38/44 Oxidoreductases (1)
• CPC Class Codes: A61K 38/446 Superoxide dismutase (1.15)
• CPC Class Codes: A61K 8/447 containing sulfur
• CPC Class Codes: A61K 8/64 Proteins; Peptides; Derivatives or degradation products thereof
• CPC Class Codes: A61K 8/678 Tocopherol, i.e. vitamin E
• CPC Class Codes: A61P 9/06 Antiarrhythmics
• CPC Class Codes: A61Q 19/00 Preparations for care of the skin
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• CPC Class Codes: C07K 2319/10 containing a tag for extracellular membrane crossing, e.g. TAT or VP22
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2510/02 : Cells for production

Abstract

The invention relates to compositions comprising cell culture medium conditioned by cells grown in three-dimensional culture. The cells used to condition the medium may be genetically modified to alter the concentration of growth factors and antioxidants in the medium. The conditioned cell medium (conditioned medium) may be used for at least one of cosmetic applications, cosmeceutical applications, and pharmaceutical applications, among other things. The invention also relates to proteins comprising a heterologous sequence that enhances cell penetration. The invention also relates to cells comprising DNA encoding such proteins. Methods for preparing the inventive compounds are also provided.

Claims

1. A composition comprising:

conditioned cell culture media, or an extract thereof, comprising at least one culture-derived growth factor, wherein the at least one growth factor comprises at least one of vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFβ

), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), interleukin-3 (IL-3), IL-6, and IL-8;

at least one culture-derived antioxidant, wherein the at least one antioxidant comprises at least one of glutathione, glutathione peroxidase, glutathione reductase, glutathione disulfide, catalase, superoxide dismutase, alpha-tocopherol, gamma-tocopherol, ubiquinol-9, ubiquinone 9, ascorbic acid, cysteine, and cystine; and

at least one culture-derived soluble collagen; and

an appropriate carrier.

2. The composition of claim 1, wherein at the least one growth factor comprises a genetically-engineered growth factor.

3. The composition of claim 2, wherein the genetically-engineered growth factor comprises at least one transport-enhanced growth factor.

4. The composition of claim 1, wherein the at least one antioxidant comprises at least one genetically-engineered antioxidant.

5. The composition of claim 4, wherein the at least one genetically-engineered antioxidant comprises at least one transport-enhanced antioxidant.

6. The composition of claim 1, wherein the appropriate carrier is a pharmaceutically-acceptable carrier.

7. The composition of claim 6, wherein the cell culture media is conditioned by eukaryotic cells in three-dimensional culture.

8. The composition of claim 7, wherein the eukaryotic cells are human fibroblasts.

9. The composition of claim 1, wherein the appropriate carrier is a cosmetically-acceptable carrier.

10. The composition of claim 1, wherein the appropriate carrier is a cosmeceutically-acceptable carrier.

11. The composition of claim 10, wherein the cell culture media is conditioned by eukaryotic cells in three-dimensional culture.

12. The composition of claim 11, wherein the eukaryotic cells are human fibroblasts.

13. The composition of claim 11, wherein the three-dimensional culture comprises a framework, a collagen matrix, a gelatin matrix, or a gel matrix.

14. The composition of claim 13, wherein the three-dimensional culture comprises a framework or a contracted collagen gel matrix.

15. The composition of claim 13, wherein the three-dimensional culture comprises a framework.

16. The composition of claim 11, wherein the composition is substantially free from phenol red.

17. The composition of claim 11, wherein the composition is substantially free from components of bovine-origin.

18. The composition of claim 11, wherein the composition is substantially free from non-human animal products.

19. The composition of claim 11, wherein at the least one growth factor comprises a genetically-engineered growth factor.

20. The composition of claim 19, wherein the genetically-engineered growth factor comprises at least one transport-enhanced growth factor.

21. The composition of claim 20, wherein the at least one transport-enhanced growth factor comprises 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, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, or SEQ ID NO;

19.

22. The composition of claim 11, wherein the at least one antioxidant comprises at least one genetically-engineered antioxidant.

23. The composition of claim 22, wherein the at least one genetically-engineered antioxidant comprises at least one transport-enhanced antioxidant.

24. The composition of claim 23, wherein the at least one transport-enhanced antioxidant comprises 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, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, or SEQ ID NO;

19.

