Note: Descriptions are shown in the official language in which they were submitted.
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STATION OF BONE GROWTH AND CARTILAGE FORMATION WITH
THROMBIN PEPTIDE DERIVATIVES
GOVERNMENT SUPPORT
The invention was supported, in whole or in part, by grant 1 R43 AR45508
Ol and 2 R44 AR45508-02 from the National Institutes of Health. The Government
has certain rights in the invention.
BACKGROUND OF THE INVENTION
Mammalian bone tissue has a remarlcable ability to regenerate and thereby
repair injuries and other defects. For example, bone growth is generally
sufficient to
I O bring about full recovery from most simple and hairline fractures.
Unfortunately,
however, there are many injuries, defects or conditions where bone growth is
inadequate to achieve an acceptable outcome. For example, bone regeneration
generally does not occur throughout large voids or spaces. Therefore,
fractures
cannot heal unless the pieces are in close proximity. If a significant aanount
of bone
tissue was lost as a result of the izijury, the healing process may be
incomplete,
resulting in undesirable cosmetic and/or mechanical outcomes. This is often
the case
with non-union fractures or with bone injuries resulting from massive trauma.
Tissue
growth is also generally inadequate in voids and segmental gaps in bone
caused, for
example, by surgical removal of tumors or cysts. In other instances, it may be
desirable to stimulate bone growth where bone is not normally found, i.e.,
ectopically. Spine fusion to relieve lower back pain where two or more
vertebrae are
induced to fuse is one example of desirable ectopic bone formation. Currently,
such
gaps or segmental defects require bone grafts for successful repair or gap
filling. The
development of effective bone graft substitutes would eliminate the need to
harvest
bone from a second surgical site for a graft procedure, thereby significantly
reducing
the discomfort experienced by the patient and risk of donor site healing
complications.
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Compounds which stimulate or induce bone growth at sites where such
growth would not normally occur if left untreated are said to be
"osteoinductive".
An osteoinductive compound would have great value as a drug to treat the
conditions described above. A number of osteoinductive proteins have been
identified, isolated and expressed using recombinant technology. Examples
include
the bone morphogeuc proteins (BMPs) disclosed in U.S Patent No. 5,902,705 and
WO 95/16035. However, the use of recombinant proteins as therapeutic agents
generally has a number of drawbacks, including the cost of manufacture, ifz
vivo
biodegradation and short shelf lives. Consequently, scientists are continuing
to
search for new osteoinductive agents which do not have the aforementioned
shortcomings.
Furthermore, unlike most tissues, cartilage does not self repair following
injury. Cartilage is an avascu1ar tissue made up largely of cartilage specific
cells, the
chondrocytes, special types of collagen, and proteoglycans. The inability of
cartilage
to self repair after injury, disease, or surgery is a major limiting factor in
rehabilitation of degrading joint surfaces and injury to meniscal cartilage.
Osteoarthritis, the major degenerative disease of weight bearing joint
surfaces, is
caused by eroding or damaged cartilage surfaces and is present in
approximately
25% of the over 50-year-old population. In the US more than 20 million people
suffer from osteoarthritis, with annual healthcare costs of more than $8.6
billion. In
addition, the cost for cartilage repair from acute joint injury (meniscal
lesions,
patellar surface damage and chondromalacia) exceeds $1 billion annually.
Therefore,
new therapeutic approaches are needed to heal lesions of cartilage caused by
degeneration or acute trauma.
SUMMARY OF THE INVENTION
It has now been found that compounds which activate the non-proteolytic
thrombin receptor are osteoinductive. For example, the compound TP508, an
agonist
of the non-proteolytic thrombin receptor, stimulates bone growth in segmental
critical size defects created in the ulna of male New Zealand rabbits (Example
2). As
shown by x-ray and confirmed by histology and mechanical testing, there was a
significant increase in bone formation induced by TP508 at doses of 100 ~,g
and 200
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~,g compared with,untreated controls. Based on these results, novel methods of
stimulating bone growth in a subject and novel implantable pharmaceutical
compositions are disclosed herein.
It has now also been found that chondrocytes isolated from articular cartilage
respond to compounds which activate the non-proteolytic thrombin cell surface
receptor (hereinafter "NPAR"). For example, chondrocytes express approximately
233,000 thrombin binding sites per cell with apparent affinities of
approximately 0.1
nM (3000 sites) and 27 nM (230,000 sites) (Example 3). In addition, the
compound
TP508, an agonist of the non-proteolytic thrombin receptor, stimulates
proliferation
of bovine chondrocytes in culture in the presence of thrombin as a co-mitogen
(Example 4A) and stimulates by itself the proliferation of rat chondrocytes
cultured
in three dimensional matrix culture (Example SA). This same TP508 compound
also stimulates proteoglycan synthesis as measured by the incorporation of 35S
sulfate in both bovine chondrocytes (Example 4B) and 3-dimensional cultures of
rat
chondrocytes (Example SB). These in vitro experiments demonstrate that NPAR
agonists can stimulate proliferation and matrix production in chondrocytes
isolated
from articular cartilage. Additional iyZ vivo experiments demonstrate that
delivering
TP508 in a sustained release formulation to rabbit trochlear grove cartilage
defects
which extend into the subchondral bone results in repair of the cartilage
defect,
including repair of subchondral bone, restoration of a normal cartilage
surface and
integration of the newly formed cartilage with uninjured cartilage outside of
the
defect area (Example 7).
Based on the results reported in the prior paragraph, novel methods of
stimulating chondrocyte growth in vivo and cartilage repair in a subject and
novel
delivery methods for delivering pharmaceutical compositions to articular
defects to
aid in surface repair and to prevent articular degradation are disclosed
herein.
One embodiment of the present invention is a method of stimulating bone
growth at a site in a subject in need of osteoinduction. The method comprises
the
step of administering a therapeutically effective amount of an agonist of the
non-
proteolytically activated thrombin receptor to the site.
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Another embodiment of the present invention is a pharmaceutical
composition comprising an implantable, biocompatible carrier and an NPAR
agonist
such as a physiologically functional equivalent of a thrombin peptide
derivative.
These methods of the present invention are directed at stimulating bone
growth in a subject and can be used at sites where bone growth would not
occur,
absent treatment with autologous bone grafts or administration of bone growth
factors. The method involves the administration of agonists of the non-
proteolytic
thrombin receptor. Such agonists include small peptides having homology to the
segment between amino acid 508 and 530 of human prothrombin. These small
peptides are inexpensive to prepare in bulk quantities and are osteoinductive
at low
dose. In addition, their lyophilized form is stable for at least thirty months
when
stored at 5° C and at 60% relative humidity.
In another aspect, the present invention is directed to a method of
stimulating
cartilage growth, regeneration or repair at a site in a subject where
cartilage growth,
repair or regeneration is needed. The method comprises the step of
administering a
therapeutically effective amomlt of an NPAR agonist, such as a physiologically
functional equivalent of a thrombin peptide derivative to the site of injury.
A further embodiment of the present invention is directed to a method of
stimulating the proliferation and expansion of chrondrocytes in vitro. The
method
comprises culturing chrondrocytes in the presence of a stimulating amount of
an
NPAR agonist.
DETAILED DESCRIPTION OF THE INVENTION
"Osteoinduction" refers to stimulating bone growth at a site within a subj ect
at which little or no bone growth would occur if the site were left untreated.
