U.S. patent application number 14/679650 was filed with the patent office on 2016-02-04 for composition and method for treating or preventing skeletal muscle fibrosis.
The applicant listed for this patent is Arnon Nagler, Mark Pines. Invention is credited to Arnon Nagler, Mark Pines.
Application Number | 20160030429 14/679650 |
Document ID | / |
Family ID | 39636474 |
Filed Date | 2016-02-04 |
United States Patent
Application |
20160030429 |
Kind Code |
A1 |
Pines; Mark ; et
al. |
February 4, 2016 |
COMPOSITION AND METHOD FOR TREATING OR PREVENTING SKELETAL MUSCLE
FIBROSIS
Abstract
A compound in combination with a pharmaceutically acceptable
carrier, the compound having a formula: wherein: R.sub.1 is a
member of the group consisting of hydrogen, halogen, nitro, benzo,
lower alkyl, phenyl, and lower alkoxy; R.sub.2 is a member of the
group consisting of hydroxy, acetoxy, and lower alkoxy; and R.sub.3
is a member of the group consisting of hydrogen and lower
alkenoxy-carbonyl; and n is either 1 or 2; and pharmaceutically
acceptable salts thereof; for use in treatment of or prevention of
skeletal muscle fibrosis and/or for inducing skeletal muscle
regeneration. ##STR00001##
Inventors: |
Pines; Mark; (Rehovot,
IL) ; Nagler; Arnon; (Jerusalem, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pines; Mark
Nagler; Arnon |
Rehovot
Jerusalem |
|
IL
IL |
|
|
Family ID: |
39636474 |
Appl. No.: |
14/679650 |
Filed: |
April 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13782325 |
Mar 1, 2013 |
9023859 |
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14679650 |
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12523954 |
Feb 15, 2010 |
8410120 |
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PCT/IL2008/000088 |
Jan 21, 2008 |
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13782325 |
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60885896 |
Jan 21, 2007 |
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Current U.S.
Class: |
514/266.22 ;
544/286 |
Current CPC
Class: |
A61K 31/517 20130101;
A61P 21/00 20180101 |
International
Class: |
A61K 31/517 20060101
A61K031/517 |
Claims
1. A pharmaceutical composition for treating and/or preventing
skeletal muscle fibrosis in a subject in need thereof, the
composition comprising a pharmaceutically effective amount of a
compound in combination with a pharmaceutically acceptable carrier,
the compound having a formula: ##STR00006## wherein: n is either 1
or 2 R.sub.1 is a member of the group consisting of hydrogen,
halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;
R.sub.2 is a member of the group consisting of hydroxy, acetoxy,
and lower alkoxy; and R.sub.3 is a member of the group consisting
of hydrogen and lower alkenoxy-carbonyl; and pharmaceutically
acceptable salts thereof.
2. A pharmaceutical composition according to claim 1, wherein said
compound is halofuginone.
3. A pharmaceutical composition according to claim 1, wherein said
subject is suffering from a disorder which encompasses skeletal
muscle tissue.
4. A pharmaceutical composition according to claim 3, wherein said
disorder is muscular dystrophy.
5. A pharmaceutical composition according to claim 4, wherein said
muscular dystrophy is selected from the group consisting of
Duchenne muscular dystrophy, Becker muscular dystrophy,
Emery-Dreifuss Muscular Dystrophy, Limb-Girdle Muscular Dystrophy,
Facioscapulohumeral Muscular Dystrophy, Myotonic Dystrophy,
Oculopharyngeal Muscular Dystrophy, Distal Muscular Dystrophy, and
congenital muscular dystrophy.
6. A pharmaceutical composition according to claim 3, wherein said
disorder is denervation atrophy.
7. A pharmaceutical composition according to claim 3, wherein said
skeletal muscle tissue is diaphragm muscle.
8. A pharmaceutical compositions for improving or inducing skeletal
muscle regeneration in a subject in need thereof, the composition
comprising a pharmaceutically effective amount of a compound in
combination with a pharmaceutically acceptable carrier, the
compound having a formula: ##STR00007## wherein: n is either 1 or 2
R.sub.1 is a member of the group consisting of hydrogen, halogen,
nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R.sub.2 is a
member of the group consisting of hydroxy, acetoxy, and lower
alkoxy; and R.sub.3 is a member of the group consisting of hydrogen
and lower alkenoxy-carbonyl; and pharmaceutically acceptable salts
thereof.
9. A pharmaceutical composition according to claim 8, wherein said
compound is halofuginone.
10. A pharmaceutical composition according to claim 8, wherein said
subject is suffering from a disorder which targets skeletal muscle
tissue.
11. A pharmaceutical composition according to claim 10, wherein
said disorder is muscular dystrophy.
12. A pharmaceutical composition according to claim 11, wherein
said muscular dystrophy is selected from the group consisting of
Duchenne muscular dystrophy, Becker muscular dystrophy,
Emery-Dreifuss Muscular Dystrophy, LimbGirdle Muscular Dystrophy,
Facioscapulohumeral Muscular Dystrophy, Myotonic Dystrophy,
Oculopharyngeal Muscular Dystrophy, Distal Muscular Dystrophy, and
congenital muscular dystrophy.
13. A pharmaceutical composition according to claim 10, wherein
said disorder is denervation atrophy.
14. A pharmaceutical composition according to claim 10, wherein
said skeletal muscle tissue is diaphragm muscle.
15. A method for reducing the progression of skeletal muscle
fibrosis in a subject in need thereof, the method comprising
administering a pharmaceutically effective amount of a compound in
combination with a pharmaceutically acceptable carrier, the
compound having a formula: ##STR00008## wherein: n is either 1 or 2
R.sub.1 is a member of the group consisting of hydrogen, halogen,
nitro, benzo, lower alkyl, phenyl, and lower alkoxy; R.sub.2 is a
member of the group consisting of hydroxy, acetoxy, and lower
alkoxy; and R.sub.3 is a member of the group consisting of hydrogen
and lower alkenoxy-carbonyl; and pharmaceutically acceptable salts
thereof.
16. A method according to claim 15, wherein said compound is
halofuginone.
17. A method according to claim 15, wherein said subject is
suffering from a disorder which targets skeletal muscle tissue.
18. A method according to claim 17, wherein said disorder is
muscular dystrophy.
