U.S. patent application number 12/910328 was filed with the patent office on 2011-03-31 for compositions and methods for treating progressive myocardial injury due to a vascular insufficiency.
Invention is credited to Andrew L. Pecora, Robert A. Preti.
Application Number | 20110076255 12/910328 |
Document ID | / |
Family ID | 43780632 |
Filed Date | 2011-03-31 |
United States Patent
Application |
20110076255 |
Kind Code |
A1 |
Pecora; Andrew L. ; et
al. |
March 31, 2011 |
COMPOSITIONS AND METHODS FOR TREATING PROGRESSIVE MYOCARDIAL INJURY
DUE TO A VASCULAR INSUFFICIENCY
Abstract
The described invention provides methods and regimens for
treating adverse consequences of a persistent and progressive
myocardial injury--due to a vascular insufficiency that occurs
early or late in a subject in need thereof, and progressive
myocardial injury-preventing compositions that contain a
chemotactic hematopoietic stem cell product, and, optionally, an
additional active agent.
Inventors: |
Pecora; Andrew L.; (Wyckoff,
NJ) ; Preti; Robert A.; (Ridgefield, NJ) |
Family ID: |
43780632 |
Appl. No.: |
12/910328 |
Filed: |
October 22, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12401291 |
Mar 10, 2009 |
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12910328 |
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11552396 |
Oct 24, 2006 |
7794705 |
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12401291 |
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12629361 |
Dec 2, 2009 |
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11552396 |
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11552396 |
Oct 24, 2006 |
7794705 |
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12629361 |
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61254539 |
Oct 23, 2009 |
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60734151 |
Nov 7, 2005 |
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61119552 |
Dec 3, 2008 |
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61169850 |
Apr 16, 2009 |
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
C12N 5/0647 20130101;
A61P 9/06 20180101; A61P 9/04 20180101; A61P 9/10 20180101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 35/28 20060101
A61K035/28; A61P 9/10 20060101 A61P009/10 |
Claims
1. A method of treating a progressive myocardial injury due to a
vascular insufficiency, the method comprising the steps of: (a)
acquiring a sterile nonexpanded, isolated population of autologous
mononuclear cells comprising CD34+ cells, which further contain a
subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity from a subject under sterile
conditions; (b) sterilely enriching the autologous mononuclear
cells comprising CD34+ cells, wherein the enriched CD34+ cells
which further contain a subpopulation of potent CD34+/CXCR-4+ cells
that have CXCR-4-mediated chemotactic activity are a chemotactic
hematopoietic stem cell product (c) administering parenterally
through a catheter on a plurality of infusion dates during lifetime
of subject a sterile pharmaceutical composition, the sterile
pharmaceutical composition comprising: (i) a therapeutically
effective amount of the sterile chemotactic hematopoietic stem cell
product, wherein the therapeutically effective amount of the
chemotactic hematopoietic stem cell product comprises at least
10.times.10.sup.6 CD34+ cells which further contain a subpopulation
of at least 0.5.times.10.sup.6 potent CD34+ cells expressing CXCR-4
and having CXCR-4 mediated chemotactic activity; and (ii) a
stabilizing amount of serum, wherein the stabilizing amount of
serum is greater than 20% (v/v), wherein the chemotactic
hematopoietic stem cell product is further characterized as having
the following properties for at least 24 hours when tested in vitro
after passage through a catheter: (1) retains the CXCR-4-mediated
activity of the chemotactic hematopoietic stem cell product; (2) at
least 70% of the cells are CD34+ cells; (3) is at least 70% viable;
and (4) is able to form hematopoietic colonies in vitro; (d)
optionally administering the chemotactic hematopoietic stem cell
product at a plurality of infusion dates during the subject's
lifetime; and (e) treating at least one adverse consequence of the
progressive vascular insufficiency.
2. The method according to claim 1, step (a) further comprising
freezing at least one aliquot of the nonexpanded, isolated
population of autologous mononuclear cells containing CD34+ cells,
which further contain a subpopulation of potent CD34+/CXCR-4+ cells
that have CXCR-4-mediated chemotactic activity at -86.degree. C.
and cryostoring the at least one aliquot in the vapor phase of a
liquid nitrogen freezer.
3. The method according to claim 2, step (a) further comprising (i)
thawing the at least one aliquot of the frozen sterile nonexpanded,
isolated population of autologous mononuclear cells containing
CD34+ cells which further contain a subpopulation of potent
CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity;
(ii) enriching the sterile nonexpanded, isolated population of
autologous mononuclear cells for CD34+ cells, which further contain
a subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity, wherein the sterile
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells which further contain a subpopulation of
potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic
activity is a thawed sterile chemotactic hematopoietic stem cell
product; and (iii) administering to the subject on a second
infusion date a therapeutically effective amount of the sterile
thawed sterile chemotactic hematopoietic stem cell product,
comprising (a) at least 10.times.10.sup.6 CD34+ cells, which
further contain a subpopulation of at least 0.5.times.10.sup.6
potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated
chemotactic activity; and (b) a stabilizing amount of serum,
wherein the stabilizing amount of serum is greater than 20% (v/v),
wherein the sterile thawed chemotactic hematopoietic stem cell
product is further characterized as having the following properties
for at least 24 hours following thawing of the nonexpanded,
isolated population of autologous mononuclear cells comprising
CD34+ cells, which further contain a subpopulation of potent
CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity
when tested in vitro after passage through a catheter: (1) retains
the CXCR-4-mediated activity of the subpopulation of potent
CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity;
(2) at least 70% of the cells are CD34+ cells; (3) is at least 70%
viable; and (4) is able to form hematopoietic colonies in
vitro.
4. The method according to claim 3, wherein enriching step (ii)
occurs at least 1 day after acquisition of the sterile nonexpanded,
isolated population of autologous mononuclear cells comprising
CD34+ cells from the subject.
5. The method according to claim 3, wherein the sterile chemotactic
hematopoietic stem cell product is administered parenterally
through a catheter to the subject within about 48 hours to about 72
hours of thawing step (i).
6. The method according to claim 1, wherein the nonexpanded,
isolated population of autologous mononuclear cells comprising
CD34+ cells, which further contain a subpopulation of CD34+/CXCR-4+
cells that have CXCR-4-mediated chemotactic activity, is acquired
early after an acute myocardial infarction.
7. The method according to claim 6, wherein the nonexpanded,
isolated population of autologous mononuclear cells comprising
CD34+ cells, which further contain a subpopulation of CD34+/CXCR-4+
cells that have CXCR-4-mediated chemotactic activity, is acquired
after peak inflammatory cytokine cascade production in an infarcted
area.
8. The method according to claim 1, wherein the nonexpanded,
isolated population of autologous mononuclear cells comprising
CD34+ cells, which further contain a subpopulation of CD34+/CXCR-4+
cells that have CXCR-4-mediated chemotactic activity, is acquired
late after an acute myocardial infarction.
9. The method according to claim 8, wherein the nonexpanded,
isolated population of autologous mononuclear cells comprising
CD34+ cells, which further contain a subpopulation of CD34+/CXCR-4+
cells that have CXCR-4-mediated chemotactic activity, is acquired
at least 15 days after an acute myocardial infarction.
10. The method according to claim 3, wherein the sterile thawed
chemotactic hematopoietic stem cell product is further
characterized as having the following properties for at least 48
hours following thawing of the nonexpanded, isolated population of
autologous mononuclear cells when tested in vitro after passage
through a catheter: (i) is able to form hematopoietic colonies; and
(ii) retains at least 2% of the CXCR-4-mediated activity of the
subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity.
11. The method according to claim 3, wherein the sterile thawed
chemotactic hematopoietic stem cell product is further
characterized as having the following properties for at least 72
hours following thawing of the nonexpanded, isolated population of
autologous mononuclear cells when tested in vitro after passage
through a catheter: (i) is able to form hematopoietic colonies; and
(ii) retains at least 2% of the CXCR-4-mediated activity of the
subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity.
12. The method according to claim 1, wherein the vascular
insufficiency is an ischemia.
13. The method according to claim 12, wherein the ischemia is a
myocardial ischemia.
14. The method according to claim 12, wherein the ischemia is a
transient ischemia.
15. The method according to claim 12, wherein the ischemia is a
chronic myocardial ischemia.
16. The method according to claim 1, wherein the vascular
insufficiency is a vascular insufficiency after an acute myocardial
infarction resulting from underlying disease.
17. The method according to claim 16, wherein the ischemia is a
peri-infarct border zone ischemia.
18. The method according to claim 16, wherein a first infusion date
comprises a specific time interval defined by a first time and a
second time, and wherein the first time is after peak inflammatory
cytokine cascade production in an infarcted area and the second
time is before myocardial scar formation in the infarcted area.
19. The method according to claim 18, wherein the first time of the
first infusion date is at least about 5 days post-infarction.
20. The method according to claim 18, wherein the first time of the
first infusion date is about 5 days post-infarction and the second
time is about 14 days post-infarction.
21. The method according to claim 16, wherein the method treats
cardiomyocyte cell death in the peri-infarct border zone, relative
to controls.
22. The method according to claim 16, wherein the method treats
hypoperfusion in the peri-infarct border zone, relative to
controls.
23. The method according to claim 16, wherein the method treats
myocardial hibernation in the peri-infarct border zone, relative to
controls.
24. The method according to claim 16, wherein the method decreases
infarct area, relative to controls.
25. The method according to claim 16, wherein the method decreases
infarct mass, relative to controls.
26. The method according to claim 16, wherein the progressive
myocardial injury is a progressive decline in heart muscle function
following the acute myocardial infarction.
27. The method according to claim 1, wherein step (e) comprises
treating at least one adverse consequence of an acute myocardial
infarction selected from premature death, recurrent myocardial
infarction, development of congestive heart failure, development of
significant arrhythmias, development of acute coronary syndrome,
worsening of congestive heart failure, worsening of significant
arrhythmias, and worsening of acute coronary syndrome.
28. The method according to claim 1, wherein the progressive
myocardial injury is heart failure.
29. The method according to claim 1, wherein the catheter is a flow
control catheter.
30. The method according to claim 1, wherein the catheter is a
balloon dilatation catheter.
31. The method according to claim 1, wherein the catheter has an
internal diameter of at least about 0.36 mm.
32. The method according to claim 1, wherein the administering step
(c) is through the catheter into myocardium.
33. The method according to claim 1, wherein the administering step
(c) is through the catheter intravascularly.
34. The method according to claim 1, wherein the pharmaceutical
composition further includes at least one compatible active
agent.
35. The method according to claim 34, 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, a hematopoietic stem cell mobilizing agent, a tyrosine
kinase receptor agonist, an anti-anginal agent, a vasoactive agent,
an anticoagulant agent, a fibrinolytic agent, and a
hypercholesterolemic agent.
36. The method according to claim 35, wherein the tyrosine kinase
receptor agonist is human neuregulin 1.
37. A regimen for treating a progressive myocardial injury due to a
vascular insufficiency in a revascularized subject, which comprises
(a) administering parenterally through a catheter on a plurality of
infusion dates during lifetime of the subject a sterile
pharmaceutical composition comprising a sterile chemotactic
hematopoietic stem cell product, wherein the sterile chemotactic
hematopoietic stem cell product comprises (i) a nonexpanded,
isolated population of autologous mononuclear cells enriched for
CD34+ cells, which further contain a subpopulation of potent
CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity,
wherein the therapeutically effective amount of the chemotactic
hematopoietic stem cell product comprises at least
10.times.10.sup.6 CD34+ cells which further contain a subpopulation
of at least 0.5.times.10.sup.6 potent CD34+ cells expressing CXCR-4
and having CXCR-4-mediated chemotactic activity; and (ii) a
stabilizing amount of serum, wherein the stabilizing amount of
serum is greater than 20% (v/v), wherein the chemotactic
hematopoietic stem cell product is further characterized as having
the following properties for at least 24 hours following
acquisition of the chemotactic hematopoietic stem cell product when
tested in vitro after passage through a catheter: (1) retains the
CXCR-4-mediated activity of the subpopulation of potent
CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity;
(2) at least 70% of the cells are CD34+ cells; (3) is at least 70%
viable; and (4) is able to form hematopoietic colonies in vitro;
and (b) treating at least one adverse consequence of the
progressive vascular insufficiency.
38. The regimen according to claim 37, wherein the vascular
insufficiency is an ischemia.
39. The regimen according to claim 38, wherein the ischemia is a
myocardial ischemia.
40. The regimen according to claim 38, wherein the ischemia is a
transient ischemia.
41. The regimen according to claim 38, wherein the ischemia is a
chronic myocardial ischemia.
42. The regimen according to claim 37, wherein the vascular
insufficiency is a vascular insufficiency after an acute myocardial
infarction resulting from underlying disease.
43. The regimen according to claim 37, wherein the nonexpanded,
isolated population of autologous mononuclear cells comprising
CD34+ cells, which further contain a subpopulation of CD34+/CXCR-4+
cells that have CXCR-4-mediated chemotactic activity, is acquired
early after occurrence of an acute myocardial infarction.
44. The regimen according to claim 43, wherein the nonexpanded,
isolated population of autologous mononuclear cells comprising
CD34+ cells, which further contain a subpopulation of CD34+/CXCR-4+
cells that have CXCR-4-mediated chemotactic activity, is acquired
after peak inflammatory cytokine cascade production in an infarcted
area.
45. The regimen according to claim 37, wherein the nonexpanded,
isolated population of autologous mononuclear cells comprising
CD34+ cells, which further contain a subpopulation of CD34+/CXCR-4+
cells that have CXCR-4-mediated chemotactic activity, is acquired
late after occurrence of an acute myocardial infarction.
46. The method according to claim 45, wherein the nonexpanded,
isolated population of autologous mononuclear cells comprising
CD34+ cells, which further contain a subpopulation of CD34+/CXCR-4+
cells that have CXCR-4-mediated chemotactic activity, is acquired
at least 15 days after occurrence of an acute myocardial
infarction.
47. The regimen according to claim 42, wherein a first infusion
date comprises a specific time interval defined by a first time and
a second time, and wherein the first time is after peak
inflammatory cytokine cascade production in an infarcted area and
the second time is before myocardial scar formation in the
infarcted area.
48. The regimen according to claim 47, wherein the first time of
the first infusion date is at least about 5 days
post-infarction.
49. The regimen according to claim 47, wherein the first time of
the first infusion date is about 5 days post-infarction and the
second time is about 14 days post-infarction.
50. The regimen according to claim 42, wherein the a first infusion
date is at least 5 days after occurrence of an acute myocardial
infarction.
51. The regimen according to claim 42, wherein a second infusion
date is at least 30 days after occurrence of an acute myocardial
infarction.
52. The regimen according to claim 37, wherein the ischemia is a
peri-infarct border zone ischemia.
53. The regimen according to claim 52, wherein step (b) comprises
treating cardiomyocyte cell death in the peri-infarct border zone,
relative to controls.
54. The regimen according to claim 52, wherein step (b) comprises
treating hypoperfusion in the peri-infarct border zone, relative to
controls.
55. The regimen according to claim 52, wherein step (b) comprises
treating myocardial hibernation in the peri-infarct border zone,
relative to controls.
56. The regimen according to claim 52, wherein step (b) comprises
decreasing infarct area, relative to controls.
57. The regimen according to claim 52, wherein step (b) comprises
decreasing infarct mass, relative to controls.
58. The regimen according to claim 52, wherein step (b) comprises
treating at least one adverse consequence of the acute myocardial
infarction selected from premature death, recurrent myocardial
infarction, development of congestive heart failure, development of
significant arrhythmias, development of acute coronary syndrome,
worsening of congestive heart failure, worsening of significant
arrhythmias, and worsening of acute coronary syndrome.
59. The regimen according to claim 42, wherein the progressive
myocardial injury is a progressive decline in heart muscle function
following the acute myocardial infarction.
60. The regimen according to claim 37, wherein the progressive
myocardial injury is heart failure.
61. The regimen according to claim 37, wherein the catheter is a
flow control catheter.
62. The regimen according to claim 37, wherein the catheter is a
balloon dilatation catheter.
63. The regimen according to claim 37, wherein the catheter has an
internal diameter of at least about 0.36 mm.
64. The regimen according to claim 37, wherein the composition is
administered through the catheter into myocardium.
65. The regimen according to claim 37, wherein the composition is
administered through the catheter intravascularly.
66. The regimen according to claim 37, wherein the pharmaceutical
composition further includes at least one compatible active
agent.
67. The regimen according to claim 66, 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, a hematopoietic stem cell mobilizing agent, a tyrosine
kinase receptor agonist, an anti-anginal agent, a vasoactive agent,
an anticoagulant agent, a fibrinolytic agent, and a
hypercholesterolemic agent.
68. The regimen according to claim 67, wherein the tyrosine kinase
receptor agonist is human neuregulin 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of
U.S. application Ser. No. 11/552,396 (filed Oct. 24, 2006), which
issued as U.S. Pat. No. 7,794,705, U.S. Ser. No. 12/401,291 (filed
Mar. 10, 2009), which is a divisional application of application
Ser. No. 11/552,396, U.S. provisional application 61/119,552 (filed
Dec. 3, 2008) and U.S. 61/169,850 (filed Apr. 16, 2009). Each of
these applications are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The described invention relates to compositions comprising a
chemotactic hematopoietic stem cell product and methods of use
thereof for treating early or late adverse consequences of vascular
insufficiency.
BACKGROUND OF THE INVENTION
The Cardiac Cycle
[0003] The term "cardiac cycle" is used to refer to all or any of
the mechanical events related to the coronary blood flow or blood
pressure that occurs from the beginning of one heartbeat to the
beginning of the next: Blood pressure increases and decreases
throughout the cardiac cycle. The frequency of the cardiac cycle is
the heart rate. Every single `beat` of the heart involves live
major stages: (1) "late diastole," which is when the semilunar
valves close, the atrioventricular (Av) valves open and the whole
heart is relaxed; (2) "atrial systole," which is when the
myocardium of the left and right atria are contracting, AV valves
open and blood flows from atrium to ventricle; (3) "isovolumic
ventricular contraction," which is when the ventricles begin to
contract, AV and semilunar valves close, and there is no change in
volume; (4) "ventricular ejection," which is when the ventricles
are empty but still contracting and the semilunar valves are open;
and (5) "isovolumic ventricular relaxation," when pressure
decreases, no blood is entering the ventricles, the ventricles stop
contracting and begin to relax, and the semilunar valves are shut
because blood in the aorta is pushing them shut. The cardiac cycle
is coordinated by a series of electrical impulses that are produced
by specialized heart cells found within the sino-atrial node and
the atrioventricular node.
Coronary Blood Flow
[0004] The flow of blood through the coronary arteries is
pulsatile, with characteristic phasic systolic and diastolic flow
components. Systolic flow, which relates to the contraction or
pumping phase of the heart cycle, has rapid, brief, retrograde
responses. Diastolic flow, which relates to the relaxation or
filling phase of the heart cycle, occurs during the relaxation
phase after myocardial contraction, with an abrupt increase above
systolic levels and a gradual decline parallel with that of aortic
diastolic pressures. Intramural coronary blood volume changes
during each heartbeat, with the myocardium accommodating the volume
change brought about by muscular contraction. Coronary venous flow
is out of phase with coronary arterial flow, occurring
predominantly in systole and nearly absent during diastole.
[0005] For each heartbeat, blood pressure varies between systolic
and diastolic pressures. The term "systolic pressure" refers to the
peak pressure in the arteries, which occurs near the end of the
cardiac cycle when the ventricles are contracting. The term
"diastolic pressure" refers to the minimum pressure in the
arteries, which occurs near the beginning of the cardiac cycle when
the ventricles are filled with blood.
[0006] Coronary blood flow not only is phasic but also varies with
the type of vessel and location in the myocardium. Coronary
arterioles appear to have specialized regulatory elements along
their length that operate "in series" in an integrated manner. A
system of multiple functional "valves" permits fine control of the
coronary circulation. The smallest arterioles dilate during
metabolic stress, resulting in reduced microvascular resistance and
increased myocardial perfusion. Stenosis or narrowing of a blood
vessel produces resistance to blood flow related directly to the
morphologic features of the stenosis. As the upstream arteriolar
pressure decreases due to a fall in distending pressure across the
stenosis, myogenic dilation of slightly larger arterioles upstream
occurs and causes an additional decrease in resistance. Increased
flow in the largest arterioles augments shear stress and triggers
flow-mediated dilation, further reducing the resistance of this
network.
[0007] The arterial and venous pulsatile flow characteristics of
the heart are dependent on intramyocardial compliance. The term
"compliance" refers to a measure of the tendency of a hollow organ
to resist recoil toward its original dimensions upon removal of a
distending or compressing force. The higher the compliance the more
elastic the material. Compliance is calculated using the following
equation, where .DELTA.V is the change in volume, and .DELTA.P is
the change in pressure:
C=.DELTA.V/.DELTA.P
[0008] The capacity of the heart as a reservoir is controlled by
resistance arterioles to coronary vascular inflow. Outlet
resistance is related to intramural cardiac veins. The
intramyocardial capillary resistance influences both arterial and
venous responses but predominantly acts in concert with outlet
resistance.
[0009] Approximately 75% of total coronary resistance occurs in the
arterial system, which comprises conductance (R1), prearteriolar
(R2) and arteriolar and intramyocardial capillary vessels (R3).
Normal epicardial coronary arteries in humans typically are 0.3 to
5 mm in diameter, and do not offer appreciable resistance to blood
flow. Normally, large epicardial vessel resistance (R1) is trivial
until atherosclerotic obstructions compromise the lumen.
Precapillary arterioles (R2), 100 to 500 .mu.m in size) are
resistive vessels connecting epicardial to myocardial capillaries
and are the principal controllers of coronary blood flow. They
contribute approximately 25% to 35% of total coronary resistance.
Distal precapillary arteriolar vessels (<100 .mu.m in diameter),
the main site of metabolic regulation of coronary blood flow, are
responsible for 40-50% of coronary flow resistance. The dense
network of about 4000 capillaries per square millimeter ensures
that each myocyte is adjacent to a capillary. Capillaries are not
uniformly patent (meaning open; affording free passage), because
precapillary sphincters regulate flow according to the needs of the
myocardium.
[0010] Several conditions, such as left ventricular hypertrophy,
myocardial ischemia, or diabetes, can impair the microcirculatory
resistance (R3), blunting the maximal absolute increase in coronary
flow in times of increased oxygen demand.
Ischemia
[0011] The myocardium depends almost entirely on aerobic
metabolism, since oxygen stores in the heart are meager. Myocardial
oxygen supply rises and falls in response to the oxygen (energy)
demands of the myocardium. The term "autoregulation" refers to the
ability to maintain myocardial perfusion at constant levels in the
face of changing driving forces. Autoregulation maintains coronary
perfusion at relatively constant levels over a wide range of mean
aortic pressure. When aortic pressure exceeds its upper or lower
limits, coronary blood flow precipitously declines or increases
proportionately.
[0012] The heart needs to be supplied with a sufficient quantity of
oxygen to prevent underperfusion. When reduced perfusion pressure
distal to stenoses is not compensated by autoregulatory dilation of
the resistance vessels, ischemia, meaning a lack of blood supply
and oxygen, occurs. Because the zone least supplied generally is
the farthest out, ischemia generally appears in areas farthest away
from the blood supply.
[0013] After total or near-total occlusion of a coronary artery,
myocardial perfusion occurs by way of collaterals, meaning vascular
channels that interconnect epicardial arteries. Collateral channels
may form acutely or may preexist in an under-developed state before
the appearance of coronary artery disease. Preexisting collaterals
are thin-walled structures ranging in diameter from 20 .mu.m to 200
.mu.m, with a variable density among different species. Preexisting
collaterals normally are closed and nonfunctional, because no
pressure gradient exists to drive flow between the arteries they
connect. After coronary occlusion, the distal pressure drops
precipitously and preexisting collaterals open virtually
instantly.
[0014] The term "myocardial ischemia" refers to a decrease in blood
supply and oxygen to the cells of the myocardium. The development
of myocardial ischemia has been attributed to two mechanisms: (1)
increased myocardial oxygen demand, and (2) decreased myocardial
perfusion and oxygen delivery. (Willerson, J. T. et al., J. Am.
Coll. Cardiol. 8(1): 245-50 (1986)). Myocardial ischemia generally
appears first and is more extensive in the subendocardial region,
since these deeper myocardial layers are farthest from the blood
supply, with greater need for oxygen.
[0015] Transient ischemia, hibernating myocardium, and myocardial
infarction are clinically different conditions.
[0016] Transient Ischemia. The term "transient ischemia" as used
herein refers to a reversible (meaning that the myocytes survive
the insult) narrowing of a coronary artery at rest or with exercise
where there is no thrombus or plaque rupture but where blood supply
cannot be met. Every time the heart's oxygen demand increases, an
imbalance between oxygen demand and supply is created. Transient
ischemia produces a cascade of events beginning with metabolic and
biochemical alterations leading to impaired ventricular relaxation
and diastolic dysfunction, impaired systolic function, and
electrocardiographic abnormalities with ST segment alterations,
followed by increased end-diastolic pressure with left ventricular
dyssynchrony, hypokineses, akinesis, and dyskinesis, and lastly
painful symptoms of angina. Even though ischemic myocytes
experience physiological and metabolic changes within seconds of
the cessation of coronary flow, resulting in T wave and sometimes
ST segment abnormalities (but without senna enzyme elevation), no
cell death results from the ischemia. Kloner, R. A. and Jennings, R
B, Circulation 104: 2981-89 (2001). Once blood flow is
re-established, a complete recovery of myocyte contractile function
takes place.
[0017] Although angina pectoris (chest pain) may be a symptom of
transient ischemia, by and large transient ischemia is silent
(meaning ST-segment depression of at least 1 mm is present without
associated symptoms, e.g., chest pain) in 79% of subjects. In most
patients with stable angina, for example, physical effort or
emotion, with a resultant increase in heart rate, blood pressure,
or contractile state, or any combination thereof, increases
myocardial oxygen demand without an adequate delivery in oxygen
delivery through tightly narrowed (stenosed) coronary arteries.
More than 40% of patients with stable angina treated with one or
more antianginal drugs have frequent episodes of silent ischemia,
which has been shown to predict a higher risk of coronary events
and cardiac death. Deedwania, P C, Carbajal, E V, Arch. Intern.
Med. 150: 2373-2382 (1991).
[0018] Chronic Myocardial ischemia. The term "chronic myocardial
ischemia (CMI)" as used herein refers to a prolonged subacute or
chronic state of myocardial ischemia due to narrowing of a coronary
blood vessel in which the myocardium "hibernates", meaning that the
myocardium downregulates or reduces its contractility, and hence
its myocardial oxygen demand, to match reduced perfusion, thereby
preserving cellular viability and preventing myocardial necrosis.
This hibernating myocardium is capable of returning to normal or
near-normal function on restoration of an adequate blood supply.
Once coronary blood flow has been restored to normal or near normal
and ischemia is resolved, however, the hibernating myocardium still
does not contract. This flow-function mismatch resulting in a slow
return of cardiac function after resolution of ischemia has been
called stunning. The length of time for function to return is quite
variable, ranging from days to months, and is dependent on a number
of parameters, including the duration of the original ischemic
insult, the severity of ischemia during the original insult, and
the adequacy of the return of the arterial flow. A number of
studies have provided evidence for inflammation in hibernating
myocardium. Heusch, G. et al., Am. J. Physiol. Heart Circ. Physiol.
288: 984-99 (2005). A study conducted in a porcine model of
myocardial hibernation in which the mean rest (left anterior
descending coronary artery (LAD) coronary blood flow was reduced to
about 60% of baseline for a period of 24 hours to four weeks,
detected apoptotic myocytes in all experimental pigs in the
hibernating regions supplied by the stenotic LAD, suggesting that
functional downregulation may not be adequate to prevent gradual,
ongoing myocyte death through apoptosis in hibernating myocardium.
Chen, C. et al., J. Am. Coll. Cardiol. 30: 1407-12 (1997).
[0019] Acute Myocardial Infarction (AMI). Another type of insult
occurs during AMI. AMI is an abrupt change in the lumen of a
coronary blood vessel which results in ischemic infarction, meaning
that it continues until heart muscle dies. On gross inspection,
myocardial infarction can be divided into two major types:
transmural infarcts, in which the myocardial necrosis involves the
full or nearly full thickness of the ventricular wall, and
subendocardial (nontransmural) infarcts, in which the myocardial
necrosis involves the subendocardium, the intramural myocardium, or
both, without extending all the way through the ventricular wall to
the epicardium. There often is total occlusion of the vessel with
ST segment elevation because of thrombus formation within the lumen
as a result of plaque rupture. The prolonged ischemic insult
results in apoptotic and necrotic cardiomyocyte cell death. See
Kajstura, J., et al., Lab Invest. 74: 86-107 (1996). Necrosis
compromises the integrity of the sarcolemmal membrane and
intracellular macromolecules such that serum cardiac markers, such
as cardiac-specific troponins and enzymes, such as serum creatine
kinase (CK), are released. In addition, the patient may have
electrocardiogram (ECG) changes because of full thickness damage to
the muscle. An ST-Elevation Myocardial Infarction (STEMI) is a
larger injury than a non-ST-elevation myocardial infarction.
ST-segment elevation and Q waves on the ECG, two features highly
indicative of myocardial infarction, are seen in only about half of
myocardial infarction cases on presentation.
[0020] AMI remains common with a reported annual incidence of 1.1
million cases in the United States alone (Antman, E. M., Braunwald,
E., Acute Myocardial Infarction, in Principles of Internal
Medicine, 15th Ed., Braunwald, E. et al., Eds., New York:
McGraw-Hill (2001)). Preclinical and clinical data demonstrate that
following a myocardial infarction, the acute loss of myocardial
muscle cells and the accompanying peri-infarct border zone
hypo-perfusion result in a cascade of events causing an immediate
diminution of cardiac function, with the potential for long term
persistence. The extent of myocardial cell loss is dependent on the
duration of coronary artery occlusion, existing collateral coronary
circulation and the condition of the cardiac microvasculature. Paul
et al., Am. Heart J. 131: 710-15 (1996); Pfeffer, M. A., Braunwald,
E., Circulation 81: 1161-72 (1990); Sheilban, I. e. al., J. Am.
Coll. Cardiol. 38: 464-71 (2001); Braunwald E., Bristow, M. R.,
Circulation 102: IV-14-23 (2000); Rich et al., Am. J. Med. 92:7-13
(1992); Ren et al., J. Histochem. Cytochem. 49: 71-79 (2002);
Hirai, T. et al., Circulation 79: 791-96 (1989); Ejiri, M. et al.,
J. Cardiology 20: 31-37 (1990). Because myocardial cells have
virtually no ability to regenerate, myocardial infarction leads to
permanent cardiac dysfunction due to contractile-muscle cell loss
and replacement with nonfunctioning fibrotic scarring.
Frangogiannis, N. G., et al., Cardiovascular Res. 53(1): 31-47
(2002). Moreover, compensatory hypertrophy of viable cardiac muscle
leads to microvascular insufficiency that results in further demise
in cardiac function by causing myocardial muscle hibernation and
apoptosis of hypertrophied myocytes in the peri-infarct border
zone.
[0021] Among survivors of myocardial infarction, residual cardiac
function is influenced by the extent of ventricular remodeling
(meaning changes in size, shape, and function, typically a
progressive decline in function, of the heart after injury).
Alterations in ventricular topography (meaning the shape,
configuration, or morphology of a ventricle) occur in both
infarcted and healthy cardiac tissue after myocardial infarction.
Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990).
Ventricular dilatation (meaning a stretching, enlarging or
spreading out of the ventricle) causes a decrease in global cardiac
function and is affected by the infarct size, infarct healing and
ventricular wall stresses. Recent efforts to minimize remodeling
have been successful by limiting infarct size through rapid
reperfusion (meaning restoration of blood flow) using thromobolytic
agents, and mechanical interventions, including, but not limited
to, placement of a stent, along with reducing ventricular wall
stresses by judicious use of pre-load therapies and proper
after-load management. Id. Regardless of these interventions, a
substantial percentage of patients experience clinically relevant
and long-term cardiac dysfunction after myocardial infarction.
Sheiban, I. et al., J. Am. Coll. Cardiol. 38: 464-71 (2001).
Despite revascularization of the infarct related artery circulation
and appropriate medical management to minimize ventricular wall
stresses, a significant percentage of these patients experience
ventricular remodeling, permanent cardiac dysfunction, and
consequently remain at an increased lifetime risk of experiencing
adverse cardiac events, including death. Paul et al., Am. Heart J.
131: 710-15 (1996); Pfeffer, M. A., Braunwald, E., Circulation 81:
1161-72 (1990).
[0022] At the cellular level, immediately following a myocardial
infarction, transient generalized cardiac dysfunction uniformly
occurs. In the setting of a brief (i.e., lasting three minutes to
five minutes) coronary artery occlusion, energy metabolism is
impaired, leading to demonstrable cardiac muscle dysfunction that
can persist for up to 48 hours despite immediate reperfusion. This
so-called "stunned myocardium phenomenon" occurs subsequent to or
after reperfusion and is thought to be a result of reactive oxygen
species. The process is transient and is not associated with an
inflammatory response. Frangogiannis, N. G., et al., Cardiovascular
Res. 53(1): 31-47 (2002). After successful revascularization,
significant recovery from stunning occurs within three to four
days, although complete recovery may take much longer. Boli, R.,
Prog. Cardiovascular Disease 40(6): 477-515 (1998); Sakata, K. et
al., Ann. Nucleic Med. 8: 153-57 (1994); Wollert, K. C. et al.,
Lancet 364: 141-48 (2004).
[0023] Coronary artery occlusion of more significant duration,
i.e., lasting more than five minutes, leads to myocardial ischemia
(i.e. an insufficient blood flow to the heart's muscle mass) and is
associated with a significant inflammatory response that begins
immediately after reperfusion and can last for up to several weeks.
Frangogiannis, N. G., et al., Cardiovascular Res. 53(1): 31-47
(2002); Frangogiannis, N. G. et al., Circulation 98: 687-798
(1998).
[0024] The inflammatory process following reperfusion is complex.
Initially it contributes to myocardial damage but later leads to
healing and scar formation. This complex process appears to occur
in two phases. In the first so-called "hot" phase (within the first
five days), reactive oxygen species (in the ischemic myocardial
tissue) and complement activation generate a signal chemotactic for
leukocytes (chemotaxis is the directed motion of a motile cell,
organism or part towards environmental conditions it deems
attractive and/or away from surroundings it finds repellent) and
initiate a cytokine cascade. Lefer, D. J., Granger, D. N., Am. J.
Med. 4:315-23 (2000); Frangogiannis, N. G., et al., Circulation
7:699-710 (1998). Mast cell degranulation, tumor necrosis factor
alpha (TNF.alpha.) release, and increased interleukin-6 (IL-6),
intercellular adhesion molecule 1 ("ICAM-1" or CD-54, a receptor
typically expressed on endothelial cells and cells of the immune
system), selectin (L, E and P) and integrin (CD11a, CD11b and CD18)
expression all appear to contribute to neutrophil accumulation and
degranulation in ischemic myocardium. Frangogiannis, N. G. et al.,
Circulation 7: 699-710 (1998), Kurrelmeyer, K. M, et al., Proc.
Natl. Acad. Sci. USA. 10: 5456-61 (2000); Lasky, L. A., Science
258: 964-69 (1992); Ma, X. L., et al., Circulation 88(2): 649-58
(1993); Simpson, P. J. et al., J. Clin. Invest. 2: 624-29 (1998).
Neutrophils contribute significantly to myocardial cell damage and
death through microvascular obstruction and activation of
neutrophil respiratory burst pathways after ligand-specific
adhesion to cardiac myocytes. Entman, M. L., et al., J. Clin.
Invest. 4: 1335-45 (1992). During the "hot" phase, angiogenesis is
inhibited due to the release of angiostatic substances, including
interferon gamma-inducible protein (IP 10). Frangogiannis, N. G.,
et al., FASEB J. 15: 1428-30 (2001).
[0025] In the second phase, the cardiac repair process begins
(about day 6 to about day 14), which eventually leads to scar
formation (about day 14 to about day 21) and subsequent ventricular
remodeling (about day 21 to about day 90). Soon after reperfusion,
monocytes infiltrate the infarcted myocardium. Attracted by
complement (C5a), transforming growth factor B1 ("TGF-B1") and
monocyte chemotactic protein 1 ("MCP-1"), monocytes differentiate
into macrophages that initiate the healing process by scavenging
dead tissue, regulating extracellular matrix metabolism, and
inducing fibroblast proliferation. Birdshall, H. H., et al.,
Circulation 3: 684-92 (1997). Secretion of interleukin 10 (IL-10)
by infiltrating lymphocytes also promotes healing by
down-regulating inflammatory cytokines and influencing tissue
remodeling. Frangogiannis, N. G. et al., J. Immunol. 5:2798-2808
(2000). Mast cells also appear to be involved in the later stages
of myocardial repair by participating in the formation of fibrotic
scar tissue. Stem Cell Factor (SCF) is a potent attractor of mast
cells. SCF mRNA has been shown to be up-regulated in ischemic
myocardial segments in a canine model of myocardial infarction and
thus may contribute to mast cell accumulation at ischemic
myocardial sites. Franigogiannis. N. G. et al., Circulation 98:
687-798 (1998). Mast cell products (including TGF-B, basic
fibroblast growth factor (bFGF), vascular endothelial growth factor
(VEGF) and gelatinases A and B) induce fibroblast proliferation,
influence extracellular matrix metabolism, and induce angiogenesis.
Fang, K. C., et al., J. Immunol. 162: 5528-35 (1999); Takeshi, S.,
et al., Cardiology 93: 168-74 (2000).
[0026] Following a myocardial infarction, neoangiogenesis occurs
after the "hot" phase of the inflammatory process subsides (about
day 5) coincident with rising levels of VEGF (VEGF peaks at about
day 7 and gradually subsides to baseline at about day 14 to about
day 21). During this phase of the healing process, endothelial
precursor cells (EPCs) are mobilized and recruited to the infarct
site. Shinitani, S., et al., Circulation 103: 2776-79 (2001).
Without being limited by theory, it has been suggested that the
chemokine stromal cell derived factor-1 (SDF-1), which is the
ligand for the CXCR-4 chemokine receptor expressed by CD34+ cells,
also plays a role in homing of cells to areas of ischemic damage.
Ceredini, D. J., et al., Nature Medicine 10: 858-63 (2004); Askari,
A., et al., Lancet 362: 697-703 (2003); Yamaguchi, J. et al.,
Circulation 107: 1322-34 (2003). While it is known that SDF-1 plays
a role in hematopoiesis and is involved in migration, homing and
survival of hematopoietic progenitors, and while SDF-1 has been
implicated in ischemic neovascularization in vivo by augmenting EPC
recruitment to ischemic sites (Yamaguchi et al. Circulation
107:1322-1328 (2003), SDF-1's role in neoangiogenesis is not
certain. There is suggestive evidence implicating SDF-1. For
example, SDF-1 gene expression is upregulated during hypoxia, a
deficiency of oxygen in the tissues, by hypoxia inducible factor-1.
Furthermore, CD34+ cells are capable of homing to areas of
ischemia, rich in SDF-1, including infarcted myocardium. Askari et
al., Lancet 362: 697-703 (2003). Moreover, virtually all
CD34+CXCR-4+ cells co-express VEGF-2 and therefore migrate in
response to VEGF as well as SDF-1. Peichev M., et al., Blood 95:
952-58 (2000). CD34+CXCR-4+VEGF-1 cells, once recruited, are
capable of contributing to neoangiogenesis. Yamaguchi, J. et al.,
Circulation 107: 1322-34 (2003).
[0027] The Peri-Infarct Border Zone
[0028] The zone of dysfunctional myocardium produced by coronary
artery occlusion extends beyond the infarct region to include a
variable boundary of adjacent normal appearing tissue. (Hu, Q., et
al., Am. J. Physiol. Heart Circ. Physiol. 291: H648-657 (2006)).