Sites
which could therapeutically benefit from the induction of bone growth are
referred
to as "in need of osteoinduction". Examples include non-union fractures or
other
severe or massive bone trauma. It is noted that bone growth normally occurs at
bone
injuries such as simple or hairline fractures and well opposed complex
fractures with
minimal gaps without the need for further treatment. Such injuries are not
considered to be "in need of osteoinduction".
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Simple fracture repair appears to be quite different from the induction of
bone formation required to fill non-union fractures, segmental gaps or bone
voids
caused, for example, by removal of a bone tumor or cyst. Segmental gaps larger
than
0.5 cm generally are in need of osteoinduction, whereas segmented gaps larger
than
0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm or 1.5 cm typically are in need of
osteoinduction. These cases require bone grafting or induction of new bone
growth
generally employing some type of matrix or scaffolding to serve as a bone
growth
substitute. Induced bone growth can also be therapeutically beneficial at
certain sites
within a subj ect (referred to as "ectopic" sites) where bone tissue would not
normally be found, such as a site in need of a bone graft or bone fusion.
Fusions are
commonly used to treat lower back pain by physically coupling one or more
vertebrae to its neighbor. The bone created by such a fusion is located at a
site not
normally occupied by bone tissue. Osteoinduction at these ectopic sites can
act as a
"graft substitute" whereby induced bone growth between the vertebrae takes the
place of a graft and obviates the need for a second operation to harvest bone
for the
grafting procedure. Induction of bone growth is also needed for treating
acquired and
congenital craniofacial and other skeletal or dental anomalies (see e.g.,
Glowacki et
al., Lancet 1: 959 (1981)); performing dental and periodontal reconstructions
where
lost bone replacement or bone augmentation is required such as in a jaw bone;
and
supplementing alveolar bone loss resulting from periodontal disease to delay
or
prevent tooth loss (see e.g., Sigurdsson et al., J. Periodontol., 66: 511
(1995)).
In addition, sites in need of cartilage growth, repair or regeneration are
found
in subjects with osteoarthritis. Osteoarthritis or degenerative joint disease
is a slowly
progressive, irreversible, often monoarticular disease characterized by pain
and loss
of function. The underlying cause of the pain and debilitation is the
cartilage
degradation that is one of the major symptoms of the disease. Hyaline
cartilage is a
flexible tissue that covers the ends of bones and lies between joints such as
the knee.
It is also found in between the bones along the spine. Cartilage is smooth,
allowing
stable, flexible movement with minimal friction, but is also resistant to
compression
and able to distribute applied loads. As osteoarthritis progresses, surfaces
of
cartilage and exposed underlying bone become irregular. Instead of gliding
smoothly, boney joint surfaces rub against each other, resulting in stiffness
and pain.
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Regeneration of damaged cartilage and the growth of new cartilage at these
arthritic
sites would relieve the pain and restore the loss of function associated with
osteoarthritis.
Cartilage damage can also occur from trauma resulting from injury or
surgery. Sports injuries are a common cause of cartilage damage, particularly
to
joints such as the knee. Traumatic injury to cartilage can result in the same
type of
functional impairment. Therefore, sites in a subject with cartilage that has
been
damaged by trauma or disease are in need of treatment to restore or promote
the
growth of cartilage.
Applicants have discovered that compounds which stimulate or activate the
NPAR, NPAR agonists, are osteoinductive. Applicants have further discovered
that
compounds which stimulate or activate NPAR can stimulate chondrocytes to
proliferate. Chondrocytes are cells which make up about 1% of the volume of
cartilage and which replace degraded matrix molecules to maintain the correct
volume and mechanical properties of the tissue.
Applicants have also found that compounds which stimulate or activate
NPAR stimulate proteoglycan synthesis in chondrocytes. Proteoglycan is a major
cartilage component. Based on these results, Applicants delivered the NPAR
agonist
TP508, prepared in a sustained release formulation, to defects in rabbit
trochlear
grove cartilage and discovered that the peptide stimulated repair of the
defect that
included formation of new cartilage with a normal cartilage surface. The
peptide
also stimulated layering and integration of this new cartilage into adjacent,
uninjured
cartilage and restoration of the subchondral bone. It is concluded that NPAR
agonists can induce cartilage growth and repair when administered to sites
needing
cartilage growth and/or repair.
NPAR is a high-affinity thrombin receptor present on the surface of most
cells. This NPAR component is largely responsible for high-affinity binding of
thrombin, proteolytically inactivated thrombin, and thrombin derived peptides
to
cells. NPAR appears to mediate a number of cellular signals that are initiated
by
thrombin independent of its proteolytic activity. An example of one such
signal is
the upregulation of annexin V and other molecules identified by subtractive
hybridization (see Sower, et. al., Experimental Cell ReseaYCh 247:422 (1999)).
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NPAR is therefore characterized by its high affinity interaction with thrombin
at cell
surfaces and its activation by proteolytically inactive derivatives of
thrombin and
thrombin derived peptide agonists as described below. NPAR activation can be
assayed based on the ability of its agonists to stimulate cell proliferation
when added
to fibroblasts in the presence of submitogenic concentrations of thrombin or
molecules that activate protein kinase C or compete with lzsl-thrombin for
high
affinity binding to thrombin receptors, as disclosed in US Patent Nos.
5,352,664 and
5,500,412 and in Glenn et al., J. Peptide Research 1:65 (1988).
NPAR is to be distinguished from other thrombin binding proteins and the
cloned family of proteolytically-activated receptors for thrombin, including
the
receptors PART, PAR2, PAR3 and PAR4. PAR1 possesses a specific thrombin
cleavage site that allows thrombin cleavage to expose a new amino-terminus
domain
that acts as a tethered ligand folding baclc onto itself inducing its
activation (see, Vu,
et al., Cell. 64:1057 (1991)). PAR2 has a similar mechanism for activation,
but is
principally activated by trypsin-like enzymes (see, Zhong, et al., J. Biol.
Chem.
267:16975 (1992)). PAR3 also has a similar mechanism of activation and appears
to
function as a second thrombin receptor in platelets (see, Ishihara, et al.,
Natuf°e.
386:502 (1997)). PAR4 has been detected in mouse megakaryocytes and studies
suggest that it also functions in human platelets (see, Kahn, et al., Nature
394:690
(1998)). In contrast with these PAR receptors, activation of NPAR requires no
proteolytic cleavage.
Several lines of evidence indicate that NPAR is distinct from PAR receptors:
(1) a population of cells has been isolated that express fully functional PART
receptors, but are non-responsive to thrombin due to a defect in the NPAR
signal
transduction pathway (see, Kim, et al., J. Cell. Physiol. 160:573 (1994)); (2)
neutrophils bind'z5I thrombin with high affinity and their chemotaxis is
stimulated
by proteolytically inactivated thrombin or NPAR agonists (see, Ramakrishnan
and
Carney, Mol. Biol. Cell 4:1993 (1993)), yet they do not express PART (see
Jenkins,
et al., J. Cell Sci. 108:3059 (1995)); (3) IIC9 fibroblasts over-express PART,
but do
not bind thrombin with high affinity (see, Kim, D. Ph.D. Dissertation. The
University of Texas Medical Branch at Galveston, 1995; and Low, et al.,
"Cancer
Cells 3/Growth Factors and Transformation", Cold Spring Harbor Laboratory, New
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York); and (4) NPAR agonists have distinct effects on gene expression from
those
of the PAR receptor agonist peptides (see, Sower, et. al., Experimental Cell
Reseal°ch 247: 422 (1999).
