U.S. patent application number 17/373330 was filed with the patent office on 2022-04-07 for compositions and methods for treating the vertebral column.
The applicant listed for this patent is BioMimetic Therapeutics, LLC. Invention is credited to Charles Hart, Samuel E. Lynch, Dan Perrien, Conan Young.
Application Number | 20220105246 17/373330 |
Document ID | / |
Family ID | 1000006038558 |
Filed Date | 2022-04-07 |
United States Patent
Application |
20220105246 |
Kind Code |
A1 |
Hart; Charles ; et
al. |
April 7, 2022 |
COMPOSITIONS AND METHODS FOR TREATING THE VERTEBRAL COLUMN
Abstract
The present invention relates to compositions and methods useful
for treating structures of the vertebral column, including
vertebral bodies. In one embodiment, a method for promoting bone
formation in a vertebral body comprising providing a composition
comprising a PDGF solution and a biocompatible matrix and applying
the composition to at least one vertebral body. Promoting bone
formation in a vertebral body, according to some embodiments, can
increase bone volume, mass, and/or density leading to an increase
in mechanical strength of the vertebral body treated with a
composition of the present invention.
Inventors: |
Hart; Charles; (Brentwood,
TN) ; Lynch; Samuel E.; (Franklin, TN) ;
Young; Conan; (Franklin, TN) ; Perrien; Dan;
(Murfreesboro, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioMimetic Therapeutics, LLC |
Franklin |
TN |
US |
|
|
Family ID: |
1000006038558 |
Appl. No.: |
17/373330 |
Filed: |
July 12, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14853901 |
Sep 14, 2015 |
11058801 |
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17373330 |
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12631731 |
Dec 4, 2009 |
9161967 |
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14853901 |
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PCT/US2008/065666 |
Jun 3, 2008 |
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12631731 |
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PCT/US2007/003582 |
Feb 9, 2007 |
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12631731 |
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11704685 |
Feb 9, 2007 |
7799754 |
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12631731 |
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61026835 |
Feb 7, 2008 |
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60933202 |
Jun 4, 2007 |
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60817988 |
Jun 30, 2006 |
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60859809 |
Nov 17, 2006 |
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60817988 |
Jun 30, 2006 |
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60859809 |
Nov 17, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/0452 20130101;
A61L 27/48 20130101; A61K 47/02 20130101; A61K 9/14 20130101; A61K
47/42 20130101; A61L 2430/38 20130101; A61L 27/227 20130101; A61L
27/425 20130101; A61L 2300/414 20130101; A61L 2300/252 20130101;
A61K 9/0024 20130101; A61L 2300/202 20130101; A61K 49/0433
20130101; A61L 27/54 20130101; A61L 2300/44 20130101; A61L 2400/06
20130101; A61L 2300/45 20130101; A61L 27/40 20130101; A61K 38/1858
20130101 |
International
Class: |
A61L 27/48 20060101
A61L027/48; A61K 9/00 20060101 A61K009/00; A61K 49/04 20060101
A61K049/04; A61L 27/54 20060101 A61L027/54; A61K 47/02 20060101
A61K047/02; A61K 47/42 20060101 A61K047/42; A61L 27/40 20060101
A61L027/40; A61K 9/14 20060101 A61K009/14; A61K 38/18 20060101
A61K038/18; A61L 27/22 20060101 A61L027/22; A61L 27/42 20060101
A61L027/42 |
Claims
1-14. (canceled)
15. A method for increasing bone density in at least one vertebral
body of a patient comprising: applying to the vertebral body a
composition comprising: a) a biocompatible matrix having
incorporated therein a solution of platelet derived growth factor
(PDGF) at a concentration in a range of about 0.1 mg/ml to about
1.0 mg/ml in a buffer, wherein the biocompatible matrix comprises
(i) particles of a porous calcium phosphate in a range of about 100
.mu.m to about 3 mm in size or (ii) (a) particles of a porous
calcium phosphate in a range of about 100 .mu.m to about 3 mm in
size and (b) collagen, wherein the calcium phosphate comprises
interconnected pores and a porosity greater than 50%, and b) b) a
contrast agent.
16. The method of claim 15, wherein the biocompatible matrix
comprises the particles of porous calcium phosphate and the
collagen.
17. The method of claim 16, wherein the collagen comprises Type I
collagen.
18. The method of claim 16, wherein the weight ratio of calcium
phosphate:collagen is about 80:20.
19. The method of claim 15, wherein the PDGF is at a concentration
of about 0.3 mg/ml.
20. The method of claim 15, wherein the PDGF comprises PDGF-BB.
21. The method of claim 20, wherein the PDGF-BB comprises
recombinant human PDGF-BB (rhPDGF-BB) or a fragment thereof.
22. The method of claim 21, wherein the rhPDGF-BB comprises at
least 65% of intact rhPDGF-BB.
23. The method of claim 21, wherein the fragment of rhPDGF-BB is
selected from the group consisting of amino acid sequences 1-31,
1-32, 33-108, 33-109 and 1-108 of the entire B chain.
24. The method of claim 15, wherein the calcium phosphate comprises
particles in a range of about 100 .mu.m to about 300 .mu.m in
size.
25. The method of claim 15, wherein the calcium phosphate comprises
particles in a range of about 1000 .mu.m to about 2000 .mu.m in
size.
26. The method of claim 15, wherein the calcium phosphate comprises
particles in a range of about 250 .mu.m to about 1000 .mu.m in
size.
27. The method of claim 15, where the calcium phosphate comprises
.beta.-tricalcium phosphate.
28. The method of claim 16, wherein the composition is
flowable.
29. The method of claim 28, wherein applying the composition to the
vertebral body comprises injecting the composition into the
vertebral body.
30. The method of claim 15, wherein the method increases bone
volume and/or mass in the vertebral body.
31. The method of claim 15, wherein the contrast agent is chosen
from a cationic contrast agent, an anionic contrast agent, a
nonionic contrast agents, or a mixture thereof.
32. The method of claim 15, wherein the contrast agent is a
radiopaque contrast agent.
33. The method of claim 32, wherein the radiopaque contrast agent
is
(S)--N,N'-bis[2-hydroxy-1-(hydroxymethyl)-ethyl]-2,4,6-triiodo-5-lactamid-
oisophthalamide or a derivative thereof.
34. The method of claim 15, further comprising locally or
systemically administering to the patient a vitamin or an
osteoclast inhibitor.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Application No. PCT/US2008/065666, filed on Jun. 3, 2008, which
claims priority of U.S. Provisional Patent Application Ser. Nos.
60/933,202, filed Jun. 4, 2007, and 61/026,835 filed Feb. 7, 2008,
all of which are incorporated herein by reference in their
entirety. This application is also a continuation-in-part of
International Application No. PCT/US2007/003582 and U.S. patent
application Ser. No. 11/704,685, both of which were filed on Feb.
9, 2007, and both of which claim priority of U.S. Provisional
Patent Application Ser. Nos. 60/817,988, filed Jun. 30, 2006, and
60/859,809, filed Nov. 17, 2006, all of which are incorporated
herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods
useful for treating structures of the vertebral column, including
vertebral bodies.
BACKGROUND OF THE INVENTION
[0003] Musculoskeletal problems are pervasive throughout the
population in all age groups and in both sexes. Half of Americans
will need services for fractures at some point in their lifetimes
according to a widely published article presented at the 2003
annual meeting of the American Academy of Orthopedic Surgeons
(AAOS). More than $10 billion per year is spent in the U.S. on
hospital care associated with fracture treatment according to this
report.
[0004] Vertebral compression fractures (VCFs) are the most common
osteoporotic fractures, occurring in about 20% of post-menopausal
women (Eastell et al., J Bone Miner Res 1991; 6:207-215). It is
estimated that 700,000 VCFs occur annually, and only 250,000 of
these are diagnosed and treated. Because these fractures are left
untreated, osteoporosis may remain untreated and progress rapidly.
Post-menopausal women have a 5-fold increased risk of sustaining
another vertebral fracture within the coming year and 2-fold
increased risk of other fragility fractures, including hip
fractures (Klotzbuecher et al, J Bone Miner Res, 2000;
15:721-739).
[0005] VCFs occur when there is a break in one or both of the
vertebral body end plates, usually due to trauma, causing failure
of the anterior column and weakening the vertebrae from supporting
the body during activities of daily living. Vertebral compression
fractures caused by osteoporosis can cause debilitating back pain,
spinal deformity, and height loss. Both symptomatic and
asymptomatic vertebral fractures are associated with increased
morbidity and mortality. With the number of aged people at risk for
osteoporosis is expected to increase dramatically in the coming
decades, accurate identification of VCFs and treatment intervention
is necessary to reduce the enormous potential impact of this
disease on patients and health care systems.
[0006] Traditionally, VCFs caused by osteoporosis have been treated
with bed rest, narcotic analgesics, braces, and physical therapy.
Bed rest, however, leads to accelerated bone loss and physical
deconditioning, further aggravating the patient as well as
contributing to the problem of osteoporosis. Moreover, the use of
narcotics can worsen the mood and mentation problem that may
already be prevalent in the elderly. Additionally, brace wear is
not well-tolerated by the elderly. Although the current treatments
of osteoporosis such as hormone replacement, bisphosphonates,
calcitonin, and parathyroid hormone (PTH) analogs deal with
long-term issues, except for calcitonin, they provide no immediate
benefit in terms of pain control once a fracture occurs
(Kapuscinski et al., Master Med. Pol. 1996; 28:83-86).
[0007] Recently, minimally invasive treatments for vertebral body
compression fractures, vertebroplasty and kyphoplasty, have been
developed to address the issues of pain and fracture stabilization.
Vertebroplasty is the filling of a fractured vertebral body with
the goals of stabilizing the bone, preventing further collapse, and
eliminating acute fracture pain. Vertebroplasty, however, does not
attempt to restore vertebral height and/or sagittal alignment. In
addition, because there is no void in the bone, vertebral filling
is performed under less control with less viscous cement and, as a
consequence, filler leaks are common.
[0008] Kyphoplasty is a minimally invasive surgical procedure with
the goal of safety, improving vertebral height and stabilizing VCF.
Guided by x-ray images, an inflatable bone tamp is inflated in the
fractured vertebral body. This compacts the inner cancellous bone
as it pushes the fractured cortices back toward their normal
position. Fixation can then be done by filling the void with a
biomaterial under volume control with a more viscous cement.
Although kyphoplasty is considered a safe and effective treatment
of vertebral compression fractures, biomechanical studies
demonstrate that cement augmentation places additional stress on
adjacent levels. In fact, this increased stiffness can decrease the
ultimate load to failure of adjacent vertebrae by 8 to 30% and
provoke subsequent fractures (Berlemann et al., J Bone Joint
Surgery BR, 2002; 84:748-52). Compression fracture of one or more
vertebral bodies subsequent to vertebroplasty or kyphoplasty is
referred to herein as a "secondary vertebral compression
fracture."
[0009] In a recent clinical study, a higher rate of secondary
vertebral compression fracture was observed after kyphoplasty
compared with historical data for untreated fractures. Most of
these occurred at an adjacent level within 2 months of the index
procedure. After this two-month period, there were only occasional
secondary vertebral compression fractures which occurred at remote
levels. This study confirmed biomechanical studies showing that
cement augmentation places additional stress on adjacent level.
(Fribourg et al., Incidence of subsequent vertebral fracture after
kyphoplasty, Spine, 2004; 20; 2270-76).
[0010] Given the increased incidence of the use of minimally
invasive surgical techniques for the treatment of vertebral
compression fractures, and the predisposition of adjacent vertebrae
to undergo secondary compression fracture, an unmet clinical need
exists to prophylactically treat and prevent secondary VCFs.
SUMMARY OF THE INVENTION
[0011] The present invention provides compositions and methods
useful for treating structures of the vertebral column, including
vertebral bodies. In some embodiments of the present invention,
compositions are provided for promoting bone formation in a
vertebral body. In other embodiments, compositions and methods are
provided for preventing or decreasing the likelihood of vertebral
compression fractures. In another embodiment, methods and
compositions are provided for preventing or decreasing the
likelihood of secondary vertebral compression fractures associated
with vertebroplasty and/or kyphoplasty. The present compositions
and methods can be useful in treating vertebral bodies of
compromised patients, such as those with osteoporosis, diabetes, or
other diseases or conditions.
[0012] In one aspect, a composition for promoting bone formation in
a vertebral body comprises a solution comprising platelet derived
growth factor (PDGF) and biocompatible matrix, wherein the solution
is disposed or incorporated in the biocompatible matrix. In some
embodiments, the PDGF is absorbed by the biocompatible matrix. In
other embodiments, the PDGF is adsorbed onto one or more surfaces
of the biocompatible matrix. In a further embodiment, the PDGF is
absorbed by the biocompatible matrix and adsorbed onto one or more
surfaces of the biocompatible matrix.
[0013] In some embodiments, PDGF is present in the solution in a
concentration ranging from about 0.01 mg/ml to about 10 mg/ml, from
about 0.05 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about
1.0 mg/ml, or from about 0.2 mg/ml to about 0.4 mg/mi. The
concentration of PDGF within the solution may be within any of the
concentration ranges stated above.