This ischemic, but viable, perinfarct zone of tissue separates the
central zone of progressive necrosis from surrounding normal
myocardium. The peri-infarct zone does not correlate with enzymatic
parameters of infarct size and is substantially larger in small
infarcts. Stork, A., et al., European Radiol. 16(10): 2350-57
(2006).
[0029] Ischemia due to edema and compression of the blood vessels
in the border zone may be very important to outcome after an AMI.
It is known, for example, that after an AMI, transient ischemia
occurs in the border zones, and that percutaneous coronary
interventions, which open up the infarct-related artery, can
adversely affect the health of the peri-infarct border zones. It
has been suggested that intermediate levels of mean blood flow can
exist as the result of admixture of peninsulas of ischemic tissue
intermingled with regions of normally perfused myocardium at the
border of an infarct. (Flu, Q., et al., Am. J. Physiol. Heart Circ
Physiol. 291: H648-657 (2006)). However, the boundary of the
intermingled coronary microvessels, which in dogs is no more than 3
mm in width, cannot explain the relatively broad region of
dysfunctional myocardium surrounding an infarct. Murdock, R H, Jr.,
et al., Cir. Res. 52: 451-59 (1983); Buda, A J, et al., J. Am.
Coll. Cardiol. 8: 150-58 (1986). Progressive dysfunction of this
peri-infarct myocardium over time may contribute to the transition
from compensated remodeling to progressive heart failure after an
AMI.
[0030] Heart Failure
[0031] Heart failure is a complex clinical syndrome that arises
secondary to abnormalities of cardiac structure and/or function
that impair the ability of the left ventricle to fill or eject
blood. See Hunt, S. J. Am. Coll. Cardiol. 46: e1-e82 (2005). It is
a progressive condition where the heart muscle weakens and cannot
pump blood efficiently. Patients may be categorized as having heart
failure with depressed ejection fraction ("EF") (referred to as
"systolic failure"), or having heart failure with a normal EF or
heart failure with a preserved EF (referred to as "diastolic
failure"). Patients may have significant abnormalities of left
ventricle (LV) contraction and relaxation and yet have no symptoms,
in which case they are referred to as having "asymptomatic heart
failure". When a patient with chronic heart failure deteriorates,
the patient is referred to as having "decompensated heart failure",
or, if the symptoms arise abruptly, as having "acute decompensated
heart failure".
[0032] The various diagnostic criteria used to determine the
presence of heart failure are shown in the following Table (V. L.
Roger, Intl. J. Environ. Res. Public Health 7(4): 1807-30
(2010)):
TABLE-US-00001 European Society Gothenburg Score.sup.4
Framingham.sup.1 Boston.sup.2 of Cardiology.sup.3 n Criteria/method
of assessment MAJOR CATEGORY I: 1. Symptoms of CARDIAC SCORE
CRITERIA: History heart failure (at rest History of heart Self-
Paroxysmal Rest dyspnea or during exercise) disease (1-2 pts)
report nocturnal dyspnea (4 pts) and Angina (1-2 pts) Self- or
orthopnea Orthopnea (4 pts) 2. Objective report Neck vein
Paroxysmal evidence of cardiac Edema (1 pt) Self- distension
nocturnal dysfunction (at rest) report Rales dyspnea (3 pts) and
Nocturnal Dyspnea Self- Cardiomegaly Dyspnea on 3. Response to (1
pt) report Acute pulmonary walking on level treatment directed
Rales (1 pt) Physical edema S3 gallop (2 pts) towards heart failure
exam Increased venous Dyspnea on (in cases where Atrial
fibrillation ECG pressure .gtoreq.16 cm climbing (1 pt) diagnosis
is in (1 pt) water CATEGORY doubt). PULMONARY SCORE Circ. time
.gtoreq.25 sec II: Physical Criteria 1 and 2 History of Chronic
Self- Hepatojugular examination should be fulfilled in
bronchitis/asthma report reflux Heart rate all cases (1-2 pts)
MINOR abnormality (1-2 pts) Cough, phlegm, or Self- CRITERIA:
wheezing (1 pt) report Ankle edema Jugular venous Night cough
pressure Rhonchi (2 pts) Physical Dyspnea on elevation (1-2 exam
exertion pts) Cardiac and pulmonary score Hepatomegaly Lung
crackles are calculated and used to Pleural effusion (1-2 pts)
differentiate Cardiac form Vital capacity Wheezing (3 pts)
pulmonary dyspnea decreased 1/3 Third heart from maximum sound (3
pts) Tachycardia rate CATEGORY of .gtoreq.120/min) III: Chest MAJOR
OR radiography MINOR Alveolar CRITERION: pulmonary Weight loss
.gtoreq.4.5 kg edema (4 pts) in 5 days in Interstitial response to
pulmonary treatment edema (3 pts) HEART Bilateral pleural FAILURE:
effusions (3 pts) present with 2 Cardiothoracic major or 1 major
ratio .gtoreq.0.50 (3 and 2 minor pts) criteria Upper-zone flow
redistribution (2 pts) HEART FAILURE: Definite 8-12 pts, possible
5-7 pts, unlikely 4 pts or less .sup.1McKee PA, Castelli WP,
McNamara PM, Kannel WB. The natural history of congestive heart
failure: the Framingham study. N. Engl. J. Med. 285: 1441-1446
(1971) .sup.2Carlson K J, Lee DC Goroll AH, Lehy M, Johnson RA, an
analysis of physicians' reasons for prescribing long-term digitalis
therapy in outpatients. J. Chronic Dis. 38: 733-39 (1985)
.sup.3Guidelines for the diagnosis of heart failure The Task Force
on Heart Failure of the European Society of Cardiology. Eur. Heart
J. 16: 741-751 (1995) .sup.4Eriksson H, Caidahl K, Larsson B,
Ohlson LO, Welin L, Wilhelmsen L. Svardsudd K. Cardiac and
pulmonary causes of dyspnoea - validation of a scoring test for
clinical-epidemiological use: the Study of Men Born in 1913. Eur.
Heart J. 8: 1007-1014 (1987)[
[0033] The prognosis of heart failure is poor with reported
survival estimates of 50% at 5 years and 10% at 10 years; left
ventricular dysfunction is associated with an increase in the risk
of sudden death. Id.
[0034] To date, no ideal therapy exists for preventing the long
term adverse consequences of vascular insufficiency, particularly
vascular insufficiency after myocardial infarction. Large vessel
revascularization (meaning the successful placement of a stent) is
insufficient in addressing increased demands posed by compensatory
myocardial hypertrophy. As a result, infarct extension and fibrous
replacement commonly occur, regardless of large vessel
revascularization, appropriate medical management of ventricular
wall stresses, and potential natural, albeit suboptimal, CD34+
cell-mediated neoangiogenesis (one of theories relating to the
underlying cause of myocardial infarction is that the ability to
mobilize these cells may be biologically limited).
[0035] Intense interest has developed in evaluating the ability of
endothelial and myocardial precursor cells to limit damage to the
myocardium after infarction and to limit or prevent ventricular
remodeling. Significant preclinical data and some clinical data
demonstrate the safety and potential of cell therapy using a
variety of cell precursors (particularly hematopoietic cells) to
contribute to neoangiogenesis, limited cardiac myogenesis
(principally by fusion), and muscle preservation in the myocardial
infarct zone. See, e.g., Jackson, et al., J. Clin. Invest. 107:
1395-1402 (2001); Edelberg, J. M., et al., Cir. Res. 90: e89-e93
(2002); Schichinger. V. et al., New Engl. J. Med. 355 (12): 1210-21
(2006) (using bone marrow-derived progenitor cells); Assmus, B. et
al., New Engl. J. Med. 355 (12) 1222-32 (2006) (using bone
marrow-derived progenitor cells), but see Lunde, K. et al., New
Eng. J. Med. 355 (12): 1199-209 (2006) (using bone marrow-derived
progenitor cells).
[0036] Bone marrow consists of a variety of precursor and mature
cell types, including hematopoietic cells (the precursors of mature
blood cells) and stromal cells (the precursors of a broad spectrum
of connective tissue cells), both of which appear to be capable of
differentiating into other cell types. Wang, J. S. et al., J.
Thorac. Cardiovasc. Surg. 122: 699-705 (2001); Tomita, S. et al.,
Circulation 100 (Suppl. II): 247-256 (1999); Saito, T. et al.,
Tissue Eng. 1: 327-43 (1995). Unmodified (i.e., not fractionated)
marrow or blood-derived cells have been used in several clinical
studies, for example, Hamano, K. et al., Japan Cir. J. 65: 845-47
(2001); Strauer. B. E., et al., Circulation 106: 1913-18 (2002);
Assmus, et al., Circulation 106: 3009-3017 (2002); Dobert, N. et
al., Fur. J. Nuel. Med. Mol. Imaging, 8: 1146-51 (2004); Wollert,
K. C. et al., Lancet 364: 141-48 (2004). Since the mononuclear
fraction of bone marrow contains stromal cells, hematopoietic
precursors, and endothelial precursors, the relative contribution
of each of these populations to the observed effects, if any,
remains unknown.
[0037] CD34 is a hematopoietic stem cell antigen selectively
expressed on hematopoietic stem and progenitor cells derived from
human bone marrow, blood and fetal liver. Yin et al., Blood 90:
5002-5012 (1997); Miaglia, S. et al., Blood 90: 5013-21 (1997).
Cells that express CD34 are termed CD34+. Stromal cells do not
express CD34 and are therefore termed CD34-. CD34+ cells isolated
from human blood may be capable of differentiating into
cardiomyocytes, endothelial cells, and smooth muscle cells in vivo.
See Yeh, et al., Circulation 108: 2070-73 (2003). CD34+ cells
represent approximately 1% of bone marrow derived nucleated cells;
CD34 antigen also is expressed by immature endothelial cell
precursors (mature endothelial cells do not express CD34). Peichev,
M. et al., Blood 95: 952-58 (2000). In vitro, CD34+ cells derived
from adult hone marrow give rise to a majority of the
granulocyte/macrophage progenitor cells (CFU-GM), some
colony-forming units-mixed (CFU-Mix) and a minor population of
primitive erythroid progenitor cells (burst forming units,
erythrocytes or BFU-E). Yeh, et al., Circulation 108: 2070-73
(2003). CD34+ cells also may have the potential to differentiate
into, or to contribute to, the development of new myocardial
muscle, albeit at low frequency.
[0038] Techniques have been developed using immunomagnetic bead
separation to isolate a highly purified and viable population of
CD34+ cells from bone narrow mononuclear cells. Sec U.S. Pat. Nos.
5,536,475, 5,035,994, 5,130,144, 4,965,205, the contents of each of
which is incorporated herein by reference. Two clinical studies
support the clinical application of bone marrow derived CD34+ cells
after myocardial infarction. See C. Stamm, et al., Lancet 361:
45-46 (2003); Herenstein, B. et al., Blood Supplement, Abs. 2696
(2004).
Animal Models
[0039] Peripheral artery disease (PAD), also called peripheral
vascular disease (PVD), is modeled by the hind limb model of
ischemia in which the femoral artery of the mouse is tied off to
simulate peripheral artery disease. PAD, which commonly affects the
arteries supplying the leg and includes all diseases caused by the
obstruction of large arteries in the arms and legs, can result from
atherosclerosis, inflammatory processes leading to stenosis, an
embolism or thrombus formation. Restriction of blood flow due to
arterial stenosis or occlusion often leads patients to complain of
muscle pain on walking (intermittent claudication). Any further
reduction in blood flow causes ischemic pain at rest. This
condition is called chronic limb ischemia, meaning the demand for
oxygen cannot be sustained when resting. Ulceration and gangrene
may then supervene in the toes, which are the furthest away from
the blood supply, and can result in loss of the involved limb if
not treated.
[0040] Therapies for limb ischemia have the goals of collateral
development and blood supply replenishment. Bone marrow derived
CD34+ mononuclear cells have been tested in such hindlimb ischemia
models, but the hindlimb ischemia model does not model what takes
place in the heart. A preferred therapy after AMI would stop cells
from dying during recovery that leads, to reverse remodeling and
failure, or replace the dying cells with cardiomyocytes.
[0041] The closest animal model, the pig model, is not a good model
of human disease because (i) all experiments generally are done in
nonatherosclerotic animals, (ii) the animals are not treated with
angioplasty, (iii) normal pigs do not embolize blood vessels; (iv)
circulation of the pig is not exactly the same as human; and (iv)
the peri-infarct border zone may not be the same.
[0042] A marginal improvement in angina symptoms recently was
reported when CD34+ cells were mobilized with G-CSF, apheresed
after 5 days, and then injected into an ischemic area of the heart
based on Noga mapping. [Northwestern University (2009, Apr. 1).
Adult Stem Cell Injections May Reduce Pain And Improve Walking In
Severe Angina Patients. ScienceDaily. Retrieved Oct. 21, 2010, from
http://www.sciencedaily.com-heleases/2009/03/090330091706.htm) Data
from a phase I trial conducted by the present inventors has
provided evidence that subjects treated with at least
10.times.10.sup.6 isolated autologous CD34+ hematopoietic stem
cells containing a subpopulation of at least 0.5.times.10.sup.6
potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated
chemotactic activity (n=9) experienced significant improvement in
resting perfusion rates at 6 months compared to subjects receiving
5 million cells (n=6) and control (n=15), as measured by the SPECT
Total Severity Score (-256 versus +13, p=0.01). U.S. Patent
Applications 61/169,850 and 61/119,552, incorporated herein by
reference.
[0043] The described invention is a therapy for preventing the
long-term adverse consequences of vascular insufficiency,
particularly vascular insufficiency that produces expansion of the
myocardial infarct area after an AMI progressing to heart failure.
It is proposed that administration of a potent dose of a
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated
chemotactic activity administered early or late after occurrence of
an AMI can result in a reduction in major adverse cardiac events,
including, but not limited to, premature death, recurrent
myocardial infarction, the development of congestive heart failure,
significant arrhythmias, and acute coronary syndrome, and the
worsening of congestive heart failure, significant arrhythmias, and
acute coronary syndrome.
SUMMARY OF THE INVENTION
[0044] The described invention provides progressive compositions
and methods to treat adverse consequences of a progressive
myocardial injury due to a vascular insufficiency. According to
some embodiments, the vascular insufficiency occurs early after an
acute myocardial infarction resulting from underlying disease.
According to some embodiments, the vascular insufficiency occurs
late after an acute myocardial infarction resulting from underlying
disease.
[0045] According to one aspect, the described invention provides a
method of treating a progressive myocardial injury due to a
vascular insufficiency, the method comprising the steps: (a)
acquiring a sterile nonexpanded, isolated population of autologous
mononuclear cells comprising CD34+ cells, which further contain a
subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity from a subject under sterile
conditions; (b) sterilely enriching the CD34+ cells which further
contain a subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity from the sterile nonexpanded,
isolated population of autologous mononuclear cells comprising
CD34+ cells, wherein the enriched CD34+ cells which further contain
a subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity are a chemotactic
hematopoietic stem cell product; (c) administering parenterally
through a catheter on a plurality of infusion dates during lifetime
of subject a sterile pharmaceutical composition, the sterile
pharmaceutical composition comprising: (i) a therapeutically
effective amount of the sterile chemotactic hematopoietic stem cell
product, wherein the therapeutically effective amount of the
chemotactic hematopoietic stem cell product comprises at least
10.times.10.sup.6 CD34+ cells which further contain a subpopulation
of at least 0.5.times.10.sup.6 potent CD34+ cells expressing CXCR-4
and having CXCR-4 mediated chemotactic activity; and (ii) a
stabilizing amount of serum, wherein the stabilizing amount of
serum is greater than 20% (v/v), wherein the chemotactic
hematopoietic stem cell product is further characterized as having
the following properties for at least 24 hours when tested in vitro
after passage through a catheter: (1) retains the CXCR-4-mediated
activity of the chemotactic hematopoietic stem cell product; (2) at
least 70% of the cells are CD34+ cells; (3) is at least 70% viable;
and (4) is able to form hematopoietic colonies in vitro; (d)
optionally administering the chemotactic hematopoietic stem cell
product at a plurality of infusion dates during the subject's
lifetime; and (e) treating at least one adverse consequence of the
progressive vascular insufficiency.
[0046] According to one embodiment, step (a) further comprises
freezing at least one aliquot of the nonexpanded, isolated
population of autologous mononuclear cells containing CD34+ cells,
which further contain a subpopulation of potent CD34+/CXCR-4+ cells
that have CXCR-4-mediated chemotactic activity at -86.degree. C.
and cryostoring the at least one aliquot in the vapor phase of a
liquid nitrogen freezer.
[0047] According to another embodiment, step (a) further comprises
(i) thawing the at least one aliquot of the frozen sterile
nonexpanded, isolated population of autologous mononuclear cells
containing CD34+ cells which further contain a subpopulation of
potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic
activity; (ii) enriching the sterile nonexpanded, isolated
population of autologous mononuclear cells for CD34+ cells, which
further contain a subpopulation of potent CD34+/CXCR-4+ cells that
have CXCR-4-mediated chemotactic activity, wherein the sterile
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic
activity, is a thawed sterile chemotactic hematopoietic stem cell
product; and (iii) administering to the subject on a second
infusion date a therapeutically effective amount of the sterile
thawed sterile chemotactic hematopoietic stem cell product,
comprising (a) at least 10.times.10.sup.6 CD34+ cells, which
further contain a subpopulation of at least 0.5.times.10.sup.6
potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated
chemotactic activity; and (b) a stabilizing amount of serum,
wherein the stabilizing amount of serum is greater than 20% (v/v),
wherein the sterile thawed chemotactic hematopoietic stem cell
product is further characterized as having the following properties
for at least 24 hours following thawing of the nonexpanded,
isolated population of autologous mononuclear cells comprising
CD34+ cells, which further contain a subpopulation of potent
CD34+/CXCR-4-+ cells that have CXCR-4-mediated chemotactic activity
when tested in vitro after passage through a catheter: (1) retains
the CXCR-4-mediated activity of the subpopulation of potent
CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity;
(2) at least 70% of the cells are CD34+ cells; (3) is at least 70%
viable; and (4) is able to form hematopoietic colonies in
vitro.
[0048] According to another embodiment, enriching step (ii) occurs
from at least 1 day to at least 40 years after acquisition of the
sterile nonexpanded, isolated population of autologous mononuclear
cells comprising CD34+ cells from the subject.
[0049] According to another embodiment, the sterile chemotactic
hematopoietic stem cell product is administered parenterally
through a catheter to the subject within about 48 hours to about 72
hours of thawing step (i).
[0050] According to another embodiment, the nonexpanded, isolated
population of autologous mononuclear cells comprising CD34+ cells,
which further contain a subpopulation of CD34+/CXCR-4+ cells that
have CXCR-4-mediated chemotactic activity, is acquired early after
an acute myocardial infarction.
[0051] According to another embodiment, the nonexpanded, isolated
population of autologous mononuclear cells comprising CD34+ cells,
which further contain a subpopulation of CD34+/CXCR-4+ cells that
have CXCR-4-mediated chemotactic activity, is acquired after peak
inflammatory cytokine cascade production in an infarcted area.
[0052] According to another embodiment, the nonexpanded, isolated
population of autologous mononuclear cells comprising CD34+ cells,
which further contain a subpopulation of CD34+/CXCR-4+ cells that
have CXCR-4-mediated chemotactic activity, is acquired late after
an acute myocardial infarction.
[0053] According to another embodiment, the nonexpanded, isolated
population of autologous mononuclear cells comprising CD34+ cells,
which further contain a subpopulation of CD34+/CXCR-4+ cells that
have CXCR-4-mediated chemotactic activity, is acquired from at
least 15 days to at least 40 years after occurrence of an acute
myocardial infarction.
[0054] According to another embodiment, the sterile thawed
chemotactic hematopoietic stem cell product is further
characterized as having the following properties for at least 48
hours following thawing of the nonexpanded, isolated population of
autologous mononuclear cells when tested in vitro after passage
through a catheter: (i) is able to form hematopoietic colonies; and
(ii) retains at least 2% of the CXCR-4-mediated activity of the
subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity.
[0055] According to another embodiment, the sterile thawed
chemotactic hematopoietic stem cell product is further
characterized as having the following properties for at least 72
hours following thawing of the nonexpanded, isolated population of
autologous mononuclear cells when tested in vitro after passage
through a catheter: (i) is able to form hematopoietic colonies; and
(ii) retains at least 2% of the CXCR-4-mediated activity of the
subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity.
[0056] According to another embodiment, the vascular insufficiency
is an ischemia. According to another embodiment, the ischemia is a
myocardial ischemia. According to another embodiment, the ischemia
is a transient ischemia. According to another embodiment, the
ischemia is a chronic myocardial ischemia. According to another
embodiment, the vascular insufficiency is a vascular insufficiency
after an acute myocardial infarction resulting from underlying
disease. According to another embodiment, the ischemia is a
peri-infarct border zone ischemia.
[0057] According to another embodiment, a first infusion date
comprises a specific time interval defined by a first time and a
second time, wherein the first time is after peak inflammatory
cytokine cascade production in an infarcted area and the second
time is before myocardial scar formation in the infarcted area.
According to another embodiment, the first time of the first
infusion date is at least about 5 days post-infarction. According
to another embodiment, the first time of the first infusion date is
about 5 days post-infarction and the second time is about 14 days
post-infarction.
[0058] According to another embodiment, the method treats
cardiomyocyte cell death in the peri-infarct border zone, relative
to controls. According to another embodiment, the method treats
hypoperfusion in the peri-infarct border zone, relative to
controls. According to another embodiment, the method treats
myocardial hibernation in the peri-infarct border zone, relative to
controls. According to another embodiment, the method decreases
infarct area, relative to controls. According to another
embodiment, wherein the method decreases infarct mass, relative to
controls.
[0059] According to another embodiment, the progressive myocardial
injury is a progressive decline in heart muscle function following
the acute myocardial infarction.
[0060] According to another embodiment, step (c) comprises treating
at least one adverse consequence of an acute myocardial infarction
selected from premature death, recurrent myocardial infarction,
development of congestive heart failure, development of significant
arrhythmias, development of acute coronary syndrome, worsening of
congestive heart failure, worsening of significant arrhythmias, and
worsening of acute coronary syndrome.
[0061] According to another embodiment, the progressive myocardial
injury is heart failure.
[0062] According to another embodiment, the catheter is a flow
control catheter.
[0063] According to another embodiment, the catheter is a balloon
dilatation catheter.
[0064] According to another embodiment, the catheter has an
internal diameter of at least about 0.36 mm.
[0065] According to another embodiment, administering step (c) is
through the catheter into myocardium. According to another
embodiment, administering step (c) is through the catheter
intravascularly.
[0066] According to another embodiment, the pharmaceutical
composition further includes at least one compatible active agent.
According to another embodiment, the active agent is selected from
the group consisting of an angiotensin converting enzyme inhibitor,
a beta-blocker, a diuretic, an anti-arrhythmic agent, a
hematopoietic stem cell mobilizing agent, a tyrosine kinase
receptor agonist, an anti-anginal agent, a vasoactive agent, an
anticoagulant agent, a fibrinolytic agent, and a
hypercholesterolemic agent.
[0067] According to another embodiment, the tyrosine kinase
receptor agonist is human neuregulin 1.
[0068] According to another aspect, the described invention
provides a regimen for treating a progressive myocardial injury due
to a vascular insufficiency in a revascularized subject, which
comprises (a) administering parenterally through a catheter on a
plurality of infusion dates during lifetime of the subject a
sterile pharmaceutical composition comprising a sterile chemotactic
hematopoietic stem cell product, wherein the sterile chemotactic
hematopoietic stem cell product comprises (i) a nonexpanded,
isolated population of autologous mononuclear cells enriched for
CD34+ cells, which further contain a subpopulation of potent
CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity,
wherein the therapeutically effective amount of the chemotactic
hematopoietic stem cell product comprises at least
10.times.10.sup.6 CD34+ cells which further contain a subpopulation
of at least 0.5.times.10.sup.6 potent CD34+ cells expressing CXCR-4
and having CXCR-4 mediated chemotactic activity; and (ii) a
stabilizing amount of serum, wherein the stabilizing amount of
serum is greater than 20% (v/v), wherein the chemotactic
hematopoietic stem cell product is further characterized as having
the following properties for at least 24 hours following
acquisition of the chemotactic hematopoietic stem cell product when
tested in vitro after passage through a catheter: (1) retains the
CXCR-4-mediated activity of the subpopulation of potent
CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity;
(2) at least 70% of the cells are CD34+ cells; (3) is at least 70%
viable; and (4) is able to form hematopoietic colonies in vitro;
and (b) treating at least one adverse consequence of the
progressive vascular insufficiency.
[0069] According to one embodiment, the vascular insufficiency is
an ischemia. According to another embodiment, the ischemia is a
myocardial ischemia. According to another embodiment, the ischemia
is a transient ischemia. According to another embodiment, the
ischemia is a chronic myocardial ischemia. According to another
embodiment, the vascular insufficiency is a vascular insufficiency
after an acute myocardial infarction resulting from underlying
disease.
[0070] According to another embodiment, the nonexpanded, isolated
population of autologous mononuclear cells comprising CD34+ cells,
which further contain a subpopulation of CD34+/CXCR-4+ cells that
have CXCR-4-mediated chemotactic activity, is acquired early after
occurrence of an acute myocardial infarction.
[0071] According to another embodiment, the nonexpanded, isolated
population of autologous mononuclear cells comprising CD34+ cells,
which further contain a subpopulation of CD34+/CXCR-4+ cells that
have CXCR-4-mediated chemotactic activity, is acquired after peak
inflammatory cytokine cascade production in an infarcted area.
[0072] According to another embodiment, the nonexpanded, isolated
population of autologous mononuclear cells comprising CD34+ cells,
which further contain a subpopulation of CD34+/CXCR-4+ cells that
have CXCR-4-mediated chemotactic activity, is acquired late after
occurrence of an acute myocardial infarction.
[0073] According to another embodiment, the nonexpanded, isolated
population of autologous mononuclear cells comprising CD34+ cells,
which further contain a subpopulation of CD34+/CXCR-4+ cells that
have CXCR-4-mediated chemotactic activity, is acquired from at
least 15 days to at least 40 years after occurrence of an acute
myocardial infarction.
[0074] According to another embodiment, a first infusion date
comprises a specific time interval defined by a first time and a
second time, and wherein the first time is after peak inflammatory
cytokine cascade production in an infarcted area and the second
time is before myocardial scar formation in the infarcted area.
[0075] According to another embodiment, the first time of the first
infusion date is at least about 5 days post-infarction. According
to another embodiment, the first time of the first infusion date is
about 5 days post-infarction and the second time is about 14 days
post-infarction. According to another embodiment, the first
infusion date is at least 5 days after occurrence of an acute
myocardial infarction. According to another embodiment, a second
infusion date is at least 30 days after occurrence of an acute
myocardial infarction.
[0076] According to another embodiment, the ischemia is a
peri-infarct border zone ischemia. According to another embodiment,
step (b) comprises treating cardiomyocyte cell death in the
peri-infarct border zone, relative to controls. According to
another embodiment, step (b) comprises treating hypoperfusion in
the peri-infarct border zone, relative to controls.
[0077] According to another embodiment, step (b) comprises treating
myocardial hibernation in the peri-infarct border zone, relative to
controls. According to another embodiment, step (b) comprises
decreasing infarct area, relative to controls. According to another
embodiment, step (b) comprises decreasing infarct mass, relative to
controls.
[0078] According to another embodiment, step (b) comprises treating
at least one adverse consequence of the acute myocardial infarction
selected from premature death, recurrent myocardial infarction,
development of congestive heart failure, development of significant
arrhythmias, development of acute coronary syndrome, worsening of
congestive heart failure, worsening of significant arrhythmias, and
worsening of acute coronary syndrome.
[0079] According to another embodiment, the progressive myocardial
injury is a progressive decline in heart muscle function following
the acute myocardial infarction. According to another embodiment,
the progressive myocardial injury is heart failure.
[0080] According to another embodiment, the catheter is a flow
control catheter. According to another embodiment, the catheter is
a balloon dilatation catheter. According to another embodiment, the
catheter has an internal diameter of at least about 0.36 mm.
[0081] According to another embodiment, the composition is
administered through the catheter into myocardium. According to
another embodiment, the composition is administered through the
catheter intravascularly.
[0082] According to another embodiment, the pharmaceutical
composition further includes at least one compatible active agent.
According to another embodiment, the active agent is selected from
the group consisting of an angiotensin converting enzyme inhibitor,
a beta-blocker, a diuretic, an anti-arrhythmic agent, a
hematopoietic stem cell mobilizing agent, a tyrosine kinase
receptor agonist, an anti-anginal agent, a vasoactive agent, an
anticoagulant agent, a fibrinolytic agent, and a
hypercholesterolemic agent.
[0083] According to another embodiment, the tyrosine kinase
receptor agonist is human neuregulin 1.
BRIEF DESCRIPTION OF THE FIGURES
[0084] FIG. 1 shows that the functional viability of the
chemotactic hematopoietic cell product of the invention at 72 hours
is equivalent to that at 48 hours.
[0085] FIG. 2 shows the migratory efficiency of the formulated
chemotactic hematopoietic stem cell product comprising CD34+ cells
of the invention.
[0086] FIG. 3 shows the improved stability of CD34+ cells
formulated in human serum.
DETAILED DESCRIPTION
[0087] The present invention describes compositions and methods for
preventing early or late adverse consequences of vascular
insufficiency, including, but not limited to, vascular
insufficiency that occurs early or late after an acute myocardial
infarction resulting from underlying disease.
GLOSSARY
[0088] The term "administer" and its various grammatical forms as
used herein means to give or to apply. The term "administering" as
used herein includes in vivo administration, as well as
administration directly to tissue ex vivo. Generally, compositions
may be administered systemically either parenterally or topically
in dosage unit formulations containing conventional nontoxic
pharmaceutically acceptable carriers, adjuvants, and vehicles as
desired, or may be locally administered by means such as, but not
limited to, injection, implantation, grafting, topical application,
or parenterally. A means of administering cells may include, but is
not limited to, infusion.
[0089] As used herein, the term "aliquot" refers to a portion of a
total amount.
[0090] As used herein, the term "angiogenesis" refers to the
process of formation and development of blood vessels.
[0091] The terms "apoptosis" or "programmed cell death" refer to a
highly regulated and active process that contributes to biologic
homeostasis comprised of a series of biochemical events that lead
to a variety of morphological changes, including blebbing, changes
to the cell membrane, such as loss of membrane asymmetry and
attachment, cell shrinkage, nuclear fragmentation, chromatin
condensation, and chromosomal DNA fragmentation, without damaging
the organism.
[0092] The term "c-kit" refers to a protein on the surface of some
cells that binds to stem cell factor (a substance that causes
certain types of cells to grow). Altered forms of this receptor may
be associated with some types of cancer.
[0093] The term "cardiac biomarkers" refers to enzymes, proteins
and hormones associated with heart function, damage or failure that
are used for diagnostic and prognostic purposes. Different
biomarkers have different times that their levels rise, peak, and
fall within the body, allowing them to be used not only to track
the progress of a heart attack but to estimate when it began and to
monitor for recurrence. Some of the tests are specific for the
heart while others also are elevated by skeletal muscle damage.
Current cardiac biomarkers include, but are not limited to CK
(creatine phosphokinase or creatine kinase) and CK-MB (creatine
kinase-myoglobin levels (to help distinguish between skeletal and
heart muscle)), troponin (blood levels of troponin 1 or T will
remain high for 1-2 weeks after a heart attack; troponin generally
is not affected by damage to other muscles), myoglobin (to
determine whether muscle, particularly heart muscle, has been
injured), and BNP (brain natriuretic peptide) or NT-proBNP
(N-terminal prohormone brain natriuretic peptide (to help diagnose
heart failure and grade the severity of that heart Failure).
[0094] The term "cardiac catheterization" refers to a procedure in
which a catheter is passed through an artery to the heart, and into
a coronary artery. This procedure produces angiograms (i.e., x-ray
images) of the coronary arteries and the left ventricle, the
heart's main pumping chamber, which can be used to measure
pressures in the pulmonary artery, and to monitor heart
function.
[0095] The term "CD34+ cells" as used herein refers to
hematopoietic stem and progenitor cells derived from human bone
marrow that "are positive for" i.e., "express", a hematopoietic
stem cell antigen, at least a subpopulation of which express
CXCR-4, and that can migrate to areas of injury. The chemotactic
hematopoietic stem cell product of the described invention that is
enriched for CD34+ cells does not co-express VEGF-2 (<1%).
[0096] The term "CD38" refers to a protein marker present on
macrophages, dendritic cells, and activated B and NK cells, which
may mediate the adhesion between lymphocytes and endothelial
cells.
[0097] The terms "CD45" and "common leukocyte antigen" refer to a
protein tyrosine phosphatase (PTP) located in hematopoietic cells
except erythrocytes and platelets.
[0098] The term "CD59" refers to a glycosylphosphatidylinositol
(GPI)-linked membrane glycoprotein which protects human cells from
complement-mediated lysis.
[0099] The term "CXCR-4" as used herein refers to a
G-protein-linked chemokine receptor.
[0100] The term "cytokine" as used herein refers to small soluble
protein substances secreted by cells which have a variety of
effects on other cells. Cytokines mediate many important
physiological functions including growth, development, wound
healing, and the immune response. They act by binding to their
cell-specific receptors located in the cell membrane, which allows
a distinct signal transduction cascade to start in the cell, which
eventually will lead to biochemical and phenotypic changes in
target cells. Generally, cytokines act locally. They include type I
cytokines, which encompass many of the interleukins, as well as
several hematopoietic growth factors; type II cytokines, including
the interferons and interleukin-10; tumor necrosis factor
("TNF")-related molecules, including TNF.alpha. and lymphotoxin;
immunoglobulin super-family members, including interleukin 1
("IL-1"); and the chemokines, a family of molecules that play a
critical role in a wide variety of immune and inflammatory
functions. The same cytokine can have different effects on a cell
depending on the state of the cell. Cytokines often regulate the
expression of, and trigger cascades of, other cytokines.
[0101] The term "colony stimulating factor" refers to a cytokine
responsible for controlling the production of white blood cells.
Types of colony stimulating factors include granulocyte colony
stimulating factor (G-CSF), macrophage colony stimulating factor
(M-CSF), and granulocyte macrophage colony stimulating factor
(GM-CSF).
[0102] The term "hematopoietic stem cell" refers to a cell isolated
from the blood or from the bone marrow that can renew itself,
differentiate to a variety of specialized cells, mobilize out of
the bone marrow into the circulating blood, and can undergo
programmed cell death (apoptosis). In some embodiments of the
described invention, hematopoietic stem cells derived from human
subjects express at least one type of cell surface marker,
including, but not limited to, CD34, CD38, HLA-DR, c-kit, CD59,
Sca-1, Thy-1, and/or CXCR-4, or a combination thereof.
[0103] "HLA-DR" refers to a human class II histocompatibility
antigen present on several cell types, including antigen-presenting
cells, B cells, monocytes, macrophages, and activated T cells.
[0104] The term "interleukin" as used herein refers to a cytokine
secreted by white blood cells as a means of communication with
other white blood cells.
[0105] The terms "VEGF-1" or "vascular endothelial growth factor-1"
are used interchangeably herein to refer to a cytokine that
mediates numerous functions of endothelial cells including
proliferation, migration, invasion, survival, and permeability.
VEGF is believed to be critical for angiogenesis.
[0106] The term "chemokine" as used herein refers to a class of
chemotactic cytokines that signal leukocytes to move in a specific
direction. The terms "chemotaxis" or "chemotactic" refer to the
directed motion of a motile cell or part along a chemical
concentration gradient towards environmental conditions it deems
attractive and/or away from surroundings it finds repellent.
[0107] The term "complete blood count" (CBC) refers to a laboratory
test that provides detailed information about the amount and the
quality of each of the blood cell types. It usually includes a
measurement of each of the three major blood cells (red blood
cells, white blood cells, and platelets) and a measure of the
hemoglobin and hematocrit. "Hemoglobin" (HGB) refers to the number
of grams of hemoglobin in a deciliter of blood (g/dL). Normal
hemoglobin levels in healthy adult human subjects are about 14 g/dL
to about 18 g/dL for men and about 12 g/dL to about 16 g/dL for
women. As a rough guideline, hemoglobin generally should be about
one-third the hematocrit. "Red Blood Cell Count" (RBC) refers to
the total number of red blood cells in a quantity of blood. Normal
ranges in human subjects are about 4.5 million cells/mm.sup.3 to
about 6.0 million cells/mm.sup.3 for men and about 4.0 million
cells/mm.sup.3 to about 5.5 million cells/mm.sup.3 for women.
"White Blood Cell Count" (WBC) refers to the total number of while
blood cells or leukocytes in a quantity of blood. Normal ranges in
human subjects are about 4.3.times.103 cells/mm3 to about
10.8.times.103 cells/mm3. "Hematocrit" (HCT) refers to the
proportion of red blood cells as a percentage of total blood
volume. A normal hematocrit for human subjects is about 40% to
about 55% for men and about 35% to about 45% for women.
[0108] The term "disease" or "disorder", as used herein, refers to
an impairment of health or a condition of abnormal functioning. The
term "syndrome," as used herein, refers to a pattern of symptoms
indicative of some disease or condition. The term "condition", as
used herein, refers to a variety of health states and is meant to
include disorders or diseases caused by any underlying mechanism or
disorder, injury, and the promotion of healthy tissues and
organs.
[0109] As used herein, the term "early" refers to being or
occurring at or near the beginning of a period of time or series of
events. As used herein, the term "late" refers to being or
occurring at an advanced period of time or stage of a series of
events.
[0110] As used herein, the term "enriching" or "purifying" refers
to increasing the fraction of cells of one type over the fraction
of that type in a starting preparation. Cells may be enriched using
any of the various markers expressed, or not expressed, on certain
cells in combination with suitable separation techniques. Suitable
separation techniques include, but are not limited to,
immunomagnetic bead separation, affinity chromatography, density
gradient centrifugation, and flow cytometry.
[0111] As used herein, the term "nonexpanded" refers to not being
increased or amplified in number of cells by in vitro culture.
[0112] As used herein, the term "inflammation" refers to a response
to infection and injury in which cells involved in detoxification
and repair are mobilized to the compromised site by inflammatory
mediators. Inflammation often is characterized by a strong
infiltration of leukocytes at the site of inflammation,
particularly neutrophils (polymorphonuclear cells). These cells
promote tissue damage by releasing toxic substances at the vascular
wall or in uninjured tissue.
[0113] Regardless of the initiating agent, the physiologic changes
accompanying acute inflammation encompass four main features: (1)
vasodilation, which results in a net increase in blood flow, is one
of the earliest s physical responses to acute tissue injury; (2) in
response to inflammatory stimuli, endothelial cells lining the
venules contract, widening the intracellular junctions to produce
gaps, leading to increased vascular permeability, which permits
leakage of plasma proteins and blood cells out of blood vessels;
(3) inflammation often is characterized by a strong infiltration of
leukocytes at the site of inflammation, particularly neutrophils
(polymorphonuclear cells). These cells promote tissue damage by
releasing-toxic substances at the vascular wall or in uninjured
tissue; and (4) fever, produced by pyrogens released from
leukocytes in response to specific stimuli.
[0114] During the inflammatory process, soluble inflammatory
mediators of the inflammatory response work together with cellular
components in a systemic fashion in the attempt to contain and
eliminate the agents causing physical distress. The terms
"inflammatory" or immuno-inflammatory" as used herein with respect
to mediators refers to the molecular mediators of the inflammatory
process. These soluble, diffusible molecules act both locally at
the site of tissue damage and infection and at more distant sites.
