Hybrid matrix implants and explants

• US 6,419,920 B1
• Filed: 10/05/1999
• Issued: 07/16/2002
• Est. Priority Date: 10/25/1995
• Status: Expired due to Fees

• Current Assignee: Shire Human Genetic Therapies, Inc. (Shire plc)
• Original Assignee: Transkaryotic Therapies Incorporated (Shire plc)
• Inventors: Mineau-Hanschke, Rochelle
• Primary Examiner(s): Nguyen, Dave T.
• Assistant Examiner(s): Nguyen, Quang
• Application Number:
  US09/413,715
• Time in Patent Office: 1,015 Days
• Field of Search: 424/93.21, 424/422, 435/455, 435/320.1, 435/69.1, 435/69.3, 435/69.52, 435/382, 435/325
• US Class Current: 424/93.21
• CPC Class Codes: A61F 2310/00365 Proteins; Polypeptides; Degradation products thereof
• 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 9/0024 Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue compositions for intravenous administration, normal injectable solutions or dispersions for, e.g. subcutaneous administration A61K9/0019; brain implants A61K9/0085; (coated) prostheses, catheters or stents A61L
• CPC Class Codes: A61L 2300/414 Growth factors
• CPC Class Codes: A61L 2300/426 Immunomodulating agents, i.e. cytokines, interleukins, interferons
• CPC Class Codes: A61L 2300/428 Vitamins, e.g. tocopherol, riboflavin
• CPC Class Codes: A61L 2300/64 Animal cells
• CPC Class Codes: A61L 27/24 Collagen
• CPC Class Codes: A61L 27/38 containing added animal cells organs or tissue containing native cells A61L27/36
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: A61L 27/3808 Endothelial cells
• CPC Class Codes: A61L 27/3813 Epithelial cells, e.g. keratinocytes, urothelial cells
• CPC Class Codes: A61L 27/3839 characterised by the site of application in the body materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60
• CPC Class Codes: A61L 27/3886 comprising two or more cell types
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: A61L 27/44 having a macromolecular matrix
• CPC Class Codes: A61L 27/54 Biologically active materials, e.g. therapeutic substances A61L27/227 takes precedence
• CPC Class Codes: C07K 14/61 Growth hormone [GH], i.e. somatotropin

Abstract

A composition having a body of matrix material made up of insoluble collagen fibrils, and disposed therewithin

(a) a plurality of vertebrate cells;

(b) a plurality of microspheres; and

(c) an agent such as a factor that promotes vascularization, a cytokine, a growth factor, or ascorbic acid.

Claims

1. A composition comprising a body of matrix material comprising insoluble collagen fibrils, there being embedded within the body of matrix material

(a) a population of cultured vertebrate cells genetically engineered to express a polypeptide;

(b) a plurality of microspheres; and

(c) an agent selected from the group consisting of a factor which promotes vascularization, a cytokine, a growth factor and ascorbic acid.

2. The composition of claim 1, wherein the agent is associated with a solid substrate embedded in the body of the matrix material.

3. The composition of claim 2, wherein the solid substrate comprises heparin or heparan sulfate proteoglycan.

4. The composition of claim 2, wherein the solid substrate comprises agarose with hepain or heparan sulfate proteoglycan bound thereto.

5. The composition of claim 4, wherein the solid substrate further comprises calcium alginate.

6. The composition of claim 2, wherein the solid substrate further comprises calcium alginate.

7. The composition of claim 2 wherein the solid substrate comprises a substance selected from the group consisting of collagen, gelatin, ethylene-vinyl acetate, polylactide/glycolic acid co-polymer, fibrin, sucrose octasulfate, dextran, polyethylene glycol, an alginate, polyacrylamide, cellulose, latex, and polyhydroxyethylmethacrylate.

8. The composition of claim 7, wherein heparin or heparan sulfate proteoglycan is bound to the substance.

9. The composition of claim 1, wherein the composition further comprises a second agent selected from the group consisting of a factor which promotes vascularization, a cytokine, and a growth factor.

10. The composition of claim 2, wherein the solid substrate is in the form of beads.

11. The composition of claim 2, wherein the solid substrate is in the form of threads.

12. The composition of claim 1, wherein the agent is basic fibroblast growth factor (bFGF).

13. The composition of claim 1, wherein the agent is selected from the group consisting of acidic fibroblast growth factor (aFGF), endothelial cell growth factor, platelet-derived growth factor (PDGF), endothelial cell stimulating angiogenesis factor (ESAF), leukotriene C₄, a prostaglandin, insulin-like growth factor 1 (IGF-1), granulocyte colony stimulating factor (G-CSF), angiogenin, transforming growth factor-α

(TGF-α

), transforming growth factory-β

(TGF-β

), ascorbic acid, epidermal growth factor (EGF), and oncostatin M.

