Note: Descriptions are shown in the official language in which they were submitted.
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CONCENTRATED PROTEIN PREPARATIONS OF
BONE MORPHOGENETIC PROTEINS AND METHODS OF USE
THEREOF
Field of the Invention
(0001] The invention relates to heretofore undescribed preparations of highly
concentrated, aqueous preparations of proteins typically insoluble at
physiological
conditions (for example, physiological pH and ionic strength). More
specifically,
the invention relates to highly concentrated protein preparations of Bone
Morphogenetic Proteins and their therapeutic uses.
Backeround
100021 Protein-based therapeutics have proven highly effective for a variety
of
disorders, injuries and diseases. Apart from the performance challenges
associated
with protein-based therapeutics, numerous other types of challenges arise
during the
development of such therapeutics, including protein processing considerations
such
as ease and cost of manufacturing, stability and shelf-life, as well as modes
of
administration, dosages and form of effective dosage, to name but a few.
[0003] Certain therapeutically significant proteins are particularly difficult
because
of their inherent insolubility under physiological conditions, such as but not
limited
to physiological pH. This insolubility is exacerbated when highly concentrated
aqueous solutions are desirable. One class of proteins which is inherently
insoluble
under physiological conditions is the TGF-beta superfamily of cysteine knot
proteins.
A similarly behaving subfamily includes the Bone Morphogenetic Proteins
(BMPs),
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for example, BMP-2, BMP-7 (also known as OP-1), GDF-5 (also Icnown as CDMP-
I and MP-52), GDF-6 (also known as BMP-13 and CDMP-2).
[0004) It is an object of the present invention to provide highly concentrated
aqueous protein preparations of such proteins, including but not limited to,
BMPs. It
is a further object to provide methods of treatment of skeletal and non-
skeletal
disorders, injuries and diseases using such preparations.
Summary of the Invention
[0005] The present invention is based on the discovery that heretofore
undescribed
aqueous preparations of proteins which are highly concentrated, i.e., at least
about
two- to about five-fold, preferably about three-fold, more concentrated than
those
known in the art, can be readily accomplished using the methods and reagents
disclosed herein. Briefly, aqueous protein solutions of at least about 4
mg/ml, can
be accomplished using a specified combination of ionic strength, pH and
buffering
systems; and, this solubility matrix of specific solubilization conditions
results in
highly efficient manufacture of such protein preparations with a high rate of
recovery. Of particular significance, the present invention's highly
concentrated
protein preparations permit the clinician to administer locally or
systemically
effective doses of proteins in minimal administration volumes, thereby
allowing
administration to physiologically-constrained sites such as intrajoint or
intra-
intervertebrai disc sites.
(0006] In one aspect, the invention is an aqueous preparation of Bone
Morphogenetic Protein (BMP) at high concentrations. As contemplated herein
below, other similar proteins can be used in the preparations of the present
invention.
In a preferred embodiment, the protein preparation comprises an aqueous
carrier and
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bone morphogenetic protein solubilized in said carrier at a concentration of
at least
about 10 mg/ml. Certain preferred embodiments have a protein concentration
greater than about 20 mg/ml; greater than about 25 mg/ml; greater than about
30
mg/ml; and, greater than about 40 mg/mi. In certain embodiments, the protein
concentration is in the range of about 10 to 60 mg/ml.
[0007] In one preferred protein preparation, the aqueous carrier has an ionic
strength of at least about 10 mM. In another, the aqueous carrier has an ionic
strength of no more than about 100 mM. In yet another, the aqueous carrier has
an
ionic strength of no more than about 10 mM; no more than about 20 mM; and, no
more than about 50 mM.
[0008) Certain preferred embodiments of the protein preparation of the present
invention comprise an aqueous carrier having a pH of at least about 2. Others
have a
pH of no more than about 5. More preferred embodiments have a pH of about 3.
In
certain embodiments, the pH is in the range from about 2 to about S.
[00091 In accordance with the present invention, a preferred protein
preparation
has a protein concentration of about 20 - 60 mg/ml; an ionic strength of about
0 - 50
mM; and a pH of about 2.5 - 4. A more preferred preparation comprises the
protein
described herein as BMP-7 in a concentration of more than 20 mg/ml; an ionic
strength of about 10 mM; and a pH of about 3.5.
[0010] Regarding each of the foregoing embodiments, the protein preparation is
solubilized at a temperature of about 25 C. The protein preparations of the
present
invention are preferably homogeneous solutions, i.e., preferably the solutions
contain no protein precipitate.
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[00111 In yet other embodiments, the protein preparations further comprise a
stabilizing excipient, such as but not limited to excipients selected from the
group
consisting of: sugars, polyols, surfactants, and any combination thereof. In
certain
other embodiments, the protein preparations comprise an aqueous carrier which
is a
buffer of the single acidic group type selected from the group consisting of:
potassium phosphate, proprionic acid, lactic acid, trifluoroacetic acid and
acetic
acid; or the two acidic group type selected from the group consisting of:
sodium
glutamate and sodium succinate. As contemplated herein, the preparations of
the
present invention can be lyophilized or can be a reconstituted lyophilate.
[00121 More generally, a protein suitable for use in the high concentration
preparations of the present invention can be mono- or dimeric; can have a
molecular
weight of about 25-50 kd; and can have a pI range of about 5-10. Generally,
proteins which are most suitable are hydrophobic proteins having poor
solubility
under physiological conditions, such as but not limited to physiological pH.
[00131 As described in detail elsewhere herein, certain preferred protein
preparations can comprise a protein known as a cysteine knot protein.
Exemplary
cysteine knot proteins include PDGF, VEGF and NGF. In other preferred
embodiments, the protein preparations comprise a member of the TGF-beta
superfamily of proteins. In more preferred embodiments, the protein
preparations
comprise a member of the BMP subfamily of the TGF-beta superfamily of
proteins.
Particularly preferred BMPS include one or more of BMP-2, BMP-4, BMP-5, BMP-
6, BMP-7, GDF-5, GDF-6 GDF-7, further including sequence variants of any one
of
the foregoing. Variants contemplated herein include but are not limited to a
protein
having at least about 50% amino acid sequence identity with a member of the
BMP
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subfamily within the conserved C-terminal cysteine-rich domain. More
particularly
preferred BMPs are BMP-2, GDF-5, GDF-6 and GDF-7. Most particularly
preferred is BMP-7.
100141 In another aspect, the present invention provides methods of treating
disorders, injuries and diseases of skeletal and non-skeletal tissues. For
example,
one preferred method of treating a skeletal tissue disorder, injury or disease
comprises the step of administering to a subject in need thereof any one of
the
aforementioned protein preparations, wherein said protein preparation is in a
dose
effective to treat said skeletal tissue disorder, injury or disease. In
certain prefecred
methods, the skeletal tissue is mineralized; in others it is non-mineralized
skeletal
tissue.
[00151 Preferred embodiments can effectively treat a skeletal tissue disorder,
injury or disease selected from the group consisting of metabolic bone
disease,
osteoarthritis, osteochondral disease, rheumatoid arthritis, osteoporosis,
bone
fractures, Paget's disease, periodontitis, and dentinogenesis. Other preferred
embodiments can effectively treat a non-mineralized skeletal tissue disorder,
injury
or disease selected from the group consisting of osteoarthritis, osteochondral
disease
or defect, chondral disease or defect, rheumatoid arthritis, trauma-induced
and
inflammation-induced cartilage degeneration, age-related cartilage
degeneration,
articular cartilage injuries and diseases, full thickness cartilage defects,
superficial
cartilage defects, sequelae of systemic lupus erythematosis, sequelae of
sclerodenma,
periodontal tissue regeneration, hierniation and rupture of intervertebral
discs,
degenerative diseases of the intervertebral disc (for example, degenerative
disc
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disease), osteocondrosis, and injuries and diseases of ligament, tendon,
synovial
capsule, synovial membrane and meniscal tissues.
[00161 Certain other preferred embodiments can effectively treat tissue injury
selected from the group consisting of: trauma-induced and inflammation-induced
cartilage degeneration, articular cartilage injuries, full thickness cartilage
defects,
superficial cartilage defects, hierniation and rupture of intervertebral
discs,
degeneration of intervertebral discs due to an injury(s), and injuries of
ligament,
tendon, synovial capsule, synovial membrane and meniscal tissues.
100171 In another embodiment, the invention provides a method of treating a
non-
skeletal tissue, comprising the step of administering to a subject in need
thereof one
or more of the foregoing protein preparations, wherein said protein
preparation is in
a dose effective to treat said non-skeletal tissue disorder, injury or
disease. Such a
method is effective to treat a disorder, injury or disease of a non-skeletal
tissue
selected from the group consisting of liver disease, liver resection,
hepatectomy,
renal disease, chronic renal failure, central nervous system ischemia or
trauma,
neuropathy, motor neuron injury, dendritic cell deficiencies and
abnormalities,
Parkinson's disease, ophthalmic disease, ocular scarring, retinal scarring,
ulcerative
diseases of the gastrointestinal tract, fibrosis, fibrotic disorders,
sclerodetma, and
pulmonary fibrosis.
(00181 A most preferred disease for treatment in accordance with the present
invention is osteoarthritis. In osteoarthritis and other disorders, injuries
or diseases
ofjoint tissues, an effective dose of a protein preparation can be
administered to the
synovial space such as but not limited to that of a knee, hip or articulating
joint. In
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one preferred embodiment, the protein preparation is administered via direct
injection into the synovial space.
[0019] Another most preferred disease for treatment in accordance with the
present invention is degenerative disc disease, also referred to herein as IVD
disease.
In the case of this disease, an effective dose can be administered to the
intradiscal
space, for example, via direct injection into the nucleus pulposus or annulus
fibrosus.