[0014] In some embodiments of the present invention, PDGF comprises
PDGF homodimers and heterodimers, including PDGF-AA, PDGF-BB,
PDGF-AB, PDGF-CC, PDGF-DD, and mixtures and derivatives thereof. In
one embodiment, PDGF comprises PDGF-BB. In another embodiment PDGF
comprises a recombinant human (rh) PDGF such as recombinant human
PDGF-BB (rhPDGF-BB).
[0015] In some embodiments of the present invention, PDGF comprises
PDGF fragments. In one embodiment rhPDGF-B comprises the following
fragments: amino acid sequences 1-31, 1-32, 33-108, 33-109, and/or
1-108 of the entire B chain. The complete amino acid sequence
(1-109) of the B chain of PDGF is provided in FIG. 15 of U.S. Pat.
No. 5,516,896. It is to be understood that the rhPDGF compositions
of the present invention may comprise a combination of intact
rhPDGF-B (1-109) and fragments thereof. Other fragments of PDGF may
be employed such as those disclosed in U.S. Pat. No. 5,516,896. In
some embodiments, rhPDGF-BB comprises at least 65% of intact
rhPDGF-B (1-109).
[0016] A biocompatible matrix, according to some embodiments of the
present invention, comprises a bone substituting agent (also called
a scaffolding material herein) and optionally a biocompatible
binder. Bone substituting agents, in some embodiments, comprise
calcium phosphate including amorphous calcium phosphate,
monocalcium phosphate monohydrate (MCPM), monocalcium phosphate
anhydrous (MCPA), dicalcium phosphate dihydrate (DCPD), dicalcium
phosphate anhydrous (DCPA), octacalcium phosphate (OCP),
.alpha.-tricalcium phosphate, .beta.-TCP, hydroxyapatite (OHAp),
poorly crystalline hydroxapatite, tetracalcium phosphate (TTCP),
heptacalcium decaphosphate, calcium metaphosphate, calcium
pyrophosphate dihydrate, calcium pyrophosphate, carbonated calcium
phosphate, hydroxyapatite, or derivatives or mixtures thereof. In
some embodiments, bone substituting agents comprise calcium sulfate
or demineralized bone such as dried cortical or cancellous
bone.
[0017] In another aspect, the present invention provides a
composition for promoting bone formation in a vertebral body
comprising a PDGF solution disposed in a biocompatible matrix,
wherein the biocompatible matrix comprises a bone scaffolding
material and a biocompatible binder. The PDGF solution may have a
concentration of PDGF as described above. A bone scaffolding
material, in some embodiments, comprises calcium phosphate. In an
embodiment, calcium phosphate comprises .beta.-TCP. In one aspect,
biocompatible matrices may include calcium phosphate particles with
or without biocompatible binders or bone allograft such as
demineralized freeze dried bone allograft (DFDBA), mineralized
freeze dried bone allograft (FDBA), or particulate demineralized
bone matrix (DBM). In another aspect, biocompatible matrices may
include bone allograft such as DFDBA, DBM, or other bone allograft
materials including cortical bone shapes, such as blocks, wedges,
cylinders, or particles, or cancellous bone particles of various
shapes and sizes.
[0018] Moreover, a biocompatible binder, according to some
embodiments of the present invention, comprises proteins,
polysaccharides, nucleic acids, carbohydrates, synthetic polymers,
or mixtures thereof. In one embodiment, a biocompatible binder
comprises collagen. In another embodiment, a biocompatible binder
comprises hyaluronic acid.
[0019] In another aspect the present invention provides a
composition for preventing or decreasing the likelihood of
vertebral compression fractures, including secondary vertebral
compression fractures. In some embodiments, a composition for
preventing or decreasing the likelihood of vertebral compression
fractures comprises a solution comprising PDGF and a biocompatible
matrix wherein the solution is disposed in the biocompatible
matrix. In other embodiments, a composition for preventing or
decreasing the likelihood of vertebral compression fractures
comprises a PDGF solution disposed in a biocompatible matrix,
wherein the biocompatible matrix comprises a bone scaffolding
material and a biocompatible binder. In embodiments of a
composition for preventing or decreasing the likelihood of
vertebral compression fractures, a PDGF solution may have a
concentration of PDGF as described above. Moreover, a bone
scaffolding material, in some embodiments, comprises calcium
phosphate. In an embodiment, calcium phosphate comprises
.beta.-tricalcium phosphate. A biocompatible binder, according to
some embodiments of the present invention, comprises proteins,
polysaccharides, nucleic acids, carbohydrates, synthetic polymers,
or mixtures thereof. In one embodiment, a biocompatible binder
comprises collagen. In another embodiment, a biocompatible binder
comprises collagen, such as bovine collagen.
[0020] In some embodiments of the present invention, compositions
for promoting bone formation in vertebral bodies and compositions
for preventing or reducing the likelihood of vertebral compression
fractures further comprise at least one contrast agent. Contrast
agents, according to embodiments of the present invention, are
substances operable to at least partially provide differentiation
of two or more bodily tissues when imaged. Contrast agents,
according to some embodiments, comprise cationic contrast agents,
anionic contrast agents, nonionic contrast agents, or mixtures
thereof. In some embodiments, contrast agents comprise radiopaque
contrast agents. Radiopaque contrast agents, in some embodiments,
comprise iodo-compounds including
(S)--N,N'-bis[2-hydroxy-1-(hydroxymethyl)-ethyl]-2,4,6-triiodo-5-lactamid-
oisophthalamide (Iopamidol) and derivatives thereof.
[0021] In another aspect, the present invention provides a kit
comprising a biocompatible matrix in a first package and a solution
comprising PDGF in a second package. In some embodiments, the
biocompatible matrix comprises a scaffolding material, a
scaffolding material and a biocompatible binder, and/or bone
allograft such as DFDBA or particulate DBM. In one embodiment, the
scaffolding material comprises a calcium phosphate, such as
.beta.-TCP. Moreover, in some embodiments, the solution comprises a
predetermined concentration of PDGF. The concentration of the PDGF
can be predetermined according to the surgical procedure being
performed, such as promoting or accelerating bone growth in a
vertebral body or preventing or decreasing the likelihood of
secondary vertebral compression fractures. Moreover, in some
embodiments, the biocompatible matrix can be present in the kit in
a predetermined amount. The amount of biocompatible matrix provided
by a kit can be dependent on the surgical procedure being
performed. In some embodiments, the second package containing the
PDGF solution comprises a syringe. A syringe can facilitate
disposition of the PDGF solution in the biocompatible matrix. Once
the PDGF solution has been disposed in the biocompatible matrix, in
some embodiments, the resulting composition can placed in a second
syringe and/or cannula and delivered to a vertebral body.
[0022] The present invention also provides methods of producing
compositions for promoting bone formation in vertebral bodies and
preventing or decreasing the likelihood of compression fractures of
vertebral bodies, including secondary vertebral compression
fractures. In one embodiment, a method for producing such
compositions comprises providing a solution comprising PDGF,
providing a biocompatible matrix, and disposing the solution in the
biocompatible matrix. In some embodiments, a method of producing
compositions for promoting bone formation in a vertebral body and
preventing or decreasing the likelihood of compression fracture in
a vertebral body further comprises providing a contrast agent and
disposing the contrast agent in the biocompatible matrix.
[0023] In another aspect, the present invention provides methods
for promoting or accelerating bone formation in a vertebral body
comprising providing a composition comprising a PDGF solution
disposed in a biocompatible matrix and applying an effective amount
of the composition to at least one vertebral body. Applying the
composition to at least one vertebral body, in some embodiments,
comprises injecting the composition into the at least one vertebral
body.
[0024] In another aspect, the present invention provides methods
comprising preventing or decreasing the likelihood of vertebral
compression fractures, including secondary vertebral compression
fractures. Preventing or decreasing the likelihood of vertebral
compression fractures, according to embodiments of the present
invention comprises providing a composition comprising a PDGF
solution disposed in a biocompatible matrix and applying an
effective amount of the composition to at least one vertebral body.
In some embodiments, applying the composition to at least one
vertebral body comprises injecting the composition into the at
least one vertebral body. In one embodiment, the composition is
applied to a second vertebral body, in some instances an adjacent
vertebral body, subsequent to a vertebroplasty or kyphoplasty of a
first vertebral body. In some embodiments, a composition comprising
a PDGF solution disposed in a biocompatible matrix is applied to at
least one high risk vertebral body. "High risk vertebral bodies"
(HVB), as used herein, refer to vertebral bodies of vertebrae T5
through T12 as well as L1 through L4, which are at the greatest
risk of undergoing secondary vertebral compression fracture.
[0025] In some embodiments of methods of the present invention, the
biocompatible matrix comprises a bone scaffolding material. In some
embodiments, the biocompatible matrix comprises a bone scaffolding
material and a biocompatible binder.
[0026] In some embodiments, methods for promoting bone formation in
vertebral bodies and preventing or decreasing the likelihood of
compression fractures of vertebral bodies further comprise
providing at least one pharmaceutical composition in addition to
the composition comprising a PDGF solution disposed in a
biocompatible matrix and administering the at least one
pharmaceutical composition locally and/or systemically. The at
least one pharmaceutical composition, in some embodiments,
comprises vitamins, calcium supplements, or any osteoclast
inhibitor known to one of skill in the art, including
bisphosphonates. In some embodiments, the at least one
pharmaceutical composition is administered locally. In such
embodiments, the at least one pharmaceutical composition can be
incorporated into the biocompatible matrix or otherwise disposed in
and around a vertebral body. In other embodiments, the at least one
pharmaceutical composition is administered systemically to a
patient. In one embodiment, for example, the at least one
pharmaceutical composition is administered orally to a patient. In
another embodiment, the at least one pharmaceutical composition is
administered intravenously to a patient.
[0027] Accordingly, it is an object of the present invention to
provide a composition comprising PDGF useful in promoting bone
formation in vertebral bodies.
[0028] It is another object of the present invention to provide a
composition comprising PDGF useful in strengthening vertebral
bodies.
[0029] It is another object of the present invention to provide a
composition comprising PDGF useful in strengthening vertebral
bodies of patients with osteoporosis.
[0030] It is another object of the present invention to provide a
composition comprising PDGF useful in preventing or decreasing the
likelihood of vertebral compression fractures, including secondary
vertebral compression fractures.
[0031] Another object of the present invention is to provide
methods for promoting bone formation in vertebral bodies using
compositions comprising PDGF.
[0032] A further object of the present invention is to provide
methods of preventing or decreasing the likelihood of vertebral
compression fractures, including secondary vertebral compression
fractures, using compositions comprising PDGF.
[0033] These and other embodiments of the present invention are
described in greater detail in the detailed description which
follows. These and other objects, features and advantages of the
present invention will become apparent after a review of the
following detailed description of the disclosed embodiments and
claims.
BRIEF DESCRIPTION OF THE FIGURES
[0034] FIG. 1 illustrates a syringe and related apparatus
penetrating tissue overlaying a vertebral body to deliver a
composition of the present invention to the vertebral body
according to an embodiment of the present invention.
[0035] FIG. 2 is a radiograph illustrating injection of a
composition into a vertebral body according to an embodiment of the
present invention.
[0036] FIG. 3 illustrates vertebrae receiving compositions of the
present invention according to one embodiment of the present
invention.
[0037] FIG. 4 illustrates percent change in volumetric bone mineral
density for vertebral bodies receiving a composition comprising 1.0
mg/ml of rhPDGF-BB disposed in a .beta.-TCP/collagen matrix in
comparison with vertebral bodies receiving a composition comprising
20 mM sodium acetate buffer disposed in a .beta.-TCP/collagen
matrix according to one embodiment of the present invention.
[0038] FIG. 5 illustrates percent change in volumetric bone mineral
density for vertebral bodies receiving a composition comprising 1.0
mg/ml of rhPDGF-BB disposed in a .beta.-TCP/collagen matrix in
comparison with vertebral bodies receiving a composition comprising
20 mM sodium acetate buffer disposed in a .beta.-TCP/collagen
matrix according to one embodiment of the present invention.
DETAILED DESCRIPTION
[0039] The present invention provides compositions and methods
useful for treating structures of the vertebral column, including
vertebral bodies. According to embodiments described herein, the
present invention provides compositions for promoting bone
formation in a vertebral body and compositions for preventing or
decreasing the likelihood of vertebral compression fractures,
including secondary vertebral compression fractures. In one
embodiment, the compositions comprise a solution comprising PDGF
and a biocompatible matrix, wherein the solution is disposed in the
biocompatible matrix. In another embodiment, the compositions
comprise a PDGF solution disposed in a biocompatible matrix,
wherein the biocompatible matrix comprises a bone scaffolding
material and a biocompatible binder. In one aspect, biocompatible
matrices include calcium phosphate particles with or without
biocompatible binders or bone allograft such as DFDBA or
particulate DBM. In another aspect, biocompatible matrices may
include DFDBA or DBM.
[0040] Turning now to components that can be included in various
embodiments of the present invention, compositions of the present
invention comprise a solution comprising PDGF.
PDGF Solutions
[0041] PDGF plays an important role in regulating cell growth and
migration. PDGF, as with other growth factors, binds with the
extracellular domains of receptor tyrosine kinases. The binding of
PDGF to these transmembrane proteins activate the kinase activity
of their catalytic domains located on the cytosolic side of the
membrane. By phosphorylating tyrosine residues of target proteins,
the kinases induce a variety of cellular processes that include
cell growth and extracellular matrix production.
