U.S. patent application number 11/750128 was filed with the patent office on 2007-11-29 for avian follistatin product.
This patent application is currently assigned to Celldyne Biopharma, LLC. Invention is credited to Richard Paul GREEN III, Richard Paul SR. Green.
Application Number | 20070275036 11/750128 |
Document ID | / |
Family ID | 38749799 |
Filed Date | 2007-11-29 |
United States Patent
Application |
20070275036 |
Kind Code |
A1 |
GREEN III; Richard Paul ; et
al. |
November 29, 2007 |
AVIAN FOLLISTATIN PRODUCT
Abstract
Described herein are avian follistatin products, and methods for
producing such products, in which such products are effective for a
variety of conditions, including increasing muscle mass. Avian
follistatin products described herein are packaged as dietary
supplements or nutritional supplements are useful in muscle
regeneration.
Inventors: |
GREEN III; Richard Paul;
(San Antonio, TX) ; Green; Richard Paul SR.;
(Joplin, MO) |
Correspondence
Address: |
WILSON SONSINI GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Assignee: |
Celldyne Biopharma, LLC
San Antonio
TX
|
Family ID: |
38749799 |
Appl. No.: |
11/750128 |
Filed: |
May 17, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60895405 |
Mar 16, 2007 |
|
|
|
60801266 |
May 18, 2006 |
|
|
|
Current U.S.
Class: |
424/439 ;
424/581; 514/10.1; 514/10.2; 514/11.2; 514/16.5; 514/16.6;
514/17.9; 514/19.3; 514/3.7; 514/3.8; 514/5.5; 514/7.3; 514/8.9;
514/9.8; 514/9.9 |
Current CPC
Class: |
A23L 15/00 20160801;
A23V 2002/00 20130101; A61K 35/57 20130101; A23L 33/17 20160801;
A61K 38/1709 20130101; A61K 35/57 20130101; A61K 38/1709 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A23V 2002/00
20130101; A23V 2200/316 20130101; A23V 2250/542 20130101; A23V
2200/316 20130101 |
Class at
Publication: |
424/439 ;
514/012; 424/581 |
International
Class: |
A61K 47/00 20060101
A61K047/00; A61K 35/54 20060101 A61K035/54; A61K 38/22 20060101
A61K038/22 |
Claims
1. A sports nutrition composition comprising follistatin and avian
egg yolk membrane, wherein the avian egg yolk membrane is
substantially dehydrated and free of viable pathogens.
2. The sports nutrition composition of claim 1, wherein the avian
egg yolk membrane is from a fertilized egg.
3. The sports nutrition composition of claim 1 comprising avian egg
yolk.
4. The sports nutrition composition of claim 1, wherein the avian
egg yolk membrane is from a chicken egg, duck egg, or ostrich
egg.
5. The sports nutrition composition of claim 4, wherein the avian
egg yolk membrane is from a chicken egg.
6. The sports nutrition composition of claim 1, further comprising
a pharmaceutically acceptable ingredient.
7. A method for increasing muscle mass, comprising administering to
a subject the sports nutrition composition of claim 1.
8. A process for making a follistatin-containing sports nutrition
composition, the method comprising sterilizing an avian egg yolk or
avian egg yolk membrane, wherein (i) the avian egg yolk or avian
egg yolk membrane is from a fertilized avian egg; or (ii) the avian
egg yolk or avian egg yolk membrane is supplemented with a
composition containing follistatin.
9. The process of claim 8, wherein the avian egg yolk or avian egg
yolk membrane is from a fertilized avian egg.
10. The process of claim 8, wherein the avian egg yolk is
dehydrated prior to the sterilization.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of a provisional patent
application filed on May 18, 2006, entitled "Fertilized egg protein
powder (naturally occurring follistatin-based myostatin inhibitor
produced in the form of a desiccated fertile egg protein powder for
human or animal consumption) and naturally occurring follistatin
extracted, isolated, and purified from fertile eggs." This
application also claims the benefit of Provisional Patent
Application No. 60/895,405, filed Mar. 16, 2007, entitled "Process
for the Preparation of Avian Follistatin Product." The contents of
these applications are incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] Described herein are products containing follistatin,
methods for preparing such products, methods for formulating such
products and methods of using such products for the regulation of
muscle growth.
BACKGROUND OF THE INVENTION
[0003] Wasting of skeletal muscle is a serious health condition
that accompanies many conditions, diseases or disorders. Wasting of
skeletal muscle also accompanies ageing. One of the most
devastating but least-discussed aspects of age-related decline is
the onset of frailty, i.e., the progressive loss of robustness in
multiple tissues and organ systems. On the other side of the scale,
many athletes benefit from an increase in muscle mass.
SUMMARY OF THE INVENTION
[0004] Strategies for muscle regeneration that lead to increased
body mass are needed. Described herein are products, methods of
making such products, and uses of such products to facilitate
muscle regeneration and increase muscle mass. Processes for the
preparation of avian follistatin product (non-albumen fecundated
avian ovum decoction standardized to specific ng/ml follistatin)
are described.
[0005] Described herein are compositions comprising follistatin and
an egg product. In some embodiments, the composition is a sports
nutrition product; in other embodiments, a dietary supplement
product; in other embodiments, a nutraceutical product; in other
embodiments, a health nutrition product; in other embodiments, an
over-the-counter drug product; in other embodiments, a prescription
drug product; and in other embodiments, a veterinarian (pet or
livestock) drug product.
