Note: Descriptions are shown in the official language in which they were submitted.
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Description
Field of the Invention:
The present invention relates to a chemical composition consisting of a
solution of water and/or a
lipid as a solvant, with a myostatin inhibitor as the solute. The purpose of
this chemical
composition is to inhibit Myostatin (GDF-8) activity by injecting said
chemical composition.
Myostatin is a hormone that limits muscle growth; myostatin inhibitors can be
used for muscle
enhancement (including inducing hypertrophy and/or hyperplasia) as well as
improving muscle
function (including decreasing atrophy and/or increasing endurance, force
and/or strength). This
chemical composition has applications in the treatment of musculoskeletal and
neurodegenerative disorders among others, performance enhancement among
athletes, as well as
enhancing muscle in livestock.
Background:
10003] Molecular advances have provided greater understanding of skeletal
muscle diseases,
such as muscular dystrophy (MD), beginning with the discovery of the
dystrophin gene and its
gene product. There are nine types of MD, each a genetic degenerative disease
primarily
affecting voluntary muscles. Duchenne muscular dystrophy (DMD) affects 1 in
3500 live male
births. The progressive muscle weakness and degeneration usually lead to loss
of ambulation and
wheelchair dependency in the early teens. Death occurs anytime after age 18
due to respiratory
infection usually complicated by cardiac failure. The disease is caused by
mutations in the
dystrophin gene, which encodes a large (427 kDa) cyto skeletal protein in both
skeletal and
cardiac muscle. The dystrophin gene is the largest gene identified to date. It
shows one of the
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highest sporadic mutation rates, with 1 of every 10,000 germ cells showing de
novo mutations.
Thus, the high incidence (1/3500), de novo mutations, early morbidity and
fatality, and the lack
of effective treatment require urgency in the search for novel therapeutics.
Since DMD was
described more than 140 years ago, the life-span of the DMD patient has only
been marginally
prolonged and the quality of life may not have changed significantly improved
despite
tremendous advances in medicine. Few treatments have been added to the current
repertoire. In
the muscular dystrophies, only corticosteroids have altered the natural
history of disease and
indeed the mechanism of action of corticosteroids in DMD remains unknown. In
fact, the known
myopathic effects of steroids might predict a deleterious effect but instead
there is a paradoxical
response resulting in an increase in muscle mass contrasting with the effects
in normal muscle.
Unfortunately the benefit of steroids comes at a high cost in terms of side
effects (bone loss,
cataracts, delayed puberty, weight gain and hypertension) providing compelling
reasons to find
other approaches.
100041 Amyotrophic Lateral Sclerosis (ALS) is another disease that results in
loss of muscle
and/or muscle function. First characterized by Charcot in 1869, it is a
prevalent, adult-onset
neurodegenerative disease affecting nearly 5 out of 100,000 individuals. ALS
occurs when
specific nerve cells in the brain and spinal cord that control voluntary
movement gradually
degenerate. Within two to five years after clinical onset, the loss of these
motor neurons leads to
progressive atrophy of skeletal muscles, which results in loss of muscular
function resulting in
paralysis, speech deficits, and death due to respiratory failure. The genetic
defects that cause or
predispose ALS onset are unknown, although missense mutations in the SOD-1
gene occurs in
approximately 10% of familial ALS cases, of which up to 20% have mutations in
the gene
encoding Cu/Zn superoxide dismutase (SOD1), located on chromosome 21. SOD-1
normally
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functions in the regulation of oxidative stress by conversion of free radical
superoxide anions to
hydrogen peroxide and molecular oxygen. To date, over 90 mutations have been
identified
spanning all exons of the SOD-1 gene. Some of these mutations have been used
to generate lines
of transgenic mice expressing mutant human SOD-1 to model the progressive
motor neuron
disease and pathogenesis of ALS.
100051 Another musculoskeletal disorder, inclusion body myositis (IBM), was
originally named
by Yunis and Samaha in 1971 to describe a patient with a chronic inflammatory
myopathy who
had intranuclear and intracytoplasmic tubular filaments within muscle fibers
on electron
microscopy. Many clinical and pathologic studies over more than three decades
have supported
this condition as a disorder distinct from other idiopathic inflammatory
myopathies. Although
incidence and prevalence statistics need further refinement, it is
unequivocally the most common
acquired muscle disease occurring after age of 50, with an estimated
prevalence at 4-
9:1,000,000. More men are affected than women by a ratio of greater than 2:1.
