COMPOSITIONS AND METHODS OF VASCULAR INJURY REPAIR

• US 20070105217A1
• Filed: 10/24/2006
• Published: 05/10/2007
• Est. Priority Date: 11/07/2005
• Status: Active Grant

• Current Assignee: Caladrius Biosciences, Inc. (Lisata Therapeutics, Inc.)
• Original Assignee: Amorcyte, Inc. (Lisata Therapeutics, Inc.)
• Inventors: Pecora, Andrew, Preti, Robert
• Granted Patent: US 7,794,705 B2
• Time in Patent Office: Days
• US Class Current: 435/343
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors

Abstract

The present invention relates to pharmaceutical compositions comprising a chemotactic hematopoietic stem cell product comprising an enriched population of CD34+ cells containing a subpopulation of cells having chemotactic activity, methods of preparing these compositions and use of these compositions to treat or repair vascular injury, including infarcted myocardium.

Claims

1. A pharmaceutical composition for repairing a vascular injury in a subject comprising:

(a) a therapeutically effective amount of a sterile isolated chemotactic hematopoietic stem cell product, the chemotactic hematopoietic stem cell product comprising an enriched population of isolated CD34+ cells having a subpopulation of potent cells having chemotactic activity; and

(b) a stabilizing amount of serum,

wherein the composition is administered to the subject parenterally through a catheter, and wherein the subpopulation of potent cells having chemotactic activity when passed through the catheter remains potent.

2. The pharmaceutical composition according to claim 1, wherein the enriched population of isolated CD34+ cells is purified from bone marrow acquired from the subject.

3. The pharmaceutical composition according to claim 1, wherein the enriched population of isolated CD34+ cells is at least 70% pure.

4. The pharmaceutical composition according to claim 1, wherein the enriched population of isolated CD34+ cells is at least 70% viable for at least about 24 hours following acquisition of the enriched population of isolated CD34+ cells.

5. The pharmaceutical composition according to claim 1, wherein the enriched population of isolated CD34+ cells is at least 70% viable for at least about 48 hours following acquisition of the enriched population of isolated CD34+ cells.

6. The pharmaceutical composition according to claim 1, wherein the enriched population of isolated CD34+ cells is at least 70% viable for at least about 72 hours following acquisition of the enriched population of isolated CD34+ cells.

7. The composition according to claim 1, wherein the therapeutically effective amount of the chemotactic hematopoietic stem cell product comprises a minimum number of isolated CD34+ hematopoietic stem cells containing a subpopulation of at least 0.5×

10⁶ potent CD34+ cells having CXCR-4 mediated chemotactic activity.

8. The composition according to claim 17 wherein the enriched CD34+ cells are capable of forming hematopoietic colonies in vitro for at least about 24 hours following acquisition of the enriched CD34+ cells.

9. The composition according to claim 1, wherein the enriched CD34+ cells are capable of forming hematopoietic colonies in vitro for at least about 48 hours following acquisition of the enriched CD34+ cells.

10. The composition according to claim 1, wherein the enriched CD34+ cells are capable of forming hematopoietic colonies in vitro for at least about 72 hours following acquisition of the enriched CD34+ cells.

11. The composition according to claim 1, wherein the serum is human serum autologous to the subject or synthetic serum.

12. The composition according to claim 17 wherein the stabilizing amount of serum is at least about 10% (v/v).

13. The composition of claim 1, wherein at least 2% of the chemotactic activity of the subpopulation of potent cells is retained by the subpopulation of potent cells for at least 24 hours following acquisition of the enriched population of isolated CD34+ cells.

14. The composition of claim 1, wherein at least 2% of the chemotactic activity of the subpopulation of potent cells is retained by the subpopulation of potent cells for at least 48 hours following acquisition of the enriched population of isolated CD34+ cells.

15. The composition of claim 1, wherein at least 2% of the chemotactic activity of the subpopulation of potent cells is retained by the subpopulation of potent cells for at least 72 hours following acquisition of the enriched population of isolated CD34+ cells.

16. The composition according to claim 1, wherein the enriched population of isolated CD34+ cells containing a subpopulation of potent cells having chemotactic activity is at least 70% pure, is at least about 70% viable, and is able to form hematopoietic colonies in vitro for at least about 24 hours following acquisition of the enriched population.

17. The composition according to claim 1, wherein the enriched population of isolated CD34+ cells containing a subpopulation of potent cells having chemotactic activity is at least 70% pure, is at least about 70% viable, and is able to form hematopoietic colonies in vitro for at least about 48 hours following acquisition of the enriched population.