Some inflammatory mediators are activated by the inflammatory
process, while others are synthesized and/or released from cellular
sources in response to acute inflammation or by other soluble
inflammatory mediators. Examples of inflammatory mediators of the
inflammatory response include, but are not limited to, plasma
proteases, complement, kinins, clotting and fibrinolytic proteins,
lipid mediators, prostaglandins, leukotrienes, platelet-activating
factor (PAF), peptides and amines, including, but not limited to,
histamine, serotonin, and neuropeptides, proinflammatory cytokines,
including, but not limited to, interleukin-1, interleukin-4,
interleukin-6, interleukin-S, tumor necrosis factor (TNF),
interferon-gamma, and interleukin 12.
[0115] The term "in-date" refers to the time interval between
completion of acquiring a preparation comprising an enriched
population of potent CD34+ cells from a subject under sterile
conditions and initiating sterilely purifying potent CD34+ cells
from the preparation. The term "out-date" refers to the time
interval between completion of acquiring a preparation comprising
an enriched population of potent CD34+ cells from a subject under
sterile conditions and infusing the formulated pharmaceutical
composition comprising a chemotactic hematopoietic cell product
into the subject.
[0116] The terms "infuse" or "infusion" as used herein refer to the
introduction of a fluid other than blood into a blood vessel of a
subject, including humans, for therapeutic purposes.
[0117] The "infusion solution" of the described invention without
autologous serum contains phosphate buffered saline (PBS)
supplemented with 25 USP units/ml of heparin and 1% human serum
albumin (HSA). In some embodiments, the infusion solution is
supplemented with serum. In some embodiments, the serum is
autologous.
[0118] The term "injury" refers to damage or harm caused to the
structure or function of the body of a subject caused by an agent
or force, which may be physical or chemical. The term "vascular
injury" refers to injury to the vasculature (i.e., the vascular
network, meaning the network of blood vessels or ducts that convey
fluids, such as, without limitation, blood or lymph). The term
"myocardial injury" refers to injury to the myocardium of the
heart.
[0119] The term "macrophage" as used herein refers to a
mononuclear, actively phagocytic cell arising from monocytic stem
cells in the bone marrow. These cells are widely distributed in the
body and vary in morphology and motility. Phagocytic activity
typically is mediated by serum recognition factors, including
certain immunoglobulins and components of the complement system,
but also may be nonspecific. Macrophages also are involved in both
the production of antibodies and in cell-mediated immune responses,
particularly in presenting antigens to lymphocytes. They secrete a
variety of immunoregulatory molecules.
[0120] The terms "microbe" or "microorganism" are used
interchangeably herein to refer to an organism too small to be seen
clearly with the naked eye, including, but not limited to,
microscopic bacteria, fungi (molds), algae, protozoa, and
viruses.
[0121] The term "modulate" as used herein means to regulate, alter,
adapt, or adjust to a certain measure or proportion.
[0122] The term "myocardial infarction" refers to death or
permanent damage to heart muscle. Most heart attacks are caused by
blockage of coronary arteries that interrupts flow of blood and
oxygen to the heart muscle, leading to death of heart cells in that
area. The damaged heart muscle loses its ability to contract,
leaving the remaining heart muscle to compensate for the weakened
area. The described invention includes steps related to evaluating
the suitability of subjects for treatment according to the
described invention by using tests to look at the size, shape, and
function of the heart as it is beating, to detect changes to the
rhythm of the heart, and to detect and evaluate damaged tissues and
blocked arteries. Examples of such tests include, but are not
limited to, electrocardiography, echocardiography, coronary
angiography, and nuclear ventriculography. Cardiac biomarkers also
are used to evaluate the suitability of subjects for treatment
according to the described invention.
[0123] The term "necrosis" refers to the premature death of cells
and living tissue induced by external factors, such as infection,
toxins or trauma. Necrotic tissue undergoes chemical reactions
different from those of apoptotic tissue. Necrosis typically begins
with cell swelling, chromatin digestion, disruption of the plasma
membrane and of organelle membranes. Damage to the lysosome
membrane can trigger release of lysosomal enzymes, destroying other
parts of the cell. Late necrosis is characterized by extensive DNA
hydrolysis, vacuolation of the endoplasmic reticulum, organelle
breakdown and cell lysis. The release of intracellular content
after plasma membrane rupture is the cause of inflammation in
necrosis. Released lysosomal enzymes can trigger a chain reaction
of further cell death. Necrosis of a sufficient amount of
contiguous tissue can result in tissue death or gangrene.
[0124] The term "perfusion" as used herein refers to the process of
nutritive delivery of arterial blood to a capillary bed in
biological tissue. Perfusion ("F") can be calculated with the
formula F=((PA-Pv)/R) wherein PA is mean arterial pressure; Pv is
mean venous pressure, and R is vascular resistance. Tissue
perfusion can be measured in vivo, by, for example, but not limited
to, magnetic resonance imaging (MRI) techniques. Such techniques
include using an injected contrast agent and arterial spin labeling
(ASL) (wherein arterial blood is magnetically tagged before it
enters into the tissue of interest and the amount of labeling is
measured and compared to a control recording).
[0125] The term "persisting" as used herein refers to that which is
never-ceasing or indefinitely continuous.
[0126] As used herein, the term "potent" or "potency" refers to the
necessary biological activity of the chemotactic hematopoietic stem
cell product of the described invention, i.e., potent cells of the
described invention remain viable, are capable of mediated
mobility, and are able to grow, i.e., to form hematopoietic
colonies in an in vitro CFU assay.
[0127] The term "progenitor cell" as used herein refers to an
immature cell in the bone marrow that may be isolated by growing
suspensions of marrow cells in culture dishes with added growth
factors. Progenitor cells mature into precursor cells that mature
into blood cells. Progenitor cells are referred to as
colony-farming units (CFU) or colony-forming cells (CFC). The
specific lineage of a progenitor cell is indicated by a suffix,
such as, but not limited to, CFU-E (erythrocytic), CFU-GM
(granulocytic/macrophage), and CFU-GEMM (pluripotent hematopoietic
progenitor).
[0128] The term "progressive" as used herein refers to that which
gradually advances in extent.
[0129] The term "repair" as used herein as a noun refers to any
correction, reinforcement, reconditioning, remedy, making up for,
making sound, renewal, mending, patching, or the like that restores
function. When used as a verb, it means to correct, to reinforce,
to recondition, to remedy, to make up for, to make sound, to renew,
to mend, to patch or to otherwise restore function. In some
embodiments "repair" includes full repair and partial repair.
[0130] The term "reverse" as used herein refers to a change to the
contrary, or to a turning backward in nature or effect.
[0131] The term "Sca-1" or "stem cell antigen-1" refers to a
surface protein component in a signaling pathway that affects the
self-renewal ability of mesenchymal stem cells.
[0132] The term "stem cells" refers to undifferentiated cells
having high proliferative potential with the ability to self-renew
that can generate daughter cells that can undergo terminal
differentiation into more than one distinct cell phenotype.
[0133] The term "stent" is used to refer to a small tube used to
prop open an artery. The stent is collapsed to a small diameter,
put over a balloon catheter, inserted through a main artery in the
groin (femoral artery) or arm (brachial artery) and threaded up to
the narrowed/blocked section of the artery. When it reaches the
right location, the balloon is inflated slightly to push any plaque
out of the way and to expand the artery (balloon angioplasty). When
the balloon is inflated, the stent expands, locks in place and
forms a scaffold to hold the artery open. The stent stays in the
artery permanently. In certain subjects, a stent reduces the
renarrowing that occurs after balloon angioplasty or other
procedures that use catheters. A stent also may help restore normal
blood flow and keep an artery open if it has been torn or injured
by the balloon catheter. Reclosure (restenosis) may be a problem
with the stent procedure. Drug-eluting stents are stents coated
with drugs that are slowly released. These drugs may help keep the
blood vessel from reclosing.
[0134] The terms "subject" and "Patients" are used interchangeably
herein and include animal species of mammalian origin, including
humans.
[0135] The term "Thy-1" refers to the Ig superfamily cell surface
glycoprotein Thy-1 expressed on immune cells and neurons of rodents
and humans, which is hypothesized to function in cell adhesion and
signal transduction in T cell differentiation, proliferation, and
apoptosis.
[0136] As used herein the terms "treat" or "treating" are used
interchangeably to include abrogating, substantially inhibiting,
slowing or reversing the progression of a condition, substantially
ameliorating clinical or aesthetical symptoms of a condition,
substantially preventing the appearance of clinical or aesthetical
symptoms of a condition, and protecting from harmful or annoying
stimuli. Treating further refers to accomplishing one or more of
the following: (a) reducing the severity of the disorder; (b)
limiting development of symptoms characteristic of the disorder(s)
being treated; (c) limiting worsening of symptoms characteristic of
the disorder(s) being treated; (d) limiting recurrence of the
disorder(s) in patients that have previously had the disorder(s);
and (e) limiting recurrence of symptoms in patients that were
previously asymptomatic for the disorder(s).
[0137] The term "vascular insufficiency" refers to insufficient
blood flow.
[0138] The described invention provides progressive myocardial
injury-preventing pharmaceutical compositions and methods to treat
or prevent a progressive myocardial injury due to a vascular
insufficiency that occurs early or late. The terms "formulation"
and "composition" are used interchangeably herein to refer to a
product of the described invention that comprises all active and
inert ingredients. The term "active" refers to the ingredient,
component or constituent of the compositions of the described
invention responsible for the intended therapeutic effect. The
terms "pharmaceutical formulation" or "pharmaceutical composition"
as used herein refer to a formulation or composition that is
employed to prevent, reduce in intensity, cure or otherwise treat a
target condition or disease.
[0139] In one aspect of the described invention, the hematopoietic
stem cells of the described invention can migrate, meaning that
they can move from one place, location or area to another. In one
embodiment, hematopoietic stem cell migration is driven by CXCR-4
chemotaxis.
Compositions
[0140] The progressive myocardial injury-preventing pharmaceutical
composition of the described invention comprises a chemotactic
hematopoietic stem cell product, the chemotactic hematopoietic stem
cell product comprising a nonexpanded, isolated population of
autologous mononuclear cells enriched for CD34+ cells, which
further contain a subpopulation of potent CD34+CXCR-4+ cells that
have chemotactic activity. In some embodiments, this chemotactic
activity is mediated by SDF-1, and/or CXCR-4. According to some
embodiments, the chemotactic hematopoietic stem cell product is
prepared by isolating or purifying CD34+ hematopoietic stem cells
from bone marrow, umbilical cord blood, peripheral blood, mobilized
peripheral blood, umbilical cord, or adipose tissue harvested from
the subject. According to some embodiments, the chemotactic
hematopoietic stem cell product is prepared by isolating or
purifying CD34+ hematopoietic stem cells from mobilized peripheral
blood. Treatment with hematopoietic growth factors has been shown
to cause a marked rise in the number of hematopoietic progenitor
cells in the peripheral blood as measured by the presence of CD34+
cells or as measured in a colony formation assay as CFUs. Such
mobilized-peripheral blood hematopoietic stem cells (HSCs) have
been used for transplantation, immunotherapy, and cardiovascular
regenerative medicine. Colony stimulating factors, for example, are
agents used for hematopoietic stem cell mobilization. Examples of
colony stimulating factors include, without limitation, G-CSF,
GM-CSF, and pharmaceutically acceptable analogs and derivatives
thereof. For example, filgrastim, a G-CSF analog produced by
recombinant technology, is marketed under the brand names
Neupogen.RTM. (Amgen); Religrast.RTM. (Reliance Life Sciences),
Nugraf.RTM. (Zenotech Laboratories, Ltd., and Neukine.RTM. (Intas
Biopharmaceuticals).
[0141] According to some embodiments, the nonexpanded, isolated
population of autologous mononuclear cells comprising CD34+ cells
which further contain a subpopulation of potent CD34+/CXCR-4+ cells
that have CXCR-4-mediated chemotactic activity can be acquired from
the subject at any time. According to some embodiments, the
nonexpanded, isolated population of autologous mononuclear cells is
acquired early after an AMI. According to some such embodiments,
the nonexpanded, isolated population of autologous mononuclear
cells is acquired 4 days, 5 days, 6 days, 7 days, 8 days, 9 days,
10 days, 11 days. 12 days, 13 days, 14 days or more after the
occurrence of an AMI. According to some embodiments, the
nonexpanded, isolated population of autologous mononuclear cells
comprising CD34+ cells which further contain a subpopulation of
potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic
activity is acquired late after the occurrence of an AMI. According
to some such embodiments, the nonexpanded, isolated population of
autologous mononuclear cells is acquired at least 15 days, at least
16 days, at least 17 days, at least 18 days, at least 19 days, at
least 20 days, at least 21 days, at least 22 days, at least 23
days, at least 24 days, at least 25 days, at least 26 days, at
least 27 days, at least 28 days, at least 29 days, at least 30
days, at least 60 days, at least 90 days, at least 120 days, at
least 150 days, at least 180 days, or more from the AMI. According
to some embodiments, the nonexpanded, isolated population of
autologous mononuclear cells comprising CD34+ cells which further
contain a subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity is acquired at least 1 month,
at least 2 months, at least 3 months, at least 4 months, at least 5
months, at least 6 months, at least 7 months, at least 8 months, at
least 9 months, at least 10 months, at least 11 months, at least 12
months, at least 16 months, at least 24 months, at least 30 months,
at least 36 months, at least 42 months, at least 48 months, at
least 54 months, at least 60 months, at least 66 months, at least
72 months, at least 78 months, at least 84 months, at least 90
months, at least 96 months, at least 102 months, at least 108
months, at least 114 months, at least 120 months, at least 126
months, at least 132 months, at least 138 months, at least 144
months, at least 150 months, at least 156 months, at least 162
months, at least 168 months, at least 174 months, at least 180
months, at least 186 months, at least 192 months, at least 198
months, at least 204 months, at least 210 months, at least 216
months, at least 222 months, at least 228 months, at least 234
months, at least 240 months or more after occurrence of an AMI.
According to some such embodiments, the nonexpanded, isolated
population of autologous mononuclear cells comprising CD34+ cells
which further contain a subpopulation of potent CD34+/CXCR-4+ cells
that have CXCR-4-mediated chemotactic activity is acquired at least
3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10
years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years
17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23
years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years,
30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36
years 37 years, 38 years, 39 years, 40 years or more after
occurrence of an AMI. According to some embodiments, the
nonexpanded, isolated population of autologous mononuclear cells
comprising CD34+ cells which further contain a subpopulation of
potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic
activity, once acquired, is frozen at -86.degree. C. and cryostored
in the vapor phase of a liquid nitrogen freezer as a plurality of
aliquots for later usage.
[0142] According to the described invention, at least 70% of potent
cells in the nonexpanded, isolated population of autologous
mononuclear cells enriched for CD34+ cells, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
chemotactic activity are CD34+ cells. In some embodiments, at least
75% of cells in the nonexpanded, isolated population of autologous
mononuclear cells enriched for CD34+ cells containing a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
chemotactic activity are CD34+ cells. In some embodiments, at least
80% of potent cells in the nonexpanded, isolated population of
autologous mononuclear cells enriched for CD34+ cells, which
further contain a subpopulation of potent CD34+ cells expressing
CXCR-4 and having chemotactic activity are CD34+ cells. In some
embodiments, at least 85% of potent cells in the nonexpanded,
isolated population of autologous mononuclear cells enriched for
CD34+ cells, which further contain a subpopulation of potent CD34+
cells expressing CXCR-4 and having chemotactic activity are CD34+
cells. In some embodiments, at least 90% of potent cells in the
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+ cells expressing CXCR-4 and having chemotactic
activity are CD34+ cells. In some embodiments, at least 95% of
potent cells in the nonexpanded, isolated population of autologous
mononuclear cells enriched for CD34+ cells, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
chemotactic activity are CD34+ cells.
[0143] According to another embodiment, at least about 70% of the
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+ cells expressing CXCR-4 and having chemotactic
activity are viable for at least about 24 hours following
acquisition of the chemotactic hematopoietic stem cell product.
According to another embodiment, at least about 75% of the
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+ cells expressing CXCR-4 and having chemotactic
activity are viable for at least about 24 hours following
acquisition of the chemotactic hematopoietic stem cell product.
According to another embodiment, at least about 80% of the
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+ cells expressing CXCR-4 and having chemotactic
activity are viable for at least 24 hours following acquisition of
the chemotactic hematopoietic stem cell product. According to
another embodiment, at least about 85% of the nonexpanded, isolated
population of autologous mononuclear cells enriched for CD34+
cells, which further contain a subpopulation of potent CD34+ cells
expressing CXCR-4 and having chemotactic activity are viable for at
least 24 hours following acquisition of the chemotactic
hematopoietic stem cell product. In some embodiments, at least
about 90% of the nonexpanded, isolated population of autologous
mononuclear cells enriched for CD34+ cells, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
chemotactic activity are viable for at least 24 hours following
acquisition of the chemotactic hematopoietic stem cell product. In
some embodiments, at least about 95% of the nonexpanded, isolated
population of autologous mononuclear cells enriched for CD34+
cells, which further contain a subpopulation of potent CD34+ cells
expressing CXCR-4 and having chemotactic activity are viable for at
least about 24 following acquisition of the chemotactic
hematopoietic stem cell product.
[0144] According to another embodiment, at least about 70% of the
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+ cells expressing CXCR-4 and having chemotactic
activity are viable for at least about 48 hours following
acquisition of the chemotactic hematopoietic stem cell product.
According to another embodiment, at least about 75% of the
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+ cells expressing CXCR-4 and having chemotactic
activity are viable for at least about 48 hours following
acquisition of the chemotactic hematopoietic stem cell product.
According to another embodiment, at least about 80% of the
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+ cells expressing CXCR-4 and having chemotactic
activity are viable for at least 48 hours following acquisition of
the chemotactic hematopoietic stem cell product. According to
another embodiment, at least about 85% of the nonexpanded, isolated
population of autologous mononuclear cells enriched for CD34+
cells, which further contain a subpopulation of potent CD34+ cells
expressing CXCR-4 and having chemotactic activity are viable for at
least 48 hours following acquisition of the chemotactic
hematopoietic stem cell product. In some embodiments, at least
about 90% of the nonexpanded, isolated population of autologous
mononuclear cells enriched for CD34+ cells containing a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
chemotactic activity are viable for at least 48 hours following
acquisition of the chemotactic hematopoietic stem cell product. In
some embodiments, at least about 95% of the nonexpanded, isolated
population of autologous mononuclear cells enriched for CD34+
cells, which further contain a subpopulation of potent CD34+ cells
expressing CXCR-4 and having chemotactic activity are viable for at
least about 48 following acquisition of the chemotactic
hematopoietic stem cell product.
[0145] According to another embodiment, at least about 70% of the
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+ cells expressing CXCR-4 and having chemotactic
activity are viable for at least about 72 hours following
acquisition of the chemotactic hematopoietic stem cell product.
According to another embodiment, at least about 75% of the
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+ cells expressing CXCR-4 and having chemotactic
activity are viable for at least about 72 hours following
acquisition of the chemotactic hematopoietic stem cell product.
According to another embodiment, at least about 80% of the
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+ cells expressing CXCR-4 and having chemotactic
activity are viable for at least 72 hours following acquisition of
the chemotactic hematopoietic stem cell product. According to
another embodiment, at least about 85% of the nonexpanded, isolated
population of autologous mononuclear cells enriched for CD34+
cells, which further contain a subpopulation of potent CD34+ cells
expressing CXCR-4 and having chemotactic activity are viable for at
least 72 hours following acquisition of the chemotactic
hematopoietic stem cell product. In some embodiments, at least
about 90% of the nonexpanded, isolated population of autologous
mononuclear cells enriched for CD34+ cells, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
chemotactic activity are viable for at least 72 hours following
acquisition of the chemotactic hematopoietic stem cell product. In
some embodiments, at least about 95% of the nonexpanded, isolated
population of autologous mononuclear cells enriched for CD34+
cells, which further contain a subpopulation of potent CD34+ cells
expressing CXCR-4 and having chemotactic activity are viable for at
least about 72 following acquisition of the chemotactic
hematopoietic stem cell product.
[0146] According to another embodiment, the nonexpanded, isolated
population of autologous mononuclear cells enriched for CD34+
cells, which further contain a subpopulation of potent CD34+ cells
having chemotactic activity can form hematopoietic colonies in
vitro for at least about 24 hours following acquisition from the
subject of the chemotactic hematopoietic stem cell product.
According to another embodiment, the nonexpanded, isolated
population of autologous mononuclear cells enriched for CD34+
cells, which further contain a subpopulation of potent CD34+ cells
expressing CXCR-4 and having chemotactic activity can form
hematopoietic colonies in vitro for at least about 48 hours
following acquisition from the subject of the chemotactic
hematopoietic stem cell product. According to another embodiment,
the nonexpanded, isolated population of autologous mononuclear
cells enriched for CD34+ cells, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
chemotactic activity can form hematopoietic colonies in vitro for
at least about 72 hours following acquisition from the subject of
the chemotactic hematopoietic stem cell product.
[0147] According to another embodiment, the progressive myocardial
injury-preventing composition further comprises at least about 10
million isolated CD34+ cells acquired from the subject, which
further contain a subpopulation of potent CD34+ cells expressing
CXCR-4 and having CXCR-4-mediated chemotactic activity. According
to another embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 11 million isolated
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity. According to another
embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 12 million isolated
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity. According to another
embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 13 million isolated
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity. According to another
embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 14 million isolated
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity. According to another
embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 15 million isolated.
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity. According to another
embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 20 million isolated
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity. According to another
embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 30 million isolated
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity. According to another
embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 40 million isolated
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity. According to another
embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 50 million isolated
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity. According to another
embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 60 million isolated
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity. According to another
embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 70 million isolated
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity. According to another
embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 80 million isolated
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity. According to another
embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 90 million isolated
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity. According to another
embodiment, the progressive myocardial injury-preventing
composition further comprises at least about 100 million isolated
CD34+ cells acquired from the subject, which further contain a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity.
[0148] For use in the present invention, CD34+ cells may be
enriched/selected by any techniques known to the skilled artisan.
For example, in some embodiments, the isolated population of
autologous mononuclear cells comprising CD34+ cells is enriched for
cells expressing CD34 cell antigen and CXCR-4 cell antigen by
fluorescence activated cell sorting (FACS). In some embodiments,
the isolated population of autologous mononuclear cells comprising
CD34+ cells are enriched/selected by positive or negative
immunoseparation techniques. In some embodiments, isolation and/or
purification of hematopoietic stem cells from the isolated
population of autologous mononuclear cells comprising CD34+ cells
is based on cell fractionation methods based on size and cell
density, efflux of metabolic dyes, or resistance to cytotoxic
agents. In one embodiment, for example, the isolated population of
autologous mononuclear cells comprising CD34+ cells in is enriched
for CD34+ cells/selected using a monoclonal anti-CD34 antibody and
an immunomagnetic separation technique.
[0149] The isolated CD34+ cells may be identified, quantified and
characterized by techniques known in the art. For example, in some
embodiments, the percentage of CD34+ cells in the isolated
population of autologous mononuclear cells comprising CD34+ cells
and in the chemotactic hematopoietic stem cell product can be
determined by FACS analysis. According to another embodiment, CD34
protein expression is quantified by Western blot. The term "Western
blot" refers to a method for identifying proteins in a complex
mixture; proteins are separated electrophoretically in a gel
medium; transferred from the gel to a protein binding sheet or
membrane; and the sheet or membrane containing the separated
proteins exposed to specific antibodies which bind to locate, and
enable visualization of protein(s) of interest. For example,
monoclonal anti-CD34 antibody can be used to detect CD34 protein
adhered to a membrane in situ.
[0150] According to another embodiment, the expression of CD34 mRNA
and DNA in the isolated CD34+ cells may be quantified. The term
"Northern blot" as used herein refers to a technique in which RNA
from a specimen is separated into its component parts on a gel by
electrophoresis and transferred to a specifically modified paper
support so that the mRNA is fixed in its electrophoretic positions.
CD34 related sequences are identified using probes comprising a
reporter molecule, such as, without limitation, a radioactive
label. According to another embodiment, the level of CD34 and/or
CXCR-4 expression is/are determined by quantitative or
semi-quantitative PCR or real time PCR ("RT-PCR") techniques. The
abbreviation "PCR" refers to polymerase chain reaction, which is a
technique for amplifying the quantity of DNA, thus making the DNA
easier to isolate, clone and sequence. See, e.g., U.S. Pat. Nos.
5,656,493, 5,333,675, 5,234,824, and 5,187,083, each of which is
incorporated herein by reference. Real-time PCR is a method of
simultaneous DNA quantification and amplification, whereby DNA is
specifically amplified by polymerase chain reaction (PCR), and
after each round of amplification, the DNA is quantified.
[0151] According to another embodiment, the isolated CD34+
hematopoietic stem cells of the chemotactic hematopoietic stem cell
product of the described invention contain a subpopulation of CD34+
cells expressing CXCR-4 and having CXCR-4 mediated chemotactic
activity. According to another embodiment, the hematopoietic stem
cell product of the described invention comprises a minimum number
of isolated CD34+ hematopoietic stem cells such that a
subpopulation of at least 0.5.times.106 CD34+ cells expressing
CXCR-4 and having CXCR-4 mediated chemotactic activity is present.
According to another embodiment, at least about 2% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 3% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 4% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 5% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 6% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 7% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 8% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 9% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 10% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 11% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 12% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 13% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 14% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 15% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 16% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 17% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 18% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 19% of the
CXCR-4 Mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 20% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 21% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 22% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 23% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 24% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 25% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 26% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 27% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 28% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 29% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 30% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 31% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 32% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 33% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 34% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 24 hours
following acquisition of the chemotactic hematopoietic stem cell
product.
[0152] According to another embodiment, at least about 2% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 3% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 4% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 5% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 6% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 7% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 8% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 9% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 10% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 11% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 12% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 13% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 14% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 15% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 16% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 17% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 18% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 19% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 20% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 21% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 22% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 23% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 24% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 25% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 26% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 27% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 28% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 29% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 30% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 31% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 32% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 33% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+ cells.
According to another embodiment, at least about 34% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 48 hours
following acquisition of the enriched population of CD34+
cells.
[0153] According to another embodiment, at least about 2% of the
CXCR-4 mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 72 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 3% of the
CXCR-4 mediated chemotactic activity of the isolated CD34.sup.+
cells containing a subpopulation of potent CD34+ cells expressing
CXCR-4 and having chemotactic activity is retained for at least 72
hours following acquisition of the chemotactic hematopoietic stem
cell product. According to another embodiment, at least about 4% of
the CXCR-4 mediated chemotactic activity of the isolated CD34.sup.+
cells containing a subpopulation of potent CD34+ cells expressing
CXCR-4 and having chemotactic activity is retained for at least 72
hours following acquisition of the chemotactic hematopoietic stem
cell product. According to another embodiment, at least about 5% of
the CXCR-4 mediated chemotactic activity of the isolated CD34.sup.+
cells containing a subpopulation of potent CD34+ cells expressing
CXCR-4 and having chemotactic activity is retained for at least 72
hours following acquisition of the chemotactic hematopoietic stem
cell product. According to another embodiment, at least about 6% of
the CXCR-4 mediated chemotactic activity of the isolated CD34.sup.+
cells containing a subpopulation of potent CD34+ cells expressing
CXCR-4 and having chemotactic activity is retained for at least 72
hours following acquisition of the chemotactic hematopoietic stem
cell product. According to another embodiment, at least about 7% of
the CXCR-4 mediated chemotactic activity of the isolated CD34.sup.+
cells containing a subpopulation of potent CD34+ cells expressing
CXCR-4 and having chemotactic activity is retained for at least 72
hours following acquisition of the chemotactic hematopoietic stem
cell product. According to another embodiment, at least about 8% of
the CXCR-4 mediated chemotactic activity of the isolated CD34.sup.+
cells containing a subpopulation of potent CD34+ cells expressing
CXCR-4 and having chemotactic activity is retained for at least 72
hours following acquisition of the chemotactic hematopoietic stem
cell product. According to another embodiment, at least about 9% of
the CXCR-4 mediated chemotactic activity of the isolated CD34.sup.+
cells containing a subpopulation of potent CD34+ cells expressing
CXCR-4 and having chemotactic activity is retained for at least 72
hours following acquisition of the chemotactic hematopoietic stem
cell product. According to another embodiment, at least about 10%
of the CXCR-4 mediated chemotactic activity of the isolated
CD34.sup.+ cells containing a subpopulation of potent CD34+ cells
expressing CXCR-4 and having chemotactic activity is retained for
at least 72 hours following acquisition of the chemotactic
hematopoietic stem cell product. According to another embodiment,
at least about 11% of the CXCR-4 mediated chemotactic activity of
the isolated CD34.sup.+ cells containing a subpopulation of potent
CD34+ cells expressing CXCR-4 and having chemotactic activity is
retained for at least 72 hours following acquisition of the
chemotactic hematopoietic stem cell product. According to another
embodiment, at least about 12% of the CXCR-4 mediated chemotactic
activity of the isolated CD34.sup.+ cells containing a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
chemotactic activity is retained for at least 72 hours following
acquisition of the chemotactic hematopoietic stem cell product.
According to another embodiment, at least about 13% of the CXCR-4
mediated chemotactic activity of the isolated CD34.sup.+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 72 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 14% of the
CXCR-4 mediated chemotactic activity of the isolated CD34.sup.+
cells containing a subpopulation of potent CD34+ cells expressing
CXCR-4 and having chemotactic activity is retained for at least 72
hours following acquisition of the chemotactic hematopoietic stem
cell product. According to another embodiment, at least about 15%
of the CXCR-4 mediated chemotactic activity of the isolated
CD34.sup.+ cells containing a subpopulation of potent CD34+ cells
expressing CXCR-4 and having chemotactic activity is retained for
at least 72 hours following acquisition of the chemotactic
hematopoietic stem cell product. According to another embodiment,
at least about 16% of the CXCR-4 mediated chemotactic activity of
the isolated CD34.sup.+ cells containing a subpopulation of potent
CD34+ cells expressing CXCR-4 and having chemotactic activity is
retained for at least 72 hours following acquisition of the
chemotactic hematopoietic stem cell product. According to another
embodiment, at least about 17% of the CXCR-4 mediated chemotactic
activity of the isolated CD34+ cells containing a subpopulation of
potent CD34+ cells expressing CXCR-4 and having chemotactic
activity is retained for at least 72 hours following acquisition of
the chemotactic hematopoietic stem cell product. According to
another embodiment, at least about 18% of the CXCR-4 mediated
chemotactic activity of the isolated CD34.sup.+ cells containing a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
chemotactic activity is retained for at least 72 hours following
acquisition of the chemotactic hematopoietic stem cell product.
According to another embodiment, at least about 19% of the CXCR-4
mediated chemotactic activity of the isolated CD34.sup.+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 72 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 20% of the
CXCR-4 mediated chemotactic activity of the isolated CD34.sup.+
cells containing a subpopulation of potent CD34+ cells expressing
CXCR-4 and having chemotactic activity is retained for at least 72
hours following acquisition of the chemotactic hematopoietic stem
cell product. According to another embodiment, at least about 21%
of the CXCR-4 mediated chemotactic activity of the isolated
CD34.sup.+ cells containing a subpopulation of potent CD34+ cells
expressing CXCR-4 and having chemotactic activity is retained for
at least 72 hours following acquisition of the chemotactic
hematopoietic stem cell product. According to another embodiment,
at least about 22% of the CXCR-4 mediated chemotactic activity of
the isolated CD34.sup.+ cells containing a subpopulation of potent
CD34+ cells expressing CXCR-4 and having chemotactic activity is
retained for at least 72 hours following acquisition of the
chemotactic hematopoietic stem cell product. According to another
embodiment, at least about 23% of the CXCR-4 mediated chemotactic
activity of the isolated CD34.sup.+ cells containing a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
chemotactic activity is retained for at least 72 hours following
acquisition of the chemotactic hematopoietic stem cell product.
According to another embodiment, at least about 24% of the CXCR-4
mediated chemotactic activity of the isolated CD34.sup.+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 72 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 25% of the
CXCR-4 mediated chemotactic activity of the isolated CD34.sup.+
cells containing a subpopulation of potent CD34 cells expressing
CXCR-4 and having chemotactic activity is retained for at least 72
hours following acquisition of the chemotactic hematopoietic stem
cell product. According to another embodiment, at least about 26%
of the CXCR-4 mediated chemotactic activity of the isolated
CD34.sup.+ cells containing a subpopulation of potent CD34+ cells
expressing CXCR-4 and having chemotactic activity is retained for
at least 72 hours following acquisition of the chemotactic
hematopoietic stem cell product. According to another embodiment,
at least about 27% of the CXCR-4 mediated chemotactic activity of
the isolated CD34.sup.+ cells containing a subpopulation of potent
CD34+ cells expressing CXCR-4 and having chemotactic activity is
retained for at least 72 hours following acquisition of the
chemotactic hematopoietic stem cell product. According to another
embodiment, at least about 28% of the CXCR-4 mediated chemotactic
activity of the isolated CD34.sup.+ cells containing a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
chemotactic activity is retained for at least 72 hours following
acquisition of the chemotactic hematopoietic stem cell product.
According to another embodiment, at least about 29% of the CXCR-4
mediated chemotactic activity of the isolated CD34.sup.+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 72 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, at least about 30% of the
CXCR-4 mediated chemotactic activity of the isolated CD34.sup.+
cells containing a subpopulation of potent CD34+ cells expressing
CXCR-4 and having chemotactic activity is retained for at least 72
hours following acquisition of the chemotactic hematopoietic stem
cell product. According to another embodiment, at least about 31%
of the CXCR-4 mediated chemotactic activity of the isolated
CD34.sup.+ cells containing a subpopulation of potent CD34+ cells
expressing CXCR-4 and having chemotactic activity is retained for
at least 72 hours following acquisition of the chemotactic
hematopoietic stem cell product. According to another embodiment,
at least about 32% of the CXCR-4 mediated chemotactic activity of
the isolated CD34.sup.+ cells containing a subpopulation of potent
CD34+ cells expressing CXCR-4 and having chemotactic activity is
retained for at least 72 hours following acquisition of the
chemotactic hematopoietic stem cell product. According to another
embodiment, at least about 33% of the CXCR-4 mediated chemotactic
activity of the isolated CD34.sup.+ cells containing a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
chemotactic activity is retained for at least 72 hours following
acquisition of the chemotactic hematopoietic stem cell product.
According to another embodiment, at least about 34% of the CXCR-4
mediated chemotactic activity of the isolated CD34.sup.+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 72 hours
following acquisition of the chemotactic hematopoietic stem cell
product.
[0154] According to another embodiment, at least an average of
about 17% of the CXCR-4 mediated chemotactic activity of the
isolated CD34+ cells containing a subpopulation of potent CD34+
cells expressing CXCR-4 and having chemotactic activity is retained
for at least 24 hours following acquisition of the chemotactic
hematopoietic stem cell product. According to another embodiment,
at least an average of about 17% of the CXCR-4 mediated chemotactic
activity of the isolated CD34+ cells containing a subpopulation of
potent CD34+ cells expressing CXCR-4 and having chemotactic
activity is retained for at least 48 hours following acquisition of
the chemotactic hematopoietic stem cell product. According to
another embodiment, at least an average of about 17% of the CXCR-4
mediated chemotactic activity of the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity is retained for at least 72 hours
following acquisition of the chemotactic hematopoietic stem cell
product. According to another embodiment, the isolated CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having chemotactic activity in the chemotactic hematopoietic
cell product retain at least about 2% of the CXCR-4 mediated
chemotactic activity for at least 72 hours following acquisition of
the chemotactic hematopoietic stem cell product.
[0155] According to another embodiment, the pharmaceutical
composition of the invention further comprises serum at a
concentration of at least 10% expressed as ml/100 cc final volume
of the progressive myocardial injury-preventing composition.
According to another embodiment, the pharmaceutical composition of
the invention further comprises serum at a concentration of at
least 11% expressed as ml/100 cc final volume of the progressive
myocardial injury-preventing composition. According to another
embodiment, the pharmaceutical composition of the invention further
comprises serum at a concentration of at least 12% expressed as
ml/100 cc final volume of the progressive myocardial
injury-preventing composition. According to another embodiment, the
pharmaceutical composition of the invention further comprises serum
at a concentration of at least 13% expressed as ml/100 cc final
volume of the progressive myocardial injury-preventing composition.
According to another embodiment, the pharmaceutical composition of
the invention further comprises serum at a concentration of at
least 14% expressed as ml/100 cc final volume of the progressive
myocardial injury-preventing composition. According to another
embodiment, the pharmaceutical composition of the invention further
comprises serum at a concentration of at least 15% expressed as
ml/100 cc final volume of the progressive myocardial
injury-preventing composition. According to another embodiment, the
pharmaceutical composition of the invention further comprises serum
at a concentration of at least 16% expressed as ml/100 cc final
volume of the progressive myocardial injury-preventing composition.
According to another embodiment, the pharmaceutical composition of
the invention further comprises serum at a concentration of at
least 17% expressed as ml/100 cc final volume of the progressive
myocardial injury-preventing composition. According to another
embodiment, the pharmaceutical composition of the invention further
comprises serum at a concentration of at least 18% expressed as
ml/100 cc final volume of the progressive myocardial
injury-preventing composition. According to another embodiment, the
pharmaceutical composition of the invention further comprises serum
at a concentration of at least 19% expressed as ml/100 cc final
volume of the progressive myocardial injury-preventing composition.
According to another embodiment, the pharmaceutical composition of
the invention further comprises serum at a concentration of at
least 20% expressed as ml/100 cc final volume of the progressive
myocardial injury-preventing composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 21% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 22% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 23% expressed as,
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 24% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 25% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 26% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 27% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 28% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 29% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 30% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 31% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 32% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 33% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 34% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 35% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 36% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 37% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 38% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 39% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 40% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 41% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 42% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 43% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 44% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 45% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 46% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 47% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 48% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 49% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 50% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 51% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 52% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 53% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 54% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 55% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 56% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 57% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 58% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 59% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 60% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 61% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 62% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 63% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 64% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 65% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 66% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the progressive myocardial injury-preventing composition
is at least about 67% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the minimum
concentration of serum present in the progressive myocardial
injury-preventing composition is at least about 68% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the minimum concentration of serum present in the
progressive myocardial injury-preventing composition is at least
about 69% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the minimum concentration of serum
present in the composition is at least about 70% expressed as
ml/100 cc final volume of the composition.
[0156] According to another embodiment, the serum is autologous.
According to another embodiment, the serum is a synthetic or
recombinant serum.
[0157] According to another embodiment, the maximum concentration
of serum present in the progressive myocardial injury-preventing
composition of the described invention is about 70% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the maximum concentration of serum present in the
progressive myocardial injury-preventing composition of the
described invention is about 69% expressed as ml/100 cc final
volume of the composition. According to another embodiment, the
maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 68% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
67% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 66% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 65% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
64% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 63% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 62% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
61% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 60% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 59% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
58% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 57% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 56% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
55% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 54% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 53% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
52% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 51% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 50% expressed as ml/100 cc final volume of the
composition.
[0158] According to another embodiment, the maximum concentration
of serum present in the progressive myocardial injury-preventing
composition of the described invention is about 49% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the maximum concentration of serum present in the
progressive myocardial injury-preventing composition of the
described invention is about 48% expressed as ml/100 cc final
volume of the composition. According to another embodiment, the
maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 47% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
46% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 45% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 44% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
43% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 42% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 41% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
40% expressed as ml/100 cc final volume of the composition.
[0159] According to another embodiment, the maximum concentration
of serum present in the progressive myocardial injury-preventing
composition of the described invention is about 39% expressed as
ml/100 cc final volume of the composition. According to another
embodiment, the maximum concentration of serum present in the
progressive myocardial injury-preventing composition of the
described invention is about 38% expressed as ml/100 cc final
volume of the composition. According to another embodiment, the
maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 37% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
36% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 35% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 34% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
33% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 32% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 31% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
30% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 29% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 28% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
27% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 26% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 25% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
24% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 23% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 22% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
21% expressed as ml/100 cc final volume of the composition.
