U.S. patent application number 14/292658 was filed with the patent office on 2014-12-04 for compositions and methods for using platelet-rich plasma for drug discovery, cell nuclear reprogramming, proliferation or differentiation.
The applicant listed for this patent is Allan Mishra. Invention is credited to Allan Mishra.
Application Number | 20140356893 14/292658 |
Document ID | / |
Family ID | 51985524 |
Filed Date | 2014-12-04 |
United States Patent
Application |
20140356893 |
Kind Code |
A1 |
Mishra; Allan |
December 4, 2014 |
COMPOSITIONS AND METHODS FOR USING PLATELET-RICH PLASMA FOR DRUG
DISCOVERY, CELL NUCLEAR REPROGRAMMING, PROLIFERATION OR
DIFFERENTIATION
Abstract
Methods are described for using compositions containing
platelet-rich plasma for the treatment of a variety of tissue
lesions, and the use of compositions containing platelet-rich
plasma to accelerate or complete the differentiation of a cell
line. Particularly, use of platelet-rich plasma in conjunction with
cell differentiation.
Inventors: |
Mishra; Allan; (Menlo Park,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mishra; Allan |
Menlo Park |
CA |
US |
|
|
Family ID: |
51985524 |
Appl. No.: |
14/292658 |
Filed: |
May 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61830754 |
Jun 4, 2013 |
|
|
|
Current U.S.
Class: |
435/29 ;
435/377 |
Current CPC
Class: |
C12N 2502/115 20130101;
C12N 5/0644 20130101; C12N 2506/1307 20130101; C12N 2502/11
20130101; C12N 5/0696 20130101; C12N 5/0622 20130101 |
Class at
Publication: |
435/29 ;
435/377 |
International
Class: |
C12N 5/074 20060101
C12N005/074 |
Claims
1. A method of producing induced pluripotent stem (iPS) cells
comprising: obtaining fibroblast cells from skin tissue from an
individual, administering a growth factor selected from the group
consisting of vascular endothelial growth factor (VEGF),
granulocyte colony stimulating factor (G-CSF), plerixafor,
sargramostim, .gamma.-tocotrienol, vitamin E, ancestim and bone
morphogenic growth factor to the individual, isolating
platelet-rich plasma (PRP) from whole blood of the individual, and
treating the fibroblast cells of the individual with the isolated
PRP.
2. The method of claim 1, wherein the whole blood is obtained from
spleen or bone marrow.
3. The method of claim 1, wherein the platelet-rich plasma
comprises a platelet concentration of 151,000/microliter to
7,000,000/microliter or higher, and further comprising treating the
fibroblast cells of the individual with a bone marrow concentrate
and fraction of adipose tissue.
4. The method of claim 1, wherein the platelet-rich plasma
comprises a platelet concentration of 151,000/microliter to
7,000,000/microliter or higher, and further comprising CD34+ cells
at a concentration 1-3.times.10.sup.9 per liter to
100.times.10.sup.9 per liter or higher.
5. The method of claim 1, wherein the platelet-rich plasma
comprises a platelet concentration of 151,000/microliter to
7,000,000/microliter or higher, and further comprising
stromal-derived factor -1 (SDF-1) in a concentration of 100 pg/ml
to 5000 pg/ml.
6. The method of claim 1, further comprising measuring the potency
of platelet rich plasma by visually observing it over time by at
least one method selected from the group consisting of cellular
movement, microfluidics, and chemotaxis.
7. The method of claim 1, wherein the PRP is used in drug discovery
to identify new targets.
8. The method of claim 1, wherein the PRP is used for nuclear
reprogramming, cell proliferation, or differentiation.
9. The method of claim 1, further comprising administrating
granulocyte colony-stimulating factor (G-CSF) or any other molecule
that stimulates the production of stem cells by the body prior to
the preparation of platelet-rich plasma from any source.
10. A composition comprising: cells from an individual, and
platelet-rich plasma (PRP) isolated from whole blood of the
individual, wherein the cells of the individual are treated with
the PRP to produce induced pluripotent stem (iPS) cells.
11. The composition of claim 10, wherein the cells are fibroblast
cells from skin tissue from the individual.
12. The composition of claim 10, further comprising a growth factor
selected from the group consisting of vascular endothelial growth
factor (VEGF), granulocyte colony stimulating factor (G-CSF),
plerixafor, sargramostim, .gamma.-tocotrienol, vitamin E, ancestim
and bone morphogenic growth factor to the individual.
13. The composition of claim 10, wherein the whole blood is
obtained from spleen or bone marrow.
14. The composition of claim 10, wherein the composition comprises
the platelet-rich plasma comprising a platelets concentration of
151,000/microliter to 7,000,000/microliter or higher and comprises
at least one cell selected from the group consisting of embryonic
stem cells, induced pluripotent stem cells, adult stem cells, and
progenitor cell lines.
15. The composition of claim 14, wherein the adult stem cells
comprise mesenchymal stem cells.
16. The composition of claim 10, wherein the composition comprises
the platelet-rich plasma comprising a platelet concentration of
151,000/microliter to 7,000,000/microliter or higher and comprises
progenitor or precursor stem cell lines.
17. The composition of claim 16, wherein the progenitor or
precursor stem cell lines comprise mesenchymal stem precursor
lines.
18. The composition of claim 10, wherein the composition comprises
the platelet-rich plasma comprising a platelet concentration of
151,000/microliter to 7,000,000/microliter or higher and comprises
CD34+ cells at a concentration 1-3.times.10.sup.9 per liter to
100.times.10.sup.9 per liter or higher.
19. The composition of claim 15, wherein the composition comprises
the platelet-rich plasma comprising a platelets in a concentration
of 151,000/microliter to 7,000,000/microliter or higher and
comprises stromal-derived factor -1 (SDF-1) in a concentration of
100 pg/ml to 5000 pg/ml.
20. The composition of platelet rich plasma comprising the platelet
rich plasma derived from induced pluripotent stem cells at a
platelet concentration of 151,000/microliter to
7,000,000/microliter or higher and pluripotent stem cells in any
concentration or form.
Description
RELATED APPLICATIONS
[0001] This application claims the priority of Provisional
Application No. 61/830,754, filed Jun. 04, 2013 which is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] Embodiments of the invention related to platelet-rich plasma
(PRP) and platelet compositions and their use.
[0004] 2. Description of the Related Art
[0005] Cell therapy is rapidly evolving in medicine. A variety of
cell lines including embryonic stem cells (ESC), induced
pluripotent stem cells (iPSCs), adult stem cells (ASCs) and
progenitor cells (PCs) derived from a variety of tissues are being
employed in preclinical and now clinical trials to treat disease. A
consistent issue with the use of any type of cell line, however, is
differentiating it to an appropriate type of cell. These
pluripotent or multipotent cells need to be driven to a specific
cell line in order for them to be used for a specific disease or
disorder.
[0006] Specifically, iPSCs can be created by overexpressing key
transcriptional factors. Landmark work by Yamanaka and coworkers
(U.S. Pat. No. 8,278,104) and others have confirmed that somatic
cells can be transformed into pluripotent cells via these methods.
Lentiviruses, retroviruses, adenoviruses, plasmids and transposable
elements have all been used to create these cells lines. This
methodology, however, may result in the integration of viral DNA
into the genome.
[0007] Cells lines can be differentiated into mature cell types
such as a muscle cells, a skin cell or an astrocyte, a mature type
of brain cell. A variety of growth factors and other cytokines can
be used to push immature cell lines into differentiated cell
types.
[0008] Unfortunately, "genetic or epigenetic errors may be
introduced during nuclear reprogramming, and the generation of
tissues from pluripotent stem cells employs laboratory methods for
cell generation and expansion that increase the risk of genetic
instability, epigenetic modification, and the generation of
tumorigenic cells. These issues may be addressed in part by using
non-viral reprogramming methods." (Volz et al 2012)
SUMMARY OF THE INVENTION
[0009] Blood is comprised of Red Blood Cells (RBC), White Blood
Cells (WBC), Plasma, and Platelets. Platelet-rich plasma (PRP) is a
fractionation of whole blood containing concentrated platelets and
white blood cells and which may include high quantities of
cytokines such as vascular endothelial growth factor (VEGF),
transforming growth factor beta (TGF-.beta.), and platelet-derived
growth factor (PDGF). Platelets are responsible for blood clotting
and when activated, release growth factors and other bioactive
molecules which are involved in stimulating the healing of bone and
soft tissue. For example, platelets release VEGF and basic
fibroblast growth factor from alpha granules and adenosine
diphosphate (ADP), adenosine triphosphate (ATP), and ionized
calcium from dense granules. White blood cells (WBCs), also known
as leukocytes, are involved in defending the body against both
infectious disease and foreign materials. The two most common types
of white blood cells are the lymphocytes and neutrophils.
Lymphocytes secrete factors, lymphokines, which modulate the
functional activities of many other types of cells and are often
present at sites of chronic inflammation. Neutrophils, which are
the most abundant white blood cell type in mammals, are recruited
to the site of injury within minutes following trauma. Neutrophils
form an essential part of the innate immune system, playing a role
in inflammation.
