[0001] This application claims priority to United States provisional
application No. 60/474,603, filed on June 2, 2003, which is incorporated
herein by reference in its entirety.
Field of the Invention
[0002] The present invention relates to the field of clinical
pathophysiology, and more particularly to methods for treating neuromuscular
disorders, such as muscular dystrophies. The invention also relates to
pharmaceutical formulations containing corticosteroids and inhibitors of
growth and differentiation.
Background of the Invention
[0003] Muscular dystrophies (MD) are progressive inherited
neuromuscular disorders that are characterized by muscle wasting and
weakness (Emery (2002) The Lancet, 359:687-695). Many forms of muscular
dystrophies are fatal and currently incurable.
[0004] Duchenne muscular dystrophy (DMD) is the most common
X-linked neuromuscular disease. The disease is caused by mutations in the
DMD gene coding for dystrophin. Alteration or absence of this protein results
in abnormal sarcolemmal membrane tearing. An abnormal variation in
diameter of muscle fibers (atrophic and hypertrophic fibers) in proximal
muscles and ongoing muscle damage are hallmarks of the disease.

THERAPEUTIC AND PROPHYLACTIC METHODS
FOR NEUROMUSCULAR DISORDERS
[0001] This application claims priority to United States provisional
application No. 60/474,603, filed on June 2, 2003, which is incorporated
herein by reference in its entirety.
Field of the Invention
[0002] The present invention relates to the field of clinical
pathophysiology, and more particularly to methods for treating neuromuscular
disorders, such as muscular dystrophies. The invention also relates to
pharmaceutical formulations containing corticosteroids and inhibitors of
growth and differentiation.
Background of the invention
[0003] Muscular dystrophies (MD) are progressive inherited
neuromuscular disorders that are characterized by muscle wasting and
weakness (Emery (2002) The Lancet, 359:687-695). Many forms of muscular
dystrophies are fatal and currently incurable.
[0004] Duchenne muscular dystrophy (DMD) is the most common
X-linked neuromuscular disease. The disease is caused by mutations in the
DMD gene coding for dystrophin. Alteration or absence of this protein results
in abnormal sarcolemmal membrane tearing. An abnormal variation in
diameter of muscle fibers (atrophic and hypertrophic fibers) in proximal
muscles and ongoing muscle damage are hallmarks of the disease.

Damaged muscle releases the intracellular enzyme creatine kinase (CK). As
a result, the serum CK levels in DMD patients are characteristically high (up to
10 times the normal). The pathophysiologic cascade is compounded by
tissue inflammation, myofiber necrosis and replacement of muscle with
fibrofatty tissue.
[0005] Another allelic variant of the DMD gene causes a milder form
of MD known as Becker muscular dystrophy (BMD). BMD is clinically similar
to DMD but the onset of symptoms occurs later in life.
[0006] Many pharmacological agents have been tried in MD but none
has proved effective in arresting the course of the disease. The current
modality of treatment is still in the realm of physical medicine and
rehabilitation.
[0007] A number of trials using corticosteroids (e.g., prednisone
and/or its derivatives) have demonstrated improvement in individuals with MD,
particularly in the short-term. Although the exact mechanism by which
corticosteroids alleviate the disease phenotype is unclear, corticosteroids are
thought to act by reducing inflammation, suppressing the immune system,
improving calcium homeostasis, upregulating expression of compensatory
proteins, and increasing myoblast proliferation (Khurana et al. (2003) Nat.
Rev. Drug Discovery 2:279-386). However, corticosteroids administered over
time can induce muscle atrophy, which primarily affects proximal muscles—
the very same muscles that are affected in DMD and BMD. The

corticosteroid-induced muscle and other side effects may limit the long-term
effectiveness of corticosteroid therapy.
[0008] GDF-8 is a member of the TGF-β superfamily and functions as
a negative regulator of muscle growth. Similarly to other members of the
superfamily, GDF-8 is synthesized as a precursor molecule, but prior to
secretion, it is cleaved into the N-terminal inhibitory propeptide and C-terminal
the active mature GDF-8. Propeptide may remain bound to GDF-8 thereby
inhibiting the biological activity of mature GDF-8. Propeptide must dissociate
from the complex for GDF-8 to bind to activin type II receptor (ActRHB). Upon
binding, ActRHB initiates a signaling cascade, ultimately leading to the
inhibition of myoblast progression. Antibody-mediated inhibition of GDF-8 in
vivo has been shown to significantly increase skeletal muscle size in normal
adult mice (Whittemore et al. (2003) BBRC, 300:965-971) and to alleviate the
dystrophic phenotype in the mdx mouse model of DMD (Bogdanovich et al.
(2002) Nature, 420(28):418-421).
SUMMARY OF THE INVENTION
[0009] It is one of the objects of the present invention to provide
methods and compositions for treating disorders characterized by or
associated with a risk of diminution of muscle function. Additional objects of
the invention will be set forth in part in the following description, and in part
will be understood from the description, or may be learned by practice of the
invention.