18. The composition according to claim 1, wherein the enriched population of isolated CD34+ cells containing a subpopulation of potent cells having chemotactic activity is at least 70% pure, is at least about 70% viable, and is able to form hematopoietic colonies in vitro for at least about 72 hours following acquisition of the enriched population.

19. The composition according to claim 1, wherein the subject is a revascularized myocardial infarction patient.

20. The composition according to claim 1, wherein the composition is administered through the catheter intravascularly to an infarct-related artery.

21. The composition according to claim 1, wherein the catheter is a flow control catheter.

22. The composition according to claim 1, wherein the catheter is a balloon catheter.

23. The composition according to claim 1, wherein the catheter has an internal diameter of at least 0.36 mm.

24. The composition according to claim 20, wherein the composition is administered through the catheter intravascularly by means of an intravascular delivery apparatus.

25. The composition according to claim 17 wherein the composition is administered through the catheter into myocardium.

26. The composition according to claim 17 wherein the pharmaceutical composition further comprises at least about 0.5% albumin.

27. The composition according to claim 1, wherein the pharmaceutical composition further includes at least one compatible active agent.

28. The composition according to claim 27, wherein the active agent is selected from the group consisting of an angiotensin converting enzyme inhibitor, a beta-blocker, a diuretic, an anti-arrhythmic agent, an anti-anginal agent, a vasoactive agent, an anticoagulant agent, a fibrinolytic agent, and a hypercholesterolemic agent.

29. The composition according to claim 1, wherein sterility of the chemotactic hematopoietic cell product is confirmed by a method comprising the steps:

(a) centrifuging the cell product to form a separated cell product comprising a pellet comprising the enriched population of isolated CD34+ cells and a supernatant;

(b) removing the supernatant of the separated cell product without disturbing the cell pellet of the separated cell product.

(c) analyzing the sterility of the supernatant of the separated cell product; and

(d) thereby determining the sterility of the cell pellet of the separated cell product.

30. A method of preparing a pharmaceutical composition for intravascular delivery to a subject having a vascular injury, the pharmaceutical composition comprising a sterile chemotactic hematopoietic stem cell product comprising an enriched population of isolated CD34+ cells containing a subpopulation of potent cells having chemotactic activity, the method comprising the steps:

(a) acquiring a preparation comprising an enriched population of potent CD34+cells from the subject under sterile conditions by a chemotactic cell acquisition process,

(b) optionally transporting the preparation to a processing facility;

(c) sterilely purifying CD34+ cells containing a subpopulation of potent cells having chemotactic activity from the preparation;

(d) sterilely formulating the purified CD34+ cells containing a subpopulation of potent cells having chemotactic activity to form the chemotactic hematopoietic cell product;

(e) sterilely formulating the sterile chemotactic hematopoietic stem cell product to form a pharmaceutical composition;

(e) assessing sterility of the pharmaceutical composition;

(f) releasing the sterile pharmaceutical composition as eligible for infusion into the subject;

(g) loading a therapeutic amount of the pharmaceutical composition into an intravascular delivery apparatus, and

(h) optionally transporting the delivery apparatus containing the therapeutically effective amount of the sterile pharmaceutical composition to a cardiac catheterization facility for intravascular infusion into the subject.

31. The method according to claim 30, optionally further comprising the step of storing the acquired preparation between steps (a) and (b).

32. The method according to claim 30, wherein purifying step (c) is initiated within about 12 hours to about 24 hours of completion of acquiring step (a).

33. The method according to claim 30, wherein purifying step (c) is initiated within about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours of completion of acquiring step (a).

34. The method according to claim 30, wherein releasing step (f) proceeds only if the sterile formulated cell product is to be infused into the subject within about 48 hours to about 72 hours of completion of acquiring step (a).

35. The method according to claim 30, wherein releasing step (f) proceeds only if the sterile formulated cell product is to be infused into the subject within about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours or about 72 hours of completion of acquiring step (a).

36. The method according to claim 30, wherein purifying step (c) is initiated within about 12 hours to about 24 hours of completion of acquiring step (a), and releasing step (f) proceeds only if the sterile pharmaceutical composition is to be infused into the subject within about 48 hours to about 72 hours of completion of acquiring step (a).

