Note: Descriptions are shown in the official language in which they were submitted.
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Description
METHODS FOR ADMINISTERING FGF18
BACKGROUND OF THE INVENTION
The fibroblast growth factor (FGF) family consists of at least twenty
three distinct members (Basilico et al., Adv. Cancer Res. 59:115-165, 1992 and
Fernig
et al., Prog. Growth Factor Res. 5(4):353-377, 1994) which generally act as
mitogens
for a broad spectrum of cell types. FGF18 was identified as a member of the
FGF
family which was most closely related to FGF8 and FGF17. Activities associated
with
FGF18 included stimulation of mesenchymal lineage cells, in particular cardiac
myocytes, osteoblasts and chondrocytes (U.S. Patent No. 6,352,971). FGF18 has
binds
and activates FGFR4 and the "IIIc" splice variants of FGFR3 and FGFR2. It has
been
shown that FGFR3 plays a role in bone growth. Mice made homozygous null for
the
FGFR3 (-/-) resulted in postnatal skeletal abnormalities (Colvin et al.,
Nature Genet.
12:309-397, 1996 and Deng et al., Cell 84:911-921, 1996). The mutant phenotype
suggests that in normal mice, FGFR-3 plays a role in regulation of chondrocyte
cell
division in the growth plate region of the bone (Goldfarb, Cytokine and Growth
Factor
Rev. 7:311-325, 1996). FGF receptor mutations are also found in human
chondrodysplasia and craniosynostosis syndromes (Ornitz and Marie, Genes and
Development 16: 1446-1465, 2002).
Bone remodeling is the dynamic process by which tissue mass and
skeletal architecture are maintained. The process is a balance between bone
resorption
and bone formation, with two cell types thought to be the major players. These
cells are
the osteoblast and osteoclast. Osteoblasts synthesize and deposit matrix to
become new
bone. The activities of osteoblasts and osteoclasts are regulated by many
factors,
systemic and local, including growth factors.
When bone resorption exceeds bone formation, a net loss in bone results,
and the propensity for fractures is increased. Decreased bone formation is
associated
with aging and certain pathological states. In the U.S. alone, there are
approximately
1.5 million fractures annually that are attributed to osteoporosis. The impact
of these
fractures on the quality of the patient's life is immense. Associated costs to
the health
care system in the U.S. are estimated to be $5-$10 billion annually, excluding
long-term
care costs.
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Other therapeutic applications for growth factors influencing bone
remodeling include, for example, the treatment of injuries which require the
proliferation of osteoblasts to heal, such as fractures, as well as
stimulation of
mesenchymal cell proliferation and the synthesis of intramembraneous bone
which have
been indicated as aspects of fracture repair (Joyce et al. 36th Annual
Meeting,
Orthopaedic Research Society, February 5-8, 1990. New Orleans, LA).
Replacement of damaged articular cartilage caused either by injury or
disease is a major challenge for physicians, and available treatments are
considered
unpredictable and effective for only a limited time. Virtually all the
currently available
treatments for cartilage damage focus on relief of pain, with little or no
emphasis on
regeneration of damaged tissues. Therefore, the majority of younger patients
either do
not seek treatment or are counseled to postpone treatment for long as
possible. When
treatment is required, the standard procedure' is a total joint replacement or
microfracture, a procedure that involves penetration of the subchondral bone
to
stimulate fibrocartilage deposition by chondrocytes. While deposition of
fibrocartilage
is not a functional equivalent of articular cartilage, it is at the present
the best available
treatment because there has been little success in replacing articular
cartilage. Two
approaches to stimulating deposition of articular cartilage that are being
investigated
are: stimulating chondrocyte activity in vivo and ex vivo expansion of
chondrocytes and
their progenitors for transplantation (Jackson et al., Arthroscopy: The J. of
Arthroscopic
and Related Surg. 12:732-738, 1996). In addition, regeneration or repair of
elastic
cartilage is valuable for treating injuries and defects to ear and nose. Any
growth factor
with specificity for chondrocytes lineage cells that stimulates those cells to
grow,
differentiate or induce cartilage production would be valuable for
maintaining, repairing
or replacing articular cartilage.
Administration of proteins generally requires a formulation that
prolongs the half-life or biological activity of the active protein by
increasing the
resistance to proteolytic degradation or aggregation. Delivery of a protein
therapeutic
composition can also be difficult when the site for therapeutic action is
preferably
limited to a specific location in the body. The present invention provides
formulations
of FGF18 that will be easier to administer and more effective, and other uses
that
should be apparent to those skilled in the art from the teachings herein.