[0020] Generally, the methods of the present invention involve an
administering
step wherein the administration can be local or systemic. In a preferred
method, an
effective dose of protein is about 1 mg to 30 mg in about 100 microliters to 3
ml. A
more preferred method uses a preparation of protein selected from the group
consisting of: BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, GDF-5, GDF-6 GDF-7,
and sequence variants of any one of the foregoing. A most preferred method
uses a
preparation of BMP-7.
[0021] In other embodiments, the present methods can be performed pre- or post-
surgery. In certain other embodiments, the administering step can be performed
more than once.
[00221 In other embodiments of the present invention, the above-described
methods further comprise a step of admixing said protein preparation with a
suitable
matrix material prior to administration. In yet other embodiments, the above-
described methods further comprise a step of coating an implantable medical
device
with one or more of the previously described protein preparations.
[0023) In yet another aspect, the present invention contemplates a kit
comprising a
lyophilized bone morphogenetic protein and a reconstitution diluent, wherein
the
protein and the diluent are in separate containers, and wherein the amounts of
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diluent an protein are sufficient only for preparation of solubilized protein
at a
concentration of at least about 10 mg/ml. In certain embodiments, the kit
further
comprises a matrix. In others, the kit further comprises an implantable device
suitable for coating with the protein.
In a further aspect, the invention provides a kit for the treatment of a
disorder, injury
or disease (such as but not limited to those disorders, injuries and/or
diseases
described elsewhere herein) which comprises a lyophilized protein, preferably
a
bone morphogenetic protein, and a reconstitution diluent. In a preferred
embodiment, the protein and the diluent are in separate containers and further
the kit
comprises a plurality of separate containers each containing an amount of
diluent,
such that the amounts of diluent in a kit are sufficient to prepare
solubilized protein
preparations ranging in concentration from at least about 2 mg/ml to about 60
mg/ml
for use in the treatment of a disorder, injury or disease. In a further
embodiment, the
concentration and volume of solubilized protein preparations are customized
for
treatment of the particular target disorder, injury or disease.
[0024] In another aspect, the present invention provides a method for
manufacturing a concentrated form of lyophilized recombinant bone
morphogenetic
protein comprising the steps of: (1) providing a processing solution of
recombinant
bone morphogenetic protein, the weight per volume of protein in said
processing
solution being less than the weight per volume specified for lyophilization;
(2)
adjusting the weight per volume of protein in said processing solution to
produce a
lyophilization solution, the weight per volume of protein in said
lyophilization
solution being greater than that of the processing solution; (3) filling a
vial suitable
for lyophilization with a specified volume of said lyophilization solution;
and, (4)
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lyophilizing said lyophilization solution to manufacture a concentrated form
of
lyophilized recombinant bone morphogenetic protein. In a related embodiment,
the
method further comprises the step of reconstituting said lyophilized
recombinant
bone morphogenetic protein in a reconstitution diluent wherein the volume of
diluent is the same as the volume in step 3 identified above. In another
related
embodiment, the method further comprises the step of reconstituting said
lyophilized
recombinant bone morphogenetic protein in a reconstitution diluent wherein the
volume of diluent is less than the volume in step 3 identified above. In
certain
preferred embodiments of the kit, the concentrated form is achieved post-
processing
and contains solubilized bone morphogenetic protein in a concentration of at
least 10
mg/ml. In a most preferred embodiment, a kit comprises a concentrated form of
recombinant bone morphogenetic protein prepared according to the method set
forth
immediately above.
100251 In yet another aspect, the present invention provides a method for
preparing
a concentrated form of bone morphogenetic protein comprising the steps of:
(1) providing a processing solution of bone morphogenetic protein, the
weight per volume of protein in said processing solution being the same as
the weight per volume specified for lyophilization;
(2) filling a vial suitable for lyophilization with a specified volume of
said processing solution thereby producing a lyophilization solution;
(3) lyophilizing said lyophilization solution; and,
(4) packaging the lyophilized lyophilization solution together with a
specified volume of reconstitution diluent, the volume of said diluent being
less than the specified fill volume of step 3.
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[0026] In one preferred embodiment of this method, the method further
comprises
the step of adjusting the weight per volume of protein in the processing
solution to
produce an adjusted lyophilization solution, the weight per volume of protein
in said
adjusted lyophilization solution being greatei than that of the processing
solution.
[00271 In yet another aspect, the present invention provides a method for
preparing
a concentrated form of protein in lyophilized or other reconstitutible non-
liquid form
comprising the steps of
(1) providing a processing solution of protein, the weight per volume of
protein in said processing solution being the same as the weight per volume
specified for lyophilization or other reconsitutible non-liquid form;
(2) providing a vial suitable for lyophilization (or other form) for
containing a specified volume of said processing solution thereby producing
a lyophilization (or other form) solution;
wherein the weight per volume of protein in each of the processing and
lyophilization (or other form) solution is at least 2 mgtml.
[0028] In a preferred embodiment, this method can further comprise the step of
providing a lyophilized form of the lyophilization solution or a
reconstitutible form
of the other non-liquid form. In another preferred embodiment, the method can
further comprise the step of providing a specified volume of diluent for
rehydrating
the lyophilized form or reconstituting the other reconstitutible non-liquid
form, the
volume of said diluent being sufficient to permit preparation of an aqueous
protein
preparation having at least 2 mg/ml; or further comprising the step of
providing
instructions for rehydrating the lyophilized form or reconstituting the other
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reconstitutible non-liquid form so as to product an aqueous protein
preparation
having at least 2 mg/ml.
[0029] The present invention contemplates that a protein preparation prepared
in
accordance with any one of the methods described herein can be used to treat a
disorder, injury or disease such as but not limited to those disorders,
injuries and/or
diseases described elsewhere herein.
Figures
[0030] Figure 1 illustrates certain exemplary aqueous protein preparations in
accordance with the present invention.
Detailed Description
[00311 The present invention is based on the discovery that heretofore
undescribed
aqueous preparations of proteins which are highly concentrated, i.e., at least
about
two- to about five-fold, preferably about three-fold, more concentrated than
those
known in the art, can be readily accomplished using the methods and materials
disclosed herein. Briefly, aqueous protein solutions of at least about 4
mg/ml, can
be accomplished using a specified combination of ionic strength, pH and
buffering
systems; and, this solubility matrix of specific solubilization conditions
results in
highly efficient manufacture of such protein preparations with a high rate of
recovery. Of particular significance, the present invention's highly
concentrated
protein preparations permit the clinician to administer locally or
systemically
effective doses of proteins in minimal administration volumes, thereby
allowing
administration to physiologically-constrained sites such as intrajoint or
intra-
intervertebral disc sites.
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Protein Preparation Considerations
(00321 In certain preferred embodiments of the present invention, protein
concentrations range from about 10 mg/ml to about 60 mg/ml. In certain other
preferred embodiments, the protein concentration is at least about 4 mg/ml but
not
more than about 100 mg/ml. A particularly preferred range is about 20 to about
40
mg/mi. A more preferred range is about 10 to about 30 mg/ml. A most preferred
range is about 5 to about 30 mg/ml. In a currently most preferred embodiment,
the
concentration is at least about 10 mg/ml. A most preferred concentration is
about 15
mg/mi. In an even more currently preferred embodiment, the concentration is
about
20 mg/m1. In another, it is greater than about 20 mg/ml. In yet another, it is
greater
than about 25 mg/ml. In the case of BMP-7, a most preferred concentration
range is
about 10 to about 40 mg/ml and a most preferred concentration is about 20
mg/ml.
Preferably, no protein precipitate is observed in any of the foregoing protein
preparations of the present invention when held at room temperature (about 25
C).
And, the foregoing protein preparations are stable in solubilized forms as
well as
lyophilized forms when prepared and forrnulated in accordance with the
teachings
set forth herein.
(0033] In certain preferred embodiments of the above-described protein
preparations, ionic strength ranges from at least about 10 mM to no more than
about
100 mM. In certain other preferred embodiments, ionic strength is at least
about 20
mM but not more than about 50 mM. A particularly preferred range is about 25
mM
to about 40 mM. A more preferred range is about 10 mM to about 20 mM. A most
preferred range is about 0 to about 20 mM. In a cun:ently most preferred
embodiment, the ionic strength is about 10 mM. In an even more currently
preferred
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embodiment, the ionic strength is about 5 mM. A most preferred ionic strength
is
about 0 mM. In the case of BMP-7, a most preferred range is about 0 to about
50
mM and a most preferred ionic strength is about 10 mM. Preferably, no
precipitate
is observed in any of the foregoing protein preparations when held at room
temperature (about 25 C).
[0034] In one preferred protein preparation, the aqueous carrier has an ionic
strength of at least about 10 mM. In another, the aqueous carrier has an ionic
strength of in the range of 0 to about 100 mM. In yet another, the aqueous
carrier
has an ionic strength from 0 to about 10 mM; from 0 to about 20 mM; and, from
0 to
about 50 mM.
[0035] In certain preferred embodiments of the above-described protein
preparations, preferred pH ranges are from at least about 2 to no more than
about 5.
In certain other preferred embodiments, pH is at least about 2 but not more
than
about 4. A particularly preferred range is about 3 to about 4. A more
preferred
range is about 2.5 to about 3.5. A most preferred range is about 2.5 to about
4. In a
currently most preferred embodiment, the pH is about 2.5. In an even more
currently preferred embodiment, the pH is about 3. A most prefenred pH is
about
3.5. In the case of BMP-7, a most preferred pH range is about 2.4 to about 4
and a
most preferred pH is about 3.5. No precipitate is observed in any of the
foregoing
protein preparations when held at temperatures ranging from about 4 C to about
C.
[0036] In certain preferred embodiments of the above-described protein
preparations, preferred pI ranges are from at least about 5 to no more than
about 10.
In certain other preferred embodiments, pI is at least about 7 but not more
than about
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9. A particularly preferred range is about 6.5 to about 9. A more preferred
range is
about 7.5 to about 8.5. A most preferred range is about 6.5 to about 10. In an
even
more currently preferred embodiment, the pl is about 6.5 to about 9. In the
case of
BMP-7, a most preferred pI range is about 6.5 to about 9 and a most preferred
pI is
about 9. Preferably, no proetin precipitate is observed in any of the
foregoing
protein preparations when held at room temperature (about 25 C).