[0010] Platelet-rich plasma (PRP) is a bioactive fraction of whole
blood that has been used to treat a variety of connective tissue
disorders including chronic tennis elbow. Within PRP are dozens of
molecules that could be used to help differentiate cell lines. By
using an autologous fraction of whole blood to create PRP, a viral
free method of differentiating cells is provided. A specific
example is given where a dedifferentiated cell line is shown to
differentiate into a specific type of cell. The compositions and
methods herein described do not limit the invention to this cell
line but it should be realized that the methods and compositions
could be used in variety of cell lines including but not limited to
ESC, iPSCs, ASCs (derived from any source) and any line of
progenitor cells. Furthermore, platelet-rich plasma may be used to
create these lines by isolating the appropriate factors and then
using these factors to reprogram mature cells into pluripotent
cells. These cells could then be differentiated with a different
set of factors within the PRP to achieve the desired cell line.
This invention would eliminate the need for viral derived vectors
or other methods of artificially stimulating nuclear reprograming
or differentiation. Importantly, the use of PRP would enhance the
yield of any reprograming or differentiation method. Imbedded in
this invention are novel compositions and methods including
specific fractions of whole blood containing platelets, white blood
cells, stem cells and specific growth factors. Methods for using
these compositions for small molecule or drug discovery are also
disclosed. After using PRP to differentiate cell lines, PRP may
also be used in combination with these lines therapeutically. PRP
may further be used in combination with a genetically engineered
small molecule or drug to enhance the effects of PRP or the small
molecule. Therapeutic applications include but are not limited to
neurologic, cardiac, cardiovascular, peripheral vascular,
neoplastic, musculoskeletal, metabolic, inflammatory, infectious,
urologic, gynecologic, dermatologic (including cosmetic issues such
as wrinkles and baldness), allergic or immunologic, ophthalmologic,
obstetric, pediatric, surgical, internal medicine or orthopedic
applications. Treatment of specific problems include but are not
limited to cartilage degeneration in any joint (knee, hip,
shoulder, spine), tendinopathy, bone related problems,
cardiovascular injuries and disorders such as acute or chronic
heart failure, an acute heart attack and peripheral vascular
disease. For clarification, the compositions and methods in this
disclosure are examples and are meant to be inclusive of other
problems and disorders.
[0011] In accordance with one aspect, a method of producing induced
pluripotent stem (iPS) cells comprising obtaining fibroblast cells
from skin tissue from an individual, administering a growth factor
selected from the group consisting of vascular endothelial growth
factor (VEGF), granulocyte colony stimulating factor (G-CSF),
plerixafor, sargramostim, .gamma.-tocotrienol, vitamin E, ancestim
and bone morphogenic growth factor to the individual, isolating
platelet-rich plasma (PRP) from whole blood of the individual, and
treating the fibroblast cells of the individual with the isolated
PRP. In some embodiments, the method wherein the whole blood is
obtained from spleen or bone marrow. In some embodiments, the
method wherein the platelet-rich plasma comprises a platelet
concentration of 151,000/microliter to 7,000,000/microliter or
higher, and further comprising treating the fibroblast cells of the
individual with a bone marrow concentrate and fraction of adipose
tissue. In some embodiments, the method wherein the platelet-rich
plasma comprises a platelet concentration of 151,000/microliter to
7,000,000/microliter or higher, and further comprising CD34+ cells
at a concentration 1-3.times.10.sup.9 per liter to
100.times.10.sup.9 per liter or higher. In some embodiments, the
method wherein the platelet-rich plasma comprises a platelets in a
concentration of 151,000/microliter to 7,000,000/microliter or
higher, and further comprising stromal-derived factor -1 (SDF-1) in
a concentration of 100 pg/ml to 5000 pg/ml. In some embodiments,
the method further comprising measuring the potency of platelet
rich plasma by visually observing it over time by at least one
method selected from the group consisting of cellular movement,
microfluidics, and chemotaxis. In some embodiments, the method
wherein the PRP is used in drug discovery to identify new targets.
In some embodiments, the method wherein the PRP is used for nuclear
reprogramming, cell proliferation, or differentiation. In some
embodiments, the method further comprising administrating
granulocyte colony-stimulating factor (G-CSF) or any other molecule
that stimulates the production of stem cells by the body prior to
the preparation of platelet-rich plasma from any source.
[0012] In accordance with another aspect, a composition comprising
cells from an individual, and platelet-rich plasma (PRP) isolated
from whole blood of the individual, wherein the cells of the
individual are treated with the PRP to produce induced pluripotent
stem (iPS) cells. In some embodiments, the composition wherein the
cells are fibroblast cells from skin tissue from the individual. In
some embodiments, the composition further comprising a growth
factor selected from the group consisting of vascular endothelial
growth factor (VEGF), granulocyte colony stimulating factor
(G-CSF), plerixafor, sargramostim, .gamma.-tocotrienol, vitamin E,
ancestim and bone morphogenic growth factor to the individual. In
some embodiments, the composition wherein the whole blood is
obtained from spleen or bone marrow. In some embodiments, the
composition wherein the composition comprises the platelet-rich
plasma comprising a platelets concentration of 151,000/microliter
to 7,000,000/microliter or higher and comprises at least one cell
selected from the group consisting of embryonic stem cells, induced
pluripotent stem cells, adult stem cells, and progenitor cell
lines. In some embodiments, the composition wherein the adult stem
cells comprise mesenchymal stem cells. In some embodiments, the
composition wherein the composition comprises a platelet-rich
plasma comprising a platelet concentration of 151,000/microliter to
7,000,000/microliter or higher and comprises progenitor or
precursor stem cell lines. In some embodiments, the composition
wherein the progenitor or precursor stem cell lines comprise
mesenchymal stem precursor lines. In some embodiments, the
composition wherein the composition comprises a platelet-rich
plasma comprising a platelet concentration of 151,000/microliter to
7,000,000/microliter or higher and comprises CD34+ cells at a
concentration 1-3.times.10.sup.9 per liter to 100.times.10.sup.9
per liter or higher. In some embodiments, the composition wherein
the composition comprises a platelet-rich plasma comprising a
platelets in a concentration of 151,000/microliter to
7,000,000/microliter or higher and comprises stromal-derived factor
-1 (SDF-1) in a concentration of 100 pg/ml to 5000 pg/ml.
[0013] In accordance with another aspect, the composition of
platelet rich plasma comprising the platelet rich plasma derived
from induced pluripotent stem cells at a platelet concentration of
151,000/microliter to 7,000,000/microliter or higher and
pluripotent stem cells in any concentration or form. In accordance
with another aspect, the method of preparing platelet rich plasma
and storing it in a reduced and or increased oxygen concentration
device.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] These and other feature of this invention will now be
described with reference to the drawings of preferred embodiments
which are intended to illustrate and not to limit the
invention.
[0015] FIG. 1 shows Glioblastoma cells (012705) cultured with
(right panels) and without (left panels) 10% PRP for 10 days at
100.times. and 200.times. magnification.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0016] Platelet-rich Plasma Preparation and Compositions
[0017] The term "platelet-rich plasma" or "PRP" as used herein is a
broad term which is used in its ordinary sense and is a
concentration of platelets greater than the peripheral blood
concentration suspended in a solution of plasma, or other excipient
suitable for administration to a human or non-human animal
including, but not limited to isotonic sodium chloride solution,
physiological saline, normal saline, dextrose 5% in water, dextrose
10% in water, Ringer solution, lactated Ringer solution, Ringer
lactate, Ringer lactate solution, and the like. PRP compositions
may be an autologous preparation from whole blood taken from the
subject to be treated or, alternatively, PRP compositions may be
prepared from a whole blood sample taken from a single donor source
or from whole blood samples taken from multiple donor sources. In
general, PRP compositions comprise platelets at a platelet
concentration that is higher than the baseline concentration of the
platelets in whole blood. In some embodiments, PRP compositions may
further comprises WBCs at a WBC concentration that is higher than
the baseline concentration of the WBCs in whole blood. As used
herein, baseline concentration means the concentration of the
specified cell type found in the patient's blood which would be the
same as the concentration of that cell type found in a blood sample
from that patient without manipulation of the sample by laboratory
techniques such as cell sorting, centrifugation or filtration.
Where blood samples are obtained from more than one source,
baseline concentration means the concentration found in the mixed
blood sample from which the PRP is derived without manipulation of
the mixed sample by laboratory techniques such as cell sorting,
centrifugation or filtration.
[0018] In some embodiments, PRP compositions comprise elevated
concentrations of platelets and WBCs and lower levels of RBCs and
hemoglobin relative to their baseline concentrations. In some
embodiments of PRP composition, only the concentration of platelets
is elevated relative to the baseline concentration. Some
embodiments of PRP composition comprise elevated levels of
platelets and WBCs compared to baseline concentrations. In some
embodiments, PRP compositions comprise elevated concentrations of
platelets and lower levels of neutrophils relative to their
baseline concentrations. Some embodiments of PRP composition
comprise elevated levels of platelets and neutrophil-depleted WBCs
compared to their baseline concentrations. In some embodiments of
PRP, the ratio of lymphocytes and monocytes to neutrophils is
significantly higher than the ratios of their baseline
concentrations.