19. A method according to claim 18, wherein said muscular dystrophy
is selected from the group consisting of Duchenne muscular
dystrophy, Becker muscular dystrophy, Emery-Dreifuss Muscular
Dystrophy, Limb-Girdle Muscular Dystrophy, Facioscapulohumeral
Muscular Dystrophy, Myotonic Dystrophy, Oculopharyngeal Muscular
Dystrophy, Distal Muscular Dystrophy, and congenital muscular
dystrophy.
20. A method according to claim 17, wherein said disorder is
denervation atrophy.
21. A method according to claim 17, wherein said skeletal muscle
tissue is diaphragm muscle.
22. A method for improving or inducing skeletal muscle regeneration
in a subject in need thereof, the method comprising administering a
pharmaceutically effective amount of a compound in combination with
a pharmaceutically acceptable carrier, the compound having a
formula: ##STR00009## wherein: n is either 1 or 2 R.sub.1 is a
member of the group consisting of hydrogen, halogen, nitro, benzo,
lower alkyl, phenyl, and lower alkoxy; R.sub.2 is a member of the
group consisting of hydroxy, acetoxy, and lower alkoxy; and R.sub.3
is a member of the group consisting of hydrogen and lower
alkenoxy-carbonyl; and pharmaceutically acceptable salts
thereof.
23. A method according to claim 22, wherein said compound is
halofuginone.
24. A method according to claim 22, wherein said subject is
suffering from a disorder which targets skeletal muscle tissue.
25. A method according to claim 24, wherein said disorder is
muscular dystrophy.
26. A method according to claim 25, wherein said muscular dystrophy
is selected from the group consisting of Duchenne muscular
dystrophy, Becker muscular dystrophy, Emery-Dreifuss Muscular
Dystrophy, Limb-Girdle Muscular Dystrophy, Facioscapulohumeral
Muscular Dystrophy, Myotonic Dystrophy, Oculopharyngeal Muscular
Dystrophy, Distal Muscular Dystrophy, and congenital muscular
dystrophy.
27. A pharmaceutical composition according to claim 24, wherein
said disorder is denervation atrophy.
28. A pharmaceutical composition according to claim 24, wherein
said skeletal muscle tissue is diaphragm muscle.
29. A method according to claim 22, wherein the improving of
skeletal muscle regeneration occurs through inhibiting the
TGF.beta. pathway and/or by inhibiting the Myostatin
Smad3-dependent pathway.
30. Use of a compound having a formula: ##STR00010## wherein: n is
either 1 or 2 R.sub.1 is a member of the group consisting of
hydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower
alkoxy; R.sub.2 is a member of the group consisting of hydroxy,
acetoxy, and lower alkoxy; and R.sub.3 is a member of the group
consisting of hydrogen and lower alkenoxy-carbonyl; and
pharmaceutically acceptable salts thereof; in the manufacture of a
medicament for reducing the progression of skeletal muscle fibrosis
in a subject in need thereof.
31. The use according to claim 30, wherein said compound is
halofuginone.
32. The use according to claim 30, wherein said subject is
suffering from a disorder which targets skeletal muscle tissue.
33. The use according to claim 32, wherein said disorder is
muscular dystrophy.
34. The use according to claim 33, wherein said muscular dystrophy
is selected from the group consisting of Duchenne muscular
dystrophy, Becker muscular dystrophy, Emery-Dreifuss Muscular
Dystrophy, Limb-Girdle Muscular Dystrophy, Facioscapulohumeral
Muscular Dystrophy, Myotonic Dystrophy, Oculopharyngeal Muscular
Dystrophy, Distal Muscular Dystrophy, and congenital muscular
dystrophy.
35. The use according to claim 32, wherein said disorder is
denervation atrophy.
36. The use according to claim 32, wherein said skeletal muscle
tissue is diaphragm muscle.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority
under 35 U.S.C. .sctn.120 to U.S. Ser. No. 13/782,325, entitled
COMPOSITION AND METHOD FOR TREATING OR PREVENTING SKELETAL MUSCLE
FIBROSIS filed on Mar. 1, 2013, which claims priority from U.S.
National application Ser. No. 12/523,954 entitled COMPOSITION AND
METHOD FOR TREATING OR PREVENTING SKELETAL MUSCLE FIBROSIS filed on
Feb. 15, 2010, which is the National Stage Entry of PCT/IL08/00088
filed Jan. 21, 2008, which claims priority from U.S. Provisional
Application Ser. No. 60/885,896 entitled COMPOSITION AND METHOD FOR
TREATING OR PREVENTING SKELETAL MUSCLE FIBROSIS filed Jan. 21,
2007.
FIELD OF THE INVENTION
[0002] The present invention relates to a method of treating
fibrosis, and more particularly to treatment, prevention or
amelioration of skeletal muscle fibrosis by use of halofuginone or
related compounds.
BACKGROUND OF THE INVENTION
[0003] Muscle fibrosis is a phenomenon that frequently occurs in
diseased or damaged muscle. It is characterized by the excessive
growth of fibrous tissue, which usually results from the body's
attempt to recover from injury. Fibrosis impairs muscle function
and causes weakness. The amount of muscle function loss generally
increases with the extent of fibrosis. Fibrosis is usually
progressive and can contribute to the patient's inability to carry
out ordinary tasks of independent living, such as grasping objects
or walking. Fibrosis commonly occurs as a result of muscular
dystrophy, as well as due to other afflictions, such as denervation
atrophy, a degradation of muscle tissue caused by loss of neural
contact to a muscle. For some types of muscular dystrophy, such as
Duchenne, fibrosis can result in death as the muscles of the
diaphragm are affected (the diaphragm is a skeletal muscle which is
involuntary rather than voluntary).
[0004] Muscular dystrophies are a heterogeneous group of genetic
disorders characterized by the progressive loss of muscle strength
and integrity. Dystrophic muscle shows variation in muscle fiber
size, infiltration of connective and fatty tissue, and centrally
located nuclei. The membranes of the fibers are fragile and
extensive damage occurs, leading to necrosis and muscle
wasting.
[0005] Victims of muscular dystrophies, particularly Becker
muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD),
frequently suffer from increasing skeletal muscle fibrosis as the
disease progresses.
