U.S. patent application number 12/088067 was filed with the patent office on 2009-02-05 for dry platelet composition.
This patent application is currently assigned to LifeCell Corporation. Invention is credited to Jerome Connor, John R. Harper, Christopher T. Wagner.
Application Number | 20090035289 12/088067 |
Document ID | / |
Family ID | 37900439 |
Filed Date | 2009-02-05 |
United States Patent
Application |
20090035289 |
Kind Code |
A1 |
Wagner; Christopher T. ; et
al. |
February 5, 2009 |
DRY PLATELET COMPOSITION
Abstract
The invention features a dry platelet composition and methods of
making and using the freeze-dried platelet composition.
Inventors: |
Wagner; Christopher T.;
(Easton, PA) ; Connor; Jerome; (Doylestown,
PA) ; Harper; John R.; (Jamison, PA) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
LifeCell Corporation
Branchburg
NJ
|
Family ID: |
37900439 |
Appl. No.: |
12/088067 |
Filed: |
September 26, 2006 |
PCT Filed: |
September 26, 2006 |
PCT NO: |
PCT/US2006/037741 |
371 Date: |
August 19, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60720851 |
Sep 26, 2005 |
|
|
|
Current U.S.
Class: |
424/93.72 ;
435/325 |
Current CPC
Class: |
A61K 35/16 20130101;
A61K 45/06 20130101; A61K 31/5575 20130101; A01N 1/0226 20130101;
A61K 35/19 20130101; A61K 31/522 20130101; A61K 31/5578 20130101;
Y02A 50/471 20180101; A61K 31/4965 20130101; A01N 1/0221 20130101;
A61K 33/26 20130101; Y02A 50/30 20180101; A61K 9/19 20130101; A61K
31/7076 20130101; A61P 17/02 20180101; A61K 35/19 20130101; A61K
2300/00 20130101; A61K 35/16 20130101; A61K 2300/00 20130101; A61K
31/7076 20130101; A61K 2300/00 20130101; A61K 31/4965 20130101;
A61K 2300/00 20130101; A61K 33/26 20130101; A61K 2300/00 20130101;
A61K 31/5578 20130101; A61K 2300/00 20130101; A61K 31/5575
20130101; A61K 2300/00 20130101; A61K 31/522 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/93.72 ;
435/325 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 5/00 20060101 C12N005/00; A61P 17/02 20060101
A61P017/02 |
Goverment Interests
[0001] The research described in this application was supported by
contract number W81XWH-05-1-0077 from the Department of Defense.
Thus, the government has certain rights in the invention.
Claims
1. A dry platelet composition, the composition comprising: a
plurality of dry platelets; and one or more inhibitors of platelet
activation.
2. The composition of claim 1, wherein the one or more inhibitors
of platelet activation are selected from effectors of the cyclic
adenosine monophosphate (cAMP) second messenger system, sodium
channel inhibitors, and effectors of the cyclic guanosine 5'
monophosphate (cGMP) second messenger system.
3. The composition of claim 1, wherein the one or more inhibitors
of platelet activation comprise adenosine, amiloride, and sodium
nitroprusside.
4. The composition of claim 3, wherein, after hydration of the
composition, the concentration in the composition: of adenosine is
about 10 .mu.M to about 1 mM; of amiloride is about 0.1 mM to about
10 mM; and of sodium nitroprusside is about 2.5 .mu.M to about 250
.mu.M.
5. The composition of claim 2, wherein the effectors of the cAMP
second messenger system are selected from the group consisting of
iloprost, prostacyclin, prostaglandin E.sub.2, forskolin, cholera
toxin, isoproterenol, 8-bromo cyclic adenosine monophosphate,
dibutyl cyclic adenosine monophosphate, theophylline,
isobutylmethyl xanthine, thyrotropin, and auranofin.
6. The composition of claim 2, wherein the sodium channel
inhibitors are selected from the group consisting of amiloride
analogues, bepridil, flecamide, saxitoxin, benzamil, and
prajnalium.
7. The composition of claim 2, wherein the effectors of the cGMP
second messenger system are selected from the group consisting of
L-arginine, nitrous oxide, SIN-1, SIN-1A, atrial natriuretic
factor, vasopressin, oxytocin, and glyceril trinitrate.
8. The composition of claim 1, further comprising one or more
cryoprotective agents
9. The composition of claim 8, wherein the cryoprotective agents
are selected from the group consisting of dimethylsulfoxide,
maltodextrin, dextran, hydroxyethyl starch, glucose, polyvinyl
pyrrolidone, mannitol, and combinations thereof.
10. The composition of claim 1, further comprising dry blood
plasma.
11. The composition of claim 1, further comprising one or more
extracellular matrix (ECM) components.
12. The composition of claim 11, wherein the one or more ECM
components are selected from the group consisting of collagen,
elastin, fibronectin, fibrillin, laminin, decorin, fibromodulin,
hyaluronic acid, and a proteoglycan.
13. The composition of claim 11, wherein the ECM components are in
particles of particulate acellular tissue matrix.
14. The composition of claim 13, wherein the particulate acellular
tissue matrix is particulate acellular dermal matrix.
15. The composition of claim 1, wherein hydration of the dry
platelet composition results in a rehydrated platelet composition
with substantially the same level of at least one platelet function
possessed by a sample of fresh platelets from which the dry
platelet composition was derived.
16. The composition of claim 15, wherein the at least one platelet
function is the ability to aggregate.
17. The composition of claim 15, wherein the at least one platelet
function is the ability to release one or more growth factors or
chemokines.
18. The composition of claim 17, wherein the growth factors or
chemokines are selected from the group consisting of transforming
growth factor-.beta. (TGF-.beta.), members of platelet derived
growth factor (PDGF) family, epidermal growth factor (EGF), members
of vascular endothelial growth factor (VEGF) family, and thymosin
.beta.4.
19. The composition of claim 15, wherein the at least one platelet
function is the ability to induce cell proliferation.
20. The composition of claim 19, wherein the cell proliferation is
fibroblast proliferation.
21. The composition of claim 1, wherein the platelets are human
platelets.
22. A method of making a freeze-dried platelet composition, the
method comprising: providing a sample comprising platelets; making
a mixture comprising the platelets and one or more inhibitors of
platelet activation; and drying the mixture.
23. The method of claim 22, wherein the one or more inhibitors of
platelet activation are selected from effectors of the cAMP second
messenger system, sodium channel inhibitors, and effectors of the
cGMP second messenger system.
24. The method of claim 22, wherein the one or more inhibitors of
platelet activation comprise adenosine, amiloride, and sodium
nitroprusside.
25. The method of claim 24, wherein, in the mixture, the
concentration of adenosine is about 10 .mu.M to about 1 mM, the
concentration of amiloride is about 0.1 mM to about 10 mM, and the
concentration of sodium nitroprusside is about 2.5 .mu.M to about
250 .mu.M.
26. The method of claim 23, wherein the effector of the cAMP second
messenger system is selected from the group consisting of iloprost,
prostacyclin, prostaglandin E.sub.2, forskolin, cholera toxin,
isoproterenol, 8-bromo cyclic adenosine monophosphate, dibutyl
cyclic adenosine monophosphate, theophylline, isobutylmethyl
xanthine, thyrotropin, and auranofin.
27. The method of claim 23, wherein the sodium channel inhibitor is
selected from the group consisting of amiloride analogues,
bepridil, flecamide, saxitoxin, benzamil, and prajnalium.
28. The method of claim 23, wherein the effector of the cGMP second
messenger system is selected from the group consisting of
L-arginine, nitrous oxide, SIN-1, SIN-1A, atrial natriuretic
factor, vasopressin, oxytocin, and glyceril trinitrate.
29. The method of claim 22, wherein the mixture further comprises
one or more cryoprotective agents.
30. The method of claim 29, wherein the one or more cryoprotective
agents are selected from the group consisting of dimethyl
sulfoxide, maltodextrin, dextran, hydroxyethyl starch, glucose,
polyvinyl pyrrolidone, mannitol, and combinations thereof.
31. The method of claim 22, wherein the mixture further comprises
one or more extracellular matrix (ECM) components.
32. The method of claim 31, wherein the one or more ECM components
are selected from the group consisting of collagen, elastin,
fibronectin, fibrillin, laminin, decorin, fibromodulin, hyaluronic
acid, and a proteoglycan.
33. The method of claim 31, wherein the ECM components are in
particles of particulate acellular tissue matrix.
34. The method of claim 33, wherein the particulate acellular
tissue matrix is particulate acellular dermal matrix.
35. The method of claim 22, wherein the mixture further comprises
blood plasma.
36. The method of claim 22, wherein drying the mixture comprises
freeze-drying the mixture.
37.-38. (canceled)
39. A method of treatment, the method comprising: identifying a
subject that has a wound that will, or is likely to, benefit from
administration of platelets; and applying the dry platelet
composition of claim 1 to the wound.
40. A method of treatment, the method comprising: identifying a
subject that has a wound that will, or is likely to, benefit from
administration of platelets; rehydrating the dry platelet
composition of claim 1 to generate a rehydrated platelet
composition; and applying the rehydrated platelet composition to
the wound.