One example of an NPAR agonist is a thrombin peptide derivative, i.e., a
polypeptide with no more than about fifty amino acids, preferably no more than
about thirty amino acids and having sufficient homology to the fragment of
human
thrombin corresponding to prothrombin amino acids 508-530 (SEQ ID NO. 5) that
the polypeptide activates NPAR. The thrombin peptide derivatives described
herein
preferably have between about 12 and 23 amino acids, more preferably between
about 19 and 23 amino acids. One example of a thrombin peptide derivative
comprises a moiety represented by Structural Formula (I):
Asp-Ala-R
R is a serine esterase conserved domain. Serine esterases, e.g., trypsin,
thrombin
chymotrypsin and the like, have a region that is highly conserved. "Serine
esterase
conserved domain" refers to a polypeptide having the amino acid sequence of
one of
these conserved regions or is sufficiently homologous to one of these
conserved
regions such that the thrombin peptide derivative retains NPAR activating
ability.
In one embodiment, the serine esterase conserved sequence has the amino
acid sequence of SEQ ID NO. 1 (Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val) or a
C-terminal truncated fragment of a polypeptide having the amino acid sequence
of
SEQ ID NO 1. It is understood, however, that zero, one, two or three amino
acids in
the serine esterase conserved sequence can differ from the corresponding amino
acid
in SEQ ID NO 1. Preferably, the amino acids in the serine esterase conserved
sequence which differ from the corresponding amino acid in SEQ ID NO 1 are
conservative substitutions, and are more preferably highly conservative
susbstitutions. A "C-terminal truncated fragment" refers to a fragment
remaining
after removing an amino acid or block of amino acids from the C-terminus, said
fragment having at least six and more preferably at least nine amino acids.
More preferably, the serine esterase conserved sequence has the amino acid
sequence of SEQ ID NO 2 (Cys-Xl-Gly-Asp-Ser-Gly-Gly-Pro-XZ Val; Xl is Glu or
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Gln and X2 is Phe, Met, Leu, His or Val) or a C-terminal truncated fragment
thereof
having at least six amino acids, preferably at least nine amino acids.
h1 a preferred embodiment, the thrombin peptide derivative comprises a
serine esterase conserved sequence and a polypeptide having a more specific
thrombin amino acid sequence Arg-Gly-Asp-Ala (SEQ m NO 3). One example of a
thrombin peptide derivative of this type comprises Arg-Gly-Asp-Ala-Cys-Xl-Gly-
Asp-Ser-Gly-Gly-Pro-XZ Val (SEQ m NO 4). Xl and Xz are as defined above.
When the thrombin peptide derivative comprises SEQ m NO 4, it preferably has
the
amino acid sequence of SEQ m NO 5 (Ala-Gly-Try-Lys-Pro-Asp-Glu-Gly-Lys-Arg-
Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val) or an N terminal
truncated fragment thereof, provided that zero, one, two or three amino acids
at
positions 1-9 in the thrombin peptide derivative differ from the amino acid at
the
corresponding position of SEQ m NO 5. Preferably, the amino acids in the
thrombin peptide derivative which differ from the corresponding amino acid in
SEQ
m NO 5 are conservative substitutions, and are more preferably highly
conservative
susbstitutions. An "N terminal truncated fragment" refers to a fragment
remaining
after removing an amino acid or block of amino acids from the N terminus,
preferably a block of no more than six amino acids, more preferably a block of
no
more than three amino acids.
A physiologically functional equivalent of a thrombin derivative peptide
encompasses molecules which differ from thrombin derivatives in particulars
which
do not affect the function of the peptide as an NPAR agonist. Such particulars
may
include, but are not limited to, amino acid substitutions, as described
herein, and
modifications, for example, amidation of the carboxyl terminus, acylation of
the
amino terminus, conjugation of the polypeptide to a physiologically inert
carrier
molecule, or sequence alterations in accordance with the serine esterase
conserved
sequences.
Physiologically functional equivalents of the thrombin derivative peptides
are also within the scope of the invention. For example, such peptides can be
amidated at the carboxyl terminus, acylated at the amino terminus or both. In
particular embodiments, the amino acid sequence of SEQ m NO.: 3 is represented
as the following physiologically functional equivalents: Ala-Gly-Try-Lys-Pro-
Asp-
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Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH2
(SEQ m NO.: 6), Ac-Ala-Gly-Try-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-
Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ >D NO.: 7) or Ac-Ala-Gly-Try-
Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-
Phe-Val-NHZ (SEQ m NO.: 8) ("Ac" is an acetyl group).
Amidation of the carboxyl terminus can be accomplished by any method
known in the art. Thus the C-terminal amino acid is represented in Structural
Formula (In:
-NH-CHR-C(O)-NR1R2
wherein Rl and Rz individually are selected from the groups of H, C1- C6
alkyl and, Rl and RZ together with the nitrogen to which they are bound form a
non-
aromatic heterocyclic ring such as pyrrolidinyl, piperazinyl, morphilinyl or
piperdinyl. Rl and RZ are preferably H. "-Val-NHZ" means -NH-CH[-CH-(CH3)a]-
CONH2.
Acylation of the amino terminus can be accomplished by any method known
in the art. Thus, the N-terminal amino is represented in the Structural
Formula (ff~:
Rl-C(O)-NH-CHR-C(O)-
wherein R is the amino acid side chain and Rl is a C1- C6 alkyl branched and
straight chained. R is preferably methyl (-CH3).
TP508 is an example of a physiologically functional equivalent of a thrombin
peptide derivative and has the amino acid sequence of SEQ m NO 6.
A "conservative substitution" is the replacement of an amino acid with
another amino acid that has the same net electronic charge and approximately
the
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same size and shape. Amino acids with aliphatic or substituted aliphatic amino
acid
side chains have approximately the same size when the total number carbon and
heteroatoms in their side chains differs by no more than about four. They have
approximately the same shape when the number of branches in the their side
chains
differs by no more than one. Amino acids with phenyl or substituted phenyl
groups
in their side chains are considered to have about the same size and shape.
Listed
below are five groups of amino acids. Replacing an amino acid in a polypeptide
with
another amino acid from the same group results in a conservative substitution:
Group I: glycine, alanine, valine, leucine, isoleucine, serine, threonine,
cysteine, and non-naturally occurring amino acids with C1-C4 aliphatic
or C1-C4 hydroxyl substituted aliphatic side chains (straight chained or
monobranched).
Group II: glutamic acid, aspartic acid and non-naturally occurring amino
acids with carboxylic acid substituted C1-C4 aliphatic side chains
(unbranched or one branch point).
Group III: lysine, ornithine, arginine and non-naturally occurnng amino
acids with amine or guanidino substituted C1-C4 aliphatic side chains
(unbranched or one branch point).
Group 1V: glutamine, asparagine and non-naturally occurring amino
acids with amide substituted C1-C4 aliphatic side chains (unbranched or
one branch point).
Group V: phenylalanine, phenylglycine, tyrosine and tryptophan.
A "highly conservative substitution" is the replacement of an amino acid
with another amino acid that has the same functional group in the side chain
and
nearly the same size and shape. Amino acids with aliphatic or substituted
aliphatic
amino acid side chains have nearly the same size when the total number carbon
and
heteroatoms in their side chains differs by no more than two. They have nearly
the
same shape when they have the same number of branches in the their side
chains.
Example of highly conservative substitutions include valine for leucine,
threonine
for serine, aspartic acid for glutamic acid and phenylglycine for
phenylalanine.
Examples of substitutions which are not highly conservative include alanine
for
valine, alanine for serine and aspartic acid for serine.