[0006] The most common form of muscle dystrophy is the X-linked
recessive DMD, a severely penetrating allelic manifestation which
affects 1 in 3500 live males at birth; about a third of cases occur
as de novo mutations in the infant (Emery A E. (1991) Neuromusc.
Disord. 1: 19-29).
[0007] Usually the disease is diagnosed at 4-5 years of age and by
8-10 years, deterioration of the patient's condition necessitates
wheelchair use. By their early teens, further neurological and
cardiological symptoms are apparent. Progression of muscle
degeneration and worsening clinical symptoms, lead to death in the
late teens or early twenties, typically as a result of
cardio-pulmonary complications due to fibrosis of the
diaphragm.
[0008] The leading causes of death in DMD victims, respiratory and
heart failure, result from weakness in diaphragm and myocardium
muscles that are most affected by fibrosis (Finsterer, (2003)
Cardiology 99:1-19). Fibrosis is characterised by an increase in
extra-cellular matrix (ECM) constituents especially collagen type
I. Both in DMD and Congenital muscular dystrophy (CMD), an increase
in type I and III collagens were observed in the skeletal muscle
(Hantai et al. (1985) Connect Tissue Res. 13:273-81 and Dunace, et
al. (1980) Nature 284:470-472) leading to fibrosis, which
correlated with muscle destruction (Zhao, et al. (2003) J. Patho.
201:149-59). The cardiac involvement in DMD is characterized
pathologically by degeneration and fibrosis of the myocardium,
probably due to myofibroblast activity, centering around the
posterolateral wall of the left ventricle.
[0009] BMD is a less severe condition than DMD, characterized by
slowly progressive muscle weakness of the legs and pelvis, again
due to fibrosis of the muscles (although for BMD the skeletal
muscles are more greatly affected). The advance of fibrosis often
causes ever greater loss of mobility and a reduced life expectancy.
At some point, the patient may become too weak to walk and takes to
a wheelchair.
[0010] Both BMD and DMD are associated with defects in the
dystrophin gene, the gene responsible for the production of
dystrophin protein, which is a vital part of the
dystrophin-glycoprotein complex. DMD is characterized by the near
absence of dystrophin protein in skeletal muscles, while BMD
results from different mutations in the same gene, resulting in
decreased or damaged dystrophin. The presence of some dystrophin
protects the muscles of those with BMD from degenerating as badly
or as quickly as those of DMD victims.
[0011] The dystrophin-glycoprotein complex connects the actin
cytoskeleton of myofibres to the extracellular matrix (ECM) and is
therefore integral to the contractile structure of muscle (Yue Y,
et al. (2003) Circulation, 108:1626-32 and Michele et al. (2003) J.
Biol. Chem 278:15457-60). The preliminary stage of DMD is
characterized by the presence of focal groups of necrotic
myofibres, muscle hypertrophy and abnormally high levels of muscle
creatine kinase (CK). In the pathological phase, repeated cycles of
degeneration exhaust the regenerative capacity of muscle-specific
progenitor cells (satellite cells) and fibrotic mechanisms cause
the progressive replacement of the muscle tissue with collagenous
connective tissue (Rafael et al., 1997). These processes lead to
joint contraction, loss of ambulation and death from respiratory or
cardiac failure (Wells, et al. (2002) Neuromuscle Disord. 12 Suppl
1:S11-22).
[0012] The perfect solution for DMD and BMD patients would be to
place a normal copy of the dystrophin gene into muscle cells, and
hence restore sufficient protein expression to improve structure
and function (Khurana, et al. (2003) Nat Rev Drug Discov.
2:379-90). At 3.0 MB the dystrophin gene is vast, and successful
therapy would require massive and sustained gene transfer (Hoffman,
et al. (1987) Cell 51:919-28 and Skuk, et al. (2002) Curr. Opin.
Neurol. 15:563-9 and Thioudellet, et al. (2002) Neuromuscul Disord.
12 Suppl 1:S49-51). Muscle fibrosis is a major obstacle in gene
therapy since it hampers gene delivery.
[0013] An alternative to replacing the faulty gene is to modulate
its expression by employing antisense oligonucleotides that alter
RNA stability, or splicing (Lu Qi, et al. (2003) Nat. Med.
9(8):1009-14 and Rando T A. (2002) Am. J. Phy. Med. Rehabil.
81(11Suppl):S175-86), thereby resulting in the production of a
functional protein. Transplantation of muscle precursor cells
(myoblast transfer) has also been explored as a method for
restoring dystrophin protein to dystrophic muscle (Law P K et al.
(1997) Transplant Proc. 29(4):2234-7). This technique is
constrained by the difficulties associated with treating large
volumes of muscle with long-lasting effect. An alternative approach
is to up-regulate the expression of an endogenous protein that
effects some functional replacement (Krag T O, et al. (2001) Acta
Physiol Scand. 171:349-58). However, all of these treatments are
ineffective unless the progression of the underlying fibrotic
condition can be halted or at least ameliorated somewhat.
[0014] The crucial role of collagen in fibrosis has prompted
attempts to develop agents that inhibit or modulate its
accumulation. Several unique post-transcriptional enzymes of the
collagen biosynthesis pathway appear to be attractive targets for
reducing the formation of collagen fibers or for the accumulation
of fibers with altered properties (Prockop D J, (1995) Annu Rev
Biochem. 64:403-34).
[0015] The major disadvantage of these inhibitors is that they are
not collagen-type specific and may inhibit the biosynthesis of
other collagens with serious toxic consequences.
[0016] To date there is no effective therapy for reducing skeletal
muscle fibrosis. No treatment which affects fibrotic tissue without
adversely affecting healthy muscle tissue or other body functions
is currently known. The only treatment to have shown clinical
efficacy is a prednisone/prednisolone treatment that results in a
modest increase in strength, and delays, but does not halt, the
progress of the disease (Backman, et al. Neuromuscul Disord.
5:233-41 and Dubowitz, (2002) Neuromuscul Disord. 12:113-6).
[0017] There is thus a widely recognized need for, and it would be
highly advantageous to have, a method of preventing or retarding
the build up of skeletal muscle fibrosis that accompanies disorders
such as Duchenne and Becker muscular dystrophies and other muscle
dystrophies with extensive fibrosis, as well as to reduce the
effect on muscles of the diaphragm for Duchenne muscular
dystrophy.