[00371 The protein preparations of the present invention can be formulated
using
acid buffers. Specifically, single acidic buffers as well as double acidic
buffers can
be used successfully. The aqueous carrier comprises a buffer of the single
acidic
group type selected from the group consisting of: potassium phosphate,
proprionic
acid, lactic acid, trifloroacetic acid and acetic acid; or the two acidic
group type
selected from the group consisting of= sodium glutamate and sodium succinate.
More preferred acids include lactic acid, TFA, potassium phosphate. Lactic
acid is
most preferred. Other acids, such as citric acid, ascorbic acid, and sodium
phosphate
can also be readily employed when practicing the present invention.
[00381 Certain embodiments of the protein preparations of the present
invention
further comprise a stabilizing excipient selected from the group consisting of
sugars,
polyols and surfactants. Glucose, sucrose, raffinose, trehalose, lactose are
among
the preferred sugars. Lactose is prefened. Trehalose is most preferred.
Mannitol
and sorbitol are among the preferred polyols. Mannitol is most preferred.
Preferable concentrations of sugars or polyols range from about 0% to 10%;
most
preferably 2.5% to 10%. Tween 80, Tween 20, and Pluronic F-68 are among the
preferred non-ionic surfactants useful as stabilizing excipients. Tween 80 and
Tween 20 are most preferred. Concentrations of these surfactants range from
about
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0.01% to about 0.1%0. Generally, the range of non-ionic surfactant
concentrations
useful herein is about 0.005% to about 10%. Combinations of any one or more of
the foregoing stabilizing excipients are contemplated herein.
Bone Morphogenetic Proteins (BMPs) and Other Preferred Proteins
100391 The present invention contemplates usefut and preferred proteins to
have
one or more of the following features: The protein is mono- or dimeric. The
protein
is basic. The protein exhibits a pI of about 5 to about 10; preferably about 6
to about
9 or about 5 to about 7 or about 7.5 to about 9. The protein has a molecular
weight
of about 25 kd to about 50 kd. The protein is hydrophobic. The protein is
insoluble
under physiological conditions, especially at concentrations in excess of
about 4
mg/mi. The present invention contemplates one such type of preferred proteins
known as cysteine knot proteins (See, McDonald et al., A Structural
Superfamily of
Growth Factors Containing a Cystine Knot Motif, Cell 73: 421-424 (1993)). The
present invention further contemplates another more preferred type of such
proteins
known as the TGF-beta superfamily of proteins. And, the present invention
further
contemplates another most preferred type of such proteins known as the Bone
Morphogenetic Proteins (BMPs).
100401 As stated above, BMPs are a preferred exemplary protein for purposes of
the present invention. BMPs belong to the TGF-R superfamily. The TGF-0
superfamily proteins are cytokines characterized by six-conserved cysteine
residues.
The human genome contains about 42 open reading frames encoding TGF-0
superfamily proteins. The TGF-(i subfamily includes, but is not limited to,
TGFs
(e.g., TGF-01, TGF-02, and TGF-03), activins (e.g., activin A) and inhibins,
macrophage inhibitory cytokine-1 (MIC- 1), Mullerian inhibiting substance,
anti-
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Mullerian hormone, and filial cell line derived neurotrophic factor (GDNF).
Structurally, such proteins are homo or heterodimers expressed as large
precursor
polypeptide chains containing a hydrophobic signal sequence, an N-terminal pro
region of several hundred amino acids, and a mature domain comprising a
variable
N-terminal region and a highly conserved C-terminal region containing
approximately 100 amino acids with a characteristic cysteine motif having a
conserved six or seven cysteine skeleton. These structurally-related proteins
have
been identified as being involved in a variety of developmental events. As
used
herein, "TGF-R subfamily," "TGF-as," "TGF-0 ligands" and grammatical
equivalents thereof refer to the TGF-[i subfamily members, unless specifically
indicated otherwise.
[00411 The TGF-P superfamily proteins can at least be divided into the BMP
subfamily and the TGF-(i subfamily based on sequence similarity and the
specific
signaling pathways that they activate. The BMP subfamily includes, but is not
limited to, BMP-2, BMP-3 (osteogenin), BMP-3b (GDF-10), BMP-4 (BMP-2b),
BMP-5, BMP-6, BMP-7 (OP-1, osteogenic protein-1), BMP-8 (OP-2), BMP-8B
(OP-3), BMP-9 (GDF-2), BMP-10, BMP-11 (GDF-11), BMP-12 (GDF-7), BMP-13
(GDF-6, CDMP-2), BMP-15 (GDF-9), BMP-16, GDF-1, GDF-3, GDF-5 (CDMP-1,
MP-52), and GDF-8 (myostatin). For purposes of the present invention,
preferred
superfamily proteins include BMP-2, -4, -5, -6 and -7 and GDF-5, -6, and -7,
as well
as MP-52. Particularly preferred proteins include BMP-2, BMP-7 and GDF-5, -6,
and -7. A most preferred exemplary BMP is BMP-7. Preferred BMPs can have a
six- or seven-cysteine conserved region in their C-terminal region. BMPs are
also
present in other animal species. Furthermore, there is allelic variation in
BMP
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sequences among different members of the human population, and there is
species
variation among BMPs discovered and characterized to date. As used herein,
"BMP
subfamily," "BMPs," "BMP ligands" and grammatical equivalents thereof refer to
the BMP subfamily members, unless specifically indicated otherwise.
[0042] The TGF-0 superfamily is in tum a subset of the cysteine knot cytokine
superfamily. Additional members of the cysteine knot cytokine superfamily
include,
but are not limited to, platelet derived growth factor (PDGF), vascular
endothelial
growth factor (VEGF), placenta growth factor (PIGF), noggin, neurotrophins
(BDNF, NT3, NT4, and (3NGF), gonadotropin, follitropin, lutropin, interleukin-
17,
and coagulogen.
[0043] Publications describing certain of these preferred proteins, as well as
their
chemical and physical properties, include: BMP-7 and OP-2 (U.S. Pat. No.
5,011,691; U.S. Pat. No. 5,266,683; Ozkaynak et al., EMBO J., 9, pp. 2085-2093
(1990); OP-3 (W094/10203 (PCT US93/10520)), BMP-2, BMP-4, (W088/00205;
Wozney et al. Science, 242, pp. 1528-1534 (1988)), BMP-5 and BMP-6, (Celeste
et
al., PNAS, 87, 9843-9847 (1991)), Vgr-1 (Lyons et al., PNAS, 86, pp. 4554-4558
(1989)); DPP (Padgett et al. Nature, 325, pp. 81-84 (1987)); Vg-1 (Weeks,
Cell, 51;
pp. 861-867 (1987)); BMP-9 (W095/33830 (PCT/US95/07084); BMP-10
(W094/26893 (PCT/US94/05290); BMP-11 (W094/26892 (PCT/US94/05288);
BMP-12 (W095/16035 (PCT/US94/14030); BMP-13 (W095/16035
(PCT/US94/14030); GDF-1 (W092/00382 (PCT/US91/04096) and Lee et al. PNAS,
88, pp. 4250-4254 (1991); GDF-8 (W094/21681 (PCT/US94/03019); GDF-9
(W094/15966 (PCT/US94/00685); GDF-10 (W095/10539 (PCT/US94/11440);
GDF-11 (W096/01845 (PCT/US95/08543); BMP-15 (W096/36710
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(PCT/US96/06540); MP- 121 (W096/01316 (PCT/EP95/02552); GDF-5 (CDMP- 1,
MP52) (W094/15949 (PCT/US94/00657) and W096/14335 (PCT/US94/12814)
and W093/16099 (PCT/EP93/00350)); GDF-6 (CDMP-2, BMP13) (W095/01801
(PCT/US94/07762) and W096/14335 and W095/10635 (PCT/US94/14030)); GDF-
7 (CDMP-3, BMP12) (W095110802 (PCT/US94/07799) and W095/10635
(PCT/US94/14030)) The above publications are incorporated herein by reference.
100441 The term "morphogenic protein" refers to a protein belonging to the TGF-
f3
superfamily of proteins which has true morphogenic activity. For instance,
such a
protein is capable of inducing progenitor cells to proliferate and/or to
initiate a
cascade of events in a differentiation pathway that leads to the formation of
cartilage,
bone, tendon, ligament, neural or other types of differentiated tissue,
depending on
local environmental cues. Thus, morphogenic proteins useful in this invention
can
behave differently in different surroundings. In certain embodiments, a
morphogenic protein of this invention can be a homodimer species or a
heterodimer
species. The term "osteogeniaprotein (OP)" refers to a morphogenic protein
that is
also capable of inducing a progenitor cell to form cartilage and/or bone. The
bone
can be intramembranous bone or endochondral bone. Most osteogenic proteins are
members of the BMP subfamily and are thus also BMPs. However, the converse
can not be true. According to this invention, a BMP identified by DNA sequence
homology or amino acid sequence identity must also have demonstrable
osteogenic
or chondrogenic activity in a functional bioassay to be an osteogenic protein.
Appropriate bioassays are well known in the art; a particularly useful
bioassay is the
heterotopic bone formation assay (see, U.S. Pat. No. 5,011,691; U.S. Pat. No.
5,266,683, for example).
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[0045) Structurally, BMPs are dimeric cysteine knot proteins. Each BMP
monomer comprises multiple intramolecular disulfide bonds. An additional
intermolecular disulfide bond mediates dimerization in most BMPs. BMPs may
form homodimers. Some BMPs may form heterodimers. BMPs are expressed as
pro-proteins comprising a long pro-domain, one or more cleavage sites, and a
mature domain. The pro-domain is believed to aid in the correct folding and
processing of BMPs. Furthermore, in some but not all BMPs, the pro-domain may
noncovalently bind the mature domain and may act as an inhibitor (e.g., Thies
et al.