Typically IBM is
a sporadic disorder with insidious onset, and distinctive clinical and
histopathological features
(sIBM). Inflammation is prominent, helping to distinguish it from the group of
inherited
disorders (hIBM). These include autosomal recessive and dominant conditions
with pathologic
features resembling sIBM without inflammatory infiltration. Perhaps the best
characterized
hIBM is associated with mutations of UDP-N-acetylglucosamine 2-epimerase/N-
acetylmannosamine kinase (GNE) [6].
100061 The clinical features of sIBM include an average time of about 6 years
from symptom
onset to diagnosis. Difficulty with ambulation and frequent falls can be
attributed to weak knee
extensor muscles. Early involvement of the quadriceps and forearm flexor
muscle compartment
is typical. This is accompanied by a scooped-out appearance of the medial
aspect of the forearms
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and thin, atrophic quadriceps muscles. Quadriceps atrophy and weakness is
directly related to
loss of safe ambulation. Frequent, sudden falls occur in three-fourths of
cases and are the prime
reason for wheelchair use. Only a minority are totally wheelchair dependent (-
15%). Mean time
between symptom onset and wheelchair use ranged from 13 8 (6-32) years.
Clinical dysphagia
is common with estimates reaching as high as 66%. Finger flexor weakness can
be detected early
in the course of illness. About 40% of patients have facial muscle weakness.
In the only
prospective study of decline of muscle strength, the investigators documented
a 4% decline in 6
months (0.66% per month) based on quantitative myometry using maximal
isometric contraction
testing. A similar rate of decline was also found in another study using
manual muscle testing.
Natural history studies document a fairly consistent, slow rate of decline in
most patients with
sIBM.
100071 Therapy for sIBM has focused on the underlying inflammatory response
with either
immunosuppressive or immunomodulating drugs. Uncontrolled trials with
corticosteroids,
cyclophosphamide, chlorambucil, azathioprine and cyclosporin, and methotrexate
fail to show
convincing evidence of benefit. An initial pilot study of IVIG in IBM showed
encouraging
results but a subsequent randomized controlled trial with WIG was negative. A
prospective,
double-blind, placebo-controlled, 6 month trial of weekly IM injections of 30
lig of n-interferon-
1 a showed no benefit. A recently published open-label trial using etanercept
showed increased
hand grip strength without any other functional benefits. Considering the
complexity of
pathogenic factors, it may be sometime before an effective treatment can
target both autoimmune
and myodegenerative factors. Attention has shifted to growth modulatory
approaches. In a 3
month, randomized, placebo-controlled, crossover study of oxandrolone, a
synthetic anabolic
steroid, a borderline significant improvement in overall strength was
reported, as measured by
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quantitative myometry. The study was limited by small sample size and short
duration of
treatment. Exercise is another means of growth modulation and this has been
tried in
inflammatory muscle diseases. An increase in isometric torque and an
improvement in functional
activities was reported in five patients with sIBM following a 12-week
isotonic training program
for the knee extensors and flexors and the elbow and wrist flexors. The small
sample size
disallows any conclusion but exercise had no deleterious effects based on
muscle biopsies
performed pre- and post-exercise.
[0008] Research has shown that skeletal muscle utilizes a regulatory mechanism
to control tissue
mass. In a screen for novel members of the transforming growth factor-13 (TGF-
13) superfamily of
growth and differentiation factors, myostatin [previously called growth and
differentiation
factor-8 (GDF-8)] was identified and has subsequently been shown to be a
negative regulator of
muscle formation. Myostatin is expressed in the myotome compartment of
developing somites at
E9.5 with expression continuing throughout adulthood, predominantly in
skeletal muscles and
adipose tissue. Myostatin is synthesized in a precursor form that undergoes
two proteolytic
processing events to remove the N-terminal signal sequence and the C-terminal
fragment, which
possess receptor-binding activity. Following proteolytic processing, the
propeptide and the
disulfide linked C-terminal dimer remain bound noncovalently in a latent
complex. Myostatin
can be activated by dissociation of the propeptide after proteolytic cleavage
by a
metalloproteinase of the bone morphogenic (BMP)/tolloid family. The
dissociated C-terminal
fragment is thus the biologically active species. For a review of the
biosynthesis and signaling
pathway of myostatin, see Lee, Ann Rev Cell Dev Biol, 20: 61-86 (2004).