37. The method according to claim 30, wherein purifying step (c) is initiated within about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours of completion of acquiring step (a), and releasing step (f) proceeds only if the sterile pharmaceutical composition is to be infused into the subject within about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours or about 72 hours of completion of acquiring step (a).

38. The method according to claim 30, wherein the chemotactic cell acquisition process in step (a) is a mini-bone marrow harvesting technique.

39. The method according to claim 30, wherein acquiring step (a) of the mini-bone marrow harvesting technique further comprises the steps:

(i) preloading harvesting syringes with heparin prior to harvesting bone marrow from the subject;

(ii) aspirating the bone marrow from a left posterior iliac crest and a right posterior iliac crest of the subject using the harvesting syringes and a mini-bone marrow harvest technique to form harvested bone marrow; and

(iii) infusing the harvested bone marrow into a collecting bag.

40. The method according to claim 38, wherein the harvesting syringes in step (i) and the collecting bag in step (iii) contain a preservative free heparinized solution.

41. The method according to claim 39, wherein the final concentration of heparin in the preservative free heparinized solution is about 20 units per ml to about 25 units per ml.

42. The method according to claim 30, wherein transporting step (b) further comprises the steps:

(i) placing the harvested bone marrow in a collection bag;

(ii) placing the collection bag in a secondary bag;

(ii) preparing an open shipping container;

(iii) placing the secondary bag containing the collection bag in the open shipping container;

(iv) sealing the shipping container; and

(v) shipping the shipping container to the processing facility.

43. The method according to claim 30, wherein the enriched population of isolated CD34+cells in step (c) is at least 70% pure.

44. The method according to claim 30, wherein the enriched population of isolated CD34+cells is at least 70% viable for at least about 24 hours following acquisition of the enriched population of isolated CD34+ cells in step (a).

45. The method according to claim 30, wherein the enriched population of isolated CD34+ cells is at least 70% viable for at least about 48 hours following acquisition of the enriched population of isolated CD34+ cells in step (a).

46. The method according to claim 30, wherein the enriched population of isolated CD34+cells is at least 70% viable for at least about 72 hours following acquisition of the enriched population of isolated CD34+ cells in step (a).

47. The method according to claim 30, wherein the enriched CD34+ cells are capable of forming hematopoietic colonies in vitro for at least about 24 hours following acquisition of the enriched CD34+ cells in step (a).

48. The method according to claim 30, wherein the enriched CD34+ cells are capable of forming hematopoietic colonies in vitro for at least about 48 hours following acquisition of the enriched CD34+ cells in step (a).

49. The method according to claim 30, wherein the enriched CD34+ cells are capable of forming hematopoietic colonies in vitro for at least about 72 hours following acquisition of the enriched CD34+ cells in step (a).

50. The method according to claim 30, wherein at least 2% of the chemotactic activity of the subpopulation of potent cells is retained by the subpopulation of potent cells for at least 24 hours following acquisition of the enriched population of isolated CD34+ cells in step (a).

51. The method according to claim 30, wherein at least 2% of the chemotactic activity of the subpopulation of potent cells is retained by the subpopulation of potent cells for at least 48 hours following acquisition of the enriched population of isolated CD34+ cells in step (a).

52. The method according to claim 30, wherein at least 2% of the chemotactic activity of the subpopulation of potent cells is retained by the subpopulation of potent cells for at least 72 hours following acquisition of the enriched population of isolated CD34+ cells in step (a).

53. The method according to claim 30, wherein the enriched population of isolated CD34+ cells containing a subpopulation of potent cells having chemotactic activity is at least 70% pure, is at least about 70% viable, and is able to form hematopoietic colonies in vitro for at least about 24 hours following acquisition of the enriched population of isolated CD34+ cells in step (a).

54. The method according to claim 30, wherein the enriched population of isolated CD34+cells containing a subpopulation of potent cells having chemotactic activity is at least 70% pure, is at least about 70% viable, and is able to form hematopoietic colonies in vitro for at least about 48 hours following acquisition of the enriched population of isolated CD34+ cells in step (a).

55. The method according to claim 30, wherein the enriched population of isolated CD34+cells containing a subpopulation of potent cells having chemotactic activity is at least 70% pure, is at least about 70% viable, and is able to form hematopoietic colonies in vitro for at least about 72 hours following acquisition of the enriched population of isolated CD34+ cells in step (a).