According to another embodiment, the maximum concentration of serum
present in the progressive myocardial injury-preventing composition
of the described invention is about 20% expressed as ml/100 cc
final volume of the composition. According to another embodiment,
the maximum concentration of serum present in the progressive
myocardial injury-preventing composition of the described invention
is about 15% expressed as ml/100 cc final volume of the
composition. According to another embodiment, the maximum
concentration of serum present in the progressive myocardial
injury-preventing composition of the described invention is about
10% expressed as ml/100 cc final volume of the composition.
[0160] In some embodiments, the progressive myocardial
injury-preventing composition may be formulated with an excipient,
carrier or vehicle including, but not limited to, a solvent. The
terms "excipient", "carrier", or "vehicle" as used herein refers to
carrier materials suitable for formulation and administration of
the chemotactic hematopoietic stem cell product described herein.
Carriers and vehicles useful herein include any such materials know
in the art which are nontoxic and do not interact with other
components. As used herein the phrase "pharmaceutically acceptable
carrier" refers to any substantially non-toxic carrier useable for
formulation and administration of the composition of the described
invention in which the chemotactic hematopoietic stem cell product
of the described invention will remain stable and bioavailable.
[0161] The pharmaceutically acceptable carrier must be of
sufficiently high purity and of sufficiently low toxicity to render
it suitable for administration to the mammal being treated. It
further should maintain the stability and bioavailability of an
active agent. The pharmaceutically acceptable carrier can be liquid
or solid and is selected, with the planned manner of administration
in mind, to provide for the desired bulk, consistency, etc., when
combined with an active agent and other components of a given
composition. For example, the pharmaceutically acceptable carrier
may be, without limitation, a binding agent (e.g., pregelatinized
maize starch, polyvinylpyrrolidone or hydroxypropyl
methylcellulose, etc.), a filler (e.g., lactose and other sugars,
microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl
cellulose, polyacrylates, calcium hydrogen phosphate, etc.), a
lubricant (e.g., magnesium stearate, talc, silica, colloidal
silicon dioxide, stearic acid, metallic stearates, hydrogenated
vegetable oils, corn starch, polyethylene glycols, sodium benzoate,
sodium acetate, etc.), a disintegrant (e.g., starch, sodium starch
glycolate, etc.), or a wetting agent (e.g., sodium lauryl sulfate,
etc.). Other suitable pharmaceutically acceptable carriers for the
compositions of the described invention include, but are not
limited to, water, salt solutions, alcohols, polyethylene glycols,
gelatins, amyloses, magnesium stearates, talcs, silicic acids,
viscous paraffins, hydroxymethylcelluloses, polyvinylpyrrolidones
and the like. Such carrier solutions also can contain buffers,
diluents and other suitable additives. The term "buffer" as used
herein refers to a solution or liquid whose chemical makeup
neutralizes acids or bases without a significant change in pH.
Examples of buffers envisioned by the described invention include,
but are not limited to, Dulbecco's phosphate buffered saline (PBS),
Ringer's solution, 5% dextrose in water (D5W), and
normal/physiologic saline (0.9% NaCl). In some embodiments, the
infusion solution is isotonic to subject tissues. In some
embodiments, the infusion solution is hypertonic to subject
tissues. Compositions of the described invention that are for
parenteral administration may include pharmaceutically acceptable
carriers such as sterile aqueous solutions, non-aqueous solutions
in common solvents such as alcohols, or solutions in a liquid oil
base.
[0162] In some embodiments, the carrier of the progressive
myocardial injury-preventing composition of the described invention
may include a release agent such as a sustained release or delayed
release carrier. In such embodiments, the carrier may be any
material capable of sustained or delayed release of the active to
provide a more efficient administration, e.g., resulting in less
frequent and/or decreased dosage of the composition, improve ease
of handling, and extend or delay effects on diseases, disorders,
conditions, syndromes, and the like, being treated, prevented or
promoted. Non-limiting examples of such carriers include liposomes,
microsponges, microspheres, or microcapsules of natural and
synthetic polymers and the like. Liposomes may be formed from a
variety of phospholipids such as cholesterol, stearylamines or
phosphatidylcholines.
[0163] The progressive myocardial injury-preventing compositions of
the described invention may be administered parenterally in the
form of a sterile injectable aqueous or oleaginous suspension. The
term "parenteral" or "parenterally" as used herein refers to
introduction into the body by way of an injection (i.e.,
administration by injection), including, but not limited to,
infusion techniques. In some embodiments, the progressive
myocardial injury-preventing composition of the described invention
comprising a chemotactic hematopoietic stem cell product is
delivered to the subject by means of a balloon catheter adapted for
delivery of the fluid compositions (i.e., compositions capable of
flow) into a selected anatomical structure.
[0164] The sterile progressive myocardial injury-preventing
composition of the described invention may be a sterile solution or
suspension in a nontoxic parenterally acceptable diluent or
solvent. A solution generally is considered as a homogeneous
mixture of two or more substances; it is frequently, though not
necessarily, a liquid. In a solution, the molecules of the solute
(or dissolved substance) are uniformly distributed among those of
the solvent. A suspension is a dispersion (mixture) in which a
finely-divided species is combined with another species, with the
former being so finely divided and mixed that it does not rapidly
settle out. In everyday life, the most common suspensions are those
of solids in liquid water. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, and
isotonic sodium chloride (saline) solution. In some embodiments,
hypertonic solutions are employed. In addition, sterile, fixed oils
conventionally are employed as a solvent or suspending medium. For
parenteral application, suitable vehicles consist of solutions,
e.g., oily or aqueous solutions, as well as suspensions, emulsions,
or implants. Aqueous suspensions may contain substances, which
increase the viscosity of the suspension and include, for example,
sodium carboxymethyl cellulose, sorbitol and/or dextran.
[0165] Additional progressive myocardial injury-preventing
compositions of the described invention readily may be prepared
using technology, which is known in the art, such as described in
Remington's Pharmaceutical Sciences, 18th or 19th editions,
published by the Mack Publishing Company of Easton, Pa., which is
incorporated herein by reference.
[0166] As used herein the terms "therapeutically effective",
"myocardial injury preventing amount", "vascular insufficiency
repairing amount", "adverse consequence preventing amount", adverse
consequence-reversing amount", or "pharmaceutically effective
amount" refer to the amount of the compositions of the invention
that result in a therapeutic or beneficial effect following its
administration to a subject. The vascular insufficiency repairing,
myocardial injury repairing, therapeutic, adverse consequence
reversing or pharmaceutical effect may be curing, minimizing,
preventing or ameliorating a disease or disorder, or may have any
other vascular insufficiency-repairing, myocardial
injury-repairing, adverse consequence reversing, or pharmaceutical
beneficial effect. The concentration of the substance is selected
so as to exert its vascular insufficiency-repairing, myocardial
injury-repairing, adverse consequence reversing, therapeutic, or
pharmaceutical effect, but low enough to avoid significant side
effects within the scope and sound judgment of the physician. The
effective amount of the composition may vary with the age and
physical condition of the biological subject being treated, the
severity of the condition, the duration of the treatment, the
nature of concurrent therapy, the timing of the infusion, the
specific compound, composition or other active ingredient employed,
the particular carrier utilized, and like factors.
[0167] A skilled artisan may determine a pharmaceutically effective
amount of the inventive compositions by determining the dose in a
dosage unit (meaning unit of use) that elicits a given intensity of
effect, hereinafter referred to as the "unit dose." The term
"dose-intensity relationship" refers to the manner in which the
intensity of effect in an individual recipient relates to dose. The
intensity of effect generally designated is 50% of maximum
intensity. The corresponding dose is called the 50% effective dose
or individual ED50. The use of the term "individual" distinguishes
the ED50 based on the intensity of effect as used herein from the
median effective dose, also abbreviated ED50, determined from
frequency of response data in a population. "Efficacy" as used
herein refers to the property of the compositions of the described
invention to achieve the desired response, and "maximum efficacy"
refers to the maximum achievable effect. The amount of the
chemotactic hematopoietic stem cell product in the pharmaceutical
compositions of the described invention that will be effective in
the treatment of a particular disorder or condition will depend on
the nature of the disorder or condition, and may be determined by
standard clinical techniques. (See, for example, Goodman and
Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Joel G. Harman,
Lee E. Limbird, Eds.; McGraw Hill, New York, 2001; THE PHYSICIAN'S
DESK REFERENCE, Medical Economics Company, Inc., Oradell, N.J.,
1995; and DRUG FACTS AND COMPARISONS, FACTS AND COMPARISONS, INC.,
St. Louis, Mo., 1993), each of which is incorporated by reference
herein. The precise dose to be employed in the formulations of the
described invention also will depend on the route of administration
and the seriousness of the disease or disorder, and should be
decided according to the judgment of the practitioner and each
subject's circumstances.
[0168] According to another embodiment, the pharmaceutical
compositions according to the described invention contain a minimum
number of CD34+ hematopoietic stem cells having a subpopulation of
at least 0.5.times.10.sup.6 CD34+ cells expressing CXCR-4 and
having CXCR-4 mediated chemotactic activity per dosage unit for
parenteral administration at the physician's discretion. According
to another embodiment, it is envisioned that subjects can benefit
from multiple administrations of the pharmaceutical compositions
according to the described invention comprising a minimum number of
CD34+ hematopoietic stem cells having a subpopulation of at least
0.5.times.10.sup.6 CD34+ cells expressing CXCR-4 and having CXCR-4
mediated chemotactic activity.
[0169] In another aspect of the described invention, the
progressive myocardial injury-preventing pharmaceutical
compositions of the described invention may further include one or
more compatible active ingredients, which are aimed at providing
the progressive myocardial injury-preventing composition with
another pharmaceutical effect in addition to that provided by the
sterile chemotactic hematopoietic stem cell product of the
described invention. "Compatible" as used herein means that the
active ingredients of such a composition are capable of being
combined with each other in such a manner so that there is no
interaction that would substantially reduce the efficacy of each
active ingredient or the composition under ordinary use conditions.
In some embodiments, the combination therapy comprises
administering to a subject in need thereof a progressive myocardial
injury-preventing pharmaceutical composition comprising a sterile
chemotactic hematopoietic stem cell product of the described
invention combined with an agent selected from the group consisting
of an angiotensin converting enzyme (ACE) inhibitor, a
beta-blocker, a diuretic, an anti-arrhythmic agent, a hematopoietic
stem cell mobilizing agent, a tyrosine kinase receptor agonist, an
anti-anginal agent, a vasoactive agent or inotrope, an
anticoagulant agent, a fibrinolytic agent, and a
hypercholesterolemic agent. According to some embodiments, the
tyrosine kinase receptor agonist is neuregulin 1. According to some
embodiments, the neuregulin 1 is a recombinant protein. According
to some embodiments, the hematopoietic stem cell mobilizing agent
is a colony stimulating factor. According to some such embodiments,
the hematopoietic stem cell mobilizing agent comprises G-CSF,
GM-CSF, or a pharmaceutically acceptable analog or derivative
thereof. According to some embodiments, the hematopoietic stem cell
mobilizing agent is a recombinant analog or derivative of a colony
stimulating factor. According to some embodiments, the
hematopoietic stem cell mobilizing agent is filgrastim.
[0170] In some embodiments, the composition of the described
invention further comprises about 0.5% to about 5% albumin. In some
embodiments, the minimum amount of albumin is about 0.5% expressed
as ml/100 cc volume of the composition. In some embodiments, the
minimum amount of albumin is about 0.75% expressed as ml/100 cc
volume of the composition. In some embodiments, the minimum amount
of albumin is about 1.0% expressed as ml/100 cc volume of the
composition. In some embodiments, the minimum amount of albumin is
about 1.25% expressed as ml/100 cc volume of the composition. In
some embodiments, the minimum amount of albumin is about 1.5%
expressed as ml/100 cc volume of the composition. In some
embodiments, the minimum amount of albumin is about 1.75% expressed
as ml/100 cc volume of the composition. In some embodiments, the
minimum amount of albumin is about 2.0% expressed as ml/100 cc
volume of the composition. In some embodiments, the minimum amount
of albumin is about 2.5% expressed as ml/100 cc volume of the
composition. In some embodiments, the minimum amount of albumin is
about 2.75% expressed as ml/100 cc volume of the composition. In
some embodiments, the minimum amount of albumin is about 3.0%
expressed as ml/100 cc volume of the composition. In some
embodiments, the minimum amount of albumin is about 3.5% expressed
as ml/100 cc volume of the composition. In some embodiments, the
minimum amount of albumin is about 4.0% expressed as ml/100 cc
volume of the composition. In some embodiments, the minimum amount
of albumin is about 4.5% expressed as ml/100 cc volume of the
composition. In some embodiments, the minimum amount of albumin is
about 5.0% expressed as ml/100 cc volume of the composition.
[0171] In some embodiments, the maximum amount of albumin in the
compositions of the described invention is about 5.0% expressed as
ml/100 cc volume of the composition. In some embodiments, the
maximum amount of albumin in the compositions of the described
invention is about 4.75% expressed as ml/100 cc volume of the
composition. In some embodiments, the maximum amount of albumin in
the compositions of the described invention is about 4.5% expressed
as ml/100 cc volume of the composition. In some embodiments, the
maximum amount of albumin in the compositions of the described
invention is about 4.0% expressed as ml/100 cc volume of the
composition. In some embodiments, the maximum amount of albumin in
the compositions of the described invention is about 4.25%
expressed as ml/100 cc volume of the composition. In some
embodiments, the maximum amount of albumin in the compositions of
the described invention is about 4.0% expressed as ml/100 cc volume
of the composition. In some embodiments, the maximum amount of
albumin in the compositions of the described invention is about
3.75% expressed as ml/100 cc volume of the composition. In some
embodiments, the maximum amount of albumin in the compositions of
the described invention is about 3.5% expressed as ml/100 cc volume
of the composition. In some embodiments, the maximum amount of
albumin in the compositions of the described invention is about
3.25% expressed as ml/100 cc volume of the composition. In some
embodiments, the maximum amount of albumin in the compositions of
the described invention is about 3.0% expressed as ml/100 cc volume
of the composition. In some embodiments, the maximum amount of
albumin in the compositions of the described invention is about
2.75% expressed as ml/100 cc volume of the composition. In some
embodiments, the maximum amount of albumin in the compositions of
the described invention is about 2.0% expressed as ml/100 cc volume
of the composition. In some embodiments, the maximum amount of
albumin in the compositions of the described invention is about
1.75% expressed as ml/100 cc volume of the composition. In some
embodiments, the maximum amount of albumin in the compositions of
the described invention is about 1.5% expressed as ml/100 cc volume
of the composition. In some embodiments, the maximum amount of
albumin in the compositions of the described invention is about
1.25% expressed as ml/100 cc volume of the composition. In some
embodiments, the maximum amount of albumin in the compositions of
the described invention is about 1% expressed as ml/100 cc volume
of the composition. In some embodiments, the albumin is human
albumin. In some embodiments the albumin is recombinant human
albumin.
[0172] Methods of the Described Invention
[0173] In another aspect, the described invention provides a method
of preparing a progressive myocardial injury-preventing
pharmaceutical composition comprising a sterile chemotactic
hematopoietic stem cell product for treating a subject in need
thereof. The method comprises the steps of
[0174] (1) acquiring a sterile nonexpanded, isolated population of
autologous mononuclear cells comprising CD34+ cells, which further
contain a subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity from the subject under sterile
conditions by a chemotactic cell acquisition process;
[0175] (2) optionally freezing at least one aliquot of the
nonexpanded, isolated population of autologous mononuclear cells of
step (1) at -86.degree. C. and cryostoring the at least one aliquot
in the vapor phase of a liquid nitrogen freezer; and thawing the at
least one aliquot of step (2) when needed;
[0176] (3) sterilely purifying the CD34+ cells from the sterile
nonexpanded, isolated population of autologous mononuclear cells
comprising CD34+ cells of (1) or (2) so as to yield a chemotactic
hematopoietic stem cell product comprising the nonexpanded,
isolated population of autologous mononuclear cells enriched for
CD34+ cells, which further contain a subpopulation of potent CD34+
cells that express CXCR-4 and that have CXCR-4-mediated chemotactic
activity;
[0177] (4) sterilely formulating the sterile chemotactic
hematopoietic stem cell product to form a sterile pharmaceutical
composition;
[0178] (5) confirming sterility of the pharmaceutical
composition;
[0179] (6) releasing the sterile pharmaceutical composition as
eligible for infusion into the subject;
[0180] (7) loading a therapeutically effective amount of the
pharmaceutical composition into a chemotactic hematopoietic stem
cell product delivery apparatus; and
[0181] (8) optionally transporting the delivery apparatus
containing the therapeutically effective amount of the sterile
pharmaceutical composition comprising the sterile chemotactic
hematopoietic stem cell product to a cardiac catheterization
facility for infusion into the subject.
[0182] According to some embodiments, the nonexpanded, isolated
population of autologous mononuclear cells can be acquired from the
subject at any time. According to some embodiments, the
nonexpanded, isolated population of autologous mononuclear cells is
acquired early after an AMI. According to some such embodiments,
the nonexpanded, isolated population of autologous mononuclear
cells is acquired 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,
11 days, 12 days, 13 days, 14 days or more after the occurrence of
an AMI. According to some embodiments, the nonexpanded, isolated
population of autologous mononuclear cells is acquired late after
the occurrence of an AMI. According to some such embodiments, the
nonexpanded, isolated population of autologous mononuclear cells is
acquired at least 15 days, at least 16 days, at least 17 days, at
least 18 days, at least 19 days, at least 20 days, at least 21
days, at least 22 days, at least 23 days, at least 24 days, at
least 25 days, at least 26 days, at least 27 days, at least 28
days, at least 29 days, at least 30 days, at least 60 days, at
least 90 days, at least 120 days, at least 150 days, at least 180
days or more after the occurrence of the AMI. According to some
embodiments, the nonexpanded, isolated population of autologous
mononuclear cells is acquired at least 1 month, at least 2 months,
at least 3 months, at least 4 months, at least 5 months, at least 6
months, at least 7 months, at least 8 months, at least 9 months, at
least 10 months, at least 11 months, at least 12 months, at least
16 months, at least 24 months, at least 30 months, at least 36
months, at least 42 months, at least 48 months, at least 54 months,
at least 60 months, at least 66 months, at least 72 months, at
least 78 months, at least 84 months, at least 90 months, at least
96 months, at least 102 months, at least 108 months, at least 114
months, at least 120 months, at least 126 months, at least 132
months, at least 138 months, at least 144 months, at least 150
months, at least 156 months, at least 162 months, at least 168
months, at least 174 months, at least 180 months, at least 186
months, at least 192 months, at least 198 months, at least 204
months, at least 210 months, at least 216 months, at least 222
months, at least 228 months, at least 234 months, at least 240
months or more after occurrence of an AMI. According to some
embodiments, the nonexpanded, isolated population of autologous
mononuclear cells is acquired at least at least 3 years, 4 years, 5
years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12
years, 13 years, 14 years, 15 years, 16 years 17 years, 18 years,
19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25
years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years,
32 years, 33 years, 34 years, 35 years, 36 years 37 years, 38
years, 39 years, 40 years or more after occurrence of an AMI.
[0183] According to one embodiment, step (3) is initiated within
about 12 hours to about 24 hours of completion of acquiring step
(1). According to some embodiments, releasing step (6) 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 (1). According to another embodiment, step (3) is
initiated within about 12 hours to about 24 hours of completion of
acquiring step (I), and releasing step (6) 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 (1).
[0184] According to some embodiments, releasing step (6) proceeds
only if the sterile formulated cell product is to be infused into
the subject within about 48 hours to about 72 hours of thawing of
the at least one frozen aliquot of optional step (2). According to
another embodiment, step (3) is initiated within about 12 hours to
about 24 hours of thawing of the at least one frozen aliquot of
optional step (2), and releasing step (6) proceeds only if the
sterile formulated cell product is to be infused into the subject
within about 48 hours to about 72 hours of thawing of the at least
one frozen aliquot of optional step (2).
[0185] According to some embodiments, a frozen aliquot of step (2)
is thawed at least 10 days, at least 11 days, at least 12 days, at
least 13 days, at least 14 days, at least 15 days, at least 16
days, at least 17 days, at least 18 days, at least 19 days, at
least 20 days, at least 21 days, at least 22 days, at least 23
days, at least 24 days, at least 25 days, at least 26 days, at
least 27 days, at least 28 days, at least 29 days, at least 30
days, at least 60 days, at least 90 days, at least 120 days, at
least 150 days, or at least 180 days, from the date the
nonexpanded, isolated population of autologous mononuclear cells is
acquired from the subject in step (1). According to some
embodiments, the frozen aliquot of step (4) is thawed at least 1
month, at least 2 months, at least 3 months, at least 4 months, at
least 5 months, at least 6 months, at least 7 months, at least 8
months, at least 9 months, at least 10 months, at least 11 months,
at least 12 months, at least 16 months, at least 24 months, at
least 30 months, at least 36 months, at least 42 months, at least
48 months, at least 54 months, at least 60 months, at least 66
months, at least 72 months, at least 78 months, at least 84 months,
at least 90 months, at least 96 months, at least 102 months, at
least 108 months, at least 114 months, at least 120 months, at
least 126 months, at least 132 months, at least 138 months, at
least 144 months, at least 150 months, at least 156 months, at
least 162 months, at least 168 months, at least 174 months, at
least 180 months, at least 186 months, at least 192 months, at
least 198 months, at least 204 months, at least 210 months, at
least 216 months, at least 222 months, at least 228 months, at
least 234 months or at least 240 months from the date the
nonexpanded, isolated population of autologous mononuclear cells is
acquired from the subject in step (1). According to some
embodiments, the frozen aliquot of step (2) is thawed at least 3
years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10
years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years
17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23
years, 24 years. 25 years, 26 years, 27 years, 28 years, 29 years,
30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36
years 37 years, 38 years, 39 years, 40 years or more from the date
the nonexpanded, isolated population of autologous mononuclear
cells is acquired from the subject in step (1).
[0186] According to such embodiments, the chemotactic hematopoietic
stem cell product produced from the frozen aliquot is further
characterized as having the following properties for at least 24
hours following thawing when tested in vitro after passage through
a catheter: (1) retains at least 10%, at least 20%, at least 30%,
at least 40%, at least 50%, at least 60%, at least 70,%, at least
80%, at least 90%, or 100% of the CXCR-4-mediated activity of the
of the subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity; (2) at least 70% of the cells
are CD34+ cells; (3) is at least 70% viable; and (4) is able to
form hematopoietic colonies in vitro.
[0187] According to another embodiment, step (5), i.e., the step of
assessing sterility of the pharmaceutical composition, further
comprises the steps of (i) centrifuging the sterile chemotactic
hematopoietic stem cell product comprising potent CD34+/CXCR-4+
cells to form a cell pellet and a supernatant, the cell pellet
comprising the potent CD34+/CXCR-4+ cells; (ii) sterilely removing
the supernatant without disturbing the cell pellet; and (iii)
analyzing whether the supernatant is contaminated by a microbe
thereby determining the sterility of the cell pellet.
[0188] According to one embodiment, in step (1), the chemotactic
cell acquisition process is a mini-bone marrow harvest technique
used to acquire the nonexpanded isolated population of autologous
mononuclear cells comprising potent CD34+/CXCR-4+ cells from the
bone marrow of the subject under sterile conditions. For the bone
marrow harvest technique, step (1) of the method further comprises
the steps: (i) preloading harvesting syringes with heparin prior to
harvesting bone marrow from a 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.
In one embodiment, the harvesting syringes in step (i) and the
collecting bag in step (iii) contain a preservative free
heparinized solution comprising 0.9% normal saline. The final
concentration of heparin in the heparinized saline solution is
about 20 units per ml to about 25 units per ml.
[0189] Optionally, according to one embodiment of the method, the
harvested bone marrow is transported to a processing facility
different from the facility from which the bone marrow was
harvested. According to one embodiment, the method for transporting
the harvested bone marrow to the processing facility comprises the
steps (a) placing the harvested bone marrow in a collection bag;
(b) placing the collection bag in a secondary bag; (c) placing the
secondary bag containing the collection bag in a shipping container
comprising an interior compartment containing frozen wet ice and at
least one sheet of bubble wrap; (d) affixing a temperature tag
monitor to the interior compartment of the shipping container; (e)
sealing the shipping container; and (f) shipping the shipping
container to the processing facility.
[0190] In another aspect, the described invention provides a method
for treating or preventing progressive myocardial injury due to a
vascular insufficiency that occurs early or late. The method
comprising the steps: (a) evaluating whether the subject qualifies
for therapy with the pharmaceutical composition of the described
invention; (b) preparing the pharmaceutical composition comprising
a chemotactic hematopoietic stem cell product; (c) loading the
pharmaceutical composition into a chemotactic hematopoietic stem
cell product delivery apparatus; (d) delivering a therapeutically
effective amount of the pharmaceutical composition to the subject;
and (e) monitoring the subject's cardiac function. According to one
embodiment, in step (d) the therapeutically effective amount of the
pharmaceutical composition is delivered to the subject
intravascularly (meaning inside a blood vessel). According to
another embodiment, the vascular insufficiency that occurs early or
late is an ischemia. According to some such embodiments, the
ischemia is a myocardial ischemia. According to some such
embodiments, the ischemia is a transient myocardial ischemia.
According to some such embodiments, the ischemia is a chronic
myocardial ischemia. According to some such embodiments, the
ischemia is a peri-infarct border zone ischemia. According to one
embodiment, the vascular insufficiency that occurs early or late is
a vascular insufficiency after an acute myocardial infarction
resulting from underlying disease. According to some such
embodiments, the progressive myocardial injury is heart
failure.
[0191] According to one embodiment of the described invention, the
subject in need thereof is a revascularized myocardial infarction
patient. The term "revascularized" as used in this embodiment
refers to the successful placement of a stent. Clinical
evaluations, for example, of coronary insufficiency using
non-laboratory tests, cardiac catheterization, measurement of
inflammatory cytokines, and measurement of cardiac biomarkers may
be used to determine the appropriate time to administer the
pharmaceutical compositions in accordance with the methods of the
described invention. According to n some embodiments, detection of
peak inflammatory cytokine cascade production enables the
administration to be tailored to the therapeutic window most
crucial for the particular subject. According to some embodiments,
peak inflammatory cytokine cascade production is determined by
measuring the levels of the appropriate cytokine(s) in the plasma
and or urine. According to other embodiments, the level(s) of the
appropriate cytokine(s) is/are measured immunochemically, for
example, by a sandwich enzyme immunoassay, by enzyme-linked
immunosorbent assays (ELISA) or by multiplex bead kits.
[0192] According to some embodiments, the composition is
administered at a first infusion date. According to one embodiment,
the first infusion date is a time after an inflammatory cytokine
cascade production peaks. According to some embodiments, the first
infusion date at which the composition is administered to a
revascularized myocardial infarction patient is about 5 days to
about 14 days post-infarction. In some embodiments, the minimum
first infusion date in which to administer the composition to a
revascularized myocardial infarction patient is about 5, 6, 7, 8,
9, 10, 11, 12, 13, or 14 days post-infarction. According to some
embodiments, the maximum first infusion date in which to administer
the composition to a revascularized myocardial infarction patient
is about 14, 12, 11, 10, 9, 8, 7, 6, or 5 days post-infarction.
[0193] According to some embodiments, the composition is
administered multiple times, or as needed in the judgment of the
treating physician. According to one such embodiment, the
composition is administered at the first infusion date, and
optionally at a second infusion date, a third infusion date, a
fourth infusion date, a fifth infusion date, a sixth infusion date,
a seventh infusion date, an eighth infusion date, a ninth infusion
date, a tenth infusion date, and so on.
[0194] According to some embodiments, the first infusion date at
which the composition is administered to a revascularized subject
suffering from a vascular insufficiency that occurs early or late
after a myocardial infarction resulting from underlying disease
comprises a specific time interval defined by a first time and a
second time, wherein the first time is after peak inflammatory
cytokine cascade production in the infarcted area and the second
time is before myocardial scar formation in the infarcted area.
[0195] According to some embodiments, the first infusion date is at
least about one day, at least about two days, at least about three
days, at least about four days, at least about five days, at least
about six 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, at least about 15 days, at least about 16 days, at least
about 17 days, at least about 18 days, at least about 19 days, at
least about 20 days, at least about 21 days, at least about 22
days, at least about 23 days, at least about 24 days, at least
about 25 days, at least about 26 days, at least about 27 days, at
least about 28 days, at least about 29 days, at least about 30 days
or more after occurrence of an AMI. According to some embodiments,
the second infusion date is at least about 1 month, at least about
2 months, at least about 3 months, at least about 4 months, at
least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 0 months, at least about 11 months, at least about 12 months,
at least about 13 months, at least about 14 months, at least about
15 months, at least about 16 months, at least about 17 months, at
least about 18 months, at least about 19 months, at least about 20
months, at least about 21 months, at least about 22 months, at
least about 23 months, at least about 24 months, at least about 30
months, at least about 36 months, at least about 42 months, at
least about 48 months, at least about 54 months, at least about 60
months, at least about 66 months, at least about 72 months, at
least about 78 months, at least about 84 months, at least about 90
months, at least about 96 months, at least about 102 months, at
least about 108 months, at least about 114 months, at least about
120 months, at least about 126 months, at least about 132 months,
at least about 138 months, at least about 144 months, at least
about 150 months, at least about 156 months, at least about 16;
months, at least about 168 months, at least about 174 months, at
least about 180 months, at least about 186 months, at least about
192 months, at least about 198 months, at least about 204 months,
at least about 20 months, at least about 216 months, at least about
222 months, at least about 228 months, at least about 234 months,
at least about 240 months or more after occurrence of an AMI.
According to some embodiments, the first infusion date is at least
3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10
years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years
17 years. 18 years, 19 years, 20 years, 21 years, 22 years. 23
years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years,
30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36
years 37 years, 38 years, 39 years, 40 years or more after
occurrence of an AMI.
[0196] According to some embodiments, the third infusion date is at
least about one day, at least about two days, at least about three
days, at least about four days, at least about five days, at least
about six 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, at least about 15 days, at least about 16 days, at least
about 17 days, at least about 18 days, at least about 19 days, at
least about 20 days, at least about 21 days, at least about 22
days, at least about 23 days, at least about 24 days, at least
about 25 days, at least about 26 days, at least about 27 days, at
least about 28 days, at least about 29 days, at least about 30 days
or more after occurrence of an AMI. According to some embodiments,
the third infusion date is at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 0
months, at least about 11 months, at least about 12 months, at
least about 13 months, at least about 14 months, at least about 15
months, at least about 16 months, at least about 17 months, at
least about 18 months, at least about 19 months, at least about 20
months, at least about 21 months, at least about 22 months, at
least about 23 months, at least about 24 months, at least about 30
months, at least about 36 months, at least about 42 months, at
least about 48 months, at least about 54 months, at least about 60
months, at least about 66 months, at least about 72 months, at
least about 78 months, at least about 84 months, at least about 90
months, at least about 96 months, at least about 102 months, at
least about 108 months, at least about 114 months, at least about
120 months, at least about 126 months, at least about 132 months,
at least about 138 months, at least about 144 months, at least
about 150 months, at least about 156 months, at least about 162
months, at least about 168 months, at least about 174 months, at
least about 180 months, at least about 186 months, at least about
192 months, at least about 198 months, at least about 204 months,
at least about 20 months, at least about 216 months, at least about
222 months, at least about 228 months, at least about 234 months,
at least about 240 months after occurrence of an AMI. According to
some such embodiments, the third infusion date is at least 3 years,
4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11
years, 12 years, 13 years, 14 years, 15 years, 16 years 17 years,
18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24
years, 25 years, 26 years. 27 years, 28 years, 29 years, 30 years,
31 years, 32 years, 33 years, 34 years, 35 years, 36 years 37
years, 38 years, 39 years, 40 years or more after occurrence of an
AMI.
[0197] According to some embodiments, the fourth infusion date is
at least about one day, at least about two days, at least about
three days, at least about four days, at least about five days, at
least about six 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, at least about 15 days, at least about 16 days, at
least about 17 days, at least about 18 days, at least about 19
days, at least about 20 days, at least about 21 days, at least
about 22 days, at least about 23 days, at least about 24 days, at
least about 25 days, at least about 26 days, at least about 27
days, at least about 28 days, at least about 29 days, at least
about 30 days or more after occurrence of an AMI. According to some
embodiments, the fourth infusion date is at least about 1 month, at
least about 2 months, at least about 3 months, at least about 4
months, at least about 5 months, at least about 6 months, at least
about 7 months, at least about 8 months, at least about 9 months,
at least about 0 months, at least about 11 months, at least about
12 months, at least about 13 months, at least about 14 months, at
least about 15 months, at least about 16 months, at least about 17
months, at least about 18 months, at least about 19 months, at
least about 20 months, at least about 21 months, at least about 22
months, at least about 23 months, at least about 24 months, at
least about 30 months, at least about 36 months, at least about 42
months, at least about 48 months, at least about 54 months, at
least about 60 months, at least about 66 months, at least about 72
months, at least about 78 months, at least about 84 months, at
least about 90 months, at least about 96 months, at least about 102
months, at least about 108 months, at least about 114 months, at
least about 120 months, at least about 126 months, at least about
132 months, at least about 138 months, at least about 144 months,
at least about 150 months, at least about 156 months, at least
about 162 months, at least about 168 months, at least about 174
months, at least about 180 months, at least about 186 months, at
least about 192 months, at least about 198 months, at least about
204 months, at least about 20 months, at least about 216 months, at
least about 222 months, at least about 228 months, at least about
234 months, at least about 240 months or more after occurrence of
an AMI. According to some such embodiments, the third infusion date
is at least 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9
years, 10 years, 11 years, 12 years, 13 years, 14 years. 15 years,
16 years 17 years, 18 years. 19 years, 20 years, 21 years, 22
years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years,
29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35
years, 36 years 37 years, 38 years, 39 years, 40 years or more
after occurrence of an AMI.
[0198] According to some embodiments, the fifth infusion date is at
least about one day, at least about two days, at least about three
days, at least about four days, at least about five days, at least
about six 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, at least about 15 days, at least about 16 days, at least
about 17 days, at least about 18 days, at least about 19 days, at
least about 20 days, at least about 21 days, at least about 22
days, at least about 23 days, at least about 24 days, at least
about 25 days, at least about 26 days, at least about 27 days, at
least about 28 days, at least about 29 days, at least about 30 days
or more after occurrence of an AMI. According to some embodiments,
the fifth infusion date is at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 0
months, at least about 11 months, at least about 12 months, at
least about 13 months, at least about 14 months, at least about 15
months, at least about 16 months, at least about 17 months, at
least about 18 months, at least about 19 months, at least about 20
months, at least about 21 months, at least about 22 months, at
least about 23 months, at least about 24 months, at least about 30
months, at least about 36 months, at least about 42 months, at
least about 48 months, at least about 54 months, at least about 60
months, at least about 66 months, at least about 72 months, at
least about 78 months, at least about 84 months, at least about 90
months, at least about 96 months, at least about 102 months, at
least about 108 months, at least about 114 months, at least about
120 months, at least about 126 months, at least about 132 months,
at least about 138 months, at least about 144 months, at least
about 150 months, at least about 156 months, at least about 162
months, at least about 168 months, at least about 174 months, at
least about 180 months, at least about 186 months, at least about
192 months, at least about 198 months, at least about 204 months,
at least about 20 months, at least about 216 months, at least about
222 months, at least about 228 months, at least about 234 months,
at least about 240 months or more after occurrence of an AMI.
According to some embodiments, the first infusion date is at least
3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10
years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years
17 years, 18 years, 19 years, 20 years. 21 years, 22 years, 23
years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years,
30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36
years 37 years, 38 years, 39 years, 40 years or more after
occurrence of an AMI.
[0199] According to some embodiments, the sixth infusion date is at
least about one day, at least about two days, at least about three
days, at least about four days, at least about five days, at least
about six 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, at least about 15 days, at least about 16 days, at least
about 17 days, at least about 18 days, at least about 19 days, at
least about 20 days, at least about 21 days, at least about 22
days, at least about 23 days, at least about 24 days, at least
about 25 days, at least about 26 days, at least about 27 days, at
least about 28 days, at least about 29 days, at least about 30 days
or more after occurrence of an AMI. According to some embodiments,
the sixth infusion date is at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 0
months, at least about 11 months, at least about 12 months, at
least about 13 months, at least about 14 months, at least about 15
months, at least about 16 months, at least about 17 months, at
least about 18 months, at least about 19 months, at least about 20
months, at least about 21 months, at least about 22 months, at
least about 23 months, at least about 24 months, at least about 30
months, at least about 36 months, at least about 42 months, at
least about 48 months, at least about 54 months, at least about 60
months, at least about 66 months, at least about 72 months, at
least about 78 months, at least about 84 months, at least about 90
months, at least about 96 months, at least about 102 months, at
least about 108 months, at least about 114 months, at least about
120 months, at least about 126 months, at least about 132 months,
at least about 138 months, at least about 144 months, at least
about 150 months, at least about 156 months, at least about 162
months, at least about 168 months, at least about 174 months, at
least about 180 months, at least about 186 months, at least about
192 months, at least about 198 months, at least about 204 months,
at least about 20 months, at least about 216 months, at least about
222 months, at least about 228 months, at least about 234 months,
at least about 240 months or more after occurrence of an AMI.
According to some such embodiments, the third infusion date is at
least 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9
years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years,
16 years 17 years, 18 years, 19 years, 20 years, 21 years, 22
years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years,
29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35
years, 36 years 37 years, 38 years, 39 years, 40 years or more
after occurrence of an AMI.
[0200] According to some such embodiments, the seventh infusion
date is at least about one day, at least about two days, at least
about three days, at least about four days, at least about five
days, at least about six 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, at least about 15 days, at least
about 16 days, at least about 17 days, at least about 18 days, at
least about 19 days, at least about 20 days, at least about 21
days, at least about 22 days, at least about 23 days, at least
about 24 days, at least about 25 days, at least about 26 days, at
least about 27 days, at least about 28 days, at least about 29
days, at least about 30 days or more after occurrence of an AMI.
According to some embodiments, the seventh infusion date is at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, at least
about 6 months, at least about 7 months, at least about 8 months,
at least about 9 months, at least about 0 months, at least about 11
months, at least about 12 months, at least about 13 months, at
least about 14 months, at least about 15 months, at least about 16
months, at least about 17 months, at least about 18 months, at
least about 19 months, at least about 20 months, at least about 21
months, at least about 22 months, at least about 23 months, at
least about 24 months, at least about 30 months, at least about 36
months, at least about 42 months, at least about 48 months, at
least about 54 months, at least about 60 months, at least about 66
months, at least about 72 months, at least about 78 months, at
least about 84 months, at least about 90 months, at least about 96
months, at least about 102 months, at least about 108 months, at
least about 114 months, at least about 120 months, at least about
126 months, at least about 132 months, at least about 138 months,
at least about 144 months, at least about 150 months, at least
about 156 months, at least about 162 months, at least about 168
months, at least about 174 months, at least about 180 months, at
least about 186 months, at least about 192 months, at least about
198 months, at least about 204 months, at least about 20 months, at
least about 216 months, at least about 222 months, at least about
228 months, at least about 234 months, at least about 240 months or
more after occurrence of an AMI. According to some such
embodiments, the third infusion date is at least 3 years, 4 years,
5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12
years, 13 years, 14 years, 15 years, 16 years 17 years, 18 years,
19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25
years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years,
32 years, 33 years, 34 years, 35 years, 36 years 37 years, 38
years, 39 years, 40 years or more after occurrence of an AMI.