[0010] The present invention is based, in part, on the discovery and
demonstration that, in a mouse model of DMD, treatment by administration of
a neutralizing anti-GDF-8 antibody and prednisone is more effective in
increasing muscle mass and strength relative to treatment with prednisone
alone. The invention is further based, in part, on the discovery and
demonstration that administration of anti-GDF-8 antibody with prednisone
reduces prednisone-induced muscle atrophy.
[0011] Accordingly, the present invention provides methods for
treating neuromuscular disorders in mammals. The disclosed methods
include administering to a subject susceptible to or having a neuromuscular
disorder therapeutically effective amounts of at least one GDF-8 inhibitor and
at least one corticosteroid so as to maintain desirable levels of muscle
integrity or function as assessed by, for example, serum concentration of
creatine kinase (CK), muscle histology, tissue imaging, activities of daily
living, muscle strength and/or mass. The populations treated by the methods
of the invention include, but are not limited to, patients having or at risk of
developing muscular dystrophy such as, for example, DMD or BMD, and
subjects undergoing corticosteroid therapy for these or other disorders.
[0012] The invention further provides methods of treating muscle
weakness and methods of treating corticosteroid-induced muscle atrophy.
The invention includes methods of treating cardiomyopathy.
[0013] Methods of administration and compositions used in the
methods of the inventions are provided. In the disclosed methods, a GDF-8

inhibitor and a corticosteroid are administered concurrently or over alternating
overlapping or non-overlapping intervals.
[0014] GDF-8 inhibitors, used in the methods of the present invention,
include, but are not limited to, antibodies to GDF-8; antibodies to GDF-8
receptors; soluble GDF-8 receptors and fragments thereof (e.g., ActRlIB
fusion polypeptides as described in U.S. Patent Application No. 10/689,677,
including soluble ActRlIB receptors in which ActRlIB is joined to the Fc portion
of an immunoglobulin); GDF-8 propeptide and modified forms thereof (e.g., as
described in WO 02/068650 or U.S. Patent Application No. 10/071,499,
including forms in which GDF-8 propeptide is joined to the Fc portion of an
immunoglobulin and/or form in which GDF-8 is mutated at an aspartate (asp)
residue, e.g., asp-99 in murine GDF-8 propeptide and asp-100 in human
GDF-8 propeptide); a small molecule inhibitor of GDF-8; follistatin (e.g., as
described in U.S. Patent No. 6,004,937) or follistatin-domain-containing
proteins (e.g., GASP-1 or other proteins as described in U.S. Patent
Application Nos. 10/369,736 and 10/369,738); and modulators of
metalloprotease activity that affect GDF-8 activation, as described in U.S.
Patent Application No. 10/662,438.
[0015] In some embodiments, the GDF-8 inhibitor is a monoclonal
antibody or a fragment thereof that blocks GDF-8 binding to its receptor.
Nonlimiting illustrative embodiments include a nonhuman monoclonal
anti-GDF-8 antibody, e.g., murine monoclonal antibody JA-16 (as described in
U.S. Patent Application No. 10/253,532; ATCC Deposit No. PTA-4236);

derivatives thereof, e.g., humanized antibody; and fully human monoclonal
anti-GDF-8 antibodies (e.g., Myo29, Myo28, and Myo22, as described in U.S.
Patent Application No. 10/688,925; ATCC Deposit Nos. PTA-4741, PTA-4740,
and PTA-4739, respectively) or derivatives thereof.
[0016] Corticosteroids, used in the method of the invention include,
but are not limited to, beclomethasone dipropionate, budesonide, Cortisol,
dexamethasone, fluticason propionate, mometasone furoate, prednisone,
triamcinolone acetonide, and derivatives thereof.
[0017] It is to be understood that both the foregoing general
description and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention as claimed.
BRIEF DESCRIPTION OF THE FIGURES
[0018] Figures 1A and 1B depict results of a histological analysis of
diaphragm muscle from mdx mice treated for four weeks with anti-GDF-8
neutralizing antibody JA-16 (60 mg/kg, once weekly) and prednisone (2
mg/kg, 3 times a week), prednisone alone, or vehicle control alone. Figure 1A
shows severity of muscle fiber atrophy on a 0-4 scale at the end of the trial.
Figure 1B shows percentage of affected (atrophied) muscle fibers at the end
of the trial. Each bar represents a single mouse.

DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0019] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions are set forth
throughout the detailed description.
[0020] The term "antibody," as used herein, refers to an
immunoglobulin or a part thereof and encompasses any polypeptide
comprising an antigen-binding site regardless of the source, method of
production, and other characteristics. As a non-limiting example, the term
"antibody" includes human, orangutan, mouse, rat, goat, sheep, and chicken
antibodies. The term includes but is not limited to polyclonal, monoclonal,
monospecific, polyspecific, non-specific, humanized, single-chain, chimeric,
synthetic, recombinant, hybrid, mutated, and CDR-grafted antibodies. For the
purposes of the present invention, it also includes, unless otherwise stated,
antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other
antibody fragments that retain the antigen-binding function.
[0021] Antibodies can be made, for example, via traditional hybridoma
techniques (Kohler and Milstein (1975) Nature, 256:495-499), recombinant
DNA methods (U.S. Patent No. 4,816,567), or phage display techniques using
antibody libraries (Clackson et al. (1991) Nature, 352: 624-628; Marks et al.
(1991) J. Mol. Biol., 222: 581-597). For various other antibody production
techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold
Spring Harbor Laboratory, 1988.

[0022] The term "antigen-binding domain" refers to the part of an
antibody molecule that comprises the area specifically binding to or
complementary to a part or all of an antigen. Where an antigen is large, an
antibody may only bind to a particular part of the antigen. The epitope or
antigenic determinant is a portion of an antigen molecule that is responsible
for specific interactions with the antigen-binding domain of an antibody. An
antigen-binding domain may be provided by one or more antibody variable
domains (e.g., a so-called Fd antibody fragment consisting of a VH domain).
An antigen-binding domain comprises an antibody light chain variable region
(VL) and an antibody heavy chain variable region (VH)..
[0023] The term "anti-GDF-8 antibody," or "antibody to GDF-8,"
refers to any antibody that specifically binds to at least one epitope of GDF-8.
The terms "GDF-8 receptor antibody" and "antibody to a GDF-8 receptor"
refer to any antibody that specifically binds to at least one epitope of a GDF-8
receptor, such as ActRIIB. The term "neutralizing antibody" refers to an
antibody that is a GDF-8 inhibitor.
[0024] The term "specific interaction," or "specifically binds," or the
like, means that two molecules form a complex that is relatively stable under
physiologic conditions. The term is also applicable where, e.g., an antigen-
binding domain is specific for a particular epitope, which may be present on a
number of antigens. Specific binding is characterized by a high affinity and a
low to moderate capacity. Nonspecific binding usually has a low affinity with a
moderate to high capacity. Typically, the binding is considered specific when

the affinity constant Ka is higher than 106 M-1 than 107 M-1 or preferably
higher than 108 M-1. If necessary, non-specific binding can be reduced
without substantially affecting specific binding by varying the binding
conditions. Such conditions are known in the art, and a skilled artisan using
routine techniques can select appropriate conditions. The conditions are
usually defined in terms of concentration of antibodies, ionic strength of the
solution, temperature, time allowed for binding, concentration of non-related
molecules (e.g., serum albumin, milk casein), etc.
[0025] The term "muscle function" refers to the ability of muscle to
perform a physiologic function, such as contraction as measured by the
amount of force generated during either twitch or tetanus. Other methods for
assessing muscle function are well known in the art and include, but are not
limited to, measurements of muscle mass, grip strength, serum CK level,
activities of daily living, motion or strength tests, tissue histology (e.g., E&A
staining, or collagen III staining), or tissue imaging. Nonlimiting illustrative
methods for assessing muscle function are set forth in the Examples.
[0026] The term "GDF-8" refers to a specific growth and differentiation
factor-8 and, where appropriate, factors that are structurally or functionally
related to GDF-8, for example, BMP-11 and other factors belonging to the
TGF-β superfamily. The term refers to the full-length unprocessed precursor
form of GDF-8 as well as the mature and propeptide forms resulting from
post-translational cleavage. The term also refers to any fragments and
variants of GDF-8 that maintain at least some biological activities associated

with mature GDF-8, as discussed herein, including sequences that have been
modified. The present invention relates to GDF-8 from all vertebrate species,
including, but not limited to, human, bovine, chicken, mouse, rat, porcine,
ovine, turkey, baboon, and fish (for sequence information, see, e.g.,
McPherron et al. (1997) Proc. Nat. Acad. Sci. U.S.A., 94:12457-12461).
[0027] The term "mature GDF-8" refers to the protein that is cleaved
from the carboxy-terminal domain of the GDF-8 precursor protein. The
mature GDF-8 may be present as a monomer, homodimer, or in a GDF-8
latent complex. Depending on conditions, mature GDF-8 may establish
equilibrium between any or all of these different forms. In its biologically
active form, the mature GDF-8 is also referred to as "active GDF-8."
[0028] The term "GDF-8 propeptide" refers to the polypeptide that is
cleaved from the amino-terminal domain of the GDF-8 precursor protein. The
GDF-8 propeptide is capable of binding to the propeptide binding domain on
the mature GDF-8.
[0029] The term "GDF-8 latent complex" refers to the complex of
proteins formed between the mature GDF-8 homodimer and the GDF-8
propeptide. It is believed that two GDF-8 propeptides associate with two
molecules of mature GDF-8 in the homodimer to form an inactive tetrameric
complex. The latent complex may include other GDF inhibitors in place of or
in addition to one or more of the GDF-8 propeptides.
[0030] The term "GDF-8 activity" refers to one or more of
physiologically growth-regulatory or morphogenetic activities associated with