56. The method according to claim 30, wherein the therapeutically effective amount of the chemotactic hematopoietic stem cell product in the pharmaceutical composition of step (g) comprises a minimum number of CD34+hematopoietic stem cells containing a subpopulation of at least 0.5×

10⁶ CD34+potent cells having CXCR-4 mediated chemotactic activity.

57. The method according to claim 30, wherein the confirming sterility step (e) further comprises the steps of

(i) centrifuging the formulated cell product to form separated cell product comprising a cell pellet comprising the enriched population of isolated CD34+ cells and a supernatant,

(ii) sterilely removing the supernatant of the separated cell product without disturbing the cell pellet of the separated cell product;

(iii) analyzing whether the supernatant of the separated cell product is contaminated by a microbe; and

(iv) thereby determining the sterility of the cell pellet of the separated cell product.

58. The method according to claim 30, wherein the intravascular delivery apparatus of step

(g) comprises

(i) an infusion syringe attached to a sterile four-way stopcock, wherein the infusion syringe contains the pharmaceutical composition.

(ii) a flushing syringe attached to the sterile four-way stopcock containing a flushing solution;

and

(iii) a catheter attached to the delivery apparatus by the sterile four-way stopcock for delivery of the pharmaceutical composition to the subject.

59. The method according to claim 58, wherein the catheter is a flow control catheter.

60. The method according to claim 58, wherein the catheter is a balloon dilatation catheter.

61. The method according to claim 58, wherein the catheter has an internal diameter of at least 0.36 mm.

62. The method according to claim 30, wherein the subject in need thereof is a revascularized myocardial infarction patient.

63. The method according to claim 62, wherein the pharmaceutical composition is delivered to the subject by infusion of the composition through the catheter to an infarct related artery.

64. The method according to claim 62, wherein the pharmaceutical composition is administered through the catheter into myocardium.

65. A method of treating or repairing a vascular injury in a subject in need thereof the method comprising the step of administering to the subject parenterally through a catheter a sterile pharmaceutical composition comprising:

(a) a therapeutically effective amount of a sterile chemotactic hematopoietic stem cell product, the chemotactic hematopoietic stem cell product comprising an enriched population of isolated CD34+ cells containing a subpopulation of potent cells having chemotactic activity; and

(b) a stabilizing amount of serum,

wherein the subpopulation of potent cells having chemotactic activity when passed through the catheter remains potent.

66. The method according to claim 65, wherein the enriched population of isolated CD34+ cells is at least 70% pure.

67. The method according to claim 65, wherein the enriched population of isolated CD34+ cells is at least 70% viable for at least about 24 hours following acquisition of the enriched population of isolated CD34+ cells.

68. The method according to claim 65, wherein the enriched population of isolated CD34+ cells is at least 70% viable for at least about 48 hours following acquisition of the enriched population of isolated CD34+ cells.

69. The method according to claim 65, wherein the enriched population of isolated CDS4+cells is at least 70% viable for at least about 72 hours following acquisition of the enriched population of isolated CD34+ cells.

70. The method according to claim 65, wherein the therapeutically effective amount of the chemotactic hematopoietic stem cell product comprises a minimum number of isolated CD34+ hematopoietic stem cells containing a subpopulation of at least 0.5×

10⁶ potent CD34+ cells having CXCR-4 mediated chemotactic activity.

71. The method according to claim 65, wherein the enriched CD34+ cells are capable of forming hematopoietic colonies in vitro for at least about 24 hours following acquisition of the enriched CD34+ cells.

72. The method according to claim 65, wherein the enriched CD34+ cells are capable of forming hematopoietic colonies in vitro for at least about 48 hours following acquisition of the enriched CD34+ cells.

73. The method according to claim 65, wherein the enriched CD34+ cells are capable of forming hematopoietic colonies in vitro for at least about 72 hours following acquisition of the enriched CD34+ cells.

74. The method according to claim 65, wherein at least 2% of the chemotactic activity of the subpopulation of potent cells is retained by the subpopulation of potent cells for at least 24 hours following acquisition of the enriched population of isolated CD34+ cells.

75. The method according to claim 65, wherein at least 2% of the chemotactic activity of the subpopulation of potent cells is retained by the subpopulation of potent cells for at least 48 hours following acquisition of the enriched population of isolated CD34+ cells.

76. The method according to claim 65, wherein at least 2% of the chemotactic activity of the subpopulation of potent cells is retained by the subpopulation of potent cells for at least 72 hours following acquisition of the enriched population of isolated CD34+ cells.