(2001) Growth Factors 18:251-259).
[0046] BMPs are naturally expressed as pro-proteins comprising a long pro-
domain, one or more cleavage sites, and a mature domain. This pro-protein is
then
processed by the cellular machinery to yield a dimeric mature BMP molecule.
The
pro-domain is believed to aid in the correct folding and processing of BMPs.
Furthermore, in some but not all BMPs, the pro-domain may noncovalently bind
the
mature domain and may act as a chaperone, as well as an inhibitor (e.g., Thies
et. Al.
(2001) Growth Factors, 18:251-259).
[00471 As further contemplated herein, the term "BMP" refers to a protein
belonging to the BMP subfamily of the TGF-0 superfamily of proteins defined on
the basis of DNA homology and amino acid sequence identity. According to this
invention, a protein belongs to the BMP subfamily when it has at least 50%
amino
acid sequence identity with a known BMP subfamily member within the conserved
C-terminal cysteine-rich domain that characterizes the BMP subfamily. Members
of
the BMP subfamily can have less than 50% DNA or amino acid sequence identity
overall. As used herein, the term "BMP"also embraces proteins which are amino
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acid sequence variants, domain-swapped variants, and/o truncations and active
fragments of naturally occurring bone morphogenetic proteins, as well as
heterodimeric proteins formed from two different monomeric BMP peptides, such
as
BMP-2/7; BMP-4/7: BMP-2/6; BMP-2/5; BMP-4/7; BMP-4/5; and BMP-4/6
heterodimers. Suitable BMP variants and heterodimers include those set forth
in US
2006/0235204; WO 05/097825; WO 00/020607; WO 00/020591; WO 00/020449;
WO 05/113585; WO 95/016034 and W093/009229.
[0048] As contemplated herein, useful BMPs include those containing sequences,
which are homologues or variants, that share at least 50%, preferably at least
60%,
more preferably at least 70% and most preferably at least 85%, amino acid
sequence
identity with the C-terminal cysteine domain of BMP-2, BMP4, BMP-5, BMP-6,
BMP-7, GDF-5, GDF-6, or GDF-7. As contemplated herein, preferred BMPs
include biologically active variants of any such BMPs, including variants
containing
conservative amino acid substitutions. All that is required by the present
invention
is that these variants retain biological activity comparable to the native
form. As
used herein, the term "BMP related protein" or "BMP related proteins" means
any
one or all of the foregoing proteins.
[0049) Proteins useful herein also include any known naturally occurring
native
proteins exhibiting one or more of the above-identified preferable features,
including
allelic, phylogenetic counterparts and other variants thereof which exhibit
one or
more of the aforementioned properties. Variants include forms having varying
glycosylation patterns, varying N-termini, and active truncated or mutated
forms of a
native protein. Useful proteins also include those that are biosynthetically
produced
(e.g., "muteins" or "mutant proteins"). Moreover, the proteins contemplated
herein
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include biologically active variants of any of the above-listed proteins,
including
variants containing conservative amino acid changes as described elsewhere
herein;
and osteogenically active proteins having the conserved seven-cysteine
skeleton or
domain as defined below. For instance, useful osteogenic proteins also include
those containing sequences that share at least 70% amino acid sequence
homology
with the C-terminal seven-cysteine domain of BMP-7. To determine the percent
homology of a candidate amino acid sequence to that seven-cysteine domain, the
candidate sequence and the sequence of the domain are aligned. The alignment
can
be made with, e.g., the dynamic programming algorithm described in Needleman
et
al., J. Mol. Biol. 48:443 (1970), and the Align Program, a commercial software
package produced by DNAstar, Inc. The teachings by both sources are
incorporated
by reference herein. An initial alignment can be refined by comparison to a
multi=
sequence alignment of a family of related proteins. Once the alignment between
the
candidate sequence and the seven-cysteine domain is made and refined, a
percent
homology score is calculated. The aligned amino acid residues of the two
sequences
are compared sequentially for their similarity to each other. Similarity
factors
include similar size, shape and electrical charge. One particularly preferred
method
of determining amino acid similarities is the PAM250 matrix_ described in
Dayhoff
et al., Atlas of Protein Sequence and Structure 5:345-352 (1978 & Supp.),
herein
incorporated by reference. A similarity score is fiust calculated as the sum
of the
aligned pairwise amino acid similarity scores. Insertions and deletions are
ignored
for the purposes of percent homology and identity. Accordingly, gap penalties
are
not used in this calculation. The raw score is then normalized by dividing it
by the
geometric mean of the scores of the candidate sequence and the seven-cysteine
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domain. The geometric mean is the square root of the product of these scores.
The
normalized raw score is the percent homology.
(00501 Furthetmore, other useful proteins also include those containing
sequences
that share greater than 60% identity with the seven-cysteine domain of the BMP
subfamily. In certain preferred embodiments, useful osteogenic proteins
include
those having an amino acid sequence sharing at least 70% (e.g., at least 80%)
sequence homology or "similarity" with all or part of a naturally occurring
reference
morphogenic protein. A preferred reference protein is human BMP-7. Other known
osteogenic proteins can also be used as a reference sequence. In one
embodiment, a
candidate amino acid sequence can be aligned with a reference amino acid
sequence
by using the method of Needleman et al., J. Mol. Biol. 48:443-453 (1970),
implemented conveniently by computer programs such as the Align program
(DNAstar, Inc.). Intetnal gaps and amino acid insertions in the candidate
sequence
are ignored for purposes of calculating the level of homology or identity
between the
candidate and reference sequences. "Amino acid sequence homology" is
understood
herein to include both amino acid sequence identity and similarity. Homologous
sequences share identical and/or similar amino acid residues, where similar
residues
are conservative substitutions for, or "allowed point mutations" of,
corresponding
amino acid residues in an aligned reference sequence. Thus, a candidate
polypeptide
sequence that shares 70% amino acid homology with a reference sequence is one
in
which any 70% of the aligned residues are either identical to, or are
conservative
substitutions of, the corresponding residues in a reference sequence. Certain
particularly prefetred morphogenic polypeptides share at least 60% (e.g., at
least
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65%) amino acid sequence identity with the C-terminal seven-cysteine domain of
human BMP-7.
[00511 As used herein, "conservative substitutions" are residues that are
physically
or functionally similar to the corresponding reference residues. That is, a
conservative substitution and its reference residue have similar size, shape,
electric
charge, chemical properties including the ability to fonm covalent or hydrogen
bonds,
or the like. Prefetred conservative substitutions are those fulfilling the
criteria
defined for an accepted point mutation in Dayhoff et al. (1978), 5 Atlas of
Protein
Sequence and Structure, Suppl. 3, Ch. 22, pp. 354-352, Natl. Biomed. Res.
Found.,
Washington, D.C. 20007. Examples of conservative substitutions are
substitutions
within the following groups: (a) valine, glycine; (b) glycine, alanine; (c)
valine,
isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine,
glutamine; (f)
serine, threonine; (g) lysine, arginine, methionine; and (h) phenylalanine,
tyrosine.
The term "conservative variant" or "conservative variation" also includes the
use of
a substituting amino acid residue in place of an amino acid residue in a given
parent
amino acid sequence, where antibodies specific for the parent sequence are
also
specific for, i.e., "cross-react" or "imuno-react" with, the resulting
substituted
polypeptide sequence.
Manufacture of Concentrated Protein Preparations: General Considerations
[0052] The methods of the present invention increase ease of protein
processing
during manufacturing, while reducing in-process product losses and yielding a
high
quality, final desired high concentration protein preparation of the present
invention.
Such preparations evidence higher recoveries, reduced aggregation and permit
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improved fill-level accuracy as compared with art-recognized methods routinely
employed by scientists and process engineers.
[0053) An exemplary method follows which describes processing (preparation,
fill,
lyophilization, and finish operations) a preferred protein, BMP-7, at low
concentrations (5 20 mg/mL), while yielding a product that can be
reconstituted to a
desired target higher protein concentration (20 to 40 mg/mL). This methodology
of
the present invention allows execution of all manufacturing operations at a
lower
protein concentration-this increases ease of processing as the viscosity of
the
solution handled is lower and it also reduces in-process losses during
manufacturing
(since a fixed volume lost contains a lower mass of protein due to the lower
concentration used during processing). The lower concentration used during
processing also allows for the product to be stored and processed at
refrigerated
temperatures (2 to 8 C), without resulting in a dramatic increase in viscosity
as
would otherwise be observed if high protein concentrations were used during
processing.
[00541 Usually drug products are processed and filled at the same
concentration as
the final desired target. For example, if the target protein concentration for
administration is 40 mg/mL, the protein preparation (in the presence of
appropriate
stabilizing excipients) would typically be filled at 40 mg/mL and the freeze-
dried
product reconstituted with the same volume of diluent as the fill volume prior
to
lyophilization (freeze-drying).
[00551 In the methodology disclosed herein, a different approach is used. For
example, if it is desired to have 1 mL of protein solution at 40 mg/mL for
administration, the processing (upstream manufacturing) would be conducted at
a
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lower concentration, say 20 mg/mL. The fill would be 2 mL at 20 mg/mL, and the
product would be reconstituted with 1 mL reconstitution diluent following
freeze-
drying. _ Hence, the manufacturing (processing) operations are conducted at a
lower
concentration than the fmal target yielding the following benefits: (a) this
increases
ease of processing as the viscosity of the solution handled is lower, (b) it
also
reduces in-process losses during manufacturing (since a fixed volume lost
contains a
lower mass of protein due to the lower concentration used during processing),
(c) the
lower concentration used during processing also allows for the in-process
intermediates to be stored (and/or processed) at refrigerated temperatures (2
to 8 C),
without resulting in a dramatic increase in viscosity as would otherwise be
observed
if high protein concentrations were used during processing (See Figure 1).