active GDF-8 protein. For example, active GDF-8 is a negative regulator of
skeletal muscle mass. Active GDF-8 can also modulate the production of
muscle-specific enzymes (e.g., creatine kinase), stimulate myoblast
proliferation, and modulate preadipocyte differentiation to adipocytes.
Exemplary procedures for measuring GDF-8 activity in vivo and in vitro are
found in U.S. Patent Application No. 10/688,925, for example.
[0031] As used herein, "GDF-8 inhibitor" generally refers to any
compound that downregulates the activity of GDF-8, and includes any agent
capable of inhibiting activity, expression, processing, or secretion of GDF-8.
A GDF-8 inhibitor may, for example, affect stability of or conversion of the
precursor molecule to the active, mature form; interfere with the binding of
GDF-8 to one or more receptors; or interfere with intracellular signaling of the
GDF-8 receptor ActRIIB. Such inhibitors include proteins, antibodies,
peptides, peptidomimetics, ribozymes, anti-sense oligonucleotides, double-
stranded RNA, and other small molecules, which specifically inhibit GDF-8.
Such inhibitors are said to "inhibit," "neutralize," or "reduce" the biological
activity of GDF-8.
[0032] The terms "neutralize," "neutralizing," "inhibitory," and their
cognates refer to a reduction in the activity of GDF-8 by a GDF-8 inhibitor,
relative to the activity of GDF-8 in the absence of the same inhibitor. The
reduction in activity is preferably at least about 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, or higher. The methods for assessing neutralizing or
inhibitory biological activity of GDF-8 inhibitors are known in the art, and can

be performed, for example, using the ActRIIB assay (e.g., as described in
Whittemore et al. (2003) BBRC, 300:965-97; or U.S. Patent Application No.
10/253,532) and the RGA assays (as described in Thies (2001) Growth
Factors, 18:251-259 or U.S. Patent Application No. 10/253,532).
[0033] The term "therapeutically effective dose," or
"therapeutically effective amount," refers to that amount of a compound that
results in prevention, reduction in the risk of occurrence, or amelioration of
symptoms in a patient, or a desired biological outcome, e.g., improved muscle
function, delayed onset of clinical symptoms, etc. The effective amount can
be determined as described in the subsequent sections.
[0034] The terms "treatment," "therapeutic method," and their
cognates refer to treatment or prophylactic/preventative measures. Those in
need of treatment may include individuals already having a particular medical
disorder as well as those who may ultimately acquire the disorder. Treatment
includes any reduction in any symptom of a disorder described in this
application. In addition to a reduction or lessening of symptoms, treatment
also includes maintaining a patient's current status when worsening is
expected, or preventing the occurrence of a symptom in an individual in which
the onset of a symptom, disorder, or disease is expected. Treatment may
include a decrease or reduction in one or more physiologic function from
normal. It may also include a decrease compared to expected symptoms or
expected progression of the condition, disorder, or disease.

II. Components for Use in the Methods of the Invention
[0035] In the methods of the present invention, one or more GDF-8
inhibitors are used in combination with one or more corticosteroids.
A. GDF-8 Inhibitors
[0036] GDF-8 inhibitors, used in the methods of the present invention,
include, but are not limited to, antibodies to GDF-8; antibodies to GDF-8
receptors; soluble GDF-8 receptors and fragments thereof (e.g., ActRIIB
fusion polypeptides as described in U.S. Patent Application No. 10/689,677,
including soluble ActRIIB receptors in which ActRIIB is joined to the Fc portion
of an immunoglobulin); GDF-8 propeptide and modified forms thereof (e.g., as
described in WO 02/068650 or U.S. Patent Application No. 10/071,499,
including forms in which GDF-8 propeptide is joined to the Fc portion of an
immunoglobulin and/or forms in which GDF-8 is mutated at an aspartate (asp)
residue, e.g., asp-99 in murine GDF-8 propeptide and asp-100 in human
GDF-8 propeptide); follistatin (e.g., as described in U.S. Patent No.
6,004,937) or follistatin-domain-containing proteins (e.g., GASP-1 or other
proteins as described in U.S. Patent Application Nos. 10/369,736 and
10/369,738); and modulators of metalloprotease activity that affect GDF-8
activation, as described in U.S. Patent Application No. 10/662,438.
[0037] In some embodiments, the GDF-8 inhibitor is a monoclonal
antibody or a fragment thereof that blocks GDF-8 binding to its receptor.
Nonlimiting illustrative embodiments include a nonhuman monoclonal
anti-GDF-8 antibody, e.g., murine monoclonal antibody JA-16 (as described in
U.S. Patent Application No. 10/253,532; ATCC Deposit No. PTA-4236);