[0019] The PRP formulation may include platelets at a level of
between about 1.01 and about 2 times the baseline, about 2 and
about 3 times the baseline, about 3 and about 4 times the baseline,
about 4 and about 5 times the baseline, about 5 and about 6 times
the baseline, about 6 and about 7 times the baseline, about 7 and
about 8 times the baseline, about 8 and about 9 times the baseline,
about 9 and about 10 times the baseline, about 11 and about 12
times the baseline, about 12 and about 13 times the baseline, about
13 and about 14 times the baseline, or higher. In some embodiments,
the platelet concentration may be between about 4 and about 6 times
the baseline. Typically, a microliter of whole blood comprises at
least 140,000 to 150,000 platelets and up to 400,000 to 500,000
platelets. The PRP compositions may comprise about 500,000 to about
7,000,000 platelets per microliter. In some instances, the PRP
compositions may comprise about 500,000 to about 700,000, about
700,000 to about 900,000, about 900,000 to about 1,000,000, about
1,000,000 to about 1,250,000, about 1,250,000 to about 1,500,000,
about 1,500,000 to about 2,500,000, about 2,500,000 to about
5,000,000, or about 5,000,000 to about 7,000,000 platelets per
microliter.
[0020] The WBC concentration is typically elevated in PRP
compositions. For example, the WBC concentration may be between
about 1.01 and about 2 times the baseline, about 2 and about 3
times the baseline, about 3 and about 4 times the baseline, about 4
and about 5 times the baseline, about 5 and about 6 times the
baseline, about 6 and about 7 times the baseline, about 7 and about
8 times the baseline, about 8 and about 9 times the baseline, about
9 and about 10 times the baseline, or higher. The WBC count in a
microliter of whole blood is typically at least 4,100 to 4,500 and
up to 10,900 to 11,000. The WBC count in a microliter of the PRP
composition may be between about 8,000 and about 10,000; about
10,000 and about 15,000; about 15,000 and about 20,000; about
20,000 and about 30,000; about 30,000 and about 50,000; about
50,000 and about 75,000; and about 75,000 and about 100,000.
[0021] Among the WBCs in the PRP composition, the concentrations
may vary by the cell type but, generally, each may be elevated. In
some variations, the PRP composition may comprise specific
concentrations of various types of white blood cells. The relative
concentrations of one cell type to another cell type in a PRP
composition may be the same or different than the relative
concentration of the cell types in whole blood. For example, the
concentrations of lymphocytes and/or monocytes may be between about
1.1 and about 2 times baseline, about 2 and about 4 times baseline,
about 4 and about 6 times baseline, about 6 and about 8 times
baseline, or higher. In some variations, the concentrations of the
lymphocytes and/or the monocytes may be less than the baseline
concentration. The concentrations of eosinophils in the PRP
composition may be less than baseline, about 1.5 times baseline,
about 2 times baseline, about 3 times baseline, about 5 times
baseline, or higher.
[0022] In whole blood, the lymphocyte count is typically between
1,300 and 4,000 cells per microliter, but in other examples, the
lymphocyte concentration may be between about 5,000 and about
20,000 per microliter. In some instances, the lymphocyte
concentration may be less than 5,000 per microliter or greater than
20,000 per microliter. The monocyte count in a microliter of whole
blood is typically between 200 and 800. In the PRP composition, the
monocyte concentration may be less than about 1,000 per microliter,
between about 1,000 and about 5,000 per microliter, or greater than
about 5,000 per microliter. The eosinophil concentration may be
between about 200 and about 1,000 per microliter elevated from
about 40 to 400 in whole blood. In some variations, the eosinophil
concentration may be less than about 200 per microliter or greater
than about 1,000 per microliter.
[0023] In certain variations, the PRP composition may contain a
specific concentration of neutrophils. The neutrophil concentration
may vary between less than the baseline concentration of
neutrophils to eight times than the baseline concentration of
neutrophils. In some embodiments, the PRP composition may include
neutrophils at a concentration of 50-70%, 30-50%, 10-30%, 5-10%,
1-5%, 0.5-1%, 0.1-0.5% of levels of neutrophils found in whole
blood or even less. In some embodiments, neutrophil levels are
depleted to 1% or less than that found in whole blood. In some
variations, the neutrophil concentration may be between about 0.01
and about 0.1 times baseline, about 0.1 and about 0.5 times
baseline, about 0.5 and 1.0 times baseline, about 1.0 and about 2
times baseline, about 2 and about 4 times baseline, about 4 and
about 6 times baseline, about 6 and about 8 times baseline, or
higher. The neutrophil concentration may additionally or
alternatively be specified relative to the concentration of the
lymphocytes and/or the monocytes. One microliter of whole blood
typically comprises 2,000 to 7,500 neutrophils. In some variations,
the PRP composition may comprise neutrophils at a concentration of
less than about 1,000 per microliter, about 1,000 to about 5,000
per microliter, about 5,000 to about 20,000 per microliter, about
20,000 to about 40,000 per microliter, or about 40,000 to about
60,000 per microliter. In some embodiments, neutrophils are
eliminated or substantially eliminated. Means to deplete blood
products, such as PRP, of neutrophils is known and discussed in
U.S. Pat. No. 7,462,268, which is incorporated herein by
reference.
[0024] Several embodiments are directed to PRP compositions in
which levels of platelets and white blood cells are elevated
compared to whole blood and in which the ratio of monocytes and/or
lymphocytes to neutrophils is higher than in whole blood. The ratio
of monocytes and/or lymphocytes to neutrophils may serve as an
index to determine if a PRP formulation may be efficaciously used
as a treatment for a particular disease or condition. PRP
compositions where the ratio of monocytes and/or lymphocytes to
neutrophils is increased may be generated by either lowering
neutrophils levels, or by maintaining neutrophil levels while
increasing levels of monocytes and/or lymphocytes. Several
embodiments relate to a PRP formulation that contains 1.01 times,
or higher, baseline platelets in combination with a 1.01 times, or
higher, baseline white blood cells with the neutrophil component
depleted at least 1% from baseline.
[0025] In some embodiments, the PRP compositions may comprise a
lower concentration of red blood cells (RBCs) and/or hemoglobin
than the concentration in whole blood. The RBC concentration may be
between about 0.01 and about 0.1 times baseline, about 0.1 and
about 0.25 times baseline, about 0.25 and about 0.5 times baseline,
or about 0.5 and about 0.9 times baseline. The hemoglobin
concentration may be depressed and in some variations may be about
1 g/dl or less, between about 1 g/dl and about 5 g/dl, about 5 g/dl
and about 10 g/dl, about 10 g/dl and about 15 g/dl, or about 15
g/dl and about 20 g/dl. Typically, whole blood drawn from a male
patient may have an RBC count of at least 4,300,000 to 4,500,000
and up to 5,900,000 to 6,200,000 per microliter while whole blood
from a female patient may have an RBC count of at least 3,500,000
to 3,800,000 and up to 5,500,000 to 5,800,000 per microliter. These
RBC counts generally correspond to hemoglobin levels of at least
132 g/L to 135 g/L and up to 162 g/L to 175 g/L for men and at
least 115 g/L to 120 g/L and up to 152 g/L to 160 g/L for
women.
[0026] In some embodiments, PRP compositions contain increased
concentrations of growth factors and other cytokines. In several
embodiments, PRP compositions may include increased concentrations
of one or more of: platelet-derived growth factor, transforming
growth factor beta, fibroblast growth factor, insulin-like growth
factor, insulin-like growth factor 2, vascular endothelial growth
factor, epidermal growth factor, interleukin-8, keratinocyte growth
factor, and connective tissue growth factor. In some embodiments,
the platelets collected in PRP are activated by thrombin and
calcium chloride to induce the release of these growth factors from
alpha granules.
[0027] In some embodiments, a PRP composition is activated
exogenously with thrombin and/or calcium to produce a gel that can
be applied to an area to be treated. The process of exogenous
activation, however, results in immediate release of growth
factors. Other embodiments relate to activation of PRP via in vivo
contact with collagen containing tissue at the treatment site. The
in vivo activation of PRP results in slower growth factor release
at the desired site.
Methods of Making
[0028] The PRP composition may comprise a PRP derived from a human
or animal source of whole blood. The PRP may be prepared from an
autologous source, an allogenic source, a single source, or a
pooled source of platelets and/or plasma. To derive the PRP, whole
blood may be collected, for example, using a blood collection
syringe. The amount of blood collected may depend on a number of
factors, including, for example, the amount of PRP desired, the
health of the patient, the severity or location of the tissue
damage and/or the MI, the availability of prepared PRP, or any
suitable combination of factors. Any suitable amount of blood may
be collected. For example, about 1 cc to about 150 cc of blood or
more may be drawn. More specifically, about 27 cc to about 110 cc
or about 27 cc to about 55 cc of blood may be withdrawn. In some
embodiments, the blood may be collected from a patient who may be
presently suffering, or who has previously suffered from,
connective tissue damage and/or an MI. PRP made from a patient's
own blood may significantly reduce the risk of adverse reactions or
infection.
[0029] In an exemplary embodiment, about 55 cc of blood may be
withdrawn into a 60 cc syringe (or another suitable syringe) that
contains about 5 cc of an anticoagulant, such as a citrate dextrose
solution. The syringe may be attached to an apheresis needle, and
primed with the anticoagulant. Blood (about 27 cc to about 55 cc)
may be drawn from the patient using standard aseptic practice. In
some embodiments, a local anesthetic such as anbesol, benzocaine,
lidocaine, procaine, bupivicaine, or any appropriate anesthetic
known in the art may be used to anesthetize the insertion area.