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Other NPAR agonists include small organic molecules which bind and
activate NPAR. Agonists of this type can be conveniently identified with high
through-put screening, e.g., with assays that assess the ability of molecules
to
stimulate cell proliferation when added to fibroblasts in the presence of
submitogenic concentrations of thrombin or molecules that activate protein
kinase C
as disclosed in US Patent Nos. 5,352,664 and 5,500,412. The entire teachings
for
US Patent Nos. 5,352,664 and 5,500,412 are incorporated herein by reference.
The term "NPAR agonist" also includes compounds and combinations of
compounds known to activate NPAR. Examples are disclosed in US Patent Nos.
5,352,664 and 5,500,412 and include thrombin, DIP-alpha-thrombin and the
combination of DIP-alpha-thrombin with phorbol myristate acetate.
An implantable biocompatible carrier for use in the pharmaceutical
compositions described herein functions as a suitable delivery or support
system for
the NPAR agonist utilized to stimulate bone growth. A biocompatible carrier
should
be non-toxic, non-inflammatory, non-irmnunogenic and devoid of other undesired
reactions at the implantation site. Suitable carriers also provide for release
of the
active ingredient and preferably for a slow, sustained release over time at
the
implantation site.
Suitable carriers include porous matrices into which bone progenitor cells
may migrate. Osteogenic cells can often attach to such porous matrices, which
can
then serve as a scaffolding for bone and tissue growth. For certain
applications, the
Garner should have sufficient mechanical strength to maintain its three
dimensional
structure and help support the immobilization of the bone segments being
united or
grafted together. Porous matrices which provide scaffolding for tissue growth
can
accelerate the rate of bone growth and are said to be "osteoconductive".
Osteoconductive carriers are highly preferred for use in the pharmaceutical
compositions described herein.
Examples of suitable osteoconductive carriers include collagen (e.g., bovine
dermal collagen); fibrin, calcium phosphate ceramics (e.g., hydroxyapatite and
tricalcium phosphate), calcium sulfate, guanidine-extracted allogenic bone and
combinations thereof. A number of suitable carriers are commercially
available,
such as COLLOGRAFT (Collagen Corporation, Palo Alto, CA), which is a mixture
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of hydroxyapatite, tricalcium phosphate and fibrillar collagen, and INTERPOR.E
(Interpore International, Irvine CA), which is a hydroxyapatite biomatrix
formed by
the conversion of marine coral calcium carbonate to crystalline
hydroxyapatite.
A number of synthetic biodegradable polymers can serve as osteoconductive
carriers with sustained release characteristics. Descriptions of these
polymers can be
found in Behravesh et al., Clinical Orthopaedics 367: S 118 (1999) and Lichun
et al.,
Polymeric Delivery Vehicles for Bone Growth Factors in "Controlled Drug
Delivery
- Designing Technologies for the Future" Park and Mrsny eds., American
Chemical
Society, Washington, DC (2000). The entire teachings of these references are
incorporated herein by reference. Examples of these polymers include poly a-
hydroxy esters such as polylactic acid/polyglycolic acid homopolymers and
copolymers, polyphosphazenes (PPHOS), polyanhydrides and polypropylene
fumarates).
Polylactic acid/polyglycolic acid (PLGA) homo and copolymers are well
known in the art as sustained release vehicles. The rate of release can be
adjusted by
the skilled artisan by variation of polylactic acid to polyglycolic acid ratio
and the
molecular weight of the polymer (see Anderson, et al., Adv. Drug Deliv. Rev.
28: 5
(1997), the entire teachings of which are incorporated herein by reference).
The
incorporation of polyethylene glycol) into the polymer as a blend to form
microparticle carriers allows further alteration of the release profile of the
active
ingredient (see Cleek et al., J. Control Release 48:259 (1997), the entire
teachings of
which are incorporated herein by reference). Ceramics such as calcium
phosphate
and hyroxyapatite can also be incorporated into the formulation to improve
mechanical qualities.
PPHOS polymers contain alternating nitrogen and phosphorous with no
carbon in the polymer backbone, as shown below in Structural Formula (IV):
R
N P
R'
n
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The properties of the polymer can be adjusted by suitable variation of side
groups R
and R' that are bonded to the polymer backbone. For example, the degradation
of
and drug release by PPHOS can be controlled by varying the amount of
hydrolytically unstable side groups. With greater incorporation of either
imidazolyl
or ethylglycol substituted PPHOS, for example, an increase in degradation rate
is
observed (see Laurencin et al., JBiomed Mater. Res. 27: 963 (1993), the entire
teachings of which are incorporated herein by reference), thereby increasing
the rate
of drug release.
Polyanhydrides, shown in Structural Formula (V), have well defined
degradation and release characteristics that can be controlled by including
varying
amounts of hydrophobic or hydrophilic monomers such as sebacic acid and 1,3-
bis(p-carboxyphenoxy)propane (see Leong et al., J. Biomed. Mater. Res. 19: 941
(1985), the entire teachings of which are incorporated herein by reference).
To
improve mechanical strength, anhydrides are often copolymerized with imides to
form polyanhydride-co-imides. Examples of polyanhydride-co-imides that are
suitable for orthopaedic applications are poly(trimellitylimido-glycine-co-1,6-
bis(carboxyphenoxy)hexane and pyromellityimidoalanine:1,6-bis(p-
carboxyphenoxy)hexane copolymers.
- C- R- C
O
n
(V)
Polypropylene fiunarates) (PPF) are highly desirable biocompatible
implantable carriers because they are an injectable, ira situ polymerizable,
biodegradable material. "Injectable" means that the material can be injected
by
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syringe through a standard needle used for injecting pastes and gels. PPF,
combined
with a vinyl monomer (N vinyl pyrrolidinone) and an initiator (benzoyl
peroxide),
forms an injectable solution that can be polymerized ira situ. It is
particularly suited
for filling skeletal defects of a wide variety of sizes and shapes (see Suggs
et al.,
Macromolecules 30:4318 (1997), Peter et al., J. Biomater. Sci. Poly,. Ed.
10:363
(1999) and Yaszemski et al., Tissue Ehg. 1:41 (1995), the entire teachings of
which
are incorporated herein by reference). The addition of solid phase components
such
as (3-tricalcium phosphate and sodium chloride can improve the mechanical
properties of PPF polymers (see Peter et al., J. Biomed. Mater. Res. 44: 314
(1999),
the entire teachings of which are incorporated herein by reference).
The pharmaceutical compositions of the present invention can be
administered by implantation at a site in need of osteoinduction.
"Implantation" or
"administration at a site" means in sufficient proximity to the site in need
of
treatment so that osteoinduction occurs (e.g., bone growth in the presence of
the
NPAR agonist but little or no growth in its absence) at the site when the NPAR
agonist is released from the pharmaceutical composition.
The pharmaceutical compositions can be shaped as desired in anticipation of
surgery or shaped by the physician or technician during surgery. It is
preferred to
shape the matrix to span a tissue defect and to take the desired form of the
new
tissue. In the case of bone repair of a non-union defect, for example, it is
desirable to
use dimensions that span the non-union. In bone formation procedures, the
material
is slowly absorbed by the body and is replaced by bone in the shape of or very
nearly
the shape of the implant. Alternatively, the pharmaceutical compositions can
be
administered to the site in the form of microparticles or microspheres. The
microparticles are placed in contact or in close proximity to the site in need
of
osteoinduction either by surgically exposing the site and applying the
microparticles
on or in close proximity to the site by painting, pipetting, spraying,
injecting or the
like. Microparticles can also be delivered to the site by endoscopy or by
laparoscopy. The preparation of PLGA microparticles and their use to stimulate
bone growth are described in Examples 1 and 2.