DETAILED DESCRIPTION OF THE INVENTION
Prior to setting forth the invention in detail, it may be helpful to the
understanding thereof to define the following terms:
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The term "affinity tag" is used herein to denote a polypeptide segment
that can be attached to a second polypeptide to provide for purification or
detection of
the second polypeptide or provide sites for attachment of the second
polypeptide to a
substrate. In principal, any peptide or protein for which an antibody or other
specific
,binding agent is available can be used as an affinity tag. Affinity tags
include a poly-
histidine tract, protein A (Nilsson et al.,, EMBO J. 4:1075, 1985; Nilsson et
al., Methods
Enz mol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene
67:31,
1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA
82:7952-
4, 1985), substance P, FlagTM peptide (Hopp et al., Biotechnology 6:1204-10,
1988),
streptavidin binding peptide, or other antigenic epitope or binding domain.
See, in
general, Ford et al., Protein Expression and Purification 2: 95-107, 1991.
DNAs
encoding affinity tags are available from commercial suppliers (e.g.,
Pharmacia
Biotech, Piscataway, NJ).
The term "allelic variant" is used herein to denote any of two or more
alternative forms of a gene occupying the same chromosomal locus. Allelic
variation
arises naturally through mutation, and may result in phenotypic polymorphism
within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or
may encode polypeptides having altered amino acid sequence. The term allelic
variant
is also used herein to denote a protein encoded by an allelic variant of a
gene.
The terms "amino-terminal" and "carboxyl-terminal" are used herein to
denote positions within polypeptides. Where the context allows, these terms
are used
with reference to a particular sequence or portion of a polypeptide to denote
proximity
or relative position. For example, a certain sequence positioned carboxyl-
terminal to a
reference sequence within a polypeptide is located proximal to the carboxyl
terminus of
the reference sequence, but is not necessarily at the carboxyl terminus of the
complete
polypeptide.
The term "hyaluronic acid" are used herein to include derivatives of
hyaluronic acid that include esters of hyaluronic acid, salts of hyaluronic
acid and also
includes the term hyaluronan. The designation also includes both low and high
molecular weight forms of hyaluronans and crosslinked hyaluronans or hylans.
Examples of such hyaluronans are Synvisc (Genzyme Corp. Cambridge, MA),
ORTHOVISC (Anika Therapeutics, Woburn, MA), and HYALGAN (Sanofi-
Synthelabo Inc., Malvern, PA)
The term "isolated", when applied to a polynucleotide, denotes that the
polynucleotide has been removed from its natural genetic milieu and is thus
free of
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other extraneous or unwanted coding sequences, and is in a form suitable for
use within
genetically engineered protein production systems. Such isolated molecules are
those
that are separated from their natural environment and include cDNA and genomic
clones. Isolated DNA molecules of the present invention are free of other
genes with
which they, are ordinarily associated, but may include naturally occurring 5'
and 3'
untranslated regions such as promoters and terminators. The identification of
associated regions will be evident to one of ordinary skill in the art (see
for example,
Dynan and Tijan, Nature 316:774-78, 1985).
An "isolated" polypeptide or protein is a polypeptide or protein that is
found in a condition other than its native environment, such as apart from
blood and
animal tissue. In a preferred form, the isolated polypeptide is substantially
free of other
polypeptides, particularly other polypeptides of animal origin. It is
preferred to provide
the polypeptides in a highly purified form, i.e. greater than 95% pure, more
preferably
greater than 99% pure. When used in this context, the term "isolated" does not
exclude
the presence of the same polypeptide in alternative physical forms, such as
dimers or
alternatively glycosylated or derivatized forms.
The term "ortholog" denotes a polypeptide or protein obtained from one
species that is the functional counterpart of a polypeptide or protein from a
different
species. Sequence differences among orthologs are the result of speciation.
A "polynucleotide" is a single- or double-stranded polymer of
deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
Polynucleotides include RNA and DNA, and may be isolated from natural sources,
synthesized in vitro, or prepared from a combination of natural and synthetic
molecules.
Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides
("nt"), or kilobases ("kb"). Where the context allows, the latter two terms
may describe
polynucleotides that are single-stranded or double-stranded. When the term is
applied
to double-stranded molecules it is used to denote overall length and will be
understood
to be equivalent to the term "base pairs". It will be recognized by those
skilled in the
art that the two strands of a double-stranded polynucleotide may differ
slightly in length
and that the ends thereof may be staggered as a result of enzymatic cleavage;
thus all
nucleotides within a double-stranded polynucleotide molecule may not be
paired. Such
unpaired ends will in general not exceed 20 nt in length.