[0201] According to some such embodiments, the eighth infusion date
is at least about one day, at least about two days, at least about
three days, at least about four days, at least about five days, at
least about six 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, at least about 15 days, at least about 16 days, at
least about 17 days, at least about 18 days, at least about 19
days, at least about 20 days, at least about 21 days, at least
about 22 days, at least about 23 days, at least about 24 days, at
least about 25 days, at least about 26 days, at least about 27
days, at least about 28 days, at least about 29 days, at least
about 30 days or more after occurrence of an AMI. According to some
embodiments, the eighth infusion date is at least about 1 month, at
least about 2 months, at least about 3 months, at least about 4
months, at least about 5 months, at least about 6 months, at least
about 7 months, at least about 8 months, at least about 9 months,
at least about 0 months, at least about 11 months, at least about
12 months, at least about 13 months, at least about 14 months, at
least about 15 months, at least about 16 months, at least about 17
months, at least about 18 months, at least about 19 months, at
least about 20 months, at least about 21 months, at least about 22
months, at least about 23 months, at least about 24 months, at
least about 30 months, at least about 36 months, at least about 42
months, at least about 48 months, at least about 54 months, at
least about 60 months, at least about 66 months, at least about 72
months, at least about 78 months, at least about 84 months, at
least about 90 months, at least about 96 months, at least about 102
months, at least about 108 months, at least about 114 months, at
least about 120 months, at least about 126 months, at least about
132 months, at least about 138 months, at least about 144 months,
at least about 150 months, at least about 156 months, at least
about 162 months, at least about 168 months, at least about 174
months, at least about 180 months, at least about 186 months, at
least about 192 months, at least about 198 months, at least about
204 months, at least about 20 months, at least about 216 months, at
least about 222 months, at least about 228 months, at least about
234 months, at least about 240 months or more after occurrence of
an AMI. According to some such embodiments, the third infusion date
is at least 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9
years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years,
16 years 17 years, 18 years, 19 years, 20 years, 21 years, 22
years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years,
29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35
years, 36 years 37 years, 38 years, 39 years, 40 years or more
after occurrence of an AMI.
[0202] According to some such embodiments, the ninth infusion date
is at least about one day, at least about two days, at least about
three days, at least about four days, at least about five days, at
least about six 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, at least about 15 days, at least about 16 days, at
least about 17 days, at least about 18 days, at least about 19
days, at least about 20 days, at least about 21 days, at least
about 22 days, at least about 23 days, at least about 24 days, at
least about 25 days, at least about 26 days, at least about 27
days, at least about 28 days, at least about 29 days, at least
about 30 days or more after occurrence of an AMI. According to some
embodiments, the ninth infusion date is at least about 1 month, at
least about 2 months, at least about 3 months, at least about 4
months, at least about 5 months, at least about 6 months, at least
about 7 months, at least about 8 months, at least about 9 months,
at least about 0 months, at least about 11 months, at least about
12 months, at least about 13 months, at least about 14 months, at
least about 15 months, at least about 16 months, at least about 17
months, at least about 18 months, at least about 19 months, at
least about 20 months, at least about 21 months, at least about 22
months, at least about 23 months, at least about 24 months, at
least about 30 months, at least about 36 months, at least about 42
months, at least about 48 months, at least about 54 months, at
least about 60 months, at least about 66 months, at least about 72
months, at least about 78 months, at least about 84 months, at
least about 90 months, at least about 96 months, at least about 102
months, at least about 108 months, at least about 114 months, at
least about 120 months, at least about 126 months, at least about
132 months, at least about 138 months, at least about 144 months,
at least about 150 months, at least about 156 months, at least
about 162 months, at least about 168 months, at least about 174
months, at least about 180 months, at least about 186 months, at
least about 192 months, at least about 198 months, at least about
204 months, at least about 20 months, at least about 216 months, at
least about 222 months, at least about 228 months, at least about
234 months, at least about 240 months or more after occurrence of
an AMI. According to some such embodiments, the third infusion date
is at least 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9
years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years,
16 years 17 years, 18 years, 19 years, 20 years, 21 years, 22
years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years,
20 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35
years, 36 years 37 years, 38 years, 39 years, 40 years or more
after occurrence of an AMI.
[0203] According to some such embodiments, the tenth infusion date
is at least about one day, at least about two days, at least about
three days, at least about four days, at least about five days, at
least about six 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, at least about 15 days, at least about 16 days, at
least about 17 days, at least about 18 days, at least about 19
days, at least about 20 days, at least about 21 days, at least
about 22 days, at least about 23 days, at least about 24 days, at
least about 25 days, at least about 26 days, at least about 27
days, at least about 28 days, at least about 29 days, at least
about 30 days or more after occurrence of an AMI. According to some
embodiments, the tenth infusion date is at least about 1 month, at
least about 2 months, at least about 3 months, at least about 4
months, at least about 5 months, at least about 6 months, at least
about 7 months, at least about 8 months, at least about 9 months,
at least about 0 months, at least about 11 months, at least about
12 months, at least about 13 months, at least about 14 months, at
least about 15 months, at least about 16 months, at least about 17
months, at least about 18 months, at least about 19 months, at
least about 20 months, at least about 21 months, at least about 22
months, at least about 23 months, at least about 24 months, at
least about 30 months, at least about 36 months, at least about 42
months, at least about 48 months, at least about 54 months, at
least about 60 months, at least about 66 months, at least about 72
months, at least about 78 months, at least about 84 months, at
least about 90 months, at least about 96 months, at least about 102
months, at least about 108 months, at least about 114 months, at
least about 120 months, at least about 126 months, at least about
132 months, at least about 138 months, at least about 144 months,
at least about 150 months, at least about 156 months, at least
about 162 months, at least about 168 months, at least about 174
months, at least about 180 months, at least about 186 months, at
least about 192 months, at least about 198 months, at least about
204 months, at least about 20 months, at least about 216 months, at
least about 222 months, at least about 228 months, at least about
234 months, at least about 240 months or more after occurrence of
an AMI. According to some such embodiments, the third infusion date
is at least 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9
years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years,
16 years 17 years, 18 years, 19 years, 20 years, 21 years, 22
years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years,
29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35
years, 36 years 37 years, 38 years, 39 years, 40 years or more
after occurrence of an AMI, and so on.
[0204] According to some embodiments, the chemotactic hematopoietic
stem cell product of the composition administered at the second,
third, fourth, fifth, sixth, seventh, eighth, ninth and/or tenth
infusion date is prepared from a frozen and thawed aliquot of a
nonexpanded, isolated population of autologous mononuclear cells
containing CD34+ cells.
[0205] According to some embodiments, the chemotactic hematopoietic
stem cell product delivery apparatus used to deliver the
pharmaceutical composition of the described invention to a subject
in need thereof comprises an infusion syringe, a flushing syringe,
a four-way stopcock, and a balloon catheter. In one embodiment, the
intravascular delivery comprises (a) an infusion device attached to
a sterile four-way stopcock containing the pharmaceutical
composition comprising the chemotactic hematopoietic stem cell
product; (b) a flushing device attached to the sterile four-way
stopcock, the flushing device containing a flushing solution, and
(c) a catheter attached to the delivery apparatus by the sterile
four-way stopcock. According to one embodiment, the infusion device
is a syringe made of any suitable material. The body and handle of
suitable four way stopcocks may be made of the same or a different
material. Examples of suitable four-way stopcocks includes, without
limitation, a stopcock having a polycarbonate body/polycarbonate
handle, a stopcock having a polyethylene body/polyethylene handle,
a stopcock having a polycarbonate body/polyethylene handle, or a
disposable stopcock. According to some embodiments, a device is
further attached to the stopcock to regulate the pressure exerted
on the delivered solution. According to some embodiments, an
integral flush device or syringe is attached to the stopcock.
According to one embodiment, the catheter is a balloon catheter.
The term "balloon catheter" refers to a type of "soft" thin
flexible tube having an inflatable "balloon" at its tip, which is
used during a catheterization procedure to enlarge a narrow opening
or passage within the body. The deflated balloon catheter is
positioned, inflated to perform the necessary procedure, and
deflated again to be removed.
[0206] The viability and potential efficacy of the chemotactic
hematopoietic stem cell product of the described invention
comprising potent CD34+/CXCR-4+ cells depends on the cells
maintaining their potency as they pass through a catheter. The
catheter used in the methods of the described invention has an
internal diameter of at least 0.36 mm. Any type of catheter having
an internal diameter of at least 0.36 mm may be effective in
delivering the pharmaceutical compositions of the described
invention.
[0207] For example, a flow control catheter, which slows drainage
of blood through the coronary artery vasculature, allows the cells
time to transit through the blood vessel wall and into the
tissue.
[0208] In some embodiments, the catheter is a balloon catheter. For
example, without limitation, the following balloon dilatation
catheters available from Cordis, Boston Scientific, Medtronic and
Guidant having an internal diameter of about 0.36 mm have been
validated (see Table 1).
TABLE-US-00002 TABLE 1 Balloon catheter validated for infusion of
selected CD34.sup.+ cells through the IRA Name and Balloon Lumen
Internal Manufacturer Model No. Dimensions Diameter Cordis Raptor
OTW 15 mm .times. 3.0 mm 0.36 mm (0.14 in.) 579-130 Boston OTW
Maverick 15 mm .times. 3.0 mm 0.36 mm (0.14 in.) Scientific
20620-1530 Medtronic OTW Sprinter 15 mm .times. 3.0 mm 0.36 mm
(0.14 in.) SPR 3015W Guidant Voyager OTW 15 mm .times. 3.0 mm 0.36
mm (0.14 in.) 1009443-15
[0209] In addition, catheters have been described having a fluid
delivery port adjacent to the balloon such that the balloon may be
inflated against a vessel wall to isolate the delivery site from
hemodynamics opposite the balloon from the port, which may be
located distally of the balloon. Additionally, balloon catheters
have been disclosed having lumens ending in side ports disposed
proximally to the balloon catheter; these balloon catheters
generally may be referred to as "balloon/delivery" catheters,
although particular references may use different descriptors. See,
e.g., U.S. Pat. No. 5,415,636 to Forman, incorporated by reference
herein.
[0210] According to some embodiments, the method of treating or
preventing a progressive myocardial injury due to a vascular
insufficiency that occurs early or late comprises administering the
progressive myocardial injury-preventing pharmaceutical composition
via balloon catheterization into an artery at a first infusion
date. In some embodiments, following angioplasty, a delivery
balloon catheter is inserted via a femoral artery into a desired
coronary artery, such as the left anterior descending coronary
artery. Some medical conditions may require both a balloon catheter
and a fluid delivery catheter to facilitate treatment.
[0211] According to some embodiments, a catheter is used to
directly inject cells into the myocardium.
Treatment Regimens
[0212] According to another aspect, the described invention
provides a regimen for treating a progressive myocardial injury due
to a vascular insufficiency that occurs early or late, which
comprises:
[0213] (a) first administering to the subject on a first infusion
date a first sterile pharmaceutical composition parenterally
through a catheter, the first sterile pharmaceutical composition of
(a) comprising: (i) a therapeutically effective amount of a first
sterile chemotactic hematopoietic stem cell product, wherein the
first chemotactic hematopoietic stem cell product comprises a
nonexpanded, isolated population of autologous mononuclear cells
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic
activity, wherein the therapeutically effective amount of the first
chemotactic hematopoietic stem cell product comprises at least
10.times.10.sup.6 CD34+ cells containing at least
0.5.times.10.sup.6 potent CD34+ cells expressing CXCR-4 and having
CXCR-4 mediated chemotactic activity; (ii) a stabilizing amount of
serum, wherein the stabilizing amount of serum is greater than 20%
(v/v), wherein the chemotactic hematopoietic stem cell product is
further characterized as having the following properties for at
least 24 hours following acquisition of the chemotactic
hematopoietic stem cell product when tested in vitro after passage
through a catheter: (1) retains the CXCR-4-mediated activity of the
subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity; (2) at least 70% of the cells
are CD34+ cells; (3) is at least 70% viable; and (4) is able to
form hematopoietic colonies in vitro;
[0214] (b) second, administering at a second infusion date a second
sterile pharmaceutical composition comprising a therapeutic amount
of a second chemotactic hematopoietic stem cell product; wherein
the therapeutically effective amount of the second chemotactic
hematopoietic stem cell product comprises at least
10.times.10.sup.6 CD34+ cells which further contain a subpopulation
of at least 0.5.times.10.sup.6 potent CD34+ cells expressing CXCR-4
and having CXCR-4 mediated chemotactic activity; (ii) a stabilizing
amount of serum, wherein the stabilizing amount of scrum is greater
than 20% (v/v), wherein the second chemotactic hematopoietic stem
cell product is further characterized as having the following
properties fur at least 24 hours when tested in vitro after passage
through a catheter: (1) retains the CXCR-4-mediated activity of the
subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity; (2) at least 70% of the cells
are CD34+ cells; (3) is at least 70% viable; and (4) is able to
form hematopoietic colonies in vitro; and
[0215] (c) third, optionally administering at a third infusion date
a sterile pharmaceutical composition comprising a third chemotactic
hematopoietic stem cell product comprising at least
10.times.10.sup.6 isolated CD34+ cells, which further contain a
subpopulation of at least 0.5.times.10.sup.6 potent CD34+ cells
expressing CXCR-4 and having CXCR-4 mediated chemotactic activity;
(ii) a stabilizing amount of serum, wherein the stabilizing amount
of serum is greater than 20% (v/v), wherein the third chemotactic
hematopoietic stem cell product is further characterized as having
the following properties for at least 24 hours when tested in vitro
after passage through a catheter: (1) retains the CXCR-4-mediated
activity of the subpopulation of potent CD34+/CXCR-4+ cells that
have CXCR-4-mediated chemotactic activity; (2) at least 70% of the
cells are CD34+ cells; (3) is at least 70% viable; and (4) is able
to form hematopoietic colonies in vitro, such that the regimen
improves at least one measure of cardiac function.
[0216] According to some embodiments, at least one aliquot of the
sterile nonexpanded, isolated population of autologous mononuclear
cells comprising CD34+ cells, which further contain a subpopulation
of potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic
activity acquired from the subject under sterile conditions is
frozen at -86.degree. C. and cryostored at least one aliquot in the
vapor phase of a liquid nitrogen freezer until needed. At that
time, the at least one aliquot of the frozen nonexpanded, isolated
population of autologous mononuclear cells containing CD34+ cells
which further contain a subpopulation of potent CD34+/CXCR-4+ cells
that have CXCR-4-mediated chemotactic activity is thawed and
enriched for CD34+ cells, which further contain a subpopulation of
potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic
activity. This frozen and thawed nonexpanded, isolated population
of autologous mononuclear cells enriched for CD34+ cells which
further contain a subpopulation of potent CD34+/CXCR-4+ cells that
have CXCR-4-mediated chemotactic activity constitutes a thawed
sterile chemotactic hematopoietic stem cell product.
[0217] According to some embodiments, the thawed sterile
chemotactic hematopoietic stem cell product can be used in step
(b), step (c), or steps (b) and step (c) of the regimen.
[0218] The term "regimen" as used herein refers to a course or plan
of treatment to preserve or restore the health off subject
suffering from a progressive myocardial injury due to a vascular
insufficiency that occurs early or late.
[0219] According to one embodiment of the regimen, the thawed
sterile chemotactic hematopoietic stem cell product, when passed
through the catheter and tested in vitro, (i) is able to Form
hematopoietic colonies; and (ii) retains at least 2% of the
CXCR-4-mediated activity of the subpopulation of potent
CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity,
for at least 48 hours following thawing of the cryostored
nonexpanded, isolated population of autologous mononuclear cells
comprising CD34+ cells, which further contain a subpopulation of
potent CD34+/CXCR-4-+ cells that have CXCR-4-mediated chemotactic
activity. According to another embodiment, the thawed chemotactic
hematopoietic stem cell product, when passed through the catheter
and tested in vitro, (i) is able to form hematopoietic colonies;
and (ii) retains at least 2% of the CXCR-4-mediated activity of the
subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity for at least 72 hours
following thawing of nonexpanded, isolated population of autologous
mononuclear cells comprising CD34+ cells, which further contain a
subpopulation of potent CD34+/CXCR-4-+cells that have
CXCR-4-mediated chemotactic activity.
[0220] According to another embodiment, the first infusion date of
(a) is at least is at least about one day, at least about two days,
at least about three days, at least about four days, at least about
five days, at least about six 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, at least about 15 days, at least
about 16 days, at least about 17 days, at least about 18 days, at
least about 19 days, at least about 20 days, at least about 21
days, at least about 22 days, at least about 23 days, at least
about 24 days, at least about 25 days, at least about 26 days, at
least about 27 days, at least about 28 days, at least about 29
days, at least about 30 days or more after occurrence of an AMI.
According to some embodiments, the first infusion date of (a) is at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, at least
about 6 months, at least about 7 months, at least about 8 months,
at least about 9 months, at least about 0 months, at least about 11
months, at least about 12 months, at least about 13 months, at
least about 14 months, at least about 15 months, at least about 16
months, at least about 17 months, at least about 18 months, at
least about 19 months, at least about 20 months, at least about 21
months, at least about 22 months, at least about 23 months, at
least about 24 months, at least about 30 months, at least about 36
months, at least about 42 months, at least about 48 months, at
least about 54 months, at least about 60 months, at least about 66
months, at least about 72 months, at least about 78 months, at
least about 84 months, at least about 90 months, at least about 96
months, at least about 102 months, at least about 108 months, at
least about 114 months, at least about 120 months, at least about
126 months, at least about 132 months, at least about 138 months,
at least about 144 months, at least about 150 months, at least
about 156 months, at least about 162 months, at least about 168
months, at least about 174 months, at least about 180 months, at
least about 186 months, at least about 192 months, at least about
198 months, at least about 204 months, at least about 20 months, at
least about 216 months, at least about 222 months, at least about
228 months, at least about 234 months, at least about 240 months or
more after occurrence of an AMI. According to some embodiments, the
first infusion date is at least 3 years, 4 years, 5 years, 6 years,
7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years,
14 years, 15 years, 16 years 17 years, 18 years, 19 years, 20
years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years,
27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33
years, 34 years, 35 years, 36 years 37 years, 38 years, 39 years,
40 years or more after occurrence of an AMI.
[0221] According to another embodiment, the second infusion date of
(b) is at least about is at least about one day, at least about two
days, at least about three days, at least about four days, at least
about five days, at least about six 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, at least about 15 days, at least
about 16 days, at least about 17 days, at least about 18 days, at
least about 19 days, at least about 20 days, at least about 21
days, at least about 22 days, at least about 23 days, at least
about 24 days, at least about 25 days, at least about 26 days, at
least about 27 days, at least about 28 days, at least about 29
days, at least about 30 days or more after occurrence of an AMI.
According to some embodiments, the third infusion date of (c) is at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, at least
about 6 months, at least about 7 months, at least about 8 months,
at least about 9 months, at least about 0 months, at least about 11
months, at least about 12 months, at least about 13 months, at
least about 14 months, at least about 15 months, at least about 16
months, at least about 17 months, at least about 18 months, at
least about 19 months, at least about 20 months, at least about 21
months, at least about 22 months, at least about 23 months, at
least about 24 months, at least about 30 months, at least about 36
months, at least about 42 months, at least about 48 months, at
least about 54 months, at least about 60 months, at least about 66
months, at least about 72 months, at least about 78 months, at
least about 84 months, at least about 90 months, at least about 96
months, at least about 102 months, at least about 108 months, at
least about 114 months, at least about 120 months, at least about
126 months, at least about 132 months, at least about 138 months,
at least about 144 months, at least about 150 months, at least
about 156 months, at least about 162 months, at least about 168
months, at least about 174 months, at least about 180 months, at
least about 186 months, at least about 192 months, at least about
198 months, at least about 204 months, at least about 20 months, at
least about 216 months, at least about 222 months, at least about
228 months, at least about 234 months, at least about 240 months or
more after occurrence of an AMI. According to some embodiments, the
first infusion date is at least 3 years, 4 years, 5 years, 6 years,
7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years,
14 years, 15 years, 16 years 17 years, 18 years, 19 years, 20
years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years,
27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33
years, 34 years, 35 years, 36 years 37 years, 38 years, 39 years,
40 years or more after occurrence of an AMI.
[0222] According to another embodiment, the third infusion date of
(c) is at least about is at least about one day, at least about two
days, at least about three days, at least about four days, at least
about five days, at least about six 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, at least about 15 days, at least
about 16 days, at least about 17 days, at least about 18 days, at
least about 19 days, at least about 20 days, at least about 21
days, at least about 22 days, at least about 23 days, at least
about 24 days, at least about 25 days, at least about 26 days, at
least about 27 days, at least about 28 days, at least about 29
days, or at least about 30 days after occurrence of an AMI.
According to some embodiments, the third infusion date of (e) is at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, at least
about 6 months, at least about 7 months, at least about 8 months,
at least about 9 months, at least about 0 months, at least about 11
months, at least about 12 months, at least about 13 months, at
least about 14 months, at least about 15 months, at least about 16
months, at least about 17 months, at least about 18 months, at
least about 19 months, at least about 20 months, at least about 21
months, at least about 22 months, at least about 23 months, at
least about 24 months, at least about 30 months, at least about 36
months, at least about 42 months, at least about 48 months, at
least about 54 months, at least about 60 months, at least about 66
months, at least about 72 months, at least about 78 months, at
least about 84 months, at least about 90 months, at least about 96
months, at least about 102 months, at least about 108 months, at
least about 114 months, at least about 120 months, at least about
126 months, at least about 132 months, at least about 138 months,
at least about 144 months, at least about 150 months, at least
about 156 months, at least about 162 months, at least about 168
months, at least about 174 months, at least about 180 months, at
least about 186 months, at least about 192 months, at least about
198 months, at least about 204 months, at least about 20 months, at
least about 216 months, at least about 222 months, at least about
228 months, at least about 234 months, at least about 240 months or
more after occurrence of an AMI. According to some embodiments, the
first infusion date is at least 3 years, 4 years, 5 years, 6 years,
7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years,
14 years, 15 years, 16 years 17 years, 18 years, 19 years, 20
years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years,
27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33
years, 34 years, 35 years, 36 years 37 years, 38 years, 39 years,
40 years or more after occurrence of an AMI.
[0223] According to another embodiment, the vascular insufficiency
that occurs early or late is an ischemia. According to another
embodiment, the ischemia is a myocardial ischemia. According to
another embodiment, the ischemia is a transient ischemia. According
to another embodiment, the ischemia is a chronic myocardial
ischemia. According to another embodiment, the ischemia is a
peri-infarct border zone ischemia. According to another embodiment,
the catheter is a flow control catheter. According to another
embodiment, the catheter is a balloon dilatation catheter.
According to another embodiment, the catheter has an internal
diameter of at least about 0.36 mm. According to another
embodiment, the composition is administered through the catheter
into myocardium. According to another embodiment, the composition
is administered through the catheter intravascularly. According to
another embodiment, the pharmaceutical composition further includes
at least one compatible active agent. According to another
embodiment, the active agent is selected from the group consisting
of an angiotensin converting enzyme inhibitor, a beta-blocker, a
diuretic, an anti-arrhythmic agent, a hematopoietic stem cell
mobilizing agent, a tyrosine kinase receptor agonist, an
anti-anginal agent, a vasoactive agent, an anticoagulant agent, a
fibrinolytic agent, and a hypercholesterolemic agent. According to
another embodiment, the tyrosine kinase receptor agonist is human
neuregulin 1. According to some embodiments, the hematopoietic stem
cell mobilizing agent is a colony stimulating factor. According to
some such embodiments, the hematopoietic stem cell mobilizing agent
comprises G-CSF, GM-CSF, or a pharmaceutically acceptable analog or
derivative thereof. According to some embodiments, the
hematopoietic stem cell mobilizing agent is a recombinant analog or
derivative of a colony stimulating factor. According to some
embodiments, the hematopoietic stem cell mobilizing agent is
filgrastim.
[0224] According to another embodiment, the vascular insufficiency
that occurs early or late is a vascular insufficiency after an
acute myocardial infarction resulting from underlying disease.
According to some such embodiments, the first infusion date
comprises a specific time interval defined by a first time and a
second time, wherein the first time is after peak inflammatory
cytokine cascade production in the infarcted area and the second
time is before myocardial scar formation in the infarcted area.
According to another embodiment, in step (a), the first time of the
first infusion date is at least about 5 days post-infarction.
According to another embodiment, in step (a) the first time of the
first infusion date is about 5 days post-infarction and the second
time is about 14 days post-infarction. According to another
embodiment, the regimen treats cardiomyocyte cell death in the
peri-infarct border zone, relative to controls. According to
another embodiment, the regimen treats hypoperfusion in the
peri-infarct border zone, relative to controls. According to
another embodiment, the regimen treats myocardial hibernation in
the peri-infarct border zone, relative to controls. According to
another embodiment, the regimen decreases infarct area, relative to
controls. According to another embodiment, the regimen decreases
infarct mass, relative to controls. According to another
embodiment, the progressive, myocardial injury is a progressive
decline in heart muscle function following the acute myocardial
infarction. According to another embodiment, the progressive
myocardial injury is heart failure.
[0225] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges which may
independently be included in the smaller ranges also is encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either both of those included limits
are also included in the invention.
[0226] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the described
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0227] As used herein and in the appended claims, the singular
forms "a", "and", and "the" include plural referents unless the
context clearly dictates otherwise. All technical and scientific
terms used herein have the same meaning.
[0228] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the described invention is not entitled to antedate such
publication by virtue of prior invention. Further, the dates of
publication provided may be different from the actual publication
dates which may need to be confirmed independently.
EXAMPLES
[0229] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the described invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Phase I Clinical Trial
Example 1
Selection of Eligible Subjects
[0230] Subjects/patients presenting with symptoms and clinical
findings suggestive of a myocardial infarction will receive
emergency diagnostic and clinical management according to
institutional guidelines. If a transmural (meaning through the
wall) myocardial infarction is confirmed, the time of first
symptoms and the time of successful stent placement will be
recorded. Revascularized subjects will receive appropriate medical
management to reduce ventricular wall stresses according to
institutional guidelines. The term "revascularized" as used in this
embodiment, refers to the successful placement of a stent.
[0231] All types of stents, including drug-eluting stents (e.g.,
paclitaxel or sirolimus) are acceptable for use in the
revascularization of the infarct related artery ("IRA"). Previous
studies employing balloon catheters to infuse cell products have
reported no limits for reference vessel diameter for the placement
of the stent. Since this study is designed to distribute the cell
product into the IRA circulation, and in an attempt to limit the
potential for damage to very small vessels, the described invention
requires that stents be placed prior to infusion of the chemotactic
hematopoietic stem cell product of the described invention.
[0232] Stent-related drug effects occur predominantly at the site
of contact of the stent with the vessel wall. Consequent to balloon
dilatation, there is limited blood flow across the stent during
cell infusion, and therefore no significant adverse drug-mediated
effect on the CD34+ cells in the chemotactic hematopoietic stem
cell product is expected. Moreover, prior clinical studies have
shown that by 96 hours after drug-eluting stent placement, whole
blood levels of either paclitaxel or sirolimus are below the limits
of detection. Therefore, tissue levels in the myocardial sites to
which the infused CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity are intended to migrate are
expected to be inconsequential. See Sousa, J. et al., Circulation
107: 2274-79, 2383-89 (2003).
[0233] During revascularization, a subject's cardiac function and
perfusion will be assessed by standard methods. Relevant measures
of cardiac function include assessment of global and regional
ejection fraction, ventricular volumes, resting and stress
perfusion, segmented wall motion, and, following a myocardial
infarction, infarct size.
[0234] The term "diastole" refers to the normal postsystolic
dilation of the heart cavities during which they fill with blood.
The term "systole" refers to contraction of the heart, especially
of the ventricles, by which the blood is driven through the aorta
and pulmonary artery to traverse the systemic and pulmonary
circulations, respectively.
[0235] The term "ejection fraction" ("EF") refers to the percentage
of blood emptied from the ventricle during contraction More
specifically, it is the fraction of the end-diastolic volume that
is ejected with each beat; that is, it is stroke volume (SV)
divided by end-diastolic volume (EDV). The volume of blood within a
ventricle immediately before a contraction is known as the
end-diastolic volume, while the volume of blood left in a ventricle
at the end of contraction is known as end-systolic volume. The
difference between end-diastolic and end-systolic volumes is the
stroke volume, the volume of blood ejected with each beat. In a
healthy 70-kg (154-lb) male, the SV is approximately 70 ml and the
left ventricular EDV is 120 ml, giving an ejection fraction of
70/120, or 0.58 (58%). An EF within the range of from 55-60% is
considered normal. The ejection fraction of the right ventricle
("RVEF") normally is equal to that of the left ventricle ("LVEF")
within narrow limits.
[0236] Other measures of cardiac function include assessment of the
stroke volume index and velocity of circumferential fiber
shortening. Strauer, et al., Circulation 106: 1913-18 (2002).
Stroke volume (SV) is the amount of blood the left ventricle ejects
in one beat, measured in milliliters per beat (ml/beat). SV can be
indexed to a patient's body size by dividing SV by Body Surface
Area (BSA) to yield the Stroke Index (SI).
[0237] Assessment of repair of infarcted myocardium also has
included evaluation of peri-infarct region perfusion using thallium
scintigraphy. Id. The term "perfusion" refers to the process of
nutritive delivery of arterial blood to a capillary bed in
biological tissue. Perfusion ("F") may be calculated using the
formula F=(Pa-Pv)/R, where Pa is mean arterial pressure. Pv is mean
venous pressure, and R is vascular resistance:
[0238] Magnetic resonance imaging (MRI) is a useful tool for
assessing cardiac function and viability (infarct size) in this
setting. See Yin, A, et al., Blood 90: 5002-5012 (1997).
[0239] The day after successful stenting, subjects will be assessed
for study eligibility and, if appropriate, will be offered informed
consent to participate in the study. Subjects exhibiting symptoms
for no more than three (3) days prior to successful stent placement
will be assessed, prior to discharge, for study eligibility.
Subjects found to meet eligibility criteria (see infra) will be
offered informed consent to participate.
[0240] Consented subjects will have a study entry SPECT no sooner
than 96 hours after stent placement. Subjects are eligible to
proceed on study if the LVEF is less than or equal to 50% on
echocardiography and a segmental ventricular wall abnormality is
observed in the IRA. Eligible subjects immediately can complete
baseline cardiac function and perfusion assessment.
[0241] Specifically, baseline cardiac function includes:
[0242] Cardiac Perfusion. Perfusion will be assessed using a
routine Technetium (Tc-99m) Sestamibi radionuclide scan at rest and
after intravenous adenosine. The Emory Cardiac Toolbox will be used
for image quantification. Evaluation will use a 17-segment model. A
core review lab will assess the perfusion studies with the
interpreter blinded to the study cohort. Improvements in perfusion
will be expressed in semi-quantitative terms (yes/no). The
percentage of patients observed to have improvement in perfusion
will be compared between dose cohorts.
[0243] MRI. Regional and global wall motion, infarct size, and left
ventricular ("LV") volumes will be measured using MRI. Subjects
will receive Gadolinium contrast during scanning. MRI scan will use
the breath holding technique. Steady state precession imaging to
obtain global and regional LV function will be performed as will
Gadolinium imaging. Left ventricular end systolic and diastolic
volumes, LVEF, LV end diastolic dimension, wall thickness in
systole and diastole of the infarcted region, and infarct size will
be reported using the AHA/AVV 17-segment model with transmural
extent of the infarct reported as <25%, 26%-50%, 51%-75% and
>76%. A core review laboratory will assess MRI with the
interpreter blinded to the study cohort.
[0244] To be selected for this study, subjects must meet all of the
following clinical criteria ("inclusion criteria"): [0245] Age:
18-75 years; [0246] Acute ST segment elevation myocardial
infarction meeting ACC/AHA criteria, with symptoms of chest pain
within 3 days of admission. Criteria include (ST elevation>1 mm
in limb leads or 2 mm in two or more precordial leads and increased
levels of troponin, creatine kinase MB (CPK MB) or both), New York
Heart Association (NYHA) heart failure class (to be recorded) of I,
II or III; [0247] Eligible for percutaneous coronary intervention
(PCI); [0248] Eligible for MRI; [0249] Eligible for Single Proton
Emission Computed Tomography (SPECT) [0250] Subject must be able to
provide informed written consent and must be willing to participate
in all required study follow-up assessments; [0251] Subjects must
have a hemoglobin content (Hgb)>10 grams/dL, white blood cell
count (WBC)>3500 cells/mm.sup.3, a platelet count>100,000
cells/mm.sup.3 and an international normalized ratio (INR, a blood
coagulation test)<2.0 the day before the bone marrow collection;
[0252] Subjects must have a serum creatinine<2.5, total
bilirubin<2.0 within 7 days of the bone marrow collection;
[0253] IRA and target lesion must be clearly identifiable when
disease is present in more than one vessel; [0254] Successful
reperfusion and intracoronary stent placement, with Thrombolysis In
Myocardial Infarction (TIMI) 2 or 3 flow and IRA with <20%
stenosis after revascularization; [0255] Subjects must be deemed
eligible to receive conscious sedation, mini-bone marrow harvest,
and second catheterization for Chemotactic Hematopoietic Stem Cell
Product infusion; [0256] The type of stent used and time and date
inserted must be recorded; [0257] Drug eluting stents should be
limited to paclitaxel or sirolimus types; [0258] Included subjects
must have an expected survival of at least one year and must not
have multiple vessel disease after revascularization, or be
expected to require intervention within 6 months of study
entry.
[0259] Subjects who satisfy any one of the following criteria do
not qualify for, and will be excluded from, the study ("exclusion
criteria"): [0260] Subjects who are not candidates for percutaneous
intervention, conscious sedation, MRI, SPECT imaging or mini-bone
marrow harvest; [0261] History of sustained chest pain unrelieved
by nitrates, occurring 4 or more days before revascularization;
[0262] Subjects who fail to re-perfuse the infarct related coronary
artery or to have successful stent placement; [0263] Subjects
presenting with cardiogenic shock (systolic pressure<80 on
vasopressors or intra aortic counterpulsation); [0264] Subjects
with a side branch of the target lesion>2 mm and with ostial
narrowing>50% diameter stenosis after revascularization; [0265]
Subjects unable to receive aspirin, clopidogrel or ticlopidine;
[0266] Subjects receiving warfarin must have an INR less than or
equal to 2; the term INR refers to INR International Normalized
Ratio, which is a system established by the World Health
Organization (WHO) and the International Committee on Thrombosis
and Hemostasis for reporting the results of blood coagulation
(clotting) tests; [0267] Subjects with severe aortic stenosis;
[0268] Subjects with severe immunodeficiency states (e.g. AIDS);
[0269] Subjects with cirrhosis requiring active medical management;
[0270] Subjects with malignancy requiring active treatment (except
basal cell skin cancer); [0271] Subjects with documented active
alcohol and/or other substance abuse; [0272] Females of child
bearing potential unless a pregnancy test is negative within 7 days
of the mini-bone marrow harvest; [0273] Subjects with ejection
fractions greater than 50% on study entry by SPECT (96 to 144 hours
after stent placement); [0274] Subjects with less than three months
of planned anti-platelet therapy post index procedure; [0275]
Subjects with multi vessel disease after revascularization
requiring subsequent planned intervention during the next 6 months;
[0276] Subjects with participation in an ongoing investigational
trial; [0277] Subjects with active bacterial infection requiring
systemic antibiotics.
[0278] Baseline assessments of cardiac function and cardiac
perfusion will be obtained one day prior to the planned mini-bone
marrow harvest and infusion of the chemotactic hematopoietic stem
cell product (see infra). A mini-bone marrow harvest ("MMH") will
be performed the day following baseline assessment of cardiac
function and cardiac perfusion.
Example 2
Cardiac Catheterization
[0279] Sterile Preparation and Draping
[0280] The subject will be brought into the Cardiac Catheterization
Laboratory after the investigator has obtained an informed consent.
The subject will receive a sterile preparation and draping in the
Cardiac Catheterization Laboratory.
[0281] Cardiac Catheterization
[0282] Vascular access will be obtained by standard technique using
right or left groin. A sheath will be placed in the femoral artery
or the right or left brachial artery. Coronary arteriographic
examination will be performed by obtaining standard views of both
right and left coronary arteries. Multiple views will be obtained
to identify the previously stented infarct related artery. All
subjects will receive standard medications during the
catheterization procedure in accordance with routine practice.
Example 3
Acquisition Process For Acquiring Chemotactic Hematopoietic Stem
Cell Product that is then Enriched for CD34+ Cells
[0283] While it is contemplated that any acquisition process
appropriate for acquiring the chemotactic hematopoietic stem cell
product comprising potent CD34+ cells is within the scope of the
described invention, the following example illustrates one such
process referred to herein as a mini-bone marrow harvest
technique.
[0284] Preparation of Harvesting Syringes
[0285] Prior to the bone marrow harvest, forty 10 cc syringes
loaded with about 2-ml of a preservative free heparinized saline
solution (about 100 units/ml to about 125 units/ml, APP Cat. No.
42592B or equivalent) will be prepared under sterile conditions.
Heparin will be injected via a sterile port into each of two 100-ml
bags of sterile 0.9% normal saline solution ("Normal Saline",
Hospira Cat. No. 7983-09 or equivalent) following removal of 10 cc
to 12.5 cc of normal saline from each hag, resulting in a final
heparin concentration of about 100 units/ml (U/ml) to about 125
units/ml (U/ml). 2-ml of the preservative free heparin solution
(about 100 U/ml to about 125 U/ml) will be loaded under sterile
conditions into each of the forty 10 cc syringes, which then are
capped and placed into a sterile bag for transport to the
harvesting site.
[0286] Subjects will be prepared for bone marrow harvest after
written informed consent is obtained as detailed in Example 1.
Conscious sedation will be provided using standard institutional
procedures and guidelines. Bone marrow harvest will be conducted
under sterile conditions. The term "sterile conditions" as used
herein includes proper scrubbing and gowning with a sterile mask
and gloves worn by the harvesting attending and assistant. The
harvesting procedure can be performed outside of an operating room
as follows: after sterile prepping and draping, each iliac crest
should be anaesthetized with a 1% lidocaine solution using a
minimum of 10-ml for each crest. The area of anesthesia should be a
circular area no less than 10 cm in diameter. The harvesting needle
is inserted until the iliac crest is punctured. The cap and stylet
is removed and 2-ml of marrow is harvested into the 10-ml
harvesting syringe containing 2-ml of the heparin solution. The
syringe then is removed and placed on the sterile field. After
re-inserting the stylet, the harvesting needle is advanced slightly
and then rotated 90.degree.. The stylet is then removed and an
additional 2-ml of marrow is drawn into the harvesting syringe
retrieved from the sterile field. This procedure is repeated two
more times until the harvesting syringe contains 8-ml of marrow for
a total of 10-ml of heparinized marrow at a final heparin
concentration of about 20 U/ml to about 25 U/ml. Finally the full
harvesting syringe is handed to the harvesting assistant and shaken
and infused in the sterile collecting bag as described below. The
harvesting physician then takes the other harvesting needle that
had been flushed previously with the heparin solution and repeats
this process.
[0287] The full harvesting syringe is infused in the sterile
collecting bag as follows. The harvesting assistant is handed the
full harvesting syringe and empties it in the 500-ml collecting bag
though the sterile adaptor attached to the bag. Then the harvesting
needle is flushed with the heparin solution in the flushing syringe
and returned to the sterile field.
[0288] The harvesting process is repeated on one iliac crest until
about 19 syringes have been collected and emptied in the collecting
bag. The same process is repeated on the other iliac crest until
another about 19 syringes have been filled. A total of thirty-eight
8 ml aspirations from both iliac crest (ideally 19 from each iliac
crest) will result in 302-ml of bone marrow harvested in a final
volume of 380 ml at a heparin concentration of about 20 U/ml to
about 25 U/ml.
[0289] The collecting bag is sealed by tying off the connecting
tube three times and then clamped distal to the ties. The bag is
appropriately labeled "Human Bone Marrow Collection" and the
results of the harvesting procedure, including final volume
collected and any procedure related complication, are recorded on
the Mayo Clinical Risk Score (MCRS) case report form. The completed
label is affixed to the bone marrow bag. The bag then is placed in
a sterile carrying bag to be transported to the processing
facility.