25. A cosmeceutical composition comprising:

cell culture media or an extract thereof, conditioned by incubation with a three-dimensional culture, wherein the conditioned media or extract comprises at least one culture-derived growth factor, the at least one growth factor comprising at least one of;

vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFβ

), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), interleukin-3 (IL-3), IL-6, and IL-8; and

at least one culture-derived antioxidant, the at least one antioxidant comprising at least one of;

glutathione, glutathione peroxidase, glutathione reductase, glutathione disulfide, catalase, superoxide dismutase, alpha-tocopherol, gamma-tocopherol, ubiquinol-9, ubiquinone 9, ascorbic acid, cysteine, and cystine; and

a cosmeceutically-acceptable carrier.

26. The composition of claim 25, wherein at the least one growth factor comprises a genetically-engineered growth factor.

27. The composition of claim 26, wherein the genetically-engineered growth factor comprises at least one transport-enhanced growth factor.

28. The composition of claim 27, wherein the at least one transport-enhanced growth factor comprises 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, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, or SEQ ID NO;

19.

29. The composition of claim 25, wherein the at least one antioxidant comprises at least one genetically-engineered antioxidant.

30. The composition of claim 29, wherein the at least one genetically-engineered antioxidant comprises at least one transport-enhanced antioxidant.

31. The composition of claim 30, wherein the at least one transport-enhanced antioxidant comprises 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, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, or SEQ ID NO;

19.

32. The composition of claim 25, wherein said at least one growth factor comprises KGF.

33. The composition of claim 32, wherein the three-dimensional fibroblast culture is treated with an amount of IL-1α

sufficient to enhance the expression of KGF.

34. The composition of claim 25, wherein the three-dimensional fibroblast culture is treated with an amount of PDGF sufficient to enhance the expression of VEGF.

35. The composition of claim 25, wherein the composition is substantially free from phenol red.

36. The composition of claim 25, wherein the composition is substantially free from components of bovine-origin.

37. The composition of claim 25, wherein the composition is substantially free from non-human animal products.

38. The composition of claim 25, wherein the three-dimensional culture comprises a framework, a collagen matrix, a gelatin matrix, or a gel matrix.

39. The composition of claim 38, wherein the three-dimensional culture comprises a framework or a contracted collagen gel matrix.

40. The composition of claim 39, wherein the three-dimensional culture comprises a framework.

41. The composition of claim 25, wherein the cell culture media is conditioned by eukaryotic cells in three-dimensional culture.

42. The composition of claim 41, wherein the eukaryotic cells are human fibroblasts.

43. The composition of claim 25, wherein the at least one growth factor comprises at least one of glutathione, alpha-tocopherol, gamma-tocopherol, or cysteine.

44. The composition of claim 25, further comprising at least one culture-derived soluble collagen.

61. The composition of claim 25, further comprising at least one culture-derived soluble collagen.

45. A method for preparing a cosmeceutical composition comprising:

combining a pre-conditioned medium with a three-dimensional culture under appropriate conditions to generate a conditioned medium comprising at least one culture-derived growth factor, the at least one growth factor comprising at least one of;

vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFβ

), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), interleukin-3 (IL-3), IL-6, and IL-8; and

at least one culture-derived antioxidant, the at least one antioxidant comprising at least one of;

glutathione, glutathione peroxidase, glutathione reductase, glutathione disulfide, catalase, superoxide dismutase, alpha-tocopherol, gamma-tocopherol, ubiquinol-9, ubiquinone 9, ascorbic acid, cysteine, and cystine; and

combining the conditioned medium with a cosmeceutically-acceptable carrier to form a cosmeceutical composition.

47. The method of claims 45 or 46, wherein the at least one growth factor comprises at least one genetically-engineered growth factor.

48. The method of claim 47, wherein the at least one genetically-engineered growth factor comprises at least one transport-enhanced growth factor.

49. The method of claims 45 or 46, wherein the at least one antioxidant comprises at least one genetically-engineered antioxidant.

50. The method of claim 49, wherein the at least one genetically-engineered antioxidant comprises at least one transport-enhanced antioxidant.

52. The method of claims 45 or 46, wherein the pre-conditioned medium is conditioned by eukaryotic cells in three-dimensional culture.

53. The method of claim 52, wherein the eukaryotic cells are human fibroblasts.

54. The method of claims 45 or 46, wherein the three-dimensional culture comprises a framework, a collagen matrix, a gelatin matrix, or a gel matrix.

55. The method of claim 54, wherein the three-dimensional culture comprises a framework or a contracted collagen gel matrix.