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100091 Myostatin is conserved among species, especially in its C-terminal
fragment which is
identical across human, rat, murine, porcine, turkey and chicken species.
Mutations within
myostatin have been shown to be linked to the double muscling phenotype in
cattle [Grobet et
al., Nat Genet, 17: 71-74 (1997); Kambadur et al., Genome Res, 7:910-915
(1997); and
McPherron and Lee, Proc Natl Acad Sci USA, 94:12457-12461 (1997)] and gross
muscle
hypertrophy in human subjects [Schuelke et al., N Eng J Med, 350: 2682-2688
(2004)]. Forced
muscle atrophy has even been achieved with recombinant myostatin
administration or over-
expression of myostatin [Zitnmers et al., Science, 296(5572): 1486-1488
(2002)]. Histology of
muscles from myostatin null mice shows increased muscle mass resulting from
hyperplasia and
hypertrophy of the muscle with less fat and connective tissues. The hypothesis
that it may be
beneficial to block, remove, or reduce myostatin to promote regeneration and
reduce fibrosis in
MD has been explored in animal studies. Wagner et al., Ann Neurol, 52: 832-836
(2002)
describes data obtained from crossing myostatin null mutant mice with mdx mice
(which are
models for dystrophin deficiency) showing that mdx mice lacking myostatin were
stronger and
more muscular than their mdx counterparts. In addition, Bogdanovich et al.,
Nature, 420: 418-
421(2002) report that when a neutralizing antibody to myostatin was
administered to 4 week old
mdx mice by intraperitoneal injection, an increase in body weight, muscle
mass, muscle size and
absolute muscle strength along with a significant decrease in muscle
degeneration and
concentrations of serum creatine kinase was observed. Similarly, Whittemore et
al., Biochem
Biophys Res Commun, 300: 965-971 (2003) describes that myostatin neutralizing
antibodies
increase muscle mass in adult mice. Tobin and Celeste, Curr Opin Pharma, 5:
328-332 (2005)
reviews the myostatin pathway as well as studies testing the effects of
reducing myostatin
expression/activity.
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[00101 Another review article, Wagner, Curr Opin Rheumatol, 17: 720-724
(2005), lists various
therapeutic approaches of inhibiting myostatin that have been considered for
treating human
disease. For example, Wyeth has developed a humanized, anti-myostatin antibody
called MY0-
029 for clinical trials for treatment of muscular dystrophy in adult patients.
The review article
states the antibody or similar agent will hopefully be tested in other
indications such as
inflammatory myopathies, cachexia and sarcopenia. The author also notes that a
number of
endogenous inhibitors of myostatin, including the myostatin propeptide,
follistatin, FLRG and
GASP-1 could be modified for use as therapeutic agents. The review refers to
two articles
describing the effects of modified propeptide on muscle in mice, Wolfinan et
al., Proc Natl Acad
Sci US, 100: 15842-15846 (2003) and Bogdanovich et al., FASEB J, 19: 543-549
(2004).
[0011] The Wagner review article states that there is significant data that
follistatin is an in vivo
inhibitor of myostatin and refers to the results of studies described in Lee
and McPherron, Proc
Natl Acad Sci USA, 98(16): 9306-9311(2001) and Amthor et al., Developmental
Biol., 270: 19-
30 (2004) to support that statement. Follistatin is a secreted protein that
inhibits the activity of
TGF-13 family members such as GDF-11/BMP-11. Follistatin-344 is a follistatin
precursor that
undergoes peptide cleavage to form the circulating Follistatin-315 isoform
which includes a C-
terminal acidic region. It circulates with myostatin propeptide in a complex
that includes two
other proteins, follistatin related gene (FLRG) and GDF associated serum
protein (GASP-1).