Example 4
Preparation of the Bone Marrow Product for Transportation
[0290] In one embodiment, the harvested bone marrow is transported
to the processing facility as follows. When the clinical site is
prepared to ship the bone marrow preparation, 24-hour notice will
be provided to the processing facility. The processing laboratory
will make shipping arrangements at the earliest possible time for
pickup for same day delivery to the processing laboratory.
Immediately after the bone marrow is collected, the bone marrow
product will be placed in the supplied shipping container. The
shipping container contains two small blocks of frozen wet ice on
the bottom and a sheet of bubble wrap on top of the wet ice. The
bone marrow product is placed into a secondary bag and the
secondary bag is placed on top of the bubble wrap. A temperature
tag monitor (a sensor used to monitor the internal temperature) is
affixed to the interior of the box. Another layer of bubble wrap
then is placed on top of the product before the shipping container
is sealed off.
Example 5
Selection of CD34+ Cells from the Harvested Bone Marrow Product
[0291] CD34+ cells will be isolated from the harvested bone marrow
product. In one embodiment, CD34+ cells will be isolated using the
anti-CD34 monoclonal antibody (Mab), Dynabeads.RTM. M-450 Sheep
anti-Mouse IgG, and PR34+ (TM) Stem Cell Releasing Agent components
of the Isolex 300i Magnetic Cell Selection System (Baxter
Healthcare Corp. Cat. No. 4R9734) as described in U.S. Pat. Nos.
5,536,475, 5,035,994, 5,130,144, 4,965,204, 5,968,753, 6,017,719,
6,251,295, 5,980,887, 6,676,937, U.S. Published Application No.
2003/0232050, and the Isolex 300i Package Insert, each of which is
incorporated herein by reference. This operating system has been
adapted for isolation of CD34+ cells from bone marrow according to
the described invention.
[0292] Upon arrival at the processing laboratory, the harvested
bone marrow product (in the collecting bag) is inspected
immediately and the bag checked for any leakage. The collection
should be free flowing with no apparent clumps and should not be
hemolyzed. The collection will not be used if the integrity of the
bag has been breached in any way.
[0293] The bone marrow product should be processed within about 12
hours to about 24 hours of inspection. A 300-ml or 400-ml transfer
pack container is obtained, and a plasma transfer set is attached
to the sampling port of the container. The bone marrow product is
transferred from the collecting bag to the transfer pack container.
The pooled bone marrow collection product is mixed thoroughly by
inverting the container twenty (20) times.
[0294] The pooled bone marrow collection product then is sampled
for analysis. In one embodiment, a total volume of 2.0 ml of the
product is removed and aliquoted as follows: 0.3 ml is used for a
duplicate run of Complete Blood Count (CRC) using a hematology
analyzer; 0.2-ml is dispensed into a 75.times.100-mm glass tube for
the detection of Gram positive and Gram negative bacteria by Gram
Stain (Gram Stain Kit, VWR, Cat. NO. BB231401); as a sterility
check, 0.6-ml is dispensed into a Tryptic Soy Broth (TSB) (VWR,
Cat. No. 29446-184) bottle for aerobic bacteria growth assay,
0.6-ml is dispensed into a Fluid Thioglycollate Media (FTM) (VWR
Cat. #29446-138) bottle for anaerobic bacteria growth assay, and
0.3-ml is used in flow analysis for CD34+ cell enumeration and cell
viability.
[0295] The collection is weighed on an electronic scale, and the
appropriate tare weight of the collection bag recorded. The
relationship of the volume of the bone marrow product to the weight
of the product can be expressed as
Volume (ml)=[Weight (gm) of product-Tare weight of bag (gm)]/1.06
(gm/ml) (Formula 1)
[0296] The number of Total Nucleated Cells (TNC) in the bone marrow
product is calculated using the white blood cell (WBC) count
obtained from the CRC according to the following relationship:
WBC/.mu.l.times.1000.times.Product volume (ml) (Formula 2)
[0297] The number of CD34+ cells in the bone marrow product is
calculated from the following relationship:
Total CD34+cells in the bone marrow product=Number of
CD34+cell/.mu.l.times.1,000.times.Product volume (ml) (Formula
3)
[0298] The Red Blood Cell (RBC) volume of the bone marrow
collection product is calculated from the following
relationship:
RBC volume (ml)=Product volume (ml).times.Hematocrit (%)/100
(Formula 4),
[0299] If the collection contains more than 20 ml of RBC, red blood
cell depletion is required. RBCs are depleted by centrifugation.
Centrifugation at 1000.times.g for 20 minutes at ambient
temperature is performed to separate the buffy coat from the RBCs.
The term "huffy coat" refers to a thin grayish white fraction of a
blood sample that contains most of the white blood cells
(leukocytes). Immediately after centrifugation, a 60 ml syringe is
connected to the bottom of the centrifugation bag and the RBCs are
removed. More than one syringe may be needed to collect all the
packed RBC. The RBC depleted bone marrow product then is washed to
remove fat contents.
[0300] A 1-ml syringe is used to remove 0.3-ml of the RBC-depleted
bone marrow cell product through the transfer set attached to the
product bag and a CBC performed. The TNC of the RBC depleted bone
marrow product is determined from the relationship:
Total TNC of the RBC depleted product=WBC/.mu.l of RBC depleted
product.times.1000.times.180-ml (Formula 5)
[0301] The TNC recovery of the RBC depleted product, which must be
at least 80% of the original product count, is calculated from the
relationship:
TNC recovery=TNC of the RBC depleted product/TNC of the unprocessed
product.times.100% (Formula 6)
[0302] The total RBC volume is calculated as described supra; the
RBC volume in the RBC depleted product should be less than
<20-ml.
[0303] In one embodiment according to the described invention, the
Isolex 300i system is used to process the RBC-depleted product or
the bone marrow product whose RBC volume is <20 ml according to
the following processing steps:
[0304] (i) The bone marrow is washed automatically to remove
platelets;
[0305] (ii) CD34 positive (CD34+) cells are labeled specifically
for selection by incubation with the Isolex 300i CD34 monoclonal
antibody (Mab);
[0306] (iii) Unbound reagent is removed by washing the cell
suspension with buffer solution;
[0307] (iv) Sensitized CD34+ cells (meaning CD34+ cells labeled
with CD34 Mab) are captured by Dynabeads M-450 Sheep anti-Mouse
IgG;
[0308] (v) A selection column is used to separate the
magnetically-labeled Dynabeads having captured CD34.sup.+ cells
from unwanted cells, which are washed through the selection column
and collected in the Negative Fraction Bag; and
[0309] (vi) PR34+ Stem Cell Releasing Agent releases CD34+ cells
from the column, and the CD34.sup.+ cells are collected in the End
Product Bag. The system performs several washing steps, disposing
of most of the liquid into the Buffer Waste Bag.
[0310] The Isolex.RTM. selected CD34+ fraction is assayed as
follows to determine WBC and CD34+ cell yields. The volume of the
CD34 Positive Fraction is determined by mixing the cells in the End
Product Bag; the bag is gently massaged by hand to ensure even cell
distribution. A transfer set is inserted into the sampling port of
the End Product Bag and a 60-ml syringe attached. The cell
suspension is withdrawn into the syringe (maximum 50-ml at a time)
in order to measure the total volume.
[0311] A 3-ml or 5-ml syringe is used to remove a 2.0-ml sample
from the End Product Bag through the transfer set for quality
control testing. The aliquoted volumes of the samples and the
analyses performed on those samples are as previously described,
i.e., CBC: 0.3-ml; Gram stain: 0.3-ml; CD34+ cell enumeration and
cell viability: 0.2-ml.
[0312] The total TNC of the CD34 Positive Fraction is calculated
from the relationship:
Total TNC of the Positive Fraction=WBC/.mu.l of the Positive
Fraction.times.1000.times.Volume of the Positive Fraction (Formula
7)
[0313] The TNC recovery of the Positive Fraction, which must be
less than 5% of the original product count, is calculated from the
following relationship:
TNC recovery=Total TNC of the Positive Fraction/Total TNC of the
unprocessed product.times.100% (Formula 8)
[0314] The total number of viable CD34+ cells in the Positive
Fraction is determined from the following relationship:
Total CD34+cells in the Positive Fraction=Number of
CD34+cells/.mu.l of the final product.times.1,000.times.Final
product volume (ml) (Formula 9)
[0315] The CD34+ cell recovery of the Positive Fraction s
calculated from the following relationship:
CD34+cell recovery=Total CD34+cells of the Positive Fraction Total
CD34+ cells of the unprocessed product.times.100% (Formula 10).
Example 6
Preparation of Selected CD34+ Cells for Transfusion
[0316] Samples of the chemotactic hematopoietic stem cell product
will be removed to be assayed for WBC count, by flow cytometry (for
CD34+ cell enumeration and viability), Gram stain, and
sterility.
[0317] CD34+ cells are characterized by flow cytometric analysis
featuring CD34bright and CD45dim fluorescence by double labeling
with anti-CD34 and anti-CD45 antibodies (Beckman Coulter, PN
IM3630). CD34+ cells and CD45+ cell viability is determined by
excluding the dying cells which take up the intercalating DNA dye
7-aminoactinomycin D (7AAD). See Brocklebank A M, Sparrow R L.
Cytometry. 2001; 46:254-26 (2001); Barnett D, et al. Br. J.
Haematol. 106:1059-1062 (1999); Sutherland, et al., J Hematotherapy
5:213-226 (1996), and U.S. Pat. Nos. 4,520,110; 4,859,582;
5,055,556; European Patent No. 76.695; Canadian Patent No.
1,179,942 (PE, APC); U.S. Pat. No. 4,876,190 (PerCP); U.S. Pat.
Nos. 5,268,486; 5,486,616; 5,569,587; 5,569,766; 5,627,027 (Cy);
U.S. Pat. Nos. 4,714,680; 4,965,204; 5,035,994 (CD34); U.S. Pat.
No. 5,776,709 (Lyse/no-wash method); U.S. Pat. Nos. 5,723,218 and
5,187,288 (TruCOUNT Tubes), the contents of each of which is
incorporated by reference herein in its entirety.
[0318] Any flow cytometer or an equivalent device can be used for
conducting analysis of CD34+ cell enumeration and viability. In one
embodiment, the processing laboratory employs a BD FACSCalibur.TM.
flow cytometer and BD FACSComp.TM. software is used for instrument
setup and monitoring. A template and a panel of legend labels are
preinstalled for acquisition and analysis. Prior to use, the
reagents, namely CD45FITC/CD34PE, Stem-Count Fluorospheres,
Concentrated Ammonium Chloride Lysing Solution, and 7AAD Viability
Dye, are brought to ambient temperature. CD34+ cell controls are
run as a positive control to affirm that the instrument is set up
for analyzing CD34+ cells, and the results are compared with the
manufacturer's pre-determined CD34 percent range.
[0319] The unprocessed bone marrow product and Isolex processed
chemotactic hematopoietic stem cell products may be analyzed by
many different procedures. In one embodiment, or example,
immediately upon receiving the sample, if the WBC count of the
sample is greater than 2.times.107 cells per ml, the sample is
diluted with Sheath fluid to achieve a cell count of about
2.times.107 WBC per ml. 100 .mu.l of the diluted product is
aliquoted into two 15.times.100 mm tubes. Using a micropipetter, 20
.mu.l of CD45FITC/CD34 PE and 7-AAD viability dye reagent are added
into each tube and the samples gently vortexed. The tubes are
covered with aluminum foil and left at ambient temperature for 15
to 20 minutes. RBCs are lysed by adding 1.5 ml of 1.times. Lysing
Solution to each tube, vortexing gently. The tubes are incubated
for ten minutes at ambient temperature, protected from light. The
samples are stored at about 2.degree. C.-about 8.degree. C. (i.e.,
on an ice bath) protected from light until data acquisition is
performed. Data acquisition must be performed within one hour of
adding the lysing buffer. Before data acquisition, Stem-Count
Fluorospheres are resuspended by end-over-end rotation (10 times).
100 .mu.l of Fluorospheres is added to each tube and gently
vortexed taking care not to generate air bubbles. The absolute
count of CD34+ cells in the product is calculated from the
relationship:
Number of viable CD34+cells per .mu.l of product=LCD34.times.FAC
(Formula 11)
[0320] where LCD34 is the averaged number of events for Live
CD34+/All CD 45+; "FAC" is Fluorospheres Assayed Concentration; and
F is the averaged number of Fluorosphere singlets counted.
[0321] The volume of CD34+Positive Fraction is calculated to obtain
the number of CD34+ cells required for the required dosing. The
Required Positive Fraction Volume (ml) is defined as:
The Requested CD34+cell dosage/(Total CD34+cells per .mu.l in the
Positive Fraction.times.1,000). (Formula 12)
[0322] An appropriate number of cells is dispensed into a 50 ml
conical tube and centrifuged at 500.times.g for 10 minutes. The
supernatant is removed using a 30 ml serological pipette and
disposed of as waste while exercising care not to disperse the cell
pellets at the bottom of the tubes during this process. The
infusion solution (20 ml) is added into the CD34+Cell Positive
Fraction tube and the cells dispersed using a 10 ml serological
pipette by repeat pipetting. The resuspended cells are centrifuged
for 10 minutes at 500 g. A 30 ml serological pipette is used
(without disturbing the cell pellet) to transfer the
supernatant/infusion solution into a 50 ml conical tube with a
label "Positive Fraction Supernatant" affixed. The tube containing
the supernatant is vortexed to homogenize the solution. A 10 ml
serological pipette is used to transfer 10 ml of the homogenized
supernatant back to the CD34+ Cell Positive Fraction tube. The
remaining 10 ml of suspension in the Supernatant tube will be used
for sterility testing (5 ml each into a TSB (Trypticase Soy Broth)
bottle and an FTM (Fluid Thioglycollate) bottle). The cells in the
CD34+ Cell Positive Fraction are resuspended by slowly withdrawing
and aspirating through a blunt end needle affixed to a 10 ml
syringe (Infusion Syringe) several times. The cell suspension is
withdrawn into the syringe, any air bubbles re aspirated off, and
the blunt end needle removed. The infusion syringe is attached to
the injection port of a 4-way stopcock.
[0323] The chemotactic hematopoietic stem cell product of the
described invention will be released for infusion only if it meets
the following criteria: [0324] CD34.sup.+ cell purity of at least
about 70%, 75%, 80%, 85%, 90% or 95%; [0325] A negative Gram stain
result for the selected positive fraction; [0326] Endotoxin Levels:
less than about 0.5 endotoxin units/ml; [0327] Viable CD34.sup.+
cell yield of the "Chemotactic hematopoietic stem cell product"
meets the required dosing as per the treatment cohort; [0328]
CD34.sup.+ cells are at least about 70%, 75%, 80%, 85%, 90% or 95%
viable by 7-AAD; [0329] USP sterility result for "Positive Fraction
Supernatant": negative (14 days later); and [0330] Bone marrow
CD34.sup.+ cell selection was initiated within about 12 hours to
about 24 hours of completion of bone marrow harvest.
[0331] Sterility assessment on the stem cell product including gram
staining and endotoxin will be performed prior to product release
for infusion. USP sterility (bacterial and fungal) culture will be
performed and the results will be reported to the principal
investigator. In the event of a positive USP sterility result, the
subject and attending physician on call will be notified
immediately, provided with identification and sensitivity of the
organism when available, and documentation of appropriate
anti-microbial treatment and treatment outcome will be recorded by
the investigative site and the sponsor.
[0332] After meeting these release criteria, the chemotactic
hematopoietic stem cell product will be released for infusion and
packaged for transportation to the catheterization facility. A
sample also will be sent for in vitro testing.
[0333] According to some embodiments, product will be released only
if CD34+ cell selection is initiated within 12 hours to about 24
hours of completion of bone marrow harvest and only if it is to be
infused within about 48 hours to about 72 hours of completion of
bone marrow harvest.
[0334] According to some embodiments, the nonexpanded, isolated
population of autologous mononuclear cells containing CD34+ cells,
which further contain potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity is divided into aliquots,
which are frozen at -86.degree. C. and cryostored in the vapor
phase of a liquid nitrogen freezer for subsequent administration.
Each of these aliquots can be used to prepare a thawed chemotactic
hematopoietic stem cell product as follows. The frozen nonexpanded,
isolated population of autologous mononuclear cells are thawed at a
sufficient time before planned administration the sterile
nonexpanded, isolated population of autologous mononuclear cells
comprising CD34+ cells, which further contain a subpopulation of
potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic
activity will be enriched for CD34+ cells, which further contain a
subpopulation of potent CD34+/CXCR-4+ cells that have
CXCR-4-mediated chemotactic activity so as to yield the thawed
chemotactic hematopoietic stem cell product. Samples of this thawed
chemotactic hematopoietic stem cell product will be removed to be
assayed for WBC count, by flow cytometry (for CD34+ cell
enumeration and viability), Gram stain, and sterility. The thawed
chemotactic hematopoietic stem cell product will be released for
infusion within about 48 hours to about 72 hours of thawing of the
frozen aliquot of the sterile nonexpanded, isolated population of
autologous mononuclear cells comprising CD34+ cells.
Example 7
Formulation of the Chemotactic Hematopoietic Stem Cell Product
Comprising CD34+ Cells
[0335] The chemotactic hematopoietic stem cell product is
formulated in 10-ml of saline (0.9% Sodium Chloride, Injection,
USP, Hospira, Cat#7983-09) supplemented with 1% HSA (Human Albumin
USP, Alpha, Cat. #521303) ("Infusion Solution") and more than 20%
autologous serum. In addition, there may be some trace amount of
materials (quantities not determined) in the Chemotactic
hematopoietic stem cell product that are used and left over during
the product processing. These materials include: Dulbecco's
Phosphate Buffered Saline-Ca++, Mg++ Free (D-PBS) (Baxter, Cat. #
EDR9865), Sodium Citrate (Baxter/Fenwal, Cat. #4B7867), Hetastarch
(Abbott Laboratories, Cat. #0074-7248-03), IVIg (Gammagard.RTM.
Immune Globulin Intravenous, Baxter, Cat. #060384) and the reagents
in the Isolex.RTM. 300i Stem Cell Reagent Kit (Baxter, Cat.
#4R9734) including anti-CD34 monoclonal antibody, stem cell
releasing agent and Sheep anti-mouse magnetic beads.
Example 8
Transporting Chemotactic Hematopoietic Stem Cell Product to the
Catheterization Facility
[0336] According to the original plan, the chemotactic
hematopoietic stem cell product that met the release criteria was
to be loaded into a sterile 10 cc syringe in a Class 100 biological
safety cabinet located within a controlled aseptic environment,
e.g., at minimum, a Class 100,000 cell processing facility; class
10,000 is preferable, but not required. The chemotactic
hematopoietic stem cell product was suspended in 10-ml PBS
supplemented with HSA and the container labeled in accordance with
release criteria. The original plan called for four dosing cohorts
consisting of live subjects each in each cohort. The first was to
receive about 5.times.10.sup.6 CD34+ cells, the second about
10.times.10.sup.6 CD34+ cells, the third about 20.times.10.sup.6
CD34+ cells and the fourth about 30.times.10.sup.6 CD34+ cells.
Subjects in the higher dosing cohorts with inadequate CD34+ cell
quantities to meet the assigned cohort dose were to be added to a
prior cohort at the greatest possible CD34+ cell dose. The loaded
infusion syringe was attached to a four-way stopcock along with a
flushing syringe, capped and have safety guards applied to prevent
leakage. The delivery apparatus was sealed in a double sterile bag
and placed in a secure transportation box for transportation to the
cardiac catheterization facility. Following release of the
chemotactic hematopoietic stem cell product and cohort assignment,
the chemotactic hematopoietic stem cell product was shipped to the
catheterization site for direct infarct-related artery infusion
("intravascular administration").
Example 9
Intra-Coronary Infusion of Chemotactic Hematopoietic Stem Cell
Product
[0337] Upon notification from the cell processing facility that the
chemotactic hematopoietic stem cell product had been released for
infusion (see supra), the subject/patient was scheduled to arrive
at the catheterization facility at a time to coincide with the
arrival of the chemotactic hematopoietic stem cell product.
[0338] Cardiac enzymes (brain natriuretic peptide (BNP), troponin
and CPK MB), complete blood counts, a full chemistry panel (renal
and liver function test) and an EKG were performed just prior to
chemotactic hematopoietic stem cell product infusion. Clinical
assessment of the stage of heart failure according to the New York
Heart Association's (NYHA) functional classification system was
recorded.
[0339] Upon receipt of the chemotactic hematopoietic stem cell
product and final quality assurance release (by facsimile) for
infusion, the subject 1 did undergo cardiac catheterization as
detailed above. Coronary arteriography was performed to assess for
patency (meaning openness, freedom from blockage) of the infarct
related artery and Thrombolysis in Myocardial Infarction (TIMI)
angiographic flow. A balloon catheter over a wire was placed in the
stented segment of the infarct related artery. Any appropriate
balloon dilatation catheter having an internal diameter of at least
about 0.36 mm compatible with the chemotactic hematopoietic stem
cell product infusion can be used. After positioning, the balloon
wire was removed. The chemotactic hematopoietic stem cell product
delivery apparatus was removed from the transportation case.
[0340] The delivery apparatus was in a sterile bag and had safety
blocks attached to the infusion syringe (containing the chemotactic
hematopoietic stem cell product) and the flushing syringe. The
apparatus consisted of the infusion syringe (containing 10 ml of
the chemotactic hematopoietic stem cell product) and the flushing
syringe (containing 6 ml of flushing solution) wherein both were
attached to a sterile four-way stopcock. The entire delivery
apparatus was shaken gently to resuspend the CD34+ cells in the
infusion solution. The flushing syringe was used to eliminate all
air bubbles in the apparatus (to prevent air emboli) and the
delivery apparatus then attached to the balloon dilatation catheter
via the stopcock.
[0341] Delivery of the chemotactic hematopoietic stem cell product
to the subject by infusion proceeded as follows. First, with the
stopcock open between the flushing syringe (6 ml solution) and the
central lumen of the balloon catheter, 1 ml of flushing solution
was infused (after removal of the guard) into the central lumen of
the catheter over 15 seconds. Second, the balloon was inflated at
two atmospheres of pressure within the stent to avoid damage to the
coronary artery endothelium and then the stopcock valve adjusted to
allow infusion of the chemotactic hematopoietic stem cell product
distal to the inflated balloon (after removal of the guard). With
the balloon inflated, about 3 cc to about 4 cc from the infusion
syringe was infused by hand over a period of about 30 seconds to
about 45 seconds (to be timed and documented). The balloon remained
inflated to allow adhesion of the CD34+ cells and to prevent back
flow for a total of about 2 minutes to about 3 minutes (including
the time for infusion). In between infusions, the balloon remain
deflated for 3 minutes to allow, restoration of blood flow
(reperfusion). It was expected that 3 infusions will be required to
empty the infusion syringe. Third, upon completion of infusing the
chemotactic hematopoietic stem cell product and with the balloon
deflated, the valve on the stopcock was adjusted to allow filling
of the infusion syringe from the flushing syringe. Finally, with
the balloon inflated (about 2 minutes to about 3 minutes), the 4 ml
of flushing solution now in the infusion syringe was infused over a
period of about 30 seconds to about 45 seconds to dislodge any
residual CD34+ cells from the syringe and catheter into the IRA
circulation. The catheter then was removed.
[0342] An infusion-related ischemia (inadequate blood flow)
assessment was performed during the first 24 hours after
chemotactic hematopoietic stem cell product infusion. An EKG at
about 12 hours and at about 24 hours and analytical chemistry of
cardiac enzymes (BNP, troponin and CPK MB) about every 8 hours for
about 24 hours was obtained. Arrhythmia assessment (24 hour Holter
monitor) was performed immediately post-chemotactic hematopoietic
stem cell product infusion.
[0343] All subjects were provided with digital thermometers and a
log book to record twice daily temperatures for 30 days post
infusion of the chemotactic hematopoietic stem cell product.
Subjects were instructed to notify the investigator site
immediately for temperatures recorded above 100.5.degree. F. Rapid
follow-up with appropriate cultures and radiographic assessments
was performed according to routine clinical standards. Documented
bacterial infections, if any, were reported to the IRB and the
FDA.
[0344] Additional follow-up visits for safety assessments included
visits at 1 week and 2 weeks after product administration. Visit
assessments included a comprehensive medical history and physical
examination, EKG, complete blood counts, full chemistry panel
(renal and liver function test), and measure of serum cardiac
markers (BNP, troponin and CPK MB). Clinical assessment of NYHA
functional class was recorded on week 1 and 2. At 4 weeks post
chemotactic hematopoietic stem cell product infusion, an EKG and
cardiac enzymes (BNP, troponin and CPK MB) was obtained. A 24
Holter monitor was used to assess for arrhythmias. Clinical
assessment of NYHA functional class was recorded. Treadmill
exercise testing using a symptom limiting Bruce protocol was
performed as well.
[0345] At about 3 months and about 6 months post chemotactic
hematopoietic stem cell product infusion at the first infusion
time, a 24 hour Halter monitor was performed. Clinical assessment
of NYHA functional class was recorded. At about 6 months post
chemotactic hematopoietic stem cell product infusion, a symptom
limited treadmill exercise testing using the Bruce protocol was
recorded.
[0346] A safety assessment at about 12 months post chemotactic
hematopoietic stem cell product infusion will include a
comprehensive medical history and physical examination. EKG,
complete blood counts, full chemistry panel (renal and liver
function test), and measure of serum cardiac markers (BNP, troponin
and CPK MB). A 24 hour Holter monitor will be performed. Clinical
assessment of NYHA functional class will be recorded.
[0347] Statistical Analysis
[0348] A paired design, where each subject serves as his or her own
control, was used in some embodiments. Differences between before
and after treatment, per subject, were analyzed for each of the
four numeric cardiac functions (i.e., myocardial contractility; end
systolic volume, end diastolic volume; and perfusion). Linear
regression analysis was used to assess the significance of
increased dosing levels. The null hypothesis is that the slope of
the regression line. (dosing level serving as the independent
variable and the "after" minus the "before" difference serving as
the dependant variable) is equal to zero. The power of rejecting a
false null hypothesis is 0.68 at the 0.05 alpha level of
significance for a high correlation of 0.5 between dosing and
improvement in cardiac function. The 95% confidence interval about
the slope of the regression line was used to assess the medical
significance of the increase in dosing level. If the slope of the
regression line was not significantly different from zero but the
intercept of the regression line is different from zero, then all
treatment groups was combined and a paired t-test will be performed
to assess the overall treatment effectiveness. The null hypothesis
is that the mean of the differences is equal to zero. A Wilcoxon
signed-ranks test also was performed as an additional test to
determine the treatment effectiveness. This test is more powerful
(rejecting a false null hypothesis) than a t-test if the
observations are not normally distributed. The power of the t-test
is 0.79 for rejecting a false null hypothesis at the alpha level of
0.05 and the treatment having a medium size effect (an effect large
enough to be discernable by the naked eye). The medical
significance of the treatment effect size was determined by
computing a 95% confidence interval about the mean of the
differences (the true mean of the differences will lay in this
interval in 95% of tested samples).
[0349] To assess improvement in perfusion, logistic regression was
used with dosing level as the independent variable and perfusion
change (1=yes, 0=no) as the dependant variable. Odds ratios of the
four dosing levels was computed separately with 5.0.times.106 cells
serving as the index group.
[0350] A binomial test was used to assess the significance of CD
34+ cell dosing on perfusion. It was expected that there would be
no spontaneous improvement in a perfusion defect if present on the
baseline perfusion scan. Therefore, any clinically significant
improvement in a perfusion defect when assessed at 6 months and
compared to baseline was considered a treatment effect.
[0351] A concurrent group (non-treated controls) meeting
eligibility but not receiving CD34+ cells was evaluated similar to
the treated group and assessed for significant improvement in
cardiac function/perfusion. Each study site alternated accrual of
treated and non-treated controls. A coin flip was used to determine
the initial (treated or non-treated) subject sequence at each site.
Comparison of outcomes between treated and non-treated groups was
made. The core lab was blinded regarding treatment or
no-treatment.
[0352] An assessment was performed to determine if a correlation
existed between clinical outcome and cell content (CD34+) and/or in
vitro colony growth (CFU-GM, CFU-GEMM, BFU-E), CXCR-4 mobility, and
CXCR-4 surface antigen expression.
[0353] As originally planned, a total of 20 subjects were to
receive the chemotactic hematopoietic cell product of the described
invention. There were to be four dose cohorts (about
5.times.10.sup.6, about 10.times.10.sup.6, about 20.times.10.sup.6,
and about 30.times.10.sup.6 CD34+ cells). If the chemotactic
hematopoietic stem cell product content in any subject was not
sufficient for the assigned cohort, that subject was reassigned to
a prior cohort at the greatest possible dose. Subjects having fewer
than 5.times.10.sup.6 CD34+ cells available for infusion were
removed from the study, did not undergo repeat catheterization and
were not counted as part of the 20-subject study group. In
addition, if the chemotactic hematopoietic cell product of the
described invention did not meet release criteria, the subject did
not receive the cell product and was not counted as a study
candidate to be replaced by the next subject. In any cohort dosing
group, if a subject experienced an acute (meaning immediate to
about 7 days post infusion) unexpected toxicity considered to
(probably) be a result of the cell product infusion, dose
escalation was halted and 3 additional subjects were accrued to
that dose level. If no other unexpected toxicity was observed, then
dose escalation resumed, however the total of 20 subjects was not
exceeded. If another toxicity occurs at that dose level, then all
subsequent subjects were accrued to the next lower dose level.
[0354] The chemotactic hematopoietic stem cell product of the
described invention was not administered to any subject in the
higher dose cohort until all the subjects from the prior dose
cohort had completed their follow-up assessments two weeks after
product administration.
Example 10
Experimental Results of Preliminary Studies
[0355] A series of preliminary preclinical studies were performed
in an attempt to accomplish the following goals:
[0356] (1) Optimize the manufacturing process for the Mini
bone-Marrow Harvest (MMH);
[0357] (2) Evaluate the stability of the inbound MMH, product and
the outbound hematopoietic cell product.
[0358] (3) Evaluate the internal diameter allowance and safety of
the catheters;
[0359] (4) Evaluate the compatibility of the cell product with the
catheters intended to be used in the study; and
[0360] (5) Evaluate the suitability of using the supernatant of the
final hematopoietic cell product to represent the final
hematopoietic cell product for stability testing.
[0361] Study 1: Optimizing The Manufacturing Process for the Mini
Bone-Marrow Harvest (MMH)
[0362] The effect of key manufacturing variables on the yield of
viable CD34 cells from representative bone marrow products was
evaluated. A total of six (6) volunteer donors over the age of 45
(based on a range of 45-57) and three under 30 years of age (based
a range of 21-28) agreed to donate an average of 45 ml (based on a
range of 31 ml-54 ml) bone marrow and provided written Informed
Consent for the procedure. The marrow aspiration technique employed
was identical to that to be performed for the clinical scale MMH
(see Example 3, supra). As shown in Table 2, the cell counts of
nucleated cell (NC) and CD34+ cells of Mini bone-Marrow Harvest
("MMH") derived cells collected from volunteer donors appeared to
be age related.
TABLE-US-00003 TABLE 2 Effect of donor age on nucleated cell yield
of the MMH. Donor age group Over 45 (45-57) Under 30 (23-28) Volume
of CD34 cells Volume of CD34 cells Donor MMH (ml) Viability (%)
(10.sup.5 per ml) MMH (ml) Viability (%) (10.sup.5 per ml) 1 31.30
83.85 1.27 48.00 96.90 7.98 2 43.50 97.42 3.89 50.60 96.28 11.60 3
51.50 85.74 1.37 39.90 87.17 5.99 4 47.50 80.95 1.76 -- -- -- 5
53.70 98.21 5.58 -- -- -- 6 44.90 96.36 4.48 -- -- -- Avg. 45.40
90.42 3.06 46.17 93.45 8.52
[0363] The average cell count of the bone marrow products from
older donors (N=6) was 28.4.times.106 (based on a range of
15.8.times.10.sup.6-49.5.times.10.sup.6) nucleated cells per ml
["NC/ml"], with an average viability, as determined by 7-AAD dye
exclusion and flow cytometry, of 90.42% (based on a range of
80.95%-98.21%) and CD34+ content of 3.06.times.10.sup.5/ml (based
on a range of 1.27.times.10.sup.5/ml-5.58.times.10.sup.5/ml). In
the younger subject group (N=3), the average cell count collected
from marrow aspiration was 46.2.times.10.sup.6 NC/ml (based on a
range of 39.9.times.10.sup.6 NC/ml-50.6.times.10.sup.6 NC/ml), with
an average 7-AAD viability of 93.5% (based on a range of
87.17%-96.90%) and total CD34.sup.+ content of
8.5.times.10.sup.5/ml (based on a range of
5.99.times.10.sup.5CD34.sup.+ cells/ml-11.60.times.10.sup.5
CD34.sup.+ cells/ml).
[0364] Red Cell Depletion and CD34 Selection
TABLE-US-00004 TABLE 3 CD34+ cell recovery after RBC depletion of
MMH from older age group (4557) donors. Donor 1 2 3 4 5 Average
Method of RBC Heta- Buffy Buffy Buffy Buffy -- depletion starch
coat coat coat coat CD34.sup.+ cell % in MMH: 1.09 1.64 1.63 1.45
1.99 1.58 Pre-RBC depletion CD34.sup.+ cell % in MMH: 1.33 1.55
1.51 1.61 1.84 1.57 Post-RBC depletion CD34.sup.+ cell recovery
65.68 92.36 80.66 78.79 81.67 79.83 post RBC depletion (%)
[0365] As shown in Table 3, following red cell depletion of the
MMH-derived bone marrow products collected from the older donors,
an average of 79.83% (based on a range of 65.68%-92.36%) of the
CD34 cells from the initial MMH was recovered. There was no
significant difference between the initial CD34 cell purity (1.58%,
based on a range of 1.09%-1.99%) and that following red cell
depletion (1.57%, based on a range of 1.33%-1.84%). Assay methods
to quantify chemotaxis are well known in the art, and a wide
variety of techniques are used to evaluate chemotactic ability of a
variety of cell types. Furthermore, cell migration assays are
commercially available.
[0366] The assay used for the determination of in vitro migratory
activity of CD34+ cells mediated by CXCR-4, which is adapted from
an assay described in Jo et at (J. Clin. Invest. 105: 101-1
(2000)), relies on transmembrane migration of CD34+ cells.
Transmembrane migration of CD34+ cells from the upper chamber to
the lower chamber of a transwell polystyrene plate (6.5 mm
diameter, 5 um pore size, Costar) is induced by SDF-1 placed in the
lower chamber. The number of migrated viable CD34+ cells in the
lower chamber then is determined by flow cytometry analysis using
CD34/CD45 antibodies and 7-AAD. Control spontaneous migration of
CD34+ cells is performed without SDF-1 in the lower chamber.
[0367] The subpopulation of potent cells that (i) express CXCR-4
and (ii) have CXCR-4 mediated chemotactic activity, expressed
VEGFR-2 at very low levels (mean 0.84%, range 0 to 2.39%). Because
the subpopulation of potent CD34+ cells co-expresses CXCR-4,
{CXCR-4 co-expression; mean 60.63%, median 52% range 31-98% of
CD34+ cells, capable of migrating in an SDF-1 gradient} while less
than 2.5% of the CD34+ cells co-expresses VEGFR-2, functionally,
these cells are VEGFR-2-, i.e., VEGFR-2 is not what drives the
cells into the peri-infarct zone.
TABLE-US-00005 TABLE 4 CD34+ cell recovery, purity, CXCR-4
migratory activity, viability and hematopoietic CFU growth
immediately after Isolex processing of MMH from older age group
(age 45-age 57) donors. Donor 1 2 3 4 5 Average Storage time
(hours) 0 0 0 12 10.50 -- at 4.degree. C.-8.degree. C. CD34.sup.+
cell recovery (%) 32.36 29.09 15.31 43.60 40.20 32.11 CD34.sup.+
cell purity (%} 76.76 73.64 71.66 72.52 72.01 73.32 CD34.sup.+ cell
viability 98.49 93.80 97.38 98.28 98.39 97.27 CD34.sup.+ cell
CXCR-4 22.10 2.60 22.00 19.90 19.70 17.26 migratory activity (%)
Hematopoietic CFU/100 27.5 25.0 18.9 17.0 21.00 21.9 CF34.sup.+
cells cultured
[0368] As shown in Table 4, following CD34 selection using the
Isolex system, which includes immunomagnetic Dynabeads.RTM. and
anti-CD34 mAb, an average of 32.11% (based on a range of
1.5.31%-43.60%) of the CD34 cells was recovered with an average
purity of 73.32% (based on a range of 71.66%-73.64%) and an average
viability of 97.27% (based on a range of 93.80%-98.49%). In
addition, these CD34+ cells displayed an average of 17.26% (based
on a range of 2.60%-22.10%) CXCR-4 migratory ability immediately
after selection and were capable of generating hematopoietic
colonies (21.89 colonies/1C CD34 cells plated (based on a range of
17.0 colonies/100 CD34+ cells plated-27.5 colonies/100 CD34+ cells
plated) in MethoCult culture.
[0369] Study 2: Evaluation of the Stability of the Inbound
Mini-Bone Marrow Harvest and of the Outbound Chemotactic
Hematopoietic Cell Product
[0370] A series of experiments, using healthy volunteers, was
performed in order to evaluate the stability of the inbound MMH and
of the outbound chemotactic hematopoietic stem cell product of the
described invention. Assessment of the functional viability of the
inbound and outbound products was evaluated by cell viability
(7-AAD), SDF-1/CXCR-4 mediated CD34.sup.+ cell migration, and the
ability to form hematopoietic colonies in methylcellulose (CFU
colony forming ability).
[0371] To evaluate the inbound product stability for shipping and
logistic purposes and for coordination with clinical schedules, MMH
products were stored at 4.degree. C. to 8.degree. C. as indicated.
To evaluate the outbound product stability for shipping and
logistic purposes, the chemotactic hematopoietic stem cell product
comprising isolated CD34+ cells enriched following MMH was stored
at 4.degree. C. to 8.degree. C. as indicated.
[0372] In preliminary studies, cells either were processed
immediately or maintained at 4-8.degree. C. for 12 hours prior to
processing to evaluate the impact of shipping and logistic duration
on the manufacture of a suitable cell product for infusion. Despite
the duration of storage prior to processing (inbound product
expiration), the results did not vary significantly (data not
shown).
[0373] In another series of experiments, cells were stored at about
4.degree. C. to about 8.degree. C. for 12 hours and about 24 hours
prior to reassessment to simulate products infused at about 36
hours and at about 48 hours, respectively, following MMH.
TABLE-US-00006 TABLE 5 CD34+ cell viability, growth and CXCR-4
migratory activity 13-13.5 hours after Isolex processing of MMH.
Donor 1 2 Average CD34+ cell viability (%) 97.59 96.90 97.24 CD34+
cell CXCR-4 migratory activity (%) 7.70 7.50 7.60 Hematopoietic
CFU/100 CD34+ cells cultured 18.00 25.00 21.5
[0374] As shown in Table 5, the isolated CD34+ cells of the
chemotactic hematopoietic stem cell product had an average
viability of 97.24% (based on a range of 96.90%-97.59%) and average
CXCR-4-mediated migratory capacity of 7.60% (based on a range of
7.50%-7.70%). As shown in Table 6, after storage for an average of
26.3 hours (based on a range of 26.0 h-26.5 h), these cells had an
average viability of 96.81% (based on a range of 96.39%-97.22%) and
an average CXCR-4-mediated migratory capacity of 4.75% (based on a
range of 4.50%-5.00%). Further, the cells still maintained their
ability to generate hematopoietic colonies in vitro.
TABLE-US-00007 TABLE 6 CD34+ cell viability, growth and CXCR-4
migratory activity 26.0-26.5 hours after Isolex processing of MMH.