Quinazolinone Derivatives
[0018] Quinazolinone derivatives were first taught in U.S. Pat. No.
3,320,124 to American Cyanamid as a treatment for the intestinal
parasitic disease, coccidiosis. Halofuginone,
(7-bromo-6-chloro-3-[3-(3-hydroxy-2-piperidinyl)-2-oxopropyl]-4(3H)-quina-
zolinone), an analog of a plant alkaloid originally isolated from
the plant Dichroa febrifuga, was described as the preferred
quinazolinone derivative. Subsequently, U.S. Pat. Nos. 4,824,847;
4,855,299; 4,861,758 and 5,215,993 all relate to the coccidiocidal
properties of halofuginone.
[0019] More recently, it was disclosed in U.S. Pat. No. 5,449,678
that these quinazolinone derivatives are unexpectedly useful for
the treatment of a fibrotic condition such as scleroderma and
graft-versus-host disease (GVHD). This disclosure provided
compositions of a specific inhibitor comprising a therapeutically
effective amount of a pharmaceutically active compound of the
formula:
##STR00002##
wherein: n=1-2 R.sub.1 is a member of the group consisting of
hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower
alkoxy; R.sub.2 is a member of the group consisting of hydroxy,
acetoxy and lower alkoxy; and R.sub.3 is a member of the group
consisting of hydrogen and lower alkenoxy-carbonyl.
Pharmaceutically acceptable salts thereof are also included. Of
this group of compounds, halofuginone has been found to be
particularly effective for the disclosed treatment.
[0020] The clinical potential of halofuginone in anti-fibrotic
therapy has also been described in Pines, et al. Drug of the Future
21:569-599 and Pines, et al. (1997) Gen. Pharmaco. 30:445-450 and
Pines, et al. (2000) Drug Develop. Res. 50, 371-378). Halofuginone,
an inhibitor of collagen type I synthesis has been found to inhibit
the gene expression of collagen type 1, but not of type II (Granot,
et al. Biochim Biophys Acta 1156: 107-112) or type III (Choi, et
al. (1995) Arch Surg 130:257-261).
[0021] U.S. Pat. No. 5,891,879 further discloses that the
quinazolinone derivatives are effective in treating restenosis. The
two earlier-described conditions, scleroderma and graft-versus-host
disease, are associated with excessive collagen deposition, which
can be inhibited by halofuginone. Restenosis is characterized by
smooth muscle cell proliferation and extracellular matrix
accumulation within the lumen of affected blood vessels in response
to a vascular injury (Choi et al., Arch. Surg., 130:257-261
(1995)). One hallmark of such smooth muscle cell proliferation is a
phenotypic alteration, from the normal contractile phenotype to a
synthetic one. Type I collagen has been shown to support such a
phenotypic alteration, which can be blocked by halofuginone (Choi
et al., Arch. Surg., 130: 257-261, (1995); U.S. Pat. No.
5,449,678).
[0022] Notably, the in vitro action of halofuginone does not always
predict its in vivo effects. For example, as demonstrated in U.S.
Pat. No. 5,449,678, halofuginone inhibits the synthesis of collagen
type I in bone chrondrocytes in vitro. However, chickens treated
with halofuginone were not reported to have an increased rate of
bone breakage, indicating that the effect is not seen in vivo. In
addition, even though halofuginone inhibits collagen synthesis by
fibroblasts in vitro, it promotes wound healing in vivo (WO
01/17531). Thus, the exact behavior of halofuginone in vivo cannot
always be accurately predicted from in vitro studies.
[0023] Quinazolinone-containing pharmaceutical compositions,
including halofuginone, have been disclosed and claimed as
effective for treating malignancies (U.S. Pat. No. 6,028,075), for
prevention of neovascularization (U.S. Pat. No. 6,090,814), as well
as for treating hepatic fibrosis (U.S. Pat. No. 6,562,829),
pulmonary fibrosis (WO 98/43642) and renal fibrosis (WO 02/094178),
scleroderma and a variety of other serious diseases, exhibit
excessive production of connective tissue, which results in the
destruction of normal tissue architecture and function.
[0024] WO 00/09070 relates to a method for treating and preventing
fibrotic process, which results from pathophysiological responses
to tissue trauma, preferably cardiac fibrosis.
[0025] In most animal models of fibrosis, regardless of the tissue,
halofuginone has a minimal effect on collagen content in the
non-fibrotic animals, whereas it exhibits a profound inhibitory
effect in the fibrotic organs. This suggests a different regulation
of the low level house-keeping expression of collagen type I genes
on the one hand and the over-expression induced by the fibrogenic
stimulus which is usually an aggressive and a rapid process, on the
other.
Muscle Tissue
[0026] Muscle is a very specialized tissue that has both the
ability to contract and the ability to conduct electrical impulses.
Muscles are classified both functionally as either voluntary or
involuntary, and structurally as either striated or smooth. From
this, there emerge three types of muscles: smooth muscle
(involuntary), skeletal voluntary muscle (voluntary and
involuntary) and cardiac muscle. Skeletal and cardiac muscle are
called striated muscle because of their striped appearance under a
microscope.
[0027] Skeletal muscle may be of the voluntary or involuntary
muscle type, being innervated by neurons that originate from the
somatic or voluntary branch of the nervous system, providing
willful control of the skeletal muscles, or, as in the case of the
diaphragm muscles, being controlled by efferent nerves from the
respiratory centre which pass down the spinal cord to the
diaphragm. Skeletal muscle cells are long multi-nucleated
cylinders, which acquired this characteristic because they develop
from the fusion of small single cells into long units. The cells
may vary in diameter, averaging between 100 and 150 microns.
[0028] Skeletal muscle cells are independent cells separated from
one another by connective tissue and must each be stimulated by
axons of a neuron. All the cells innervated by branches from the
same neuron will contract at the same time and are referred to as a
motor unit. Motor units vary in size: large motor units with more
than 100 cells are typical of the slow acting postural muscles.
Very small motor units with around 10 cells or so are typical of
fast acting muscles with very precise control such as those which
move the eye. Most human muscles have a mixture of motor units of
different sizes.