[0056] By way of another example as illustrative of the present invention, if
a
target volume of V mL is desired at a protein concentration of C mg/mL for
administration, the protein preparation/fill/finish operations can be
conducted at C/N
mg/mL with a fill volume of V*N mL, with final reconstitution of the
lyophilized
product with V mL reconstitution diluent, where N (N > 1) is a suitable
scaling
factor, which can be appropriately chosen based on the viscosity and
processability
of the protein solution. Preferably, N equals about 2. A preferred range is N
equals
about 2 to about 4; another more preferred range is N equals about 2 to about
10.
[0057] This methodology allows execution of all manufacturing operations at a
lower protein concentration-this increases ease of processing as the viscosity
of the
solution handled is lower and it also reduces in-process losses during
manufacturing
(since a fixed volume lost contains a lower mass of protein due to the lower
concentration used during processing). The lower concentration used during
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processing also allows for the product to be stored and processed at
refrigerated
temperatures (2 to 8 C), without resulting in a dramatic increase in viscosity
as
would otherwise be observed if high protein concentrations were used during
processing.
[0058[ In addition, the above advantages also serve to yield a product with
higher
quality attributes (for example, if processing is done at a lower protein
concentration
then critical physicochemical protein attributes such as aggregation tend to
be lower
than if the protein was processed at a higher concentration) and lower cost of
goods
(lower COGS due to smaller mass loss of protein and a higher quality product,
as
discussed above).
[0059[ The above-described protocols have been successfully implemented to
manufacture protein preparations ranging from about 10 to about 60 mg/ml.
Using a
titration matrix of varying ionic strengths, pHs and protein concentrations
(mg/mi),
it was discovered that highly concentrated preparations of protein in aqueous
carriers
are possible without risk of precipitation. Generally, as ionic strength is
decreased,
pH is contemporaneously decreased thereby maintaining a solubilized protein in
an
aqueous carrier comprising one or more of the buffer systems described
elsewhere
herein. That is, using a titration matrix in which ionic strength and pH are
simultaneously titrated downward, it is possible to achieve conditions
suitable for
maintaining solubilized protein at heretofore undescribed high concentrations
in an
aqueous carrier. As described elsewhere, certain preferred combinations of
protein
concentration, pH and ionic strength using certain preferred buffering systems
result
in a highly stable, aqueous preparation of concentrated protein unlike
anything
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previously described in the art. Stabilizing excipients can be used optionally
as
described earlier.
[0060[ In yet another manufacturing aspect, the present invention also
provides a
method for preparing a concentrated form of bone morphogenetic protein
comprising the steps of
(1) providing a processing solution of bone morphogenetic protein, the
weight per volume of protein in said processing solution being the same as
the weight per volume specified for lyophilization;
(2) filling a vial suitable for lyophilization with a specified volume of
said processing solution thereby producing a lyophilization solution;
(3) lyophilizing said lyophilization solution; and,
(4) packaging the lyophilized lyophilization solution together with a
specified volume of reconstitution diluent, the volume of said diluent being
less than the specified fill volume of step 3.
[00611 In one preferred embodiment of this method, the method further
comprises
the step of adjusting the weight per volume of protein in the processing
solution to
produce an adjusted lyophilization solution, the weight per volume of protein
in said
adjusted lyophilization solution being greater than that of the processing
solution.
[00621 In still another manufacturing aspect, the present invention also
provides a
method for preparing a concentrated form of protein in lyophilized or other
reconstitutible non-liquid form comprising the steps of:
(1) providing a processing solution of protein, the weight per volume of
protein in said processing solution being the same as the weight per volume
specified for lyophilization or other reconsitutible non-liquid form;
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(2) providing a vial suitable for lyophilization (or other fonn) for
containing a specified volume of said processing solution thereby producing
a lyophilization (or other form) solution;
wherein the weight per volume of protein in each of the processing and
lyophilization (or other form) solution is at least 2 mg/ml.
[00631 In a preferred embodiment, this method can further comprise the step of
providing a lyophilized form of the lyophilization solution or a
reconstitutible form
of the other non-liquid form. In another preferred embodiment, the method can
further comprise the step of providing a specified volume of diluent for
rehydrating
the lyophilized form or reconstituting the other reconstitutible non-liquid
form, the
volume of said diluent being sufficient to permit preparation of an aqueous
protein
preparation having at least 2 mg/ml; or further comprising the step of
providing
instructions for rehydrating the lyophilized form or reconstituting the other
reconstitutible non-liquid form so as to product an aqueous protein
preparation
having at least 2 mg/ml.
100641 In summary, the present invention contemplates that a protein
preparation
prepared in accordance with any one of the methods taught herein can be used
to
treat a disorder, injury or disease such as but not limited to those
disorders, injuries
and/or diseases described elsewhere herein.
Kits
100651 The present invention also provides kits useful for the treatment of
skeletal
or non-skeletal tissue disorders, injuries or diseases. The kits are
particularly useful
for treating joints impacted by disease, especially osteoarthritis and
osteochondral
disease; and for treating intervertebral discs affected by injury or disease.
In a
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prefeffed embodiment, the kits of the present invention comprise one or more
protein preparations, and one or more reconstitution diluents. Optionally, a
kit of
the present invention can further comprise one or more additional biologically
active
agents. In a particularly preferred embodiment, the protein is a BMP. In a
still more
particularly preferred embodiment, the protein is BMP-7. The kits of the
present
invention can also comprise a matrix for admixture with the protein for local
implantation at a site of injury or disease; or an implantable medical device
suitable
for coating with the protein prior to implantation. In a further aspect, the
invention
also provides a kit for the treatment of a disorder, injury or disease (such
as but not
limited to those disorders, injuries and/or diseases described elsewhere
herein)
which comprises a lyophilized protein, preferably a bone morphogenetic
protein,
and a reconstitution diluent. In a preferred embodiment, the protein and the
diluent
are in separate containers and further the kit comprises a plurality of
separate
containers each containing an amount of diluent, such that the amounts of
diluent in
a kit are sufficient to prepare solubilized protein preparations ranging in
concentration from at least about 2 mg/ml to about 60 mg/mI for use in the
treatment
of a disorder, injury or disease. In a further embodiment, the concentration
and
volume of solubilized protein preparations are customized for treatment of the
particular target disorder, injury or disease.
Therapeutic Interventions and Methods of Treatment
[0066] In the case of skeletal disorders, a number of factors can cause or
contribute to cartilage degeneration in mammals, including trauma and
inflammatory disease. Damage to cells resulting from the effects of
inflammatory
response has been implicated as the cause of reduced cartilage function or
loss of
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cartilage function in diseases of the joints (e.g., rheumatoid arthritis (RA)
and
osteoarthritis (OA)). In addition, autoimmune diseases such as systemic lupus
erythematosis (SLE) and sclerodenna can also be characterized by a degradation
of
connective tissue. In the case of some cartilage degenerative diseases such as
osteoarthritis (OA), the mechanisms that turn the normal aging of articular
cartilage
into the pathological OA process are currently unknown. Each of the foregoing
diseases can be effectively treated with the materials and methods of the
present
invention.
100671 As stated earlier, the BMP preparations of the invention can be used
effectively to treat skeletal diseases or injuries. For example, the
preparations can be
used to treat a bone fracture, such as an open fracture or a closed fracture.
For the
treatment of a closed fracture, the preparation is preferably injected at the
fracture
site. For open fractures, critical size defects or persistent nonunions, the
preparations can be administered by surgical implantation at the fracture
site. In
both cases, the preparation can be administered alone, or in combination with
a
suitable carrier, matrix or scaffold, such as a bone cement, a calcium
phosphate
material, a gel material or a collagen matrix. Suitable carriers, matrices and
scaffolds include those disclosed in US Patent Nos. 6,919,308; 6,949,251; and
7,041,641.
[0068] In a preferred embodiment, the BMP preparations of the invention can be
used to treat a disease or injury resulting in cartilage degradation or a
cartilage
defect. For example, the preparations can be applied to a cartilage defect
site, such
as a degenerative intervertebral disc, or other fibrocartilaginous tissue,
including a
tendon, a ligament or a meniscus. Such methods are set out in U.S. Patent No.
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6,958,149. The preparations of the invention can also be used to treat a
defect or
degeneration of articular cartilage, as set forth in published PCT application
WO
05/115438, such as the cartilage lining of ajoint, such as a synovial joint,
including
a knee, an elbow, a hip, or a shoulder. In this embodiment, the preparation is
preferably injected into the synovial space of the joint. In another
embodiment, the
preparations of the invention are used to treat an articular cartilage defect
site, such
as a chondral defect or an osteochondral defect, in a joint. Such articular
cartilage
defects can be the result of a disease process, such as osteoarthritis or
rheumatoid
arthritis, or due to injury of the joint. In this embodiment, the preparation
can be
injected into the joint space or it can be surgically implanted. For example,
the
preparation can be placed within the defect either alone or in combination
with one
or more additional active agents, a supporting matrix or scaffold, or marrow
stromal
cells. The preparation can, optionally, be covered with a suitable covering,
for
example a muscle flap or a bioresorbable membrane, such asa collagen membrane.
100691 As will be appreciated by those skilled in the art, the concentration
of the
compounds described in a therapeutic composition will vary depending upon a
number of factors, including without limitation the dosage of the drug to be
administered and the route of administration. The preferred dosage of drug to
be
administered also is likely to depend on variables including, but not limited
to, the
type and extent of a disease, tissue loss or defect, the overall health status
of the
particular patient, the relative biological efficacy of the compound selected,
the
preparation of the compound, the presence and types of excipients in the
preparation,
and the route of administration. The present invention may be provided to an
individual where typical doses range from about 10 ng/kg to about 1 g/kg of
body
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weight per day; with a preferred dose range being from about 0.1 mg/kg to 100
mg/kg of body weight, and with a more particularly preferred dosage range of
10-
1000 g/dose. In a particularly preferred embodiment, a dose of 10-1000 g of
a
BMP-7 is administered to an individual afflicted with osteoarthritis.