[0042] In one aspect, a composition provided by the present
invention comprises a solution comprising PDGF and a biocompatible
matrix, wherein the solution is disposed or incorporated in the
biocompatible matrix. In some embodiments, PDGF is present in the
solution in a concentration ranging from about 0.01 mg/ml to about
10 mg/ml, from about 0.05 mg/ml to about 5 mg/ml, or from about 0.1
mg/ml to about 1.0 mg/ml. PDGF may be present in the solution at
any concentration within these stated ranges including the upper
limit and lower limit of each range. In other embodiments, PDGF is
present in the solution at any one of the following concentrations:
about 0.05 mg/ml; about 0.1 mg/ml; about 0.15 mg/ml; about 0.2
mg/ml; about 0.25 mg/ml; about 0.3 mg/ml; about 0.35 mg/ml; about
0.4 mg/ml; about 0.45 mg/ml; about 0.5 mg/ml, about 0.55 mg/ml,
about 0.6 mg/ml, about 0.65 mg/ml, about 0.7 mg/ml; about 0.75
mg/ml; about 0.8 mg/ml; about 0.85 mg/ml; about 0.9 mg/ml; about
0.95 mg/ml; or about 1.0 mg/ml. In some embodiments, PDGF is
present in the solution in a concentration ranging from about 0.2
mg/ml to about 2 mg/ml, from about 0.3 mg/ml to about 3 mg/ml, from
about 0.4 mg/ml to about 4 mg/ml, or from about 0.5 mg/ml to about
5 mg/ml. It is to be understood that these concentrations are
simply examples of particular embodiments, and that the
concentration of PDGF may be within any of the concentration ranges
stated above including the upper limit and the lower limit of each
range.
[0043] Various amounts of PDGF may be used in the compositions of
the present invention. Amounts of PDGF that could be used include
amounts in the following ranges: about 1 .mu.g to about 50 mg,
about 10 .mu.g to about 25 mg, about 100 .mu.g to about 10 mg, and
about 250 .mu.g to about 5 mg.
[0044] The concentration of PDGF or other growth factors in
embodiments of the present invention can be determined by using an
enzyme-linked immunoassay as described in U.S. Pat. Nos. 6,221,625,
5,747,273, and 5,290,708, or any other assay known in the art for
determining PDGF concentration. When provided herein, the molar
concentration of PDGF is determined based on the molecular weight
(MW) of PDGF dimer (e.g., PDGF-BB; MW about 25 kDa).
[0045] In some embodiments of the present invention, PDGF comprises
PDGF homodimers and heterodimers, including PDGF-AA, PDGF-BB,
PDGF-AB, PDGF-CC, PDGF-DD, and mixtures and derivatives thereof. In
one embodiment, for example, PDGF comprises PDGF-BB. In another
embodiment PDGF comprises a recombinant human PDGF, such as
rhPDGF-BB. In some embodiments, PDGF comprises mixtures of the
various homodimers and/or heterodimers. Embodiments of the present
invention contemplate any combination of PDGF-AA, PDGF-BB, PDGF-AB,
PDGF-CC, and/or PDGF-DD.
[0046] PDGF, in some embodiments, can be obtained from natural
sources. In other embodiments, PDGF can be produced by recombinant
DNA techniques. In other embodiments, PDGF or fragments thereof may
be produced using peptide synthesis techniques known to one of
ordinary skill in the art, such as solid phase peptide synthesis.
When obtained from natural sources, PDGF can be derived from
biological fluids. Biological fluids, according to some
embodiments, can comprise any treated or untreated fluid associated
with living organisms including blood
[0047] Biological fluids, in another embodiment, can also comprise
blood components including platelet concentrate (PC), apheresed
platelets, platelet-rich plasma (PRP), plasma, serum, fresh frozen
plasma (FFP), and buffy coat (BC). Biological fluids, in a further
embodiment, can comprise platelets separated from plasma and
resuspended in a physiological fluid.
[0048] When produced by recombinant DNA techniques, a DNA sequence
encoding a single monomer (e.g., PDGF B-chain or A-chain), in some
embodiments, can be inserted into cultured prokaryotic or
eukaryotic cells for expression to subsequently produce the
homodimer (e.g. PDGF-BB or PDGF-AA). In other embodiments, a PDGF
heterodimer can be generated by inserting DNA sequences encoding
for both monomeric units of the heterodimer into cultured
prokaryotic or eukaryotic cells and allowing the translated
monomeric units to be processed by the cells to produce the
heterodimer (e.g. PDGF-AB). Commercially available GMP recombinant
PDGF-BB can be obtained commercially from Novartis Corporation
(Emeryville, Calif.). Research grade rhPDGF-BB can be obtained from
multiple sources including R&D Systems, Inc. (Minneapolis,
Minn.), BD Biosciences (San Jose, Calif.), and Chemicon,
International (Temecula, Calif.). In some embodiments, monomeric
units can be produced in prokaryotic cells in a denatured form,
wherein the denatured form is subsequently refolded into an active
molecule.
[0049] In embodiments of the present invention, PDGF comprises PDGF
fragments. In one embodiment rhPDGF-B comprises the following
fragments: amino acid sequences 1-31, 1-32, 33-108, 33-109, and/or
1-108 of the entire B chain. The complete amino acid sequence
(1-109) of the B chain of PDGF is provided in FIG. 15 of U.S. Pat.
No. 5,516,896. It is to be understood that the rhPDGF compositions
of the present invention may comprise a combination of intact
rhPDGF-B (1-109) and fragments thereof. Other fragments of PDGF may
be employed such as those disclosed in U.S. Pat. No. 5,516,896. In
accordance with one embodiment, the rhPDGF-BB comprises at least
60% of intact rhPDGF-B (1-109). In another embodiment, the
rhPDGF-BB comprises at least 65%, 75%, 80%, 85%, 90%, 95%, or 99%
of intact rhPDGF-B (1-109).
[0050] In some embodiments of the present invention, PDGF can be
purified. Purified PDGF, as used herein, comprises compositions
having greater than about 95% by weight PDGF prior to incorporation
in solutions of the present invention. The solution may be any
pharmaceutically acceptable solution. In other embodiments, the
PDGF can be substantially purified. Substantially purified PDGF, as
used herein, comprises compositions having about 5% to about 95% by
weight PDGF prior to incorporation into solutions of the present
invention. In one embodiment, substantially purified PDGF comprises
compositions having about 65% to about 95% by weight PDGF prior to
incorporation into solutions of the present invention. In other
embodiments, substantially purified PDGF comprises compositions
having about 70% to about 95%, about 75% to about 95%, about 80% to
about 95%, about 85% to about 95%, or about 90% to about 95%, by
weight PDGF, prior to incorporation into solutions of the present
invention. Purified PDGF and substantially purified PDGF may be
incorporated into scaffolds and binders.
[0051] In a further embodiment, PDGF can be partially purified.
Partially purified PDGF, as used herein, comprises compositions
having PDGF in the context of platelet rich plasma (PRP), fresh
frozen plasma (FFP), or any other blood product that requires
collection and separation to produce PDGF. Embodiments of the
present invention contemplate that any of the PDGF isoforms
provided herein, including homodimers and heterodimers, can be
purified or partially purified. Compositions of the present
invention containing PDGF mixtures may contain PDGF isoforms or
PDGF fragments in partially purified proportions. Partially
purified and purified PDGF, in some embodiments, can be prepared as
described in U.S. patent application Ser. No. 11/159,533
(Publication No: 20060084602).
[0052] In some embodiments, solutions comprising PDGF are formed by
solubilizing PDGF in one or more buffers. Buffers suitable for use
in PDGF solutions of the present invention can comprise, but are
not limited to, carbonates, phosphates (e.g. phosphate buffered
saline), histidine, acetates (e.g. sodium acetate), acidic buffers
such as acetic acid and HCl, and organic buffers such as lysine,
Tris buffers (e.g. tris(hydroxymethyl)aminoethane),
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and
3-(N-morpholino) propanesulfonic acid (MOPS). Buffers can be
selected based on biocompatibility with PDGF and the buffer's
ability to impede undesirable protein modification. Buffers can
additionally be selected based on compatibility with host tissues.
In one embodiment, sodium acetate buffer is used. The buffers may
be employed at different molarities, for example, about 0.1 mM to
about 100 mM, about 1 mM to about 50 mM, about 5 mM to about 40 mM,
about 10 mM to about 30 mM, or about 15 mM to about 25 mM, or any
molarity within these ranges. In some embodiments, an acetate
buffer is employed at a molarity of about 20 mM.
[0053] In another embodiment, solutions comprising PDGF are formed
by solubilizing lyophilized PDGF in water, wherein prior to
solubilization the PDGF is lyophilized from an appropriate
buffer.
[0054] Solutions comprising PDGF, according to embodiments of the
present invention, can have a pH ranging from about 3.0 to about
8.0. In one embodiment, a solution comprising PDGF has a pH ranging
from about 5.0 to about 8.0, from about 5.5 to about 7.0, or from
about 5.5 to about 6.5, or any value within these ranges. The pH of
solutions comprising PDGF, in some embodiments, can be compatible
with the prolonged stability and efficacy of PDGF or any other
desired biologically active agent. PDGF is more stable in an acidic
environment. Therefore, in accordance with one embodiment the
present invention comprises an acidic storage formulation of a PDGF
solution. In accordance with this embodiment, the PDGF solution
preferably has a pH from about 3.0 to about 7.0 or from about 4.0
to about 6.5. The biological activity of PDGF, however, can be
optimized in a solution having a neutral pH range. Therefore, in a
further embodiment, the present invention comprises a neutral pH
formulation of a PDGF solution. In accordance with this embodiment,
the PDGF solution preferably has a pH from about 5.0 to about 8.0,
from about 5.5 to about 7.0, or from about 5.5 to about 6.5. In
accordance with a method of the present invention, an acidic PDGF
solution is reformulated to a neutral pH composition, wherein such
composition is then used to treat bone and promote bone growth
and/or healing. In accordance with some embodiments of the present
invention, the PDGF utilized in the solutions is rh-PDGF-BB. In a
further embodiment, the pH of the PDGF containing solution may be
altered to optimize the binding kinetics of PDGF to a matrix
substrate or linker. If desired, the pH of the material
equilibrates to adjacent material, the bound PDGF may become
labile.
[0055] The pH of solutions comprising PDGF, in some embodiments,
can be controlled by the buffers recited herein. Various proteins
demonstrate different pH ranges in which they are stable. Protein
stabilities are primarily reflected by isoelectric points and
charges on the proteins. The pH range can affect the conformational
structure of a protein and the susceptibility of a protein to
proteolytic degradation, hydrolysis, oxidation, and other processes
that can result in modification to the structure and/or biological
activity of the protein.
[0056] In some embodiments, solutions comprising PDGF can further
comprise additional components, such as other biologically active
agents. In other embodiments, solutions comprising PDGF can further
comprise cell culture media, other stabilizing proteins such as
albumin, antibacterial agents, protease inhibitors [e.g.,
ethylenediaminetetraacetic acid (EDTA), ethylene
glycol-bis(beta-aminoethylether)-N, N,N',N'-tetraacetic acid
(EGTA), aprotinin, .epsilon.-aminocaproic acid (EACA), etc.] and/or
other growth factors such as fibroblast growth factors (FGFs),
epidermal growth factors (EGFs), transforming growth factors
(TGFs), keratinocyte growth factors (KGFs), insulin-like growth
factors (IGFs), hepatocyte growth factors (HGFs), bone
morphogenetic proteins (BMPs), or other PDGFs including
compositions of PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and/or
PDGF-DD.
[0057] In addition to solutions comprising PDGF, compositions of
the present invention also comprise a biocompatible matrix in which
to dispose the PDGF solutions and may also comprise a biocompatible
binder either with or without addition of a biocompatible
matrix.
Biocompatible Matrix
[0058] Bone Scaffolding Material
[0059] A biocompatible matrix, according to embodiments of the
present invention, comprises a bone scaffolding material. It is to
be understood that the terms bone scaffolding material and bone
substituting agent are used interchangeably in the present
application. The bone scaffolding material provides the framework
or scaffold for new bone and tissue growth to occur. In some
embodiments, a bone scaffolding material has multidirectional and
interconnected pores of varying diameters. In some embodiments, a
bone scaffolding material comprises a plurality of pockets and
non-interconnected pores in addition to the interconnected pores. A
bone scaffolding material, in some embodiments, is one that can
permanently or temporarily replace bone. Following implantation,
the bone scaffolding material can be retained by the body or it can
be resorbed by the body and replaced by bone.
[0060] A bone scaffolding material, in some embodiments, comprises
at least one calcium phosphate. In other embodiments, a bone
scaffolding material can comprise a plurality of calcium
phosphates. Calcium phosphates suitable for use as a bone
scaffolding material, in embodiments of the present invention, have
a calcium to phosphorus atomic ratio ranging from 0.5 to 2.0. In
some embodiments, a bone scaffolding material comprises an
allograft such as DFDBA, FDBA, or particulate DBM. In some
embodiments, a bone scaffolding material comprises mineralized bone
allograft, mineralized bone, mineralized deproteinized xenograft,
or demineralized bone.