[0029] Skeletal muscles have distinct stripes or striations that
identify them and are related to the organization of protein
myofilaments inside the cell. Skeletal muscle cells are associated
with a type of stem cell known as a satellite cell. These cells are
believed to aid in recovery of muscle fibers from damage and can
contribute their nuclei to replace and supplement the nuclei of the
damaged cells. This occurs in response to the "microtears" produced
by strenuous exercise and results in increased production of
proteins and myofibrils.
[0030] Voluntary muscles comprise a variety of fiber types which
are specialized for particular tasks. Most voluntary muscles
contain a mixture of fiber types although one type may
predominate.
[0031] Type 1 or slow oxidative fibers have a slow contraction
speed and a low myosin ATPase activity. These cells are specialized
for steady, continuous activity and are highly resistant to
fatigue. Their motor neurons are often active, with a low firing
frequency. These cells are thin (high surface to volume ratio) with
a good capillary supply for efficient gas exchange. They are rich
in mitochondria and myoglobin, which gives them a red color. They
are built for aerobic metabolism and prefer to use fat as a source
of energy. These are the marathon runner's muscle fibers.
[0032] Type 2A or fast oxidative-glycolytic fibers have a fast
contraction speed and a high myosin ATPase activity. They are
progressively recruited when additional effort is required, but are
still very resistant to fatigue. Their motor neurons show bursts of
intermittent activity. These cells are thin (high surface to volume
ratio) with a good capillary supply for efficient gas exchange.
They are rich in mitochondria and myoglobin which gives them a red
color. They are built for aerobic metabolism and can use either
glucose or fats as a source of energy. These are general purpose
muscle fibers which give the edge in athletic performance, but they
are more expensive to operate than type 1.
[0033] Type 2B or fast glycolytic fibers have a fast contraction
speed and a high myosin ATPase activity. They are only recruited
for brief maximal efforts and are easily fatigued. Their motor
neurons transmit occasional bursts of very high frequency impulses.
These are large cells with a poor surface to volume ratio and their
limited capillary supply slows the delivery of oxygen and removal
of waste products. They have few mitochondria and little myoglobin,
resulting in a white color (e.g. chicken breast). They generate ATP
by the anaerobic fermentation of glucose to lactic acid. These are
sprinter's muscle fibers, no use for sustained performance.
[0034] Cardiac muscle is the muscle found in the heart. It is
composed of much shorter cells than skeletal muscle that branch to
connect to one another. These connections are by means of gap
junctions called intercalated disks that allow an electrochemical
impulse to pass to all the connected cells. This causes the cells
to form a functional network called a syncytium in which the cells
work as a unit. Many cardiac muscle cells are myogenic which means
that the impulse arises from the muscle, not from the nervous
system. This causes the heart muscle and the heart itself to beat
with its own natural rhythm. But the autonomic nervous system
controls the rate of the heart and allows it to respond to stress
and other demands. As such the heart is said to be involuntary.
[0035] The cardiac muscle has a number of unique features that
reflect its function of pumping blood. [0036] The myofibrils of
each cell (and cardiac muscle is made of single cells--each with a
single nucleus) are branched. [0037] The branches interlock with
those of adjacent fibers by adherens junctions. These strong
junctions enable the heart to contract forcefully without ripping
the fibers apart. [0038] The action potential that triggers the
heartbeat is generated within the heart itself. Motor nerves (of
the autonomic nervous system) do run to the heart, but their effect
is simply to modulate--increase or decrease--the intrinsic rate and
the strength of the heartbeat. Even if the nerves are destroyed (as
they are in a transplanted heart), the heart continues to beat.
[0039] The action potential that drives contraction of the heart
passes from fiber to fiber through gap junctions.
[0040] Due to the numerous structural and functional differences
between various muscle types, the effect of an active
pharmaceutical ingredient on a particular muscle type cannot be
predicted with any degree of reliability according to the effect of
that ingredient on a different muscle type.
SUMMARY OF THE INVENTION
[0041] The present invention successfully addresses the
shortcomings of the presently known methods of treating and/or
preventing skeletal muscle fibrosis by providing compositions and
methods comprising a quinizolinone derivative.
[0042] As used herein, the quinazolinone derivative comprises a
compound having a formula:
##STR00003##
[0043] wherein:
[0044] R.sub.1 is a member of the group consisting of hydrogen,
halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;
R.sub.2 is a member of the group consisting of hydroxy, acetoxy,
and lower alkoxy; and R.sub.3 is a member of the group consisting
of hydrogen and lower alkenoxy-carbonyl; and n is either 1 or 2;
and pharmaceutically acceptable salts thereof.
[0045] According to one aspect of the present invention there is
provided a pharmaceutical composition for treating and/or
preventing and/or at least reducing the rate of progression of
skeletal muscle fibrosis in a subject in need thereof, the
composition comprising a pharmaceutically effective amount of a
quinizolinone derivative and pharmaceutically acceptable salts
thereof; and a pharmaceutically acceptable carrier.
[0046] According to another aspect of the present invention there
is provided a pharmaceutical composition for improving skeletal
muscle regeneration in a subject in need thereof, the composition
comprising a pharmaceutically effective amount of a quinazolinone
derivative and pharmaceutically acceptable salts thereof; and a
pharmaceutically acceptable carrier.
[0047] According to yet another aspect of the present invention
there is provided a method for reducing the progression of skeletal
muscle fibrosis in a subject in need thereof, the method comprising
administering a pharmaceutically effective amount of a
quinazolinone derivative and pharmaceutically acceptable salts
thereof; and a pharmaceutically acceptable carrier.
[0048] According to still another aspect of the present invention
there is provided a method for improving and/or inducing skeletal
muscle regeneration in a subject in need thereof, the method
comprising administering a pharmaceutically effective amount of a
quinazolinone derivative, and pharmaceutically acceptable salts
thereof; and a pharmaceutically acceptable carrier. The improving
of skeletal muscle regeneration optionally occurs through
inhibiting the TGF.beta. pathway and/or by inhibiting the Myostatin
Smad3-dependent pathway.
[0049] According to an additional aspect of the present invention
there is provided the use of a quinazolinone derivative, and
pharmaceutically acceptable salts thereof; in the manufacture of a
medicament for reducing the progression of skeletal muscle fibrosis
in a subject in need thereof.
[0050] According to further features in preferred embodiments of
the invention described below, quinazolinone derivative is
optionally and preferably halofuginone.