derivatives thereof, e.g., humanized antibodies; and fully human monoclonal
anti-GDF-8 antibodies (e.g., Myo29, Myo28, and Myo22, as described in U.S.
Patent Application No. 10/688,925; ATCC Deposit Nos. PTA-4741, PTA-4740,
and PTA-4739, respectively), or derivatives thereof.
[0038] In some embodiments, the GDF-8 inhibitor blocks GDF-8 from
binding to its receptor, by binding to GDF-8 or to the GDF-8 receptor. In
various embodiments, the GDF-8 inhibitor is an anti-GDF-8 antibody that has
the affinity to GDF-8, expressed as an affinity constant (Ka), wherein Ka is at
least 105 M-1,106 M-1, 107 M-1 108 M-1,109 M-1, 1010 M-1, 1011 M-1, or 1012 M-1.
Also contemplated for use in humans are inhibitors that are humanized forms
and derivatives of nonhuman antibodies derived from any vertebrate species
described in patent applications cited herein, or in Antibody Engineering, ed.
Borrebaeck, 2nd ed., Oxford University Press, 1995; and Antibodies: A
Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988.
B. Corticosteroids
[0039] Corticosteroids used in the methods of the present invention,
include, but are not limited to, beclomethasone dipropionate, budesonide,
Cortisol, dexamethasone, fluticason propionate, prednisone., mometasone
furoate, triamcinolone acetonide, and derivatives thereof.
[0040] Pharmaceutically acceptable salts of compounds disclosed
herein can also be used.
[0041] Corticosteroids are available commercially in various
pharmaceutical formulations (Physician's Desk Reference (PDR) 2003, 57th
ed., Medical Economics Company, 2002). For example, oral formulations are

commercially available for cortisone, hydrocortisone (Cortef®), prednisone
(Deltasone®, Meticorten®, Orasone®), prednisolone (Delta-Cortef®,
Pediapred®, Prelone®), triamcinolone (Aristocort®, Kenacort®),
methylprednisolone (Medrol®), dexamethasone (Decadron®, Dexone®,
Hexadrol®), betamethasone (Celestone®), and deflazacort (Calcort®). Other
formulations of these and other corticosteroids can be used in the methods of
the invention.
C. Therapeutic and Prophylactic Methods
[0042] The invention provides method of treating mammalian
subjects, including methods to treat loss of muscle function, muscle
weakness, and/or corticosteroid-induced muscle atrophy.
[0043] Methods of the invention comprise administering to the
mammal a therapeutically effective amount of at least one GDF-8 inhibitor and
a therapeutically effective amount of at least one corticosteroid in the amounts
and for a period of time sufficient to treat at leapt one of loss of muscle
function, muscle mass, muscle weakness, muscle atrophy, or
cardiomyopathy. The methods can be used for treating neuromuscular
disorders such as muscular dystrophies. In some embodiments, muscle
function is improved relative to the same treatment either in the absence of
the GDF-8 inhibitor or the corticosteroid. The muscles treated include, but are
not limited to, gastrocnemius, tibialis, anterior, quadriceps, extensor digitorum,
cardiac muscle, or diaphragm muscle.
[0044] Neuromuscular disorders include, but are not limited to, any
acute or chronic disease or disorder that compromises muscle function,

causes muscular injury, or otherwise causes a diminution in muscle mass
and/or function. A wide variety of diseases or disorders is known and
includes, for example, muscular dystrophies such Duchenne muscular
dystrophy, Becker muscular dystrophy, Emery Dreifuss muscular dystrophy,
limb girdle muscular dystrophy, rigid spine syndrome, Ullrich syndrome,
Fukuyama muscular dystrophy, Walker-Warburg syndrome, muscle-eye-brain
disease, facioscapulohumeral muscular dystrophy, congenital muscular
dystrophy, myotonic dystrophy (Steinert's disease), nondystrophic myotonia,
periodic paralyses spinal muscular atrophy, familial amytrophic lateral
sclerosis, hereditary motor and sensory neuropathy, Charcot-Marie-Tooth
disease, chronic inflammatory neuropathy, distal myopathy,
myotubular/centronuclear myopathy, nemaline myopathy, mini core disease,
central core disease, desminopathy, inclusion body myositis, mitochondrial
myopathy, congenital myasthenic syndrome, post-polio muscle dysfunction,
and disorders described in Emery (2002) The Lancet, 359:687-695; and
Khurana et al. (2003) Nat. Rev. Drug Disc, 2:379-386. Patients may exhibit
mild, moderate or severe muscle weakness, muscle wasting, and effects on
independent ambulation associated with such a disorder. Patients having or
at risk for developing these disorder will benefit from GDF-8 inhibitor and a
corticosteroid.
[0045] In general, a patient who will benefit from coadministration of a
GDF-8 inhibitor and a corticosteroid is one who exhibits a 2-10-fold or higher
increase in the serum CK activity, a positive family history, an abnormal