[0030] The PRP may be prepared in any suitable way. For example,
the PRP may be prepared from whole blood using a centrifuge. The
whole blood may or may not be cooled after being collected.
Isolation of platelets from whole blood depends upon the density
difference between platelets and red blood cells. The platelets and
white blood cells are concentrated in the layer (i.e., the "buffy
coat") between the platelet depleted plasma (top layer) and red
blood cells (bottom layer). For example, a bottom buoy and a top
buoy may be used to trap the platelet-rich layer between the upper
and lower phase. This platelet-rich layer may then be withdrawn
using a syringe or pipette. Generally, at least 60% or at least 80%
of the available platelets within the blood sample can be captured.
These platelets may be resuspended in a volume that may be about 3%
to about 20% or about 5% to about 10% of the sample volume.
[0031] In some examples, the blood may then be centrifuged using a
gravitational platelet system, such as the Cell Factor Technologies
GPS System.RTM. centrifuge. The blood-filled syringe containing
between about 20 cc to about 150 cc of blood (e.g., about 55 cc of
blood) and about 5 cc citrate dextrose may be slowly transferred to
a disposable separation tube which may be loaded into a port on the
GPS centrifuge. The sample may be capped and placed into the
centrifuge. The centrifuge may be counterbalanced with about 60 cc
sterile saline, placed into the opposite side of the centrifuge.
Alternatively, if two samples are prepared, two GPS disposable
tubes may be filled with equal amounts of blood and citrate
dextrose. The samples may then be spun to separate platelets from
blood and plasma. The samples may be spun at about 2000 rpm to
about 5000 rpm for about 5 minutes to about 30 minutes. For
example, centrifugation may be performed at 3200 rpm for extraction
from a side of the separation tube and then isolated platelets may
be suspended in about 3 cc to about 5 cc of plasma by agitation.
The PRP may then be extracted from a side port using, for example,
a 10 cc syringe. If about 55 cc of blood may be collected from a
patient, about 5 cc of PRP may be obtained.
[0032] As the PRP composition comprises activated platelets, active
agents within the platelets are released. These agents include, but
are not limited to, cytokines (e.g., IL-1B, IL-6, TNF-A),
chemokines (e.g., ENA-78 (CXCL5), IL-8 (CXCL8), MCP-3 (CCL7),
MIP-1A (CCL3), NAP-2 (CXCL7), PF4 (CXCL4), RANTES (CCL5)),
inflammatory mediators (e.g., PGE2), and growth factors (e.g.,
Angiopoitin-1, bFGF, EGF, FGF, HGF, IGF-I, IGF-II, PDAF, PDEGF,
PDGF AA and BB, TGF-.beta. 1, 2, and 3, and VEGF).
[0033] The PRP composition may be delivered as a liquid, a solid, a
semi-solid (e.g., a gel), an inhalable powder, or some combination
thereof. When the PRP is delivered as a liquid, it may comprise a
solution, an emulsion, a suspension, etc. A PRP semi-solid or gel
may be prepared by adding a clotting agent (e.g., thrombin,
epinephrine, calcium salts) to the PRP. The gel may be more viscous
than a solution and therefore may better preserve its position once
it is delivered to target tissue. In some embodiments, the delivery
to the target tissue can include delivery to a treatment area in
the body as well as incorporation into cell cultures or suspensions
as described herein. In some embodiments, the PRP composition is
delivered without a clotting agent.
[0034] In some instances, it may be desirable to deliver the PRP
composition as a liquid and have it gel or harden in situ. For
example, the PRP compositions may include, for example, collagen,
cyanoacrylate, adhesives that cure upon injection into tissue,
liquids that solidify or gel after injection into tissue, suture
material, agar, gelatin, light-activated dental composite, other
dental composites, silk-elastin polymers, Matrigel.RTM. gelatinous
protein mixture (BD Biosciences), hydrogels and/or other suitable
biopolymers. Alternatively, the above mentioned agents need not
form part of the PRP mixture. For example, the above mentioned
agents may be delivered to the target tissue before or after the
PRP has been delivered to the target tissue to cause the PRP to
gel. In some embodiments, the PRP composition may harden or gel in
response to one or more environmental or chemical factors such as
temperature, pH, proteins, etc.
[0035] The PRP may be buffered using an alkaline buffering agent to
a physiological pH. The buffering agent may be a biocompatible
buffer such as HEPES, TRIS, monobasic phosphate, monobasic
bicarbonate, or any suitable combination thereof that may be
capable of adjusting the PRP to physiological pH between about 6.5
and about 8.0. In certain embodiments, the physiological pH may be
from about 7.3 to about 7.5, and may be about 7.4. For example, the
buffering agent may be an 8.4% sodium bicarbonate solution. In
these embodiments, for each cc of PRP isolated from whole blood,
0.05 cc of 8.4% sodium bicarbonate may be added. In some
embodiments, the syringe may be gently shaken to mix the PRP and
bicarbonate.
[0036] As noted above, the PRP composition may comprise one or more
additional agents, diluents, solvents, or other ingredients.
Examples of the additional agents include, but are not limited to,
thrombin, epinephrine, collagen, calcium salts, pH adjusting
agents, materials to promote degranulation or preserve platelets,
additional growth factors or growth factor inhibitors, NSAIDS,
steroids, anti-infective agents, and mixtures and combinations of
the foregoing.
[0037] In some embodiments, the PRP compositions may comprise a
contrast agent for detection by an imaging technique such as
X-rays, magnetic resonance imaging (MRI), or ultrasound. Examples
of such contrast agents include, but are not limited to, X-ray
contrast (e.g., IsoVue), MRI contrast (e.g., gadolinium), and
ultrasound contrast.
[0038] The PRP composition may be modified using a filtration
device and/or cell sorter. The filtration device may use vacuum
and/or gravity to remove a portion of the platelet, WBCs, and/or
RBCs. In some variations, a cell sorter may receive a CBC input
from an automated blood analyzer and/or a gene chip reader. A user
may select or confirm one or more modifications to be made to the
PRP composition. Of course, the cell sorter may be used with whole
blood, portions of whole blood, and/or PRP. The cell sorter may
sort the PRP composition based on electric charge, density, size,
deformation, fluorescence, or the like. Examples of cell sorters
include the BD FACSAria.RTM. cell sorter, the Cytopeia InFlux.RTM.
cell sorter, those manufactured by Beckman Coulter, the Cytonome
Gigasort.RTM. cell sorter, and the like.
PRP Compositions According to Embodiments of the Invention
[0039] PRP is prepared by any method including but not limited to
cell sorting, centrifugation, gravity filtration or other methods.
The composition described herein can be created by a cell sorter
device that uses electric charge, density, size, deformation,
fluorescence or other means to achieve the composition. As part of
this invention a variety of methods and compositions are claimed as
novel.
[0040] Granulocyte-colony stimulating factor (GCSF) can mobilize
cells from bone marrow including platelets. Pretreating patients
with GCSF will enhance the value of PRP by increasing the number of
platelets and other reparative cells. This may be especially true
in patients with low platelet counts or in older patients that have
lower cell counts. GCSF or other molecules that mobilize cells are
given once, twice or many times prior to drawing whole blood from
any source as described above. A preferred protocol would be to
give GCSF for several days prior to the PRP isolation.
[0041] One specific method is to administer granulocyte
colony-stimulating factor (G-CSF, also known as pegfilgrastim) or
any other molecule that stimulates the production of stem cells by
the body to a patient prior to the preparation of PRP. 5-50
micrograms per kilogram per day is administered to a patient for
1-10 days. PRP is then prepared from whole blood of the patient.
The whole blood may be from any source including bone marrow and
spleen. In a preferred embodiment, 10 micrograms per kilogram per
day of G-CSF is administered subcutaneously to a patient for 1-10
days. PRP is then prepared from whole blood of the patient from any
source including bone marrow or the spleen. Additional medications
or newer molecules that stimulate blood or stem cell production
could be added or substituted. Embodiments of the invention are
directed to the use of a molecule to stimulate endogenous
production of desired cells prior to preparing the PRP or bone
marrow concentrate to be used therapeutically or for the
reprogramming, proliferation, or differentiation of cells. Examples
of other potential molecules include but are not limited to:
plerixafor, sargramostim, gamma-tocotrienol, vitamin E and
ancestim. As science evolves, newer molecules that help mobilize
desired cells will be developed. It is anticipated by this filing
that these newer molecules would be included in the list that could
be administered prior to the production of PRP or other bioactive
blood fractions.
[0042] By giving a patient such a mobilizing drug prior to
preparing PRP, it should be noted that the composition will change
compared to without such a cell mobilization drug. Therefore, a
variety of compositions are claimed to be novel and useful.
[0043] In alternate embodiments, the molecule, such as G-CSF is
administered at the same time that the PRP is administered or the
molecule, such as G-CSF, is administered after the administration
of PRP for a period to be determined depending on the treatment.