Donor 1 2 Average CD34+ cell viability (%) 97.22 96.39 96.81
CD34.sup.+CXCR-4.sup.+ cell CXCR-4 migratory activity 4.50 5.00
4.75 (%) Hematopoietic CFU/100 CD34+ cells cultured 28.00 14.00
21.00
[0375] Thus, an average of 13.3 hours (based on a range of 13.0
h-13.5 h) after CD34+ cell selection, representing 26.0-26.5 hr
post-MMH, the CD34+ cell population had an average viability of
97.24% (based on a range of 96.90% 97.59%), with average CXCR-4
mediated migratory capacity of 7.60% (based on a range of
7.50%-7.70%). At an average of 26.3 hours (based on a range of 26.0
h-26.5 h) following MMH, the average viability of the cells was
96.81% (based on a range of 96.39%-97.2%) and maintained an average
CXCR-4-mediated migratory capacity of 4.75% (based on a range of
4.50%-5.00%).
[0376] Formulation of the composition of the described invention
comprising this product occurred an average of 8 hours
(8.63.+-.1.80 N=4) hours after MMH collection, and infusion
occurred within 24 hours of MMH.
TABLE-US-00008 TABLE 7 CD34+ cell viability as a function of time
after MMH: 12-hour in-dating and 48 hour outdating (all time points
measured from completion of MMH.) Time (h) CD34.sup.+ cell
viability (%) after MMH (SD) A B C I) Average (SD) 98.22 97.13
97.60 99.00 97.99 (0.29) 24 95.32 97.76 -- -- 96.54 (1.73) 33 91.92
96.32 95.90 80.00 91.04 (7.62)
[0377] In a subsequent experiment, four (4) MMH products (A-D) were
collected and stored at 4.degree. C. for an average of 12.8 hours
(based on a range of 12.5 h-13.0 h) before the CD34+ cells were
isolated by the Isolex procedure. This group, representing the "12
hour in-date" group (meaning that the product was formulated within
the in-date time of about 12 hours), was evaluated for functional
viability out-date at "24 hours" (22.9 h.+-.1.63, N=4), "33 hours"
(33.38.+-.1.11, N=2), and "48 hours" (48.33.+-.0.82, N=4) post MMH
harvest. The data, summarized in Tables 7-9, demonstrate that
following MMH, the chemotactic hematopoietic stem cell product
comprising enriched CD34+ cells maintains 1) high viability
(>90.0% average viability, Table 7), 2) 76.85% (.+-.21.66) of
their SDF-1/CXCR-4 mediated migratory ability (Table 8), and 3)
their ability to form hematopoietic colonies in vitro (Table 9),
respectively.
[0378] Table 8 shows SDF-1/CXCR-4 mediated CD34+ cell migration (%
migrating CD34+ cells) as a function of time after MMH: 12-hour
in-dating and 48-hour outdating (all time points measured from
completion of MMH). For the purpose of determining the impact of
time post-MMH on the migratory ability of the CD34+ cells, time
point "X" was considered the reference point, as this was
determined to represent the earliest time point following MMH at
which cells reasonably could be expected to be returned to the
subject in a finished formulation. The remaining migratory activity
at the following time points (Y=33 hours, Z=48 hours) was
calculated as percent migratory ability remaining following the 24
hour (X) time point.
TABLE-US-00009 TABLE 8 SDF-1/CXCR-4 mediated CD34+CXCR-4+ cell
migration (% migrating CD34+ cells) as a function of time after
MMH: 12-hour in-dating and 48-hour outdating (all time points
measured from completion of MMH). Time (h) Migrating
CD34.sup.+CXCR-4.sup.+ cells (%) after MMH A B C D Average (SD) 24
(X) 20.00 18.50 21.50 36.00 24 (8.09) % Remaining 100.00 100.00
100.00 100.00 100.00 (0) 33 (Y) 21.80 10.50 -- -- 16.15 (7.99) *%
Remaining 109.00 56.76 -- -- 82.88 (36.94) 48 (Z) 8.80 17.00 17.50
31.00 18.58 (9.19) .sup.@% Remaining 44.00 91.89 81.40 86.00 75.85
(21.66) *= (Y / X) .times. 100% .sup.@= (Z / X) .times. 100%
[0379] Table 9 shows the number of colony forming units (CFU) per
100 viable CD34+ cells plated as a function of time after MMH:
12-hour in-dating and 48 hour-out-dating (all time points measured
from completion of MMH).
TABLE-US-00010 TABLE 9 CFU per 100 viable CD34+cells plated as a
function of time after MMH Time (h) # of CFU per 100 viable
CD34.sup.+ cells plated after MMH A B C D Average (SD) 24 13.00
30.00 37.00 39.00 29.75 (11.81) 33 12.00 34.00 -- -- 23.00 (15.56)
48 15.00 30.00 20.00 8.00 28.25 (14.57)
[0380] In an attempt to extend both the in-date and out-date
stability parameters for the chemotactic hematopoietic stem cell
product of the described invention comprising CD34+ cells from
12-hours (in-date) and from 48-hours (out-date) (12/48),
respectively, to 24-hours (in-date) and 72-hours (outdate) (24/72),
respectively, C1).sub.34 cells were purified about 12 hours after
MMH harvest (12 hour in-date) and about 24 hours after MMH harvest
(24 hour in-date) and analyzed for functional viability at about 48
hours and at about 72 hours total time from MMH to time of
testing/anticipated infusion (48 hour out-date and 72 hour
out-date, respectively). Specifically, the functional viability
characteristics of two MMH/chemotactic hematopoietic stem cell
products of the described invention were evaluated at 48 hours and
72 hours. The resulting data were further compared to the same
indices derived at the previous 12/48 time points (Tables 7-9).
[0381] Tables 10-12 show that at 33 hours (based on 32.5.+-.0.71,
N=2), 48 hours (based on one data point at 49 hours), and at 72
hours (based on 72.5 h.+-.0.71, N=2), the isolated CD34+ cells of
the chemotactic hematopoietic stem cell product of the described
invention maintained 1) over 90% viability (Table 10), 2)
102.19.+-.32.69% of their SDF-1/VEGF/CXCR-4 mediated migratory
ability (Table 11), and 3) their ability to generate hematopoietic
colonies in vitro (Table 12).
TABLE-US-00011 TABLE 10 CD34+ cell viability as a function of time
after MMH: 24-h in-dating and 72-h outdating (all time points
measured from completion of MMH) CD34.sup.+ cell viability (%) Time
(h) after Average MMH A B (SD) 33 98.00 99.00 98.50 (0.71) 48 --
97.00 97.00 (--) 72 91.00 97.00 94.00 (4.24)
TABLE-US-00012 TABLE 11 SDF-1/CXCR-4 mediated CD34+ cell migration
(% population of migrated CD34+ cells as a function of time after
MMH): 24-h in-dating and 72-h outdating (all time points measured
from completion of MMH) Time (h) after Migrating CD34.sup.+ cells
(%) MMH Average (SD) A B (range) 33 8.20 14.05 11.13 (2.93) %
Remaining 100.00 100.00 100.00 (0.00) 48 -- 18.61 18.61 (--) %
Remaining -- 132.46 132.46 (--) 72 5.70 18.95 12.33 (6.63) %
Remaining 69.51 134.88 102.19 (32.69)
[0382] The % remaining ratios in Table 11 were determined as in
Table 8 above.
TABLE-US-00013 TABLE 12 Number of CFU per 100 viable CD34+ cells
plated as a function of time after MMH: 24-h in-dating and 72-h
outdating (all time points measured from completion of MMH) # of
CFU per 100 viable CD34.sup.+ cells Time (h) plated after MMH
Average (SD) A B (range) 33 26.00 28.50 22.25 (1.25) 48 -- 16.80
16.80 (--) 72 14.50 27.50 21.00 (6.5)
[0383] Further evaluation of the functional viability parameters of
the chemotactic hematopoietic stem cell product comprising isolated
CD34+ cells of the described invention ("clinical product") at 8
hours (8.6 h.+-.1.80, N=4), 12 hours (12.87 h.+-.1.92, N=4), 32
hours (one time point at 33.5 h), 48 hours (47.50 h.+-.2.5, N=2),
and 72 hours (71.5 h.+-.0.50, N=2) after MMH shows that after 72
hours, the product retains its 1) viability (Table 13), 2)
SDF-1/CXCR-4 mediated migratory ability (Table 14) and 3) ability
to form hematopoietic colonies in vitro (Table 15), equivalent to
the 24-hour time point.
TABLE-US-00014 TABLE 13 Clinical Product Experience: CD34+ cell
viability as a function of time after MMH. Time (h) CD34.sup.+ cell
viability (%) after MMH A B C D Average (SD) 8 98.30 99.08 90.00
96.45 95.96 (4.12) 12 98.89 96.96 99.00 99.43 98.57 (1.10) 33 --
93.42 -- -- 93.42 48 -- 93.15 91.58 -- 92.37 (1.11) 72 -- 91.25
89.25 -- 90.30 (1.48)
TABLE-US-00015 TABLE 14 Clinical Product Experience: SDF-1/CXCR-4
mediated CD34.sup.+ cell migration (% migrating CD34.sup.+ cells as
a function of time after MMH) Time (h) Migrating CD34.sup.+ cells
(%) after MMH A B C D Average (SD) 12 (X) 14.31 13.08 9.74 31.73
17.97 (11.34) % Remaining 100.0 100.0 100.0 100.0 100.0 (0) 33 (Y)
-- 6.17 -- -- 6.17 *% Remaining -- 47.17 -- -- 47.17 48 (Y) -- 4.88
8.21 -- 6.55 (2.35) *% Remaining -- 37.30 84.29 -- 60.79 (23.49) 72
(Y) -- 3.7 6.6 -- 5.15 (2.05) *% Remaining -- 28.29 21.19 -- 24.74
(3.55) *= (Y / X) .times. 100%
[0384] All remaining ratios were calculated as in Table 8
above.
TABLE-US-00016 TABLE 15 Clinical Product Experience: # of CFU per
100 viable CD34+ cells plated as a function of time after MMH # of
CFU per 100 viable CD34.sup.+ cells plated Time (h) Average after
MMH A B C D (SD) 12. 98.14 33.30 24.00 22.50 44.49 (36.09) 33 --
16.50 -- -- 16.5 48 -- 19.56 20.50 -- 20.03 (0.66) 72 -- 20.45
21.19 -- 20.82 (1.10)
[0385] Based on these data, extension of the in-dating to 24 hours
(from 12-hours) and the out-dating to 72 hours (from 48 hours) for
the CD34+ cell clinical product of the described invention is
justified.
[0386] FIG. 1 indicates the equivalence of the functional viability
of the chemotactic hematopoietic cell product of the described
invention at 72 hours to the same indices evaluated at 48
hours.
[0387] Study 3: Catheter Safety.
[0388] The viability and potential efficacy of the chemotactic
hematopoietic stem cell product of the described invention
comprising potent CD34+ cells depends on the cells maintaining
their potency as they pass through a catheter. The catheter used in
the methods of the described invention has an internal diameter of
at least 0.36 mm. Any type of catheter having an internal diameter
of at least 0.36 mm may be effective in delivering the
pharmaceutical compositions of the described invention.
[0389] In one embodiment, the catheter is a balloon catheter.
Balloon catheter safety studies were conducted to determine whether
high cell concentrations and repeated perfusions adversely affect
cell viability, cell recovery or catheter integrity. Non-mobilized
peripheral blood progenitors were used in order to obtain an
adequate number of cells to perform the analysis. Catheters were
assessed for infusion of the cell product of the described
invention comprising selected CD34+ cells through the IRA. None of
the 0.36 mm internal diameter catheters tested adversely affected
CD34+ selected cell viability, growth in culture, or mobility in
CXCR-4 assays.
TABLE-US-00017 TABLE 16 Viability of CD34.sup.+ cells before and
after infusions through the catheters. Viability (%) Catheter
Condition 1 2 3 4 5 -- Pre-infusion 81.45 Raptor After 1st infusion
84.29 70.94 87.89 88.02 84.68 After 2nd infusion 83.00 87.44 86.39
79.91 83.18 Sprinter After 1st infusion 93.39 91.09 84.13 88.28
81.68 After 2nd infusion 91.89 91.08 84.88 77.65 77.73 Voyager
After 1st infusion 94.21 86.21 83.08 77.53 69.68 After 2nd infusion
88.03 84.71 79.27 78.11 76.80 Maverick After 1st infusion 90.00
89.76 90.79 85.49 81.31 After 2nd infusion 90.94 87.38 81.98 80.09
85.47
[0390] As shown in Table 16, in all catheters tested, average CD34+
cell viability was at or above 70% following passage through the
catheters.
[0391] To demonstrate that infusion of the CD34+ cell product does
not pose any safety breach of the catheter used and that a
significant percentage of cell product does not adhere to the
interior walls of the catheter, catheters were challenged with
repeat infusions of a CD34+ cell product having a considerably
higher cell concentration than that used clinically. Four brands of
catheters (Sprinter, Voyager, Maverick and Raptor) were evaluated
using 5 catheters of each type. Non-mobilized apheresis products
were used in order to obtain an adequate number of cells to perform
the analysis. A cell concentration greater than three times that
planned as treatment doses for the trial, i.e., 160.times.106
nucleated cells containing CD34+ cells in 10 ml of infusion
solution, was passed twice through each catheter. The average CD34+
cell recovery was 100.59% (based on a range of 76.99% to 228.70%)
following passage through the catheters.
[0392] All twenty catheters were tested for integrity using a
methylene blue dye leak test after two perfusions with the
nucleated cells. There was no evidence of leakage and the contact
points and catheter tips were normal upon inspection.
[0393] As shown in Tables 17a and 17b, the effect on the cells of
their perfusion through a catheter appears to be independent of
catheter model and make among those catheters tested and was
independent of the amount of time the cells were stored either
prior to processing and/or after CD34+ cell selection and prior to
perfusion, resulting in a final formulation containing an average
recovery of 96.0% (range 80.8%-102.2%) of the CD34+ cells (Table
17b) and 86.36% of the CD45+ cells perfused through the catheter.
Further, the average viability of the cells was 96.5% (range
92.5%-98.6%, N=16); the cells maintained both CXCR-4 migratory
capacity (data not shown) and their ability to form hematopoietic
colonies in methylcellulose (average 25.8 CFU/100 cells seeded
(range 21.0%-30.5%)
TABLE-US-00018 TABLE 17a CD45 cell recovery and viability after
being infused through the catheters. 1 2 3 4 5 Average R'd Re- R'd
R'd Re- R'd R'd R'd Catheter Condition Recovery viab covery viab
Recovery viab covery viab Recovery viab Recovery viab Raptor After
1.sup.st 69.68% -1.35% 78.67% 2.08% 72.14% -4.55% 80.54% 1.83%
73.21% -2.13% 74.85% -0.82% infusion (30.83%) (2.53%) After
2.sup.nd 97.91% -8.55% 81.84% -4.76% 142.98% 3.28% 107.82% -8.48%
94.08% 0.08% 104.93% -3.69% infusion (47.60%) (4.94%) Sprinter
After 1.sup.st 76.74% -0.60% 68.56% 4.01% 72.63% 5.29% 73.61% 6.06%
66.83% 8.31% 71.67% 4.61% infusion (29.48%) (3.51%) After 2.sup.nd
78.82% 2.86% 85.40% 0.98% 90.29% -1.02% 82.22% 6.50% 91.61% 0.00%
85.67% 1.86% infusion (35.30%) (2.76%) Voyager After 1.sup.st
87.38% 1.58% 83.93% -0.36% 103.58% 0.93% 95.82% 4.52% 131.55%
-4.39% 100.45 0.46% infusion (44.39%) (2.91%) After 2.sup.nd 82.70%
7.01% 69.34% 15.90% 69.54% 10.40% 89.04% 0.27% 69.03% 7.50% 75.93%
8.22% infusion (32.11%) (6.09%) Maverick After 1.sup.st 73.97%
1.58% 87.01% 0.42% 78.31% 0.69% 75.53% 2.61% 77.22% 2.95% 78.41%
1.65% infusion (32.33%) (1.21%) After 2.sup.nd 152.35% -5.06%
73.44% 2.78% 80.85% -3.92% 97.10% -2.97% 91.11% -2.07% 98.97%
-2.25% infusion (49.11%) (2.85%) Average of all 86.36% 1.26%
catheters: .sup.aRecovery of CD45+ cells = (# of CD45 cells after
infusion + # of CD45 before infusion) .times. 100% .sup.bReduction
of CD45+ cell viability = [1 - (CD45+ cell viability % after
infusion + CD45+ cell viability % before infusion)] .times.
100%
TABLE-US-00019 TABLE 17b CD34 cell recovery and viability after
being infused through the catheters. 1 2 3 Catheter R'd R'd R'd
used Condition Recovery.sup.a viab.sup.b Recovery viab Recovery
viab Raptor After 1.sup.st 116.49% -3.48% 121.62% 12.91% 110.89%
-7.91% infusion After 2.sup.nd 91.66% 1.53% 85.18% -23.26% 122.47%
1.71% infusion Sprinter After 1.sup.st 89.19% -14.66% 83.34%
-11.83% 102.72% -3.29% infusion After 2.sup.nd 103.52% 1.61% 99.82%
0.01% 82.11% -0.89% infusion Voyager After 1.sup.st 81.02% -15.67%
96.08% -5.84% 90.16% -2.00% infusion After 2.sup.nd 106.48% 6.56%
81.66% 1.74% 95.04% 4.58% infusion Maverick After 1.sup.st 76.99%
-10.50% 101.79% -10.21% 98.62% -11.46% infusion After 2.sup.nd
228.70% -1.05% 88.66% 2.65% 103.35% 9.70% infusion 4 5 Average
Catheter R'd R'd Recovery R'd viab used Condition Recovery viab
Recovery viab (SD) (SD) Raptor After 1.sup.st 97.55% -8.06% 96.14%
-3.97% 108.54% -2.10% infusion (45.46%) (7.79%) After 2.sup.nd
111.33% 9.21% 98.96% 1.78% 101.92% -1.81% infusion (43.73%)
(11.14%) Sprinter After 1.sup.st 84.57% -8.39% 88.65% -0.28% 89.69%
-7.69% infusion (37.26%) (6.16%) After 2.sup.nd 114.87% 12.05%
100.45% 4.84% 100.15% 3.52% infusion (42.22%) (4.90%) Voyager After
1.sup.st 82.73% 4.82% 89.32% 14.46% 87.86% -0.85% infusion (36.28%)
(10.13%) After 2.sup.nd 94.81% -0.75% 91.01% -10.23% 93.80% 0.38%
infusion (39.12%) (5.86%) Maverick After 1.sup.st 112.58% -4.96%
98.05% 0.18% 97.21% -7.39% infusion (41.34%) (5.34%) After 2.sup.nd
89.35% 6.31% 117.63% -5.12% 125.54% 2.50% infusion (73.48%) (5.33%)
Average of 100.59% -1.68% all catheters: .sup.aRecovery of
CD34.sup.+ cells = (# of CD34 cells after infusion + # of CD34
before infusion) .times. 100% .sup.bReduction of CD34.sup.+ cell
viability = [1 - (CD34.sup.+ cell viability % after infusion +
CD34.sup.+ cell viability % before infusion)] .times. 100%
[0394] Collectively these experiments demonstrate that the serial
passage of a chemotactic hematopoietic stem cell product comprising
CD34+ cells through a cardiac catheter with an internal diameter of
at least about 0.36 mm does not adversely affect either catheter
integrity or CD34+ cell potency, i.e., CD34+ cell viability, CFU
colony growth, or CD34+CXCR+ mediated migratory
capacity/mobility.
[0395] Study 4: Compatibility of the Cell Product with the
Catheters
[0396] To further test the compatibility of the chemotactic
hematopoietic stem cell product comprising CD34+ cells with each of
the catheters that may be used for delivery of the cell product in
the study, cell products were tested after multiple passages
through each catheter type to evaluate the effects of extreme
conditions of stress that would be greater than those expected
during the treatment protocol.
[0397] At 48 hours post-MMH harvest, the chemotactic hematopoietic
stem cell product comprising a range of about 5.73.times.106 CD34+
cells to about 21.10.times.106 CD34+ cells (i.e., dosages
reflective of the treatment cohort) obtained from individual donors
was infused sequentially through three catheters of the same brand,
one type of catheter for each donor (Sprinter, Voyager or
Maverick), and the cell product assessed for CD34+ cell recovery,
colony formation and viability.
TABLE-US-00020 TABLE 18 CD34+ cell recovery and sterility after
sequential infusions through the catheters. Catheter used Condition
Parameter Sprinter Voyager Maverick Pre-infusion CD34.sup.+ cell
yield 9.72 .times. 10.sup.6 2.11 .times. 10.sup.7 5.73 .times.
10.sup.6 After 1.sup.st CD34.sup.+ cell recovery 111% 103% 99%
catheter After 2.sup.nd CD34.sup.+ cell recovery 94% 104% 97%
catheter After 3.sup.rd CD34.sup.+ cell recovery 99% 99% 106%
catheter Sterility (aerobic and Negative Negative Negative
anaerobic microbes)
[0398] As shown in Table 18, viable, colony forming cells were
recovered in all experiments for all three catheters tested (cell
recovery 99%, 99% and 106%).
[0399] As shown in Table 19, the average viability of the CD34+
cells after passing through the third catheter was 94.000% (based
on a range of 93.55%-94.40%) versus 96.01% (based on range of
94.18%-97.93%) of the pre-infusion cell product.
TABLE-US-00021 TABLE 19 CD34+ cell viability after sequential
infusions through the catheters. CD34.sup.+ cell viability
Condition Sprinter Voyager Maverick Average Pre-infusion 94.18%
95.91% 97.93% 96.01% After 1st catheter 94.73% 96.31% 95.45% 95.50%
After 2.sup.nd Catheter 95.34% 95.72% 95.01% 95.36% After 3rd
catheter 93.55% 94.40% 94.04% 94.00%
[0400] As shown in Table 20, colony forming unit (CFU) growth
derived from the CD34+ cells after passing through the third
catheter was 95.27% (based on a range of 43.47%-163.64%) of the
infusion product (i.e., the infused chemotactic hematopoietic stem
cell product comprising CD34+ cells).
TABLE-US-00022 TABLE 20 CFU growth of CD34+ cells after sequential
infusions through the catheters. CFU per 100 CD34.sup.+ cells
cultured Condition Sprinter Voyager Maverick Pre-infusion 30.5 11.5
11.0 After 1st catheter 22.0 14.0 22.0 After 2nd catheter 20.5 4.0
19.0 After 3rd catheter 24.0 5.0 18.0 Recovery from the pre- 78.69%
43.47% 163.64% infused product after the 3rd catheter Average
recovery 95.27%
[0401] To determine the effect of catheter perfusion on CD34+ cell
mobility and ability to grow in culture, a series of experiments
were performed where MMH cells obtained from healthy donors were
stored at 4.degree. C. for 12 or 24 hours before initiation of
Isolex processing. Isolated CD34+ cell product that had been stored
for about 12 hours pre-Isolex processing then were stored at
4.degree. C. until about 36 hours had elapsed from the end of
processing, for a total of about 48 hours post MMH. At that time
they were assessed for SDF-1/CXCR-4 mobility and CFU growth pre and
post perfusion through a 0.36 mm inner diameter (i.d.) cardiac
balloon catheter. Similarly, cells that were stored pre-Isolex
processing for 24 hours then were stored at 4.degree. C. until 48
hours had elapsed from the end of Isolex processing, for a total of
72 hours, and then assessed.
TABLE-US-00023 TABLE 21 12 inbound/48 outbound and 48 hour
inbound/72 hour outbound from MMH: SDF-1/CXCR-4 mobility (%
population of migrated CD34+ cells) and CFU (per 100 viable CD34+
plated) pre catheter perfusion ("PRE") and post catheter perfusion
("POST") Time (h) after MMH SDF-1/CXCR-4 mobility (%) // # of CFU
per Inbound/ 100 viable CD34.sup.+ cells plated outbound A B C D E
12/48 2.7 // 14 8.8 // 15 15.8 // 16 -- -- PRE 12/48 3.4 // 15 18.9
// 13 17.6 // 8 -- -- POST 24/72 -- -- -- 34 // 37 18.9 // 27.5 PRE
24/72 34 // 43 23.5 // 24 POST
[0402] The results in Table 21 demonstrate that neither
CD34+CXCR-4-mediated cell mobility nor the cell's ability to grow
in culture at any of the time points tested was affected adversely
by perfusion through a catheter having an internal diameter of at
least 0.36 mm.
[0403] The Stabilizing Effect of Serum
[0404] The following data confirm the importance of the stabilizing
effect of serum to the migratory capability of the selected CD34+
cells.
[0405] As shown in Table 22, no CXCR-4 migratory activity was
observed for all samples tested including the pre-catheter infusion
samples when the composition comprising a chemotactic hematopoietic
stem cell product was formulated without serum.
TABLE-US-00024 TABLE 22 Chemotaxis of CD34+ cells after sequential
infusions through the catheters in the absence of serum. Migration
(%) Condition Sprinter Voyager Maverick Pre-infusion 0.0 0.0 0.1
After 1st catheter 0.0 0.0 0.0 After 2nd catheter 0.0 0.0 0.1 After
3rd catheter 0.0 0.0 0.0
[0406] FIGS. 2 and 3 further illustrate that Isolex selected CD34+
cells retain their migratory capacity longer when formulated in the
presence of human serum. Following Isolex processing, the bone
marrow derived hematopoietic stem cell product comprising selected
CD34+ cells was formulated either in (1) phosphate buffered saline
(Dulbecco's phosphate buffered saline, Ca++, Mg++ Free (Baxter Cat.
No. EDR9865) ("PBS") containing 1% human serum albumin, 25 U/ml of
heparin sodium and various concentrations (about 0%, about 10%,
about 20%, or about 70%) of autologous serum; or (2) normal saline
(0.9%) containing 1% human serum albumin, 25 U/ml of heparin sodium
and (about 0% or about 10%) autologous serum. SDF-1/CXCR-4 mediated
CD34+ cell migratory capacity was evaluated at different times
during final product storage (at 2.degree. C.-8.degree. C.) and
after passing the cells through the catheter at the same rate and
duration as anticipated by the clinical protocol. None of these
formulations affected CD34+ cell viability or the recovery of CD34+
cells after they had been passed through the catheter.
[0407] Regardless of whether the chemotactic hematopoietic cell
products comprising selected CD34+ cells was (i) formulated either
in PBS-serum or in saline-serum and (ii) either passed through the
catheter immediately or passed through the catheter after a
prolonged stability testing storage interval at about 4.degree. C.
to about 8.degree. C., they maintained an average of 96.6%
viability (range 92.5%-98.6%) and an average CXCR-4-mediated
migratory capacity of 11.4% (range 2.4%-30.6%), representing a
total time from harvest to mobility analysis of up to 48 hours.
[0408] As shown in FIG. 2 panel (a), cells formulated in PBS alone
at about 25 hours retained about 10% of their CXCR-4 migratory
capacity, which dropped off to near 0 at about 48 hours. As shown
in panel (b), cells formulated in normal saline alone retained
little, if any, of their migratory capacity. As shown in panels (c)
and (d), cells formulated with PBS containing at least about 10%
serum retained about 10-15% of their migratory capacity for up to
about 55 hours (c), while cells formulated with saline and at least
about 10% serum retained about 20% of their migratory capacity for
up to about 50 hours. As shown in panels (e) and (f), cells
retained a higher migratory capacity for a longer duration in PBS
supplemented with even higher concentrations of serum.
[0409] As shown in FIG. 3, the product of the described invention
comprising selected CD34+ cells when formulated in 10% serum,
retained 14.25%, <1%, 6%, and 5.8% of its CD34+CXCR4-mediated
migratory capacity about 24, about 32, about 48 and about 56 hours
after harvest, respectively. FIG. 3 further shows that the product
of the described invention comprising selected CD34+ cells when
formulated in 20% serum retained 18.25%, 10.25%, 17% and 11% of its
CD34+-CXCR-4-mediated migratory capacity about 24, about 32, about
48 and about 56 hours after harvest, respectively. The term
"stabilizing amount" as used herein therefore refers to the amount
of serum that, when included in the formulation of the product of
the described invention comprising selected CD34+ cells, enables
these cells to retain their CXCR-4 mediated chemotactic activity
and hematopoietic colony forming ability.
[0410] As shown in Table 23, CD34+CXCR-4+ cells obtained from
healthy volunteers and from patients to which autologous serum was
added maintained their motility out to 72 hours. CD34+ cells were
isolated from the bone marrow of healthy volunteers and of patients
by the mini-bone marrow harvest procedure as described in Example 3
under identical conditions; and the chemotactic hematopoietic stem
cell product was created as described in Examples 4 and 5. The
products then were formulated with or without >20% autologous
serum, and tested at 24, 48 and 72 hours. As shown in column 2,
CXCR-4 cell mobility of CD34+CXCR-4+ cells obtained from healthy
volunteers, when formulated without serum, decreased 72% after 48
hours. As shown in column 3, CXCR-4 cell mobility of CD34+CXCR4+
cells obtained from healthy, volunteers, when formulated with serum
showed no change in mean CD34+CXCR-4+ cell motility, meaning that
the serum stabilizes SDF-1/CXCR-4 motility. Column 4 shows that
CD34+CXCR-4+ cells obtained from patients showed less motility than
did cells from healthy volunteers, but that the motility of the
CD34+CXCR-4+ cells was maintained out to 72 hours.
TABLE-US-00025 TABLE 23 Mean CD34+ Cell Mobility and % Change Over
Time. Volunteers.sup..dagger. Volunteers With Patients Hours
(N).sup..dagger..dagger. Serum.sup..sctn. With Serum Mean CD34+
Cell Mobility % 24 14.6.sup.| (4) 18.1 (6) 12.8 (6) 48 3.2 (4) 19.7
(8) 4.7 (3) 72 ND.sup.# 22.1 (7) 4.6 (5) Mean % Change (range)** 48
.dwnarw.72 (.dwnarw.53-.dwnarw.84) .dwnarw.0.6
(.dwnarw.16-.uparw.28) .dwnarw.57 (.dwnarw.13-.dwnarw.93) 72 ND
.uparw.9.6 (.dwnarw.30-.uparw.85) .dwnarw.68
(.dwnarw.48-.dwnarw.86) *Hours from bone marrow aspiration
.sup..dagger.CD34+ cells suspended in PBS only
.sup..dagger..dagger.Number of individuals tested .sup..sctn.CD34+
cells suspended in PGS and autologous serum .sup.|% CD34+ migrating
to lower chamber .sup.#Not Done **Sum of % change of each
experiment/number of experiments
[0411] Study 5: Final Product Sterility Testing
[0412] Due to the limited yield of CD34+ cells obtained from a
300-ml MMH, final cell product sterility is assessed using the
supernatant removed from the final product formulation in order to
preserve cell product for infusion. Supernatant samples are loaded
into the syringes in a manner identical to that used to load the
cell product into the syringes used for infusion (see supra).
[0413] To demonstrate that such a sample is representative of the
final cell product formulation, we inoculated selected CD34+ cells
in infusion solution prior to centrifugation of the final product
with C. sporogenes (13 CFU/ml), P. aeruginosa (2 CFU/ml), S. aureus
(18 CFU/ml), A. niger (17 CFU/ml), C. albicans (3 CFU/ml) and B.
subtilis (17 CFU/ml) (See table 24). After centrifugation, the
sterility of both cell pellet and non-cell supernatant fractions
was assessed using USP aerobic and anaerobic testing.
TABLE-US-00026 TABLE 24 Bacteria and fungi used for the sterility
study. Each source microorganism vial prepared by Microbiological
Environments contained 400 microbes per ml, but the numbers of CFU
derived from each species are varied. Expected CFU/ml of Total # of
inoculated sample Microbe microbes/ml Total CFU/ml (21 ml) C.
sporogenes 400 279 13 P. aeruginosa 400 36 2 S. aureus 400 371 18
A. niger 400 356 17 C. albicans 400 62 3 B. subtilis 400 349 17
[0414] As shown in Table 25, both the cell pellet fraction and
suspension fractions from all tested samples showed outgrowth of
the inoculated microorganisms, while un-inoculated controls showed
no growth. Further, no apparent differential growth rate was
observed between testing of cell pellet fractions and the
suspension fractions for all microorganisms tested. Samples taken
before each step of the processing procedure and following the
final perfusion through the catheters all tested negative for
microbial contamination.
TABLE-US-00027 TABLE 25 14-day sterility testing of nucleated cell
(NC) samples inoculated with specific species of microorganism (400
microbes in 21-m1 NC sample). Sample with microbe Medium Inoculated
type Sample fraction Test 1 Test 2 Test 3 C. sporogenes FTM.sup.a
Cell pellet Positive Positive Positive Suspension Positive Positive
Positive S. aureus FTM Cell pellet Positive Positive Positive
Suspension Positive Positive Positive P. aeruginosa FTM Cell pellet
Positive Positive Positive Suspension Positive Positive Positive A.
niger TSB.sup.b Cell pellet Positive Positive Positive Suspension
Positive Positive Positive C. albicans TSB Cell pellet Positive
Positive Positive Suspension Positive Positive Positive B. subtilis
TSB Cell pellet Positive Positive Positive Suspension Positive
Positive Positive Positive control: C. sporogenes FTM Cell Positive
Positive control: S. aureus FTM suspension Positive Positive
control: P. aeruginosa FTM Positive Positive control: A. niger TSB
Positive Positive control: C. albicans TSB Positive Positive
control: B. subtilis TSB Positive Negative control: No microbes FTM
Cell Negative Negative control: No microbes TSB suspension Negative
.sup.aFluid thioglycollate medium .sup.bTryptic soy broth
[0415] Preclinical Study Summary
[0416] Collectively, these preclinical data indicate that the
manufacturing and testing procedures described are capable of
generating adequate numbers of viable cells with adequate stability
to withstand shipment and perfusion through the catheter in a
manner that should pose no additional safety concerns to the
subject other than those associated with the routine use of fluid
infusion through the balloon catheter.
Example 11
Preliminary Phase 1 Efficacy Data, with a Single Infusion Date
[0417] The following preliminary phase 1 efficacy data show that
within about 10.times.10.sup.6 isolated CD34+ cells, there are
enough potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity to effect a paracrine effect,
which affects immediate cell death and later changes consistent
with ventricular remodeling.
[0418] In accordance with the disclosure in Example 1, a total of
31 subjects were consented, eligible and enrolled in the study. The
31 patients enrolled in the phase 1 study were randomly assigned to
an autologous stem cell harvest treatment group or to a control
group five days after an ST elevation myocardial infarction (STEMI)
characterized by a prolonged period of hypoperfusion (meaning
blocked blood supply) Of the 31 subjects enrolled, 16 were in the
treatment group and 15 in the control group. The first subject at
each Center was randomized to either treatment or control, and each
subsequent patient was enrolled into alternating treatment or
control groups. If the subject was assigned to treatment, he/she
continued into the Treatment Phase as long as all
inclusion/exclusion criteria continued to be met. Subjects assigned
to the control group progressed to the low-up phase. There were no
significant differences between groups in any of the baseline
demographic or clinical characteristics. Patients enrolled were
from 34 to 71 years of age. 87% male, 77% white, 61% in NYHA Class
II or III and 49% in NYHA Class I. 74% experienced an infarcted
left anterior descending coronary artery, and 55% received a drug
eluting stent.
[0419] CD34+ cells were isolated from the bone marrow by the
mini-bone marrow harvest procedure as described in Example 3 within
5-8 days post stent replacement. Harvested marrow then was shipped
to the cGMP cell processing facility as described in Example 4 and
isolated as described in Example 5.
[0420] As originally planned, and as described in Example 8, there
were to be four dosing cohorts (5 million, 10 million, 15 million
and 20 million CD34+ cells) in the study. However more than 15
million cells post CD34+ selection could not be obtained reliably.
Therefore enrollment terminated at the end of cohort 3 with
15.times.106 being the highest cell dose assessed.
[0421] Following cell product release and cohort assignment, the
CD34+ cell product was shipped to the catheterization site for
direct infarct related artery infusion. Treatment infusion occurred
6-9 days post stent replacement (and within 48 hours of mini-bone
marrow harvest). Subjects were brought to the catheterization
laboratory only after the CD34+ cell product had arrived at the
facility and had received final release for infusion.
[0422] The dosing cohorts consisted of 5 subjects in cohorts 1 and
2, 6 subjects in cohort 3, and 15 control subjects. For cohort I,
the chemotactic hematopoietic stem cell product of the invention
comprised 5.times.10.sup.6 isolated CD34+ hematopoietic stem cells
containing a subpopulation of at least 0.5.times.10.sup.6 potent
CD34+ cells expressing CXCR-4 and having CXCR-4 mediated
chemotactic activity [represented as "5 M"}. For cohort 2, the
chemotactic hematopoietic stem cell product of the invention
comprised 10.times.10.sup.6 isolated CD34+ hematopoietic stem cells
containing a subpopulation of at least 0.5.times.10.sup.6 potent
CD34+ cells expressing CXCR-4 and having CXCR-4 mediated
chemotactic activity [represented as "10 M"]. For cohort 3, the
chemotactic hematopoietic stem cell product of the invention
comprised 1.times.10.sup.6 isolated CD34+ hematopoietic stem cells
containing a subpopulation of at least 0.5.times.10.sup.6 potent
CD34+ cells expressing CXCR-4 and having CXCR-4 mediated
chemotactic activity [represented as "15 M"}. Control subjects
(i.e., those not receiving CD34+ cell infusion) were not expected
to have significant improvements in cardiac function (ejection
fraction, end systolic and diastolic volumes), or infarct region
perfusion at 6 months follow up.
[0423] A sterile pharmaceutical composition of the described
invention was delivered to each subject in cohorts 1, 2, and 3
parenterally by infusion via the infarct-related artery through a
catheter seven to eleven days following the STEW. The sterile
pharmaceutical composition comprised: (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; which,
when passed through the catheter remained potent, and (b) a
stabilizing amount of serum.
[0424] Cardiac function follow-up was performed at 3 and 6 months
post-infusion. Cardiac infarct region perfusion was assessed at 6
months post infusion. Both perfusion and functional follow-up
testing was assessed by a core lab facility blinded to the study
treatment status of each subject. Comparison of these results to
baseline indices was performed. Long term follow-up visits are
conducted at 12 months and telephone interview with subjects will
be made annually at years 2 through 5. For those subjects
completing the 2-year follow-up telephone call, no serious adverse
events were reported, and thus, there have been no long term safety
events detected at this point.
[0425] The cardiac performance measures Resting Total Severity
Score (RTSS), percent infarct ("% Infarct"), End Systolic Volume
(ESV) and Ejection Fraction ("EF") were assessed at 3 months post
treatment and at 6 months post treatment and compared with controls
to assess efficacy of the compositions compared to controls.
Preliminary results are shown in Table 26. SPECT SCAN. As used
herein, a single-photon emission computerized tomography (SPECT)
scan is a type of nuclear imaging test, which uses a radioactive
substance and a special camera to create three dimensional images
of the heart to show blood flows to the heart. Generally, the
"Resting Total Severity Score (RTSS) is a score based on the amount
of dye not taken up in a SPECT SCAN. The data from Resting Total
Severity Score represents cardiac perfusion, i.e., blood flow at
the microvascular level, and muscle function. In brief, the
technetium dye used in a SPECT SCAN is taken up by the heart
muscle. If the heart muscle is healthy and takes up the dye, it
appears white. If the heart muscle is not healthy, dye uptake is
diminished or does not occur at all, and the muscle appears gray to
black.
[0426] Percent Infarct (MRI). The size of a heart attack matters
for determining how well a patient will recover from the trauma. A
patient who has suffered damage to more than 30 percent of the left
ventricle of the heart is twice as likely to die within a year from
the injury as a patient who has suffered less damage, and bigger
infarcts often require more aggressive therapy A computer method
calculates the amount of damaged tissue by comparing MRI signal
strength between damaged and undamaged tissue. Damaged heart tissue
is denser than undamaged tissue because the muscle structure has
collapsed, and MRI can distinguish between tissues of varying
density. The term "percent (%) infarct" as used herein refers to
the infarcted area compared to the rest of the heart. For purposes
of this study, a % infarct greater than 20% is considered
significant.