(00701 Cartilage repair and regeneration is one of the major obstacles in
current
orthopedics. The importance is enormous because cartilage injury and
degenerative
disorders such as osteoarthritis, intervertebral disc degeneration and
meniscal tears
are a major cause of disability among the adult population in the United
States.
(00711 Cartilage is connective tissue composed of chondrocytes embedded in an
extracellular matrix of collagen fibers, proteoglycans, and other non-
collagenous
proteins. There are two forms of cartilage--articular and non-articular.
Articular
cartilage is a thin layer of connective tissue, which covers the ends of bones
in joints.
Non-articular cartilage includes fibrocartilage and elastic cartilage and
includes
intervertebral discs, meniscus, trachea, larynx, nose, ear and ribs.
100721 The function of cartilage is to cushion load bearing, resist wear, and
allow
for almost frictionless movement ofjoints. Defects in cartilage tissue, often
caused
by trauma, abnormal wear or disease, can lead to pain and stiffness, and if
left
untreated, may progress and ultimately require replacement of the entire
joint. For
example, articular cartilage defects often lead to early degradation of the
articular
surface and may eventually result in osteochondral defects, osteoarthritis or
both.
(00731 Osteoarthritis is considered a process of attempted, but gradually
failing,
repair of damaged cartilage extracellular matrix, as the balance between
synthesis
and breakdown of matrix components is disturbed and shifted toward catabolism,
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[0074] The ability of cartilage tissue to regenerate on its own is severely
limited
due to its avascular nature. Repair of osteochondral defects, which involves
both the
cartilage tissue and the underlying bone, occurs to a limited extent promoted
by the
presence of both stem cells and growth and differentiation factors brought
into the
defect by the blood and/or marrow. In animal studies, these defects undergo
some
repair with formation of a new layer of bone and cartilage, but the
macromolecular
organization and the biochemical characteristics of the cartilage matrix are
imperfect.
Type I coliagen, rather than Type II collagen, and proteoglycans that are not
cartilage specific, such as dermatan sulfate containing proteoglycans, make up
the
repair tissue and result in fibrillations and degenerative changes over time.
And,
repair of cartilage defects that do not penetrate into the subchondral bone
does not
occur, even to a limited extent.
100751 Moreover, surgical treatment of cartilage defects is complex and has
been
demonstrated to have only limited success. For example, articular cartilage
defects
are treated with an arthroscopic approach where loose bodies are debrided and
transition areas are smoothed. However, this method alone frequently does not
provide long lasting relief of the symptoms. Knee replacements often require
resecting significant amounts of bone and often require multiple surgeries.
[0076] The meniscus is a small horseshoe shaped tissue located between the
bone
ends inside the knee joint, which acts as a shock absorber. There are two
menisci in
each knee on either side of the knee. They are usually strong in young people
and
with age become more brittle and tear more easily. Tears are extremely common
with anterior cruciate ligament (ACL) injuries. Meniscal fibrocartilage, like
articular hyaline cartilage, has a limited capacity to heal, particularly in
the middle
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and inner avascular regions. The current treatment for small tears is to leave
them
alone if they do not cause much trouble. Surgical options for treating
meniscal tears
depend on a number of factors including the nature and extent of the injury
and most
importantly, its location. Tears in the vascularized region, which is
integrated with
the highly vascularized synovium have been successfully repaired by suturing.
Partial or total meniscectomy is the normal surgical treatment for symptomatic
tears
within the avascular two thirds of the meniscus. Tears in the latter meniscus
regions
are the most common types seen clinically. Irrespective of whether open,
arthroscopic, total or partial meniscectomy are employed, osteoarthritis is a
frequent
sequela in these patients within a few years post surgery. Therefore, the
common
form of repair is to only partially remove the torn bits and to repair the
cartilage by
stapling it. Unfortunately, the healing process following this procedure is
slow.
Moreover, if the repair is not successful, then the entire torn meniscus must
subsequently be removed.
100771 The major cause of persistent and often debilitating back pain is
intervertebral disc (IVD) degeneration also known as degenerative disc disease
(DDD). As discs degenerate, they cause the adjoining vertebrae to become
compressed, often resulting in severe pain.
[00781 The IVD as a syndesmosis provides articulation between adjoining
vertebral bodies and acts as a weight bearing cushion which dissipates axially
applied spinal loads. These biomechanical functions are made possible by the
unique structure of the IVD which is composed of an outer collagen-rich
annulus
fibrosus surrounding a central hydrated proteoglycan rich gelatinous nucleus
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pulposus. Superior and inferior cartilaginous endplates, thin layers of
hyaline-like
cartilage covers the interfaces of the vertebral bodies within the disc.
[0079) Lumbar disc degeneration represents a substantial social and economic
burden to the community which is manifest principally as low back pain (LBP).
It is
estimated that as much as 80% of the population experience at least one
significant
episode of LBP during life, and approximately 2.5% of the working population
will
take some sick leave during the year as a result of LBP. The direct costs of
LBP in
modem Westenn countries has been estimated at $9 billion, most of which is
spent
on consulting general practitioners, physical therapists and other
conservative
practitioners (Williams D A et al., (1998) Health care and indemnity costs
across the
natural history of disability in occupational low back pain, Spine 23:2329-
36). Total
indirect expenditure, including surgical management may be ten times higher
(Maetzel and Li (2002) The economic burden of low back pain: a review of
studies
published between 1996 and 2001, Best Prac Res Clin Rheumatol 16:23-30; Walker
et al., (2003) The economic burden, Proceedings of the Spine Society of
Australia
Annual Scientific Meeting, Canberra, Australia).
[0080) Disc degeneration is a natural phenomenon that occurs, in most
instances,
from the time of skeletal maturity (Vernon-Roberts (1992) Age-related and
degenerative pathology of intervertebral discs and apophyseal joints, In: The
lumbar
spine and back pain. Fourth edition, Jayson M I V, Ed. Churchill Livingstone,
Edinburgh, Chapter 2, 17-41). It is consistent with advancing age but in many
cases
is also associated with pain, particularly in the lumbar spine, and restricted
mobility.
Symptoms of LBP often resolve spontaneously over time as patients modify their
lifestyles to accommodate restricted mobility. In many cases however, it
remains a
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significant factor that requires surgical intervention. The traditional "gold
standard"
surgical treatment for chronic LBP has been spinal fusion to immobilize the
one or
more painful level. Fusion is expensive because it requires prolonged
hospitalization and specialist surgical expertise, and although most of these
patients
will experience short-term pain relief there is evidence now that fusion does
not
provide the best outcome. Long-term studies suggest that spinal fusion
actually
promotes degeneration at levels adjacent to the fusion site (Lee (1988)
Accelerated
degeneration of the segment adjacent to a lumbar fusion, Spine 13:375-7.). In
the
same way that artificial prostheses were developed 50 years ago to restore
function
to arthritic and fractured hips and knees, prostheses are now being developed
with
the aim of restoring full mechanical function to discs that have become
painful and
arthritic due to chronic degeneration (Szpaalski et al (2002) V Spine
arthroplasty: a
historical review, Eur Spine J 11 :S65-S84). It is however too early to know
if any
of the myriad models undergoing trials will provide long-term benefit.
100811 Additionally, as described below, the protein preparations, preferably
the
BMP preparations of the present invention can be used to treat diseases or
injuries of
non-skeletal tissues. As further contemplated by the present invention, BMPs
are
capable of inducing the developmental cascade of bone morphogenesis and tissue
morphogenesis for a variety of tissues in mammals different from bone or bone
cartilage. This morphogenic activity includes the ability to induce
proliferation and
differentiation of progenitor cells, and the ability to support and maintain
the
differentiated phenotype through the progression of events that results in the
formation of bone, cartilage, non-mineralized skeletal or connective tissues,
and
other adult tissues.
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[00821 For example, BMPs can be used for treatment to prevent loss of and/or
increase bone mass in metabolic bone diseases. General methods for treatment
to
prevent loss of and/or increase bone mass in metabolic bone diseases using
osteogenic proteins are disclosed in U.S. Patent No. 5,674,844, the
disclosures of
which are hereby incorporated by reference. BMPs of the present invention can
be
used for periodontal tissue regeneration. General methods for periodontal
tissue
regeneration using osteogenic proteins are disclosed in U.S. Patent No.
5,733,878,
the disclosures of which are hereby incorporated by reference. BMPs can be
used
for liver regeneration. General methods for liver regeneration using
osteogenic
proteins are disclosed in U.S. Patent No. 5,849,686, the disclosures of which
are
hereby incorporated by reference. BMPs can be used for treatment of chronic
renal
failure. General methods for treatment of chronic renal failure using
osteogenic
proteins are disclosed in U.S. Patent No. 6,861,404, the disclosures of which
are
hereby incorporated by reference. BMPs can be used for enhancing functional
recovery following central nervous system ischemia or trauma. General methods
for
enhancing functional recovery following central nervous system ischemia or
trauma
using osteogenic proteins are disclosed in U.S. Patent No. 6,407,060, the
disclosures
of which are hereby incorporated by reference. BMPs can be used for inducing
dendritic growth. General methods for inducing dendritic growth using
osteogenic
proteins are disclosed in U.S. Patent No. 6,949,505, the disclosures of which
are
hereby incorporated by reference. BMPs can be used for inducing neural cell
adhesion. General methods for inducing neural cell adhesion using osteogenic
proteins are disclosed in U.S. Patent No. 6,800,603, the disclosures of which
are
hereby incorporated by reference. BMPs can be used for treatment and
prevention
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of Parkinson's disease. General methods for treatment and prevention of
Parkinson's disease using osteogenic proteins are disclosed in U.S. Patent No.
6,506,729, the disclosures of which are hereby incorporated by reference.
100831 As another example, BMPs can also be used to induce dentinogenesis. To
date, the unpredictable response of dental pulp tissue to injury is a basic
clinical
problem in dentistry. As yet another example, BMPs can induce regenerative
effects
on central nervous system (CNS) repair can be assessed using a rat brain stab
model.