[0061] Non-limiting examples of calcium phosphates suitable for use
as bone scaffolding materials comprise amorphous calcium phosphate,
monocalcium phosphate monohydrate (MCPM), monocalcium phosphate
anhydrous (MCPA), dicalcium phosphate dihydrate (DCPD), dicalcium
phosphate anhydrous (DCPA), octacalcium phosphate (OCP),
.alpha.-tricalcium phosphate, .beta.-TCP, hydroxyapatite (OHAp),
poorly crystalline hydroxapatite, tetracalcium phosphate (TTCP),
heptacalcium decaphosphate, calcium metaphosphate, calcium
pyrophosphate dihydrate, calcium pyrophosphate, carbonated calcium
phosphate, hydroxyapatite, or derivatives or mixtures thereof.
[0062] In some embodiments, a bone scaffolding material comprises a
polymeric material. A polymeric scaffold, in some embodiments,
comprises collagen, polylactic acid, poly(L-lactide),
poly(D,L-lactide), polyglycolic acid, poly(L-lactide-co-glycolide),
poly(L-lactide-co-D,L-lactide), polyacrylate, polymethacrylate,
polymethylmethacrylate, chitosan, or combinations or derivatives
thereof.
[0063] In some embodiments, a bone scaffolding material comprises
porous structure. Porosity is a desirable characteristic as it
facilitates cell migration and infiltration into the scaffolding
material so that the infiltrating cells can secrete extracellular
bone matrix. Porosity also provides access for vascularization.
Porosity also provides a high surface area for enhanced resorption
and release of active substances as well as increased cell-matrix
interaction. A bone scaffolding material, in some embodiments, can
be sized and shaped prior to use. In some embodiments. the bone
scaffolding material can be provided in a shape suitable for
implantation.
[0064] Porous bone scaffolding materials, according to some
embodiments, can comprise pores having diameters ranging from about
1 .mu.m to about 1 mm. In one embodiment, a bone scaffolding
material comprises macropores having diameters ranging from about
100 .mu.m to about 1 mm or greater. In another embodiment, a bone
scaffolding material comprises mesopores having diameters ranging
from about 10 .mu.m to about 100 .mu.m. In a further embodiment, a
bone scaffolding material comprises micropores having diameters
less than about 10 .mu.m. Embodiments of the present invention
contemplate bone scaffolding materials comprising macropores,
mesopores, and micropores or any combination thereof.
[0065] A porous bone scaffolding material, in one embodiment, has a
porosity greater than about 25% or greater than about 40%. In
another embodiment, a porous bone scaffolding material has a
porosity greater than about 50%, greater than about 60%, greater
than about 65%, greater than about 70%, greater than about 80%, or
greater than about 85%. In a further embodiment, a porous bone
scaffolding material has a porosity greater than about 90%. In some
embodiments, a porous bone scaffolding material comprises a
porosity that facilitates cell migration into the scaffolding
material.
[0066] In some embodiments, a bone scaffolding material comprises a
plurality of particles. A bone scaffolding material, for example,
can comprise a plurality of calcium phosphate particles. Particles
of a bone scaffolding material, in some embodiments, can
individually demonstrate any of the pore diameters and porosities
provided here for the bone scaffolding material. In other
embodiments, particles of a bone scaffolding material can form an
association to produce a matrix having any of the pore diameters or
porosities provided herein for the bone scaffolding material.
[0067] Bone scaffolding particles may be mm, .mu.m, or submicron
(nm) in size. Bone scaffolding particles, in one embodiment, have
an average diameter ranging from about 1 .mu.m to about 5 mm. In
other embodiments, particles have an average diameter ranging from
about 1 mm to about 2 mm, from about 1 mm to about 3 mm, or from
about 250 .mu.m to about 750 .mu.m. Bone scaffolding particles, in
another embodiment, have an average diameter ranging from about 100
.mu.m to about 300 .mu.m. In a further embodiment, bone scaffolding
particles have an average diameter ranging from about 75 .mu.m to
about 300 .mu.m. In additional embodiments, bone scaffolding
particles have an average diameter less than about 25 .mu.m, less
than about 1 .mu.m, or less than about 1 mm. In some embodiments,
scaffolding particles have an average diameter ranging from about
100 .mu.m to about 5 mm or from about 100 .mu.m to about 3 mm. In
other embodiments, bone scaffolding particles have an average
diameter ranging from about 250 .mu.m to about 2 mm, from about 250
.mu.m to about 1 mm, or from about 200 .mu.m to about 3 mm.
Particles may also be in the range of about 1 nm to about 1 .mu.m,
less than about 500 nm, or less than about 250 nm.
[0068] Bone scaffolding materials, according to some embodiments,
can be provided in a shape suitable for implantation (e.g., a
sphere, a cylinder, or a block). In other embodiments, bone
scaffolding materials are moldable, extrudable and/or injectable.
Moldable, extrudable, and injectable bone scaffolding materials can
facilitate efficient placement of compositions of the present
invention in and around vertebral bodies. In some embodiments, bone
scaffolding materials are flowable. Flowable bone scaffolding
materials, in some embodiments, can be applied vertebral bodies
through a syringe and needle or cannula. In some embodiments, bone
scaffolding materials harden in vivo.
[0069] In some embodiments, bone scaffolding materials are
bioresorbable. A bone scaffolding material, in one embodiment, can
be at least 30%, 40%, 50%, 60%, 70%, 75%, or 90% resorbed within
one year subsequent to in vivo implantation. In another embodiment,
a bone scaffolding material can be resorbed at least 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 75%, or 90% within 1, 3, 6, 9, 12, or 18
months of in vivo implantation. In some embodiments, a bone
scaffolding material is greater than 90% resorbed within 1, 3, 6,
9, 12, or 18 months of in vivo implantation. Bioresorbability will
be dependent on: (1) the nature of the matrix material (i.e., its
chemical make up, physical structure and size); (2) the location
within the body in which the matrix is placed; (3) the amount of
matrix material that is used; (4) the metabolic state of the
patient (diabetic/non-diabetic, osteoporotic, smoker, old age,
steroid use, etc.); (5) the extent and/or type of injury treated;
and (6) the use of other materials in addition to the matrix such
as other bone anabolic, catabolic and anti-catabolic factors.
Bone Scaffolding Comprising .beta.-Tricalcium Phosphate
(.beta.-TCP)
[0070] A bone scaffolding material for use as a biocompatible
matrix can comprise .beta.-TCP. .beta.-TCP, according to some
embodiments, can comprise a porous structure having
multidirectional and interconnected pores of varying diameters. In
some embodiments, .beta.-TCP comprises a plurality of pockets and
non-interconnected pores of various diameters in addition to the
interconnected pores. The porous structure of .beta.-TCP, in one
embodiment, comprises macropores having diameters ranging from
about 100 .mu.m to about 1 min or greater, mesopores having
diameters ranging from about 10 .mu.m to about 100 .mu.m, and
micropores having diameters less than about 10 .mu.m. Macropores
and micropores of the .beta.-TCP can facilitate osteoinduction and
osteoconduction while macropores, mesopores and micropores can
permit fluid communication and nutrient transport to support bone
regrowth throughout the .beta.-TCP biocompatible matrix.
[0071] In comprising a porous structure, .beta.-TCP, in some
embodiments, can have a porosity greater than 25% or greater than
about 40%. In other embodiments, .beta.-TCP can have a porosity
greater than 50%, greater than about 60%, greater than about 65%,
greater than about 70%, greater than about 75%, greater than about
80%, or greater than about 85%. In a further embodiment, .beta.-TCP
can have a porosity greater than 90%. In some embodiments,
.beta.-TCP can have a porosity that facilitates cell migration into
the .beta.-TCP.
[0072] In some embodiments, a .beta.-TCP bone scaffolding material
comprises .beta.-TCP particles. .beta.-TCP particles, in some
embodiments, can individually demonstrate any of the pore
diameters, pore structures, and porosities provided herein for
scaffolding materials.
[0073] .beta.-TCP particles, in one embodiment have an average
diameter ranging from about 1 .mu.m to about 5 mm. In other
embodiments, .beta.-TCP particles have an average diameter ranging
from about 1 mm to about 2 mm, from about 1 mm to about 3 mm, from
about 100 .mu.m to about 5 mm, from about 100 .mu.m to about 3 mm,
from about 250 .mu.m to about 2 mm, from about 250 .mu.m to about
750 .mu.m, from about 250 .mu.m to about 1 mm, from about 250 .mu.m
to about 2 mm, or from about 200 .mu.m to about 3 mm. In another
embodiment, .beta.-TCP particles have an average diameter ranging
from about 100 .mu.m to about 300 .mu.m. In some embodiments,
.beta.-TCP particles have an average diameter ranging from about 75
.mu.m to about 300 .mu.m. In some embodiments, .beta.-TCP particles
have an average diameter of less than about 25 .mu.m, less than
about 1 .mu.m, or less than about 1 mm. In some embodiments,
.beta.-TCP particles have an average diameter ranging from about 1
nm to about 1 .mu.m. In a further embodiment, .beta.-TCP particles
have an average diameter less than about 500 nm or less than about
250 nm.
[0074] A biocompatible matrix comprising a .beta.-TCP bone
scaffolding material, in some embodiments, can be provided in a
shape suitable for implantation (e.g., a sphere, a cylinder, or a
block). In other embodiments, a .beta.-TCP bone scaffolding
material can be moldable, extrudable, and/or flowable thereby
facilitating application of the matrix to vertebral bodies.
Flowable matrices may be applied through syringes, tubes, cannulas,
or spatulas.
[0075] A .beta.-TCP bone scaffolding material, according to some
embodiments, is bioresorbable. In one embodiment, a .beta.-TCP bone
scaffolding material can be at least 30%, 40%, 50%, 60%, 65%, 70%,
75%, 80%, or 85% resorbed one year subsequent to in vivo
implantation. In another embodiment, a .beta.-TCP bone scaffolding
material can be greater than 90% resorbed one year subsequent to in
viva implantation.
Bone Scaffolding Material and Biocompatible Binder
[0076] In another embodiment, a biocompatible matrix comprises a
bone scaffolding material and a biocompatible binder. Bone
scaffolding materials in embodiments of a biocompatible matrix
further comprising a biocompatible binder are consistent with those
provided hereinabove.
[0077] Biocompatible binders, according to some embodiments, can
comprise materials operable to promote cohesion between combined
substances. A biocompatible binder, for example, can promote
adhesion between particles of a bone scaffolding material in the
formation of a biocompatible matrix. In certain embodiments, the
same material may serve as both a scaffolding material. In some
embodiments, for example, polymeric materials described herein such
as collagen and chitosan may serve as both scaffolding material and
a binder.
[0078] Biocompatible binders, in some embodiments, can comprise
collagen, polysaccharides, nucleic acids, carbohydrates, proteins,
polypeptides, poly(.alpha.-hydroxy acids), poly(lactones),
poly(amino acids), poly(anhydrides), polyurethanes,
poly(orthoesters), poly(anhydride-co-imides),
poly(orthocarbonates), poly(.alpha.-hydroxy alkanoates),
poly(dioxanones), poly(phosphoesters), polylactic acid,
poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolide
(PGA), poly(lactide-co-glycolide (PLGA),
poly(L-lactide-co-D,L-lactide), poly(D,L-lactide-co-trimethylene
carbonate), polyglycolic acid, polyhydroxybutyrate (PHB),
poly(.epsilon.-caprolactone), poly(.delta.-valerolactone),
poly(.gamma.-butyrolactone), poly(caprolactone), polyacrylic acid,
polycarboxylic acid, poly(allylamine hydrochloride),
poly(diallyldimethylammonium chloride), poly(ethyleneimine),
polypropylene fumarate, polyvinyl alcohol, polyvinylpyrrolidone,
polyethylene, polymethylmethacrylate, carbon fibers, poly(ethylene
glycol), poly(ethylene oxide), poly(vinyl alcohol),
poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene
oxide)-co-poly(propylene oxide) block copolymers, poly(ethylene
terephthalate)polyamide, and copolymers and mixtures thereof.
[0079] Biocompatible binders, in other embodiments, can comprise
alginic acid, arabic gum, guar gum, xantham gum, gelatin, chitin,
chitosan, chitosan acetate, chitosan lactate, chondroitin sulfate,
N,O-carboxymethyl chitosan, a dextran (e.g., .alpha.-cyclodextrin,
.beta.-cyclodextrin, .gamma.-cyclodextrin, or sodium dextran
sulfate), fibrin glue, lecithin, phosphatidylcholine derivatives,
glycerol, hyaluronic acid, sodium hyaluronate, a cellulose (e.g.,
methylcellulose, carboxymethylcellulose, hydroxypropyl
methylcellulose, or hydroxyethyl cellulose), a glucosamine, a
proteoglycan, a starch (e.g., hydroxyethyl starch or starch
soluble), lactic acid, pluronic acids, sodium glycerophosphate,
glycogen, a keratin, silk, and derivatives and mixtures
thereof.
[0080] In some embodiments, a biocompatible binder is
water-soluble. A water-soluble binder can dissolve from the
biocompatible matrix shortly after its implantation, thereby
introducing macroporosity into the biocompatible matrix.
Macroporosity, as discussed herein, can increase the
osteoconductivity of the implant material by enhancing the access
and, consequently, the remodeling activity of the osteoclasts and
osteoblasts at the implant site.