A "polypeptide" is a polymer of amino acid residues joined by peptide
bonds, whether produced naturally or synthetically. Polypeptides of less than
about 10
amino acid residues are commonly referred to as "peptides".
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The term "promoter" is used herein for its art-recognized meaning to
denote a portion of a gene containing DNA sequences that provide for the
binding of
RNA polymerase and initiation of transcription. Promoter sequences are
commonly,
but not always, found in the 5' non-coding regions of genes.
5 A "protein" is a macromolecule comprising one or more polypeptide
chains. A protein may also comprise non-peptidic components, such as
carbohydrate
groups. Carbohydrates and other non-peptidic substituents may be added to
aprotein
by the cell in which the protein is produced, and will vary with the type of
cell.
Proteins are defined herein in terms of their amino acid backbone structures;
substituents such as carbohydrate groups are generally not specified, but may
be present
nonetheless.
The term "receptor" denotes a cell-associated protein that binds to a
bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on
the cell.
Membrane-bound receptors are characterized by a multi-peptide structure
comprising
an extracellular ligand-binding domain and an intracellular effector domain
that is
typically involved in signal transduction. Binding of ligand to receptor
results in a
.conformational change in the receptor that causes an interaction between the
effector
domain and other molecule(s) in the cell. This interaction in turn leads to an
alteration
in the metabolism of the cell. Metabolic events that are linked to receptor-
ligand
interactions include gene transcription, phosphorylation, dephosphorylation,
increases
in cyclic AMP production, mobilization of cellular calcium, mobilization of
membrane
lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of
phospholipids. In
general, receptors can be membrane bound, cytosolic or nuclear; monomeric
(e.g.,
thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric
(e.g.,
PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF
receptor, erythropoietin receptor and IL-6 receptor).
The term "secretory signal sequence" denotes a DNA sequence that
encodes a polypeptide (a "secretory peptide") that, as a component of a larger
polypeptide, directs the larger polypeptide through a secretory pathway of a
cell in
which it is synthesized. The larger polypeptide is commonly cleaved to remove
the
secretory peptide during transit through the secretory pathway.
Molecular weights and lengths of polymers determined by imprecise
analytical methods (e.g., gel electrophoresis) will be understood to be
approximate
values. When such a value is expressed as "about" X or "approximately" X, the
stated
value of X will be understood to be accurate to 10%.
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The present invention is based in part on the discovery that when
compositions of FGF18 polypeptides or proteins plus a negatively charged
carrier, such
as hyaluronic acid, are administered to a synovial joint, the stimulatory
effects of the
FGF18 are enhanced. Therefore, the present invention is directed to
compositions of
FGF18 polypeptides or proteins plus negatively charged carriers, in particular
hyaluronic acid for stimulating the proliferation of mesenchymal cells,
particularly
chondrocytes. The compositions are administered intraarticulariy to a joint ,
containing
synovial fluid.
The nucleotide sequence of the FGF18 cDNA is described in SEQ ID
NO. 1, and its deduced amino acid sequence is described in SEQ ID NO. 2. FGF18
was
originally designated zFGF5, and is fully described in commonly assigned U.S.
Patents
6,352,971 and 5,989,866. Analysis of the cDNA
encoding a FGF18 polypeptide (SEQ ID NO: 1) revealed an open reading frame
encoding 207 amino acids (SEQ ID NO: 2) comprising a mature polypeptide of 180
amino acids (residue 28 to residue 207 of SEQ ID NO: 2).
The mouse FGF18 polynucleotide sequence as shown in SEQ ID NO: 3
and corresponding amino acid sequence as shown in SEQ ID NO: 4 were found to
have
a high degree of homology to that of the human ortholog. At the amino acid
level, the
mouse and human polypeptides are approximately 98% identical, with three amino
acid
changes. Those skilled in the art will recognize that the sequences disclosed
in SEQ ED,
NO: 1 or SEQ ID NO: 3 and SEQ ID NO: 2 and SEQ ID NO: 4 represent a single
allele
of the human and mouse FGF18 gene and polypeptide, respectively, and that
allelic
variation and alternative splicing are expected to occur.
Members of the FGF family are characterized by heparin binding
domains. A putative heparin-binding domain for FGF18 has been identified in
the
region of amino acid residue 148 (Gly) to amino acid residue 169 (Gln) of SEQ
ID NO:
2 and SEQ ID NO: 4. It is postulated that receptor-mediated signaling is
initiated upon
binding of FGF ligand complexed with cell-surface heparin sulfate
proteoglycans.