variation in the diameter of muscle fibers, a deficiency in dystrophin or a
mutation in the dystrophin gene, loss of muscle mass, muscle weakness,
cardiomyopathy, and/or loss of muscle strength. The diagnostic procedures,
including the appropriate genetic testing, are described in Diagnostic Criteria
for Neuromuscular Disorders, ed. Emery, 2nd ed., Royal Society of Medicine
Press, 1997. The combination treatment can be also beneficial to subjects
undergoing corticosteroid therapy for disorders other than neuromuscular
disorders and/or subjects with a history of a long-term corticosteroid use so
long these subjects exhibit, or are at risk of diminution of muscle function such
as characterized by muscle weakness, loss of muscle mass, and/or muscle
atrophy, etc. Examples of disorders for which corticosteroid therapy is often
used include, but are nor limited to, asthma, allergy, arthritis, dermatologic
disorders (e.g., inflammatory dermatoses, eczema, psoriasis, etc), lupus
erythematosus, and other chronic inflammatory conditions.
[0046] Methods of administration and compositions used in the
methods of the inventions are provided. Administration is not limited to any
particular delivery system and may include, without limitation, parenteral
(including subcutaneous, intravenous, intramedullary, intraarticular,
intramuscular, or intraperitoneal injection) rectal, topical, transdermal, or oral
(for example, in capsules, suspensions, or tablets). Administration to an
individual may occur in a single dose or in repeat administrations, and in any
of a variety of physiologically acceptable salt forms, and/or with an acceptable
pharmaceutical carrier and/or additive as part of a pharmaceutical

composition. Physiologically acceptable salt forms and standard
pharmaceutical formulation techniques and excipients are well known to
persons skilled in the art (e.g., as described in Physician's Desk Reference
(PDR) 2003,57th ed., Medical Economics Company, 2002; and Remington:
The Science and Practice of Pharmacy, eds. Gennado et al., 20th ed,
Lippincott, Williams & Wilkins, 2000).
[0047] A GDF-8 inhibitor and a corticosteroid are administered
concurrently or consecutively over overlapping or nonoverlapping intervals. In
the sequential administration, the GDF-8 inhibitor and the corticosteroid can
be administered in any order. In some embodiments, the length of an
overlapping or nonoverlapping interval is more than 2, 4, 6, 12, 24, or 48
weeks.
[0048] For corticosteroids, the prescribing physician routinely selects
the dosage and regimen. For example, prednisone is used at about 0.1-2 mg
per kilogram of body weight per day, and most commonly at 0.5-1 mg/kg/day,
e.g., 0.75 mg/kg/day. The corticosteroid may be administered at average
weekly doses of approximately 1-14 mg/kg body weight, including
approximately 1, 2, 5, 7, 10, 12, or 15 mg/kg body weight per week, and the
prescribing physician may select a frequency of administration as appropriate.
Single dose, continuous or periodic corticosteroid administration may be
selected, including administration at hourly, daily, bi-weekly, weekly, or other
periodic intervals. Preferably, corticosteroids are administered orally or by
injection 1-4 times per day. Corticosteroid dosage may be optimized as a

combination therapy, and dosage may be lowered to reduce significant side
effects of administration.
[0049] The GDF-8 inhibitors can be administered alone or in a mixture
with a corticosteroid or another compound. GDF-8 inhibitors can be
administered at a dose of approximately from 1 µg/kg to 25 mg/kg, depending
on physiology, the severity of the symptoms and the progression of the
disease. Single dose, continuous, or periodic administration may be selected,
with intervals between GDF-8 inhibitor doses chosen from hourly, daily, bi-
weekly, weekly, bi-monthly, monthly, or other appropriate intervals. For
example, GDF-8 inhibitors such as antibodies may be administered in an
outpatient setting by weekly administration at about 0.1-10 mg/kg dose by
intravenous (IV) infusion, intraperitoneal, or subcutaneous injection. In
general, the appropriate therapeutically effective dose of a GDF-8 inhibitor is
selected by a treating clinician and would range approximately from 1 µg/kg to
20 mg/kg, from 1 µg/kg to 10 mg/kg, from 1 µg/kg to 1 mg/kg, from 10 µg/kg
to 1 mg/kg, from 10 µg/kg to 100 µg/kg, from 100 µg to 1 mg/kg, and from 500
µg/kg to 5 mg/kg. Exemplary effective doses of GDF-8 inhibitor include
approximately 0.1, 0.3, 0.5, 1, 5, 10, or 20 mg/kg/wk. Additionally, specific
dosages indicated in the Examples or in the Physician's Desk Reference
(PDR) 2003, 57th ed., Medical Economics Company, 2002, can be used.
D. Methods of Testing Compounds for Therapeutic Efficacy
[0050] The invention further provides methods for testing in an animal,
e.g., a rodent or a primate, whether a therapeutic compound is efficacious
when administered in combination with at least one GDF-8 inhibitor and at