[0081] In some embodiments, a biocompatible binder can be present
in a biocompatible matrix in an amount ranging from about 5 weight
percent to about 50 weight percent of the matrix. In other
embodiments, a biocompatible binder can be present in an amount
ranging from about 10 weight percent to about 40 weight percent of
the biocompatible matrix. In another embodiment, a biocompatible
binder can be present in an amount ranging from about 15 weight
percent to about 35 weight percent of the biocompatible matrix. In
a further embodiment, a biocompatible binder can be present in an
amount of about 20 weight percent of the biocompatible matrix. In
another embodiment, a biocompatible binder can be present in a
biocompatible matrix in an amount greater than about 50 weight
percent or 60 weight percent of the matrix. In one embodiment, a
biocompatible binder can be present in a biocompatible matrix in an
amount up to about 99 weight percent of the matrix.
[0082] A biocompatible matrix comprising a bone scaffolding
material and a biocompatible binder, according to some embodiments,
can be flowable, moldable, and/or extrudable. In such embodiments,
a biocompatible matrix can be in the form of a paste or putty. A
biocompatible matrix in the form of a paste or putty, in one
embodiment, can comprise particles of a bone scaffolding material
adhered to one another by a biocompatible binder.
[0083] A biocompatible matrix in paste or putty form can be molded
into the desired implant shape or can be molded to the contours of
the implantation site. In one embodiment, a biocompatible matrix in
paste or putty form can be injected into an implantation site with
a syringe or cannula.
[0084] In some embodiments, a biocompatible matrix in paste or
putty form does not harden and retains a flowable and moldable form
subsequent to implantation. In other embodiments, a paste or putty
can harden subsequent to implantation, thereby reducing matrix
flowability and moldability.
[0085] A biocompatible matrix comprising a bone scaffolding
material and a biocompatible binder, in some embodiments, can also
be provided in a predetermined shape including a block, sphere, or
cylinder or any desired shape, for example a shape defined by a
mold or a site of application.
[0086] A biocompatible matrix comprising a bone scaffolding
material and a biocompatible binder, in some embodiments, is
bioresorbable. A biocompatible matrix, in such embodiments, can be
resorbed within one year of in vivo implantation. In another
embodiment, a biocompatible matrix comprising a bone scaffolding
material and a biocompatible binder can be resorbed within 1, 3, 6,
or 9 months of in vivo implantation. In some embodiments, a
biocompatible matrix comprising a scaffolding material and a
biocompatible binder can be resorbed within 1, 3, or six years of
in vivo implantation. Bioresorbablity will be dependent on: (1) the
nature of the matrix material (i.e., its chemical make up, physical
structure and size); (2) the location within the body in which the
matrix is placed; (3) the amount of matrix material that is used;
(4) the metabolic state of the patient (diabetic/non-diabetic,
osteoporotic, smoker, old age, steroid use, etc.); (5) the extent
and/or type of injury treated; and (6) the use of other materials
in addition to the matrix such as other bone anabolic, catabolic
and anti-catabolic factors.
Biocompatible Matrix Comprising .beta.-TCP and Collagen
[0087] In some embodiments, a biocompatible matrix can comprise a
.beta.-TCP bone scaffolding material and a biocompatible collagen
binder. .beta.-TCP bone scaffolding materials suitable for
combination with a collagen binder are consistent with those
provided hereinabove.
[0088] A collagen binder, in some embodiments, can comprise any
type of collagen, including Type I, Type II, and Type III
collagens. In one embodiment, a collagen binder comprises a mixture
of collagens, such as a mixture of Type I and Type II collagen. In
other embodiments, a collagen binder is soluble under physiological
conditions. Other types of collagen present in bone or
musculoskeletal tissues may be employed. Recombinant, synthetic and
naturally occurring forms of collagen may be used in the present
invention.
[0089] A biocompatible matrix, according to some embodiments, can
comprise a plurality of .beta.-TCP particles adhered to one another
with a collagen binder. In some embodiments, .beta.-TCP particles
for combination with a collagen binder have an average diameter
ranging from about 1 .mu.m to about 5 mm. In other embodiments,
.beta.-TCP particles have an average diameter ranging from about 1
mm to about 2 mm, from about 1 mm to about 3 mm, from about 100
.mu.m to about 5 mm, from about 100 .mu.m to about 3 mm, from about
250 .mu.m to about 2 mm, from about 250 .mu.m to about 750 .mu.m,
from about 250 .mu.m to about 1 mm, from about 250 .mu.m to about 2
mm, or from about 200 .mu.m to about 3 mm. In another embodiment,
.beta.-TCP particles have an average diameter ranging from about
100 .mu.m to about 300 .mu.m. In some embodiments, .beta.-TCP
particles have an average diameter ranging from about 75 .mu.m, to
about 300 .mu.m. In some embodiments, .beta.-TCP particles have an
average diameter of less than about 25 .mu.m, less than about 1
.mu.m, or less than about 1 mm. In some embodiments, .beta.-TCP
particles have an average diameter ranging from about 1 nm to about
1 .mu.m. In a further embodiment, .beta.-TCP particles have an
average diameter less than about 500 nm or less than about 250
nm.
[0090] .beta.-TCP particles, in some embodiments, can be adhered to
one another by the collagen binder so as to produce a biocompatible
matrix having a porous structure. In some embodiments, the porous
structure of a biocompatible matrix comprising .beta.-TCP particles
and a collagen binder demonstrates multidirectional and
interconnected pores of varying diameters. In some embodiments, a
the biocompatible matrix comprises a plurality of pockets and
non-interconnected pores of various diameters in addition to the
interconnected pores.
[0091] In some embodiments, a biocompatible matrix comprising
.beta.-TCP particles and a collagen binder can comprise pores
having diameters ranging from about 1 .mu.m to about 1 mm. A
biocompatible matrix comprising .beta.-TCP particles and a collagen
binder can comprise macropores having diameters ranging from about
100 .mu.m to about 1 mm or greater, mesopores having diameters
ranging from about 10 .mu.m to 100 .mu.m, and micropores having
diameters less than about 10 .mu.m.
[0092] A biocompatible matrix comprising .beta.-TCP particles and a
collagen binder can have a porosity greater than about 25% or
greater than about 40%. In another embodiment, the biocompatible
matrix can have a porosity greater than about 50%, greater than
about 65%, greater than about 70%, greater than about 75%, greater
than about 80%, or greater than about 85%. In a further embodiment,
the biocompatible matrix can have a porosity greater than about
90%. In some embodiments, the biocompatible matrix can have a
porosity that facilitates cell migration into the matrix.
[0093] In some embodiments, the .beta.-TCP particles can
individually demonstrate any of the pore diameters, pore
structures, and porosities provided herein for a biocompatible
matrix comprising the .beta.-TCP and collagen binder.
[0094] A biocompatible matrix comprising .beta.-TCP particles, in
some embodiments, can comprise a collagen binder in an amount
ranging from about 5 weight percent to about 50 weight percent of
the matrix. In other embodiments, a collagen binder can be present
in an amount ranging from about 10 weight percent to about 40
weight percent of the biocompatible matrix. In another embodiment,
a collagen binder can be present in an amount ranging from about 15
weight percent to about 35 weight percent of the biocompatible
matrix. In a further embodiment, a collagen binder can be present
in an amount of about 20 weight percent of the biocompatible
matrix.
[0095] A biocompatible matrix comprising .beta.-TCP particles and a
collagen binder, according to some embodiments, can be flowable,
moldable, and/or extrudable. In such embodiments, the biocompatible
matrix can be in the form of a paste or putty. A paste or putty can
be molded into the desired implant shape or can be molded to the
contours of the implantation site. In one embodiment, a
biocompatible matrix in paste or putty form comprising .beta.-TCP
particles and a collagen binder can be injected into an
implantation site with a syringe or cannula.
[0096] In some embodiments, a biocompatible matrix in paste or
putty form comprising .beta.-TCP particles and a collagen binder
can retain a flowable and moldable form when implanted. In other
embodiments, the paste or putty can harden subsequent to
implantation, thereby reducing matrix flowability and
moldability.
[0097] A biocompatible matrix comprising .beta.-TCP particles and a
collagen binder, in some embodiments, can be provided in a
predetermined shape such as a block, sphere, or cylinder.
[0098] A biocompatible matrix comprising .beta.-TCP particles and a
collagen binder can be resorbable. In one embodiment, a
biocompatible matrix comprising .beta.-TCP particles and a collagen
binder can be at least 75% resorbed one year subsequent to in vivo
implantation. In another embodiment, a biocompatible matrix
comprising .beta.-TCP particles and a collagen binder can be
greater than 90% resorbed one year subsequent to in vivo
implantation.
[0099] A solution comprising PDGF can be disposed in a
biocompatible matrix to produce a composition for treating
structures of the vertebral column according to embodiments
described herein.
[0100] In some embodiments, compositions comprising a PDGF solution
disposed in a biocompatible matrix for promoting bone formation in
a vertebral body and preventing or reducing the likelihood of
vertebral compression fractures, as described herein, further
comprise at least one contrast agent. Contrast agents, according to
some embodiments, comprise cationic contrast agents, anionic
contrast agents, nonionic contrast agents or mixtures thereof. In
some embodiments, contrast agents comprise radiopaque contrast
agents. Radiopaque contrast agents, in some embodiments, comprise
iodo-compounds including
(S)--N,N'-bis[2-hydroxy-1-(hydroxymethyl)-ethyl]-2,4,6-triiodo-5-lactamid-
oisophthalamide (Iopamidol) and derivatives thereof.
Disposing PDGF Solution in a Biocompatible Matrix
[0101] The present invention provides methods for producing
compositions for promoting bone formation in a vertebral body and
preventing or reducing the likelihood of compression fractures of
vertebral bodies, including secondary vertebral fractures. In one
embodiment, a method for producing such compositions comprises
providing a solution comprising PDGF, providing a biocompatible
matrix, and disposing the solution in the biocompatible matrix.
PDGF solutions and biocompatible matrices suitable for combination
are consistent with those described hereinabove.
[0102] In some embodiments, a PDGF solution can be disposed in a
biocompatible matrix by soaking the biocompatible matrix in the
PDGF solution. A PDGF solution. in another embodiment, can be
disposed in a biocompatible matrix by injecting the biocompatible
matrix with the PDGF solution. In some embodiments, injecting a
PDGF solution can comprise disposing the PDGF solution in a syringe
and expelling the PDGF solution into the biocompatible matrix to
saturate the biocompatible matrix.
[0103] In some embodiments, the PDGF is absorbed into the pores of
the biocompatible matrix. In some embodiments, the PDGF is adsorbed
onto one or more surfaces of the biocompatible matrix, including
surfaces within pores of the biocompatible matrix.
[0104] The biocompatible matrix, according to some embodiments, can
be in a predetermined shape, such as a brick or cylinder, prior to
receiving a PDGF solution. Subsequent to receiving a PDGF solution,
the biocompatible matrix can have a paste or putty form that is
flowable, extrudable, and/or injectable. In other embodiments, the
biocompatible matrix can already demonstrate a flowable paste or
putty form prior to receiving a solution comprising PDGF. Flowable,
extrudable, and/or injectable forms of compositions comprising a
PDGF solution disposed in a biocompatible matrix are advantageous
for use in methods of the present application as they can applied
to vertebral bodies with syringes and/or cannulas.
[0105] In some embodiments, methods of producing compositions for
promoting bone formation in vertebral bodies and preventing or
decreasing the likelihood of compression fractures in vertebral
bodies further comprise providing at least one contrast agent and
disposing the at least one contrast agent in the biocompatible
matrix. In some embodiments, disposing at least one contrast agent
in a biocompatible matrix comprises combining the at least one
contrast agent with a PDGF solution and injecting the biocompatible
matrix with the PDGF/contrast agent solution.
[0106] In another embodiment, disposing at least one contrast agent
in a biocompatible matrix comprises combining the at least one
contrast agent with a PDGF solution and soaking the biocompatible
matrix in the PDGF/contrast agent solution. Alternatively, in some
embodiments, a contrast agent is disposed in a biocompatible matrix
independent of the PDGF solution.
[0107] Contrast agents, according to some embodiments of the
present invention, facilitate placement or application of
compositions of the present invention in and around vertebral
bodies. Contrast agents, according to some embodiments, comprise
cationic contrast agents, anionic contrast agents, nonionic
contrast agents, or mixtures thereof. In some embodiments, contrast
agents comprise radiopaque contrast agents. Radiopaque contrast
agents, in some embodiments, comprise iodo-compounds including
(S)--N,N'-bis[2-hydroxy-1-(hydroxymethyl)-ethyl]-2,4,6-triiodo--
5-lactamidoisophthalamide (Iopamidol) and derivatives thereof.