14. The composition of claim 1, wherein the agent is selected from the group consisting of vascular endothelial growth factor (VEGF), VEGF-A, VEGF-B, VEGF-C, and VEGF-D.

15. The composition of claim 1, wherein the cultured vertebrate cells are selected from the group consisting of adipocytes, astrocytes, cardiac muscle cells, chondrocytes, endothelial cells, epithelial cells, fibroblasts, gangliocytes, glandular cells, glial cells, hematopoietic cells, hepatocytes, keratinocytes, myoblasts, neural cells, osteoblasts, pancreatic beta cells, renal cells, smooth muscle cells, striated muscle cells, and precursors of any of the above.

16. The composition of claim 1, wherein the cultured vertebrate cells are human cells.

17. The composition of claim 1, wherein the polypeptide is selected from the group consisting of enzymes, hormones, cytokines, colony stimulating factors, vaccine antigens, antibodies, clotting factors, regulatory proteins, transcription factors, receptors, and structural proteins.

18. The composition of claim 1, wherein the polypeptide is Factor VIII.

19. The composition of claim 1, wherein the polypeptide is human growth hormone.

20. The composition of claim 1, wherein the polypeptide is Factor IX.

21. The composition of claim 1, wherein the polypeptide is erythropoietin.

22. The composition of claim 1, wherein the polypeptide is selected from the group consisting of VEGF, VEGF-A, VEGF-B, VEGF-C, and VEGF-D.

23. The composition of claim 1, wherein the polypeptide is insulinotropin.

24. The composition of claim 1, wherein the polypeptide is selected from the group consisting of alpha-1 antitrypsin, calcitonin, glucocerebrosidase, low density lipoprotein (LDL) receptor, IL-2 receptor, globin, immunoglobulin, catalytic antibodies, the interleukins, insulin, insulin-like growth factor 1 (IGF-1), parathyroid hormone (PTH), leptin, the nerve growth factors, basic fibroblast growth factor (bFGF), acidic FGF (aFGF), epidermal growth factor (EGF), endothelial cell growth factor, platelet derived growth factor (PDGF), transforming growth factors, endothelial cell stimulating angiogenesis factor (ESAF), angiogenin, tissue plasminogen activator (t-PA), granulocyte colony stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF).

25. The composition of claim 1, wherein the microspheres are beads of type I collagen.

26. The composition of claim 25, wherein the microspheres are porous.

27. The composition of claim 1, wherein the microspheres are beads of porous gelatin.

28. The composition of claim 1, wherein the majority of the microspheres have an approximately spherical shape and have a diameter between approximately 0.1 and approximately 2 mm.

29. The composition of claim 1, wherein the matrix material additionally comprises a substance selected from the group consisting of a second type of collagen, agarose, alginate, fibronectin, laminin, hyaluronic acid, heparan sulfate, dermatan sulfate, sulfated proteoglycans, fibrin, elastin, and tenascin.

30. The composition of claim 1, additionally comprising noncollagen fibers dispersed within the body of matrix material.

31. The composition of claim 30, wherein the noncollagen fibers comprise a material, selected from the group consisting of nylon, dacron, polytetrafluoroethylene, polyglycolic acid, polylactic/polyglycolic acid copolymer, polystyrene, polyvinylchloride, cat gut, cotton, linen, polyester, and silk.

32. The composition of claim 1, configured to be implanted into a patient.

33. The composition of claim 32, wherein the cultured vertebrate cells are derived from one or more cells removed from the patient.

34. The composition of claim 33, wherein the cultured vertebrate cells consist of a clonal population.

35. The composition of claim 1, wherein each of the plurality of microspheres consists primarily of one or more substances selected from the group consisting of collagen, polystyrene, dextran, polyacrylamide, cellulose, calcium alginate, latex, polysulfone, and glass.

36. The composition of claim 1, wherein the cultured vertebrate cells are fibroblasts.

37. The composition of claim 1, wherein the microspheres have an approximately spherical shape.

38. A method of making the composition of claim 1, comprising

forming a mixture comprising:

(a) a plurality of cultured vertebrate cells genetically engineered to express a polypeptide;

(b) a plurality of microspheres;

(c) a solution comprising soluble collagen; and

(d) an agent selected from the group consisting of a factor that promotes vascularization, a cytokine, a growth factor, and ascorbic acid;

subjecting the soluble collagen in the mixture to conditions effective to form a gel; and

exposing the gel to culture conditions which cause the gel to contract, thereby forming the body of the composition.

39. The method of claim 38, wherein the agent is associated with a solid substrate.

40. The method of claim 39, wherein the solid substrate comprises beads of agarose with heparin or heparan sulfate proteoglycan bound thereto.