[0051] According to still further features in the described
preferred embodiments, the subject in need of treatment by the
compositions and methods of the present invention is suffering from
a disorder which targets skeletal muscle tissue, such as, for
example muscular dystrophy, including Duchenne muscular dystrophy,
Becker muscular dystrophy, Emery-Dreifuss Muscular Dystrophy,
Limb-Girdle Muscular Dystrophy, Facioscapulohumeral Muscular
Dystrophy, Myotonic Dystrophy, Oculopharyngeal Muscular Dystrophy,
Distal Muscular Dystrophy, and congenital muscular dystrophy.
[0052] Alternatively, the subject may be suffering from denervation
atrophy.
[0053] Optionally, the skeletal muscle tissue targeted by the
disorder may comprise diaphragm muscle.
[0054] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the patent specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
[0055] As used herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition, substantially ameliorating clinical or aesthetical
symptoms of a condition or substantially preventing the appearance
of clinical or aesthetical symptoms of a condition.
[0056] The term "comprising" means that other steps and ingredients
that do not affect the final result can be added. This term
encompasses the terms "consisting of" and "consisting essentially
of."
[0057] The phrase "consisting essentially of" means that the
composition or method may include additional ingredients and/or
steps, but only if the additional ingredients and/or steps do not
materially alter the basic and novel characteristics of the claimed
composition or method.
[0058] The term "method" refers to manners, means, techniques and
procedures for accomplishing a given task including, but not
limited to, those manners, means, techniques and procedures either
known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0059] The term "active ingredient" refers to a pharmaceutical
agent including any natural or synthetic chemical substance that
subsequent to its application has, at the very least, at least one
desired pharmaceutical or therapeutic effect.
[0060] The term "therapeutically effective amount" or
"pharmaceutically effective amount" denotes that dose of an active
ingredient or a composition comprising the active ingredient that
will provide the therapeutic effect for which the active ingredient
is indicated.
[0061] As used herein, the term "pharmaceutically acceptable" means
approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, and more particularly
in humans. Herein, the phrases "physiologically suitable carrier"
and "pharmaceutically acceptable carrier" are interchangeably used
and refer to an approved carrier or a diluent that does not cause
significant irritation to an organism and does not abrogate the
biological activity and properties of the administered
conjugate.
[0062] As used herein, the term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered.
[0063] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
processes and administration of the active ingredients.
[0064] As used herein, the singular form "a," "an," and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0065] Throughout this disclosure, various aspects of this
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0066] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals there between.
BRIEF DESCRIPTION OF THE DRAWINGS
[0067] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0068] In the drawings:
[0069] FIG. 1 shows the effect of halofuginone on fibrosis and
myogenesis via Smad3-dependent pathways;
[0070] FIG. 2 presents the results of Sirius red staining for
collagen content and in situ hybridization of collagen al (I) gene
expression in Mdx mice and C57B control mice, in the presence and
absence of halofuginone;
[0071] FIG. 3 is a bar chart demonstrating the effect of
halofuginone on muscle fibrosis using staining and quantification
by Image analysis; and
[0072] FIG. 4 is a bar chart illustrating muscle regeneration after
halofuginone treatment.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0073] The present invention is of a method and pharmaceutical
composition for treating or preventing skeletal muscle fibrosis in
a subject, or at least reducing the progression thereof, by using a
quinazolinone derivative, preferably halofuginone. According to
preferred embodiments, the present invention relates to a method
and pharmaceutical compositions for at least reducing the
progressive loss of muscle strength and/or function associated with
skeletal muscle fibrosis in a subject. Optionally and preferably,
such at least reduction of the progressive loss of muscle strength
and/or function and/or treatment and/or prevention occurs in a
subject suffering from skeletal muscle fibrosis associated with a
disease of which directly or indirectly causes such fibrosis,
including but not limited to muscular dystrophy or denervation
atrophy. Muscular dystrophy includes Duchenne Muscular Dystrophy;
Becker Muscular Dystrophy; Emery-Dreifuss Muscular Dystrophy;
Limb-Girdle Muscular Dystrophy; Facioscapulohumeral Muscular
Dystrophy (also known as Landouzy-Dejerine); Myotonic Dystrophy;
Oculopharyngeal Muscular Dystrophy; Distal Muscular Dystrophy; and
Congenital Muscular Dystrophy.
[0074] Unexpectedly, according to preferred embodiments of the
present invention, a quinazolinone derivative, preferably
halofuginone, may optionally and preferably be used to at least
delay the progression of fibrosis of the diaphragm in a subject
suffering from a disease which affects this involuntary muscle,
including but not limited to Duchenne muscular dystrophy. More
preferably, the present invention relates to treating and/or
preventing fibrosis of the diaphragm in a subject suffering from
such a disease.
[0075] According to preferred embodiments of the present invention
and as demonstrated below, one or more types of muscle cells may
optionally and preferably be able to regenerate themselves as a
result of administration of a quinazolinone derivative, preferably
halofuginone, to a subject in need thereof.
[0076] The principles and operation of the compositions and methods
according to the present invention may be better understood with
reference to the drawings and accompanying descriptions.
[0077] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0078] Myostatin is a transforming growth factor-.beta.
(TGF-.beta.) family member that plays an essential role in
regulating skeletal muscle growth, by inhibition of the
proliferation and differentiation of myeloblasts, via inhibition of
muscle-specific MyoD and myogenin genes. Signaling occurs via
activation of Smad3 proteins (Langley, et al. (2002) J Biol Chem.
277:49831-40 and Zhang, et al. (2000) J. Biol. Chem. 275:39237-45
and Zhu, et al. (2004) Cytokine. 26(6):262-72; FIG. 1).
[0079] TGF.beta. is the major stimulator of collagen synthesis,
particularly collagen type I, in fibroblasts leading to fibrosis.
The inverse correlation between fibrosis and muscle formation in
dystrophy has been demonstrated (Wanger, et al. (2002) Ann Neurol.
52:832-6).
[0080] It has been demonstrated that halofuginone, an inhibitor of
Smad3 activity (McGaha, et al. (2002) J Invest Dermatol.