77. The method according to claim 65, wherein the enriched population of isolated CD34+cells containing a subpopulation of potent cells having chemotactic activity is at least 70% pure, is at least about 70% viable, and is able to form hematopoietic colonies in vitro for at least about 24 hours following acquisition of the enriched population.

78. The method according to claim 65, wherein the enriched population of isolated CD34+cells containing a subpopulation of potent cells having chemotactic activity is at least 70% pure, is at least about 70% viable, and is able to form hematopoietic colonies in vitro for at least about 48 hours following acquisition of the enriched population.

79. The method according to claim 65, wherein the enriched population of isolated CD34+cells containing a subpopulation of potent cells having chemotactic activity is at least 70% pure, is at least about 70% viable, and is able to form hematopoietic colonies in vitro for at least about 72 hours following acquisition of the enriched population.

80. The method according to claim 65, wherein the serum is human serum autologous to the subject or a synthetic serum.

81. The method according to claim 65, wherein the stabilizing amount of serum is at least about 10% (v/v).

82. The method according to claim 65, wherein the subject is a revascularized myocardial infarction patient.

83. The method according to claim 65, the method further comprising the step of delivering the composition intravascularly to an infarct related artery by means of an intravascular delivery apparatus.

84. The method according to claim 83, wherein the intravascular delivery apparatus comprises

(i) an infusion syringe attached to a sterile four-way stopcock, wherein the infusion syringe contains the pharmaceutical composition;

(ii) a flushing syringe attached to the sterile four-way stopcock containing a flushing solution; and

(iii) a catheter attached to the delivery apparatus by the sterile four-way stopcock for delivery of the pharmaceutical composition to the subject.

85. The method according to claim 65, wherein the catheter is a flow control catheter.

86. The method according to claim 65, wherein the catheter is a balloon dilatation catheter.

87. The method according to claim 65, wherein the catheter has an internal diameter of at least 0.36 mm.

88. The method according to claim 82, wherein the pharmaceutical composition is delivered to the subject during a specific time interval defined by a first time and a second time.

89. The method according to claim 88, wherein the first time is at least about 5 days post-infarction and the second time is less than about 14 days post-infarction.

90. The method according to claim 88, wherein the first time is after peak inflammatory cytokine cascade production in the infracted area.

91. The method according to claim 88, wherein the second time is before myocardial scar formation in the infracted area.

92. The method according to claim 65, wherein the composition is administered through the catheter intravascularly by means of an intravascular delivery apparatus.

93. The method according to claim 65, wherein the composition is administered through the catheter into myocardium.

94. The method according to claim 65, wherein the pharmaceutical composition further includes at Least one compatible active agent.

95. The method according to claim 94, wherein the active agent is selected from the group consisting of an angiotensin converting enzyme inhibitor, a beta-blocker, a diuretic, an anti-arrhythmic agent, an anti-anginal agent, a vasoactive agent, an anticoagulant agent, a fibrinolytic agent, and a hypercholesterolemic agent.

96. A method of assessing sterility of a clinically useful isolated cell product having a limited volume, the method comprising the steps:

(a) sterilely separating cellular components of the isolated cell product from noncellular components; and

(b) testing the noncellular components for microbial contamination, thereby determining the sterility of the clinically useful isolated cell product.

97. The method according to claim 96, wherein in step (a) the cellular components are separated from noncellular components by centrifugation.

98. The method according to claim 97, wherein the cellular components form a cell pellet upon centrifugation of the isolated cell product.

99. The method according to claim 97, wherein the noncellular components form a supernatant upon centrifugation of the isolated cell product.

100. The method according to claim 97, wherein the isolated cell product is a chemotactic hematopoietic stem cell product.

101. The method according to claim 97, wherein the chemotactic hematopoietic stem cell product comprises an enriched population of isolated CD34+ cells containing a subpopulation of potent cells having CXCR-4 mediated chemotactic activity.

102. The method according to claim 97, wherein after sterility of the supernatant is confirmed, a therapeutically effective amount of the sterile chemotactic hematopoietic stem cell product is administered to a subject intravascularly to treat or repair a vascular injury.

103. The method according to claim 102, wherein the sterile chemotactic hematopoietic stem cell product is administered through a catheter.

104. The method according to claim 102, wherein the subject is a revascularized myocardial infarction patient.

105. The method according to claim 102, wherein the vascular injury is a vascular insufficiency.

106. The method according to claim 105, wherein the vascular insufficiency affects at least one coronary artery.