Many FGF family members can be placed into one of two related families on the
basis
of their structures and functions. aFGF and bFGF consist of three exons
separated by
two introns of variable length. FGF-8 consists of five exons, the first three
of which
correspond to the first exon of aFGF and bFGF. All the known FGF family
members
are spliced to form single polypeptides.
Analysis of the ligand-receptor complex of FGF18 has demonstrated that
FGF18 has specificity for FGFR4 and the "IHc" splice variants of FGFR3 and
FGFR2.
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FGFR3-111c and FGFR2-111c have been identified within chondrocytes of
cartilage
tissue, and in particular, both receptors have been found within human
articular
cartilage. FGFR3 and FGFR2 have been found in the growth plate of mammals and
play important roles in the formation of endochondral and intramembranous
bone. In
particular, FGFR2 and FGFR3 play important roles in developing endochondral
and
intramembranous bone, FGFR2 is first expressed in condensing mesenchyme and
FGFR3 expression is initiated as chondrocytes differentiate and proliferate.
In
developing cranial bones, FGFR3 is found in the dura mater and periosteum,
whereas
FGFR2 is expressed in osteoprogenitor cells at the osteogenic front separating
the
sutures. FGFR2 is also expressed in traebecular bone. (Ornitz and Marie,
ibid., 2002)
Previously, it has been shown that FGF18 is a proliferative agent for
chondrocytes and
osteoblasts, depending upon both the differentiated state of these cell types
and the
mode of administration. (See, U.S. Patents 6,352,971 and 5,989,866; Ellsworth
et al.
Osteoarthritis and Cartilage, 10:308-320, 2002; Shimoaka et al., J. Bio.Chem.
277 fD
7493-500, 2002).
Osteoarthritis causes pain in the joints, is believed to be caused by a
deficiency in the production of extracellular matrix- including sulfated
proteoglycans,
hyaluronic acid (HA) and type II collagen. HA is natural high viscosity
mucopolysaccharide with alternating, (1-3) glucuronidic and, (1-4)
glucosaminidic
bonds. It is found in the umbilical cord, in vitreous humor, and synovial
fluids. For use
in the treatment methods and compositions of the present invention, any source
of HA
is appropriate, however, recombinantly-produced HA (i.e., protein produced in
bacterial, yeast, or mammalian cell culture) may be preferred over isolation
from animal
or human tissue sources in order to insure purity of the composition. In the
connective
tissue HA functions as binding and protective agent. HA fractions and salts of
HA have
been used for treatment of damaged bone joints and osteoarthritis. (See, U.S.
Patent
5,925,626; U.S. Patent 5,631,241 and EP 0,939,086.) HA is also used in
viscosuregery
and viscoupplementation and as an aid in ophthalmic surgery.
HA has been used as a component for therapeutic treatment of a variety
conditions, both using the HA as the primary therapeutic and as a component of
a
therapeutic composition useful for treatment. In experiments done by others,
HA
scaffolds were used to implant autologous chondrocytes into patients' knees,
with data
showing that symptomatic and functional improvements results. Raynauld et al.
(Osteoarthritis and Cartilage, 10(7):506-517, 2002) describe results using an
HA
formulation in conjunction with appropriate care in which clinically
effectiveness for
primary and secondary outcomes were improved over appropriate care alone.
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Generally, primary outcomes can be measured as change in the Western Ontario
and
McMaster (WOMAC) osteoarthritis index, which is a measurement of pain.
Secondary
outcomes measures will include functional disability and self-reported quality
of life. If
the therapeutic outcome includes a disease modifying agent, then joint
morphology is a
primary outcome variable, as well. (Hochberg et al., J. of Rhematolog.
24(4):792-794,
1997).
U.S. Patent 4,636,524 discloses cross-linked gels of HA, alone and mixed
with other hydrophilic polymers and containing various substances or
covalently
bonded low molecular weight substances and processes for preparing them. These
products are useful in numerous applications including cosmetic formulations
and as
drug delivery systems. HA is known to be a biologically tolerable polymer in
the sense
that it does not cause any immune or other kind of response when introduced
into a
human body,. the cross-linked HA gels can be used for various medical
applications.
The cross-linked gels modified with other polymers or low molecular weight
substances
can be used as drug delivery devices.
Canadian Letters Patent 1,240,929 teaches the combination of chondroitin
sulfate compound and a hyaluronate to protect both human and animal cell
layers and
tissue subject to exposure to trauma.