Bioactive Co-agents
[0084] The present invention also contemplates "bioactive co-agents" that can
be
co-administered with the protein preparations of the present invention
include, but
are not limited to, anabolic agents, anti-asthmatic agents, anti-infective
agents
including, for example, antiproteincterial and antimicrobial agents, anti-
inflammatory agents, antimetabolite agents, anti-neoplastic agents, anti- bone
resorption agents, anti-obesity agents, anti-pyretic and analgesic agents,
anti-
spasmodic agents, anti-thrombotic agents, antihistamines, biologicals,
bronchodilators, cytotoxic agents, diagnostic agents, erythropoietic agents,
immunomodulating agents, mineral supplements, peripheral vasodilators,
stimulants,
tissue growth agents, vitamins, or antigenic materials.
[0085] More particularly, the bioactive co-agents preferred for co-
administration
include, but are not limited to, growth factors, honnones, anti-angiogenesis
factors,
dextromethorphan, peptides, polypeptides, proteins, amino acids, hormones,
interferons, cytokines, and vaccines. Other representative bioactive co-agents
that
can be co-administered include, but are not limited to, peptide drugs, protein
drugs,
antigens, anti-infective agents such as antibiotics, antimicrobial agents,
antiviral,
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antiproteincterial, antiparasitic, antifungal substances and combination
thereof,
antiallergenics, steroidal anti-inflammatory agents, analgesics, nonsteroidal
anti-
inflammatory agents, and nutritional agents
[0086] The bioactive co-agent may also be a substance, or metabolic precursor
thereof, which is capable of promoting growth and survival of cells and
tissues, or
augmenting the activity of functioning cells, as for example, blood cells,
neurons,
muscle, bone marrow, bone cells and tissues, and the like. For example,
bioactive
co-agents that may be co-administered include without limitation a nerve
growth
promoting substance, as for example, a ganglioside, phosphatidylserine, a
nerve
growth factor, brain-derived neurotrophic factor. The bioactive co-agent may
also
be a growth factor for soft or fibrous connective tissue as, for example, a
fibroblast
growth factor, an epidermal growth factor, an endothelial cell growth factor,
a
platelet derived growth factor, an insulin-like growth factor, a periodontal
ligament
cell growth factor, to name but a few.
Examples
(0087] Example I: Osteoarthritis
[0088] A. Sheep Model for Prevention of Osteoarthritis
[0089] Sheep can be used as a model for osteoarthritis because it has been
demonstrated that progressive osteoarthritis occurs in these animals after a
single
injury impact. All sheep will receive general anesthesia and using aseptic
techniques, a 3 cm arthrotomy will allow access to both femorotibial joints. A
spring loaded mechanical device will be used to create bilateral impact
injuries to
the weight bearing region of the median femoral condyle (30 Mpa, 6 mm
diameter×2). After a routine closure of these incisions, the sheep will
receive
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an intra-articular injection in each knee of a concentrated protein
preparation of a
BMP, preferably BMP-7. The sheep will be sacrificed 12 weeks postoperatively
for
detailed assessment (paravital staining, TUNEL staining, histopathology,
cartilage,
sulfated GAG analysis, biomechanical indentation testing) of the articular
tissues.
Histological sections will be prepared for evaluation of cartilage physiology.
Sulfated glycosaminoglycan concentrations will be measured as an indicia of
cartilage physiology and health. It is expected that the control group will
exhibit
fibrillations and erosion of the surface, whereas the BMP-treated group will
show
little or no sign of damage. It is expected that the BMP-treated joints will
look
healthier and shinier than the controls indicating regeneration and repair of
the
osteoarthritic lesions.
100901 These experiments will demonstrate marked improvement, if not complete
protection with BMP.
100911 B. Guinea Pig and Rabbit Models of Osteoarthritis
(0092] The Hartley guinea pig (spontaneous) and rabbit ACL-resection (induced)
osteoarthritis models will be utilized. Fourteen guinea pigs of either 3, 6 or
9
months of age will be injected in the right knee with a concentrated solution
of BMP
at 3-week intervals for a period of 12 weeks. The left knee will serve as an
untreated control. In ten New Zealand White rabbits, the left ACL will be
resected
and will receive either an injection into the joint of BMP or a control
solution at 3-
week intervals during a 12-week evaluation period. The right knee will serve
as a
non ACL-resected nontreated control in all animals. All animals in both models
will
be evaluated for gross appearance and histologic evidence of arthritic changes
using
a modified Mankin scale to grade the severity of degeneration. It is expected
that
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the BMP-7 treatment will have a profound effect in preventing degeneration in
the
guinea pig at the early time periods. In the rabbit ACL-resected model BMP
treatment is expected to show slight improvement in the severity of
degeneration in
treated sites at the 12 week evaluation period. These results will demonstrate
that
BMP, preferably BMP-7, has some beneficial effects in preventing or slowing
early
stage arthritic changes.
100931 C. Patients having a diagnosis of osteoarthritis will be treated with a
protein preparation of the present invention. Certain patients will receive
aqueous
carrier only. In certain clinical studies, doses will be administered ranging
from
about 1 mg in about 100 microliters to about 30 mg in about 3 ml. In others,
doses
will be administered ranging from about 2 mg in about 100 microliters to about
60
mg in about 3 ml. Doses will be administered into the synovial space of
various
articulating joints, including but not limited to the knee and hip joints. It
is expected
that patients receiving at least 1 mg of a concentrated protein preparation of
a BMP,
preferably BMP-7, will exhibit amelioration of their symptoms. It is further
expected that patients receiving at least I mg of concentrated protein
preparation of
a BMP, preferably BMP-7, will exhibit repair of joint deterioration associated
with
osteoarthritis.
[00941 Example II: Degenerative Disc Disease
[0095J A. Sheep Model of Disc Repair and Regeneration
10096J Experimental induction of controlled outer annular defects in sheep
initiates a sequence of events which closely reproduces, pathologically and
biochemical, the evolution of disc degeneration in man. Compositional changes
include an alteration in the amount of, and the types of collagens synthesized
by
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cells of the lesion site (Kaapa et al 1994a, b, 1995 Kaapa E. et al. (1995)
Collagen
synthesis and types I, 111, IV, and VI collagens in an animal model of disc
degeneration, Spine 20, 59-67; Kaapa E et al., (1994) Collagens in the injured
porcine intervertebral disc, J. Orthop. Res. 12. 93-102; and Kaapa E et al.,
(1994)
Proteoglycan chemistry in experimentally injured porcine intervertebral disk,
J. Spin.
Dis. 7, 296-306) loss of large high buoyant density aggrecan type
proteoglycans and
an elevation in levels of the small DS substituted proteoglycans decorin and
biglycan in the injured disc (Melrose J. et al, (1992) A longitudinal study of
the
matrix changes induced in the intervertebral disc by surgical damage to the
annulus
fibrosus, J Orthop Res 10:665-676; Melrose J. et al., (1997) Topographical
variation
in the catabolism of aggrecan in an ovine annular lesion model of experimental
disc
degeneration J Spinal Disord 10:55-67; and Melrose J. et al., (1997) Elevated
synthesis of biglycan and decorin in an ovine annular lesion model of
experimental
disc degeneration, Eur Spine J 6:376-84). Changes in the vascular supply to
the
cartilaginous end plate (CEP) (Moore R J et al., (1992) Changes in endplate
vascularity after an outer anulus tear in the sheep, Spine 17:874-878) and
remodeling of vertebral bone adjacent to experimental annular defects (Moore R
J,
et al. (1996) Remodeling of vertebral bone after outer anular injury in sheep,
Spine
21:936-940.), changes in the biomechanical competence of "repaired" lesion
affected discs (Latham J M et al., (1994) Mechanical consequences of annular
tears
and subsequent intervertebral disc degeneration, J Clin Biomech 9:211-9), and
osteoarthritic changes in spinal facet joints (Moore R J et al., (1999)
Osteoarthrosis
of the facet joints resulting from anular rim lesions in sheep lumbar discs,
Spine,
24:519-525) as a consequence of disc degeneration have also been noted.
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(0097] The Ovine Annular Lesion Model
[0098] The sheep will be fasted for 24 h prior to surgery and anesthesia will
be
induced with an intravenous injection of I g thiopentone. A lateral plain X-
ray film
will be taken to verify normal lumbar spine anatomy. General anesthesia will
be
maintained after endotracheal intubation by 2.5% halothane and monitored by
pulse
oximetry and end tidal CO<sub>2</sub> measurement. The left flank from the ribs to
the
iliac crest will be prepared for sterile surgery. The sheep will receive an
intramuscular injection of 1200 mg penicillin. A skin incision will be made on
the
left side immediately anterior to the transverse processes of the spine and
the lumbar
spine will be exposed by blunt dissection using an anterior muscle-splitting
technique. The vascular and neural anatomy will be respected and bleeding will
be
controlled by direct pressure or electrocautery as required. A total of twelve
two
year old sheep will receive controlled annular lesions in their L1-L2, L3-L4
and L5-
L6 discs by incision through the left anterolateral annulus fibrosus parallel
and
adjacent to the cranial endplate using a #11 scalpel blade to create a lesion
measuring 4 mm long×5 mm deep. The intervening lumbar discs (L2-L3, L4-
L5) will not be incised. The incised discs will receive one of 3 therapies,
(I) no
treatment, (II) lactose solution or (III) lactose containing BMP-7. In all
sheep the
L3-L4 disc will receive an annular lesion with no treatment. In 4 sheep the L1-
L2
discs will be treated with lactose solution only and the L5-L6 disc will be
treated
with lactose plus BMP-7. In the remaining 4 sheep the treatments in the L1-L2
and
L5-L6 discs will be reversed to avoid any potential outcome bias associated
with
spinal level. A non-operated disc must remain between treated discs to allow
for
adequate anchorage of FSUs in subsequent mechanical testing (see below). A
wire
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suture will be used to identify the craniad operated level for later
identification
purposes both in X-rays and for morphological identification. Three additional
non-
operated animals will also be used as controls for the biomechanical study.