For example, for treatment of a connective tissue injury, G-CSF
with or without PRP may be administered over a period of weeks of
months on a regular basis such as once a week, twice a week, three
times per week or more as determined by the medical practitioner.
In some embodiments, the molecule, such as G-CSF is administered
before blood is taken from the patient for isolation of PRP for a
period of 1-10 days as well as at the same time as administration
of PRP to the patient and/or after administration of PRP as
described above. PRP contains cytokines such as SDF-1 that are
chemoattractive for stem cells. In some embodiments, the use of
GCSF would enhance the value of the PRP treatment by mobilizing
more cells.
[0044] In some embodiments, the PRP could also be used in
combination with other drugs or fillers such as calcium phosphate,
collagen or even with botox.
[0045] Embodiments of the invention are directed to mobilizing
cells from bone marrow using GCSF or other similar type molecules
prior to the production of PRP of any type.
Methods of Use
[0046] Embodiments of the invention are directed to the use of that
PRP for a variety of research and clinically applications including
but not limited to: [0047] Cell culture of any type. [0048]
Connective Tissue Disorders of any type including but not limited
to: tennis elbow, carpal tunnel syndrome, deQuervain's
tenosynovitis, medial epicondylitis, ulnar collateral ligament
injuries of the elbow, cartilage injuries of any type, cartilage
degeneration of any type or location, rotator cuff disease
including partial and complete tears, labral tears, biceps tendon
disorders, fractures of any type, cervical radiculopathy, acute or
chronic spinal cord injuries, disc herniations, disc degeneration,
spinal disorders of any type including spinal stenosis and facet
arthropathy, acute bony injuries including subchondral edema,
tendinopathy of any type including gluteal, quadriceps, patellar,
achilles and others, osteoarthritis of any joint, rheumatoid
arthritis, arthritis of any type, achilles tendon tears, muscle
injuries of any type or severity, or any other musculoskeletal
injury or disorder. [0049] Cardiovascular disorders of any type
including but not limited to: acute myocardial infarction, acute or
chronic heart failure and peripheral vascular disease of any grade.
[0050] Neurologic injuries or disorders of any type including
Parkinson's disease, brain cancer or Alzheimer's disease. [0051]
Wound healing. The methods outlined above could be used to
specifically treat acute or chronic wounds. [0052] Infections of
any type. [0053] Dermatologic or Cosmetic Injuries or disorders
such baldness, wrinkles, burns or even skin cancer. [0054] Internal
organ injuries or disorders such as diabetes, hernias or
fistulas.
[0055] It should be recognized that the concept is to give a cell
mobilization agent either before, during, and/ or after preparing
and using PRP as a treatment for any medical issue in humans or
animals.
[0056] The PRP composition may be delivered at any suitable dose.
In some embodiments, the dose may be between about 1 cc and about 3
cc, between about 3 cc and about 5 cc, between about 5 cc and about
10 cc, between about 10 cc and about 20 cc, or more. The dose may
be delivered according to a medical procedure (e.g., at specific
points in a procedure) and/or according to a schedule. For example,
prior to an elective cardioversion, the PRP composition may be
delivered about 24 hours, about 12 hours, about 6 hours, about 2
hours, and/ or about 1 hour before the procedure begins.
[0057] In some embodiments, the PRP composition may be delivered to
tissue damaged by ischemia or reperfusion injury. The list of
tissues includes, but is not limited to, the heart, ischemic limbs,
ischemic or damaged organs including the brain and skin. The
[0058] PRP composition may be delivered to an individual in need
thereof by injection using a syringe or catheter. The PRP
composition may also be delivered via delivery device such as a
dermal patch, a spray device, sutures, stents, screws, plates, or
some other implantable medical device such as bioresorbable tissue
patch. The PRP composition may be used as a coating or incorporated
into the delivery device. The PRP delivery device may be incubated
with PRP prior to use. Incubation times may be from a few seconds
up to any convenient time such as a few seconds to hours before
use, such as less than 1 minute, 5-10 minutes, 10 minutes to an
hour, 1-3 hours, 4-12 hours, 13-24 hours, 1-3 days, or 3-31 days.
PRP compositions may be used in conjunction with an ointment, bone
graft, or drug.
[0059] The PRP alone or in combination with a delivery device may
be conveniently stored in an appropriate chamber. In some
embodiments, the PRP and/or PRP combined delivery device may be
stored frozen and/or under reduced oxygen concentration or
increased oxygen concentration, low and/or high pH, low and/or high
pressure, low and/or high UV or other light conditions, low and/or
high temperature. Storage times may vary from such as less than 1
minute, 5-10 minutes, 10 minutes to an hour, 1-3 hours, 4-12 hours,
13-24 hours, 1-3 days, 3-31 days, or 1-12 months or 1-5 years. The
PRP composition alone or in combination with the delivery device
may then be used clinically as appropriate.
[0060] In one exemplary embodiment, a platelet rich plasma
composition is prepared and combined with a stent in an appropriate
low oxygen chamber for 1-30 minutes, preferably about 10 minutes.
The chamber is then exposed to ultraviolet light for a brief period
of time, such as 1-60 seconds, 1-5 minutes, or 5-15 minutes. The
stent is then removed from the chamber and implanted into a
patient. It is expected that this chamber will improve the biologic
activity of the platelet rich plasma and or device.
[0061] The site of delivery of the PRP composition is typically at
or near the site of tissue damage. The site of tissue damage may be
determined by well-established methods including imaging studies
and patient feedback or a combination thereof. The preferred
imaging method used may be determined based on the tissue type.
Commonly used imaging methods include, but are not limited to MRI,
X-ray, CT scan, Positron Emission tomography (PET), Single Photon
Emission Computed Tomography (SPECT), Electrical Impedance
Tomography (EIT), Electrical Source Imaging (ESI), Magnetic Source
Imaging (MSI), laser optical imaging NOGA mapping and ultrasound
techniques. The patient may also assist in locating the site of
tissue injury or damage by pointing out areas of particular pain
and/or discomfort.
[0062] The PRP compositions described herein may also be used to
treat peripheral vascular disease, strokes or other ischemic areas
such as a kidney that was damaged. PRP compositions could also be
used as a primary or secondary treatment for pulmonary disease.
[0063] In some examples, a PRP composition may be used to treat a
patient diagnosed with an acute myocardial infarction or ischemic
heart disease. Treatment with the PRP composition may occur in the
field or in the emergency room setting. Criteria for PRP
composition treatment may include positive cardiac markers,
ST-elevations, or new wall motion abnormalities identified on
echocardiogram, for example. The decision to treat with a PRP
composition, and the treatment location(s), may depend upon one or
more characteristics of the myocardial infarction. For example, a
myocardial infarction may be characterized as a ST-elevation
myocardial infarction (STEMI) or non-ST-elevation myocardial
infarction (NSTEMI), a Q-wave or non-Q-wave myocardial infarction,
and whether they are subendocardial or transmural. Myocardial
infarctions may also be characterized anatomically by cardiac wall
region and/or the suspected blockage site in the cardiac
vasculature. Myocardial infarctions may also be characterized as
anterior, lateral, inferior, posterior, septal, or
right-ventricular in location, and may involve disease or blockage
of the left-anterior descending, left circumflex, left main,
posterior-descending and right coronary arteries, for example.
[0064] In some embodiments, timing of the PRP preparation and
application may be based upon other treatments that are indicated
in a patient with a myocardial infarction or ischemic heart
disease. In some embodiments, a PRP composition may be prepared and
delivered before, during, and/or after reperfusion therapy is
performed to treat an acute myocardial infarction, a previous
myocardial infarction, or ischemic heart disease. Reperfusion
therapies may include thrombolytic therapy (such as heparin, TPA
and or other pharmacologic agents), angioplasty, stenting
(including bare metal stents and drug-eluting stents) or coronary
artery bypass graft (CABG) surgery. In some instances, reperfusion
therapy may be associated with an increased risk of an arrhythmia,
including sudden death. Also, it is believed that the etiology of
reperfusion arrhythmias or reperfusion arrhythmia risk may be
different from the arrhythmia etiologies associated with the
myocardial infarction itself For example, some reperfusion
arrhythmias may be caused by triggered activity and/or re-entry. In
some embodiments, PRP composition is prepared before or at the
start of a reperfusion procedure, but not used unless an arrhythmia
occurs during the procedure. In other embodiments, the patient may
be prophylactically pre-treated with a PRP composition before
reperfusion occurs, e.g., before guidewire passage across an
occlusion, stent positioning, stent expansion, or reestablishment
of coronary flow through a bypass segment.
[0065] In some embodiments, the PRP composition is injected into or
near an infarct site. The location of the infarct site may be
determined or approximated using various techniques. For example,
in some variations, diagnostic procedures such as an
electrophysiology study or an electrical mapping study of the heart
may be used. In other variations, one or more imaging technologies
such as MRI, X-ray, CT scan, Positron Emission tomography (PET),
Single Photon Emission Computed Tomography (SPECT), Electrical
Impedance Tomography (EIT), Electrical Source Imaging (ESI),
Magnetic Source Imaging (MSI), NOGA mapping, laser optical imaging
and ultrasound techniques may be used. Other technologies and
approaches that may be used include visual inspection during open
chest surgical procedures, localized blood flow determinations,
local electrical and structural activity, nuclear cardiology,
echocardiography, echocardiographic stress test, coronary
angiography, magnetic resonance imaging (MRI), computerized
tomography (CT) scans, and ventriculography.
[0066] PRP compositions that are formulated as gels or other
viscous fluids may be difficult to deliver via a needle or syringe.
Thus, in variations where the use of a needle or syringe is
desirable, a gelling and/or hardening agent may be optionally added
to the PRP composition in situ. One or more needles or catheters
may be configured to deliver the PRP composition and/or the gelling
or hardening agent simultaneously, or substantially simultaneously,
to the cardiac tissue. For example, if a needle is used to deliver
the PRP composition, the needle may comprise a plurality of lumens
through which the PRP composition and the agent separately travel.
Alternatively or additionally, separate needles may be used to
deliver the components to the tissue at the same time or one after
the other.
[0067] The PRP composition may be delivered minimally invasively
and/or surgically. For example, the PRP composition may be
delivered to the heart using a catheter inserted into the patient
via the femoral vein or artery, the internal jugular vein or
artery, or any other suitable vein or artery. The PRP composition
may be delivered along with one or more medical devices,
instruments, or agents to treat the MI and/or other cardiac
conditions.
[0068] To deliver a PRP composition to the ischemic tissue, a
physician may use one of a variety of access techniques. These
include surgical (e.g., sternotomy, thoracotomy, mini-thoracotomy,
sub-xiphoidal) approaches, endoscopic approaches (e.g., intercostal
and transxiphoidal) and percutaneous (e.g., transvascular,
endocardial, and pericardial) approaches. Once access has been
obtained, the composition may be delivered via epicardial,
endocardial, or transvascular approaches. The composition may be
delivered to the cardiac wall tissue or cardiac vessels in one or
more locations. This includes intra-myocardial, sub-endocardial,
and/or sub-epicardial administration.
[0069] Upon gaining access to the ischemic tissues of the heart,
the delivery device may be inserted through any appropriate vessel.
The distal end of the delivery device may be then placed against
the surface of the myocardium and one or more needles may be
advanced into tissue. Following delivery of one or more components
of the PRP composition, the needles, if any, may be retracted.
Mapping or guidance systems that rely upon voltage, ultrasound or
pressure in addition to other systems may be used in combination
with injection. The delivery device may then be repositioned for
additional delivery of one or more components of the composition or
may be removed from the patient. Incisions may then be closed using
standard techniques.
[0070] The delivery system may deliver the components of the PRP
composition in a prescribed ratio (e.g., a ratio of the lymphocytes
and the neutrophils). The prescribed ratio may be calculated
beforehand or determined on an ad hoc basis once delivery begins.
To deliver the components in the prescribed ratio, the delivery
device may include one or more gears having a corresponding gear
ratio, one or more lumens having a proportional lumen size, or any
other suitable mechanism. Some delivery devices may include one or
more mixing chambers. The multiple components may be delivered
using separate delivery devices or may be delivered one after the
other using the same delivery device.
[0071] The delivery devices may be advanced through a vessel
adjacent to the ischemic tissue to be treated. The PRP composition
may be injected directly into the ischemic tissue using a needle
and/or a needle-tip catheter. The PRP composition may alternatively
or additionally be infused into the vessel.
[0072] When the PRP compositions are delivered using one or more
catheters, any suitable catheter may be used. For example, the
catheters may include one or more lumens and staggered or flush
tips. The catheters may include needles or other devices (e.g.,
imaging devices) located at the distal end, and plungers or any
other control located at the proximal end. The catheters and/or
other delivery devices may have differently sized lumens to deliver
multiple components of the PRP composition in the prescribed ratio.
When catheters are used, a physician may navigate to the heart
using one of the routes known for accessing the heart through the
vasculature, including but not limited to navigation to a heart
chamber for epicardial, endocardial, and/or transvascular delivery
of the PRP composition.
[0073] Endocardial delivery of the PRP composition may comprise
accessing a treatment site, for example, in the left ventricle of a
heart, using a delivery device advanced percutaneously in an
anterograde approach through the superior vena cava or inferior
vena cava into the right ventricle. The delivery device may be
passed through the interatrial septum into the left atrium and then
into the left ventricle to reach treatment site. Alternatively, the
device may be advanced using a transseptal procedure, e.g., through
the intraventricular septum into the left ventricle. In another
embodiment, the PRP composition may be injected directly into the
intraventricular septum from the right ventricle. An alternative
endocardial delivery method may comprise accessing the treatment
site using a delivery device advanced percutaneously in a
retrograde approach through the aorta into the left atrium and then
into the left ventricle.
[0074] Transvascular delivery of compositions may comprise passing
the delivery device through the coronary sinus into the cardiac
venous system via the cardiac veins and, if needed, leaving the
veins by tracking through myocardial tissue. An alternative
transvascular delivery method comprises accessing a treatment site
through the aorta into a coronary artery to reach treatment
site.
[0075] A practitioner may make multiple deliveries into various
locations using a single device, make multiple deliveries into
various locations using multiple devices, make a single delivery to
a single location using a single device, or make a single delivery
to a single location using multiple devices. The delivery devices
may include at least one reusable needle or catheter. Some
embodiments may include delivery devices having an automated dosing
system (e.g., a syringe advancing system). The automated dosing
system may allow each dose to be pre-determined and dialed in (may
be variable or fixed). In some embodiments, an iontophoresis device
may be used to deliver the PRP composition into the ischemic
tissue.
[0076] It may be desirable to deliver the PRP composition to the
ischemic tissues while avoiding coincidental delivery to other
cardiac tissues or other locations adjacent to the heart. For
example, the PRP composition may gel or harden upon delivery to
prevent migration. In other embodiments, the PRP compositions may
be delivered without a gelling agent/activator such as thrombin. In
some variations, a balloon catheter may be placed in the coronary
sinus and inflated during delivery until the PRP composition has
solidified or at least partially immobilized. Other variations may
include a pressure control system on the delivery device to prevent
pressure-driven migration of the PRP composition. Backbleed may
also be prevented by keeping the needle in place for several
seconds (e.g., about 5 to about 30 seconds, or about 5 to about 120
seconds) following an injection.
[0077] Sensors may be used to direct the delivery device to a
desired location and/or to deliver the PRP composition. For
example, real-time recording of electrical activity (e.g., an ECG),
pH, oxygenation, metabolites such as lactic acid, CO2, or the like
may be used. The sensors may be one or more electrical sensors,
fiber optic sensors, chemical sensors, imaging sensors, structural
sensors, and/or proximity sensors that measure conductance. The
sensors may be incorporated into the delivery device or be separate
from the delivery device. In some embodiments, the sensors may
sense and/or monitor such things as needle insertion depth, blood
gas, blood pressure or flow, hemocrit, light, temperature,
vibration, voltage, electric current, power, and/or impedance. The
sensors may include one or more imaging systems and may be coupled
to any appropriate output device, for example, a LCD or CRT monitor
which receives and displays information.
[0078] The total volume of the PRP composition delivered to the
patient may be based on the size of the heart, the amount of the
affected ischemic tissue, and/or the desired outcome of the
procedure. For example, the total volume of composition injected
may be less than 15000 .mu.L.
[0079] The number of delivery sites in the heart may be based on
the type and location of the infarct(s), the desired location of
the PRP composition, and the distance separating the desired
locations. The number of delivery sites may range from about 1 to
about 25 sites. The distance separating delivery sites may vary
based on the desired volume of PRP to be delivered per delivery
site, the desired total volume to be delivered, and/or the
condition of the ischemic tissue. At the delivery site, the PRP
composition may be injected, infused, or otherwise disposed at or
adjacent to the ischemic tissue. The PRP composition may also be
infused into the vasculature (i.e., vessels) upstream of the target
site, so that it will flow towards the affected ischemic
tissue.
[0080] The location of the delivery sites may vary based on the
size and shape of the ischemic tissue, and the desired extent of
the treatment of the tissue. For example, the PRP composition may
be delivered into the ischemic tissue, and/or into the tissue that
bordering the ischemic tissue. Similarly, the composition may be
delivered to any combination of the regions of ischemic tissue and
other cardiac tissue.
[0081] The timing of PRP delivery relative to an acute MI may be
based on the severity of the infarction, the extent of the ischemic
tissue, the condition of the patient, and the progression of any
concurrent MI or arrhythmia treatments. The PRP composition may be
delivered at any suitable time. For example, it may be delivered
immediately after the onset of an MI, within one hour of an MI, one
to eight hours following an MI, or three to four days after an MI
after clinical stabilization of the patient when it is safer for
the patient to undergo a separate procedure. Treatment may also be
done later. The timing may be based upon the level of caspase-3 in
the blood. In some variations, the composition is delivered about
one week, about 1 to about 3 weeks, about 1 to about 6 months, or
even up to or more than about 1 year after the MI. Treatment may be
done for patients with congestive heart failure, cardiomyopathy or
other heart disorders. Other times for injecting compositions into
the ischemic tissue are also contemplated, including prior to any
potential MI, and immediately upon finding an area of ischemic
tissue. Of course, compositions may be injected into the ischemic
tissue years after an MI.