U.S. Patent 4,851,521 and European Patent Application 0,265,116, both
describe HA fractions and cross-linked esters of H.A. U.S. Patent 4,851,521
describes
esters of HA incorporated into pharmaceutical preparations as the active
ingredient and
as vehicles for ophthamological medicines for topical use and in suppositories
for a
systemic effect due to the effect of transcutaneous absorption, such as in
suppositories.
U.S. Patent Nos. 6,221,854 and 5,942,499 Cl (Reexam 4806) describe the
use of HA and basic FGF (FGF-2) for the treatment of bone. The patent teaches
an
injectable mixture that is administered into an orthotopic or intraosseous
site of desired
bone growth.
In contrast, the FGF18 polypeptide and HA compositions of the present
invention provide a method for stimulating the proliferation of chondrocytes,
in
particular differentiated chondrocytes, capable of inducing specialized cell
functions,
normally associated with terminally differentiated cells. When a composition
of FGF18
and HA was administered locally to articular cartilage, proliferation of the
cells and
concomitant synthesis of glycosaminoglycans was increased beyond the results
seen
either with FGF18 alone or HA alone. These results indicate that compositions
of
FGF18 polypeptides and HA can play a therapeutic role in maintaining or
repairing
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cartilaginous tissue, such as joints damaged by osteoarthritis, rheumatoid
arthritis or
traumatic injury.
FGF18 has been shown to increase cartilage deposition both in vivo and
in vitro. Generation of hyaline cartilage, elastic cartilage, and
fibrocartilage are
valuable both as a therapeutic and as component for biological matrices. FGF18
and
HA compositions will be useful in treating articular cartilage defects in
synovial joints
that are due to age-related superficial fibrillation, cartilage degeneration
due to
osteoarthritis, and focal chondral and osteochondral defects due to injury or
disease.
FGF18 and HA compositions will also be useful for treating joint disease
caused by
osteochondritis dissecans and degenerative joint disease. In the field of
reconstructive
and plastic surgery, FGF18 and HA compositions will be useful for autogenous
or
allogenic cartilage expansion and transfer for reconstruction of extensive
tissue defects.
Expansions of cells and induction of elastic cartilage production will be
useful for
generation and repair of ear and nose tissue.
FGF18 compositions can be applied by direct injection into the synovial
fluid of the joint or directly into the defect, either alone or complexed with
a suitable
carrier for extended release of protein. However, when FGF18 polypeptide and
HA
were delivered directly to the synovial joint, the effects of the compositions
to stimulate
chondrocytes proliferation exceeded that of FGF1 8 polypeptide or HA alone.
FGF18 can also be used to expand chondrocyte populations in culture
for autogenous or allogenic chondrocyte transplantation and then administered
with
concurrent treatment consisting of administration of FGF18 polypeptide and HA
compositions. In these procedures, for example, chondrocytes can be harvested
arthroscopically from an uninjured minor load-bearing area of the damaged
joint, and
can be cultured in the presence of FGF18 compositions to increase the number
of cells
prior to transplantation. The expanded cultures will then be admixed with
FGF18
polypeptide and HA compositions, and placed in the joint space or directly
into the
defect. FGF18 and HA compositions can be used in combination with periosteal
or
perichondrial grafts that contain cells that can form cartilage and/or help to
hold the
transplanted chondrocytes or their precursor cells in place. FGF18 and HA
compositions can be used to repair cartilage damage in conjunction with lavage
of the
joint, stimulation of bone marrow, abrasion arthroplasty, subchondral
drilling, or
microfracture of the subchondral bone. Additionally, after the growth of
cartilage due
to the administration of the FGF18 and HA composition, additional surgical
treatment
may be necessary to suitably contour the newly formed cartilage surface.
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The compositions of the present invention provide a method for
stimulating chondrocyte proliferation and cartilage production in
cartilagenous tissues
that have been damaged due to traumatic injury or chondropathy. Of particular
importance for treatment are tissues that exhibit articulated surfaces, such
as, spine,
5 shoulder, elbow, wrist, joints of the fingers, hip, knee, ankle, and the
joints of the feet.
Examples of diseases that may benefit from treatment include osteoarthritis,
rheumatoid
arthritis, other autoimmune diseases, or osteochondritis dessicans. In
addition, cartilage
malformation is often seen in forms of dwarfism in humans suggesting that
FGF18
would be useful in these patients.
10 FGF18 and HA compositions can be applied by direct injection into the
synovial space of the joint, into nearby tissues, or directly into a cartilage
defect in
combination with a carrier that exhibits a negative charge under physiological
conditions. Since FGF18 has an isoelectric point of >9.0, at physiological pH
FGF18
exhibits a net positive charge. Thus carrier molecules with an abundance of
negative
charge may bind FGF18 and enhance its activity. Such carriers include low and
high
molecular weight hyaluronans, sulfated proteoglycans, B-cyclodextrin
tetradecasulphate, hydroxyapatite, alginate microspheres, chitosans, and
methylcellulose.