[0099] Degeneration following annular incision is well established in the
sheep
(Osti 0 L et al., (1990) Volvo Award for Basic Science, Annulus tears and
intervertebral disc degeneration. An experimental study using an animal model,
Spine 15:762-7) and can be expected to show the earliest radiographic and
histochemical evidence after 12 weeks. Three months after induction of the
annular
lesions the sheep will be killed by intravenous injection of 6.5 g sodium
pentobarbitone and the lumbar spines will be radiographed to evaluate disc
calcification, excised and processed for biomechanical (n=8) and histochemical
(n=4) analyses, and, after the biomechanical testing the same discs will be
zonally
dissected for compositional analyses. It is expected that BMP-7 treated
animals will
exhibit less degeneration than untreated animals.
[01001 B. The in vivo effects of BMP-7 on the repair of intervertebral discs
are
studied in two rabbit models--one model involves stab-wounding of the annulus
fibrosus, as described in Lipson et al., Spine 6:194 (1981), and the other
model
involves intradiscal C-ABC injection, as described in Kato et al., Clin.
Orthop.
253:301 (1990). Briefly, for the stab-wounding method, an incision will be
made in
the annulus fibrosus of New Zealand White rabbits. Each rabbit will have two
discs
treated: one disc treated with BMP-7 and the other treated with aqueous
carrier. For
the intradiscal injection model, the lumbar discs of New Zealand White rabbits
will
be exposed and BMP-7 or aqueous carrier will be injected into the
intervertebral
discs. At varying times following treatment, the rabbits will be euthanized
and the
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effects of BMP-7 on the repair of the intervertebral disc space will be
evaluated by
methods well known in the art. These methods include magnetic resonance
imaging,
mechanical tests, histological analysis, and biochemical studies of the
various
extracellular matrix components in the repaired discs. It is expected that BMP-
7
treated animals will exhibit improved disc health and less degeneration than
untreated animals.
101011 C. Patients having a diagnosis of degenerative disc disease will be
treated
with a protein preparation of the present invention.
101021 It is expected that administration of the protein preparations of the
present
invention to patients presenting with lower back pain, particularly discogenic
pain
diagnosed using a discogram, will result in amelioration of such pain. In a
preferred
treatment modality, such patients will receive at least one intradiscal
injection of a
protein preparation of the present invention. Such patients will display
amelioration
of pain as compared to other patients who are not treated with a protein
preparation
of the present invention. It is expected that BMP-7 treatrnent will be
particularly
effective for such patients.
[01031 Example III: Non-skeletal Tissue Repair
[01041 It is expected that administration of the protein preparations of the
present
invention to patients having disorders, injuries or diseases of non-skeletal
tissue will
result in amelioration and/or repair of such disorders, injuries or diseases.
As
explained elsewhere herein, protein preparations containing certain preferred
morphogenic proteins will be useful for repair of soft tissues such as but not
limited
to kidney and liver, and will be useful for treatment of opthamologic and
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defects to name but a few. It is expected that administration via a systemic
route or
via local administration will result in improvements relative to untreated
subjects.
[0105] Example IV: Prophylactic Administration Post-injury
[0106] It is expected that certain disorders, injuries or diseases will
benefit from
treatment with the protein preparations of the present invention prior to
surgical
intervention. Prophylactic administration is expected to facilitate the
likelihood of
post-surgical healing and to promote restoration of a normal or near-normal
physiology. For example, in the case of a patient suffering from DDD, it is
expected
that treatment with a concentrated preparation of a BMP, preferably BMP-7,
prior to
surgery will be beneficial. In certain patients, multiple pre-surgical
treatments will
be beneficial. Patients treated prior to surgery will receive a treatment
concurrent
ti
with surgery. Certain other patients will benefit from multiple treatments
post-
surgery. In each of the foregoing situations, it is expected that treated
patients will
exhibit better disc health than untreated patients.
[0107] Example V: Preparation of BMP-7 at certain high concentrations (20 to
40
mg/mL) in certain prefen:ed aqueous carriers.
[01081 The following exemplary method increases ease of protein processing
during manufacturing, while reducing in-process product losses and yielding
the
final desired high concentration protein preparation of the present invention.
[0109] This Example outlines a method for processing (preparation, fill,
lyophilization, and finish opcrations) BMP-7 at low concentrations (< 20
mg/mL),
while yielding a product that can be reconstituted to a desired target higher
protein
concentration (20 to 40 mg/mL). This methodology allows execution of all
manufacturing operations at a lower protein concentration-this increases ease
of
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processing as the viscosity of the solution handled is lower and it also
reduces in-
process losses during manufacturing (since a fixed volume lost contains a
lower .
mass of protein due to the lower concentration used during processing). The
lower
concentration used during processing also allows for the product to be stored
and
processed at refrigerated temperatures (2 to 8 C), without resulting in a
dramatic
increase in viscosity as would.otherwise be observed if high protein
concentrations
were used during processing.
[0110] Usually drug products are processed and filled at the same
concentration as
the fmal desired target. For example, if the target protein concentration for
administration is 40 mg/mL, the protein preparation (in the presence of
appropriate
stabilizing excipients) would typically be filled at 40 mg/mL and the freeze-
dried
product reconstituted with the same volume of diluent as the fill volume prior
to
lyophilization (freeze-drying).
[0111] In the methodology disclosed in this document, a different approach is
used.
For example, if it is desired to have 1 mL of protein solution at 40 mg/mL for
administration, the processing (upstream manufacturing) would be conducted at
a
lower concentration, say 20 mg/mL. The fill would be 2 mL at 20 mg/mL, and the
product would be reconstituted with 1 mL reconstitution diluent following
freeze-
drying. Hence, the manufacturing (processing) operations are conducted at a
lower
concentration than the fmal target yielding the following benefits: (a) this
increases
ease of processing as the viscosity of the solution handled is lower, (b) it
also
reduces in-process losses during manufacturing (since a fixed volume lost
contains a
lower mass of protein due to the lower concentration used during processing),
(c) the
lower concentration used during processing also allows for the in-process
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intermediates to be stored (and/or processed) at refrigerated temperatures (2
to 8 C),
without resulting in a dramatic increase in viscosity as would otherwise be
observed
if high protein concentrations were used during processing (See Figure 1).
[0112] Likewise, in the same spirit of the invention disclosure, if a target
volume
of V mL is desired at a protein concentration of C mg/mL for administration,
the
protein preparation/filUfmish operations can be conducted at CJN mg/mL with a
fill
volume of V*N mg/mL, with final reconstitution of the lyophilized product with
V
mL water, where N (N > 1) is a suitable scaling factor, which can be
appropriately
chosen based on the viscosity and processability of the protein solution.
101131 This methodology allows execution of all manufacturing operations at a
lower protein concentration-this increases ease of processing as the viscosity
of the
solution handled is lower and it also reduces in-process losses during
manufacturing
(since a fixed volume lost contains a lower mass of protein due to the lower
concentration used during processing). The lower concentration used during
l5 processing also allows for the product to be stored and processed at
refrigerated
temperatures (2 to 8 C), without resulting in a dramatic increase in viscosity
as
would otherwise be observed if high protein concentrations were used during
processing.
101141 In addition, the above advantages also serve to yield a product with
better
quality attributes (for example, if processing is done at a lower protein
concentration
then critical physicochemical protein attributes such as aggregation tend to
be lower
than if the protein was processed at a higher concentration) and lower cost of
goods
(lower COGS due to smaller mass loss of protein and a higher quality product,
as
discussed above).
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[01151 The above-described protocol has been successfully implemented to
manufacture protein preparations ranging from about 10 to about 60 mg/ml.
Using a
titration matrix of varying ionic strengths, pHs and protein concentrations
(mg/ml),
it was discovered that highly concentrated preparations of protein in aqueous
carriers
are possible without risk of precipitation. Generally, as ioniC strength is
increased,
pH is decreased thereby maintaining a solubilized protein in an aqueous
carrier
comprising one or more of the buffer systems described elsewhere herein.
Stabilizing excipients can be used as described earlier.
101161 Example VI: 30 mg/mL BMP-7 in 5% trehalose with 0.75 mL volume
post-reconstitution.
101171 Bulk BMP-7 was obtained as a starting material at 2 mg/mL in 50 mM
acetic acid (pH = 3.0). Tangential flow filtration (or cross-flow filtration)
was
performed for protein concentration and buffer exchange. Buffer exchange was
performed against 10 mM lactate buffer (pH = 3.0) and lOX (i.e., about 10-
fold)
difiltration volume exchanges were performed to achieve a >99.9% buffer
exchange
efficiency. Trehalose (2.5% w/v) may be added either during diafiltration or
following diafiltration. In this Example, trehalose was added following
ultrafiltration and diafiltration (UF/DF). Protein concentration was adjusted
to 15
mg/mL in 10 mM lactate buffer + 2.5% w/v trehalose. Thereafter, 1.5 mL of
solution was filled into each 3 mL vial and lyophilized. Post-lyophilization
(or
freeze-drying), a pharmaceutically elegant cake was obtained as the protein
preparation which was reconstituted with 0.75 mg water-for-injection (WFI).
This
yielded a 30 mg/mL BMP-7 solution in 5% trehalose. The resulting product had
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desirable product quality attributes suitable for intended clinical uses, such
as but not
limited to, levels of aggregation and oxidation within accepted limits.
Equivalents
[01181 The invention maybe embodied in other specific forms without departing
from the spirit or essential characteristics thereof. The present embodiments
are
therefore to be considered illustrative and not restrictive, the scope of the
inventioil
being indicated by the appended claims rather than by the foregoing
description, and
all changes which come within the meaning and range of equivalency of the
claims
are therefore intended to be embraced therein.
[01191 We claim:
