METHODS FOR TREATING WOUNDS

Abstract

Novel compositions for treating wounds and promoting the healing thereof are described, including composition containing novel combinations of a carrier and recombinant platelet derived grown factor having fewer isoforms and enhanced biostability. Methods of treating wounds with novel therapeutic composition using dosing procedures leading to effective results with a minimal number of treatment applications are also described.

Claims

1. A method of treating a wound that extends through the epidermis, wherein said method comprises: (1) debriding the wound to remove necrotic or infected tissue; (2) forming a therapeutic composition consisting essentially of sterile recombinant human PDGF-BB (rhPDGF-BB) in a physiologic solution and a sterile porous biocompatible carrier, wherein the porous biocompatible carrier is a collagen sponge or collagen wound dressing, and said therapeutic composition is free from an enzyme inhibitor; (3) applying the therapeutic composition to the wound surface in an amount that is at least about 10 μg rh PDGF-BB per cm.sup.2 of treated wound surface area, wherein the carrier provides a substrate for cell attachment and vascular ingrowth as the wound heals; (4) covering the wound with a dressing; and (5) monitoring the healing of the wound during a treatment period and repeating steps (1)-(4) to retreat the wound at treatment intervals of 7 or more days, (6) wherein the wound is retreated from 2 to 20 times, and wherein each retreatment comprises applying the therapeutic composition to the wound surface in an amount that is at least 10 μg rhPDGF-BB/cm.sup.2 treated wound surface area up to 100 μg rhPDGF-BB/cm.sup.2 treated wound surface area.

2. The method of claim 1 wherein the wound extends through the epidermis for at least six weeks duration.

3. The method of claim 1 wherein the wound is an ulcer.

4. The method of claim 1 wherein the wound is a diabetic foot ulcer.

5. The method of claim 1 wherein the wound is a venous stasis ulcer.

6. The method of claim 1 wherein the wound is a pressure ulcer.

7. The method of claim 1 wherein the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB; and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.1 ml/cm.sup.3 carrier to about 1 ml/cm.sup.3 or the ratio of rhPDGF-BB to the carrier is between about 75 μg PDGF/cm.sup.3 of carrier to about 225 μg PDGF/cm.sup.3 of carrier.

8. The method of claim 1 wherein the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.25 ml solution/cm.sup.3 of carrier to about 5 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 750 μg rhPDGF-BB/cm.sup.3 of carrier.

9. The method of claim 1 wherein at least about 80% of the rhPDGF-BB on a weight basis is unclipped rhPDGF-BB.

10. The method of claim 1 wherein the porous biocompatible carrier is a collagen sponge.

11. The method of claim 1 wherein the porous biocompatible carrier is a collagen wound dressing.

12. The method of claim 1 wherein the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 50 mg of rhPDGF-BB.

13. The method of claim 1 wherein the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 25 mg of rhPDGF-BB.

14. The method of claim 1 wherein the wound is retreated from 2 to 20 times over a maximum treatment period of 2 to 20 weeks.

15. The method of claim 1 wherein the steps (1)-(4) are repeated to retreat the wound at a retreatment frequency of at least every 8 days.

16. The method of claim 1 wherein the steps (1)-(4) are repeated to retreat the wound at a retreatment frequency of at least every 10 days.

17. The method of claim 1 wherein the wound is retreated a maximum of 10 times.

18. The method of claim 1 wherein: (A) the wound extends through the epidermis for at least six weeks duration; (B) the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, wherein at least about 80% of the rhPDGF-BB on a weight basis is unclipped rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.1 ml solution/cm.sup.3 of carrier to about 1 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 225 μg rhPDGF-BB/cm.sup.3 of carrier; (C) the wound is retreated from 2 to 20 times over a maximum treatment period of 2 to 20 weeks at a retreatment frequency of at least every 8 days; and (D) the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 50 mg of rhPDGF-BB.

19. A method of treating a wound that extends through the epidermis, wherein said method comprises: (1) debriding the wound to remove necrotic or infected tissue; (2) forming a therapeutic composition consisting essentially of sterile recombinant human PDGF-BB (rhPDGF-BB) in a physiologic solution and a sterile porous biocompatible carrier, wherein the porous biocompatible carrier is a collagen sponge or collagen wound dressing; (3) applying the therapeutic composition to the wound surface in an amount that is at least about 10 μg rhPDGF-BB per cm.sup.2 of treated wound surface area, wherein the carrier provides a substrate for cell attachment and vascular ingrowth as the wound heals; (4) covering the wound with a dressing; and (5) monitoring the healing of the wound during a treatment period and repeating steps (1)-(4) to retreat the wound at treatment intervals of 8 or more days, wherein the wound is retreated from 2 to 20 times over a maximum treatment period of 2 to 20 weeks, and wherein each retreatment comprises applying the therapeutic composition to the wound surface in an amount that is at least 0 μg rhPDGF-BB/cm.sup.2 treated wound surface area up to 100 μg rhPDGF-BB/cm.sup.2 treated wound surface area.

20. The method of claim 19 wherein the wound is a wound that extends through the dermis for at least six weeks duration.

21. The method of claim 19 wherein the wound is an ulcer.

22. The method of claim 19 wherein the wound is a diabetic foot ulcer.

23. The method of claim 19 wherein the wound is a venous stasis ulcer.

24. The method of claim 19 wherein the wound is a pressure ulcer.

25. The method of claim 19 wherein the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.1 ml solution/cm.sup.3 of carrier to about 1 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 225 μg rhPDGF-BB/cm.sup.3 of carrier.

26. The method of claim 19 wherein the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.25 ml solution/cm.sup.3 of carrier to about 5 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 750 μg rhPDGF-BB/cm.sup.3 of carrier.

27. The method of claim 19 wherein the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 50 mg of rhPDGF-BB.

28. The method of claim 19 wherein the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 25 mg of rhPDGF-BB.

29. The method of claim 19 wherein the steps (1)-(4) are repeated to retreat the wound at a retreatment frequency of at least every 10 days.

30. The method of claim 19 wherein the wound is retreated a maximum of 10 times.

31. The method of claim 19 wherein: (A) the wound extends through the epidermis for at least six weeks duration; (B) the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, wherein at least about 80% of the rhPDGF-BB on a weight basis is unclipped rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.1 ml solution/cm.sup.3 of carrier to about 1 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 225 μg rhPDGF-BB/cm.sup.3 of carrier; and (C) the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 50 mg of rhPDGF-BB.

32. A method of treating a wound that extends through the epidermis: (1) debriding the wound to remove necrotic or infected tissue; (2) forming a therapeutic composition consisting essentially of sterile recombinant human PDGF-BB (rhPDGF-BB) in a physiologic solution and a sterile porous biocompatible carrier, wherein the porous biocompatible carrier is a collagen sponge or collagen wound dressing; (3) applying the therapeutic composition to the wound surface in an amount that is at least about 10 μg rhPDGF-BB per cm.sup.2 of treated wound surface area, wherein the carrier provides a substrate for cell attachment and vascular ingrowth as the wound heals; (4) covering the wound with a dressing; and (5) monitoring the healing of the wound during a treatment period and repeating steps (1)-(4) to retreat the wound at treatment intervals of 10 or more days, wherein the wound is retreated from 2 to 20 times, and wherein each retreatment comprises applying the therapeutic composition to the wound surface in an amount that is at least 10 μg rhPDGF-BB/cm.sup.2 treated wound surface area up to 100 μg rhPDGF-BB/cm.sup.2 treated wound surface area.

33. The method of claim 32 wherein the wound extends through the epidermis for at least six weeks duration.

34. The method of claim 32 wherein the wound is an ulcer.

35. The method of claim 32 wherein the wound is a diabetic foot ulcer.

36. The method of claim 32 wherein the wound is a venous stasis ulcer.

37. The method of claim 32 wherein the wound is a pressure ulcer.

38. The method of claim 32 wherein the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (1) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (2) the ratio of the rhPDGF-BB solution to the carrier is between about 0.1 ml solution/cm.sup.3 of carrier to about 1 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 225 μg rhPDGF-BB/cm.sup.3 of carrier.

39. The method of claim 32 wherein the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.25 ml solution/cm.sup.3 of carrier to about 5 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 750 μg rhPDGF-BB/cm.sup.3 of carrier.

40. The method of claim 32 wherein the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 50 mg of rhPDGF-BB.

41. The method of claim 32 wherein the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 25 mg of rhPDGF-BB.

42. The method of claim 32 wherein the wound is retreated from 2 to 20 times over a maximum treatment period of 2 to 20 weeks.

43. The method of claim 1 wherein when the rhPDGF-BB and the porous carrier are combined, the carrier is capable of entrapping the rhPDGF-BB within its pores such that the rhPDGF-BB is released over time as the carrier is absorbed by the patient's body, thereby providing controlled delivery of rhPDGF-BB at the wound over an extended period of time and simultaneously providing a matrix for new cell and tissue ingrowth.

44. A method of treating a wound that is a burn, wherein said method comprises: (1) debriding the wound to remove necrotic or infected tissue; (2) forming a therapeutic composition consisting essentially of sterile recombinant human PDGF-BB (rhPDGF-BB) in a physiologic solution and a sterile porous biocompatible carrier, wherein the porous biocompatible carrier is a collagen sponge or collagen wound dressing, and said therapeutic composition is free from an enzyme inhibitor; (3) applying the therapeutic composition to the wound surface in an amount that is at least about 10 μg rh PDGF-BB per cm.sup.2 of treated wound surface area, wherein the carrier provides a substrate for cell attachment and vascular ingrowth as the wound heals; (4) covering the wound with a dressing; and (5) monitoring the healing of the wound during a treatment period and repeating steps (1)-(4) to retreat the wound at treatment intervals of 7 or more days, (6) wherein the wound is retreated from 2 to 20 times, and wherein each retreatment comprises applying the therapeutic composition to the wound surface in an amount that is at least 10 μg rhPDGF-BB/cm.sup.2 treated wound surface area up to 100 μg rhPDGF-BB/cm.sup.2 treated wound surface area.

45. The method of claim 44 wherein the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB; and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.1 ml/cm3 carrier to about 1 ml/cm3 or the ratio of rhPDGF-BB to the carrier is between about 75 μg PDGF/cm3 of carrier to about 225 μg PDGF/cm3 of carrier.

46. The method of claim 44 wherein the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.25 ml solution/cm.sup.3 of carrier to about 5 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 750 μg rhPDGF-BB/cm.sup.3 of carrier.

47. The method of claim 44 wherein at least about 80% of the rhPDGF-BB on a weight basis is unclipped rhPDGF-BB.

48. The method of claim 44 wherein the porous biocompatible carrier is a collagen sponge.

49. The method of claim 44 wherein the porous biocompatible carrier is a collagen wound dressing.

50. The method of claim 44 wherein the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 50 mg of rhPDGF-BB.

51. The method of claim 44 wherein the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 25 mg of rhPDGF-BB.

52. The method of claim 44 wherein the wound is retreated from 2 to 20 times over a maximum treatment period of 2 to 20 weeks.