For pharmaceutical use, the compositions of the present invention are
formulated for intraarticular administration according to conventional
methods. The
dosage regiment will be determined using various patient variables
(e.g.,weight, age,
sex), as well as clinical presentation (e.g., extent of injury, site of
injury, etc.) In
general, pharmaceutical formulations will include a FGF18 protein in
combination with
a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5%
dextrose in
water or the like. Formulations may further include one or more excipients,
preservatives, solubilizers, buffering agents, albumin to prevent protein loss
on vial
surfaces, extend half-life, etc. The FGF18 and HA may be administered
separately or in
combination as a single composition. Thus, the formulations may be provided as
a
single formulation or as a multicomponent kit. Methods of formulation are well
known
in the art and are disclosed, for example, in Remington's Pharmaceutical
Sciences,
Gennaro, ed., Mack Publishing Co., Easton PA, 1990. -
Determination of dose is within the level of ordinary skill in the art. The
proteins may be administered for acute treatment, over one week or less, often
over a
period of one to three days or may be used in chronic treatment, over several
months or
years.
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In other embodiments, a pharmaceutical FGF18 and HA composition
will comprise a formulation for timed-release of the protein. Time-release
formulations
generally include a monolithic delivery device comprising biocompatible
solutions,
gels, pastes, and putties in a matrix, in which the composition is entrapped
or dissolved.
Release from such a timed-release composition occurs by diffusion through the
matrix
and/or erosion of the matrix. A reservoir system, where the pharmaceutical
composition diffuses through a membrane, may also be used.
Although administration of FGF18 and HA, in a pharmaceutically
acceptable admixture, is sufficient to provide the delivery of the
chondrogenic peptides
of the present method, there may be clinical situations where additional drugs
are
combined in the admixture. Examples of other drugs which may be clinically
indicated
include anti-inflammatory drugs such as nonspecific and specific
cyclooxygenase-2
inhibitors, non-steriodal and steroidal anti-inflammatory drugs. Some of the
nonspecific COX inhibitors that could be used in the present invention include
salicylic
acid and derivatives, such as aspirinTM or sulfasalazine, para-aminophenol
derivatives,
such as acetaminophen, indole and indene acetic acids, such as indomethacin or
sulindac, arylprpionic acids, such as ibuprofen, naproxen, or oxaprozin,
anthranilic
acids, such as mefenamic acid, enolic acids including oxicams, or alkanonoes,
such as
nabumentone. Specific COX-2 inhibitors would be diaryl-substituted fuanonoes
(Refecoxib), diaryl-substituted pyrazoles (Celecoxib), indole acetic acids
(Etodolac)
and sulfonaildes (Nimesulide). Additionally, steroids, such as dexamethazone,
prednisone, triamcinolone, or methylprednisone, are among the drugs that could
be
used. Other types of drugs suitable for the present invention would be
inhibitors of the
tumor necrosis factor family, such as EnbrelTM or TACI-Ig, IL-1 antagonists
such as
Kinaret, antagonists of IL-18 and IL-15, and immunosuppressive drugs such as
cyclosporine. In addition, FGF18 may be administered with inhibitors of the CC
(MCP-1, RANTES, MEP-lalpha, and MIP-lbeta) and CXC (IL-8 and GRO-alpha)
chemokine family.
The invention is further illustrated by the following non-limiting examples.
Example 1
Intraarticular injection of FGF18
FGF18 was lyophilized and reconstituted at the appropriate
concentration in either PBS or 0.5% hyaluronan (0.2 urn sterile filtered). A
single dose
of FGF18, vehicle (PBS), hyaluronan or the appropriate combination of FGF18
dissolved in either PBS or 0.5% hyaluronan, contained in a final volume of 5 j
l was
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injected into the intraarticular space of the left stifle (knee) of 10 week
old female
c57/B16 mice. There were seven animals per group. All dosing was performed
under
isoflurane anesthesia and 100 l of buprenorephine was administered upon
recovery for
analgesia. The animals were sacrificed 2 weeks after dosing and tissues were
taken for
routine histology.
The following dose groups were used:
Group treatment
1 no treatment
2 PBS
3 5 N.g FGF18 in PBS
4 0.5 gg FGF18 in PBS
5 0.05 g FGF18 in PBS
6 hyaluronan 0.5%
7 hyaluronan 0.5% + 5.0 pg FGF18
8 hyaluronan 0.5% + 0.5 g FGF18
9 hyaluronan 0.5% + 0.05 g FGF18
10 sham injection
A dose-dependent increase in cartilage formation was observed
following two weeks after injection of FGF18 contained in 0.5% hyaluranon..The
effective doses were 0.5 and 5 pg FGF18. Definitive evidence of chondrocyte
proliferation was seen in 7/7 animals treated with 5.0 g FGF18 in 0.5%
hyaluronan
and in 3/7 mice treated with 0.5 g FGF18 in 0.5% hyaluronan. The increase in
cartilage formation was confined to the margins of the joint in the region of
the
perichondrium, and did not appear to include the articular surface. Little or
no
chondrocyte proliferation was seen in any of the other groups. Importantly,
neither PBS
nor hyaluronan alone had any apparent effects on chondrocyte proliferation.
There
were no apparent effects on the cartilage proliferation when FGF18 was
injected in the
absence of carrier. These observations, combined with those made previously,
suggests
that the progenitor cells in the perichondrium are a primary target for FGF18.
The lack
of effect on the articular chondrocytes may be due to the failure of the
protein to
penetrate the intact articular surface because FGF18 will stimulate the growth
and
proteoglycan synthesis of primary human and porcine articular chondrocytes in
vitro.
In addition, FGF18 induced closure and mineralization of the growth
plate both in the presence and absence of hyaluranon: closure was seen in 2/4
mice
treated with 5.0 g FGF18 in PBS, 2/6 mice treated with 0.5 g FGF18 in PBS,
and
1/7 animals treated with 0.05 .tg FGF18 in PBS. This effect was more
pronounced in
the presence of hyaluronan with closure seen in 7/7 mice treated with 5.0 .tg
FGF18 in
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0.5% hyaluronan and in 5/7 animals treated with 0.5 g FGF18 in 0.5%
hyaluronan.
These data reinforce the conclusion that hyaluranon enhances the activity of
FGF18.
Inflammation was minimal and only observed at the highest doses of
FGF18 in combination with hyaluranon. No cartilage degradation was observed,
an
important point since bFGF has been reported to have both anabolic and
catabolic
effects on cartilage.
Lastly, no significant systemic effects of FGF18 were observed in
animals that received 5 g FGF18 with hyaluranon, either at distant cartilage
sites or
other tissues. However, some focal closures of the epiphysis were observed in
one
animal that received HA alone and one animal that received 5 gr FGF18 alone.
The
significance of these observations is not clear at this time.
Example 2
Treatment of Osteoarthritis Model
To evaluate whether FGF18 could generate chondral tissue and reverse
cartilage degeneration in a setting of osteoarthritis (OA), OA was induced by
creating a
meniscal tear in the knee joint of rats. In this model, damage to the meniscus
induces
progressive cartilage degeneration and osteophyte formation that mimic the
changes
that occur in spontaneous osteoarthritis.
FGF18 was dissolved in a hyaluronan carrier and was applied to the
operated knee by intra-articular injection. The repair of cartilage
degeneration was
evaluated 3 weeks later. The medial collateral ligament of each rat (n=10 rats
per
group) was transected and the medial meniscus was cut through the full
thickness to
simulate a complete tear. Three weeks after surgery, rats received intra-
articular
injections of either vehicle (0.5% hyaluronan) or vehicle containing E. coli-
derived
recombinant human FGF18 (0.1, 1.0, or 5.0 ug) twice per week for three weeks.
Four
days after the last injection, the knee joints were harvested, collected into
buffered
formalin, decalcified, and embedded in paraffin for histology. Frontal
sections of the
knee, joints were stained with toluidine blue to assess formation of chondral
tissue. An
image of the tibial plateau of each knee was captured using an Optimas image
analysis
system. Multiple sections of the right knee were analyzed microscopically and
scored
subjectively for cartilage degeneration (chondrocyte/matrix loss and
fibrillation) and
chondrophyte formation. Strict attention to zones (outside, middle, and inside
thirds of
the medial tibial plateau) was adhered to and summed to reflect total severity
of tibial
degeneration. Micrometer measurements of the total extent of the tibial
plateau
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affected by degeneration, width of tibial lesions that extended >50% of
cartilage
thickness (Tibial Cartilage Degeneration Width), lesion depth (Depth Ratio),
thickness
of the medial tibial cartilage to the tidemark, and chondrophyte size and
number were
assessed. Statistical analysis of histopathologic parameters was done by
comparing
group means using the two-tailed Student's t-test or by analysis of variance.
All
injections and scoring were performed by investigators blinded to the
treatment groups.
In this rat OA model, the degeneration of cartilage is most severe on the
outer two-thirds of the tibial plateau and reaches maximal levels at 3 weeks
following
the meniscal damage. FGF1 8 was administered from 3 to 6 weeks following
surgery to
determine if it could induce repair of the damaged cartilage. Analysis
indicated that
FGF18 induced a dose-dependent increase in cartilage hypertrophy and
overgrowth of
new cartilage around the damaged areas as well as normal cartilage in the
lateral
compartment. Specifically, the highest dose of FGF18 (5 g) resulted in a 57%
decrease (p<0.05) in cartilage degeneration scores for the outer 1/3 of the
tibial plateau
(Table 1), a 57% reduction (p<0.05) in the width of significant tibial
cartilage
degeneration (Table 2), and a 46% decrease (p<0.05) in depth ratio for any
matrix
change (Table 3) as a result of filling of the cartilage defects with repair
tissue. In
addition, FGF18 produced dose-dependent increases in medial tibial cartilage
thickness,
from 243 21 to 319 77 m (Table 4) in rats treated with vehicle or 5.0 g
of
FGF18, respectively.
The morphology of the repair tissue ranged from fibrous with
proteoglycan deposition to fibrocartilage. Repair tissue appeared to originate
from the
marginal zone areas and extended across the degraded and sometimes intact
surfaces. In
nearly all areas, repair tissue. appeared to integrate well with the margins
of the
remaining normal cartilage. Although the morphology of the repair tissue was
different
from hyaline cartilage, it appeared to effectively fill the defect and there
were virtually
no degenerative changes. In contrast to the FGF18-treated animals, rats
treated with
vehicle alone showed no signs of cartilage repair except in rare cases where
erosion of
the subchondral bone permitted influx of bone marrow stem cells. In addition
to
FGF18-mediated chondrogenesis, other changes were noted in the FGF18-treated
joints. For example, the medial tibia chondrophyte measurement at the 1 or 5 g
doses
of FGF18 was increased 50% (Table 5).
These data demonstrate that local delivery of FGF18 in a hyaluronan
carrier can increase cartilage formation and can reduce cartilage degeneration
scores in
a rat model of osteoarthritis.
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Table 1. FGF18 reduced the medial tibia cartilage degeneration scores.
Treatment Group Degeneration Score' Number of rats
5 HA alone 3.23 0.34 10
HA+0.1 ug FGF18 2.57 0.39 10
HA+1.0 ug FGF18 1.93 0.24 10
HA+5.0 ug FGF18 1.4* 0.18 10
'Mean SE Differences significant, *p=0.0008 by ANOVA.
Table 2. FGF18 reduced the size of large cartilage lesions in the medial tibia
plateau.
Treatment Group Significant Tibial Cartilage Number of Rats
Degeneration Width (um)'
HA alone 576.6 100.4 10
HA+0.1 ug FGF18 476.6 95.6 10
HA+1.0 ug FGF18 416.8 87.9 10
HA+5.0 ug FGF18 246.6* 50 10
'Mean SE. Differences significant, *p=0.015 by Student's t-test; p=0.067 by
ANOVA.
Table 3. FGF18 reduced the depth of cartilage lesions in the medial tibia
plateau.
Treatment Group Depth ratio for any matrix change' Number of rats
HA alone 0.45 0.05 10
HA+O.lug FGF18 0.36 0.04 10
HA+1.0 ug FGF18 0.34 0.04 10
HA+5.0 ug FGF18 0.24* 0.26 10
'Mean SD. Differences significant, *p=0.0 12 by ANOVA.
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Table 4. FGF18 increased the medial tibia cartilage thickness in rats with
meniscal
tear-induced osteoarthritis.
Treatment Group Cartilage thickness' (um) Number of rats
HA alone 243.8 6.7 10
HA+0.1 ug FGF18 249.2 5.3 10
HA+1.0 ug FGF18 276.9 13.5 10
HA+5.0 ug FGF18 319.1 * 24.4 10
'Mean SE. Differences significant, p<0.05 by ANOVA.
Table 5. FGF18 increased the size of medial tibia chondrophytes.
Treatment Group Chondrophyte size' (um.) Number of rats
HA alone 602.4 49.1 10
HA+0.1ug FGF18 579.0 34.8 10
HA+1.Oug FGF18 874.9 120.9 10
HA+5..Oug FGF18 913.3 51.9 10
'Mean SD. Differences significant, p<0.0005 by ANOVA.