least one corticosteroid. In some embodiments, the method of evaluating the
efficacy of a compound comprises: administering the compound to a first
animal in combination with a GDF-8 inhibitor and a corticosteroid;
administering the GDF-8 inhibitor and the corticosteroid to a second animal;
determining the level of muscle function in the first and in the second animal
after the administrations; and comparing the levels of muscle function. If the
level in the first animal is lower than the level in the second animal, it indicates
that the compound or the combination is efficacious.
[0051] In other embodiments, the compound may be evaluated for
efficacy in treatment of muscular dystrophy when administered in combination
with a GDF-8 inhibitor and/or a corticosteroid.
[0052] Several animal models are available for such evaluative
purposes. For example, the mdx model has been described, for example, by
Torres et al. (1987) Brain, 110:269-299, and Hoffman et al. (1987) Science,
238:347-350. Extremely high levels of CK are consistently noted with
dystrophin-deficiency in mdx mice and DMD humans due to sarcolemmal
damage (Bulfield et al. (1984) Proc. Natl. Acad. Sci. USA, 81:1189-1192; and
Matsuda et al. (1995) J. Biochem. (Tokyo), 118: 959-64). As another
example, two other animal models can be used: utr-/- mdx mice (Gillis (2002)
Neuromuscul. Disord., 12(1):90-84; and Deconick et al. (1997) Cell,
90:729-738) and nu-/- mdx mice (Morrison et al. (2000) Lab. Invest.,
80:881-891).

EXAMPLES
Example 1: Effect of GDF-8 neutralizing antibody on dystrophic muscle
[0053] The ability of in vivo inhibition of GDF-8 to ameliorate muscular
dystrophy was tested in the mdx mouse model of DMD. Five to seven week
old male CSTBL/10ScSn-mdx/j mice (Jackson Laboratory, Bar Harbor, ME)
were treated with weekly intraperitoneal injections of the GDF-8 neutralizing
murine antibody JA-16 (60 mg/kg, double dosing at first week, n=11), and
vehicle alone (control group, n=10) for 12 weeks. These mice were also
compared to mice of the same background strain (C57BL/10, n=12) without
the dystrophin deficiency.
[0054] The body weight was monitored before, during and after
treatment. Mice in the treatment group gained weight relative to mice in the
vehicle control group. Results are shown in Table 1..
Table 1. Total body weight (g) Average values with SEM

[0055] Mice were also subjected to a grip test after 6 and 10 weeks of
dosing. Mice in the treatment group at four and ten weeks had 9% (p = 0.09)
and 19% (p < 0.05) respectively greater grip strength than mice in the vehicle
control groups. Results are shown in Table 2.

[0056] To quantify the difference in muscle mass between treatment
and vehicle control, animals were sacrificed and quadriceps and
gastrocnemius muscles dissected out and weighed. Quadriceps muscles
from the treated group of animals weighed 13% more than controls
(0.371±0.009 vs. 0.317±0.008 g; p<0.05). Gastrocnemius muscles from the
treated group of animals weighed 17% more than controls (0.223±0.008 vs.
0.197±0.005 g; p<0.0005).
Example 2: Effect of GDF-8 neutralizing antibody and prednisone on
normal and dystrophic muscle
[0057] Male C57BL/10ScSn-mdx/j and C57BL/10 (Jackson
Laboratory, Bar Harbor, ME). Mouse monoclonal anti-GDF-8 antibody JA-16,
prednisone (P-9901, Sigma), or vehicle (peanut oil) was injected starting at
age 5-7 weeks for 4 weeks. Mice were intraperitoneally (IP) injected with
JA-16 at a dose of 60 mg/kg per week (double dosing at first week), or
subcutaneously (SC) injected with prednisone at 2 mg/kg, 3 times a week.
[0058] The body weight and grip strength were monitored before,
during and after treatment. Results are shown in Table 3 and Table 4,
respectively.

[0059] At the end of the study, mice were sacrificed and muscle
mass was assessed by dissecting and weighing the gastrocnemius and
quadriceps. Results are shown in Table 5. To confirm biological activity of
prednisone, sera from a separate cohort of mice were collected and analyzed
for IL6 and IL1β (Ani Lytics, Inc., Gaithersburg, MD). Both cytokines were
found to be reduced in the sera of mice treated with prednisone.

[0060] Therefore, the results demonstrate that in muscular dystrophy,
administration of an inhibitor of GDF-8, i.e., anti-GDF-8 antibody, and

prednisone is effective in increasing muscle mass and strength relative to
treatment with prednisone alone or vehicle.
[0061] Furthermore, in these studies the effects of JA16 plus
prednisone treatment (Example 2) were greater than the effects of treatment
with JA16 alone (Example 1). The increase in body weight compared to
vehicle after four weeks of treatment was more dramatic for JA16 plus
prednisone treatment than for JA16 treatment alone. The increase in grip
strength compared to vehicle control after four weeks of treatment with JA16
plus prednisone was greater that the increase after six or ten weeks of
treatment with JA16 alone. The increase over vehicle control in muscle mass
after four weeks of treatment with JA16 plus prednisone was also greater than
the increase after twelve weeks of treatment with JA16 alone.
Example 3: Effect of GDF-8 neutralizing antibody on
prednisone-induced muscle atrophy
[0062] In the mice treated as described in Example 2,diaphragm
muscle was histologically examined as described in Example 1. The
morphological changes were evaluated by an independent pathology lab that
had no knowledge of the treatment group assignments. Severity grades were
assigned on a scale from 0 to 4 (0 = none; 1 = minimal; 2 = mild; 3 =
moderate; and 4 = marked). Results are shown in Figure 1A (severity scores)
and Figure 1B (percentage of muscle fibers atrophied). The results show that
administration of the anti-GDF-8 antibody with prednisone reduces
prednisone-induced muscle atrophy.

Example 4: Treatment of Muscular Dystrophies
[0063] As an example of treating MD in humans, the Myo29 antibody
is administered in combination with prednisone or prednisolone. Nonlimiting
exemplary treatment regimens and outcomes are summarized in Table 6.
Other treatment regimens can be determined by a treating physician, with
ranges of the corticosteroids and GDF-8 inhibitors dosage and administration
as discussed above.

[0064] All publications and patents cited and sequences identified by
accession or database reference numbers in this disclosure are incorporated
by reference in their entirety.

WE CLAIM:
1. A pharmaceutical composition comprising at least one GDF-8 inhibitor and at
least one corticosteroid.
2. The pharmaceutical composition as claimed in claim 1, wherein the
corticosteroid is chosen from at least one of:
(a) at least one of beclomethasone dipropionate, budesonide, Cortisol,
dexamethasone, fluticason propionate, mometasone furoate, prednisone,
or triamcinolone acetonide;
(b) a derivative of at least one of beclomethasone dipropionate, budesonide,
Cortisol, dexamethasone, fluticason propionate, mometasone furoate,
prednisone, or triamcinolone acetonide; or
(c) a pharmaceutically acceptable salt of at least one of beclomethasone
dipropionate, budesonide, Cortisol, dexamethasone, fluticason propionate,
mometasone furoate, prednisone, or triamcinolone acetoniden.

3. The pharmaceutical composition as claimed in claim 2, wherein the
corticosteroid is prednisone or prednisolone.
4. The pharmaceutical composition as claimed in any one of claims 1-3,
wherein the GDF-8 inhibitor is chosen from an antibody to GDF-8, an antibody to a
GDF-8 receptor, a soluble GDF-8 receptor, a GDF-8 propeptide, a small molecule
inhibitor of GDF-8, follistatin, or a follistatin-domain-containing protein.

5. The pharmaceutical composition as claimed in claim 4, wherein the antibody
to GDF-8 is chosen from JA-16, Myo29, Myo28, or Myo22.
6. The pharmaceutical composition as claimed in claim 4, wherein the GDF-8
propeptide is mutated at an aspartate residue.
7. The pharmaceutical composition as claimed in claim 4, wherein the GDF-8
propeptide is joined to the Fc portion of an immunoglobulin.

8. The pharmaceutical composition as claimed in claim 4, wherein the GDF-8
receptor is ActRIIB.
9. The pharmaceutical composition as claimed in claim 4, wherein the GDF-8
receptor is joined to the Fc portion of an immunoglobulin.
10. The pharmaceutical composition as claimed in claim 4, wherein the GDF-8
inhibitor is follistatin.
11. The pharmaceutical composition as claimed in claim 4, wherein the
follistatin-domain-containing protein is GASP-1.
12. The pharmaceutical composition as claimed in claim 4, wherein the GDF-8
inhibitor is a small molecule inhibitor.

The instant invention discloses a pharmaceutical composition comprising at least one GDF-8 inhibitor and at least one corticosteroid.