41. The method or use of claim 40, wherein the wound is a cutaneous
wound.
42. The method of claim 41, wherein the cutaneous wound is selected
from the group consisting of a pressure ulcer, a venous stasis
ulcer, a diabetic ulcer, an arterial ulcer, an injury wound, a burn
wound, a complex soft tissue wound, a failed skin graft or flap, a
radiation-induced wound, and a gangrenous wound.
43. The method claim 40, wherein the wound is an internal
wound.
44. The method of claim 43, wherein the internal wound is selected
from the group consisting of a contusion, a fracture, a fistula, an
ulcer, and an injury wound of an internal organ.
Description
TECHNICAL FIELD
[0002] The present invention relates to dry platelet compositions,
in particular to dry platelet compositions containing one or more
inhibitors of platelet activation.
BACKGROUND
[0003] Platelets are useful in the treatment of various pathologic
conditions such as, for example, wounds, platelet deficiencies
(e.g., thrombocytopenia), various genetic or acquired
abnormalities, and severe blood loss. However, despite their high
demand, the availability of platelets has been limited, at least in
part, by their short shelf-life and the inability of current
methods to preserve normal platelet function after storage for
relatively long periods of time. There is a need therefore to
develop platelet compositions that have increased shelf-life.
SUMMARY
[0004] The invention is based, in part, on the discovery that
platelets dried and rehydrated in the presence of a
cryopreservative additive (CPA) containing three inhibitors of
platelet activation and cryoprotectants retain all, or a
substantial level, of normal function. These findings provide the
basis for a dry platelet composition (e.g., a freeze-dried platelet
composition) and methods of making a freeze-dried platelet
composition. In addition, the invention features methods of
treatment.
[0005] More specifically, the invention provides a dry platelet
composition. The composition includes: a plurality of dry
platelets; and one or more inhibitors of platelet activation. The
one or more inhibitors of platelet activation can be, for example,
effectors of the cyclic adenosine monophosphate (cAMP) second
messenger system, sodium channel inhibitors, and/or effectors of
the cyclic guanosine 5' monophosphate (cGMP) second messenger
system. The one or more inhibitors of platelet activation can
include, for example, adenosine, amiloride, and/or sodium
nitroprusside. After hydration of the composition, the
concentration in the composition: of adenosine can be about 10
.mu.M to about 1 mM; that of amiloride can be about 0.1 mM to about
10 mM; and that of sodium nitroprusside can be about 2.5 .mu.M to
about 250 .mu.M. The effectors of the cAMP second messenger system
can be, for example, iloprost, prostacyclin, prostaglandin E.sub.2,
forskolin, cholera toxin, isoproterenol, 8-bromo cyclic adenosine
monophosphate, dibutyl cyclic adenosine monophosphate,
theophylline, isobutylmethyl xanthine, thyrotropin, and/or
auranofin. The sodium channel inhibitors can be, for example,
amiloride analogues, bepridil, flecamide, saxitoxin, benzamil,
and/or prajnalium. The effectors of the cGMP second messenger
system can be, for example, L-arginine, nitrous oxide, SIN-1,
SIN-1A, atrial natriuretic factor, vasopressin, oxytocin, and/or
glyceril trinitrate. The composition can further induce one or more
cryoprotective agents, e.g., dimethylsulfoxide (DMSO),
maltodextrin, dextran, hydroxyethyl starch, glucose, polyvinyl
pyrrolidone, and/or mannitol. The composition can also further
include dry blood plasma. Moreover, the composition can further
contain one or more extracellular matrix (ECM) components. The ECM
components can be components of particles of particulate acellular
tissue matrix, e.g., particles of particulate acellular dermal
matrix. The one or more ECM components can be, for example,
collagen, elastin, fibronectin, fibrillin, laminin, decorin,
fibromodulin, hyaluronic acid, and/or a proteoglycan such as a
heparin sulfate, chondroitin sulfate, keratan sulfate, or a
dermatan sulfate proteoglycan.
[0006] Moreover, hydration of the dry platelet composition can
result in a rehydrated platelet composition with substantially the
same level of at least one platelet function possessed by a sample
of fresh platelets from which the dry platelet composition was
derived. The at least one platelet function can be the ability to
aggregate or the ability to release one or more growth factors, one
or more cytokines, or one or more chemokines. The growth factors or
chemokines can be, for example, transforming growth factor-.beta.
(TGF-.beta.), members of platelet derived growth factor (PDGF)
family, epidermal growth factor (EGF), members of vascular
endothelial growth factor (VEGF) family, and/or thymosin .beta.4.
Alternatively, the at least one platelet function can be the
ability to induce cell (e.g., fibroblast) proliferation. The
platelets of the composition can be human platelets.
[0007] Any of the platelet compositions described herein can be
used as a medicament. In addition, any of the platelet compositions
described herein can also be used in the preparation of a
pharmaceutical composition (i.e., a medicament) for the treatment
of a wound (e.g., a wound that will, or is likely to benefit from,
administration of platelets (i.e., any of the platelet compositions
described herein)) in or on a subject. The wound can be, for
example, an internal wound or a cutaneous wound and can include,
but is not limited to, any of the types of wounds described
below.
[0008] Another aspect of the invention is a method of making a
freeze-dried platelet composition. The method includes: providing a
sample that contains platelets; making a mixture containing the
platelets and one or more inhibitors of platelet activation; and
drying the mixture. The one or more inhibitors of platelet
activation can be any of those recited above. In the mixture, the
concentration of adenosine can be about 10 .mu.M to about 1 mM, the
concentration of amiloride can be about 0.1 mM to about 10 mM, and
the concentration of sodium nitroprusside can be about 2.5 .mu.M to
about 250 .mu.M. The mixture can further include one or more
cryoprotective agents such as any of those recited above. The
mixture can also further include blood plasma. Drying the mixture
can be by, for example, freeze-drying the mixture.
[0009] In another embodiment, the invention features a method of
treatment. The method includes: identifying a subject that has a
wound that will, or is likely to, benefit from administration of
platelets; and applying the above-described dry platelet
composition to the wound. An alternative method of treatment
includes: identifying a subject that has a wound that will, or is
likely to, benefit from administration of platelets; rehydrating
the above-described dry platelet composition to generate a
rehydrated platelet composition; and applying the rehydrated
platelet composition to the wound.
[0010] In both methods of treatment, the wound can be a cutaneous
wound (e.g., a pressure ulcer, a venous stasis ulcer, a diabetic
ulcer, an arterial ulcer, an injury wound, a burn wound, a complex
soft tissue wound, a failed skin graft or flap, a radiation-induced
wound, or a gangrenous wound) or an internal wound (e.g., a wound
under or below the skin). Internal wounds can include, but are not
limited to, a contusion, a fracture, a fistula, an ulcer, or an
injury wound of an internal organ.
[0011] The term "dry" as used in reference to platelet
compositions, platelets, and other components of the compositions
(e.g., blood plasma) means that the platelet compositions,
platelets, or other components of the compositions are
substantially free of water. "Substantially free of water," as used
herein, means containing less than 5 percent (e.g., less than: 4
percent; 3 percent; 1 percent; 0.5 percent; 0.2 percent; 0.1
percent; 0.01 percent; or 0.001 percent) by weight water (including
bound and unbound water).
[0012] As used herein, a "control wound" is a wound to which a
platelet composition of the invention has not been applied. Such a
control wound can be in a subject also having a wound to which a
platelet composition of the invention has been applied.
Alternatively, the control wound can be in another subject. The
control wound is preferably of the same type and size and in the
same tissue or organ as the wound to which a platelet composition
of the invention is applied.
[0013] Unless otherwise defined, 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 pertains.
Preferred methods and materials are describe below, although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention. All publications, patent applications, patents, and
other references mentioned herein are incorporated by reference in
their entirety. The materials, methods, and examples disclosed
herein are illustrative only and not intended to be limiting.
[0014] Other features and advantages of the invention, e.g., dry
platelet compositions, will be apparent from the following
description, from the drawings and from the claims.
DESCRIPTION OF DRAWINGS
[0015] FIG. 1 is a bar graph showing the recovery of the
aggregation ability of platelets that had been freeze-dried and
rehydrated in the presence or absence of a cryopreservative
additive (CPA) solution that contains inhibitors of platelet
activation and cryoprotectant agents. The platelet aggregation
response was activated by a combination of adenosine diphosphate
(10 mM) and collagen (2 .mu.g/ml). The data are presented as the
aggregation responses of the freeze-dried and rehydrated platelet
samples as percentages of the aggregation response of fresh
platelets from the same sample used for making the freeze-dried
platelets. The experiment was performed three times using platelet
rich plasma (PRP) from a separate donor for each experiment. The
data shown are the means obtained from the three experiments and
standard deviations are indicated. These means are the means of the
averages three replicates in each experimental group.
[0016] FIG. 2A is a diagrammatic representation of the
"Transwell.RTM." cell culture system used to measure proliferation
of fibroblasts in response to soluble factors released by
platelets. Fibroblast cells were seeded onto the bottom surfaces of
the wells of 24-well tissue culture plates and test platelet
materials were added to Transwell.RTM. chambers having floors
consisting of semi-permeable membranes that permit the diffusion of
soluble factors (but not whole platelets or insoluble platelet
material) from the Transwell.RTM. chambers into the culture well
where they come in contact with the fibroblasts.