In yet another alternative, the pharmaceutical composition can be partially
enclosed in a supporting physical structure such as a mesh, wire matrix,
stainless
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steel cage, threaded interbody fusion cage and the like before administering
to the
site in need of osteoinduction.
Another alternative for applying the pharmaceutical composition of the
present invention is by injection. Compositions which are injectable include
the
solutions of polypropylene fumarate) copolymers described above and pastes of
calcium phosphate ceramics (see Schmitz et al., J. Oral Maxillofaeial SuYgefy
57:1122 (1999), the entire teachings of which are incorporated herein by
reference).
Injectable compositions can be injected directly to the site in need of
osteoinduction
and can conveniently be used to fill voids and fuse bones without the need for
invasive surgery.
NPAR agonists can also be administered by means other than implantation,
for example, by applying a solution comprising the NPAR agonist in an
acceptable
pharmaceutical carrier directly to or in near proximity to the site.
Administration of a
solution can be conveniently accomplished, for example, by syringe, either
through a
surgical opening or by parenteral administration to the desired site. Standard
pharmaceutical formulation techniques may be employed such as those described
in
Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
Suitable pharmaceutical carriers for parenteral administration include, for
example,
sterile water, physiological saline, bacteriostatic saline (saline containing
about 0.9%
mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's-
lactate
and the like.
A NPAR agonist or an implantable pharmaceutical composition of the
present invention cm be used in conjuction with an implantable prosthetic
device.
For example, a therapeutically effective amount of the pharmaceutical
composition
can be disposed on the prosthetic implant on a surface region that is
implantable
adjacent to a site in need of osteoinduction. Alternatively, the prosthetic
device is
constructed so as to continuously release the implantable pharmaceutical
composition or NPAR agonist at a pre-determined rate. The prosthesis may be
made
from a material comprising metal or ceramic. Examples of prosthetic devices
include a hip device, a screw, a rod and a titanium cage for spine fusion.
Thus this invention also provides a method for stimulating bone growth by
implanting a prosthetic device into a site in need of osteoinduction in a
subject. The
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prosthetic is at least partially coated with an implantable pharmaceutical
composition described hereinabove and implanted at a site in need of
osteoinduction
and maintained at the site for a period of time sufficient to permit
stimulation of
bone growth.
NPAR agonists used in the method of the present invention directed to
regeneration of cartilage are typically administered as one component in a
pharmaceutical composition to the site in need of cartilage growth, repair or
regeneration. Administering to the site in need of treatment means that the
pharmaceutical composition containing the NPAR agonist is administered in
sufficient proximity to the site in need of treatment so that cartilage growth
or
cartilage regeneration occurs at the site (e.g., a greater amount of cartilage
growth or
better quality of cartilage growth in the presence of the NPAR agonist than in
its
absence).
In one means of administration, the pharmaceutical composition is a solution
comprising the NPAR agonist and a suitable carrier. The solution is applied
directly
to or in near proximity to the site in need of treatment. Administration of
the
solution can be conveniently accomplished, for example, intraarticularly by
syringe,
in close proximity to the damaged tissue by syringe or through a surgical
opening.
Standard pharmaceutical formulation techniques may be employed such as those
described in Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, PA. Suitable pharmaceutical carriers for include, for example,
physiological
saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl
alcohol),
phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like.
In another means of administration, the pharmaceutical composition
comprises the NPAR agonist and an implantable biocompatible carrier. A
biocompatible carrier should be non-toxic, non-inflammatory, non-immunogenic
and devoid of other undesired reactions at the implantation site. Suitable
carriers
also provide for release of the active ingredient and preferably for a slow,
sustained
release over time at the implantation site.
A number of synthetic biodegradable polymers can serve as carriers with
sustained release characteristics. Examples of these polymers include poly a-
hydroxy esters such as polylactic acid/polyglycolic acid copolymers and
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polyanhydrides.
The polylactic acid/polyglycolic acid (PLGA) homo and copolymers
discussed with regard to regeneration of bone tissue are also suitable for use
as
sustained release vehicles for the compounds utilized to treat cartilage.
Similarly,
the polyanhydrides as shown in Structural Formula (V), can be used in the
methods
of treating collagen.
The pharmaceutical compositions can be shaped as desired in anticipation of
surgery or shaped by the physician or teclnlician during surgery. It is
preferred to
shape the matrix to span a tissue defect and to take the desired form of the
new
tissue. In the case of cartilage repair of large defects, it is desirable to
use dimensions
that span the defect. After implantation, the material is slowly absorbed by
the body
and is replaced by cartilage in the shape of or very nearly the shape of the
implant.
In one aspect, the carrier is a porous matrix into which progenitor cells may
migrate. Cells can often attach to such porous matrices, which can then serve
as a
scaffolding for tissue growth and thereby accelerate the rate of bone growth.
Chondrocytes can be applied to such matrices prior to implant to further
accelerate
healing. Collagen or a collagen gel is an example of a suitable porous matrix.
In another aspect, the carrier is a viscous solution or gel that is inj
ectable
intraarticuarly or at the site in need of treatment. Hyaluronic acid is an
example of a
carrier of this type. Hyaluronic acid products are commercially available and
include
ORTHOVISC developed by Anika, SYNVISC, developed by ~iomatrix,
HYALGAN, developed by Fidia and ARTZ, developed by Seikagaku. Platonic gel
is another example of this type of carrier. Platonic gels are nontxoic block
copolymers of ethylene oxide and propylene oxide. They exhibit thermosetting
properties that allow them to exist as viscous liquids at room temperatures,
but as
gels at body temperatures. Injectable compositions can be applied directly to
the site
in need of treatment without the need for invasive surgery. Polymers of
polyethylene oxide) and copolymers of ethylene and propylene oxide are also
suitable as injectable matrices (see Cao et al., J. Biomate~~. Sci 9:475
(1998) and
Sims et al., Plast Reconstr.Sufg. 98: 843 (196), the entire teachings of which
are
incorporated herein by reference).
NPAR agonists can be used to accelerate the growth or to maintain the
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functionality of isolated chondrocytes. In one embodiment, NPAR agonists can
be
added to tissue culture medium to stimulate proliferation and provide for more
rapid
proliferation and/or to prevent apoptotic death or senescence of cells often
encountered when primary cell isolates are place in culture. In another
embodiment,
because the NPAR agonists appear to stimulate matrix production, such NPAR
agonists could be used to maintain the differentiated functionality of
chondrocytes in
culture. NFAR agonists can be used alone in standard defined tissue culture
medium
or as a supplement to tissue culture medium containing serum or other growth
factor
to provide additive or synergistic effects on the ih vitro production or
maintenance
of chondrocytes. A sufficient quantity of the NPAR agonist is added to the
culture to
provide more rapid growth or to maintain greater functionality of the
chondrocytes
than in the absence of the agonist, i.e., a "stimulatory amount". Typically,
between
about 0.1 wg/ml and about 100 ~,g/ml of NPAR agonist is used.
With respect to bone growth, a "therapeutically effective amount" is the
quantity of NPAR agonist which results in bone growth where little or no bone
growth would occur in the absence of the agonist. Typically, the agonist is
administered for a sufficient period of time to achieve the desired
therapeutic or
cosmetic effect, i.e., sufficient bone growth. The amount administered will
depend
on the amount of bone growth that is desired, the health, size, weight, age
and sex
of the subject and the release characteristics of the pharmaceutical
formulation.