56. The method of claims 45 or 46, wherein the composition is substantially-free from phenol red.

57. The method of claims 45 or 46, wherein the composition is substantially-free from components of bovine-origin.

58. The method of claims 45 or 46, wherein the composition is substantially-free from non-human animal products.

59. The method of claim 48, wherein the at least one transport-enhanced growth factor comprises 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, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, or SEQ ID NO;

19.

60. The method of claim 50, wherein the at least one transport-enhanced antioxidant comprises 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, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, or SEQ ID NO;

19.

62. The method of claims 45 or 46, wherein an extract of the conditioned medium is combined with the carrier to form the composition.

46. A method for preparing a composition comprising:

combining a pre-conditioned medium with a three-dimensional culture under appropriate conditions to generate a conditioned medium comprising at least one culture-derived growth factor, the at least one growth factor comprising at least one of;

vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFβ

), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), interleukin-3 (IL-3), IL-6, and IL-8; and

at least one culture-derived antioxidant, the at least one antioxidant comprising at least one of;

glutathione, glutathione peroxidase, glutathione reductase, glutathione disulfide, catalase, superoxide dismutase, alpha-tocopherol, gamma-tocopherol, ubiquinol-9, ubiquinone 9, ascorbic acid, cysteine, and cystine; and

combining the conditioned medium with an acceptable carrier to form a composition.

51. The method of claim 46, wherein the acceptable carrier is a pharmaceutically-acceptable carrier.

63. A growth factor comprising a heterologous peptide sequence that enhances cell penetration.

64. The growth factor of claim 63, wherein the growth factor comprises at least one of insulin, insulin-like growth factor (IGF), nerve growth factor, VEGF, keratinocyte growth factor (KGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), transforming growth factor alpha (TGFα

), transforming growth factor beta (TGFβ

), epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and interleukin-8 (IL-8); and

wherein the heterologous peptide sequence comprises at least one of the following sequences;

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, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, and SEQ ID NO;

19.

65. An antioxidant comprising a heterologous peptide sequence that enhances cell penetration.

66. The antioxidant of claim 65, wherein the antioxidant comprises at least one of cysteine, cystine, ascorbic acid, glutathione, glutathione disulfide, glutathione peroxidase, glutathione reductase, glutathione disulfide, superoxide dismutase, catalase, vitamin E, ascorbic acid, ubiquinol 9, and ubiquinone 9;

and

wherein the heterologous peptide sequence comprises at least one of the following sequences;

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, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, and SEQ ID NO;

19.

67. An extracellular matrix component comprising a heterologous peptide sequence that enhances cell penetration.

68. The extracellular matrix component of claim 67, wherein the extracellular matrix component comprises at least one of:

at least one glycoprotein, at least one proteoglycan, and at least one glycosaminoglycan; and

wherein the heterologous peptide sequence comprises at least one of the following sequences;

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, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, and SEQ ID NO;

19.

69. A cell comprising DNA encoding a growth factor comprising a heterologous peptide sequence that enhances cell penetration.

70. A cell according to claim 69, wherein the growth factor comprises at least one of insulin, insulin-like growth factor (IGF), nerve growth factor, VEGF, keratinocyte growth factor (KGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), transforming growth factor alpha (TGFα

), transforming growth factor beta (TGFβ

), epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and interleukin-8 (IL-8); and

wherein the heterologous peptide sequence comprises at least one of the following sequences;

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, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, and SEQ ID NO;

19.

71. A cell comprising DNA encoding an antioxidant comprising a heterologous peptide sequence that enhances cell penetration.

72. A cell according to claim 71, wherein the antioxidant comprises at least one of cysteine, cystine, ascorbic acid, glutathione, glutathione disulfide, glutathione peroxidase, glutathione reductase, glutathione disulfide, superoxide dismutase, catalase, vitamin E, ascorbic acid, ubiquinol 9, and ubiquinone 9;

and

wherein the heterologous peptide sequence comprises at least one of the following sequences;

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, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, and SEQ ID NO;

19.

73. A cell comprising DNA encoding an extracellular matrix component comprising a heterologous peptide sequence that enhances cell penetration.

74. A cell according to claim 73, wherein the extracellular matrix component comprises at least one of:

at least one glycoprotein, at least one proteoglycan, and at least one glycosaminoglycan; and

wherein the heterologous peptide sequence comprises at least one of the following sequences;

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, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, and SEQ ID NO;

19.