[0017] FIG. 2B is a bar and line graph showing the induction of
proliferation in fibroblasts at 24, 48, and 72 hours of exposure to
growth media alone, serum reduced medium alone, or serum reduced
medium and soluble factors released from sonicated platelets or
platelets that had been activated with 1 unit/ml of thrombin in the
Transwell.RTM. culture system described in FIG. 2A. The graph bars
represent percent increase in the amount of colored product
produced by metabolic conversion of the substrate MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)-2H-tetrazolium, inner salt] (as an indication of relative cell
number) over baseline (left y-axis) and the graph lines represent
actual cell counts (right y-axis). The horizontal dashed line
indicates the number of cells at the time of initial exposure of
the fibroblasts to platelet material (i.e., time 0). The experiment
was performed three times using PRP from a separate donor for each
experiment. The data are the means obtained from the three
experiments and standard deviations are indicated. These means are
the means of the averages of three replicates in each experimental
group.
[0018] FIG. 2C is a bar graph showing the percent induction of
fibroblast proliferation after 72 hours of exposure to soluble
factors released from sonicated and activated platelets. The data
were obtained from the observations at the 72 hour time point of
the experiment shown in FIG. 2B. The experiment was performed three
times using PRP from a separate donor for each experiment. The data
are the means obtained from the three experiments and standard
deviations are indicated. These means are the means of the averages
of three replicates in each experimental group.
[0019] FIG. 3 is a bar graph showing the percent induction of
fibroblast proliferation (calculated from data obtained using the
Transwell.RTM. cell culture system shown in FIG. 2A) in response to
fresh platelets and platelets freeze-dried and rehydrated in the
presence of CPA (F/D/R CPA). The experiment was performed three
times using PRP from a separate donor for each experiment. The data
are the means obtained from the three experiments and standard
deviations are indicated. These means are the means of the averages
of three replicates in each experimental group.
[0020] FIG. 4 is a bar graph showing the percent induction of
fibroblast proliferation (calculated from data obtained using the
Transwell.RTM. cell culture system shown in FIG. 2A) after
treatment with fresh platelets, platelets freeze-dried and
rehydrated (F/D/R) in the presence of CPA (w/CPA) or the absence of
CPA (w/o CPA), or plasma obtained by centrifugation of the platelet
rich plasma (PRP) that was the source of the platelets used to make
the freeze-dried platelet preparations or by centrifugation of the
two rehydrated freeze-dried samples. The dotted line shows the
average percent induction of fibroblast proliferation by all three
plasma samples. The experiment was performed three times using PRP
from a separate donor for each experiment. The data are the means
obtained from the three experiments and standard deviations are
indicated. These means are the means of the averages of three
replicates in each experimental group.
[0021] FIG. 5 is a bar graph showing the ability of various amounts
of platelets (freeze-dried and rehydrated in the presence of CPA)
to induce fibroblast proliferation as assessed using the
Transwell.RTM. cell culture system shown in FIG. 2A. The legend
indicates the relative amounts of platelets added to the
Transwell.RTM. chambers of the culture system. The experiment was
performed three times using PRP from a separate donor for each
experiment. The data are the means obtained from the three
experiments and standard deviations are indicated. These means are
means of three replicates in each experiment.
[0022] FIG. 6A is a series of photographs of diabetic mouse wounds
after wounding that had not been treated (NT) or had been treated
with fresh frozen platelets (FFP), platelets that had been
freeze-dried and rehydrated in the absence of CPA (FDP), or
platelets that had been freeze-dried and rehydrated in the presence
of CPA (FDP-CPA). Scale bar; 500 mm.
[0023] FIGS. 6B and 6C are bar graphs showing the percent (%)
epithelialization (FIG. 6B) and percent (%) contraction (FIG. 6C)
of the wounds shown in FIG. 6A. Scale bar, 5 mm.
[0024] FIG. 7A is a series of photomicrographs showing the
different amounts of granulation tissue deposition in histological
sections of the beds of the wounds shown in FIG. 6A. Arrows
indicate epithelial margins and boxes indicate where measurements
of tissue area and thickness were made (see FIGS. 7B and 7C). Scale
bar, 100 .mu.m.
[0025] FIGS. 7B and 7C are bar graphs showing the granulation
tissue area (FIG. 7B) and thickness (FIG. 7C) in the areas of the
wounds shown by boxes in FIG. 7A. * indicates p<0.01.
DETAILED DESCRIPTION
[0026] Platelets constitute an important therapeutic for a variety
of platelet diseases or abnormalities involving platelet deficiency
and/or defective platelet function (e.g., thrombocytopenias) as
well as for the treatment of various wounds. However, the rapid
loss of platelet viability and function during storage has greatly
complicated management of an effective inventory of platelets in
blood banks. In many settings, the limited shelf life of platelets
has drastically reduced their usage.
[0027] Current guidelines allow platelets to be stored for a
maximum of only 5 days at 20.degree. C.-24.degree. C., creating an
inventory control problem for hospital and blood banks [Lazarus et
al. (1982) Transfusion 22:39-43; Murphy (1985) Seminars in
Hematology 22:165-177]. This time restriction was established, at
least in part, because of concerns over the potential for microbial
contamination during storage of platelets at room temperature. On
the other hand, the use of various cryopreservation methods to
extend the shelf-life of platelets have not proven very effective.
Such methods result in, for example, a loss of normal platelet
discoid morphology, a loss of platelet cell number, and a reduction
in platelet functional activity [Balduni et al. (1993)
Haematologia. 78:101-104; Bock et al. (1995) Transfusion.
35:921-924]. It is desirable therefore to obtain platelets that
retain function after storage for prolonged periods of time.
[0028] The inventors found that platelets freeze-dried and
rehydrated in the presence of a cryopreservative additive (CPA)
solution containing inhibitors of platelet activation, retain their
functional properties. Platelets freeze-dried and rehydrated with
CPA exhibited increased agonist-induced aggregation compared to
platelets freeze-dried and rehydrated without CPA and retained
their ability to secrete growth factors. In the case of TGF-.beta.
(as a representative growth factor), substantially all of the
TGF-.beta.-specific antibody detected protein produced by CPA
freeze-dried and rehydrated platelets had activity. In addition,
CPA freeze-dried and rehydrated platelets secreted factors that
induced proliferation in fibroblasts, an important determinant for
normal wound closure and remodeling. In a diabetic mouse wound
model, delivery to the wound of platelets that had been
freeze-dried in the presence of CPA resulted in increased wound
healing as assessed by the degree of granulation, wound closure,
vascularity and cell proliferation.
[0029] These findings provide support for the compositions and
methods of the invention, which are described below.
Dry Platelet Compositions
[0030] The invention provides a dry platelet composition. The
composition is made by drying (e.g., freeze-drying) isolated
platelets or preparations or samples (e.g., platelet rich plasma)
containing platelets in the presence of one or more (e.g., two or
more, three of more, four or more, five or more, six or more, seven
or more, eight or more, nine or more, ten or more, or 12 or more)
inhibitors of platelet activation. As used herein, "platelet
activation" refers to a biological (e.g., thrombin-mediated) or
physical (e.g., exposure to cold temperature, e.g., 4.degree. C.)
process that lead to a change in shape (discoid to spheroid to
amorphous) of the platelet, and/or granule release from the
platelet, and or platelet aggregation. An "inhibitor of platelet
activation" is an agent that can totally prevent or partially
decrease platelet activation.
[0031] The preparations or samples containing platelets useful for
making the compositions of the invention are preferably free of
non-platelet cells. However they can contain small numbers of such
cells, e.g., blood cells such as erythrocytes, lymphocytes,
granulocytes, monocytes, and/or macrophages. They will preferably
contain less than 10% (e.g., less than: 5%; 2%; 1%; 0.1%; 0.01%;
0.001%; or 0.0001%) of any of the non-platelet cell types present
in blood from which a relevant platelet preparation or sample was
made.
[0032] The dry platelet composition of the invention contains a
plurality of dry platelets and one or more inhibitors of platelet
activation. The one or more inhibitors of platelet activation
include one or more effectors (activators or enhancers) of the
cyclic adenosine monophosphate (cAMP) second messenger system, one
or more inhibitors of sodium channels, and or one or more effectors
(activators or enhancers) of the cyclic guanosine monophosphate
(cGMP) second messenger system. Other inhibitors of platelet
activation include inhibitors of the cyclooxygenase second
messenger system, inhibitors of the lipoxygenase pathway,
inhibitors of the phospholipase pathway, inhibitors of the calcium
cascade, protease and proteinase inhibitors, and membrane
modifiers.
[0033] Effectors of the cAMP second messenger system include, for
example, adenosine, iloprost, prostacyclin, prostaglandin E.sub.2,
forskolin, cholera toxin, isoproterenol, 8-bromo cyclic adenosine
monophosphate, dibutyl cyclic adenosine monophosphate,
theophylline, isobutylmethyl xanthine, thyrotropin, and auranofin.