Typically, between about 1 ~g per day and about 1 mg per day of NPAR agonist
(preferably between about 5 ~.g per day and about 100 ~,g per day) is
administered
by continuous release or by direct application to the site in need of bone
growth.
With respect to cartilage growth, a "therapeutically effective amount" is the
quantity of NPAR agonist (or chondrocytes) which results in greater cartilage
growth or repair in the presence of the NPAR agonist than in its absence.
Alternatively or addition, a "therapeutically effective amount" is the
quantity of
NPAR agonist (or chondrocytes) which results in alleviation of the pain and/or
lack
of function associated with the cartilage damage. Typically, the agonist (or
chondrocytes) is administered for a sufficient period of time to achieve the
desired
therapeutic or effect. The amount administered will depend on the amount of
cartilage growth that is desired, the health, size, weight, age and sex of the
subject
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and the release characteristics of the pharmaceutical formulation. Typically,
between
about 0.1 wg per day and about 1 mg per day of NPAR agoiist (preferably
between
about 5 ~,g per day and about 100 wg per day) is administered by continuous
release
or by direct application to the site in need of carihage growth or repair.
Chondrocytes cultured in the presence of an NPAR agonists can also be
used to treat cartilage damage by administering a therapeutically effective
amount of
the chondrocytes to the site in need of treatment. With respect to
chondrocytes,
"therapeutically effective" also means which results in greater cartilage
growth or
repair with the treatment than in its absence. The administration of
chondrocytes to
treat cartilage damage is described in US Patent No. 4,846,835, the entire
teachings
of which are incorporated herein by reference.
A "subj ect" is preferably a human, but can also be an animal in need of
treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm
animals (e.g.,
cows, pigs, horses and the like) and laboratory animals (e.g., rats, mice,
guinea pigs
and the like).
Thrombin peptide derivatives can be synthesized by solid phase peptide
synthesis (e.g., BOC or FMOC) method, by solution phase synthesis, or by other
suitable techniques including combinations of the foregoing methods. The BOC
and
FMOC methods, which are established and widely used, are described in
Merrifiehd,
J. Ana. Chem. Soc. &x:2149 (1963); Meienhofer, Hormonal Proteins and Peptides,
C.H. Li, Ed., Academic Press, 1983, pp. 48-267; and Barany and Merrifield, in
The
Peptides, E. Gross and J. Meienhofer, Eds., Academic Press, New York, 1980,
pp.
3-285. Methods of solid phase peptide synthesis are described in Merrifiehd,
R.B.,
Scieyace, 232: 341 (1986); Carpino, L.A. and Han, G.Y., J. Org. Chem., 37:
3404
(1972); and Gauspohl, H. et al., Synthesis, 5: 315 (1992)). The teachings of
these
six articles are incorporated herein by reference in their entirety.
The invention is illustrated by the following examples which are not
intended to be limiting in any way.
EXEMPLIFICATION
Example 1 - Preparation of Pohylactic Acid/Polyg~lycolic Acid Copolyer
Microspheres of TP508
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A double emulsion technique was used to prepare microspheres of
polylactic acid/polyglycolic acid copolymer (PLGA) containing TP508.
Briefly, the matrix components were dissolved in methylene chloride and
TP508 was dissolved in water. The two were gradually mixed together
while vortexing to form a water-in-oil (W/O) emulsion. Polyvinyl alcohol
(0.3% in water) was added to the emulsion with further vortexing to form
the second emulsion (O/W), thereby forming a double emulsion: an O/W
emulsion comprised of PLGA droplets, and within those droplets, a second
disperse phase consisting of TP508 in water. Upon phase separation, the
PLGA droplets formed discrete microspheres containing cavities holding
TP508. To cause phase separation of the microspheres, a 2% isopropyl
alcohol solution was added. The particles were collected by centrifugation,
and then lyophilized to remove residual moisture. The composition of the
matrix was varied to form microspheres with different release kinetics
(Table 1).
Table 1: Composition of different microsphere formulations
Formu- PLGA , Polymer % % poly-
lation M. Wt. TP508 ethylene
glycol
A 50:50 46,700 S 0
20B 50:50 7,200 5 0
C 50:50 46,700 5 5
D 50:50 46,700 5 0
E 75:25 120,000 5 0
The mean diameter of the microspheres was measured in a Coulter
counter and the drug entrapment efficiency was measured by
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spectrophotometric assay at 276 nm following dissolution of a weighed
sample of microspheres in methylene chloride and extraction of the released
drug into water (Table 2).
Table 2: Formulation diameter and drug entrapment efficiency
Formulation Diameter, m TP508 Entra ment,
A 26.0 53.8
B 16.2 27.1
C 17.6 58.9
D 23.9 42.6
E 25.8 ~ 36.2
To measure TP508 release from the different PLGA matrices, 20 mg
of microspheres were placed in 1.0 ml of PBS contained in 1.5 ml
polypropylene microcentrifuge tubes. Tubes were incubated at 37°C and
shaken at 60 rpm. At various times, the tubes were centrifuged and the
supernatant containing released TP508 was removed and frozen for
subsequent analysis. Fresh PBS was added to the microspheres and
incubation was continued. TP508 in the supernatant was measured by
absorbance at 276 nm. For each formulation, quadruplicate release
determinations were performed. Formulations B and D showed no
detectable drug release during 28 days of incubation at 37°C. The
remaining
formulations all released detectable amounts of TP508 , although in all
cases the amount of drug released fell below detectable limits (<1 ~,g/mg
matrix/day) within 3-4 days. Formulations A and C showed the greatest
release of TP508, releasing 60-80% of the entrapped drug over 3-4 days.
The formulation with the fastest release kinetics, C , was chosen for fiuther
testing in in vivo studies.
Example 2 - PLGA Micros~heres Contaiun~ TP508 Induce Bone
Formation in Large (1.5 cml Defects in Rabbit Ulna
A 1.5 cm segmental defect was created in each ulna of 20 male New
Zealand rabbits. These bilateral ulnar osteotomies were created exactly the
same size by using a small metal guide to direct the cutting blade of the
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oscillating microsaw. Each rabbit acted as its own control; thus the left
defect was filled with microspheres that did not contain TP508, while the
right defect was filled with microspheres containing 100 or 200 ~,g TP508
(10 animals/group). The microspheres were prepared as described in
Example 1. Rabbits given bilateral uhzar osteotomies were randomly
divided into two groups. The first group received 100 ~g of TP508 in
microspheres (30 mg) in the right limb and microspheres alone in the left
limb. The second group was treated similarly, but received 200 ~,g of
TP508. These different doses were achieved by mixing TP508-containing
and TP508-devoid microspheres in different proportions. Animals were x-
rayed at two week intervals, beginning at week three, and sacrificed at nine
weeks.
100 wg of TP508 stimulated mineralization in the defect at 3 and 5
weeks post-surgery. X-rays at 7 and 9 weeks appeared similar to those
obtained at 5 weeks. Animals were sacrificed at 9 weeks post-surgery and
the ulna-radius was removed and photographed. In most cases a large
defect is still visible in ulnas from the control limbs, in contrast with the
TP508-treated limbs, in which most of the defects have successfully closed.
After sacrifice at 9 weeks post-surgery, repair strength was
measured by torsion testing (MTS-858 Minibionix machine). The results
are shown in Tables 3 and 4.
Table 3: Torsion testing of segmental defects treated with 100 ~g TP508.
Parameter Control SEM TP508,100 ~.g SEM
Ultimate 0.107 0.034 0.255+ 0.041
torque
Failure torque0.103 0.032 0.239+ 0.042
Ultimate 0.815 0.365 1.916~ 0.398
energy
Failure energy0.940 0.436 2.064~ 0.421
Stiffness 0.013 0.004 0.028~ 0.006
coeff.