Compositions Further Comprising Biologically Active Agents
[0108] Compositions of the present invention, according to some
embodiments, can further comprise one or more biologically active
agents in addition to PDGF. Biologically active agents that can be
incorporated into compositions of the present invention, in
addition to PDGF, can comprise organic molecules, inorganic
materials, proteins, peptides, nucleic acids (e.g., genes, gene
fragments, small interfering ribonucleic acids [si-RNAs], gene
regulatory sequences, nuclear transcriptional factors, and
antisense molecules), nucleoproteins, polysaccharides (e.g.,
heparin), glycoproteins, and lipoproteins. Non-limiting examples of
biologically active compounds that can be incorporated into
compositions of the present invention, including, e.g., anti-cancer
agents, antibiotics, analgesics, anti-inflammatory agents,
immunosuppressants, enzyme inhibitors, antihistamines, hormones,
muscle relaxants, prostaglandins, trophic factors, osteoinductive
proteins, growth factors, and vaccines, are disclosed in U.S.
patent application Ser. No. 11/159,533 (Publication No:
20060084602). In some embodiments, biologically active compounds
that can be incorporated into compositions of the present invention
include osteostimulatory factors such as insulin-like growth
factors, fibroblast growth factors, or other PDGFs. In accordance
with other embodiments, biologically active compounds that can be
incorporated into compositions of the present invention preferably
include osteoinductive and osteostimulatory factors such as bone
morphogenetic proteins (BMPs), BMP mimetics, calcitonin, or
calcitonin mimetics, statins, statin derivatives, fibroblast growth
factors, insulin-like growth factors, growth differentiating
factors, small molecule or antibody blockers of Wnt antagonists
(e.g. sclerostin, DKK, soluble Wnt receptors), and/or parathyroid
hormone. In some embodiments, factors also include protease
inhibitors, as well as osteoporotic treatments that decrease bone
resorption including bisphosphonates, teriparadide, and antibodies
to the activator receptor of the NF-kB ligand (RANK) ligand.
[0109] Standard protocols and regimens for delivery of additional
biologically active agents are known in the art. Additional
biologically active agents can introduced into compositions of the
present invention in amounts that allow delivery of an appropriate
dosage of the agent to the implant site. In most cases, dosages are
determined using guidelines known to practitioners and applicable
to the particular agent in question. The amount of an additional
biologically active agent to be included in a composition of the
present invention can depend on such variables as the type and
extent of the condition, the overall health status of the
particular patient, the formulation of the biologically active
agent, release kinetics, and the bioresorbability of the
biocompatible matrix. Standard clinical trials may be used to
optimize the dose and dosing frequency for any particular
additional biologically active agent.
[0110] A composition of the present invention, according to some
embodiments, can further comprise the addition of additional bone
grafting materials with PDGF including autologous bone marrow,
autologous platelet extracts, allografts, synthetic bone matrix
materials, xenografts, and derivatives thereof.
Methods of Treating Vertebral Bodies
[0111] In some embodiments, the present invention provides methods
for promoting bone formation in a vertebral body comprising
providing a composition comprising a PDGF solution disposed in a
biocompatible matrix and applying the composition to at least one
vertebral body. In some embodiments, the composition can be applied
to a plurality of vertebral bodies. Applying the composition, in
some embodiments, comprises injecting at least one vertebral body
with the composition. Compositions of the present invention, in
some embodiments, are injected into the cancellous bone of a
vertebral body. Vertebral bodies, in some embodiments, comprise
thoracic vertebral bodies, lumbar vertebral bodies, or combinations
thereof. Vertebral bodies, in some embodiments, comprise cervical
vertebral bodies, coccygeal vertebral bodies, the sacrum, or
combinations thereof.
[0112] In another aspect, the present invention provides methods
for preventing or decreasing the likelihood of vertebral
compression fractures, including secondary vertebral compression
fractures by strengthening vertebrae. Preventing or decreasing the
likelihood of vertebral compression fractures, according to
embodiments of the present invention, comprises providing a
composition comprising a PDGF solution disposed in a biocompatible
matrix and applying the composition to at least one vertebral body.
In some embodiments, applying the composition to at least one
vertebral body comprises injecting the composition into the at
least one vertebral body.
[0113] In some embodiments, a composition of the present invention
is applied to a second vertebral body subsequent to vertebroplasty
or kyphoplasty of a first vertebral body. In some embodiments, the
second vertebral body is adjacent to the first vertebral body. In
other embodiments, the second vertebral body is not adjacent to the
first vertebral body. In a further embodiment, a composition of the
present invention is applied to a third vertebral body subsequent
to vertebroplasty or kyphoplasty of a first vertebral body. In some
embodiments, the third vertebral body is adjacent to the first
vertebral body. In other embodiments, the third vertebral body is
not adjacent to the first vertebral body. Embodiments of the
present invention additionally contemplate application of
compositions provided herein to a plurality of vertebral bodies,
including high risk vertebral bodies, subsequent to vertebroplasty
or kyphoplasty of a first vertebral body. It is to be understood
that first, second, and third vertebral bodies, as used herein, do
not refer to any specific position in the vertebral column as
methods for inhibiting vertebral compression fractures, including
secondary compression fractures, can be applied to all types of
vertebral bodies including thoracic vertebral bodies, lumbar
vertebral bodies, cervical vertebral bodies, coccygeal vertebral
bodies, and the sacrum.
[0114] In some embodiments, methods for promoting bone formation in
vertebral bodies and preventing or decreasing the likelihood of
compression fractures of vertebral bodies further comprise
providing at least one pharmaceutical composition in addition to
the composition comprising a PDGF solution disposed in a
biocompatible matrix and administering the at least one
pharmaceutical composition locally and/or systemically. The at
least one pharmaceutical composition, in some embodiments,
comprises vitamins, such as vitamin D3, calcium supplements, or any
osteoclast inhibitor known to one of skill in the art, including
bisphosphonates. In some embodiments, the at least one
pharmaceutical composition is administered locally. In such
embodiments, the at least one pharmaceutical composition can be
incorporated into the biocompatible matrix or otherwise disposed in
and around a vertebral body. In other embodiments, the at least one
pharmaceutical composition is administered systemically to a
patient. In one embodiment, for example, the at least one
pharmaceutical composition is administered orally to a patient. In
another embodiment, the at least one pharmaceutical composition is
administered intravenously to a patient.
[0115] The following examples will serve to further illustrate the
present invention without, at the same time, however, constituting
any limitation thereof. On the contrary, it is to be clearly
understood that resort may be had to various embodiments,
modifications and equivalents thereof which, after reading the
description herein, may suggest themselves to those skilled in the
art without departing from the spirit of the invention.
Example 1
Preparation of a Composition Comprising a Solution of PDGF and a
Biocompatible Matrix
[0116] A composition comprising a solution of PDGF and a
biocompatible matrix was prepared according to the following
procedure.
[0117] A pre-weighed block of biocompatible matrix comprising
.beta.-TCP and collagen was obtained. The .beta.-TCP comprised
.beta.-TCP particles having an average diameter ranging from about
100 .mu.m to about 300 .mu.m. The .beta.-TCP particles were
formulated with about 20% weight percent soluble bovine type I
collagen binder. Such a .beta.-TCP/collagen biocompatible matrix
can be commercially obtained from Kensey Nash (Exton, Pa.).
[0118] A solution comprising rhPDGF-BB was obtained. rhPDGF-BB is
commercially available from Novartis Corporation at a stock
concentration of 10 mg/ml (i.e., Lot #QA2217) in a sodium acetate
buffer. The rhPDGF-BB is produced in a yeast expression system by
Chiron Corporation and is derived from the same production facility
as the rhPDGF-BB that is utilized in the products REGRANEX,
(Johnson & Johnson) and GEM 21S (BioMimetic Therapeutics) which
have been approved for human use by the United States Food and Drug
Administration. This rhPDGF-BB is also approved for human use in
the European Union and Canada. The rhPDGF-BB solution was diluted
to 0.3 mg/ml in the acetate buffer. The rhPDGF-BB solution can be
diluted to any desired concentration according to embodiments of
the present invention, including 1.0 mg/ml.
[0119] A ratio of about 3 ml of rhPDGF-BB solution to about 1 g dry
weight of the .beta.-TCP/collagen biocompatible matrix was used to
produce the composition. In the preparation of the composition, the
rhPDGF-BB solution was expelled on the biocompatible matrix with a
syringe, and the resulting composition was blended into a paste for
placement into a syringe for subsequent injection into a vertebral
body.
Example 2
Method of Inhibiting Secondary Vertebral Compression Fractures
Experimental Design and Overview
[0120] This prospective, randomized, controlled, single-center
clinical trial is to evaluate the efficacy of compositions
comprising a PDGF solution disposed in a tricalcium phosphate
matrix for inhibiting secondary compression fractures in high risk
vertebral bodies (HVBs) at the time of kyphoplasty of vertebral
compression fractures. Comparisons are made between vertebral
bodies treated with a .beta.-tricalcium phosphate+rhPDGF-BB
composition and untreated vertebral bodies. The present study is a
pilot, clinical trial to support the proof-or-principle of
.beta.-TCP+rh-PDGF-BB to prevent or decrease the likelihood of
secondary vertebral compression fractures by increased bone
formation in HVBs.
[0121] The study is performed on up to a total of 10 patients
requiring prophylactic treatment of HVBs at the time of
kyphoplasty. Potential patients are screened to determine if they
meet the inclusion and exclusion criteria If all entry criteria are
achieved, the potential patients are invited to participate in the
clinical trial. All patients considered for entry into the study
are documented on the Screening Log and reasons for exclusion are
recorded.
[0122] All patients have undergone kyphoplasty and do not have a
symptomatic VCF adjacent to the two vertebral bodies treated in
this study. The subject is not to be enrolled into the study if the
surgeon determines intraoperatively that the fracture does not meet
the fracture enrollment criteria or other fractures exist that
would preclude treatment in this protocol.
[0123] A total of 10 patients are enrolled and treated in the
present study. A first vertebral body adjacent to the kyphoplasty
of each patient is injected with 3.0 ml of a composition of
.beta.-TCP+0.3 mg/ml of rhPDGF-BB prepared in accordance with
Example 1 herein. The second vertebral body adjacent to the
kyphoplasty remains untreated and serves as a control. The treated
vertebral body may be either cranial or caudal to the kyphoplasty
and is determined randomly.
[0124] Patients are treated according to the standard protocols and
follow-up for kyphoplasty/vertebroplasty. Each patient is examined
by the surgeon at 7-14 days, and at 6, 12, 24, and 52 weeks for
clinical, radiographic and quantitative computed tomography (QCT).
All over-the-counter and prescribed medication usage is recorded.
An independent radiologist, unaware of the patients' treatment
group assignments, performs QCT analysis to assess bone density.
These measurements are documented and analyzed.
[0125] All postoperative complications and device-related adverse
events are recorded on the appropriate case report form. If a
subject experiences a subsequent VCF during the study period or
another surgical procedure for a serious adverse event or the
investigational device is removed, the subject is monitored for
safety until the end of the study. Those subjects who are
re-operated and/or have the fracture fixation hardware removed are
requested to give permission to examine the explants for
histological purposes. All patients are monitored during the
12-month trial and any subject who requests study withdrawal or is
withdrawn by the investigator is requested to provide a reason for
study discontinuance. Table 1 provides a timeline summary for the
present study.
TABLE-US-00001 TABLE 1 Study Timeline Survey Visit 1 Visit 2
Screening Surgical Visit Visit .dwnarw. .dwnarw. Visit 3 Visit 4
Visit 5 Visit 6 Visit 7 Within 21 Within 21 Post Tx Post Tx Post Tx
Post Tx Post Tx Days of Days of Follow Up Follow Up Follow Up
Follow Up Follow Up Surgery Screening .dwnarw. .dwnarw. .dwnarw.
.dwnarw. .dwnarw. Day 0 Day 7-14 Week 6 Week 12 Week 24 Week 52
.+-.3 days .+-.7 days .+-.7 days .+-.14 days
[0126] The primary endpoint is the bone density at 12 weeks
post-operatively measured by QCT scans. Secondary endpoints include
subject pain and quality of life assessments.
Surgical Protocol
[0127] After patients have been enrolled in the study, satisfying
both the inclusion and exclusion criteria, the following surgical
protocol is undertaken.
[0128] Patients are brought into an operating room (OR) in the
standard fashion, and standard methods are used to perform the
kyphoplasty procedure with methyl methacrylate cement augmentation
of the fractured vertebral body. Standard radiographs are taken of
the vertebral bodies treated with kyphoplasty and with preventative
bone augmentation treatment.
[0129] Following the kyphoplasty treatment, the investigator
identifies and qualifies the two levels to be treated with
prophylactic bone augmentation. If two (2) qualified vertebral
bodies are not available for treatment, as determined at the time
of surgery, the patient is considered a screen failure and not
enrolled into the study.
[0130] Upon identification of the two HVBs, the investigator
requests that the randomization code be opened to determine the
study treatment administered. The randomization code specifies
treatment with the .beta.-TCP+rhPDGF composition either proximally
or distally in relation to the level treated with kyphoplasty. The
other HVB remains untreated.
[0131] The .beta.-TCP+rhPDGF composition is mixed according to the
procedure provided in Example 1. Once mixed, the paste is loaded
into a syringe for injection using aseptic technique. Once the
.beta.-TCP+rhPDGF composition is mixed, the clinician waits about
10 minutes prior to implantation. A new sterile mixing device
(spatula) is used for each mix. The investigator directs the
assistant who performs the mixing to record the cumulative amount
of implanted composition, as well as the residual amount of
composition not implanted. The amount of composition is calculated
and documented using qualitative relative measurements (1/3, 2/3,
All).