Sodium channel inhibitors include, for example, amiloride,
amiloride analogues, bepridil, flecamide, saxitoxin, benzamil, and
prajnalium. Effectors of the cGMP second messenger system include,
for example, sodium nitroprusside, L-arginine, nitrous oxide, SIN-1
(3-morpholinosydnonimine), SIN-1A
(N-nitroso-N-morpholinoamino-acetonitrile), atrial natriuretic
factor, vasopressin, oxytocin, and glyceril trinitrate. Inhibitors
of the cyclooxygenase pathway can be aspirin, dipyridamole,
flurbiprofen, ticlopidine, ketoprofen, ibuprofen, indomethacin,
sulfinpyrazone, guanabenz, ursolic acid and benzohydroquinone.
Inhibitors of the lipoxygenase pathway include aspirin,
ticlopidine, ursolic acid, unbelliferone, 5,8,11,14
eicosatetraynoic acid and esculetin. Inhibitors of the
phospholipase pathway include quinacrine and mepacrine. Inhibitors
of the calcium cascade include protein kinase C effectors, calcium
channel blockers, calcium concentration modifiers, calmodulin
effectors, calcium ionophores, and ATPase stimulators. Protease and
proteinase inhibitors include heparin and apoprotinin. Membrane
modifiers include amantadine, heparin, ticlopidine, pentoxifylline,
and ajoene. Inhibitors of platelet activation are described in
greater detail in U.S. Pat. No. 5,919,614, the disclosure of which
is incorporated herein by reference in its entirety.
[0034] The dry platelet composition of the invention can include
adenosine as an effector of the cAMP second messenger system,
amiloride as a sodium channel inhibitor, and sodium nitroprusside
as an effector of the cGMP second messenger system. The
concentration of these inhibitors of platelet activation in the
solution in which the platelets are dried, or after rehydration (if
they are rehydrated), can be as follows: the concentration of
adenosine can be about 10 .mu.M to about 1 mM (e.g., about 100
.mu.M to about 1 mM or about 10 .mu.M to about 0.1 mM); the
concentration of amiloride can be about 0.1 mM to about 10 mM
(e.g., about 1 mM to about 10 mM or about 0.1 mM to about 1 mM),
and the concentration of sodium nitroprusside can be about 2.5
.mu.M to about 250 .mu.M (e.g., about 25 .mu.M to about 250 .mu.M
or about 2.5 .mu.M to about 25 .mu.M). For example, in a preferred
embodiment, the concentration of adenosine is 0.1 mM, the
concentration of amiloride is 0.25 mM, and the concentration of
sodium nitroprusside is 50 .mu.M.
[0035] The term "about" used in regard to concentrations of
inhibitors of platelet activation and cryoprotectants (see below)
indicates that the concentration of the agent referred to can vary
by up to 20% (e.g., up to: 15%; 10%; 5%; 2.5%; or 1%) of the
concentration stated.
[0036] In addition to the one or more inhibitors of platelet
activation, one or more cryoprotective agents (also referred to
herein as cryoprotectants) can be added to platelets before drying.
Such cryoprotective agents can be, for example, dimethylsulfoxide
(DMSO), maltodextrin, dextran, hydroxyethyl starch, glucose,
polyvinyl pyrrolidone, mannitol, and combinations thereof. The DMSO
concentration can be from about 0.5% to about 10% (e.g., about 1.0%
to about 10%; or about 0.5% to about 1%). In one preferred
embodiment, the concentration of DMSO can be 0.5%. Thus, where one
or more cryoprotective agents have been added to a platelet
preparation before drying, the resulting dry platelet composition
will contain the appropriate one or more cryoprotective agents.
[0037] Where an inhibitor of platelet activation or a
cryoprotective agent that is added to platelets prior to drying is
in its pure form a liquid (e.g., DMSO), the dry platelet
composition (and its rehydrated form) likely contains less of the
inhibitor of platelet activation or the cryoprotective agent than
prior to drying.
[0038] In addition to inhibitors of platelet activation and
cryoprotective agents, the dry platelet compositions of the
invention can contain one or more proteins. For example, the
compositions can contain dry blood plasma, e.g., dry blood plasma
derived from the donor of the platelets. This will inherently be
the case where the compositions are made using platelet rich plasma
(PRP) as the platelet preparation used for making the composition.
In addition, proteins in the composition can be present as dry
blood serum. Alternatively, protein can be added to the platelet
mixture prior to drying in the form of one or more (e.g., all)
isolated blood plasma-derived or blood serum-derived proteins
(e.g., albumin or gamma globulins). Blood plasma, blood serum, or
protein(s) derived from either can be from the same donor as the
platelets (i.e., autologous), one or more donors of the same
species, or one or more donors of one more other species. The
species from which these protein sources are obtained can be any of
those listed below as sources of platelets for the compositions
(see below). Moreover, blood or serum proteins can be recombinant
proteins.
[0039] The dry platelet compositions can also contain one or more
extracellular matrix (ECM) components, e.g., any types of collagen
(such as, for example, collagens I, II, III, or IV or any of
collagens V-XVII), elastin, fibronectin, laminin, decorin,
fibrillin, fibromodulin, hyaluronic acid, and/or a proteoglycan
such as a heparin sulfate, chondroitin sulfate, keratan sulfate, or
a dermatan sulfate proteoglycan. These components can be added to
the mixture containing platelets before drying or they can be added
after drying. Such ECM components can enhance wound repair by
providing a scaffold structure and local binding sites for factors
released by administered platelets. Moreover, when added to
platelet mixtures prior to drying by freeze-drying, the ECM
components (like the above described protein additives) can
substitute for a significant amount of water in the platelet
mixture, thereby reducing the amount of ice formed in freezing the
platelet mixture and hence reducing ice-mediated damage to the
platelets. The ECM components can be obtained from any of the
donors described above for blood plasma, blood serum, or proteins
derived from either. In addition, ECM that are proteins can be
recombinant proteins. The ECM components can be added, for example,
in the form of particulate acellular tissue matrix made from any of
a variety of collagen-containing tissues, e.g., dermis. Particulate
acellular tissue matrices are described in detail in U.S. Pat. No.
6,933,326, U.S. application Ser. No. 10/273,780, and U.S.
application Ser. No. 10/959,780, the disclosures of all of which
are incorporated herein by reference in their entirety.
[0040] A substantial proportion of the platelets of the dry
platelet composition regain at least one platelet function (e.g.,
at least: two; three; or four platelet functions) upon rehydration
(in vitro or in vivo). After drying and rehydration, a platelet
composition of the invention has at least 10% (e.g., at least 20%,
at least 30%, at least 40%, at least 50%, at least 60%, at least
70%, at least 80%, at least 90%, at least 95%, at least 98%, at
least 99%, or 100%) of the level of at least one platelet function
that a corresponding fresh preparation (not dried and rehydrated,
from the same donor, and containing the same number of platelets as
the platelet composition of invention) of platelets would have.
Moreover, at least 10% (e.g., at least 20%, at least 30%, at least
40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 95%, at least 98%, at least 99%, or 100%) of
the platelets of a dry platelet composition of the invention have,
upon rehydration, at least one platelet function. Relevant platelet
functions include, for example, growth factor, cytokine, and
chemokine production upon activation; the ability to stimulate cell
(e.g., fibroblast, endothelial cell, or epithelial cell (e.g.,
keratinocyte)) proliferation upon activation; and the ability to
aggregate upon activation. Assays for platelet function are known
in the art and include those described in the Examples below. The
platelet-produced growth factors, cytokines and chemokines include,
without limitation, transforming growth factor-.beta. (TGF-.beta.),
members of the platelet derived growth factor family (e.g., PDGF-A,
B, C, D, and A/B), epidermal growth factor (EGF), members of the
vascular endothelial growth factor (VEGF, VEGF-B, VEGF-C, and
VEGF-D), and thymosin-.beta.4. Additional indicia of intact
platelet function that can be tested include, without limitation,
morphology score (proportion of platelets that are discoid,
spheroid, and/or amorphous), extent of shape change (ESC),
hypotonic shock response (HSR), extent of shape change (ESC),
platelet aggregation (as measured by platelet aggregometry),
efficiency of inducing blood coagulation (as measured by
thromboelastography (TEG)), and platelet adenosine triphosphate
(ATP) levels. P-selectin expression on the surface of a platelet
indicates that it has degranulated. Degranulation can occur
without, for example, aggregation.
[0041] Assessment of platelet function in the rehydrated dry
platelet compositions can be quantitative, semi-quantitative, or
qualitative. Thus it can, for example, be measured as a discrete
value or expressed relative to a baseline or to similar
measurements in control samples (e.g., fresh platelets). Platelet
function can be assessed and expressed using any of a variety of
semi-quantitative/qualitative systems known in the art. Thus,
platelet viability and/or function can be expressed as, for
example, (a) one or more of "excellent", "good", "satisfactory",
and/or "poor"; (b) one or more of "very high", "high", "average",
"low", and/or "very low"; or (c) one or more of "++++"; "+++",
"++", "+", "+/-", and/or "-".