~p<0.05,+p<0.01
Table 4: Torsion testing of segmental defects treated with 200 ~g TP508.
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Parameter Control SEM TP508, 200 SEM
~g
Ultimate torque0.095 0.042 0.322* 0.046
Failure torque0.093 0.041 0.306* 0.046
Ultimate energy0.534 0.355 2.947* 0.543
Failure energy0.641 0.374 3.433* 0.701
Stiffness coeffØ016 0.006 0.033~ 0.004
~p < 0.05, *p < 0.005
At 100 ~.g, TP508 more than doubled the mechanical strength of the healing
defect as measured by all the parameters tested (Table 3). Even stronger
repairs were
noted in the 200-~,g group (Table 4), with most parameters being approximately
50% higher than those seen in the low dose treatment group
Example 3 Thrombin Binding to Rat Chondrocytes
Primary cultures of rat articular chondrocytes were isolated and prepared for
in vitYO analysis using established methods (see Kuettner, K E., et.al.,J.
Cell Biology
93: 743-750, 1982). Briefly, cartilage pieces were dissected from the shoulder
of
rats and the pieces were digested with trypsin for one hour and with
collagenase for
three hours in tissue culture medium (DMEM) at 37 C with stirnng. The cells
were
plated in flasks at high density (50,000 cells/cm sq.) and were culture in
DMEM
containing antibiotics an ascorbic acid at 37° C in an atmosphere of 5%
COz.
The specific binding of'zsl thrombin to chondrocytes was carried out using
established thrombin receptor binding assays as disclosed in US Patent
5,352,664
and Carnet', DH and Cunningham, DD, Cell 15:1341-1349, 1978. Briefly, highly
purified human thrombin was iodinated and added to cultures of chondrocytes
with
or without unlabeled thrombin to correct for nonspecific binding. By
incubating
cells with different concentrations of labeled thrombin and measuring the
amount of
thrombin bound to cells and the amount of free thrombin in the medium it is
possible to estimate the number of receptors per cell and the affinity of
thrombin for
that binding site.
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Scatchard analysis of the labeled thrombin binding from three separate
experiments suggest that rat chondrocytes express an average of 3000 very high
affinity binding sites (100 pM affinity) and 230,000 high affinity sites (27
nM).
Example 4A NPAR Agonist Stimulation of Bovine Chondrocyte Proliferation
Primary cultures of bovine chondrocytes were prepared using the procedure
described for rat chondrocytes in Example 1. The cultures were subcultured
into 24
well plastic dishes at a low density and placed in 1% serum. Addition of the
NPAR
agonist TP508 to these cultures at concentrations of 1.0 or 10 ~,g/ml by
itself did not
stimulate cell proliferation. In contrast, addition of these concentrations of
TP508
together with a small amount of thrombin co-mitogen, resulted in a small, but
significant (p < 0.05) increase in cell number relative to that seen in
thrombin alone
after three days in culture.
Example 4B NPAR Agonist Stimulation of Bovine Chondrocyte Proteoglycan
Synthesis
To determine the effect of NPAR agonists on proteoglycan synthesis, bovine
chondrocytes were seeded into 96 well plates at a density of 2 x 105 cells per
well
and cultured in DMEM with 10% fetal calf serum. After establishment of these
mufti-layer cultures, the medium was replaced daily with DMEM containing 1%
serum with indicated concentrations of TP508 from 1 to 100 ~,g per ml (Table
5).
After 6 days in culture with daily changes of culture medium with or without
TP508, 35S sulfate was added to the medium and incubation continued for an
additonal 24 hours. As shown in Table 5, treatment with high concentrations of
TP508 (100 wg per ml) increased 35S sulfate incorporation relative to
untreated cells
by more than 10-fold.
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Table 5. Effect of the NPAR agonist TP508 on 35S sulfate incorporation in
bovine
chondrocyte cultures.
Treatment Mean CPM Std. Dev of Mean
1 % S erum
Control 4975 3552
TP508 (l~,g/ml) 4701 2692
TP508 (10~g/ml) 6960 3265
TP508 (100~,g/ml) 81946 13783
Example SA NPAR Agonist Stimulation of Proliferation Synthesis in Cultured Rat
Articular Chondrocytes
Rat articular chondrocytes were isolated from slices of rat articualar
shoulder
cartilage utilizing trypsin and collagenase digestions as described in Example
3.
Preparations of chondrocyte "3-dimensional" alginate bead cultures were
established
using established techniques as described by Guo et. al., (Corm. Tiss. Res.
19:277-
297, 1998). Following removal of cells from tissue culture flasks with
trypsin, the
cells were suspended in an alginate gel (1.2% w/v) and slowly expressed
through a
22 gauge needle in a dropwise fashion into 102 mM CaClz. As the drops contact
the
CaClz there is a nearly instantaneous polymerization of the alginate to create
a gel
bead. The beads were then washed three times in DMEM culture medium and
transferred to 35mm dishes and maintained in culture at 37 C in a 5% C02
. atmosphere by feeding with culture medium every two days.
The effect of NPAR agonist TP508 on chondrocyte cell proliferation after
three days in 3-dimensional alginate culture was determined by removing beads
from 35 mm dishes, washing them with 0.9% saline, and dissolving the alginate
beads by adding 1 ml of 55 mM sodium citrate, 0.15 M NaCI at 37° C for
10
minutes. Cell number was determined by diluting the 1 ml of dissolved beads
1:10
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with phosphate buffered saline (PBS) and counting the cells with a Z-series
Coulter
Counter. As shown in Table 6, TP50~ by itself stimulated proliferation of
chondrocytes in 3 dimensional culture.
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Table 6. Effect of the NPAR agonist TP508 on Proliferation of Rat Chondrocytes
in 3-D Bead Culture.
Treatment Cells/bead AfterStd. dev % Increase over
3 Control
days
Control 6238 688
TP508 30nM 7463 167 19.7
TP508 300 nM 8882 148 42.4
TP508 3 ~,M 8866 4 42.1
TP508 30 ~,M 7772 258 24.6
Example SB NPAR Agonist Stimulation of Proteoglycan Synthesis in
Cultured Rat Articular Chondrocytes
To determine the effectos of the NPAR agonist TP508 on proteoglycan
synthesis, 3-dimensional alginate cultures were prepared as described above
and
assayed for incorporation of [35S]-sulfate. Bead cultures were exposed to
indicated
concentrations of TP508 as well as [35S]-sulfate (20 ~,Ci/ml) and with daily
medium
changes and were harvested on days 7 for [35S]-sulfate incorporation. At each
time
point 5 -10 beads were removed, washed 3x with 0.9% saline, dissolved by
adding
0.5 ml of 55 mM sodium citrate, 0.15 M NaCl at 37 C for 10 minutes as
described
above, and counted in a liquid scintillation counter. [35S]-sulfate
incorporation was
normalized in each sample for number of beads added. As shown in Table 7,
TP508
treatment alone at a concentration of 300 nM (about 0.7 ~.g per ml),
stimulated [35S]-
sulfate incorporation about 50% over controls. There was also a large
stimulation
by 30 ~,M TP508 (about 70 ~.g per ml), however, there was a large relative
standard
deviation in measurements at this concentration.
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Table 7. Effect of the NPAR agonist TP508 on [35S]-sulfate incorporation into
proteoglycans.