[0132] An 8 to 16 gauge JAMSHIDI.RTM. needle available from
Cardinal Health of Dublin, Ohio is inserted through an
extrapedicular approach into the vertebral bodies requiring
prophylactic treatment. The wire is passed through the
JAMSHIDI.RTM. needle and the JAMSHIDI.RTM. needle through the
stylet over the wire The appropriate mixed preparation is injected
into the subject vertebral body. Care should be taken to minimize
leakage of the paste outside of the vertebral body.
[0133] Contrast agents, according to embodiments of the present
invention, can assist in identifying the leakage of the paste
outside the vertebral body. FIG. 1 illustrates a syringe and
related apparatus penetrating tissue overlaying a vertebral body to
deliver a composition of the present invention to the vertebral
body. FIG. 2 is a radiograph illustrating injection of a
composition of the present invention into the vertebral body of the
L3 vertebra according to one embodiment.
[0134] The instrumentation is removed. Thorough irrigation and
standard wound closure techniques are employed.
Follow-up Evaluations
[0135] Patients are seen for post-operative evaluations at days
7-14, and at 6 (.+-.3 days), 12 (.+-.7 days), 24 (.+-.7 days), and
52 (.+-.14 days) weeks post-surgery. Routine evaluations and
procedures are performed during the follow-up period, as specified
in the study flowchart of Table 2 below.
TABLE-US-00002 TABLE 2 Study Flow Chart and Follow-up Assessments
Post-Treatment Follow-Up Evaluations Surgery Visit Visit Visit
Visit Screening Visit Visit 4 5 6 7 Visit 2 3 Week 6 Week 12 Week
24 Week 52 Procedure 1 Day 0 Day 7-14 .+-.3 Days .+-.7 Days .+-.7
Days .+-.14 Days Informed Consent X.sup.1 Screening Log X Medical
History X Physical Examination of Spine X X X X X X X Subject
Eligibility X X Criteria Verification Identification of High- X
Risk Vertebral Bodies Randomization X Kyphoplasty and X
Preventative Bone Augmentation Volume of Graft Material X Placed
Qualitative CT X X X X Assessments.sup.2 Plain Radiographic X X X X
X X Assessments Adverse X X X X X X Events/Complications
Concomitant Medications X X X X X X X Review .sup.1Must occur prior
to any study-specific procedures. .sup.2Quantitative Computed
Tomography (QCT) is performed according to standard protocol to
obtain BMD data which is determined by the designated
musculoskeletal radiologist.
Assessment of Effectiveness
[0136] Outcome data is collected from this study on findings
derived from radiographs, QCTs, and from direct examination of
function. The schedule of these measurements is provided in Table
3.
TABLE-US-00003 TABLE 3 Frequency of Radiographic and Functional
Assessments Study Parameters Plain film Qualitative Timepoint
radiographs CT Scans Pain Function Prior to X X X X Treatment
Immediately X Post-Treatment Day 7-14 X Week 6 X X X X Week 12 X X
X X Week 24 X X X X Week 52 X X X X
[0137] Vertebral bodies injected with a .beta.-TCP+rhPDGF
composition are expected to display increased bone mineral density
(BMD) in comparison to untreated vertebral bodies. Increased bone
mineral density in a vertebral body can render the vertebral body
less susceptible to fractures including secondary fractures induced
by kyphoplasty/vertebroplasty operations.
Example 3
Method of Inhibiting Vertebral Compression Fractures in
Osteoporotic Individuals
[0138] A method of inhibiting vertebral compression fractures in
osteoporotic individuals comprises promoting bone formation in
vertebral bodies through treatment with compositions comprising a
PDGF solution disposed in a biocompatible matrix such as
.beta.-tricalcium phosphate.
[0139] Compositions of the present invention are mixed in
accordance with that provided in Example 1. The concentration of
PDGF in the PDGF solutions ranges from 0.3 mg/ml to 1.0 mg/ml. Once
mixed, the composition is loaded into a syringe for injection using
aseptic technique. The surgeon waits about 10 minutes prior to
implantation. A new sterile mixing device (spatula) is used for
each mix.
[0140] The JAMSHIDI.RTM. needle is inserted through an
extrapedicular approach into the vertebral bodies requiring
prophylactic treatment. Vertebral bodies requiring prophylactic
treatment, in some embodiments, comprise high risk vertebral bodies
including vertebral bodies T5 through T12 and L1 through L4. The
wire is passed through the JAMSHIDI.RTM. needle and the
JAMSHIDI.RTM. needle through the stylet over the wire The mixed
composition is injected into the subject vertebral body. Care is
taken to minimize leakage of the paste outside of the vertebral
body. A plurality of vertebral bodies are treated according to the
present example. Osteoporotic patients receiving this treatment
have a lower incidence of vertebral compression fractures than
untreated osteoporotic patients.
Example 4
Evaluation of the Chronic Safety of rh-PDGF-BB Combined with
Collagen/.beta.-tricalcium Phosphate Matrix in a Rabbit
Paravertebral Implant Model
Experimental Design and Overview
[0141] This study evaluated the safety of implanting injectable
rhPDGF-BB/collagen/.beta.-TCP material in a paravertebral
intramuscular site adjacent to the spine of rabbits. The animals
were observed for signs of neurotoxicity, and the implant sites
with adjacent vertebral bodies and spinal cord were examined
histologically to document tissue-specific responses to the
material.
[0142] The study protocol and animal care was approved by the local
IACUC and conducted according to AAALAC guidelines. Twelve (12)
naive, female, albino New Zealand rabbits weighing .gtoreq.2.5 kg
were assigned to one of 4 groups: 0.3 mg/ml PDGF; 1.0 mg/ml PDGF;
rubber; or acetate buffer. PDGF treated rabbits received 0.2 cc
implants of appropriately concentrated rhPDGF-BB in matrix injected
into a 1 cm pocket in the right paravertebral muscle adjacent to
the L4-L5 vertebral bodies while high density polyethylene (HDPE)
was implanted in a similar incision in the left paravertebral
muscles near L2-L3 of the same animals. Rabbits in the sodium
acetate buffer group received sodium acetate buffer in place of the
PDGF+matrix implant, while those in the rubber group received only
rubber in the right paravertebral muscle. One rabbit in each group
was sacrificed at 29, 90, and 180 days post-surgery.
[0143] Body weights were measured prior to surgery and biweekly
following surgery for the duration of the study. Radiographs were
taken prior to surgery, immediately following surgery, and
immediately prior to sacrifice. Digital photography of the surgical
sites was performed during surgery and at the study end points.
Weekly clinical observations of the implant sites were recorded for
signs of erythema, edema, and inflammation and for signs of
neurotoxicity, such as ambulatory changes. At necropsy, each
implant site along with the adjacent vertebral body and spinal cord
were harvested en bloc, fixed in formalin, and prepared for
decalcified, paraffin embedded histopathological analysis.
Materials
[0144] The dosages of rhPDGF-BB tested in this study included 0.3
mg/ml and 1.0 mg/ml in 20 mM sodium acetate buffer, pH 6.0+/-0.5.
The matrix material consisted of 20% lyophilized bovine type I
collagen and 80% .beta.-TCP with a particle size of 100-300 .mu.m
(Kensey Nash Corporation). Negative control material consisted of
high-density polyethylene (HDPE) and positive control material
consisted of black rubber. Immediately prior to surgery, the
rhPDGF-BB and control solutions were mixed with matrix material in
a 3:1 liquid to mass ratio.
[0145] Briefly, the PDGF solution was allowed to saturate the
material for about 2 minutes then was manually mixed for about 3
minutes to generate a paste-like consistency. The homogeneous
distribution of rhPDGF-BB throughout the mixed material using this
mixing technique was confirmed by eluting the PDGF from samples of
similar mass and then quantifying the PDGF by ELISA (R&D
Systems).
Results
[0146] Following manual mixing of 0.3 mg/ml rhPDGF-BB with the
collagen/.beta.-TCP matrix, the homogeneity of rhPDGF-BB throughout
the mixed material was confirmed within +/-4% error across
samples.
[0147] All animals recovered from surgery, and at the time of this
writing, all clinical observations were reported to be normal with
no signs of neurotoxicity or abnormal wound healing at the surgical
sites. Two animals treated with sodium acetate buffer and matrix
control exhibited minor scabbing at the surgical wounds which
healed completely. One animal that received 0.3 mg/mi rhPDGF-BB
exhibited slight erythema at the surgical site 3-4 days after
surgery and then returned to normal appearance. A histopathological
analysis of test article implant sites 29 days post-surgery
indicated a mild amount of tissue in-growth into the implanted test
materials and a mild inflammatory response. No ectopic or abnormal
bone formation was observed in the vertebral bodies adjacent to the
implant sites. These findings are summarized in Table 4 and
compared with ratings for negative control HDPE implant sites.
TABLE-US-00004 TABLE 4 Summary of Histopathology Findings at
Implant Sites 29 Days After Surgery [PDGF-BB] Tissue In- Ectopic
(mg/ml) Macrophages MGCs growth Bone Exostosis 0.3 3, 1(NC) 2,
0(NC) 2, 0(NC) 0, 0(NC) 0, 0(NC) 1.0 2, 2(NC) 2, 0(NC) 2, 0(NC) 0,
0(NC) 0, 0(NC) NC = Negative Control; MGC = multinucleated giant
cells; Bioreactivity scale: 0 = Absent, 1 = Minimal/Slight, 2 =
Mild, 3 = Moderate, 4 = Marked/Severe
[0148] Preliminary evidence from this study based on clinical
observations, suggests that collagen/.beta.-tricalcium phosphate
combined with either 1.0 mg/ml, 0.3 mg/ml rhPDGF-BB, or sodium
acetate buffer does not elicit any acute or chronic neurotoxic
effects. Histopathological assessment of the implant sites 29 days
post-surgery indicated a normal and expected mild amount of tissue
in-growth into the implanted material and a mild inflammatory
response. No ectopic bone formation, exostosis, or abnormal bone
resorption was observed at any of the implant sites. Based on
observations of the animals treated in this study,
collagen/.beta.-tricalcium phosphate combined with either 1.0
mg/ml, 0.3 mg/ml rhPDGF-BB is safe to use when injected in close
proximity to the spinal column.
Example 5
Evaluation of the Safety of PDGF-BB Combined with a Bovine Type I
Collagen/.beta.-TCP Matrix for Vertebral Therapy
[0149] This study evaluated the safety of a composition comprising
rhPDGF-BB combined with a biocompatible matrix comprising
.beta.-tricalcium phosphate and type I collagen for bone
augmentation following injection of the composition into vertebral
bodies of baboons. Experimental Design
[0150] A total of 6 female baboons (Papio anubis) of 18 to 21 years
of age were studied, each baboon being assigned to one of two
treatment groups as provided in Table 5. During the study, the
animals were imaged and analyzed using radiography, quantitative
computed tomography (QCT), magnetic resonance imaging (MRI)
techniques, terminal histology and non-GLP microcomputed tomography
(microCT).
[0151] Four vertebral levels (T12, L2, L4 and L6) were investigated
in each animal. Each animal of Group 1 received an injection of
about 0.5 cc of a 1.0 mg/ml rhPDGF-BB+collagen/.beta.-TCP (matrix)
composition into each of the T12, L2 and L4 vertebral bodies. The
1.0 mg/ml rhPDGF-BB+collagen/.beta.-TCP (matrix) compositions were
prepared as set forth in Example 1 hereinabove. Each animal of
Group 11 received an injection of about 0.5 cc of a sodium acetate
buffer+collagen/.beta.-TCP (matrix) composition into each of the
T12, L2, and L4 vertebral bodies. Animals of each group I and II
additionally received an injection of about 0.5 cc of a sodium
acetate buffer into the L6 vertebral bodies. Therefore, a total of
four (4) vertebral bodies per animal received an injection. FIG. 3
summarizes the injection strategy of the present study.
Documentation of the treatments in each animal was recorded on
study forms.
[0152] Surgery was conducted using a percutaneous, fluoroscopically
guided approach. The procedure was performed similar to a
vertebroplasty, except that an injectable 1.0 mg/ml
rhPDGF-BB+collagen/.beta.-TCP matrix or appropriate control
treatment was injected. About 0.5 cc of a
rhPDGF-BB+collagen/.beta.-TCP material, control material, or buffer
was injected into each vertebral body as described above. Each
animal was provided anesthesia during the surgery.
TABLE-US-00005 TABLE 5 Summary of Treatments for Injection of
rhPDGF-BB + Collagen/ .beta.-TCP Matrix in Vertebral Bodies of
Baboons Group Dose Timepoints Analyses I 1.0 mg/ml PDGF + Pre- and
post- QCT, MRI, Clinical collagen/.beta.-TCP surgery, 1 and
Observations, Serum matrix. L6 received 3 months, Chemistry and
Hematology sodium acetate (optionally (Pre- and post-surgery,
buffer only 6 months) and at 1 month, 3 months, and 6 months
post-surgery), Body Weights, Radiography, Histology, non-GLP
microCT II 20 mM sodium Pre- and post- QCT, MRI, Clinical acetate
buffer, pH surgery, 1 and Observations, Serum 6.0 + collagen/ 3
months, Chemistry and Hematology .beta.-TCP matrix. L6 (optionally
(Pre- and post-surgery, received sodium 6 months) and at 1 month, 3
months, acetate buffer only and 6 months post-surgery), Body
Weights, Radiography, Histology, non-GLP microCT
A. Assignment to Dose Group
[0153] Three animals were assigned to treatment groups by a manual
scheme designed to achieve similar group mean body weights.