[0042] The platelets may be obtained from one or more individuals
of any of a variety of mammalian species (e.g., humans, non-human
primates (e.g., monkeys, baboons, or chimpanzees), cows, sheep,
horses, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters,
gerbils, rats, or mice) but are preferably of the same species as a
subject to which they are to be administered. The dry platelet
composition can be used in vitro or in vivo. In vitro uses of the
dry platelet composition include their use as targets for in vitro
screening assays or testing of compounds of interest for, e.g.,
those with hemostasis-promoting activity, those with
hemostasis-inhibiting activity, or those that promote wound
healing. The dry platelet composition can be rehydrated (e.g., with
a physiological solution such as normal saline or culture medium)
and plated into tissue culture dishes.
[0043] The dry platelet compositions can also be used for the in
vitro production and subsequent isolation of soluble factors that
are expressed by platelets (see above). Such factors are useful as
diagnostic tools themselves or can be used as antigens to generate
antibodies for diagnostic use. In addition, rehydrated dry
platelets of the invention can be used in in vitro drug efficacy or
toxicity assays. The dry platelet composition can also be used as
"positive controls" in procedures to develop other platelet storage
compositions.
[0044] Platelets obtained by rehydrating a dry platelet composition
of the invention can also be used to support the growth and/or
differentiation of non-platelet cells in culture. Such platelets
upon activation can promote, for example, the survival and/or
growth of fibroblast cells or other cells (e.g., fibroblast,
endothelial cell, or epithelial cell (e.g., keratinocyte)) in cell
culture. The dry platelet composition can also be used for in vitro
and in vivo basic scientific studies of platelet function.
[0045] In vivo uses of the dry platelet compositions, or of
platelets derived therefrom, include, for example, studies in
animal models (e.g., in any of the mammals listed above) or in
human subjects. Such studies may be performed, for example, in
order to assess the therapeutic and/or prophylactic efficacy of
platelets per se or of chemical compounds and biological molecules
that modulate (up or down regulate) platelet function. Other uses
of the platelet compositions of the invention include methods of
treatment (see below).
[0046] Methods of isolating platelets are well known in the art.
For example, platelets can be prepared by centrifugation of whole
blood by either the platelet-rich plasma (PRP) method or the buffy
coat method. Moreover, platelets can be collected by various
apheresis techniques that are available in the art.
Method of Making Freeze-Dried Platelet Composition
[0047] Also embodied by the invention is a method of making a
freeze-dried platelet composition. The method includes: (a)
providing a preparation or sample of platelets; (b) making a
mixture including the platelets and one or more inhibitors of
platelet activation; and (c) drying the mixture. The mixture can
also optionally contain one or more cryoprotectants, one or more
proteins (e.g., blood plasma), and/or ECM components described
above.
[0048] The preparation or sample of platelets, the inhibitors of
platelet activation, and the optional cryprotectants, proteins, and
ECM components can be any of those listed above. Drying the mixture
can be by any method known in the art, e.g., air drying, drying in
atmosphere of, or under a stream of, inert gas (e.g., nitrogen or
argon), or freeze-drying. Freeze-drying methods are well-known in
the art (see, for example, "A Guide to Freeze-drying for the
Laboratory"--an industry service publication by Labconco, 2004;
Franks (1994) Proc. Inst. Refrigeration. 91: 32-39, and U.S. Pat.
Nos. 4,619,257; 4,676,070; 4,799,361; 4,865,871; 4,964,280;
5,024,838; 5,044,165; 5,154,007; 6,194,136; 5,336,616; 5,364,756;
and 5,780,295, the disclosures of all of which are incorporated
herein be reference in their entirety).
[0049] Freeze-drying of platelets using one or more inhibitors of
platelet activation, and optionally one or more cryoprotectants,
results in minimal, if any, functional damage to the platelets.
Suitable freeze-drying equipment is available from commercial
sources, e.g., Labconco (Kansas City, Mo.) and VirTis (Gardiner,
N.Y.). Freeze-drying a liquid (e.g., water)-containing sample
involves freezing the sample and the subsequent removal of liquids
(e.g., water) from the frozen sample by a process called
sublimation. Freezing can be, for example, in the freeze-drier
apparatus or in a -80.degree. C. freezer. The sample is cooled
until the liquid in the sample has solidified (as assessed by the
visually). Freezing can be at a cooling rate of between, for
example, 1.degree. C. and 5.degree. C. per minute and is preferably
not by "snap-freezing." Freezing methods are described extensively
in the above references cited in regard to freeze-drying.
Sublimation occurs when a frozen liquid goes directly to the
gaseous state without passing through the liquid phase.
Freeze-drying may be accomplished by any of a variety of methods,
including, for example, the manifold, batch, or bulk methods.
Method of Treatment
[0050] The invention also provides a method of treatment. The
method can include identifying a subject that will, or is likely
to, benefit from administration of platelets and administering to
the subject any of the platelet compositions described above.
[0051] The dry platelet compositions can per se be administered to
the subject. In this case, rehydration of the platelets occurs in
the subject. Alternatively, the platelet compositions can be
rehydrated and then administered to the subject. In the latter
case, the a composition can optionally, prior to being
administered, be subjected to a washing process to remove all or a
substantial amount of the one or more inhibitors of platelet
activation and, if used in the relevant composition, one or more
cryoprotectants. Such washing methods are known in the art and
generally involved one or more (e.g., two, three, or four)
centrifugation steps. It is particularly desirable to perform such
washing steps where inhibitors of platelet activation and/or
cryoprotectants used are toxic. In this case, washing is performed
until none, or an acceptably low level, of the toxic components
remains in the composition.
[0052] Rehydration (and optional washing) can be with any
physiological solution (e.g., water, normal saline, tissue culture
medium, or the physiological solutions described in Examples 1 and
7 below) such that the platelets retain one or more of their
functions (see above). The dried platelet compositions can
optionally be rehydrated in the pharmaceutically acceptable carrier
in which the platelets are to be administered to an appropriate
subject (see below). Rehydration can be by rapid immersion of the
platelets in the relevant carrier or by gradual (e.g., drop-wise
addition of the carrier) to the dry platelets.
[0053] The subject that will, or is likely to, benefit from
administration of platelets can have a wound. The wound can be one
that will, or is likely to, benefit from being treated with
platelets. The wound can be a cutaneous wound that can be, or can
be a result of, a pressure ulcer, a venous stasis ulcer, a diabetic
ulcer, an arterial ulcer, an injury wound, a burn wound, a complex
soft tissue wound, a failed skin graft or flap, radiation-induced
tissue damage, and a gangrenous wound. The wound can also be an
internal wound of any internal organ or tissue, e.g.,
gastrointestinal tissue, pulmonary (e.g., lung or bronchial)
tissue, heart tissue, connective tissue (e.g., tendon, ligament,
and cartilage), bone tissue, neural (central and peripheral nerve
system) tissue, and vascular (vein and artery) tissue. Internal
wounds of interest include, without limitation, contusions,
fractures, fistulas, ulcers, or internal organ injuries (e.g.,
injury of the intestine, spleen, liver, lungs, or heart). The wound
can be caused by a trauma, including, e.g., a compound fracture, a
gunshot wound, or an abrasion from an accident.
[0054] The dry (or rehydrated) platelet composition can be
delivered to a wound immediately after it occurs or at any stage of
its natural healing process. Preferably, the platelet composition
will be delivered to the wound immediately, or soon after, the
wound is detected, or formed, in the subject. The wound that is to
be treated with the platelet composition can have varying
appearance, size, depth (i.e., stage), and color, and can include,
for example, the presence of hematomas, seromas, wound exudate,
necrotic tissue, and eschar.
[0055] The dry (or rehydrated) platelet composition can be applied
topically, i.e., directly to the wound. It can be applied to the
wound by any suitable means, such as by sprinkling or spraying the
platelets onto the wound, packing the platelets into the wound, or
by means of a surgical aid as discussed below. Sprayable aerosol
preparations can include the platelet composition in combination
with a solid or liquid inert carrier material and can be packaged
in a squeeze bottle or in admixture with a pressurized volatile,
normally gaseous propellant, e.g., a freon.
[0056] Dry platelets may be applied to the wound by means of a
surgical aid, such as, for example, a wound dressing or bandage, a
suture, a fabric, or a prosthetic device. Such aids can include,
for example, a solid physiologically acceptable substrate material
and platelets on or in (e.g., applied as a coating on or
impregnated in) the substrate material. Typically, such surgical
aids are provided in a sterile form packaged in a sterile
container. The surgical aid substrate material may be coated with
the platelets, e.g., by sprinkling dry platelets onto the material
or by impregnating the surgical aid substrate with, or applying to
its surface, a liquid suspension of fresh platelets containing the
one or more inhibitors of platelet activation (and optionally one
or more cryoprotectants) and drying (e.g., freeze-drying) the
surgical aid/platelet mixture so that the platelets adhere to the
surgical aid substrate. Alternatively, dry platelets can be adhered
to the surgical aid substrate with a suitable adhesive material, or
simply sprinkled onto the surgical aid prior to application of the
surgical aid to the subject.