53. The method of claim 44 wherein the steps (1)-(4) are repeated to retreat the wound at a retreatment frequency of at least every 8 days.

54. The method of claim 44 wherein the steps (1)-(4) are repeated to retreat the wound at a retreatment frequency of at least every 10 days.

55. The method of claim 44 wherein the wound is retreated a maximum of 10 times.

56. The method of claim 44 wherein: (A) the wound is a burn of a least six weeks duration; (B) the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, wherein at least about 80% of the rhPDGF-BB on a weight basis is unclipped rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.1 ml solution/cm.sup.3 of carrier to about 1 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 225 μg rhPDGF-BB/cm.sup.3 of carrier; (C) the wound is retreated from 2 to 20 times over a maximum treatment period of 2 to 20 weeks at a retreatment frequency of at least every 8 days; and (D) the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 50 mg of rhPDGF-BB.

57. A method of treating a wound that is a burn, wherein said method comprises: (1) debriding the wound to remove necrotic or infected tissue; (2) forming a therapeutic composition consisting essentially of sterile recombinant human PDGF-BB (rhPDGF-BB) in a physiologic solution and a sterile porous biocompatible carrier, wherein the porous biocompatible carrier is a collagen sponge or collagen wound dressing; (3) applying the therapeutic composition to the wound surface in an amount that is at least about 10 μg rhPDGF-BB per cm.sup.2 of treated wound surface area, wherein the carrier provides a substrate for cell attachment and vascular ingrowth as the wound heals; (4) covering the wound with a dressing; and (5) monitoring the healing of the wound during a treatment period and repeating steps (1)-(4) to retreat the wound at treatment intervals of 8 or more days, wherein the wound is retreated from 2 to 20 times over a maximum treatment period of 2 to 20 weeks, and wherein each retreatment comprises applying the therapeutic composition to the wound surface in an amount that is at least 10 μg rhPDGF-BB/cm.sup.2 treated wound surface area up to 100 μg rhPDGF-BB/cm.sup.2 treated wound surface area.

58. The method of claim 57 wherein the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.1 ml solution/cm.sup.3 of carrier to about 1 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 225 μg rhPDGF-BB/cm.sup.3 of carrier.

59. The method of claim 57 wherein the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.25 ml solution/cm.sup.3 of carrier to about 5 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 750 μg rhPDGF-BB/cm.sup.3 of carrier.

60. The method of claim 57 wherein the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 50 mg of rhPDGF-BB.

61. The method of claim 57 wherein the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 25 mg of rhPDGF-BB.

62. The method of claim 57 wherein the steps (1)-(4) are repeated to retreat the wound at a retreatment frequency of at least every 10 days.

63. The method of claim 57 wherein the wound is retreated a maximum of 10 times.

64. The method of claim 57 wherein: (A) the wound is a burn of at least six weeks duration; (B) the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, wherein at least about 80% of the rhPDGF-BB on a weight basis is unclipped rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.1 ml solution/cm.sup.3 of carrier to about 1 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 225 μg rhPDGF-BB/cm.sup.3 of carrier; and (C) the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 50 mg of rhPDGF-BB.

65. A method of treating a wound that is a burn: (1) debriding the wound to remove necrotic or infected tissue; (2) forming a therapeutic composition consisting essentially of sterile recombinant human PDGF-BB (rhPDGF-BB) in a physiologic solution and a sterile porous biocompatible carrier, wherein the porous biocompatible carrier is a collagen sponge or collagen wound dressing; (3) applying the therapeutic composition to the wound surface in an amount that is at least about 10 μg rhPDGF-BB per cm.sup.2 of treated wound surface area, wherein the carrier provides a substrate for cell attachment and vascular ingrowth as the wound heals; (4) covering the wound with a dressing; and (5) monitoring the healing of the wound during a treatment period and repeating steps (1)-(4) to retreat the wound at treatment intervals of 10 or more days, wherein the wound is retreated from 2 to 20 times, and wherein each retreatment comprises applying the therapeutic composition to the wound surface in an amount that is at least 10 μg rhPDGF-BB/cm.sup.2 treated wound surface area up to 100 μg rhPDGF-BB/cm.sup.2 treated wound surface area.

66. The method of claim 65 wherein the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (1) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (2) the ratio of the rhPDGF-BB solution to the carrier is between about 0.1 ml solution/cm.sup.3 of carrier to about 1 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 225 μg rhPDGF-BB/cm.sup.3 of carrier.

67. The method of claim 65 wherein the step of forming the therapeutic composition comprises combining the sterile rhPDGF-BB and the sterile porous biocompatible carrier, wherein: (i) the sterile rhPDGF-BB comprises an rhPDGF-BB solution comprising between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, and (ii) the ratio of the rhPDGF-BB solution to the carrier is between about 0.25 ml solution/cm.sup.3 of carrier to about 5 ml solution/cm.sup.3 of carrier, or the ratio of rhPDGF-BB to the carrier is between about 75 μg rhPDGF-BB/cm.sup.3 of carrier to about 750 μg rhPDGF-BB/cm.sup.3 of carrier.

68. The method of claim 65 wherein the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 50 mg of rhPDGF-BB.

69. The method of claim 65 wherein the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is less than about 25 mg of rhPDGF-BB.

70. The method of claim 65 wherein the wound is retreated from 2 to 20 times over a maximum treatment period of 2 to 20 weeks.

71. The method of claim 44 wherein when the rhPDGF-BB and the porous carrier are combined, the carrier is capable of entrapping the rhPDGF-BB within its pores such that the rhPDGF-BB is released over time as the carrier is absorbed by the patient's body, thereby providing controlled delivery of rhPDGF-BB at the wound over an extended period of time and simultaneously providing a matrix for new cell and tissue ingrowth.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 graphically shows the changes in mean wound area over time in three groups of animals included in a study as calculated by digital caliper measurements.

[0037] FIG. 2 graphically shows the changes in mean wound area over time in three groups of animals included in a study as calculated using ImageJ software.

[0038] FIG. 3 graphically shows the wound area as calculated using ImageJ software for each test animal on day 0 of a study.

[0039] FIG. 4 graphically shows the wound area as calculated using ImageJ software for each test animal on day 7 of a study.

[0040] FIG. 5 graphically shows the wound area as calculated using ImageJ software for each test animal on day 14 of a study.

[0041] FIG. 6 graphically shows the wound area as calculated using ImageJ software for each test animal on day 21 of a study.

[0042] FIGS. 7, 7A-7B graphically show that positive results are achieved with fewer applications of the therapeutic composition. FIG. 7A graphically shows the amount of wound healing (mm.sup.2) using ImageJ software per the number cumulative dosages for each of the four time point s (Day 0, Day 7, Day 14, and Day 21) for the Regranex Group 1 and rhPDGF/collagen Group 3. FIG. 7B shows the average percent healing for the Regranex Group 1, Collagen Group 2, and rhPDGF/collagen Group 3 at each of the four time points.

[0043] FIGS. 8, 8A-D graphically shows the average percent wound closure for each group over the treatment period: FIG. 8A shows Day 0; FIG. 8B shows Day 7; FIG. 8C shows Day 14; and FIG. 8D shows Day 21.

[0044] FIGS. 9-12 include a series of wound images showing the degree of healing over time for animals included in a study testing compositions in accordance with the invention.

[0045] FIG. 9 illustrates the state of the wound in 15 test subjects on day 0 of the study.

[0046] FIG. 10 illustrates the state of the wound in 13 test subjects on day 7 of the study.

[0047] FIG. 11 illustrates the state of the wound in 13 test subjects on day 14 of the study.

[0048] FIG. 12 illustrates the state of the wound in 13 test subjects on day 21 of the study.

[0049] FIGS. 13-18 include a series of photomicrographs of a cross section of the wound site on day 21 from three study animals.

[0050] FIG. 13 is a photomicrograph (2× magnification) of a study control animal treated with a collagen sponge and buffer.

[0051] FIG. 14 is a photomicrograph (10× magnification) of a study control animal treated with a collagen sponge and buffer.

[0052] FIG. 15 is a photomicrograph (2× magnification) of a study animal treated with a composition in accordance with the present invention.

[0053] FIG. 16 is a photomicrograph (10× magnification) of a study animal treated with a composition in accordance with the present invention.

[0054] FIG. 17 is a photomicrograph (2× magnification) of a study animal treated with Regranex.

[0055] FIG. 18 is a photomicrograph (10× magnification) of a study control animal treated with Regranex.

MODES FOR CARRYING OUT THE INVENTION

[0056] The present invention provides for a novel method of treating dermal wounds, such as diabetic foot ulcers (DFUs), venous stasis ulcers, pressure ulcers, burns, tramatic injuries and large surgical wounds. The present invention additionally provides for novel bioactive therapeutic compositions for use in treating such wounds, novel methods of preparing bioactive dressings useful for the treatment of wounds, and novel treatment regimens to improve patient compliance and wound healing.

[0057] The novel methods and therapeutic compositions in accordance with the present invention will enable equivalent or superior efficacy compared to prior art products in treating dermal wounds, and provide a better safety profile and improved patient compliance and convenience. The novel therapeutic compositions provided herein provide: 1) prolonged delivery of the PDGF onto the wound from each application, thus obviating the need for far more frequent applications by the patient (e.g., daily or every other day applications with prior art products); 2) a physical material such as a collagen sponge that can be applied like a Band-Aid onto the wound once every several days thus improving patient compliance; 3) stability at room temperature, eliminating the need to keep the product refrigerated; 4) a sterile product improving safety over prior art products; 5) a higher initial dose of PDGF compared to prior art products which better initiates the healing process thus reducing the need for prolonged patient use; 6) the use of an improved carrier that not only sustains the delivery of the PDGF but simultaneously provides a biological scaffold and/or open porous matrix that facilitates ingrowth of cells, blood vessels and new tissue leading to improved healing compared to prior art products which lack the ability to provide a biological matrix for cellular ingrowth; and 7) contains a more pure and potent rhPDGF-BB formulation with fewer isoforms than prior art formulations. The novel methods disclosed herein provide: 1) a higher initial dose of PDGF as compared to prior art products to better initiate the healing process, thus reducing the need for prolonged patient use; and 2) a treatment protocol that will facilitate improved patient compliance and convenience by requiring fewer periodic applications of the therapeutic composition, perhaps as few as 1 to 6 applications versus the 140 applications required by prior art products.

I. Definitions/Nomenclature

[0058] As used herein unless otherwise indicated, open terms such as “contain,” “containing,” “include,” “including,” and the like mean comprising.

[0059] Some embodiments herein contemplate numerical ranges. When a numerical range is provided, the range includes the range endpoints unless otherwise indicated. Unless otherwise indicated, numerical ranges include all values and subranges therein as if explicitly written out.

[0060] Some values herein are modified by the term “about.” In some instances, the term “about” in relation to a reference numerical value can include a range of values plus or minus 10% from that value. For example the amount “about 10” can include amounts from 9 to 11. In other embodiments, the term “about” in relation to a reference numerical value can include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

[0061] As used herein, the article “a” means one or more unless explicitly stated otherwise.