B. Assignment to Surgery Days
[0154] Animals were assigned to one of two surgery days (Day I or
Day II). For each animal, a coin flip determined assignment into
the Day I group or the Day 11 group. This process was continued
until each of the days was filled with three animals. The animal
numbers, their dosing group assignments, and surgery days were
recorded. The animal treatment groups were known to the study
monitors and study director. The radiologists and histopathologist
were blind to the treatment groups.
C. In-Vivo Observations and Measurements
Clinical Observations
[0155] Animals were observed within their cages daily throughout
the study. Recording of cageside observations was commenced after
the pre-selection criteria was completed and is continued until the
end of study. Each animal was observed for changes in general
appearance and behavior, including changes in ambulation. Each
animal was observed for evidence of menstrual cycling over the
course of the study. The cycling readings were performed non-GLP
and were recorded in the Southwest Foundation for Biomedical
Research (SFBR) animal database.
[0156] Treatment of the animals was in accordance with SFBR
standard operating procedures (SOPs), which adhere to the
regulations outlined in the USDA Animal Welfare Act (9 CFR, Parts
1, 2 and 3) and the conditions specified in The Guide for Care and
Use of Laboratory Animals (ILAR publication, 1996, National Academy
Press). The study animals were observed and were recorded at least
once daily for signs of illness or distress, including changes in
ambulation, and any such observations were reported to the
responsible veterinarian and study director.
Body Weight
[0157] Body weights were measured at the initial health check,
prior to surgery, and prior to follow up radiographs. Food was
withheld prior to sedation and subsequent body weight
measurements.
Food Consumption
[0158] Except when animals were fasting for study procedures, food
consumption was qualitatively assessed daily for each animal (as
part of the cageside observations), beginning at least 7 days prior
to surgery. Each animal was provided a full feeder of food once a
day (non-sedation days) and the amount eaten was documented as per
SFBR SOPs. On sedation days, the animals were fed once each day
when they recovered from anesthesia.
D. Fluoroscopy, Photography, Radiography, MRI and QCT Imaging
[0159] Non-GLP digital photographs were taken of the injection
sites pre-operatively, immediately post-operatively, and at 1, 3,
6, and 9 months post-operatively.
[0160] Anteroposterior and lateral radiographs were taken
pretreatment and immediately following surgery as well as at about
1, 3, 6, and 9 months post-operatively. For anteroposterior
radiographs, the animals were placed on their backs in supine
position with their legs supported. For lateral radiographs, the
animals were positioned lying on their left sides with arms and
legs supported. Energy (kV) and intensity (mA) settings for each
position and animal were recorded.
[0161] Non-GLP fluorographs were captured intraoperatively before,
during, and after injection of the test and control articles into
the vertebrae of the animals. Fluorographs were not assessed as an
outcome of this study, but enable the surgeon to accurately insert
the introduction needle into the vertebral bodies during
surgery.
[0162] Magnetic resonance imaging (MRI) was performed to image the
spine of each animal pre-operatively and within 4 to 10 days
post-operatively. MRI was additionally performed to image the spine
of each animal at about 1, 3, 6, and 9 months post-operatively. MRI
sessions consisted of T1 and T2-weighted scans.
[0163] Quantitative computed tomography imaging (QCT) was performed
to image the spine of each animal pre-operatively and within 4 to
10 days post-operatively. QCT was additionally performed to image
the spine of each animal at about 1, 3, 6, and 9 months
post-operatively. Scans consisted of a series of contiguous
cross-sectional slices of the torso from the caudal endplate of the
11th thoracic vertebrae to the cranial endplate of the sacrum.
[0164] QCT was also used to obtain 3 mm thick cross sectional
images of injected and intervening untreated vertebral bodies of
each baboon 1 week prior to surgery (pre-surgical) and at 1, 4, and
12 weeks post surgery. A total of 5-8 slices were required to fully
image each vertebral body. The DICOM (digital imaging and
communications in medicine) format images produced by the QCT were
transferred and converted to a file format for 3-dimensional
volumetric analysis using software developed by Scanco AG
(Bassersdorf, Switzerland). Volumetric bone mineral density (vBMD)
of the anterior compartment of each vertebral body was determined
by manually selecting a region of interest (ROI) that excluded the
cortical shell in each slice. The evaluation software created a
z-stack of the individual slices and ROI's before extracting the
volumetric ROI and calculating volumetric density in arbitrary
units derived from the gray-scale intensity in the images and a
percent change from the baseline scans was calculated. One-way
repeated measures ANOVA with Tukey's post-hoc test was used to
determine the presence of any statistically significant changes in
vBMD for animals of Group I and Group II from pre-surgical or 1 wk
post-surgical to the conclusion of the study.
[0165] The radiographs, MRI, and QCT images were evaluated by one
board certified clinical radiologist and one qualified associate to
provide a consensus assessment of the neuropathological,
osteopathological and surrounding soft tissue pathological outcomes
resulting from the treatments of the vertebrae. The evaluation
consisted of a qualitative examination of each image for
abnormalities of the bone and of the neural tissues and adjacent
surrounding soft tissues. The radiologist followed the Radiology
Assessment Protocol to evaluate the radiology data.
E. Clinical Pathology Evaluation
Serum Chemistry
[0166] About 3 ml whole blood was collected into containers without
anticoagulant pre-surgery and post-surgery. About 3 ml whole blood
was also collected into containers without anticoagulant at about
1, 3, 6, and 9 months post-operatively. The animals were fasted
overnight prior to blood collection for serum chemistry. Serum was
analyzed for the following parameters set forth in Table 6.
TABLE-US-00006 TABLE 6 Serum Analysis Sodium Phosphorus Potassium
Glucose Chloride Urea nitrogen (BUN) Total carbon dioxide
(bicarbonate) Creatinine Total bilirubin Total protein Alkaline
phosphatase (AP) Albumin Lactate dehydrogenase (LDH) Globulin
Aspartate aminotransferase (AST) Albumin/globulin ratio Alanine
aminotransferase (ALT) Cholesterol Gamma-glutamyltransferase (GGT)
Triglycerides Calcium BUNICREAT Ratio Anion Gap Direct Bilirubin
CPK
Hematology
[0167] About 3 ml of blood was collected in EDTA-containing tubes
pre-surgery and post-surgery. About 3 ml of blood was also
collected in EDTA-containing tubes at 1, 3, 6, and 9 months
post-surgery. The whole blood samples were analyzed for the
following parameters set forth in Table 7.
TABLE-US-00007 TABLE 7 Blood Analysis Red blood cell (RBC) counts
Mean corpuscular hemoglobin (MCH) White blood cells (WBCs) Mean
corpuscular hemoglobin (total and differential*) concentration
(MCHC) Hemoglobin concentration Mean corpuscular volume (MCV)
Hematocrit Platelet counts (Plt) RDW Abnormal blood cell morphology
*Includes polysegmented neutrophils, band cells, lymphocytes,
monocytes, basophils, eosinophils.
Serum Collection for Analysis by Sponsor or SFBR
[0168] About 14 ml of blood was collected into non-additive (i.e.,
"clot") tubes from all animals once pre-surgery and post-surgery.
About 14 ml of blood was also collected into non-additive (i.e.,
"clot") tubes from all animals at 1, 3, 6, and 9 months
post-surgery. The blood was centrifuged to obtain serum and divided
into two aliquots. The serum is stored at -70.degree. C. or
lower.
Anatomic Pathology
[0169] All animals are humanely sacrificed at the end of the study.
A gross necropsy is conducted on each animal sacrificed in a
moribund or diseased condition to determine the cause and/or nature
of the moribund or diseased condition.
Necropsy
[0170] A complete necropsy is conducted under the supervision of
the study pathologist on the sacrificed animals in a moribund or
diseased condition during the study to determine the cause and/or
nature of the moribund or diseased condition. A standard necropsy
includes an examination of external surfaces and orifices,
extremities, body cavities, and internal organ/tissues. All of the
treated vertebrae are collected and are examined for abnormalities.
A brief morphologic description of all macroscopic abnormalities is
recorded on individual necropsy forms.
Tissue Collection and Preservation
[0171] The following tissues and organs are obtained at sacrifice
and are preserved in 10% neutral-buffered formalin (except for the
eyes, which were preserved in Bouin's Solution for optimum
fixation). Each tissue or organ specimen is then embedded in
paraffin for preservation purposes and is archived at a
sponsor-approved site or used to help determine the cause of
death.
[0172] For all baboons, all treated and adjacent untreated
vertebrae (T12 thru L6) are individually harvested en bloc
including the spinal cord and spinal canal and are appropriately
identified as to the treatment received. The T12 vertebral body is
identified by leaving a minimum of 2 cm of the ribs attached to the
bone. All bone specimens are placed in formalin fixative in
preparation for plastic embedding.
[0173] Each of the soft tissues or organ specimens is embedded in
paraffin for preservation purposes and are archived. A summary of
the tissue samples to be collected is provided in Table 8.
TABLE-US-00008 TABLE 8 Summary of Tissue Samples to be Collected at
Necropsy Cardiovascular Urogenital Aorta Kidneys Heart Urinary
Bladder Digestive Ovaries Salivary Gland (mandibular) Uterus Tongue
Cervix Esophagus Vagina Stomach Skin/MusculoskeIetal Small
Intestine Skin/Mammary Gland (males and females) Duodenum Bone
(femoral head) Jejunum Bone (7th rib) Ileum Skeletal Muscle (thigh)
Large Intestine Knee joint Cecum Shoulder joint Colon Mandible
Rectum Right Foot Pancreas Left Ankle Liver Right Hand Gallbladder
Left Wrist Respiratory Spine from T12 to L7/Sacrum-separated
Trachea Nervous/Special Sense Lung (including bronchi) Eyes with
optic nerve Lymphoid/Hematopoietic Sciatic Nerve Bone Marrow
(sternum) Brain Thymus Optic chiasm Spleen Cerebrum Lymph Nodes
Cerebellum Axillary Medulla Mandibular Pons Mesenteric Spinal Cord
(thoracic) Endocrine Other Adrenals Animal Number Tattoo Pituitary
Gross Lesions Thyroid/Parathyroids* Lacrimal glands *The occasional
absence of the parathyroid gland from the routine tissue section
will not require a recut of the section.
[0174] Vertebral bodies injected with a composition comprising a
rhPDGF-BB solution disposed in a .beta.-TCP/collagen matrix
displayed the formation of normal bone with no adverse neurotoxic
effects. Moreover, soft tissues adjacent to vertebral bodies
receiving a composition comprising a rhPDGF-BB solution disposed in
a .beta.-TCP/collagen matrix did not demonstrate abnormalities
resulting from the administration of the rh-PDGF/matrix
composition.
[0175] Furthermore, vertebral bodies injected with a composition
comprising a rhPDGF-BB solution disposed in a .beta.-TCP/collagen
matrix additionally displayed increased volumetric bone mineral
densities. FIG. 4 illustrates percent change in volumetric bone
mineral density (vBMD) for vertebral bodies of animals of Group I
and Group II. Each data point in FIG. 4 represents an average of
all vertebral bodies treated within each group. For example, the
first data point in FIG. 4 for Group I is the average of nine
vertebral body measurements (T12, L2, and IA for each of three
animals in Group I) taken after injection of the rhPDGF-BB matrix
composition into the vertebral bodies. Similarly, the first data
point in FIG. 4 for Group 2 is the average of nine vertebral body
measurements (T12, L2 and L4 for each of three animals in Group II)
taken after injection of a collagen/.beta.-TCP matrix into the
vertebral bodies.
[0176] As illustrated in FIG. 4, vertebral bodies treated with a
composition comprising a rhPDGF-BB solution disposed in a
.beta.-TCP/collagen matrix (Group I) demonstrated a steady increase
in vBMD over the course of the study, the increase in vBMD becoming
statistically significant over the 1 week post-operative level by
the third month [2.64%+/-1.16 (week 1) v. 5.93%+/-1.33 (week 12);
p=0.023]. vBMD continued to increase through the sixth month of the
study before reaching a plateau at the ninth month. Vertebral
bodies treated with a composition comprising 20 mM sodium acetate
buffer disposed in the .beta.-TCP/collagen matrix (Group II),
however, did not demonstrate significant increases in vBMD over the
course of the study.
[0177] Additionally, FIG. 5 illustrates percent change in vBMD for
vertebral bodies of animals of Group I and Group II wherein the
injected .beta.-TCP/collagen matrix is subtracted from the
volumetric bone mineral density analysis. As in FIG. 4, each data
point in FIG. 5 represents an average of all vertebral bodies
treated within each group.
[0178] As illustrated in FIG. 5, vertebral bodies treated with a
rhPDGF-BB matrix composition (Group I) demonstrated increases in
vBMD. The subtraction of the .beta.-TCP/collagen matrix from the
volumetric bone mineral density analysis provided a clear
indication that vBMD increased throughout all regions of the
vertebral bodies of Group I as opposed to regions local to the
injection site of the rhPDGF-BB matrix composition.
[0179] All patents, publications and abstracts cited above are
incorporated herein by reference in their entirety. It should be
understood that the foregoing relates only to preferred embodiments
of the present invention and that numerous modifications or
alterations may be made therein without departing from the spirit
and the scope of the present invention as defined in the following
claims.
* * * * *