[0057] The surgical aid can be of any suitable shape and size and
be made of any suitable solid material, hydrophobic or hydrophilic,
which is physiologically acceptable. Sutures, for example, may be
monofilament or braided, can be biodegradable, and can be made of
materials such as, for example, nylon silk, polyester, or cotton.
Prosthetic devices, for example, include woven or extruded tubular
structures, having use in the repair of arteries, veins, ducts;
fabrics useful surgically in hernia repair and in supporting
damaged liver, kidney, or other internal organs; pins, screws, and
reinforcing plates; heart valves, artificial tendons, or cartilage
material. Bandages can be made of any suitable substrate material,
such as cotton or other fabric suitable for application to or over
a wound, can optionally include a backing material, and can
optionally include one or more adhesive regions on the face surface
thereof for securing the bandage over the wound.
[0058] The platelet compositions of the invention are administered
to subjects in pharmaceutically acceptable formulations that
include a pharmaceutically acceptable carrier. A pharmaceutically
acceptable carrier e.g., normal saline, excipient, or stabilizer,
can be added to the cells before they are administered to a
subject. The phrase "pharmaceutically acceptable" refers to
molecular entities and compositions that, at the concentration
used, are not deleterious to cells, are physiologically tolerable,
and typically do not produce an allergic or similar untoward
reaction, such as gastric upset, dizziness and the like, when
administered to a human.
[0059] Suitable formulations include, but are not limited to,
solutions, suspensions, emulsions, creams, ointments, powders,
liniments, salves, and aerosols, which are, if desired, sterilized
or mixed with auxiliary agents, e.g., preservatives, stabilizers,
wetting agents, antiseptic agents, antimicrobial agents (e.g.,
hydrogen peroxide, Betadine, or acetic acid), or buffers or salts
for influencing osmotic pressure. A wide variety of
pharmaceutically acceptable carriers, excipients or stabilizers are
known in the art [Remington's Pharmaceutical Sciences, 16th
Edition, Osol, A. Ed. 1980]. Pharmaceutically acceptable carriers,
excipients, or stabilizers include: buffers, such as phosphate,
citrate, and other non-toxic organic acid buffers; antioxidants
such ascorbic acid; low molecular weight (less than 10 residues)
polypeptides; proteins such as serum albumin, gelatin or
immunoglobulins; hydrophilic polymers such polyvinylpylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine, or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrans; chelating agents such as
EDTA; sugar alcohols such as mannitol, or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
Tween, Pluronics, or PEG.
[0060] The dosage of the platelet composition required depends on
the nature of the formulation, the nature of the wound or the type
and severity of the wound that is to be treated, the subject's
size, weight, surface area, age, and sex, other therapeutic agents
being administered, and the judgment of the attending physician.
Wide variations in the needed dosage are to be expected in view of
differing efficiencies of various routes of administration.
Platelet compositions can be applied to wounds such that about 1 ml
of rehydrated composition is applied for each about 1 cm.sup.3 of
wound. Variations in these dosage levels can be adjusted using
standard empirical routines for optimization as is well understood
in the art.
[0061] The platelet compositions can be administered to a subject
once or multiple times. Thus, the compositions can be administered
one, two, three, four, five, six, seven, eight, nine, ten, 11, 12,
13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 100, 150,
200, 250, 300, 350, 400, 450, 500, 700, 1000, or more times. Where
a plurality of administrations is made, the administrations can
separated by any appropriate time period, e.g., 30 seconds, one
minute, two minutes, three minutes, four minutes, five minutes, 10
minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, two hours,
three hours, four hours, five hours, eight hours, 12 hours, 18
hours, 24 hours, two days, three days, four days, a week, two
weeks, three weeks, a month, two months, three months, four months,
five months, six months, eight months, ten months, a year, 18
months, two years, three years, four years, or five years.
[0062] The platelets can be obtained from the individual to whom
the platelet composition is to be administered (the recipient),
i.e., the platelets can be autologous. Alternatively, they can be
from one or more individuals of the same species as the recipient,
e.g., the platelet composition can be made from a pool of platelets
samples prepared from a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or
more) of subjects, e.g., human volunteers. A recipient can also be
of a species other than the donor. In addition, platelets can be
isolated from the blood of adult, infant, or fetal blood of one or
more individuals. Recipients and donors of platelets can be of any
other species listed above.
[0063] As used herein, "therapeutic" or "therapy" means a complete
abolishment of the symptoms of a pathological condition (e.g., a
wound) or a decrease in the severity of the symptoms of the
pathological condition. "Prevention" means that symptoms of the
pathological condition are essentially absent. As used herein,
"prophylaxis" means complete prevention of the symptoms of a
pathological condition, a delay in onset of the symptoms of a
pathological condition, or a lessening in the severity of
subsequently developed pathological condition symptoms.
[0064] The following examples are meant to illustrate, not limit,
the invention.
EXAMPLES
Example 1
Preparation of Platelet Compositions Used in Examples 1-6
Collection of Human Platelet-Rich Plasma (PRP)
[0065] PRP samples in the form of random donor units (RDU) of human
platelets were purchased from an American Association of Blood
Banks (AABB) accredited blood bank, stored with agitation at room
temperature, and used within 5 days of donation. All required
donor-screening and release-testing were performed by the blood
bank in accordance with AABB requirements [MEB Technical Manual
14.sup.th ed. Bethesda, Md.: American Association of Blood Banks
(2002)].
Freeze-Drying of PRP
[0066] An aliquot of PRP was mixed with a cryopreservative additive
(CPA) solution consisting of the metabolic inhibitors amiloride,
adenosine, and sodium nitroprusside and the cryoprotectants
polyvinyl pyrrolidone (PVP), mannitol, and dimethylsulfoxide
(DMSO). These compounds were all diluted in isotonic buffer (buffer
B described in Example 7). A separate control PRP aliquot was mixed
with isotonic buffer only. The final concentration of platelets in
the treated and control PRP samples was 9.times.10.sup.5
cells/.mu.l and the final concentrations of the metabolic
inhibitors and cryoprotectants were as follows: amiloride (0.25
mM), adenosine (0.1 mM), sodium nitroprusside (50 .mu.M), polyvinyl
pyrrolidone (4% w/v), mannitol (50 mM), and dimethylsulfoxide (0.5%
v/v).
[0067] Both platelet samples were frozen at a cooling rates of
between 1.degree. C. and 5.degree. C. per minute and then
freeze-dried under standard conditions. After freeze-drying, the
PRP samples were stored for less than 5 days at -80.degree. C. and
then rehydrated by rapid whole volume addition with buffer B to the
volume prior to freeze-drying.
Example 2
Platelets Freeze-Dried and Rehydrated in the Presence of CPA
Exhibit Increased Platelet Aggregation
[0068] To determine whether platelets freeze-dried and rehydrated
with CPA are capable of mediating functions important for
hemostasis, the aggregation potential of samples freeze-dried and
rehydrated with and without CPA was studied in the presence of
adenosine disphosphate (10 .mu.M) and type I equine collagen (2
.mu.g/ml; Chrono-Log Corp, Havertown, Pa.). For these studies, in
contrast to other experiments described below, the CPA-treated and
control freeze-dried were washed once with buffer B. The
concentration of platelets in both samples was then adjusted to
3.times.10.sup.5 platelets/.mu.l with buffer B and both sample were
incubated at 37.degree. C. for one hour after which the adenosine
and collagen were added at the indicated concentrations.
Aggregation was measured with an optical aggregometer while
maintaining the sample temperature at 37.degree. C. The data are
presented as aggregation response of freeze-dried and rehydrated
platelets expressed as a percentage of the aggregation response of
fresh platelets (of the sample PRP sample used for making the
freeze-dried samples).
[0069] Platelets freeze-dried and rehydrated in the presence of CPA
showed an increase in aggregation capacity relative to control
freeze-dried platelets (FIG. 1).
Example 3
Platelet Growth Factor Release Assay
[0070] In an initial validation of an assay to measure growth
factor release from thrombin-activated platelets, fresh platelet
suspensions (that had not been frozen or freeze-dried and
rehydrated) were tested. The fresh platelet PRP samples were
diluted to a concentration of 3.times.10.sup.5 cells/.mu.l with
buffer B and activated with thrombin (1 unit/ml) for 5 minutes at
room temperature. The resulting platelet clot was centrifuged and
the supernatant was separated from the pelleted clot. The
concentrations in the supernatant of four growth factors were
measured by enzyme-linked immunosorbent assays (ELISA) using a
commercially available kit (R & D Systems, Minneapolis, Minn.)
according to the manufacturer's directions. The growth factors were
transforming growth factor-beta (TGF-.beta.), platelet derived
growth factor (PDGF A/B), epidermal derived growth factor (EGF),
and vascular endothelial growth factor (VEGF).
[0071] The data are presented as the amounts of growth factor
released in the thrombin-activated supernatant expressed as
percentages of the amount of growth factor released by sonication
of a corresponding sample of the same platelets (Table 1). The
experiment was performed three times using PRP from a separate
donor for each experiment. The data are the means obtained from the
three experiments and standard deviations are indicated. These
means are the means of the averages of three replicates in each
experimental group.