[0062] Where methods and steps described herein indicate certain events occurring in certain order, those of ordinary skill in the art will recognize that the ordering of certain steps may be modified and that such modifications are in accordance with the variations of the invention. Additionally, certain steps may be performed concurrently in a parallel process when possible, as well as performed sequentially.

[0063] The meaning of abbreviations is as follows: “C” means Celsius or degrees Celsius, as is clear from its usage, “μL” or “uL” or “U” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm” means nanometers, “mM” means millimolar, “μM” or “uM” means micromolar, “M” means molar, “mmol” means millimole(s), “μmol” or “uMol” means micromole(s)”, “g” means gram(s), “μg” or “ug” means microgram(s) and “ng” means nanogram(s), “% w/v” means weight/volume percent, “% v/v” means volume/volume percent, “HPLC” means high performance liquid chromatography, “UPLC” means ultra performance liquid chromatography, and “GC” means gas chromatography.

[0064] The term “homology” refers to the optimal alignment of sequences (either nucleotides or amino acids), which may be conducted by computerized implementations of algorithms. “Homology”, with regard to polynucleotides, for example, may be determined by analysis with BLASTN version 2.0 using the default parameters. “Homology”, with respect to polypeptides (i.e., amino acids), may be determined using a program, such as BLASTP version 2.2.2 with the default parameters, which aligns the polypeptide or fragments (and can also align nucleotide fragments) being compared and determines the extent of amino acid identity or similarity between them.

[0065] The above descriptions and methods for sequence homology are intended to be exemplary and it is recognized that this concept is well-understood in the art. Further, it is appreciated that nucleic acid sequences may be varied and still provide a functional enzyme, and such variations are within the scope of the present invention. The term “enzyme homolog” can also mean a functional variant.

[0066] As used herein, the term “carrier” is intended to refer broadly to any biologically compatible substance that can serve as a delivery vehicle for PDGF, whereas the terms “matrix” and “scaffold” are used interchangeable to refer to a carrier that acts as a substrate for cell attachment and/or vascular ingrowth as a wound heals, and/or provides a means for trapping the PDGF within its structure (such as, for example, through interconnected pores), thereby allowing for an ongoing or delayed or prolonged delivery of PDGF as a wound heals.

II. Novel Method of Treating Wounds

[0067] The present invention provides novel methods of treating of wounds. In one embodiment, a method of treating a wound comprises providing a therapeutic composition comprising a PDGF solution incorporated in a biocompatible scaffold, matrix or carrier and applying the therapeutic composition to a wound. A therapeutic composition comprising a PDGF solution incorporated in a biocompatible scaffold, matrix or carrier, for example, can be applied topically to the wound. In some embodiments, a method of treating a wound comprises multiple periodic applications of a therapeutic composition to a wound over a period of weeks.

[0068] In accordance with one aspect of the present invention, the novel treatment method for treating wounds includes the following steps: [0069] (1) debriding the wound as needed to remove necrotic or infected tissue; [0070] (2) forming a therapeutic composition comprising sterile rhPDGF-BB and a sterile porous biocompatible carrier; [0071] (3) applying the therapeutic composition containing PDGF to the wound surface, wherein the carrier provides a substrate for cell attachment and vascular ingrowth as the wound heals; [0072] (4) covering the wound with a dressing; and [0073] (5) monitoring the healing of the wound during a treatment period and repeating steps (1)-(4) at treatment intervals of 3 or more days.
The novel treatment method may further include preparing the novel therapeutic composition prior to applying it to the wound surface, wherein the composition comprises PDGF and a biological matrix. The method of preparing the composition may include: [0074] (2a) reconstituting a lyophilized (freeze-dried) sterile PDGF powder with sterile water, saline, a buffer, or a physiologic solution to provide a specific safe and therapeutic concentration of PDGF; and [0075] (1b) withdrawing the sterile PDGF solution from a vial (container) and aseptically adding it to a dry hydrophilic sterile matrix or patch in such a way that the matrix or patch is wetted with the PDGF solution.

[0076] In some embodiments, the dressing is an occlusive or semi-occlusive dressing. In some embodiments, the repeat of steps (1)-(3) may also comprise the steps of: (A) removing the dressing and cleaning the wound with saline or an appropriate antiseptic wound cleansing agent prior to applying the therapeutic composition the dressing, and (B) covering the wound with a new dressing following application of the therapeutic composition. In some embodiments, novel bioactive therapeutic compositions described herein may be used in combination with other aspects of treating wounds, including for example infection control, negative pressure wound therapy, and/or instructing the patient to avoid placing pressure on the wound site.

[0077] In accordance with one aspect of the invention, there is provided a timing schedule for periodically retreating the wound, i.e. repeating steps (2)-(4) or periodically reapplying the therapeutic composition to the wound. The actual number of retreatments and the retreating frequency (i.e., the treatment interval) should be determined based on a number factors including the severity of the wound (e.g., its grade, size and depth), the extent to which the natural wound healing environment is compromised (e.g., the vascular supply at the site, the metabolic state of the patient, the ability to off-load pressure on the site, presence of infection, diabetes stage for a DFU, degree of burn for a burn), patient's age, duration of the wound, and other co-morbidities such as smoking, obesity, uncontrolled glucose levels, patient compliance and others. The number of retreatments and the retreatment frequency should be increased for more severe wounds or for wounds with more compromised healing environments. In addition, the prescribed number of treatments and/or the retreatment frequency may be adjusted during the treatment period based on the wound's rate of healing, i.e. increase number of retreatments and/or retreatment frequency for slower healing wounds, or decrease number of retreatments and/or retreatment frequency for faster healing wounds.

[0078] In accordance with one aspect of the invention, the retreatment frequency is at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or at least about 15 days and so on up to at least about once every six weeks, or combinations thereof. In accordance with another aspect of the invention, the retreatment frequency is once every 2 to 42 days, or once every 3 to 42 days, or once every 2 to 28 days, or once every 3 to 28 days, or once every 2 to 7 days, or once every 3 to 7 days, or once every 4 to 21 days, once every 7 to 28 days, or once every 7 to 21 days, or once every 7 to 14 days, or once every 10 to 15 days, or once every 12 to 14 days. In accordance with another aspect of the invention, the retreatment frequency is once every 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 15 days, 21 days, 28 days, 30 days, 35 days, 42 days, or combinations thereof.

[0079] In accordance with one another aspect of the invention, the retreatment frequency is substantially the same over the treatment period, and the retreatment frequency is one time at least about every 2 days, at least about every 3 days, at least about every 4 days, at least about every 5 days, at least about every 6 days, at least about every 7 days, at least about every 8 days, at least about 9 every days, at least about every 10 days, at least about every 11 days, at least about every 12 days, at least about every 13 days, at least about every 14 days, or at least about every 15 days and so on up to at least about once every six weeks.

[0080] In accordance with one aspect of the invention, the wound is retreated at least 1 time, at least 2 times, at least 3 times, at least 4 times, or at least 5 times over the treatment period. In accordance with another aspect of the invention, the wound is retreated between 0 and 6 times, between 0 and 7 times, or between 0 and 8 times over the treatment period. In accordance with another aspect of the invention, the wound is treated between 1 to 8 times, or between 2 to 7 times, or between 3 to 6 times over the treatment period. In accordance with another aspect of the invention, the wound is retreated 1, 2, 3, 4, 5, 6, 7, 8, 10, or 20 times over the treatment period. In accordance with another aspect of the invention the wound is retreated between 0 and 46 times, or between 1 and 46 times, or between 0 and 20 times, or between 1 and 20 times, or between 0 and 27 times, or between 1 and 27 times.

[0081] In accordance with one aspect of the invention, the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is preferably more than 0 mg, but less than about 50 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 4 mg, or less than about 3 mg, or less than about 2 mg, or less than about 1 mg of rhPDGF-BB. In certain embodiments, the cumulative total amount of rhPDGF-BB applied to the wound during the treatment period is preferably between about 0.1 mg to about 50 mg, or about 0.5 mg to about 25 mg, or about 1 mg to about 10 mg, or about 2.5 mg to about 8 mg, or about 3 mg to about 7 mg, about 4 mg to about 6 mg.

[0082] The various retreatments may involve the same or different dosages of rhPDGF-BB, either in terms of the exact amount of rhPDGF-BB that is applied to the wound (i.e., “absolute dosage”) or in terms of the amount of rhPDGF-BB that is applied per square centimeter (cm.sup.2) of wound area (i.e., “area dosage”). In accordance with one aspect of the invention, each treatment applies an absolute dosage of between about 10 μg and about 50 mg, or between about 10 μg and about 25 mg, or between about 10 μg and about 20 mg, or between about 10 μg and about 15 mg, or between about 10 μg and about 10 mg, or between about 10 μg and about 5 mg of rhPDGF-BB or between about 10 μg and about 1 mg or rhPDGF-BB. In accordance with another aspect of the invention, each treatment applies an area dosage between about 10 μg PDGF/cm.sup.2 and about 1.0 mg PDGF/cm.sup.2, or between about 10 μg PDGF/cm.sup.2 and about 0.5 mg PDGF/cm.sup.2, or between about 10 μg PDGF/cm.sup.2 and about 0.25 mg PDGF/cm.sup.2, or between about 10 μg PDGF/cm.sup.2 and 0.1 mg PDGF/cm.sup.2, or between about 10 μg PDGF/cm.sup.2 and about 0.05 mg PDGF/cm.sup.2. In certain embodiments, each treatment with rhPDGF-BB is preferably between about 10 μg to 1000 μg PDGF/cm.sup.2, or about 0.01 mg to about 50 mg PDGF/cm.sup.2, or about 0.05 mg to about 25 mg PDGF/cm.sup.2, or about 0.1 mg to about 10 mg PDGF/cm.sup.2, or about 0.2 mg to about 2 mg PDGF/cm.sup.2. In certain embodiments, each treatment applies an area dosage that is at least about 10 μg of rhPDGF/cm.sup.2 of wound surface area, or at least about 25 μg of rhPDGF/cm.sup.2 of wound surface area, or at least about 50 μg of rhPDGF/cm.sup.2 of wound surface area, or at least about 100 μg of rhPDGF/cm.sup.2 of wound surface area, or at least about 250 μg of rhPDGF/cm.sup.2 of wound surface area, or at least about 500 μg of rhPDGF/cm.sup.2 of wound surface area. In certain embodiments, each treatment applies an area dosage that is between about 10 μg of rhPDGF/cm.sup.2 of wound surface area and about 500 μg of rhPDGF/cm.sup.2 of wound surface area, or between about 10 μg of rhPDGF/cm.sup.2 of wound surface area and about 100 μg of rhPDGF/cm.sup.2 of wound surface area, or between about 15 μg of rhPDGF/cm.sup.2 of wound surface area and about 375 μg of rhPDGF/cm.sup.2 of wound surface area, or between about 30 μg of rhPDGF/cm.sup.2 of wound surface area and about 190 μg of rhPDGF/cm.sup.2 of wound surface area, or between about 30 μg of rhPDGF/cm.sup.2 of wound surface area and about 300 μg of rhPDGF/cm.sup.2 of wound surface area.

[0083] In accordance with one aspect of the invention, the initial treatment with compositions in accordance with the present invention may be the most important treatment. PDGF facilitates the wound healing process through its effect on cell proliferation (mitogenesis) and directed cellular movement (chemotaxis) as well as re-vascularization (generating new blood vessels). Many cells have been shown to possess receptors (binding sites) for PDGF including connective tissue cells (skin, bone, cartilage, tendon and ligament), blood vessel cells and cells of the nervous system. Cells that possess receptors for PDGF respond by migrating toward the site of the wound (where PDGF is present at elevated levels as a result of applying therapeutic compositions in accordance with the present invention) and subsequently proliferating after binding PDGF. Since the PDGF receptor is degraded quickly after activation, cell proliferation is controlled and limited by the presence of locally available PDGF as well as by cell-cell interaction that leads cells to proceed from the proliferative phase of wound healing to that of matrix deposition that ultimately results in complete healing. As a result, a critical bolus of rhPDGF-BB must be applied during the initial treatment to ensure that the patient's natural wound healing process is properly activated. Therefore, in accordance with the invention the initial treatment comprises applying a therapeutic composition containing an area dosage that is at least 10 μg PDGF/cm.sup.2 wound surface area, up to 5000 μg PDGF/cm.sup.2 wound surface area, or at least 20 μg PDGF/cm.sup.2 up to 1000 μg PDGF/cm.sup.2 wound surface area, or at least 30 μg PDGF/cm.sup.2 up to 600 μg PDGF/cm.sup.2 wound surface area, or at least 40 μg PDGF/cm.sup.2 up to 400 μg PDGF/cm.sup.2 wound surface area, or at least 50 μg PDGF/cm.sup.2 up to 350 μg PDGF/cm.sup.2 wound surface area, or at least 60 μg PDGF/cm.sup.2 up to 300 μg PDGF/cm.sup.2 wound surface area, or at least 200 μg PDGF/cm.sup.2 up to 2000 μg PDGF/cm.sup.2 wound surface area. In accordance with another aspect of the invention the initial treatment comprises applying a therapeutic composition containing an area dosage that is at least 10 μg PDGF/cm.sup.2 wound surface area, or at least 20 μg PDGF/cm.sup.2 wound surface area, or at least 25 μg PDGF/cm.sup.2 wound surface area, or at least 30 μg PDGF/cm.sup.2 wound surface area, or at least 40 μg PDGF/cm wound surface area, or at least 50 μg PDGF/cm.sup.2 wound surface area, or at least 60 μg PDGF/cm.sup.2 wound surface area, or at least 70 μg PDGF/cm.sup.2 wound surface area, or at least 80 μg PDGF/cm.sup.2 wound surface area, or at least 90 μg PDGF/cm.sup.2 wound surface area, or at least 100 μg PDGF/cm.sup.2 wound surface area, or at least 250 μg PDGF/cm.sup.2 wound surface area, or at least 500 μg PDGF/cm.sup.2 wound surface area.

[0084] In accordance with another aspect of the invention, each treatment applies is between about 4 μl PDGF solution/cm.sup.3 of carrier (which may be a matrix such as a collagen sponge) to about 40 ml PDGF solution/cm.sup.3 of carrier, or between about 0.1 ml PDGF solution/cm.sup.3 of carrier to about 30 ml PDGF solution/cm.sup.3 of carrier, or between about 0.2 ml PDGF solution/cm.sup.3 of carrier to about 20 ml PDGF solution/cm.sup.3 of carrier, or between about 0.1 ml PDGF solution/cm.sup.3 of carrier to about 10 ml PDGF solution/cm.sup.3 of carrier, or between about 0.25 ml PDGF solution/cm.sup.3 of carrier to about 5 ml PDGF solution/cm.sup.3 of carrier, or between about 0.25 ml PDGF solution/cm.sup.3 of carrier to about 2.5 ml PDGF solution/cm.sup.3 of carrier, or between about 0.1 ml PDGF solution/cm.sup.3 of carrier to about 1 ml PDGF solution/cm.sup.3 of carrier, or between about 0.5 ml PDGF solution/cm.sup.3 of carrier to about 1.5 ml PDGF solution/cm.sup.3 of carrier. In certain embodiments, the PDGF solution contains about 0.3 mg/ml of rhPDGF-BB.

[0085] In accordance with another aspect of the invention, each treatment applies between about 1.2 μg PDGF/cm.sup.3 of carrier to about 12 mg PDGF/cm.sup.3 of carrier, or between about 30 μg PDGF/cm.sup.3 of carrier to about 9 mg PDGF/cm.sup.3 of carrier, or between about 60 μg PDGF/cm.sup.3 of carrier to about 6 mg PDGF/cm.sup.3 of carrier, or between about 75 μg PDGF/cm.sup.3 of carrier to about 3 mg PDGF/cm.sup.3 of carrier, or between about 75 μg PDGF/cm.sup.3 of carrier to about 1.5 mg PDGF/cm.sup.3 of carrier, or between about 75 μg PDGF/cm.sup.3 of carrier to about 750 μg PDGF/cm.sup.3 of carrier, or between about 120 μg PDGF/cm.sup.3 of carrier to about 600 μg PDGF/cm.sup.3 of carrier, or between about 150 μg PDGF/cm.sup.3 of carrier to about 450 μg PDGF/cm.sup.3 of carrier, or between about 75 μg PDGF/cm.sup.3 of carrier to about 225 μg PDGF/cm.sup.3 of carrier.

[0086] In accordance with one aspect of the invention, the initial PDGF treatment absolute dosage may be greater than the subsequent retreatment dosages. The initial PDGF treatment absolute dosage may be about 10%, about 20%, about 30%, about 40%, or about 50% higher, or up to about 300% higher than each of the subsequent retreatment PDGF dosages.

[0087] In accordance with one aspect of the invention, the method includes storing the PDGF at room temperature, generally between 16 and 32 degrees C. Prior to use it may be reconstituted with sterile water, saline, a buffer, or other physiologic solution to form a solution having the desired PDGF concentration. The solution is then added to a carrier, preferably a cell matrix (e.g., a collagen sponge) having the desired porosity in the desired volume to wet the matrix. The rhPDGF soaked matrix is then applied to the wound surface. If the wound is an external wound it is then covered with a wound dressing. This process may then be repeated in accordance with frequency and duration parameters described above until the wound is substantially healed.

III. Novel Therapeutic Compositions for Treating Wounds

[0088] The present invention also provides novel therapeutic compositions for treating wounds, which comprise sterile PDGF incorporated in a biocompatible sterile carrier, matrix or scaffold. For example, the therapeutic composition can be applied topically to a wound to facilitate the wound's healing.

[0089] In accordance with one aspect of the invention, a therapeutic composition is provided that comprises a rhPDGF-BB solution and a carrier that is preferably a biocompatible cell scaffold, wherein the rhPDGF-BB solution is disposed in or incorporated into the cell scaffold. In some embodiments, the rhPDGF-BB solution comprises between about 0.05 mg/ml to about 5 mg/ml of rhPDGF-BB, or between about 0.1 mg/ml to about 1 mg/ml of rhPDGF-BB, or between about 0.2 mg/ml to about 0.4 mg/ml of rhPDGF-BB. In accordance with one aspect of the invention, the rhPDGF-BB solution contains rhPDGF-BB at a concentration of about 0.05 mg/ml, or about 0.1 mg/ml, or about 0.2 mg/ml, or about 0.25 mg/ml, or about 0.3 mg/ml, or about 0.35 mg/ml, or about 0.4 mg/ml, or about 0.5 mg/ml, or about 0.6 mg/ml, or about 0.7 mg/ml, or about 0.8 mg/ml, or about 0.9 mg/ml, or about 1 mg/ml, or about 2 mg/ml, or about 3 mg/ml, or about 4 mg/ml, or about 5 mg/ml.

[0090] In some embodiments, the rhPDGF-BB solution is a preformulated aseptic PDGF solution comprising the elements described herein (e.g., PDGF concentration, sterile solution composition, etc.). In other embodiments the rhPDGF-BB solution is formed at the time of use, preferably by combining a sterile solution (e.g., sterile water, saline, a buffer solution, or a physiologic solution) with a sterile powder comprising or consisting essentially of lyophilized rhPDGF-BB. The sterile solution is used to reconstitute the lyophilized rhPDGF-BB. The lyophilized rhPDGF-BB is formed by lyophilizing liquid rhPDGF-BB produced by using a recombinant expression system as described further herein below under aseptic conditions.

[0091] In another aspect of the invention rhPDGF may be incorporated into carrier, preferably a sterile, biocompatible, absorbable cell scaffold, and the PDGF saturated carrier is then lyophilized to form a sterile, dry device incorporating rhPDGF. Any known technique for lyophilizing recombinant proteins may be used to lyophilize rhPDGF-BB so long as it yields a sterile powder. The resulting lyophilized rhPDGF-BB powder is capable of being stored at room temperature and still maintain at least about 80% of its bioactivity for at least about 6 months, or at least about 1 year, or at least about 2 years, or at least about 3 years. The sterile lyophilized device may then be applied directly to a wound site or wetted either by blood or other sterile solution prior to placement on the wound.

[0092] Because PDGF has a tendency to adhere to surfaces of a container, such as a vial, (particularly at higher pH's) achieving reconstitution of 100% of the lyophilized PDGF in a vial may be challenging. Therefore, in certain embodiments, additives may be included in the PDGF solution to lower its pH below about 7, or below about 6, or below about 5 or below about 4 or below about 3. Additives that may facilitate reconstituting the lyophilized PDGF include salts, carrier proteins such as albumin, or low pH solutions such as dilute acetic acid or hydrochloric acid. If the PDGF solution is too acidic, however, it could negatively impact the biocompatible scaffold. Therefore, in certain embodiments, the lyophilized PDGF is reconstituted in a solution having a pH below about 5, and once the PDGF is substantially fully reconstituted a base solution is added to increase the pH of the PDGF solution to between about 6 to about 8, or to increase it to about 7 before it is combined with the biocompatible scaffold. Such a pH adjustment step is particularly useful when the biocompatible scaffold is a collagen sponge.

[0093] The buffer solution used to reconstitute the lyophilized rhPDGF-BB may comprise, but is not limited to, water, saline, carbonates, phosphates (e.g. phosphate buffered saline), histidine, acetates (e.g. sodium acetate), acidic buffers such as acetic acid and HCl, and organic buffers such as lysine, Tris buffers (e.g. tris(hydroxymethyl)aminoethane), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and 3-(N-morpholino) propanesulfonic acid (MOPS). Preferably, the buffer solution is sterile. Buffers can be selected based on biocompatibility with PDGF and the buffer's ability to impede undesirable protein modification. Buffers can additionally be selected based on compatibility with wound tissues. In one embodiment, sodium acetate buffer is used. The buffers can be employed at different molarities, for example, about 0.1 mM to about 100 mM, about 1 mM to about 50 mM, about 5 mM to about 40 mM, about 10 mM to about 30 mM, or about 15 mM to about 25 mM, or any molarity within these ranges. In some embodiments, an acetate buffer is employed at a molarity of about 20 mM.

[0094] As noted above, the rhPDGF-BB solution is combined with carrier to form a therapeutic composition. The carrier may be a matrix or scaffold that acts as a substrate for cell attachment and/or vascular ingrowth as a wound heals, and/or provides a means for trapping the PDGF within its structure (such as, for example, through interconnected pores), thereby allowing for an ongoing or delayed or prolonged delivery of PDGF as a wound heals and the matrix or scaffold is resorbed by the body. In some embodiments, the carrier or matrix is a biocompatible, resorbable cell scaffold. The carrier or matrix may comprise natural polymers such as collagen, gelatin, fibrin, alginate, cellulose, Chitosan or fibronectin. The carrier or matrix may also comprise synthetic biocompatible polymers selected from the group of synthetic polymers such as poly(DL-lactide-co-glycolide) (PLGA), poly(DL-lactide)(PDLA), poly(L-lactide)(PLLA), poly(e-caprolactone)(PCL), polyurethane or others. The carrier or matrix may also be a mixture of such natural and synthetic polymers. In some embodiments, the matrix comprises a collagen or gelatin sponge, which may be a Type 1 collagen sponge. A collagen sponge holds the rhPDGF at the wound site and concurrently provides a scaffold for cell growth, resulting in improved user friendliness and more rapid and complete healing. In one aspect the invention, the carrier or matrix, which may be a collagen sponge, has a porosity of between about 10 microns to about 2 mm, or about 50 microns to about 1000 microns, or about 100 microns to about 500 microns. The average pore size may be between about 50 microns to about 500 microns and wherein the majority of the pores are interconnected.

[0095] In some embodiments, carrier or matrix materials are bioresorbable. A carrier or matrix material, in one embodiment, can be at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 90% or 100% resorbed within one month subsequent to its application to the wound. Bioresorbability will be dependent on: (1) the nature of the material (i.e., its chemical makeup, physical structure and size); (2) the location within the body in which the material is placed; (3) the amount of material that is used; (4) the metabolic state of the patient (diabetic/non-diabetic, smoker, old age, etc.); and (5) the extent and/or type of wound treated.

[0096] In one aspect of the invention, the rhPDGF-BB solution and the carrier should be combined in an appropriate ratio in order to form a therapeutic composition that has optimal effectiveness in healing wounds. In some embodiments, the rhPDGF-BB solution and the carrier are combined at a ratio that is between about 4 μl PDGF solution/cm.sup.3 of carrier (which may be a matrix such as a collagen sponge) to about 40 ml PDGF solution/cm.sup.3 of carrier, or between about 0.1 ml PDGF solution/cm.sup.3 of carrier to about 30 ml PDGF solution/cm.sup.3 of carrier, or between about 0.2 ml PDGF solution/cm.sup.3 of carrier to about 20 ml PDGF solution/cm.sup.3 of carrier, or between about 0.1 ml PDGF solution/cm.sup.3 of carrier to about 10 ml PDGF solution/cm.sup.3 of carrier, or between about 0.25 ml PDGF solution/cm.sup.3 of carrier to about 5 ml PDGF solution/cm.sup.3 of carrier, or between about 0.25 ml PDGF solution/cm.sup.3 of carrier to about 2.5 ml PDGF solution/cm.sup.3 of carrier, or between about 0.1 ml PDGF solution/cm.sup.3 of carrier to about 1 ml PDGF solution/cm.sup.3 of carrier, or between about 0.5 ml PDGF solution/cm.sup.3 of carrier to about 1.5 ml PDGF solution/cm.sup.3 of carrier.

[0097] In some embodiments, the rhPDGF-BB and the carrier are combined at a ratio that is between about 1.2 μg PDGF/cm.sup.3 of carrier to about 12 mg PDGF/cm.sup.3 of carrier, or between about 30 μg PDGF/cm.sup.3 of carrier to about 9 mg PDGF/cm.sup.3 of carrier, or between about 60 μg PDGF/cm.sup.3 of carrier to about 6 mg PDGF/cm.sup.3 of carrier, or between about 75 μg PDGF/cm.sup.3 of carrier to about 3 mg PDGF/cm.sup.3 of carrier, or between about 75 μg PDGF/cm.sup.3 of carrier to about 1.5 mg PDGF/cm.sup.3 of carrier, or between about 75 μg PDGF/cm.sup.3 of carrier to about 750 μg PDGF/cm.sup.3 of carrier, or between about 120 μg PDGF/cm.sup.3 of carrier to about 600 μg PDGF/cm.sup.3 of carrier, or between about 150 μg PDGF/cm.sup.3 of carrier to about 450 μg PDGF/cm.sup.3 of carrier, or between about 75 μg PDGF/cm.sup.3 of carrier to about 225 μg PDGF/cm.sup.3 of carrier.

[0098] In one aspect of the invention, the carrier is a scaffold and the rhPDGF-BB/scaffold ratio is such that when the rhPDGF-BB solution and the scaffold are combined, the scaffold is capable of entrapping at least about 20%, 30%, 40% or 50% up to at least about 100% of the rhPDGF-BB within the scaffold's pores such that the rhPDGF-BB is released over time as the scaffold is absorbed by the patient's body, thereby providing controlled delivery of rhPDGF-BB at the wound site over an extended period of time and simultaneously providing a matrix for new cell and tissue ingrowth. In some embodiments, the scaffold is capable of entrapping between about 20% to about 100%, or between about 25% to about 95%, or between 30% to about 90% of the rhPDGF-BB within the scaffold's pores. The percentages of PDGF entrapment described above are also applicable to entrapment of reconstituted lyophilized PDGF-BB.

[0099] Various amounts of rhPDGF-BB may be used in the therapeutic compositions of the present invention. In accordance with one aspect of the invention, the total amount of rhPDGF-BB included in the therapeutic composition is less than 50 mg, or less than 25 mg, or less or less than 10 mg, or less than 5 mg, or less than 2.5 mg or less than 1 mg. In accordance with another aspect of the invention the total amount of rhPDGF-BB included in the therapeutic composition is about 50 mg, or about 25 mg, or about 10 mg, or about 1.0 mg, or about 0.5 mg, or about 0.1 mg.

[0100] The concentration of PDGF in embodiments of the present invention can be determined by using an enzyme-linked immunoassay as described in U.S. Pat. Nos. 6,221,625, 5,747,273, and 5,290,708, incorporated herein by reference, or any other assay known in the art for determining PDGF concentration. The concentration of PDGF in the embodiments of the present invention is less than about 10 mg/g, or less than about 5 mg/g or less than about 1 mg/g or less than about 0.5 mg/g or less than about 0.1 mg/g or less than about 0.05 mg/ml. In another aspect of the invention the concentration of PDGF in the embodiments of the present invention is between about 0.05 mg/g to about 5 mg/g, or between about 0.1 mg/g to about 1 mg/g or between about 0.25 mg/g and about 0.5 mg/g.

[0101] The PDGF-BB used in the therapeutic composition of the present invention may be derived from any source such as natural source, synthetic source or recombinant source. In accordance with one aspect of the invention, PDGF is produced by recombinant DNA techniques. When PDGF is produced by recombinant DNA techniques, a DNA sequence encoding a single monomer (e.g., PDGF B-chain), is inserted into cultured cells for expression of the B chain monomer. The monomer is then extracted and isolated from the cell culture and refolded to form the biologically active homodimer (e.g., PDGF-BB), which may be further processed for additional purification. In accordance with one aspect of the invention, the cultured cells are prokaryotic cells or are E. coli cells. The rhPDGF-BB produced through these recombinant techniques can be purified in accordance with the techniques outlined in PCT No. WO 2005/077973, which is incorporated herein.

[0102] As noted above, prior art recombinant DNA production methods have resulted in mixtures of rhPDGF-BB fragments. In accordance with one aspect of the invention, substantially all of the rhPDGF-BB included in the therapeutic compositions described herein are intact non-clipped chains. In accordance with one aspect of the invention, the bacterial expression system is an E. coli expression system, and the resulting protein is purified using reversed phase high performance liquid chromatography, gel filtration or ion exchange chromatography, or some combination thereof, wherein the resulting rhPDGF-BB contained in the purified protein composition is at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% unclipped rhPDGF-BB on a weight basis.

[0103] In some embodiments, the rhPDGF-BB included in the therapeutic compositions of the present invention is a rhPDGF-BB that comprises or consists essentially of an amino acid sequence having at least about 90%, about 92%, about 94%, about 96%, about 98%, about 99%, or about 100% homology to SEQ ID NO. 1, which is provided below:

TABLE-US-00001 SEQ ID NO. 1: Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu Cys Lys Thr Arg Thr                  5                  10                  15                  20 Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn Ala Asn Phe Leu Val Trp                 25                  30                  35                  40 Pro Pro Cys Val Glu Val Gln Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys                 45                  50                  55                  60 Arg Pro Thr Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg Lys                 65                  70                  75                  80 Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu Ala Cys Lys Cys Glu                 85                  90                  95                  100 Thr Val Ala Ala Ala Arg Pro Val Thr                 105

[0104] In accordance with another aspect of the invention, the rhPDGF-BB included in the therapeutic compositions of the present invention comprises or consists essentially of at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% unclipped rhPDGF-BB on a weight basis. In accordance with another aspect of the invention, the rhPDGF-BB included in the therapeutic compositions of the present invention comprises or consists essentially of at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97% of rhPDGF-BB that comprises or consists essentially of an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to SEQ ID NO. 1.

[0105] In some embodiments, the components comprising the novel compositions of the present invention are provided in a kit. A kit can comprise three components: [0106] a) vial of sterile rhPDGF-BB lyophilized powder, [0107] b) vial of sterile water, a buffer, saline, or a physiologic solution, and [0108] c) a carrier.
The kit will may be stored at room temperature for up to 3 years. In some embodiments the storage is between 16 and 32 degrees C. In some embodiments, the powder included in the kit comprises a predetermined amount of PDGF. In some embodiments, the amount of PDGF is consistent with the values provided herein. In some embodiments, the carrier is included in a blister pack comprising a predetermined amount of carrier. In some embodiments, the amount of carrier is consistent with the values provided herein, and the type carrier is consistent with the materials described herein.

[0109] At the time of use, the rhPDGF-BB in the kit will be reconstituted with the sterile water, saline, buffer, or physiologic solution and the carrier will be shaped to the size of the wound. Following trimming the carrier to fit the wound, it will be soaked with the rhPDGF solution such that the solution fully saturates the interior pores of the carrier. The rhPDGF-saturated carrier will then be applied to the debrided wound and covered with a wound dressing. This process is repeated in accordance with the timing schedules described hereinabove.

IV. Methods of Treating Various Types of Wounds

[0110] The methods and compositions of the present invention are useful in treating a variety of wounds including diabetic ulcers, pressure ulcers, neuropathic ulcers, vascular ulcers, burns, accidental acute wounds and surgical wounds. Various wound classification systems exists and can be used to identify wounds that methods and compositions of the present invention are particularly useful in treating. Two such ulcer classification systems include the Wagner classification system (see, Wagner (1987) Orthopedics 10:163-72) and the University of Texas classification system (see, Lavery (1996) J Foot Ankle Surg 35:528-31). The Wagner system grades the wound by the depth of the wound and the presence of infection. It has five numeric grades: [0111] Grade 1: Superficial Diabetic Ulcer [0112] Grade 2: Ulcer extension [0113] Involves ligament, tendon, joint capsule or fascia [0114] No abscess or Osteomyelitis [0115] Grade 3: Deep ulcer with abscess or Osteomyelitis [0116] Grade 4: Gangrene to portion of forefoot [0117] Grade 5: Extensive gangrene of foot
The University of Texas classification has four numeric grades based on the depth of the wound. In addition there are four letter grades, A to D, related to infection and ischemia. The University of Texas classification system includes: [0118] Stages [0119] Stage A: No infection or ischemia [0120] Stage B: Infection present [0121] Stage C: Ischemia present [0122] Stage D: Infection and ischemia present [0123] Grading [0124] Grade 0: Epithelialized wound [0125] Grade 1: Superficial wound [0126] Grade 2: Wound penetrates to tendon or capsule [0127] Grade 3: Wound penetrates to bone or joint
A wound with a numeric grade of 3 and letter grade of D, for example, would be a wound that penetrates to bone or joint and is infected and ischemic. In accordance with one aspect of the present invention, the methods and compositions of the present invention are used to treat a wound that is either a grade 2, grade 3 or grade 4 wound under the Wagner classification system, or a grade 1, 2 or 3 wound (stages A, B, C, or D) under the University of Texas classification system.

[0128] In some embodiments, the methods and compositions described herein may be used to treat wounds such as lower extremity ulcers, and in particular foot ulcers on diabetic patients. The methods and compositions of the present invention are particularly useful in treating non-healing lower extremity diabetic ulcers which have failed to heal by about 50% after about 4 weeks of conventional therapies under the current standard of care as described above in the Background.

[0129] In some embodiments, the compositions of the present invention are used to treat burns in combination with a 1:1.5 or 1:1.3 meshed split thickness skin graft (the meshing allows the graft to cover a wider area but leaves small openings that need to heal), abdominoplasties (so-called “tummy tucks”), healing following other types of plastic and reconstructive surgeries, or post-amputation wounds.

V. Additional Therapeutic Elements

[0130] The therapeutic compositions of the present invention may include additional therapeutics elements to further facilitate healing a wound. In some embodiments, solutions comprising PDGF can further comprise additional components, such as other biologically active agents. In other embodiments, solutions comprising PDGF can further comprise cell culture media, other stabilizing proteins such as albumin, antibacterial agents, protease inhibitors [e.g., ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), aprotinin, e-aminocaproic acid (EACA), etc.] and/or other growth factors such as fibroblast growth factors (FGFs), epidermal growth factors (EGFs), transforming growth factors (TGFs), keratinocyte growth factors (KGFs), insulin-like growth factors (IGFs), or other PDGFs including compositions of PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and/or PDGF-DD. In addition, biologically active agents that can be incorporated into compositions of the present invention in addition to PDGF can comprise organic molecules, inorganic materials, proteins, peptides, nucleic acids (e.g., genes, gene fragments, small insert ribonucleic acids [si-RNAs], gene regulatory sequences, nuclear transcriptional factors, and antisense molecules), nucleoproteins, polysaccharides (e.g., heparin), glycoproteins, and lipoproteins. Additional non-limiting examples of biologically active compounds that can be incorporated into compositions of the present invention, including, e.g., anti-cancer agents, antibiotics, analgesics, anti-inflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, hormones, muscle relaxants, prostaglandins, trophic factors, growth factors, and vaccines, are disclosed in U.S. patent application Ser. No. 11/159,533 (Publication No: 20060084602).

[0131] Standard protocols and regimens for delivery of additional biologically active agents are known in the art. Additional biologically active agents can be introduced into compositions of the present invention in amounts that allow delivery of an appropriate dosage of the agent to the wound site. In most cases, dosages are determined using guidelines known to practitioners and applicable to the particular agent in question. The amount of an additional biologically active agent to be included in a composition of the present invention can depend on such variables as the type and extent of the condition, the overall health status of the particular patient, the formulation of the biologically active agent, release kinetics, and the bioresorbability of the biocompatible scaffold. Standard clinical trials may be used to optimize the dose and dosing frequency for any particular additional biologically active agent

EXAMPLES

Example 1

[0132] The efficacy of a collagen wound dressing containing 0.3 mg/ml recombinant human platelet derived growth factor-BB (rhPDGF-BB) was evaluated in the treatment of surgically induced full thickness wounds in mice rendered diabetic by a mutation in the leptin receptor (db/db).

A. Study Design

[0133] Fifteen (15) male C57/B6 (Leprdb) db/db mice with an average starting body weight of 41.46 g were obtained from Jackson Laboratory (Bar Harbor, Me.) strain code 000642. Animals were acclimatized prior to study commencement. During this period of 3 days, the animals were observed daily in order to reject animals that presented in poor condition.

[0134] During the study all animals were single housed under identical conditions in disposable cages. The study was performed in animal rooms provided with HEPA-filtered air at a temperature of 70° F.+/−5° F. and relative humidity of 50%+/−20%. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour. The room was on an automatic timer for a light/dark cycle of 12 hours on and 12 hours off with no twilight. AlphaDry® bedding was used. Bedding was changed a minimum of once per week. Cages, tops, bottles, etc. were washed with a commercial detergent and allowed to air dry. A commercial disinfectant was used to disinfect surfaces and materials introduced into the hood. Floors were swept daily and mopped a minimum of twice weekly with a commercial detergent. Walls and cage racks were sponged a minimum of once per month with a dilute bleach solution. A cage card or label with the appropriate information necessary to identify the study, dose, animal number and treatment group marked all cages. The temperature and relative humidity was recorded during the study, and the records retained. Animals were fed with a sterile Purina Labdiet® 5053 rodent diet and sterilized water was provided ad libitum.

[0135] At the commencement of the study, the fifteen (15) animals were randomly and prospectively divided into three (3) groups of five (5) animals each: [0136] Group 1—Regranex Gel 0.01% rhPDGF-BB was applied daily for 21 days as prescribed by the package insert; [0137] Group 2—a collagen wound dressing combined with buffer was applied on days 0, 7 and 14; and [0138] Group 3—a collagen wound dressing containing 0.3 mg/ml recombinant human platelet derived growth factor-BB (rhPDGF-BB) was applied on days 0, 7 and 14.
Each animal was identified by an ear punch corresponding to an individual number. On Day 0, mean starting weights were recorded, to ensure that mean starting weights were comparable among groups. A cage card was used to identify each cage or label marked with the study number (LYN-01), treatment group number, and animal numbers.

[0139] Test and control collagen+/−PDGF articles were administered topically as surgical dressings (as described below) immediately following the induction of the wound and were changed every seven (Q7) days. Regranex treated sites were treated as prescribed in the Instructions For Use (IFU) included in the product insert including daily dosing as outlined below. All dressings were applied and held in place using Tegaderm™ and secured in place outside of the wound area with benzoin. At the time of dressing change, the wound area was rinsed with saline and the rinse was collected and stored at −80° C. for future analysis of protease activity. For sites treated with the collagen wound dressing, all non-adherent collagen was gently removed from the healing wound, the site rinsed with saline and the rinse collected as described. Following removal of the dressing and collection of the rinse, the wound was measured using a caliper and photographed prior to re-application of dressing/test article. All wound areas were reported in mm.sup.2.

[0140] For photographic documentation of wound healing, the camera was mounted on a tripod at an optimal distance to ensure all photos were consistent. A ruler was placed such that it was captured in the image to allow accurate estimation of lesion size. In addition to in life measurements of the wound area, all photographs of the wounds were analyzed using Image J Software and the wound area was traced and quantitated at the conclusion of the study.

[0141] Blood glucose levels were determined prior to the start of the study and again just prior to sacrifice on Day 21 to confirm diabetic disease state. At study termination, the wound site was collected in 10% NBF and prepared for histopathology. The study design is summarized below in Table 1.

TABLE-US-00002 TABLE 1 Study Design Group Number Wound Num- of Wound Treat- Route/ Assess- ber Animals (Day 0) ment* Frequency ment 1 5 male 1.5 cm × Regranex Topical - 21 daily Daily To (db/db) 1.5 cm Gel applications; dose Day 21 applied based on open wound measurements at Day 0, 7 and 14** 2 5 male 1.5 cm × Collagen Topical - 3 weekly Every 7 (db/db) 1.5 cm wound applications; Days To dressing + Days Day 21 Buffer 0, 7 and 14*** 3 5 male 1.5 cm × PDGF Topical - 3 weekly Every 7 (db/db) 1.5 cm Bioactive applications; Days To Wound Days Day 21 Dressing 0, 7 and 14*** *All dressings were applied and held in place by Tegaderm ™ **The cm length of Regranex applied was based on open wound measurements obtained on Days 0, 7 and 14. The centimeter length of Regranex applied daily was the same for Days 0-6 and was based on measurements obtained on Day 0. The centimeter length of Regranex applied daily for Days 7-13 was based on measurements obtained on Day 7 and the centimeter length of Regranex applied on Days 14-21 was based on measurements obtained on Day 14. See “PDGF-BB Calculations” for detail. ***The volume of PDGF-BB or buffer/sterile saline that was applied to the collagen sponge at Days 0, 7 and 14 utilized the formula: 145 × cm.sup.2 open wound surface area (length [cm] × width [cm] open wound)

B. Test Articles &Vehicle Preparation

[0142] The topical formulations used in the study were Regranex Gel (0.01% rhPDGF-BB in carboxymethylcellulose gel) (Group 1); acollagen wound dressing wetted with rhPDGF-BB (Group 3); and acollagen wound dressing wetted with saline (Group 2). All dressings were covered with Tegaderm™ and secured with benzoin.

[0143] 1. Dressing Compositions

[0144] a. Group 1 rhPDGF Dosage

[0145] As described in Regranex Package Insert, “each square centimeter of ulcer surface area will require approximately 0.25 cm length of gel squeezed from 15 gram tube”. Formula: (1×w)÷4=cm length Regranex. For a 1.5 cm×1.5 cm square wound: (1.5×1.5)÷4=0.56 cm length Regranex. As described in Regranex Package Insert, “the weight of Regranex gel from 15 g tube is 0.25 g/cm length”. Regranex is 0.01% rhPDGF-BB or 100 μg/g Regranex. For 0.56 cm length of product, the weight of product is 0.14 g for a total dose of PDGF-BB of 14 μg. For sites treated with Regranex for 21 days the maximum total dose for the study period (assuming no change in open wound size from Day 0) would be 14 μg/day×21 days or 294 μg of PDGF-BB. However, at Days 7 and 14 the open wound size was determined for all Regranex treated sites and the amount of Regranex applied was recalculated using the formula above ([1×w]÷4=cm length Regranex).

[0146] b. Group 3 rhPDGF and Group 2 Saline Dosages

[0147] The concentration of rhPDGF-BB used in the study was 0.3 mg/ml or 300 μg/ml. To not exceed a total dose for the study period of 294 μg PDGF-BB (same total maximum study dose as Regranex), a total of 0.98 ml of 0.3 mg/ml PDGF-BB would be applied to the wound site over the 21-day study period. Assuming a total of 3 administrations (days 0, 7 and 14), each administration would consist of ≈327 μl PDGF-BB onto the collagen sponge representing a dose of approximately 98 μg PDGFBB/administration (slightly more than 7× the initial individual dose for Regranex treated sites). This represents a total of 145 μl per square centimeter of open wound surface area (327 μl/2.25 cm.sup.2 wound surface area).

[0148] The volume of 0.3 mg/ml PDGF-BB (Group 3) or buffer/sterile saline (Group 2) to be applied to the new collagen sponge on Days 7 and 14 was determined using the following formula:

145×cm.sup.2 open wound surface area (length [cm]×width [cm] of open wound).

[0149] C. Collagen Sponge

[0150] As described above for Group 1 treated sites, all wounds were evaluated and measured at Days 7 and 14 to record the open wound measurements for each individual site. For sites treated with a collagen sponge (Group 2 and Group 3), the sponge was measured and trimmed to fit the open wound portion of the original wound following removal of the dressing, gentle rinsing of the site and documentation of findings including measurements and photographic documentation.

C. Surgical Procedures

[0151] On Day 0, animals were anesthetized with isoflurane. The hair on the back was clipped and the skin swabbed with an aseptic solution. A template was used to mark a 1.5×1.5 cm square on the mid-back of the animal and a full thickness wound, corresponding to the template, was made by excising the skin and the panniculus carnosus. A hot water circulation pad or equivalent was placed under the animal to maintain normal body temperature during procedures, and animals recovered on a similar hot water circulation pad. Buprenorphine (0.06 mg/kg) was given by subcutaneous injections immediately after recovery from anesthesia and every 12 hours thereafter for 72 hours. Warmed Ringers solution (0.5 mL) was given by sub-cutaneous injection after the mice have recovered consciousness. The wounding of the animal was carried out under aseptic conditions. The wound site was photographed and the length and width measured immediately after excision and daily thereafter using a digital caliper. From Days 0 to 21, mice were administered test articles as listed in Table 1.

D. Study Results

[0152] 1. Animal Survival

[0153] Three animals died or were prematurely euthanized during this study (all animals from Group 1—Regranex). The first animal was found dead one day after surgery (Animal #3). The second animal (Animal #1) had to be sacrificed on Day 5 due to self-mutilating the rear flank posterior to the wound site. Animal #5 in Group 1 had to be sacrificed on day 16 as a result of losing more than 20% of its starting body weight. The following Table 2 summarizes the animal deaths/sacrifice:

TABLE-US-00003 TABLE 2 Summary of Animal Deaths/Sacrifice Day 1 Day 6 Day 16 Group 1, Animal #3 Group 1, Animal #1 Group 1, Animal #5 Found Dead Sacrificed, Exceeded 20% Self Mutilating Weight Loss

[0154] 2. Wound Measurements

[0155] The wound area was measured using a digital caliper and the length (L) and width (W) of each wound was recorded. Wound area was calculated using the formula to calculate the area of a square where A=L×W. FIG. 1 shows the area of the wounds for each group on Days 0, 7, 14 and 21. Peak wound area was recorded on Day 0 for all three groups with subsequent decreases in mean wound area on Day 7, Day 14, and Day 21. All treated groups showed a substantial decrease in wound area during the course of the study.

[0156] To provide an additional measurement and account for wounds that may not of healed in the shape of a square (and therefore not be captured in the formula used above), the inside of the wounds were also measured by tracing the inside wound edge using ImageJ Software™. FIG. 2 represents the wound area from each animal using this approach for each treatment group. FIGS. 3-6 show the average wound area for all treatment groups by evaluation day (Day 0, Day 7, Day 14, and Day 21) as a scatter plot to provide a more detailed assessment of the individual measurements recorded on those days. For animals that died during the study, the last data point is carried forward in FIGS. 3-6. FIGS. 7A-7B highlights a key aspect of the invention, namely that positive results are achieved with fewer applications of the therapeutic composition. FIG. 7A shows the amount of wound area reduction (mm.sup.2) per cumulative number of treatments at each of the four time points (Day 0, Day 7, Day 14, and Day 21) for Groups 1 and 3. FIGS. 8A-8D show the average percent of wound closure over the course of the study for each Group. For animals that died, the last data point was carried forward.

[0157] 3. Clinical Assessments

[0158] Wound images were also clinically assessed for possible differences in the degree of healing with respect to reepithelialization and formation of granulation tissue. Representative images of the wounds from each animal at each time point are shown in FIGS. 9-12 from Day 0 (FIG. 9), Day 7 (FIG. 10), Day 14 (FIG. 11) and Day 21 (FIG. 12). The raw images from each treatment show that Group 3 (rhPDGF/collagen sponge group) resulted in a demonstrable acceleration in the formation of granulation tissue and re-epithelialization compared to Group 2 (the collagen sponge control group treated with buffer). In addition, wounds treated with Regranex daily (Group 1) also showed a better wound closure rate compared to Group 2 (the buffer control+collagen). Histopathology on formalin fixed samples of the wound areas was also performed and further corroborated accelerated wound healing resulting from treatment with the rhPDGF/collagen and Regranex treated wounds compared to buffer control treated sponges. The pathology also suggests even further improvement in re-epithelialization in wounds treated with rhPDGF/collagen over Regranex treated.

[0159] Representative samples of the histopathological samples are provided in FIGS. 13-18 which include a series of photomicrographs of a cross section of the wound site on day 21 from three study animals, including Group 1—Animal 2 (FIGS. 17 and 18), Group 2—Animal 2 (FIGS. 13 and 14), and Group 3—Animal 1 (FIGS. 15 and 16). For each set of photomicrographs there is shown a 2× (FIGS. 13, 15, and 17) and a 10× magnification (FIGS. 14, 16, and 18).

[0160] Regarding Group 1, FIGS. 17 and 18 show that the wound was 100% resurfaced at Day 21, although some shearing was apparent due to the fragility of the dermal-epidermal architecture. Arrow 60 in FIG. 17 indicates the approximate location of the adjacent epidermis to the left and the wound bed to the right. FIG. 18 provides a higher magnification image from the middle of the wound. 100% epidermal resurfacing is apparent and the presence of differentiated stratification in the epidermis indicates its maturity. The neodermis still contains a high density of capillaries and new collagen formation is underway.

[0161] Regarding Group 2, the photomicrographs illustrate that a portion of the collagen sponge persisted in the wound bed for 21 days. In this wound edge the sponge appeared to obstruct the resurfacing of the epithelium. The arrow 10 in FIG. 13 indicates the wound edge. As seen in FIG. 13, some granulation tissue developed beneath the collagen sponge. Referring to FIG. 14, the collagen sponge and the granulation tissue are indicated by arrows 20 and 30, respectively. As shown, the collagen sponge appeared adherent in this region although infiltration with cells is minimal. A classic granulation tissue is shown formed beneath the sponge.

[0162] Regarding Group 3, FIGS. 15 and 16 illustrate the wound is 100% resurfaced with epidermis. The arrow 40 in FIG. 15 indicates the approximate edge of the wound bed, and the region 50 is subcutaneous fat. FIG. 16 was taken within the middle of the wound, and shows that all evidence of the original collagen sponge is gone. The wound is 100% resurfaced and is well stratified with a stratum corneum indicating maturity. The neodermis shows evidence of new collagen production and the cellularity is decreased indicating that the dermal tissue is maturing and losing the immature characteristics of granulation tissue.

E. Study Conclusions

[0163] The following conclusions were made from this study: [0164] (1) 21 applications of Regranex were given in Group 1, while only 3 applications of buffer/collagen or rhPDGF/collagen wound dressings were applied in Groups 2 and 3, respectively. [0165] (2) Three animals from Group 1 (Regranex) were either found dead or had to be euthanized during the in-life portion of the study. [0166] (3) All treated groups showed a decrease in wound area from Day 0-21 as determined by both caliper measurements and wound tracing using ImageJ software analysis. At sacrifice (Day 21), 2 of 5 Regranex treated wounds, 3 of 5 rhPDGF/collagen treated wounds and 0 of 5 collagen dressing treated wounds were healed. [0167] (4) The raw images from each treatment show that Group 3 (rhPDGF/collagen) results in a demonstrable acceleration in the formation of granulation tissue and re-epithelialization compared to the collagen wound dressing control group treated with buffer (Group 2). In addition, wounds treated with Regranex daily (Group 1) also showed a better closure rate compared to the control collagen sponge treated animals. [0168] (5) Healing as assessed by wound reepithelialization was greatest in wounds treated with three applications of rhPDGF/collagen (Group 3) compared to 21 applications of Regranex (Group 1) or three applications of the collagen wound dressing wetted with saline. [0169] (6) Three (3) weekly applications of rhPDGF/collagen (Group 3) accelerate wound closure, including granulation tissue formation and re-epithelialization compared to a collagen wound dressing (Group 2) and appear at least as effective as 21 daily doses of Regranex Gel (Group 1). [0170] (7) rhPDGF/collagen is safe and effective, promoting better healing of diabetic wounds compared to the marketed collagen wound dressing. 3 of 5 rhPDGF/collagen treated wounds completely healed, as evidenced by complete re-epithelialization, compared to 0 of 5 collagen wound dressing treated animals. [0171] (8) rhPDGF/collagen is safe and effective, promoting angiogenesis, granulation tissue formation and re-epithelialization compared to a marketed collagen wound dressing as demonstrated histologically. [0172] (9) rhPDGF/collagen, a sterile product, is highly biocompatible as demonstrated histologically. [0173] (10) rhPDGF/collagen is much easier to apply than Regranex Gel, which should improve patient compliance. [0174] (11) rhPDGF/collagen may be safer than Regranex, given that animals that received Regranex had a high mortality rate, while no such mortality was observed with rhPDGF/collagen or the collagen wound dressing.

Example 2—Prophetic

[0175] A study is conducted to demonstrate the efficacy of the novel therapeutic compositions and wound treatment methods described herein. The same study design outlined for Example 1 is also used for this study including the db/db mouse model with five test groups—a standard of care group (saline moistened gauze), a Regranex group, a collagen sponge group, and two groups utilizing treatment compositions in accordance with the present invention comprising PDGF-BB and a collagen sponge. As detailed below, however, the frequency of the dosing is changed in this study. The study is also designed so that the total dose of PDGF delivered over the course of the study in both the Regranex group and the collagen sponge/PDGF-BB groups is the same.