Treatment CPM/bead Std. dev % Increase over
Control
Control 665 24
TP508 30nM 829 87 24.7
TP508 300 nM 1008 29 51.6
TP508 3 ~,M 827 9 24.1
TP508 30 ~M 1153 519 73.3
Example 6 Preparation of Polylactic Acid/Polyglycolic Acid Copolymer
Microspheres of TP508
A double emulsion technique was used to prepare microspheres of polylactic
acid/polyglycolic acid copolymer (PLGA) containing TP508. Briefly, the matrix
components were dissolved in methylene chloride and TP508 was dissolved in
water. The two were gradually mixed together while vortexing to form a water-
in-
oil (W/O) emulsion. Polyvinyl alcohol (0.3% in water) was added to the
emulsion
with further vortexing to form the second emulsion (O/W), thereby forming a
double emulsion: an O/W emulsion comprised of PLGA droplets, and witlun those
droplets, a second disperse phase consisting of TP508 in water. Upon phase
separation, the PLGA droplets formed discrete microspheres containing cavities
holding TP508. To cause phase separation of the microspheres, a 2% isopropyl
alcohol solution was added. The particles were collected by centrifugation,
and then
lyophilized to remove residual moisture. The composition of the matrix was
varied
to form microspheres with different release kinetics (Table 8).
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Table 8. Composition of different microsphere formulations
Formu-lationPLA:PGA Polymer % % poly-ethylen~
M. Wt. TP508 glycol
A 50:50 46,700 5 0
B 50:50 7,200 5 0
5C 50:50 46,700 5 5
D 50:50 46,700 5 0
E 75:25 120,000 5 0
The mean diameter of the microspheres was measured in a Coulter counter and
the drug entrapment efficiency was measured by spectrophotometric assay at 276
nm
following dissolution of a weighed sample of microspheres in methylene
chloride and
extraction of the released drug into water (Table 9).
Table 9. Formulation diameter and drug entrapment efficiency
Formulation Diameter, m TP508 Entra ment,
A 26.0 53.8
B 16.2 27.1
C 17.6 58.9
D 23.9 42.6
E 25.8 36.2
To measure TP508 release from the different PLGA matrices, 20 mg of
microspheres were placed in 1.0 ml of PBS contained in 1.5 ml polypropylene
microcentrifuge tubes. Tubes were incubated at 37°C and shaken at 60
rpm. At
various times, the tubes were centrifuged and the supernatant containing
released
TP508 was removed and frozen for subsequent analysis. Fresh PBS was added to
the microspheres and incubation was continued. TP508 in the supernatant was
measured by absorbance at 276 nm. For each formulation, quadruplicate release
determinations were performed. Formulations B and D showed no detectable drug
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release during 28 days of incubation at 37°C. The remaining
formulations all
released detectable amounts of TP508 , although in all cases the amount of
drug
released fell below detectable limits (<1 ~,g/mg matrixlday) within 3-4 days.
Formulations A and C showed the greatest release of TP508, releasing 60-80% of
the entrapped drug over 3-4 days. Formulation C showed the fastest release
kinetics
and was chosen for testing in the rabbit cartilage defect model described in
Example
7.
Example 7 The NPAR Agonist TP508 Stimulates Cartilage
Growth in Rabbit Models
Young, male New Zealand rabbits (2-3 kilograms) (n=15) were anesthetized
and given bilateral, medial longitudinal parapatellar arthrotomies. The skin,
subcutaneous tissue and joint capsule were incised, using electrocautery to
minimize
bleeding. The joint surface was exposed by lateral dislocation of the patella.
A 3-
mm diameter, 1-2-mm deep full-thickness defect was made in the trochlear
groove
of the femur using a surgical drill and pointed stainless steel drill bit. The
aim was
to extend the defect into the subchondral plate without piercing the
subchondral
bone.
The rabbits were divided into three groups. For each rabbit, both right and
left trochlear groove defects were filled with the same treatment. For this
study,
TP508 was formulated into PLGA controlled release microspheres, prepared as
described in Example 6 (Formulation C). The microspheres were mixed with
sufficient Pluronic F68 gel (5% w/v) to bind the spheres together into a paste-
like
consistency that could easily be packed into the defect. The control group
received
PLGA microspheres without TP508 in both defects. The treated groups received
microspheres containing either 10 or 50 mg of TP508/defect. One rabbit from
each
group was sacrificed at 4 weeks, 2 from each group were sacrificed at 6 weeks
and
the remaining aiumals were sacrificed at 9 weeks. Samples were fixed and
processed for histological analysis.
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At the time of sacrifice, there appeared to be considerable fibrous
granulation tissue and no evidence of white cartilage-like material in the
control
defects. In contrast, the defect had a nearly uniform, dense, white material
filling in
the defects from the 10 ~,g treated group and 50 ~,g group. By 6 weeks post-
surgery,
the macroscopic differences between treated and control defects were not so
pronounced.
Histology of the four week samples showed that indeed the control defects
were filled with what appeared to represent early granulation tissue including
inflammatory and fibroblastic cells. In contrast, the 10 and 50 microgram
treated
defects appeared to have a large number of chondrocytes and early signs of
cartilage
formation. This effect was seen more dramatically at weelc six. Controls had a
small
amount of connective tissue, yet little evidence of cartilage repair. In
contrast, in
both the 10 ~.g and 50 ~g treated defects, there appeared to be good
integration with
hyaline cartilage forming at the top of the defect and extensive subchondral
bone
repair.
Nine-week TP508 treated defects exhibited a predominantly hyaline matrix
with evidence of significant aggrecan content as shown by positive safranin-O
staining. In most instances there was no difference in aggrecan content
between the
repair site and native tissue. Histological results were quantitatively
assessed using
a grading system adapted by Freed, et al., J. Bioyned. MateYials Res. 28: 891-
899
(1944) from the scheme of O'Driscoll, et al., J. Bone Joint Sung. 126:1448-
1452
(2000) with a maximum score of 25 for normal articular cartilage. Experimental
TP508 treated defects scored mean averages that were significantly higher than
control defects (Table 10).
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Table 10. Histology Scoring For Articular Defect Study
Milligrams of TP508 Repair Score ~ SE
0 9.4 ~ 1.6
18.6 ~ 1.4
5 50 19.8 ~ 1.0
Peptide treated defects repaired with smooth articular surfaces and were
typically well bonded at the junction between repair and native tissue. The
quality of
control repair tissue was characterized as mostly fibrocartilage with poor
quality
joint surfaces. Integration at the junction between repair and native tissue
was
10 usually poor. Overall, the quality of cartilage repaired with TP508 was
significantly
enhanced over control non-treated defects. This improved quality of repair
tissue
should lead to more durable and functional restoration of joint biomechanics
and
reduction in the incidence of osteoarthritis in patients suffering from
traumatic
cartilage injuries.
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In summation, ulnar osteotomies treated with microspheres containing the
NPAR agonist TP508 showed evidence of bone mineralization and growth whereas
in most control osteotomies that received osteoconductive microspheres, there
was
no bone growth and/or failure to fill the voided region. Mechanical testing
for
mechanical strength and stiffiiess confirmed significant effects of TP508 on
bone
formation in this model. Because TP508 induced bone formation in sites where
it
did not occur without TP508, this discovery of osteoinduction is distinct from
prior
studies, in which TP508 accelerated the rate of normal fracture healing in
fracture or
small gap defects that would heal without TP508.
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While this invention has been particularly shown and described with references
to preferred embodiments thereof, it will be understood by those skilled in
the art that
various changes in form and details may be made therein without departing from
the
scope of the invention encompassed by the appended claims.