Follistatin-317 is another follistatin precursor that undergoes peptide
cleavage to form the
membrane-bound Follistatin-288 isoform. The DNA and amino acid sequences of
the follistatin-
344 precursor are respectively set out in SEQ ID NOs: 3 and 4. The Follistatin-
288 isoform,
which lacks a C-terminal acidic region, exhibits strong affinity for heparin-
sulfate-proteoglycans,
is a potent suppressor of pituitary follicle stimulating hormone, is found in
the follicular fluid of
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the ovary, and demonstrates high affinity for the granulose cells of the
ovary. The testis also
produce Follistatin-288. The DNA and amino acid sequences of the follistatin-
317 precursor are
respectively set out in SEQ ID NOs: 5 and 6. Lack of follistatin results in
reduced muscle mass
at birth.
[0012] In the experiments described in the Lee and McPherron article,
follistatin was over-
expressed in transgenic mice. The mice showed increased muscling resulting
from a combination
of hyperplasia (increased muscle fiber number) and hypertrophy (increased
muscle fiber
diameter). The article proposes that follistatin binds the C-terminal dimer of
myostatin and, in
turn, inhibits the ability of myostatin to bind to activin type II receptors.
Transgenic mice
expressing high levels of myostatin propepetide or a dominant-negative form of
activin type II
receptor (Act RIB) were also shown to exhibit increased muscle mass in the
article.
[0013] The Amthor et al. article is stated to report that follistatin directly
binds myostatin with
high affinity, is co-expressed with myostatin in somites and prevents
myostatin-mediated
inhibition of limb muscle development in chick embryos. Indicating that the
inhibitory effects of
follistatin are not specific to myostatin evening in regard to muscle growth,
the Wagner review
article alternatively indicates that FLRG and GASP-I, which bind to and
inhibit circulating
myostatin, may prove to be specific inhibitors of myostatin for therapeutic
use. FLRG is a
protein that exhibits homology to a 10-cysteine repeat in follistatin. Hill et
al., J Biol Chem,
277(43): 40735-40741 reports that FLRG binds circulating myostatin in vivo.
[0014] Yet another review article addressing the regulation of muscle mass by
myostatin and
clinical implications is Lee, Annu Rev Cell Dev Biol., 20: 61-86 (2404).
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Brief Summary of the Invention:
[0018] The present invention is useful for muscle enhancement and/or improving
muscle
function. Administering injections of the chemical composition is effective
for treating
musculoskeletal diseases/disorders and neurodegenerative diseases/disorders in
which muscle is
adversely affected as well as treating sarcopenia, cachexia, obesity, Type II
diabetes, Pompe
disease and lysosomal storage disorders. Administering injections of the
chemical composition is
also effective for muscle enhancement livestock, including ¨ but not limited
to ¨ cattle, pigs and
fowl.
Detailed Description:
[0019] The terms "inhibitor of myostatin" and "myostatin inhibitor" are
intended to be
interchangeable herein. The terms "muscle enhancement" and "enhancing muscle"
are also
intended to be interchangeable herein and include, but are not limited to,
inducement of
hyperplasia (increased muscle fiber number), inducement of hypertrophy
(increased muscle fiber
diameter) or both. "Enhanced muscle performance" includes, but is not limited
to, one or more of
decreased atrophy, increased muscle endurance, increased muscle force and
increased muscle
strength.
[0020] Myostatin inhibitor proteins according to the invention may be peptides
or polypeptides.
The proteins may inhibit myostatin by binding myostatin [McPherron et al.,
Nature, 387(6628):
83-90 (1997)] or by binding the myostatin receptor activin lib [McPherron et
al., Nat. Genet.,
22(3): 260-264 (1999)]. Examples of proteins that inhibit myostatin by binding
to myostatin are
myostatin propeptide, follistatin [Shimasaki et al., U.S. Pat. No. 5,041,538],
other follistatin-like
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proteins (U.S. Pat. Nos. 5,942,420; 6,410,232; 6,537,966; and 6,953,662), FLRG
(SEQ ID NO:
8, the corresponding nucleotide sequence is SEQ ID NO: 7) [Hill et al., J.
Biol. Chem., 277(43):
40735-40741 (2002)] and GASP-1 (SEQ ID NO: 10, corresponding nucleotide
sequence is SEQ
ID NO: 9) [Hill et al., Mol Endocrinol, 17: 1144-1154 (2003)]. Proteins that
are myostatin
inhibitors according to the invention may be protein fragments or may be
chimeric (i.e., fusion)
proteins.