[0427] Preliminary results are shown in Tables 26 and 27. In order
to assess statistical significance, data for the control group and
the 5 M group were pooled and data for the 10 M group and 15 M
group were pooled (N=7 for each pooled group). The preliminary
results for these pooled groups are shown in Table 27. Note that
only the SPECT data reached statistical significance; the other
measures did not reach statistical significance because of the
small numbers of patients involved.
TABLE-US-00028 TABLE 26 Phase I Efficacy Data 5M, 10M, 15M and
Control Quantitative Measures of Left Ventricular Function Treated
Treated Treated (5 Million) (10 Million) (15 Million) All Treated
Cardiac Function Test Control (N = 5) (N = 5) (N = 6) (N = 15) MRI
n = 10 n = 5 n = 4 n = 2 n = 11 LVEF (%) Baseline 53 +/- 11 47 +/-
13 47 +/- 11 50 +/- 7 48 +/- 10 6 Months 54 +/- 11 47 +/- 13 54 +/-
11 50 +/- 6 50 +/- 11 Difference 1.1 +/- 7.8 -0.02 +/- 13 7 +/- 4
0.2 +/- 0.8 2.5 +/- 9 EDV (mL) Baseline 154.7 +/- 55 153.3 +/- 30
176.6 +/- 51 175.7 +/- 12 165.8 +/- 36.1 6 Months 154.1 +/- 55
176.3 +/- 53 182.4 +/- 58 180.1 +/- 41 179.2 +/- 48 Difference
-0.56 +/- 20 23.1 +/- 37 5.83 +/- 29 4.39 +/- 29 13.4 +/- 31 ESV
(mL) Baseline 76 +/- 45 81 +/- 23 97 +/- 46 88 +/- 18 88 +/- 30 6
months 74 +/- 44 95 +/- 46 87 +/- 46 91 +/- 32 91 +/- 40 Difference
-1.84 +/- 17 14 +/- 25 -9.9 +/- 10 2.7 +/- 13 3.4 +/- 22 Infarct
Size.sup.1 Baseline 17 +/- 8 18.0 +/- 8.6 33.2 +/- 14 12 +/- 1 22.8
+/- 13 (% of LV 6 months 10 +/- 9 16.2 +/- 10.9 22 +/- 12 11 +/- 2
17.5 +/- 11 Mass) Difference -7 +/- 5 -2.6 +/- 5.9 -10.9 +/- 3 -0.6
+/- 1 -5.2 +/- 6 SPECT n = 13 n = 5 n = 5 n = 4 n = 14 RTSS
Baseline 259 +/- 283 714 +/- 658 999 +/- 753 584 +/- 440 779 +/-
620 (perfusion) 6 Months 273 +/- 395 722 +/- 521 636 +/- 532 462
+/- 290 617 +/- 449 Difference 14 +/- 210 7.8 +/- 216 -363 +/- 307
-122 +/- 260 -162 +/- 293 P-Value P-Value Combined Combined Treated
P-Value All Treated Control vs Control 10 million + 15 Control + 5
million Cardiac Function Test vs. Control 10 + 15 million Control +
5 million million vs 10 + 15 million MRI n = 10 n = 6 n = 15 n = 6
LVEF (%) Baseline 51 +/- 11 48 +/- 9 6 Months 52 +/- 12 53 +/- 9
Difference 0.706 0.336 1 +/- 9 4.5 +/- 5 0.352 EDV (mL) Baseline
154 +/- 47 176.4 +/- 39.9 6 Months 161.5 +/- 53.3 181.7 +/- 48.7
Difference 0.244 0.617 7.3 +/- 28.1 5.35 +/- 25.9 0.884 ESV (mL)
Baseline 78 +/- 38 94 +/- 37 6 months 81 +/- 44 88 +/- 38
Difference 0.553 0.666 3.6 +/- 21 -5.7 +/- 17 0.341 Infarct
Size.sup.1 Baseline 17.3 +/- 8.2 26 +/- 16 (% of LV 6 months 12 +/-
9.3 19 +/- 11 Mass) Difference 0.570 0.794 -5.3 +/- 5.0 -7.5 +/-
5.7 0.450 SPECT n = 13 n = 9 n = 18 n = 9 RTSS Baseline 385 +/- 450
814 +/- 636 (perfusion) 6 Months 398 +/- 465 559 +/- 426 Difference
0.087 0.021 13 +/- 205 -256 +/- 298 0.011 .sup.1Infarct size
expressed in % of total mass (grams)
TABLE-US-00029 TABLE 27 Simple Changes in SPECT Rest Reperfusion
Severity Score by Treatment Group - 6 Month Completers Change from
P-value Baseline to 6 Months 6 Month Baseline Post-Infusion Change
Treatment Group 1 N 5 5 0.940 Mean 714.200 7.8000 Std. Deviation
657.850 216.054 Minimum, (0.000, 1787.000) (-322.000, 222.000)
Maximum Treatment Group 2-3 N 9 9 0.033 Mean 814.333 -255.7778 Std.
Deviation 635.641 297.644 Minimum, (97.000, 1868.000) (-859.000,
263.000) Maximum Control Group N 13 13 0.808 Mean 259.000 14.462
Std. Deviation 282.698 210.078 Minimum, (0.000, 858.000) (-250.000,
528.000) Maximum Control + Treatment N 18 18 0.798 Group 1 Mean
385.444 12.611 Std. Deviation 449.728 205.293 Minimum, (0.000,
1787.000) (-322.000, 528.000) Maximum Note 1: p-values estimated
from paired differences t-tests. 95% confidence interval from
t-distribution.
[0428] As for Resting Total Severity Score, Table 27 shows that for
the 5M and control pooled group, the change in Resting Total
Severity Score after 6 months was +12.6, indicating that the
infarct area grew in these patients. The Resting Total Severity
Score data further shows that patients in the 10M and 15M groups
had bigger infarct areas at risk. The 10 M and 15M group showed a
drop of 31.4% in infarct size with a p of <0.01. Based on this
data, infusion of at least 10.times.106 isolated CD34+
hematopoietic stem cells containing a subpopulation of at least
0.5.times.106 potent CD34+ cells expressing CXCR-4 and having
CXCR-4 mediated chemotactic activity results in a statistically
significant improvement in infarct area perfusion.
[0429] The RTSS data for nontreated control subjects show neither
neoangiogenesis or prevention of cell death. When subjects were
treated with a subtherapeutic dose of cells (i.e., 5.times.106
CD34+ cells containing a subpopulation of at least 0.5.times.106
potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated
chemotactic activity), RTSS data showed neither neoangiogenesis or
prevention of cell death. Improvement in RTSS was seen only in
subjects treated with 10.times.106 or more CD34+ cells containing a
subpopulation of at least 0.5.times.106 potent CD34+ cells
expressing CXCR-4 and having CXCR-4 mediated chemotactic activity.
This dose therefore is the minimal therapeutically-effective
dose.
Example 12
Multiple Administrations of Chemotactic Hematopoietic Stem Cell
Product to Subjects
[0430] The blood supply in the peri-infarct ischemic border zones
is marginal, placing the cardiomyocytes of the border zone in
jeopardy. Multiple infusions of chemotactic hematopoietic stem cell
product, by supporting cells in the border zone, can
preserve/restore viability of the peri-infarct myocardium.
[0431] According to this aspect of the described invention, a first
aliquot of the composition is administered at a first infusion
date, a second aliquot of the composition is administered at a
second infusion date, a third aliquot of the composition is
administered at a third infusion date, and so on. The scheduling of
infusion dates is determined for a given patient by the treating
practitioner according to his/her medical judgment.
[0432] According to one embodiment, the first infusion date is at
least about one day, at least about two days, at least about three
days, at least about four days, at least about five days, at least
about six 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, at least about 15 days, at least about 16 days, at least
about 17 days, at least about 18 days, at least about 19 days, at
least about 20 days, at least about 21 days, at least about 22
days, at least about 23 days, at least about 24 days, at least
about 25 days, at least about 26 days, at least about 27 days, at
least about 28 days, at least about 29 days, at least about 30 days
or more after occurrence of an AMI. According to another
embodiment, the first infusion date is at least about 1 month, at
least about 2 months, at least about 3 months, at least about 4
months, at least about 5 months, at least about 6 months, at least
about 7 months, at least about 8 months, at least about 9 months,
at least about 0 months, at least about 11 months, at least about
12 months, at least about 13 months, at least about 14 months, at
least about 15 months, at least about 16 months, at least about 17
months, at least about 18 months, at least about 19 months, at
least about 20 months, at least about 21 months, at least about 22
months, at least about 23 months, at least about 24 months, at
least about 30 months, at least about 36 months, at least about 42
months, at least about 48 months, at least about 54 months, at
least about 60 months, at least about 66 months, at least about 72
months, at least about 78 months, at least about 84 months, at
least about 90 months, at least about 96 months, at least about 102
months, at least about 108 months, at least about 114 months, at
least about 120 months, at least about 126 months, at least about
132 months, at least about 138 months, at least about 144 months,
at least about 150 months, at least about 156 months, at least
about 162 months, at least about 168 months, at least about 174
months, at least about 180 months, at least about 186 months, at
least about 192 months, at least about 198 months, at least about
204 months, at least about 20 months, at least about 216 months, at
least about 222 months, at least about 228 months, at least about
234, months, at least about 240 months or more after occurrence of
an AMI. According to some embodiments, the first infusion date is
at least 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9
years, 10 years, 11 years, 12 years, 13 years, 14 years. 15 years,
16 years 17 years, 18 years, 19 years, 20 years, 21 years, 22
years, 23 years, 24 years, 25 years, 26 years. 27 years, 28 years.
29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35
years, 36 years 37 years, 38 years, 39 years, 40 years or more
after occurrence of an AMI.
[0433] According to another embodiment, the second infusion date is
at least about one day, at least about two days, at least about
three days, at least about four days, at least about five days, at
least about six 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, at least about 15 days, at least about 16 days, at
least about 17 days, at least about 18 days, at least about 19
days, at least about 20 days, at least about 21 days, at least
about 22 days, at least about 23 days, at least about 24 days, at
least about 25 days, at least about 26 days, at least about 27
days, at least about 28 days, at least about 29 days, at least
about 30 days or more after occurrence of an AMI. According to
another embodiment, the second infusion date is at least about 1
month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, at least about 6 months,
at least about 7 months, at least about 8 months, at least about 9
months, at least about 0 months, at least about 11 months, at least
about 12 months, at least about 13 months, at least about 14
months, at least about 15 months, at least about 16 months, at
least about 17 months, at least about 18 months, at least about 19
months, at least about 20 months, at least about 21 months, at
least about 22 months, at least about 23 months, at least about 24
months, at least about 30 months, at least about 36 months, at
least about 42 months, at least about 48 months, at least about 54
months, at least about 60 months, at least about 66 months, at
least about 72 months, at least about 78 months, at least about 84
months, at least about 90 months, at least about 96 months, at
least about 102 months, at least about 108 months, at least about
114 months, at least about 120 months, at least about 126 months,
at least about 132 months, at least about 138 months, at least
about 144 months, at least about 150 months, at least about 156
months, at least about 162 months, at least about 168 months, at
least about 174 months, at least about 180 months, at least about
186 months, at least about 192 months, at least about 198 months,
at least about 204 months, at least about 20 months, at least about
216 months, at least about 222 months, at least about 228 months,
at least about 234 months, at least about 240 months or more after
occurrence of an AMI. According to some embodiments, the second
infusion date is at least 3 years, 4 years, 5 years, 6 years. 7
years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14
years, 15 years, 16 years 17 years, 18 years, 19 years, 20 years,
21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27
years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years,
34 years, 35 years, 36 years 37 years, 38 years, 39 years, 40 years
or more after occurrence of an AMI.
[0434] According to another embodiment, the third infusion date is
at least about one day, at least about two days, at least about
three days, at least about four days, at least about five days, at
least about six 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, at least about 15 days, at least about 16 days, at
least about 17 days, at least about 18 days, at least about 19
days, at least about 20 days, at least about 21 days, at least
about 22 days, at least about 23 days, at least about 24 days, at
least about 25 days, at least about 26 days, at least about 27
days, at least about 28 days, at least about 29 days, at least
about 30 days or more after occurrence of an AMI. According to
another embodiment, the third infusion date is at least about 1
month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, at least about 6 months,
at least about 7 months, at least about 8 months, at least about 9
months, at least about 0 months, at least about 11 months, at least
about 12 months, at least about 13 months, at least about 14
months, at least about 15 months, at least about 16 months, at
least about 17 months, at least about 18 months, at least about 19
months, at least about 20 months, at least about 21 months, at
least about 22 months, at least about 23 months, at least about 24
months, at least about 30 months, at least about 36 months, at
least about 42 months, at least about 48 months, at least about 54
months, at least about 60 months, at least about 66 months, at
least about 72 months, at least about 78 months, at least about 84
months, at least about 90 months, at least about 96 months, at
least about 102 months, at least about 108 months, at least about
114 months, at least about 120 months, at least about 126 months,
at least about 132 months, at least about 138 months, at least
about 144 months, at least about 150 months, at least about 156
months, at least about 162 months, at least about 168 months, at
least about 174 months, at least about 180 months, at least about
186 months, at least about 192 months, at least about 198 months,
at least about 204 months, at least about 20 months, at least about
216 months, at least about 222 months, at least about 228 months,
at least about 234 months, at least about 240 months or more after
occurrence of an AMI. According to some embodiments, the first
infusion date is at least 3 years, 4 years, 5 years, 6 years, 7
years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14
years, 15 years, 16 years 17 years, 18 years, 19 years, 20 years,
21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27
years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years,
34 years, 35 years, 36 years 37 years, 38 years, 39 years, 40 years
or more after occurrence of an AMI.
[0435] Eligible subjects/patients presenting with symptoms and
clinical findings suggestive of a myocardial infarction and
eligible for inclusion in the study will be selected as described
in Example 1 and catheterized as described in Example 2. In some
embodiments, the nonexpanded, isolated population of autologous
mononuclear cells comprising potent CD34+ cells will be acquired
from the subject/patient as described in Example 3 and, in some
embodiments, the harvested bone marrow will be transported to the
processing facility as described in Example 4. CD34+ cells will be
selected from the harvested bone marrow product as described in
Example 5.
[0436] The Isolex 300i system will be used to process the
RBC-depleted product or the bone marrow product whose RBC volume is
<20 ml according to the following processing steps: [0437] (i)
The bone marrow is washed automatically to remove platelets; [0438]
(ii) CD34 positive (CD34+) cells are labeled specifically for
selection by incubation with the Isolex 300i CD34 monoclonal
antibody (Mab); [0439] (iii) Unbound reagent is removed by washing
the cell suspension with buffer solution; [0440] (iv) Sensitized
CD34+ cells (meaning CD34+ cells labeled with CD34 Mab) are
captured by Dynabeads M-450 Sheep anti-Mouse IgG; [0441] (v) A
selection column is used to separate the magnetically-labeled
Dynabeads having captured CD34+ cells from unwanted cells, which
are washed through the selection column and collected in the
Negative Fraction Bag; and [0442] (vi) PR34+ Stem Cell Releasing
Agent releases CD34+ cells from the column, and the CD34+ cells are
collected in the End Product Bag. The system performs several
washing steps, disposing of most of the liquid into the Buffer
Waste Bag.
[0443] The Isolex.RTM. selected CD34+ fraction then will be assayed
to determine WBC and CD34+ cell yields as described in Example 6. A
first aliquot of the chemotactic hematopoietic stem cell product
containing at least 10.times.10.sup.6 CD34+ cells will be
formulated a described in Example 7, transported to the
catheterization facility as described in Example 8, and infused
into the patient as described in Example 9 at the first infusion
date. A plurality of aliquots of the nonexpanded, isolated
population of autologous mononuclear cells containing CD34+ cells,
which further contain a subpopulation of CD34+/CXCR-4+ cells that
have CXCR-4-mediated chemotactic activity will be frozen at
-86.degree. C. and cryostored in the vapor phase of a liquid
nitrogen freezer for subsequent administration. (see
"Cryopreservation Study" below).
[0444] CryoPreservation Study.
[0445] This study was conducted to evaluate the ability of the
Isolex-based portion of the chemotactic hematopoietic stem cell
product manufacturing process to effectively enrich for CD34+ cells
of the cryopreserved MMH. The protocol has been designed to
evaluate the yield, viability, functionality and stability of the
CD34+ cells derived from the enrichment of cryopreserved MMH. The
study has been designed to evaluate and describe the effect on the
chemotactic hematopoietic stem cell product of cryopreservation of
the RBC reduced MMH prior to the Isolex-based CD34 selection.
[0446] The following experimental conditions have been applied:
[0447] (1) Two (2) MMH for each of three (3) replicates in order to
provide for adequate cell yield to meet with requirements of the
experimental design; with a twenty four (24) hour interval between
MMH and commencement of RBC depletion procedure.
[0448] (2) Study control: Freshly prepared chemotactic
hematopoietic stem cell product, with full product characterization
after perfusion of the chemotactic hematopoietic stem cell product
through a catheter at 48 and 72 hours after MMH.
[0449] (3) Experimental: the chemotactic hematopoietic stem cell
product derived from cryopreserved MMH, with full product
characterization after perfusion of the chemotactic hematopoietic
stem cell product derived from cryopreserved MMH through a catheter
at 48 and 72 hours after MMH, minus the time the cryopreserved MMH
remains in storage (defined as >24 hours)
[0450] Study Design
[0451] In order to yield sufficient CD34+ cells to perform the
intended experiment, two (2) donors will be required. More than or
equal to 80 ml MMH and .gtoreq.30 ml of peripheral blood will be
collected from each donor.
[0452] In bound storage: Samples will be stored at 2 to 8.degree.
C. for twenty four (24) hours before commencing the RBC reduction
procedure.
[0453] Following RBC reduction, the MMH from both donors will be
pooled and then divided into two equal fractions. One fraction will
be used as a fresh (unfrozen) product control and the other
fraction will be used for the cryopreservation test.
[0454] For the cryopreservation test, RBC reduced MMH will be
frozen in a -86.degree. C. freezer and then cryostored in the vapor
phase (.ltoreq.-150.degree. C.) of a liquid nitrogen freezer (LNF)
using the cryoprotectant containing the liquid source Hetastarch
(6% Hetastarch in 0.9% Sodium Chloride manufactured by
Hospira).
[0455] Both control (unfrozen) and cryopreserved (after thaw)
samples will be Isolex processed essentially as described in
Example 5 above. Samples in two 10 ml syringes will be prepared
from the selected CD34+ cells. Full product characterization will
be performed at the following time points: (i) After perfusion of
the product through a catheter at 48 hours after MMH; and (ii)
after perfusion of the product through a catheter at 72 hours after
MMH. For the cryopreserved samples, the term "72 hours of
collection", for example, means the time from collection to the
time of testing, excluding the time elapsed from freezing and
cryostorage of the RBC depleted bone marrow.
[0456] Key determinants for the CD34+ cell quality of the
hematopoietic stem cell product include: (i0 CD34+ cell enumeration
and 7-AAD viability; (ii) SDF-1/CXCR-4 mediated CD34+ cell
migratory activity; (iii) expression of CXCR-4 cell surface antigen
on CD34+ cells; and (iv) growth of hematopoietic progenitor cell
colonies (CFU). This experiment will be repeated three times.
[0457] Summary Of Results
[0458] The study was conducted in accordance with the methods
described above. All deviations from methodology and materials used
are detailed in the related result sections presented below.
[0459] Table 28 summarizes the relevant information on the donors
of the bone marrow used in this study.
TABLE-US-00030 TABLE 28 Age and gender of the bone marrow donors
for the cryopreservation study. Exp 1 Exp 2 Exp 3 Donor 1 2 3 4 5 6
Age 26 26 22 62 32 24 Gender F F F F F F
[0460] Table 29 summarizes the sample volume, RBC content and the
yield, viability and purity of the cells in the pre-processed MMH
following 24-h storage in a 2-8.degree. C. refrigerator.
TABLE-US-00031 TABLE 29 Post 24 hours storage at 2-8.degree. C. -
Volume, cell yield and quality of MMH. Exp 1 Exp 2 Exp 3 Donor 1
Donor 2 Donor 3 Donor 4 Donor 5 Donor 6 Volume (ml) 117 64 106 105
103 113 WBC per .mu.l.sup.# 1.39E+04 1.26E+04 1.39E+04 1.44E+04
1.94E+04 2.45E+04 TNC.sup.# 1.62E+09 8.03E+08 1.47E+09 1.51E+09
1.99E+09 2.76E+09 HCT.sup.# 33.85% 33.40% 29.10% 27.85% 31.60%
32.60% RBC vol. (ml).sup.# 39.44 21.38 30.85 29.24 32.55 36.84
CD45+ cell viability* 91.13% 91.72% 90.58% 93.17% 94.11% 95.8%
Viable CD34+ cell 149.18 148.38 140.89 114.45 150.80 203.76 per
.mu.l* CD34+ cell viability* 94.14% 98.90% 98.35% 97.24% 98.89%
98.78% CD34+ cell purity* 1.44% 1.32% 1.23% 0.97% 1.21% 0.88%
CXCR-4 expressing 77.68% 77.03% 71.88% 64.57% 75.75% 68.36% CD34+
cells Total # of CD34+ 1.74E+07 9.50E+06 1.49E+07 1.20E+07 1.55E+07
2.30E+07 cells* .sup.#Determined by hematology analyzer *Determined
by flow cytometric analysis of CD45-FITC/CD34-PE antibodies and
7-AAD staining of the sample Determined by flow cytometric analysis
of CD34-FITC and CXCR-4-PE antibodies staining of the sample
[0461] In each of the experiments, the MMH from each pair of donors
were pooled following RBC reduction.
[0462] Table 30 presents the RBC content, viability and cell
recovery of pooled MMH after RBC reduction:
TABLE-US-00032 TABLE 30 Post RBC reduction - RBC content and cell
quality Exp 1 Exp 2 Exp 3 Donors 1 & 2 Donors 3 & 4 Donors
5 & 6 RBC volume 15.35 ml 13.80 ml 20.85 ml TNC recovery#
76.95% 85.93% 89.37% CD45+ cell viability 84.97% 93.35% 95.60% CD34
recovery# 72.89% 84.00% 88.36% CD34+ cell viability 93.99% 97.92%
98.95% CXCR-4 expressing 71.33% 64.89% 74.64% CD34+ cells #As
compared to the pre-processed samples
[0463] Following RBC reduction, each of the pooled MMH samples was
divided into two equal fractions. One was used as a fresh
(unfrozen) control and the other one was used for the
cryopreservation test.
[0464] For cryopreservation, MMH mixed with an equal volume of
chilled cryoprotectant was loaded evenly into two 250 ml Cryocyte
containers, frozen in a mechanical freezer (-86.degree. C.) and
then stored cryopreserved in the vapor phase of a LNF according to
the Protocol. Table 31 presents data obtained from post-thawed and
washed MMH:
TABLE-US-00033 TABLE 31 Post thawed & washed MMH - Cell quality
and recovery of cells Cryopreserved Sample Thawed and washed Exp 1
Exp 2 Exp 3 Storage duration prior to 10 days 8 days 8 days thaw
Wash media PBS working 2% Dextran~ 8.3% Dextran@ sol'n* RBC volume
0.39 ml 1.11 ml 0.38 ml TNC recovery# 36.11% 50.73% 28.61% CD45+
cell viability 61.85% 32.18% 43.97% CD34+ cell recovery# 52.43%
46.29% 15.72% CD34+ cell viability 94.36% 86.11% 81.76% CD34+ cell
purity 2.40% 1.29% 1.88% CXCR-4 expressing 51.42% 50.74% 37.85%
CD34+ cells Key: #As compared to the RBC reduced MMH before
cryopreservation. *PBS Working Solution contained 1% HSA and 0.41%
sodium citrate (w/v) in PBS (Ca.sup.++ and Mg.sup.++ free). Washing
of cells with this solution was performed according to that
instructed in the Protocol. ~This wash solution contained 2%
Dextran 40, 1% HSA and 0.4% Na citrate in PBS (Ca.sup.++ and
Mg.sup.++ free). The thawed sample was expanded with 200 ml of this
solution and was then washed twice each with 200 ml of this
solution. Centrifugation was set for 600 g, 10 minutes at
20.degree. C. The washed cells were resuspended with 150 ml PBS
Working Solution for Isolex process. @ This solution contained 8.3%
Dextran 40 and 4.2% HSA in saline. The washing procedure was
essentially as described for the 2% Dextran 40 wash solution.
[0465] Table 32 summarizes the CD34+ cell quality and recovery of
the chemotactic hematopoietic stem cell product prepared from the
unfrozen and cryopreserved MMH following Isolex processing.
TABLE-US-00034 TABLE 32 Post Isolex - Cell quality and recovery of
cells Exp 1 Exp 2 Exp 3 MMH source Unfrozen Frozen Unfrozen Frozen
Unfrozen Frozen CD34+ cell 47.28% 37.88% 35.94% 49.29% 44.05%
82.25% recovery# CD34+ cell viability# 99.37% 96.89% 98.97% 95.05%
98.26% 95.38% CD34+ cell purity 87.51% 83.95% 86.47% 81.91% 81.71%
50.87% Total # of viable 4.63E+06 1.95E+06 4.07E+06 2.58E+06
7.50E+06 2.20E+06 CD34+ cells #As compared to the RBC reduced
sample for unfrozen samples and post thawed and washed samples for
frozen samples.
[0466] Following Isolex processing of each RBC reduced MMH pooled
pair, two chemotactic hematopoietic stem cell product ("AMR-001")
samples with equal number of CD34+ cells, each in a 10 ml syringe,
were prepared. Both AMR-001 samples were stored at 2-8.degree. C.
for stability testing. At 48 and 72 hours from MMH (For
cryopreserved MMH samples, the time for cryostorage was not
included), a prepared AMR-001 was perfused through a balloon
dilatation catheter performed in a manner as for a clinical
AMR-001. A full CD34+ cell characterization was performed on the
perfused AMR-001 samples and the results are presented in Tables
33, 34, 35, and 36. Table 37 shows the balloon dilatation catheter
used.
TABLE-US-00035 TABLE 33 Post infusion through catheter - CD34+ cell
purity, viability and recovery Catheter perfused AMR-001 MMH source
Unfrozen Frozen Time post MMH Experiment 48 h 72 h 48 h 72 h 1
CD34+ cell recovery# 101.73% 92.32% 91.71% 69.35% CD34+ cell
viability 99.08% 98.13% 94.98% 91.80% CD34+ cell purity 85.92%
84.93% 82.94% 74.24% Total # of CD34+ cells 2.36E+06 2.14E+06
8.92E+05 6.74E+05 2 CD34+ cell recovery# 95.65% 89.20% 77.10%
74.01% CD34+ cell viability 98.29% 97.29% 89.47% 82.82% CD34+ cell
purity 81.49% 82.42% 75.30% 70.50% Total # of CD34+ cells 1.95E+06
1.81E+06 9.96E+05 9.56E+05 3 CD34+ cell recovery# 104.17% 101.99%
77.35% 79.12% CD34+ cell viability 98.46% 97.51% 86.86% 85.59%
CD34+ cell purity 83.18% 82.80% 47.81% 43.71% Total # of CD34+
cells 3.91E+06 3.83E+06 8.52E+05 8.71E+05 #As compared with the
prepared AMR-001 before perfusion
TABLE-US-00036 TABLE 34 Post infusion through catheter - CXCR-4
expressing CD34+ cells (% of total CD34+ cells). MMH source of
AMR-001 samples Exp 1 Exp 2 Exp 3 Catheter perfusion Unfrozen
Frozen Unfrozen Frozen Unfrozen Frozen 48 h post MMH 66.52% 53.31%
57.64% 41.35% 60.14% 54.16% 72 h post MMH 73.87% 53.89% 56.73%
44.07% 64.60% 50.67%
TABLE-US-00037 TABLE 35 Post infusion through catheter - Migratory
CD34+ cells (% of total viable CD34+ cells). MMH source of AMR-001
samples Catheter Exp 1 Exp 2 Exp 3 perfusion Unfrozen Frozen
Unfrozen Frozen Unfrozen Frozen 48 h post 18.81 .+-. 1.83%* 5.87
.+-. 1.98% 19.67 .+-. 10.43% 15.67 .+-. 2.24% 24.89 .+-. 1.93%
26.66 .+-. 1.53% MMH 72 h post (1.07%)# (1.51%) (1.06%) (2.19%)
(1.44%) (1.56%) MMH *SDF-1 induced migration. % of migratory CD34+
cell of total viable CD34+ cells with standard deviation of three
replicates. #Natural migration (no SDF-1 added)
TABLE-US-00038 TABLE 36 Post infusion through catheter - Number of
CFU per 100 viable CD34+ cells cultured. MMH source of AMR-001
samples Exp 1 Exp 2 Exp 3 perfusion Unfrozen Frozen Unfrozen Frozen
Unfrozen Frozen 48 h post MMH 24 15.5 31.5 14 38 15.5 72 h post MMH
20.5 0.05 62.5 12 30.5 7
TABLE-US-00039 TABLE 37 Balloon dilatation catheters used MMH
source Time of of the perfusion AMR- (Hours 001 of Balloon Exp
sample MMH) Manufacture length/dia. Catalog # Lot # Comment 1
Unfrozen 48 h Sprinter 12/3.5 mm SPR3512W 258795 Outdated 72 h
Sprinter 12/4.0 mm SPR4012W 254243 Outdated Frozen 48 h Sprinter
15/3.0 mm SPR3015W 412090 Outdated 72 h Voyager 15/3.0 mm 1009443-
8111462 -- 15 2 Unfrozen 48 h Sprinter 15/3.5 mm SPR3515W 443152
Outdated 72 h Sprinter 15/3.5 mm SPR3515W 443152 Outdated Frozen 48
h Voyager 15/3.0 mm 1009443- 8111462 -- 15 72 h Voyager 15/3.0 mm
1009443- 8092561 -- 15 3 Unfrozen 48 h Voyager 15/3.0 mm 1009443-
8111462 Reused* 15 72 h Sprinter 15/3.0 mm SPR3015W 476734 Outdated
Frozen 48 h Sprinter 15/3.0 mm SPR3015W 476734 Outdated 72 h
Sprinter 15/3.0 mm SPR3015W 476734 Outdated *Prior to be used for
the 2.sup.nd time, the catheter and the central lumen were 1.sup.st
washed and flushed with 70% isopropyl alcohol and then with sterile
PBS. The central lumen was then injected with air in order to
remove the residual liquid inside. The washing procedure was
performed inside a bio-safety cabinet.
[0467] Discussion
[0468] The aim of this study was to evaluate the quality of AMR-001
manufactured from cryopreserved MMH.
[0469] Post Isolex CD34+ cell recovery of the AMR-001 manufactured
from unfrozen MMH (Control samples) was on average 34.6.+-.4.35%
(range 30.3% to 39%) which is within the acceptance range for
manufacture of AMR-001 for clinical use. It should be noted that
the data presented above are estimated without taking account for
the cells removed for the in-process tests, therefore the actual
CD34+ cell recovery will be slightly higher than that
presented.
[0470] Post catheter CD34+ cell recovery was 100.52.+-.4.39%
(95.65% to 104.17%) at 48 hours post MMH and 94.50.+-.6.67% (89.20%
to 101.99%) at 72 hours post MMH. There was no substantial
reduction in viability (Table 33), CXCR-4 expression (Table 34),
migratory activity (Table 35) and CFU growth (Table 36) of CD34+
cells at 72 hours post MMH as compared to those monitored at 48
hours post MMH.
[0471] For the cryopreservation test, RBC reduced MMH samples were
cryopreserved according to PCT protocol for cryopreservation of
bone marrow for transplantation where MMH samples mixed with equal
volume of cryoprotectant with final concentration of 5% DMSO, 2.5%
HSA and 2.1% Hetastarch (from liquid source 6% Hetastarch, Hospira)
were frozen at -86.degree. C. and then cryostored in the vapor
phase of a LNF.
[0472] Post cryopreservation and thaw, the stability, viability,
mobility and growth in culture of Isolex selected CD34+ cells is
maintained. Thus the frozen-thawed cells meet the criteria for
clinical use.
[0473] In some embodiments, a chemotactic hematopoietic stem cell
product prepared from frozen and thawed aliquots of a sterile
nonexpanded, isolated population of autologous mononuclear cells
comprising CD34+ cells, which further contain a subpopulation of
potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic
activity will be used for infusion. Samples of thischemotactic
hematopoietic stem cell product will be removed to be assayed for
WBC count, by flow cytometry (for CD34+ cell enumeration and
viability), Gram stain, and sterility. The chemotactic
hematopoietic stem cell product will be released for infusion
within about 48 hours to about 72 hours of thawing of the sterile
nonexpanded, isolated population of autologous mononuclear cells
only if it meets the following criteria:
[0474] CD34+ cell purity of at least about 70%, 75%, 80%, 85%, 90%
or 95%;
[0475] A negative Gram stain result for the selected positive
fraction;
[0476] Endotoxin Levels: less than about 0.5 endotoxin
units/ml;
[0477] Viable CD34+ cell yield of the "Chemotactic hematopoietic
stem cell product" meets the required dosing as per the treatment
cohort;
[0478] CD34+ cells are at least about 70%, 75%, 80%, 85%, 90% or
95% viable by 7-AAD;
[0479] USP sterility result for "Positive Fraction Supernatant":
negative (14 days later).
[0480] Sterility assessment on the stem cell product including gram
staining and endotoxin will be performed prior to product release
for infusion. USP sterility (bacterial and fungal) culture will be
performed and the results will be reported to the principal
investigator. In the event of a positive USP sterility result, the
subject and attending physician on call will be notified
immediately, provided with identification and sensitivity of the
organism when available, and documentation of appropriate
anti-microbial treatment and treatment outcome will be recorded by
the investigative site and the sponsor.
[0481] The chemotactic hematopoietic stem cell product prepared
from the frozen and thawed autologous mononuclear cells will be
formulated a described in Example 7, transported to the
catheterization facility as described in Example 8, and infused
into the patient as described in Example 9.
[0482] It is proposed that administration of a potent dose of
CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity,
early or late after occurrence of an acute myocardial infarction
according to the described invention can result in a reduction in
persistent/chronic and progressive adverse cardiac events,
including, but not limited to, premature death, recurrent
myocardial infarction, the development of congestive heart failure,
significant arrhythmias, and acute coronary syndrome, and the
worsening of congestive heart failure, significant arrhythmias, and
acute coronary syndrome.
Example 13
Co-Administration of the Chemotactic Hematopoietic Stem Cell
Product and Neuregulin 1
[0483] Neuregulin 1 (NRG1) is an agonist for receptor tyrosine
kinases of the epidermal growth factor receptor family, consisting
of ErbB1,2, 3, and 4. (Fuller, S J, et al., J. Mol. Cell. Cariol.
44: 831-54 (2008). Binding of NRG1 to Erb4 increases its kinase
activity and leads to heterodimerization with erbB2 or
homodimerization with ErbB4 and stimulation of intracellular signal
transduction pathways. Id. NFRG1 receptor subunits ErbB2 and ErbB4
also are expressed in differentiated cardiomyocytes. Id. Recently
it has been shown in mice that NRG1 induces proliferation of
differentiated mononucleated cardiomyocytes in vivo by inducing
differentiated cardiomyocytes to leave proliferative quiescence.
Bersell, et al (Bersell, K. et al., Cell 138: 257-70 (2009).
Undifferentiated stem and progenitor cells did not contribute to
this proliferation. (Id). Using a mouse model in which the left
anterior descending coronary artery (LAD) of two month old mice was
ligated permanently and NRG1 administered daily one week later for
12 weeks, it was shown that administration of NRG1 for 12 weeks
resulted in a sustained improvement in myocardial function,
determined by ejection fraction, a reduced infarct scar size, and
attenuation of cardiomyocyte hypertrophy. (Id).
[0484] Following acute myocardial infarction, in addition to
necrotic cell death as a consequence of ischemic, ongoing apoptotic
cell death and cardiomyocyte hibernation collectively lead to a
decrement in cardiac function that can worsen over time and
ultimately causing major adverse cardiac events. Once lost,
cardiomyocytes are unable to significantly regenerate to restore
cardiac function. Carbon 14 dating of cardiomyocytes show the
regenerative capacity of cardiac muscle to be less than 1% annually
(Bergman O, Science. 2009; 324:98-101). The described invention
demonstrates the prevention of cardiomyocyte loss after AMI through
enhancement of perfusion and prevention of apoptosis. Further
restoration of cardiac function requires significantly increasing
the regenerative capacity of cardiomyocytes. Regenerating
cardiomyocytes will require adequate perfusion or will suffer the
consequences of ischemia including hibernation and apoptosis.
[0485] It is proposed that the combination of the described
invention with significant augmentation of the natural regenerative
capacity of cardiomyocytes would be synergistic in restoring
cardiac function after AMI and preventing major adverse cardiac
events. Co-administration therefore of the chemotactic
hematopoietic stem cell product of the described invention with
neuregulin 1 is proposed as a therapeutic capable of restoring
cardiac function after AMI through increasing perfusion, which
prevents apoptotic cardiomyocyte cell death and rescues
cardiomyocytes from hibernation, and by providing the
infrastructure needed for generation of new cardiomyocytes to
replace lost cardiomyocytes.
[0486] Recombinant human neuregulin 1 will be obtained from
commercial sources. (Cell Sciences, Novus Biologicals, R & D
Systems, Raybiotech, Inc., Shenandoah Biotechnology, Spring
Bioscience).
[0487] Increased doses of neuregulin 1 will be admixed with the
chemotactic hematopoietic stem cell product of the described
invention and tested in vitro after passage through a catheter for
product viability, sterility, purity and potency, meaning
viability, migratory capacity and CFU-growth, after storage for up
to 72 hours. If potency, purity and viability are maintained, a
preclinical experiment is proposed in which purified, sterile human
derived CD34+ cells containing a subpopulation of potent CD34+
cells expressing CXCR-4 and having CXCR-4-mediated chemotactic
activity will be infused via the tail vein in Nod SCID mice after
coronary artery ligation and relief (induced AMI model). The effect
of this treatment on cardiac perfusion, cardiac muscle function,
histopathology, apoptosis, and scarring will be assessed post
infusion and compared to controls (i.e., Nod SCID mice not
receiving cells). Prior studies have demonstrated an improvement in
perfusion, human neoangiogenesis, prevention of apoptosis, and
preserved cardiac function in treated versus control animals. Next,
increasing doses of neuregulin 1 will be added to the purified,
sterile human derived CD34+ cells containing a subpopulation of
potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated
chemotactic activity of the described invention and the results
will be compared to control animals and to animals treated with the
purified, sterile human derived CD34+ cells containing a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity of the described invention
alone.
[0488] If pre-clinical models show a potential synergistic
beneficial effect with the purified, sterile human derived CD34+
cells containing a subpopulation of potent CD34+ cells expressing
CXCR-4 and having CXCR-4-mediated chemotactic activity of the
described invention combined with neuregulin 1, a dose escalation
safety and efficacy trial in sustaining and in AMI patients is
proposed. For this study, patients will receive the purified,
sterile human derived CD34+ cells containing a subpopulation of
potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated
chemotactic activity of the invention with or without neuregulin 1.
Neuregulin 1 will be administered in increasing doses to determine
(i) the mean therapeutic dose (MTD) and (ii) whether perfusion and
cardiac function are enhanced by the combination of neuregulin 1
and the purified, sterile human derived CD34+ cells Containing a
subpopulation of potent CD34+ cells expressing CXCR-4 and having
CXCR-4-mediated chemotactic activity of the described invention
compared to the purified, sterile human derived CD34+ cells
containing a subpopulation of potent CD34+ cells expressing CXCR-4
and having CXCR-4-mediated chemotactic activity of the described
invention alone.
[0489] While the described invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the described invention. All
such modifications are intended to be within the scope of the
claims appended hereto.
* * * * *
References