[0072] As shown in Table 1, for all four growth factors,
approximately 50% of sonication-releasable growth factor was
released from fresh platelets by thrombin activation.
TABLE-US-00001 TABLE 1 Growth Factor Release from Fresh Human
Platelets Growth Factor Release TGF-.beta. 60.4% .+-. 18.8% PDGF
55.7% .+-. 16.0% EGF 50.8% .+-. 19.0% VEGF 47.0% .+-. 7.8%
Example 4
Growth Factor Release of CPA-Containing Freeze-Dried and Rehydrated
Platelets
[0073] The effect of freeze-drying and rehydration of platelets in
the presence and absence of CPA on platelet growth factor release
was determined. Data obtained for TGF-.beta. as a representative
growth factor are shown in Table 2. ELISA assays (as in Example 3)
were performed on the following samples:
(a) a supernatant obtained by centrifugation of the PRP sample used
for freeze-drying ("Fresh" "Plasma"). (b) the same PRP sample after
sonication ("Fresh" "Sonicate"); this measurement gave the total
TGF-.beta. releasable from platelets in the PRP sample plus the
TGF-.beta. in the plasma of the PRP sample. (c) supernatants
obtained by centrifugation of samples of freeze-dried and
rehydrated (in the presence and absence of CPA) PRP ("F/D/R"
"Plasma"). (d) freeze-dried and rehydrated (in the presence and
absence of CPA) PRP samples after sonication ("F/D/R" "Sonicate").
(e) supernatants obtained by centrifugation of thrombin-treated (as
in Example 3), freeze-dried and rehydrated (in the presence and
absence of CPA) PRP; the amounts detected in these supernatants
minus the amounts detected in (c) were expressed as a fraction
(percentage) of the amounts detected in (d) minus the amounts
detected in (c) ("Release").
[0074] CPA components were not washed out of the samples before
testing. The experiment was performed three times using PRP from a
separate donor for each experiment. The data are the means obtained
from the three experiments and standard deviations are indicated.
These means are the means of the averages of three replicates in
each experimental group.
TABLE-US-00002 TABLE 2 Release of TGF-.beta. by Freeze-Dried and
Rehydrated Platelets (with or without CPA) Condition TGF-.beta.
(ng/ml) PRP Fresh Plasma 18.1 .+-. 0.7 Sonicate 65.3 .+-. 11.2 PRP
without CPA PRP with CPA F/D/R Plasma 33.6 .+-. 5.5 28.2 .+-. 2.3
Sonicate 57.5 .+-. 12.0 57.1 .+-. 9.1 Release 16.9% 40.4% F/D/R;
freeze-dried and rehydrated
[0075] Freeze-drying and/or rehydration of both CPA-treated and
untreated platelets resulted in a significant level of spontaneous
leakage of TGF-.beta. compared to fresh platelet suspensions (Table
2). However, this spontaneous leakage was somewhat lower in the
platelets freeze-dried in the presence than in the absence of CPA.
Most importantly, platelets that had been freeze-dried and
rehydrated in the presence of CPA have a substantially higher
ability to release TGF-.beta. than platelets freeze-dried and
rehydrated without CPA. Similar results were observed for the other
three growth factors listed above.
Example 5
TGF-.beta. Produced by Platelets Freeze-Dried and Rehydrated in the
Presence of CPA is Active
[0076] The levels of active TGF-.beta., as measured by a cellular
assay (see below), and the levels of total TGF-.beta. protein, as
measured by ELISA, in supernatants from sonicated fresh PRP and
sonicated PRP freeze-dried and rehydrated (F/D/R) with CPA were
compared (Table 3). These supernatants were the same as some of
those shown in Table 2. The cell culture assay used to measure
TGF-.beta. activity was that described in Abe et al. [(1994) Anal.
Biochem. 216 (2):276-284], the disclosure of which is incorporated
herein by reference in its entirety. The experiment was performed
three times using PRP from a separate donor for each experiment.
The data are the means obtained from the three experiments and
standard deviations are indicated. These means are the means of the
averages of three replicates in each experiment.
[0077] Essentially all the TGF-.beta. released from sonicated fresh
platelets and sonicated platelets freeze-dried and rehydrated with
CPA was active (Table 3). The same results were obtained with
sonicated platelets freeze-dried and rehydrated without CPA.
TABLE-US-00003 TABLE 3 Measurements of TGF-.beta. Activity in Human
Platelets Total TGF-.beta. TGF-.beta. Percent of protein Activity
TGF-.beta. protein Condition (ng/ml) (ng/ml) that is active Fresh
65.3 .+-. 11.2 57.1 .+-. 2.9 87.4% Sonicate F/D/R (CPA) 57.1 .+-.
9.1 57.9 .+-. 3.0 100% Sonicate
Example 6
Cellular Proliferation Assay
Assay Design and Validation
[0078] To test for cell proliferation-inducing activity insoluble
factors released by thrombin-activated platelets, an in vitro
"Transwell.RTM." cell culture system was used. FIG. 2A is a
diagrammatic representation of this "Transwell.RTM." cell culture
system. Swiss Albino mouse 3T3 fibroblast cells were plated at a
density of 10,000 cells per well onto the bottom surfaces of
culture wells of 24-well tissue culture plates in Growth Medium
(GM; Dulbecco's Modified Eagle's Medium supplemented with 4 mM
glutamine, 405 g/L glucose, 1.5 g/L sodium bicarbonate, and 10%
calf serum (Invitrogen, Carlsbad, Calif.) and cultured for 16 hours
at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2. In
cultures to which activated or sonicated platelets (and
corresponding control cultures) were added (see below), the GM was
replaced with serum reduced medium (SRM; same as GM but
supplemented with 0.5% rather than 10% calf serum). Sonicated or
thrombin-activated (1 unit/ml for 5 minutes at 37.degree. C.)
platelets (in 75 .mu.l of 1.2.times.10.sup.6 platelets per .mu.l)
and SRM (225 .mu.l of 1.2.times.10.sup.6 platelets per .mu.l) were
added to Transwell.RTM. chambers having bottoms consisting of
semi-permeable membranes (having 8 .mu.m pores) and the chambers
were placed above the cells in appropriate culture wells such that
the bottoms of the chambers were submerged in culture media in the
culture wells (see FIG. 2A). This culture system permitted culture
media and soluble factors (but not whole platelets or insoluble
platelet material) to diffuse through the semi-permeable membranes
and contact the fibroblasts on the culture well bottoms. "Positive
control" cultures contained GM and no platelet material. The
cultures were incubated for the indicated periods of time after
which proliferation was determined by an MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)-2H-tetrazolium, inner salt] metabolic conversion assay. This
involved: (a) removal of the Transwell.RTM. chambers and culture
medium from culture wells; (b) addition to the wells of 0.5 ml
fresh SRM and 100 .mu.l of CellTiter96.RTM. Aqueous One solution
(Promega Corporation, Madison, Wis.); and (c) a further incubation
(under the same conditions described above) for 3 hours.
Supernatants (100 .mu.l) from each culture were transferred to the
wells of 96-well microtiter plates and the OD.sub.540 (as a measure
of relative cell proliferation) of each was measured using a
microtiter plate reader (BioRad, Hercules, Calif.).
[0079] Measurements of cellular proliferation were recorded in
separate culture wells every 24 hours, for a total of 72 hours
after introduction of the Transwell.RTM. chambers containing the
test platelet materials into culture vessel wells, (FIG. 2B). In
FIG. 2B, the graph vertical bars represent percent increase in MTS
levels over baseline levels (as measured at time 0, i.e., the time
at which the Transwell.RTM. chambers containing platelet materials
were added to the cultures) (left y-axis) and the graph lines
represent actual cell counts (right y-axis). The horizontal dashed
line indicates number of cells (right y-axis) at time 0. Sonicated
platelets and platelets activated with 1 unit/ml thrombin increased
fibroblast proliferation in a time-dependent fashion (compared to
cultures containing SRM only).
[0080] The cell proliferation levels obtained at 72 hours with
sonicated and thrombin-activated platelets minus the minimal level
of cell proliferation (observed in cultures containing SRM only)
were expressed as percentages of the maximal level of cell
proliferation (observed in cultures containing GM only) minus the
minimal level of cell proliferation (observed in cultures
containing SRM only). The values obtained are referred to as "%
induction" (FIG. 2C).
Soluble Factors Produced by Platelets Freeze-Dried and Rehydrated
in the Presence of CPA Retain the Ability to Induce Cellular
Proliferation
[0081] The above-described in vitro cellular proliferation assay
was used to determine the effect of freeze-drying and rehydration
in the presence of CPA on the ability of platelets to induce cell
proliferation. Platelets were mixed with CPA solution,
freeze-dried, and resuspended in buffer B as described in Example
1. The same controls described for the experiment shown in FIG. 2
were performed and the data were calculated as described for FIG.
2C. Platelet preparations which had been freeze-dried and
rehydrated with CPA exhibited approximately the same % induction of
proliferation as fresh platelets after activation with thrombin
(FIG. 3).