41. The method of claim 39, wherein the solid substrate further comprises calcium alginate.

42. The method of claim 40, wherein the solid substrate comprises calcium alginate.

43. The method of claim 39, wherein the solid substrate comprises a substance selected from the group consisting of collagen, gelatin, ethylene-vinyl acetate, polylactide/glycolic acid co-polymer, fibrin, sucrose octasulfate, dextran, polyethylene glycol, an alginate, polyacrylamide, cellulose, latex, and polyhydroxyethylmethacrylate.

44. The method of claim 43, wherein heparin or heparan sulfate proteoglycan is bound to the substance.

45. The method of claim 38, wherein the microspheres are porous collagen beads.

46. The method of claim 38, wherein the microspheres are porous gelatin beads.

47. The method of claim 38, wherein the mixture additionally comprises a substance selected from the group consisting of a second type of collagen, agarose, alginate, fibronectin, laminin, hyaluronic acid, heparan sulfate, dermatan sulfate, sulfated proteoglycans, fibrin, elastin, and tenascin.

48. The method of claim 38, wherein the solution is an acidic aqueous solution of soluble collagen, and gelation is accomplished by raising the pH of the solution.

49. The method of claim 38, wherein the gelation step takes place in a mold, so that, prior to the contracting step, the gel is in the shape of the mold.

50. The method of claim 38, wherein the cultured vertebrate cells are cultured in the presence of the microspheres prior to being mixed with the solution.

51. A method of administering a polypeptide to a patient in need thereof, comprising

providing the composition of claim 32, wherein the cultured vertebrate cells secrete the polypeptide;

and

implanting the composition in the patient, wherein the cultured vertebrate cells secrete the polypeptide after implanting the composition in the patient.

52. The method of claim 51, wherein the cultured vertebrate cells are derived from one or more cells removed from the patient, and have been genetically engineered in vitro to express and secrete the polypeptide.

53. The method of claim 51, wherein the implanting is carried out at a subcutaneous site in the patient.

54. The method of claim 51, wherein the implanting is carried out at an intraperitoneal, sub-renal capsular, inguinal, intramuscular, intraventricular, intraomental, or intrathecal site in the patient.

55. The method of claim 51, wherein the polypeptide is one which promotes wound healing, and the implanting is carried out at the site of a preexisting wound of the patient.

56. The method of claim 51, wherein each of the plurality of microspheres consists primarily of one or more substances selected from the group consisting of collagen, polystyrene, dextran, polyacrylamide, cellulose, calcium alginate, latex, polysulfone, and glass.

57. The method of claim 51, wherein each of the plurality of microspheres consists primarily of gelatin.

58. The method of claim 38, wherein the gel is formed in a flat-bottomed mold filled with the mixture to a depth of about 0.18 cm.

59. A composition produced by the method of claim 58.

60. The method of claim 38, wherein the gel is formed in a flat-bottomed cylindrical mold with an internal radius (r);

the mixture has a volume (V); and

when r is expressed in cm and V is expressed in ml, r²/V is about 1.8.

64. The composition of claim 1, wherein the polypeptide is selected from the group consisting of interferon-α

(IFN-α

), interferon-β

(IFN-β

), interferon-γ

(IFN-γ

), follicle stimulating hormone (FSH), α

-galactosidase, β

-gluceramidase, α

-iduronidase, α

-L-iduronidase, glucosamine-N-sulfatase, α

-N-acetylglucosaminidase, acetylcoenzyme A;

α

-glucosaminide-N-acetyltransferase, N-acetylglucosamine-6-sulfatase, β

-galactosidase, N-acetylgalactosamine-6-sulfatase, and β

-glucuronidase.

65. The composition of claim 1, wherein the matrix material additionally comprises a substance selected from the group consisting of heparin, cellulose, starch and dextran.

66. The method of claim 38, wherein the mixture additionally comprises a substance selected from the group consisting of heparin, cellulose, starch, and dextran.

61. A method of making a composition, the method comprising

forming a mixture comprising:

(a) a plurality of cultured vertebrate cells genetically engineered to express a polypeptide;

(b) a plurality of microspheres; and

(c) a solution comprising soluble collagen;

subjecting the soluble collagen in the mixture to conditions effective to form a gel; and

exposing the gel to culture conditions which cause the gel to contract, thereby forming the body of the composition,

wherein the gel is formed in a flat-bottomed mold filled with the mixture to a depth of about 0.18 cm.

62. A composition produced by the method of claim 61.

63. A method of making a composition, the method comprising

forming a mixture comprising:

(a) a plurality of cultured vertebrate cells genetically engineered to express a polypeptide;

(b) a plurality of microspheres; and

(c) a solution comprising soluble collagen;

subjecting the soluble collagen in the mixture to conditions effective to form a gel; and

exposing the gel to culture conditions which cause the gel to contract, thereby forming the body of the composition,

wherein the gel is formed in a flat-bottomed cylindrical mold with an internal radius (r);

the mixture has a volume (V); and

when r is expressed in cm and V is expressed in ml, r²/V is about 1.8.