A. Experimental Design

[0176] The experimental design is refined by the results of the study described in Example 1, however it is anticipated that the number of animals per group are greater (i.e. eight) and the study duration is longer, i.e. 28 days. Additionally the test and control collagen +/−PDGF articles are administered topically as wound dressings immediately following the induction of the wound and at about Day 14 for a total of two applications (Group 5) or immediately after surgery and at Days 7, 14 and 21 for a total of four applications of test articles in accordance with the novel compositions and treatment methods described herein (see Table X). Negative control (saline moistened gauze) and Regranex treated sites will undergo 28 daily administrations topically as wound dressing in accordance with the prescribed Instructions for Use.

[0177] In this Example, blood glucose levels are determined prior to the start of the study and again just prior to sacrifice on Day 28 to confirm diabetic disease state. In all other aspects, the design for this study is the same as described in Example 1. The study details are outlined in below in Table 3.

TABLE-US-00004 TABLE 3 Example 2 Study Design Dressing Changes & Group Number Wound Num- of Treat- Route/ Assess- ber Animals Wound ment Frequency ment 1 8 male Day 0 Saline Topical/28 Daily 27; (db/db) 1.5 cm × Moistened applications Daily To 1.5 cm Gauze Day 28 2 8 male Day 0 Regranex Topical/28 Daily 27; (db/db) 1.5 cm × Gel applications Daily To 1.5 cm Day 28 3 8 male Day 0 Collagen Topical/4 3; Days (db/db) 1.5 cm × Sponge applications Days 7, 14 1.5 cm 0, 7, 14 and 21 21 and 28* 4 8 male Day 0 Collagen Topical/4 3; Days (db/db) 1.5 cm × Sponge + applications Days 7, 14, 1.5 cm 326 μl 0.3 0, 7, 14 and 21 21 and mg/ml 28* PDGF-BB 5 8 male Day 0 Collagen Topical/2 1; Day (db/db) 1.5 cm × Sponge + applications Days 14, 1.5 cm 654 μl 0.3 0 and 14 21.sup.# and mg/ml 28* PDGF-BB *Wound assessment and necropsy; .sup.#Wound assessment only.

[0178] Every day for the period of the study, each animal is inspected and its survival recorded, in order to assess possible visual differences in animal responses among treatment groups. The rate of wound closure will be determined, as will the percentage of wounds completely healed at any given time point.

B. PDGF-BB Dose Calculations

[0179] The PDGF dosage used in this study is designed to mimic the actual therapeutic doses in accordance with either the present invention (Groups 4 and 5) or the actual therapeutic dose prescribed in accordance with the Regranex label (Group 2). The dosage for Group 2 is determined in the same manner as the Regranex group in Example 1. For sites treated with Regranex for 28 days the total dose of PDGF administered for the study period is 14 μg/day×28 days or 392 μg of PDGF-BB. This dose may also be expressed amount of PDGF per area of the original wound size (“area dose”) of 6.22 μg/cm.sup.2/day or 174.2 μg/cm.sup.2 of PDGF-BB.

[0180] For Groups 4 and 5, a PDGF solution is used having a PDGF concentration of 0.3 mg/ml or 300 μg/ml. To achieve a total dose for the study period of 392 μg PDGF-BB (the same total study dose as in the Regranex group), a total of 1.307 ml PDGF solution is applied to the wound site over the 28 day study period. For sites receiving dressing changes once every 7 days there are a total of 4 administrations (days 0, 7, 14 and 21). Each administration consists of 327p PDGF-BB (or buffer alone) onto the collagen sponge. With respect to dose of PDGF, each administration consists of 0.3 μg/μl×326 μl or 98 ug PDGF-BB (approximately 7× Regranex individual dose). For sites receiving just two doses, at Day 0 and 14 there are a total of 2 administrations. Each administration consists of 654 μl PDGF-BB onto the collagen sponge. Each administration would consist of 0.3 μg/μl×654 μl or 196 ug PDGF-BB (approximately 14× Regranex individual dose). These doses may also be expressed as amount of PDGF per area of the original wound size (“area dose”). With respect to Group 4, the PDGF dose is 43.5 μg/cm.sup.2/dose or 174 μg/cm.sup.2 of PDGF-BB. With respect to Group 5, the PDGF dose is about 87 μg/cm.sup.2/dose or 174 μg/cm.sup.2 of PDGF, i.e. the same cumulative dose in all groups but Groups 4 and 5 have many fewer doses.

Example 3—Prophetic

[0181] A randomized clinical trial is conducted to assess the effectiveness of various compositions of rhPDGF-BB and collagen as compared to standard of care (consisting of moist wound healing with removal of excess wound exudate, debriding necrotic tissue, off-loading of pressure, saline moistened gauze, antibiotics if needed and wound dressing) and Regranex in the treatment of chronic diabetic foot ulcers. Table 4 below summarizes the study design. For each arm of the study (1-37) the product is applied at the dosage and frequency indicated in Table 4 for up to 20 weeks or until complete wound closure. Regranex is applied in accordance with its approve US labeling. rhPDGF-BB/collagen compositions are applied in accordance with the procedures (steps 1-5) described above in paragraph 50.

[0182] The outcome measures for the study are: [0183] Incidence of complete wound closure. [0184] Time to achieve complete wound closure. [0185] Percentage reduction in total ulcer surface area at each visit. [0186] Number of ulcer recurrence observed 12 weeks after wound healing. [0187] Treatment emergent adverse events (up to 52 weeks).

[0188] The inclusion criteria for the study include: [0189] Men or women aged 18 years old or older, with type 1 or 2 diabetes mellitus [0190] Patient with a single ulcer on the treated feet [0191] Patient able and willing to provide informed consent [0192] Patient able and willing to comply with protocol visits and procedure [0193] Patient willing to use an off-loading method during the whole duration of the study [0194] Full-thickness plantar, lateral or dorsal ulcer of the extremity (below the malleolus), excluding inter-digits ulcer (web spaces), extending through the epidermis and dermis, but not involving bone, tendons, ligaments or muscles (grade IA as defined by University of Texas Diabetic Wound Classification or Grade 1 according to Wagner classification) [0195] Chronic ulcer of at least six weeks despite appropriate wound care [0196] Ulcer area measured with the formula Length×Width×0.8 following sharp debridement, of 1 to 10 cm.sup.2, both inclusive [0197] Well controlled infection or cellulitis (systemic antibiotherapy) before Baseline Visit [0198] Peripheral neuropathy as assessed by Semmes-Weinstein monofilament test or by the bio esthesimeter (vibration perception threshold) [0199] Ankle brachial pressure index >0.60 and <1.3 [0200] Women surgically sterile, post-menopausal, or agree to practice adequate contraception and have a negative pregnancy test at screening [0201] Non-nursing

[0202] The exclusion criteria for the study include: [0203] Inter digit ulcers [0204] Ulcer of other cause or origin: electrical, chemical or radiation insult, bedsores, vascular ulcer or Charcot deformities ulcers [0205] Charcot foot [0206] Wound originated from amputation bed [0207] Active ulcer infection assessed by clinical examination and radiography if necessary. Presence of necrosis, purulence or sinus tracts that cannot be removed by debridement and controlled by standard wound care [0208] Active osteomyelitis affecting the area of the target ulcer [0209] Poorly controlled diabetes (uncontrolled glycemia HbAlc %>=10%), renal failure (serum creatinine >3.0 mg/dL), poor nutritional status (albumin <3.0 g/dL or total protein <6.5 g/dL) [0210] Known connective tissue or malignant disease [0211] Concomitant treatment with corticosteroids, immunosuppressive agents, radiation therapy, or anticancer chemotherapy [0212] Use of investigational drug/device or growth factor within 30 days [0213] Topical application of any advance wound care on this wound (antiseptics, antibiotics, debriders, enzyme) within 7 days [0214] Vascular reconstruction within 8 weeks [0215] Patients expected to be noncompliant with the protocol (not available for the duration of the trial, treatment or wound care compliance), or felt to be unsuitable by the Investigator for any other reason [0216] A history of severe cerebrovascular events

TABLE-US-00005 TABLE 4 Example 4 Study Design ratio rhPDGF Max Initial Later solution/ Treatment Number of dose doses Dosage Collagen Frequency Treatments (μg/cm2) (μg/cm2) Adjust (μl/cm3) 1 Regranex daily 140 6.25 6.25 weekly 2 Standard of Care daily 140 — — — 3 rhPDGF/collagen 3 days 46 10 10 6 days 67 4 sponge 3 days 46 18.75 18.75 6 days 125 5 5 days 28 31.25 31.25 10 days 208 6 7 days 20 43.75 43.75 7 days 292 7 14 days 10 87.5 87.5 14 days 583 8 21 days 6 131.25 131.25 21 days 875 9 28 days 5 175 175 28 days 1167 10 35 days 4 218.75 218.75 35 days 1459 11 42 days 3 262.5 262.5 42 days 1750 12 3 days 46 10 10 6 days 134 13 3 days 46 18.75 18.75 6 days 250 14 5 days 28 31.25 31.25 10 days 416 15 7 days 20 43.75 43.75 7 days 584 16 14 days 10 87.5 87.5 14 days 1166 17 21 days 6 131.25 131.25 21 days 1750 18 28 days 5 175 175 28 days 2334 19 35 days 4 218.75 218.75 35 days 2918 20 42 days 3 262.5 262.5 42 days 3500 21 3 days 46 20 10 6 days 67 22 3 days 46 37.5 18.75 6 days 125 23 5 days 28 62.5 31.25 10 days 208 24 7 days 20 87.5 43.75 7 days 292 25 14 days 10 175 87.5 14 days 583 26 21 days 6 262.5 131.25 21 days 875 27 28 days 5 350 175 28 days 1167 28 35 days 4 437.5 218.75 35 days 1459 29 42 days 3 525 262.5 42 days 1750 30 3 days 46 18.75 18.75 6 days 250 31 5 days 28 31.25 31.25 10 days 370 32 7 days 20 43.75 43.75 7 days 480 33 14 days 10 87.5 87.5 14 days 890 34 21 days 6 131.25 131.25 21 days 1290 35 28 days 5 175 175 28 days 1690 36 35 days 4 218.75 218.75 35 days 2100 37 42 days 3 262.5 262.5 42 days 2500

[0217] Each of the rhPDGF/collagen sponge compositions performs better than Regranex or standard of care in at least one of the outcome measures, and/or achieves a substantially equivalent result with the application of less cumulative rhPDGF applied over the treatment period or the application of fewer treatments which leads to better patient compliance.

[0218] The embodiments, variations, and sequences described herein should provide an indication of the utility and versatility of the present invention. Other embodiments that do not provide all of the features and advantages set forth herein may also be utilized, without departing from the spirit and scope of the present invention. Such modifications and variations are considered to be within the scope of the invention.