[0082] As mentioned previously, a PRP composition may additionally
or alternatively be used in other cardiac procedures. These cardiac
procedures may include anti-arrhythmia procedures, procedures to
correct congenital heart defects, or other pathologies. Examples of
other cardiac procedures include, but are not limited to,
angioplasty, coronary artery bypass, Minimally Invasive Direct
Coronary Artery Bypass (MIDCAB), off-pump coronary artery bypass,
Totally Endoscopic Coronary Artery Bypass (TECAB), aortic valve
repair, aortic valve replacement, mitral valve repair, mitral valve
replacement, Ross procedure, Bentall procedure, pulmonary
thromboendarterectomy, transmyocardial revascularization (TMR),
valve-sparing aortic root replacement, cardiomyoplasty, Dor
procedure, heart transplantation, septal myectomy, ventricular
reduction, pericardiocentesis, pericardiectomy, atrial septostomy,
Blalock-Taussig shunt procedure, Fontan procedure, Norwood
procedure, Rastelli procedure, Maze procedure (Cox maze and
minimaze), and/or pacemaker insertion. The PRP composition may be
used to prevent an arrhythmia associated with reperfusion of the
cardiac tissue during any of the above procedures. As is known,
reperfusion may cause a spontaneous arrhythmia to occur after
cardiac surgery.
[0083] In some embodiments, PRP may be used to treat any lung
disease. Examples of lung disease include, but are not limited to:
Acute Respiratory Distress Syndrome (ARDS), Alpha-1-Antitrypsin
Deficiency, Asbestos-Related Lung Diseases, Asbestosis, Asthma,
Bronchiectasis, Bronchitis, Bronchopulmonary Dysplasia (BPD),
Chronic Bronchitis (see COPD), Chronic Obstructive Pulmonary
Disease (COPD), Collapsed Lung (see Atelectasis), Cough, Cystic
Fibrosis, Emphysema (see COPD), Hemothorax, Idiopathic Pulmonary
Fibrosis, Infant Respiratory Distress Syndrome (Respiratory
Distress Syndrome in Infants), LAM (Lymphangioleiomyomatosis), Lung
Transplant, Pleural Effusion, Pleurisy and Other Pleural Disorders,
Pneumonia, Pneumonoconiosis, Pneumothorax (see Pleurisy and Other
Disorders of the Pleura), Pulmonary Embolism, Pulmonary Arterial
Hypertension, Pulmonary Fibrosis (see Idiopathic Pulmonary
Fibrosis), Respiratory Distress Syndrome in Infants, Respiratory
Failure, Sarcoidosis, Tracheostomy, and Ventilator/Ventilator
Support. In some embodiments, PRP compositions are delivered
directly to the lung via bronchoscopy or delivering indirectly to
the lung via the heart or blood vessel. Measurements of tissue
perfusion or function may be done to evaluate the efficacy of the
treatment.
[0084] In some embodiments PRP is useful in treatment of disease
and conditions in a variety of tissues including but not limited to
heart, lung, liver, kidney, brain, spinal cord, muscle, tendon,
bone, skin, ligaments and any other body cell or tissue. Rotator
Cuff Tendinitis or Tear, Rotator Cuff Impingement Syndrome or
Bursitis, Bicipital Tendinitis, labrum tears, arthritis,
instability DeQuervaine's Tenosynovitis, arthritis, other wrist or
finger tendinitis, ligament tears or dysfunction of the fingers
Illiotibial Band Tendinitis (ITB Syndrome), Psoas Tendinitis and
bursitis, Greater Trochanteric Bursitis, Hip labrum tears,
Piriformis Syndrome, Sacroiliac Joint Dysfunction, arthritis
Patellar Tendinitis, Patellar Femoral Syndrome, chondromalacia
patella, partially torn or strained major ligaments of knee
(ACL/LCL/MCL), meniscus tears, arthritis, patellar instability
Achilles Tendinitis, Peroneal Tendinitis, arthritis, recurrent
ankle sprains, other foot or ankle tendinitis Whiplash injuries,
headaches related to the neck, arthritis Facet joint arthritis, rib
problems, and pain associated with scoliosis. In some embodiments,
PRP compositions may be used to treat disogenic spine pain or
disorders alone or in combination with other treatments.
[0085] As used herein the term "an effective amount" of an agent is
the amount sufficient to treat, inhibit, or prevent ischemia and/or
reperfusion injury associated with indications and conditions
including, but not limited to, myocardial infarction,
arteriosclerosis. stroke, septic shock, traumatic shock, and
associated with surgical procedures such as vascular interventional
procedures including angioplasty, surgery that involves restriction
of blood supply to an organ or tissue, abdominal surgery,
abdominoplasty, adenoidectomy, amputation, angioplasty,
appendectomy, arthrodesis, arthroplasty, brain surgery, cesarean
section, cholecystectomy, colon resection, colostomy, corneal
transplantation, discectomy, endarterectomy, gastrectomy, grafting
of skin or other tissues, heart transplantation, liver
transplantation, heart surgery hemicorporectomy, hemorrhoidectomy,
hepatectomy, hernia repair, hysterectomy, kidney transplantation,
laminectomy, laryngectomy, lumpectomy, lung transplantation,
mammoplasty, mastectomy, mastoidectomy, myotomy, nephrectomy,
nissen fundoplication, oophorectomy, orchidectomy, orthopedic
surgery, parathyroidectomy, penectomy, phalloplasty, pneumonectomy,
prostatectomy, radiosurgery, rotationplasty, splenectomy,
stapedectomy, thoracotomy, thrombectomy, thymectomy, thyroidectomy,
tonsillectomy, ulnar collateral ligament reconstruction,
vaginectomy, vasectomy and any surgery involving cardiac bypass,
cardiac artery bypass graft surgery and organ transplantation.
[0086] In addition to the foregoing uses for the compositions,
methods and systems described herein, it will be apparent to those
skilled in the art that other injured tissues, in addition to
injured cardiac tissue and connective tissue, would benefit from
the delivery of structural support materials to treat the injuries.
Non-limiting examples of such tissues include the stomach, to
reduce food intake and increase satiety; the abdominal wall, to
prevent and treat hernias; and the bladder to prevent or treat
incontinence. Such tissues may additionally include vascular
tissues.
[0087] In some embodiments, a specific cytokine, growth factor or
drug is administered to the patient to mobilize cells, PRP is
prepared from the patient's blood and is used to treat cartilage
injuries or disorders such as knee osteoarthritis or spinal disc
degeneration.
[0088] In some embodiments, a mobilization agent is provided
subcutaneously, intravenously or intramuscularly for 1-5 days to
the patient, PRP is prepared from the patient and injected
intramuscularly to treat the heart or into the muscle of the leg to
treat peripheral vascular disease.
[0089] In some embodiments, a mobilization agent is administered to
the patient and the PRP is prepared. This PRP is then used in an
activated or unactivated manner to treat the wound.
[0090] It is noted that the PRP may be obtained from the person to
be treated (autologous) or the PRP may be obtained from one
individual but administered to a different individual.
[0091] PRP prepared as described by any of the above methods may be
used to provide the platelet concentrations in the following
platelet formulations. Further, in some embodiments, the
compositions and components for created and utilizing the
compositions as disclosed herein can be assembled into kits to be
used by laboratory technician, doctor, medical professional, or
others assembling such compositions for use.
Platelet Formulations
Example One
[0092] Platelets Concentration: 151,000/microliter to
7,000,000/microliter or higher.
[0093] CD34+ Cell Concentration: 1-3.times.10.sup.9 per liter to
100.times.10.sup.9 per liter or higher.
[0094] Platelet and CD34+ cell concentrations may be at any level
in between the above values. The PRP could be unactivated or
activated.
Example Two
[0095] Platelets Concentration: 151,000/microliter to
7,000,000/microliter or higher.
[0096] CD34+ Cell Concentration: 1-3.times.10.sup.9 per liter to
100.times.10.sup.9 per liter or higher.
[0097] Platelet and CD34+ cell concentrations may be at any level
in between the above values. The PRP could be unactivated or
activated.
[0098] The composition of example one that includes:
[0099] Neutrophils, Lymphocytes, Monocytes, Eosinophils or
Basophils in any concentration.
Example Three
[0100] Platelet Concentration: 151,000/microliter to 7,000,000 or
higher.
[0101] Stem cells of any type in any concentration including
embryonic stem cells, induced pluripotent stem cells, adult stem
cells, and progenitor cell lines of any type.
Example Four
[0102] Platelet Concentration: 151,000/microliter to 7,000,000 or
higher.
[0103] CD34+ Cell Concentration: 1-3.times.10.sup.9 per liter to
100.times.10.sup.9 per liter or higher.
[0104] Lymphocytes, Monocytes Eosinophils or Basophils in any
concentration
[0105] without any neutrophils.
Example Five
[0106] The composition of example three or four where the
neutrophil concentration is less than 100/microliter or less than
500/microliter, or less than 1000/microliter, or less than
5000/microliter or less than 10,000/microliter, or less than
20,000, or less than 50,000/microliter or less than
100,000/microliter.
Example Six
[0107] Platelet-rich plasma derived from induced pluripotent stem
cells.
[0108] Platelet concentration: 151,000/microliter to 7,000,000 or
higher.
[0109] Pluripotent stem cells in any concentration.