[0082] Supernatants from centrifuged fresh platelets PRP, platelets
freeze-dried and rehydrated in the absence of CPA, and platelets
freeze-dried and rehydrated in the presence of CPA ("Plasma" data
in FIG. 4) and supernatants of from the same three samples after
thrombin activation were tested in an assay essentially the same as
that in the experiment depicted in FIG. 2 ("Platelets" data in FIG.
4). Platelets freeze-dried and rehydrated with CPA, but not those
freeze-dried and rehydrated without CPA, retained the ability of
fresh platelets to induce cell proliferation (FIG. 4). The dotted
line in FIG. 4 shows the average ability of plasma samples to
induce fibroblast proliferation. In addition, a PRP sample
freeze-dried in the presence of CPA was divided into several
aliquots that were stored at -80.degree. C. for various periods of
time up to 24 weeks. The samples were rehydrated at the relevant
time points and tested for their ability to induce fibroblast
proliferation in the Transwell.RTM. culture system described above.
All samples demonstrated the same ability to induce fibroblast
proliferation as a control sample that was tested without
storage.
Ability of Freeze-Dried Platelets to Induce Cellular Proliferation
is Dose-Dependent
[0083] Platelets were freeze-dried and rehydrated in the presence
of CPA and thrombin activated as described above. Various volumes
of the sample (at the same platelet concentration) were tested in
the Transwell.RTM. fibroblast proliferation assay system described
above by addition of the rehydrated platelet samples to the
Transwell.RTM. chambers (FIG. 5). Measurements were made after 72
hours of culturing in the presence of the activated platelets. The
ability of the thrombin activated freeze-dried platelets to induce
proliferation of fibroblast cells was dose-dependent on the amount
of rehydrated platelets added to the assay system.
Example 7
CPA-Treated Freeze-Dried Platelets Increase Wound Healing in a
Diabetic Mouse Wound Model
Materials and Methods
Preparation of Platelet Therapeutics
[0084] Single donor units (SDU) of human platelets were purchased
from an American Association of Blood Banks (AABB) accredited blood
bank, stored with agitation at room temperature, and used within 5
days of donation. All required donor-screening and release-testing
were performed by the blood bank in accordance with AABB
requirements [MEB Technical Manual 14.sup.th ed. Bethesda, Md.:
American Association of Blood Banks (2002)]. Each SDU was divided
into three aliquots to prepare three unique platelet-based
therapeutic materials. The first aliquot was adjusted to
1.2.times.10.sup.6 platelets/.mu.l with a CPA-containing solution
using as a solvent a physiologic buffer [buffer B; 136 mM NaCl,
11.9 mM NaHCO.sub.3, 5.6 mM glucose, 5 mM HEPES, 2.7 mM KCl, 2.0 mM
MgCl.sub.2, 0.42 mM NaH.sub.2PO.sub.4; pH 7.4] and freeze-dried,
thereby creating a CPA stably preserved freeze-dried platelet rich
plasma (FDP-CPA). The second aliquot was adjusted to
1.2.times.10.sup.6 platelets/.mu.l using Buffer B and freeze-dried
to create a freeze-dried platelet rich plasma (FDP) sample. The
third aliquot was adjusted to 1.2.times.10.sup.6 platelets/.mu.l
using buffer B, sonicated for 10 seconds to disrupt cellular
structure and release intracellular constituents, and frozen at
-80.degree. C. creating a fresh frozen platelet (FFP) sample.
Addition of the CPA protectant solution yielded a final treatment
composition of 250 .mu.M amilioride, 100 .mu.M adenosine, 50 .mu.M
sodium nitroprusside, 1% (v/v) dimethyl sulfoxide, 4% (w/v)
polyvinyl pyrrolidone (Plasdone.TM. C-15, International Specialty
Products, Wayne, N.J.), and 50 mM mannitol. All manipulations of
the platelet material were done using standard aseptic technique
and all solutions were filter sterilized using filters having 0.2
.mu.m diameter pores (Millipore, Billerica, Mass.). Platelet
concentrations were verified using a CellDyn.RTM. 1700 hematology
analyzer (Abbott Laboratories, Abbott Park, Ill.). The dried
platelet products were packed under dry nitrogen in heat-sealed
foil pouches and stored at -80.degree. C. until used.
Wound Model & Treatment Procedure
[0085] Homozygous genetically diabetic 8-12 week-old, Lep/r--db/db
male mice (strain C57BL/KsJ-Lepr.sup.db) were used under an
approved animal protocol in an AAALAC accredited facility. The day
before surgery, hair was clipped and depilated (Nair.RTM.; Church
& Dwight Co., Princeton, N.J.). On the day of the surgery (post
operative day 0; POD 0), animals were weighed and anesthetized with
60 mg/kg Nembutal.RTM. (pentobarbital sodium). A dorsal 1.0
cm.sup.2 area of skin and panniculus carnosus was excised and the
wounds were photographed. Simultaneously, the following
platelet-based treatments were prepared: the FFP samples were
thawed, while the freeze-dried samples, FDP and FDP-CPA, were
rehydrated with sterile dH.sub.2O to their original volume. The
three different platelet treatments, with equivalent platelet
concentrations, based on pre-processing determinations, were
divided into 250 .mu.l aliquots. Each aliquot was treated with 1
U/ml thrombin (Chronolog Corporation, Havertown, Pa.) just prior to
application and allowed to clot in situ, thereby facilitating
persistence of the platelet material in the wound. Fifteen wounds
in each platelet experimental group (NT (not treated), FFP, FDP and
FDP-CPA) were included. All wounds were covered with a
semi-occlusive polyurethane dressing (Tegaderm.TM., 3M, St. Paul,
Minn.). On post operative day 9 (POD 9), the animals were
euthanized and the wounds were photographed, excised, and fixed in
10% neutral-buffered folmalin solution.
Wound Closure Analysis
[0086] Digital photographs captured on POD 9 were compared with
initial photographs (POD 0) by two independent observers, who were
blinded to the treatment mode, using planimetric methods (Scion
Image, Scion Corporation, Frederick, Md.). Wound closure was
quantified by measuring contraction, re-epithelialization, and open
wound as a percentage of the original wound area. The sum of
contracted, re-epithelialized, and open wound areas equals 100% of
the original wound size [Sullivan et al. (2004) Plast. Reconstr.
Surg. 113(3):953].
Microscopic Analysis
[0087] Wound biopsies were bisected, processed, and stained
according to routine Hematoxylin and Eosin (H&E) protocols.
Digital photographs were taken of the microscopic wound sections at
40.times. magnification and panoramic cross-sectional composites of
each wound were created using Adobe Photoshop.RTM. CS Software
(Adobe Systems Incorporated, San Jose, Calif.). The digital images
were analyzed with Scion Image.TM. software (Scion Corporation,
Frederick, Md.) by two independent observers, blinded to
experimental treatment mode, to quantify the area and thickness of
granulation tissue. Capillary density was evaluated using 3 fields
per slide viewed at 200.times. magnification: one in the middle of
the lesion and one at each wound margin. The images were viewed
with Adobe Photoshop.RTM. CS Software and blood vessels in each
high-powered field were marked and counted.
Statistical Analysis
[0088] Values were expressed as means +/- standard deviation in the
text and figures. One-way analysis of variance and ad hoc Dunnetts
tests were used to determine the significance of differences
between treatment modes.
Results
Wound Closure
[0089] Wound healing occurred in all groups by a combination of
wound contration and re-epithelialization. Previous work showed
that a 1.0 square cm.sup.2 wound in a diabetic mouse reaches the
50% closure point at about 8-12 days after surgery and that, once
healed, there are no differences in either the histology or visual
appearance of wounds in diabetic and control non-diabetic mice
(data not shown). FIG. 6 shows that re-epithelialization was
similar in all treatment groups but there was significantly reduced
wound contraction in the FDP-CPA group compared to the other
groups.
Granulation Tissue
[0090] Panoramic cross-sectional digital images of each wound were
prepared to analyze granulation tissue area and thickness (FIG. 7).
Both FDP-CPA and FDP induced a significant (p<0.01) 2.3-fold
increase in granulation tissue area compared with the NT group
(FIG. 7). Treatment with FFP also stimulated the formation of
granulation tissue when compared to untreated wounds (p<0.05),
but it was visibly more edematous than either of the freeze-dried
conditions. Similar results were observed with respect to
granulation tissue thickness (FIG. 7). FDP-CPA and FDP treatments
induced significant (p<0.01) 3.1 and 3.2-fold increases in
granulation tissue thickness measured in the center of the wound,
respectively, compared with the NT group. Tissue thickness in
response to FFP treatment was also significantly elevated over NT
(p<0.01) but failed to achieve comparable results to the
freeze-dried treatment conditions.
Neovascularity
[0091] Increased tissue vascularity in response to treatment with
platelet material was evident measuring standard H&E stained
wound sections. FDP-CPA treatment resulted in significant
(p<0.01) 2.2 and 1.8-fold increases in mean vessel count per
high-power field compared with the NT group and the FFP group,
respectively. FDP treatment induced a 1.6-fold increase compared
with NT group (p<0.01).
* * * * *