118(3):461-70), is a potent inhibitor of fibrosis due to its effect
on collagen biosynthesis and degradation in variety of tissues
(Levi-Schaffer, et al. (1996) J Invest Dermatol 106:84-88). The
tissue types studied did not include skeletal muscle. Furthermore,
the prior art does not teach the use of halofuginone in
regeneration of muscle.
[0081] Preliminary experiments by the present inventors have
demonstrated a halofuginone-dependent inhibition in myostatin gene
expression in fibrotic liver. Halofuginone is to date the only
known collagen type-specific inhibitor on the transcriptional
level. Inhibition of collagen synthesis on the transcriptional
level is more effective than attempting to treat the consequences
of collagen overproduction, which is the aim of other antifibrotic
drugs.
[0082] Due to the numerous structural and functional differences
between various muscle types, the effect of an active
pharmaceutical ingredient on a particular muscle type cannot be
predicted with any degree of reliability.
[0083] Therefore, it was hypothesized that halofuginone may improve
skeletal muscle integrity and inhibit fibrosis in a subject, as a
consequence of its combined effect on inhibition of collagen type I
synthesis, increase in collagen degradation and improved muscle
regeneration by inhibiting TGF.beta. and myostatin Smad3-dependent
pathways, as shown in FIG. 1. Also, halofuginone may inhibit
myostatin gene expression in muscle as well as was shown in other
tissues causing a further increase in muscle regeneration.
[0084] Hereinafter, the term "halofuginone" is defined as a
compound having the formula:
##STR00004##
and pharmaceutically acceptable salts thereof.
[0085] The phrase "pharmaceutically acceptable salt" refers to a
charged species of the parent compound and its counter ion, which
is typically used to modify the solubility characteristics of the
parent compound and/or to reduce any significant irritation to an
organism by the parent compound, while not abrogating the
biological activity and properties of the administered
compound.
[0086] Although the specific quinazolinone derivative
"halofuginone" is referred to throughout the specification, it is
understood that other quinazolinone derivatives may be used in its
place, these derivatives having the general formula:
##STR00005##
wherein: n=1-2 R.sub.1 is a member of the group consisting of
hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower
alkoxy; R.sub.2 is a member of the group consisting of hydroxy,
acetoxy and lower alkoxy; and R.sub.3 is a member of the group
consisting of hydrogen and lower alkenoxy-carbonyl, and
pharmaceutically acceptable salts thereof.
[0087] The terms "skeletal muscle fibrosis" "muscle fibrosis" and
"fibrosis" as used herein refer to a phenomenon that frequently
occurs in diseased or damaged muscle, characterized by the
excessive growth of fibrous tissue, and impairment of muscle
function.
[0088] Hereinafter, the term "a subject" refers to a human or
animal to whom halofuginone was administered.
[0089] The term "reducing the extent of" includes both
substantially preventing the process of skeletal muscle fibrosis
from starting and slowing or halting the progression of skeletal
muscle fibrosis once it has arisen.
[0090] Compounds which are intended for the inhibition of skeletal
muscle fibrosis were tested by an in vivo model for their ability
to slow or halt the pathological process leading to deposition of
fibrotic tissue. Such experiments were conducted for the collagen
type I synthesis inhibitor halofuginone, as described in greater
detail in the Examples section below.
[0091] The mdx mouse was selected as a model to evaluate the
efficacy of halofuginone in prevention and treatment of dystrophy
by inhibiting muscle fibrosis (by decreasing collagen synthesis and
increasing collagen degradation) and delaying muscle degeneration,
and in improving muscle regeneration by inhibiting myostatin
signaling.
[0092] As shown in the Examples section below, halofuginone was
shown to be highly effective in reducing the extent of skeletal
muscle fibrosis, and in improving skeletal muscle regeneration, in
both skeletal muscle and in muscles of the diaphragm.
[0093] The present invention therefore provides a pharmaceutical
composition comprising a quinazolinone derivative, such as
halofuginone, for reducing the extent of skeletal muscle fibrosis
and for improving skeletal muscle regeneration.
[0094] The pharmaceutical composition of the present invention
comprises, in addition to the quinazolinone derivative, a
pharmaceutically acceptable carrier, and may optionally further
comprise one or more pharmaceutically acceptable excipients, such
as, for example, binding agents, stabilizers, diluents,
surfactants, flavors, and odorants.
[0095] Pharmaceutically acceptable carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions.
[0096] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes. Further techniques for formulation and administration of
active ingredients may be found in "Remington's Pharmaceutical
Sciences," Mack Publishing Co., Easton, Pa., latest edition, which
is incorporated herein by reference as if fully set forth herein.
Pharmaceutical compositions for use in accordance with the present
invention thus may be formulated in conventional manner using one
or more pharmaceutically acceptable carriers comprising excipients
and auxiliaries, which facilitate processing of the active
ingredients into preparations which, can be used pharmaceutically.
Proper formulation is dependent upon the route of administration
chosen.
[0097] The pharmaceutical composition of the present invention may
be administered by any route selected from the oral, parenteral,
transdermal, intravenous, subcutaneous, intramuscular, intranasal,
intraauricular, sublingual, rectal, transmucosal, intestinal,
intraauricular, buccal, intramedullar, intrathecal, direct
intraventricular, intraperitoneal, or intraocular routes.
Preferably, administration is by the oral or parenteral routes.
[0098] Hereinafter, the term "oral administration" includes, but is
not limited to, administration by mouth for absorption through the
gastrointestinal tract, buccal administration and sublingual
administration.
[0099] For oral administration, the active ingredients can be
formulated readily by combining the active ingredients with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the active ingredients of the invention to be
formulated as tablets, pills, dragees, capsules, liquids, gels,
syrups, slurries, powders or granules, suspensions or solutions in
water or non-aqueous media, and the like, for oral ingestion by a
patient. Pharmacological preparations for oral use can be made
using a solid excipient, optionally grinding the resulting mixture,
and processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients such as thickeners, diluents, flavorings, dispersing
aids, emulsifiers, binders or preservatives may be desirable.
[0100] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active ingredient doses.
[0101] Pharmaceutical compositions, which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0102] The term "parenteral administration" includes, but is not
limited to, administration by intravenous drip or bolus injection,
subcutaneous, or intra muscular injection. Formulations for
parenteral administration may be presented in unit dosage form,
e.g., in ampoules or in multidose containers with optionally, an
added preservative. The compositions may be suspensions, solutions
or emulsions in oily or aqueous vehicles, and may contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents.