Example Seven
[0110] The composition of example six within of any of the
compositions of examples 1-6.
Example Eight
[0111] The compositions of any of examples above where the
hemaglobin concentration is less than 10 g per deciliter;
preferably less than 5 g per deciliter, more preferably less than 1
g per deciliter, and most preferably where the hemoglobin
concentration is zero.
Example Nine
[0112] The composition of any of above examples that include or do
not include other stem cells that can be derived from whole blood,
bone marrow, adipose tissue or other sources.
Example Ten
[0113] The composition of any of the above examples with various
concentrations of other white blood cells including monocytes in a
concentration of 0, 100-500, 500-1000, 1000-5000, 5000-10, 000,
10,0000-50,000 per microliter or higher.
Example Eleven
[0114] The composition of any of the above examples with various
concentrations of other white blood cells including lymphocytes in
a concentration of 0 100-500, 500-1000, 1000-5000, 5000-10, 000,
10,0000-50,000 per microliter or higher.
Example Twelve
[0115] The composition of any of the above examples with various
concentrations of other white blood cells including neutrophils in
a concentration of 0 100-500, 500-1000, 1000-5000, 5000-10, 000,
10,0000-50,000 per microliter or higher.
Example Thirteen
[0116] The composition of any of the above examples with various
concentrations of other white blood cells including eosinophils in
a concentration of 0 100-500, 500-1000, 1000-5000, 5000-10, 000,
10,0000-50,000 per microliter or higher. Example Fourteen
[0117] The composition of any of the above examples with various
concentrations of other white blood cells including basophils in a
concentration of 0 100-500, 500-1000, 1000-5000, 5000-10, 000,
10,0000-50,000 per microliter or higher.
Example Fifteen
[0118] A composition that contains platelets in a concentration of
151,000/microliter to 7,000,000/microliter or higher that is also
shown to contain stromal-derived factor -1 (SDF-1) in a
concentration of 500 pg/ml to 5000 pg/ml, or 100-500 pg/ml or
greater than 5000 pg/ml.
Methods of Using Platelet Formulations
[0119] The platelet formulations disclosed herein may be useful in
treating damaged connective tissue, cardiac tissue, and/or lung
tissue in the same manner as described above for PRP. Platelet
formulations may also be useful in treating tissues with
compromised blood flow, such as ischemic tissue in the legs, arms,
brain or other organs. Specifically, acute or chronic limb ischemia
may be treated with platelet formulations. In some embodiments, the
platelet formulations described herein may repair tissue damage by
slowing or halting apoptosis, and that the anti-apoptotic effects
of the platelet formulations may be measured based on a decrease in
caspases in the blood, such as caspase-3. In some embodiments, the
platelet formulations may be applied in conjunction with
reperfusion therapy.
[0120] In some embodiments, the platelet formulations disclosed
herein may be useful in treating ischemia, cancer, a disease of the
immune system, a connective tissue injury, a skin disease, or a
disease of the nervous system. The platelet formulations may be
useful for the treatment of acute or chronic skin conditions such
as burns or wrinkles. The ischemia may be a brain ischemia or
cardiac ischemia. The cancer may be brain cancer, thyroid cancer,
pancreatic cancer, liver cancer, breast cancer, or prostate cancer.
Other types of cancer or neoplasia may also be treated with the
described platelet formulations. The connective tissue injury may
be a tendinosis, such as tennis elbow, rotator cuff injury, a knee
injury, a spinal injury or plantar fasciitis. The nervous system
disease may be Parkinsons' disease or other neurodegenerative
disorders such as Alzheimers or Multiple Sclerosis.
Method Example 1
Use of PRP to Obtain and/or Differentiate Pluripotent Cell
Lines
[0121] A skin biopsy (or any other appropriate tissue) is taken
from an individual and then his or her fibroblasts (or other cells)
are cultured. PRP is then prepared from the whole blood of that
individual or another individual and specific bioactive molecules
such as vascular endothelial growth factor or a bone morphogenetic
protein (or other appropriate cytokines, chemokines or growth
factors are isolated). These molecules are then administered via a
variety of methods to the fibroblasts and nuclear reprogramming to
a pluripotent cell line is achieved.
[0122] Alternatively, an established pluripotent or multipotent
cell line is treated with PRP with or without specific
fractionation to induce differentiation of that cell line.
Additional exogenous factors or methods may be added as needed to
accelerate or complete the differentiation of that cell line.
Different preparations or compositions of PRP may also be used to
accomplish the task. In some embodiments, the preparations and
compositions of PRP can be similar to but not limited to those as
described herein. PRP may further be used alone or in combination
with bone marrow concentrate, or fractions of adipose tissue. In
some embodiments, some fractions of adipose tissue can include
adipose tissue that is a source of mesenchymal stem cells and
endothelial progenitor cell or other adipose derived stem cells as
well as other cell components including preadipocytes, T cells, B
cells, mast cells, adipose tissue macrophages, and other cells
known in the art to be in fractions of adipose tissue. In some
embodiments, the cell line or stem cell line can be treated with a
composition containing platelets at a concentration of
151,000/microliter to 7,000,000/microliter or higher in combination
with bone marrow concentrate and/or any fraction of adipose
tissue.
[0123] In some embodiments, the composition can include progenitor
or precursor cells combined with the PRP composition to aid in
acceleration or completion of the differentiation of a cell line.
Progenitor or precursor cells in tissue are partially
differentiated, usually unipotent cells, which give rise to the
differentiated cells. The progenitor or precursor cells may have
lost most or all of the stem cell multipotency. Progenitor or
precursor cells can be combined with the PRP composition to
accelerate or complete the differentiation of a cell line.
Therefore, in some embodiments, the cell line or stem cell line
culture can include a composition containing platelets
concentration of 151,000/microliter to 7,000,000 or higher in
combination progenitor or precursor stem cell lines of any type
including mesenchymal stem precursor lines.
[0124] In some embodiments, the cell line or stem cell line culture
can include a composition containing platelets or PRP in
combination with embryonic stem cells, induced pluripotent stem
cells, adult stem cells (such as mesenchymal stem cells), and or
progenitor cell lines of any type. The composition containing
platelets can have a concentration of 151,000/microliter to
7,000,000 or higher.
[0125] The cells lines would be carefully monitored for
differentiation by surface markers or other measures of cell
specificity. PRP could further be used to accelerate proliferation
of the cell line. One specific example would be to create
functional human platelets from induced pluripotent stem cells
alone or then use them to create platelet-rich plasma of any
composition. The iPSC would first be differentiated into
megakaryocytes via a variety of vectors. Concentrated platelets
would then be harvested and suspended in plasma, saline or other
biologic media. In some embodiments, the PRP produced with this
method can be a composition of platelet rich plasma derived from
induced pluripotent stem cells at a platelet concentration of
151,000 to 7,000,000 or higher and pluripotent stem cells in any
concentration or form.
[0126] Further, in some embodiments, the compositions and
components for created and utilizing the compositions as disclosed
herein can be assembled into kits to be used by laboratory
technician, doctor, medical professional, or others assembling such
compositions for use.
Method Example 2
Cell Differentiation using Platelet-rich Plasma
[0127] Glioblastoma cells (012705), a cell type with severe
dedifferentiation and an anaplastic phenotype were cultured with
and without 10% PRP for 10 days. At 10 days, dramatic differences
were noted in the phenotypic expression of the cell lines.
[0128] Without PRP, the cells continued to be of an anaplastic
phenotype. With PRP, the cells differentiated into a mature
phenotype resembling an astrocyte. See Figure.
[0129] Further, in some embodiments, the compositions and
components for created and utilizing the compositions as disclosed
herein can be assembled into kits to be used by laboratory
technician, doctor, medical professional, or others assembling such
compositions for use.
Drug Discovery
[0130] Platelet-rich plasma (PRP) could be used to identify or
validate a new drug. Specific targets such as proteins, genes, RNA,
microRNA, or epigenetic modifications or markers would be tested
using whole or fractionated PRP of any type or formulation. Assays
would be developed using PRP to look for disease associations,
genetic expression, phenotypic or genotypic alternations in-vitro
or in-vivo markers. Specific fractions of PRP could also be
developed by blocking specific growth factors to create more
bioactive PRP. Importantly, novel factors within PRP could be
identified, purified and either used therapeutically or created via
genetic engineering techniques to produce a drug. Drugs could
further be tested in cell cultures that use PRP as part or all of
the growth media. Toxicity or potency of a drug could further be
measured its influence on PRP. Specifically, PRP could be used in
target identification and validation of drugs.
PRP Potency
[0131] PRP potency could be measured visually by observing over
time the chemotaxis of stem cells or other bioactive molecules. PRP
potency could also be simply measured by visually quantifying
platelet interaction with other cellular components or via the
activity of platelets to a known agonist such as calcium, collagen
or thrombin. This could be done via single observations under a
microscope or via time lapse photography to measure cellular
movement or chemotaxis. Time or completeness of platelet activation
could be another endpoint. For example, in some embodiments, the
potency of platelet rich plasma can be visually observed over time
by any method known in the art or described herein including but
not limited to cellular movement, microfluidics or chemotaxis. In
some embodiments, the measurement of PRP potency can be observed
via visual means such as a microscope or its activity in relation
to other cells
* * * * *