[0103] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acids esters such as
ethyl oleate, triglycerides or liposomes. Aqueous injection
suspensions may contain substances, which increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol or
dextran. Optionally, the suspension may also contain suitable
stabilizers or agents which increase the solubility of the active
ingredients to allow for the preparation of highly concentrated
solutions.
[0104] The dosage may vary depending upon the dosage form employed
and the route of administration utilized. The exact formulation,
route of administration and dosage can be chosen by the individual
physician in view of the patient's condition. (See e.g., Fingl, et
al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.
1).
[0105] Compositions of the present invention may, if desired, be
presented in a pack or dispenser device, such as an FDA approved
kit, which may contain one or more unit dosage forms containing the
active ingredient. The pack may, for example, comprise metal or
plastic foil, such as a blister pack. The pack or dispenser device
may be accompanied by instructions for administration. The pack or
dispenser may also be accompanied by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert.
[0106] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0107] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
Example 1
Collagen Content and Collagen .alpha.1 (I) Gene Expression (in the
Quadriceps and Tibialis Muscles
[0108] Mdx mice were treated either with halofuginone by I.P.
injection every other day (3 .mu.g/mouse) starting at 5 weeks of
age for 4 weeks, or with saline vehicle. C57B mice were used as
controls.
[0109] At the end of the 4 week period, tibialis anterior (fast)
and quadriceps (slow) muscles were removed for evaluation of
fibrosis by Sirius red staining for collagen content, and by in
situ hybridization of collagen .alpha.1(I) gene expression.
Example 2
Effect of Halofuginone on Fibrosis in the Diaphragm, Gastrochnemius
and Tibialis Muscles
[0110] Halofuginone was administered by I.P injection to mdx mice
(n=7) from 3 weeks of age every other day at a concentration of 5
.mu.g/mouse The wild type (C57/BL) and mdx mice without
halofuginone served as controls. At the age of 7 and 11 weeks the
mice were sacrificed and the diaphragm, gastrochnemius and the
tibialis were fixed for histology and samples were frozen for
further analysis. Sections were stained for collagen by Sirius red
and with methyl green as a counter stain. Image analysis using the
Image Pro software was performed for statistical evaluation of the
effect of halofuginone on collagen content. For the image analysis,
images were taken from 3 animals/group and 4 replicates from each
animal/tissue. The results are the ratio of red (R) to green color
(G).
Example 3
Effect of Halofuginone on Muscle Regeneration
[0111] Mdx mice were treated with halofuginone 5 .mu.g or 7.5 .mu.g
for 1 or 2 months starting at age of 3 weeks. The untreated mdx
mice served as controls. At the end of the experiment, the
diaphragms were taken for hematoxyline & eosine staining before
central nuclei counting. The results are the mean.+-.SE of 20
photos taken from 3 different mice. Each photo contained
approximately 120 fibers.
Results
[0112] As shown in FIG. 2, the quadriceps (slow) and tibialis
(fast) muscles of the C57B controls showed almost no cells
expressing the collagen .alpha.1(1) gene, and low levels of
collagen surrounding the fibers were observed. A significant
increase was observed both in the expression of collagen al (I)
gene and in the amount of collagen fibers in the mdx quadriceps and
tibialis muscles compared to the controls. This increase was almost
completely prevented by halofuginone treatment.
[0113] FIG. 3 demonstrates that in the wild-type mice, the level of
collagen was low in diaphragm, gastrochnemius and tibialis whereas
in the mdx mice, an increase in the collagen content was observed
in all muscles. The main increase was observed in the diaphragm;
this was already evident at 3 weeks of age and reached 9 and
15-fold increase compared to the wild type at 7 and 11 weeks,
respectively. In the gastrochnemius, an increase in the collagen
content was observed already at 3 weeks of age with further
increase at 7 weeks. At older ages, a decline in the collagen
content was observed although the level was still higher than that
in the wild type. Only a minute increase in collagen was observed
in the tibialis. In the diaphragm, halofuginone reduced the
collagen levels by 25% at 7 weeks (4 weeks of treatment) and by 53%
at 11 weeks (8 weeks of treatment). In the gastrochnemius,
halofuginone reduced the collagen content by 25% and 33% at 7 and
11 weeks, respectively. No effect of halofuginone on the collagen
content was observed in the tibialis muscle.
[0114] FIG. 4 shows the effect of halofuginone 5 .mu.g (purple) or
7.5 .mu.g (off white) on muscle regeneration, with untreated mdx
mice (blue) as control. In the wild type mice, the nucleus of each
muscle fiber is located at the periphery of the cell. In the mdx
mice small immature centrally nucleated fibers are observed,
reflecting muscle regeneration from myoblasts that results in a
balance between necrotic and regenerative processes in the early
phase of the disease. After halofuginone treatment a major
reduction in the number of diaphragm central nuclei was observed
that was dose-dependent. These results suggest that halofuginone
improved the muscle physiology and reduced the pressure on the mdx
mice muscle to regenerate.
DISCUSSION
[0115] In different muscular dystrophies and dystrophic syndromes,
different muscle types are affected by fibrosis to a different
extent. In the mdx mouse, which serves as a model for Duchenne
muscle dystrophy (DMD), the most affected muscle is the diaphragm
although other muscles are also affected. Consequently, the
greatest effect of halofuginone in mdx mice was shown with regard
to fibrotic diaphragm muscles, resulting in 53% inhibition of
fibrosis and of the fibrotic process.
[0116] Together, the above results suggest that halofuginone is a
potent inhibitor of muscle fibrosis in mdx mice by inhibiting
collagen type I synthesis and may inhibit the need for muscle
degeneration by inhibiting the myostatin pathway.
[0117] The above results further show that the extent of inhibition
of fibrosis by halofuginone is dependent upon the extent of
fibrosis. Halofuginone has been shown to inhibit fibrosis by
inhibition of collagen synthesis in every muscle that is affected
by fibrosis, but in muscles in which only minute increases in
collagen synthesis occur, the inhibition of collagen synthesis
caused by halofuginone may not be clearly demonstrable in the
context of the experiment, for example during the time period
tested.
[0118] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0119] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0120] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
* * * * *