[0006] In a further embodiment, the egg product is derived from an
avian egg. In further embodiments, the avian egg is a fertilized
avian egg. In a further embodiment, the fertilized avian egg is
from a domestic fowl selected from a turkey, chicken, duck, goose
and ostrich. In a further embodiment, the egg product is a
lyophilized, fertilized egg product. In a further embodiment, the
egg product is substantially free from egg white. In a further
embodiment, the egg product portion of the composition is from egg
yolk. In a further or alternative embodiment, the egg product
portion of the composition is from egg yolk membrane. In a further
or alternative embodiment, the composition has been sterilized from
a process comprising use of antibacterial agents, use of
sterilizing solvent (including ethanol), use of electron beam
irradiation, use of gamma irradiation, use of pasteurization, use
of x-ray irradiation, use of filtration, use of membrane
separation, or a combination thereof.
[0007] In a further embodiment, the egg product portion has been
enriched with follistatin by a protein enhancement procedure
selected from chromatography, protein extraction with solvents, ion
exchange, ultrafiltration, microfiltration, or combinations
thereof.
[0008] In a further embodiment, the levels of follistatin are
approximated or determined in the composition using a method
selected from Bradford protein assay, gel electrophoresis, mass
spectrometry, microscopy, protein immunostaining,
immunoprecipitation, a Western blot, spectrophotometry, an enzyme
assay, ELISA, follistatin standardization, or combinations
thereof.
[0009] In a further embodiment, the composition has been subjected
to a procedure selected from lipid removal, carbohydrate removal,
other protein removal (other than follistatin), sulfonimide
removal, or combinations thereof. In a further embodiment,
additional follistatin has been added to the composition (i.e.,
other than follistatin derived from the egg product). In a further
embodiment, the composition is primarily follistatin and the other
egg product portion is present in less than 50% by weight, less
than 40% by weight, less than 30% by weight, less than 20% by
weight, less than 10% by weight, less than 5% by weight and less
than 1% by weight.
[0010] In a further embodiment, the composition is used for at
least one of the following purposes: athletic performance
enhancement, as a protease inhibitor, to prevent muscle atrophy,
for anti-aging purposes, as an HIV medication, for the treatment of
Cushings disease, for the treatment of cachexia, for the prevention
of muscle wasting, for the build-up of muscle mass; for the
treatment of Addison's disease, muscular dystrophy, multiple
sclerosis, type I diabetes, type II diabetes, rheumatoid arthritis,
Crohn's disease, ulcerative colitis, irritable bowel syndrome,
other autoimmune disorders, burns, joint injury, immobilization,
coma, dislocations, gastrointestinal disorders, cancer, HIV, AIDS,
sarcopenia, spinal cord damage, nerve damage, TGF alpha and beta
disorders, ALS, vision disorders, weak muscles, ankolosing
spondilitis, anorexia, bulimia, corticosteroid use, growth factor
disorders, dysphasia, SIV, body dysmorphia, low androgen levels,
menstruation problems, infertility, hypo- or hyper-thyroidism,
catacholamine surge, low FSH, low LH, low GRH, low HCG, and viral
infections; in conjunction with surgery and/or joint replacement
operations; for the improvement of weight and/or muscle mass in
livestock; to raise follistatin levels in a mammal (including a
human); to lower myostatin levels in a mammaol (including a human);
and combinations thereof.
[0011] In a further aspect, the composition is in the form of a
powder, a tablet, a capsule, an implant, an aerosol, a pudding, a
gel, a paste, a syrup, a suspension, an enteric-coated product. In
other embodiments, the composition is suitable for oral
administration, nasal administration, inhalation administration,
buccal administration, sub-buccal administration, intramuscular
administration, intravenous administration, subcutaneous
administration, transdermal administration, or viral delivery. In
further embodiments, the composition, is micro-encapsulated,
nano-encapsulated, in a vial, suitable for Monoject administration,
suitable for Rediject administration, or suitable for air
injection. In a further embodiment, the composition is in the form
of a powder that can be mixed into a liquid to provide a drinkable
suspension. By way of example only, the powder can be mixed into
and suspended in milk, water, juice or other liquid, to form a
shake or mixed drink.
[0012] In one aspect are fertilized egg protein powders and to
naturally occurring follistatin extracted, isolated, and purified
from fertile eggs, i.e., a naturally occurring follistatin-based
myostatin inhibitor produced in the form of a desiccated fertile
egg protein powder for use. The powder is used as a dietary
supplement, sports ergogenic, novel therapy for metabolic
conditions, diseases with application to worldwide markets, wasting
diseases, obesity, and/or obesity-related conditions. Product may
be used in food, supplement, and/or encapsulated variations.
Extracted, isolated, and purified follistatin may be used as a
pharmaceutical through oral, intravenous, or sustained-release
dosing.
[0013] In another aspect is a naturally occurring follistatin-based
myostatin inhibitor produced in the form of a desiccated fertile
egg protein powder for use, including by not limited to, as a
dietary supplement, sports ergogenic, novel therapy for metabolic
conditions, diseases with application to worldwide markets, wasting
diseases, obesity, and/or obesity-related conditions.
[0014] In another aspect is the use of fertilized egg powder for
producing products such as protein shake product containing
bioactive peptides, or other products. Additionally, the
follistatin producing membranes may be mechanically separated from
the other egg components. The membranes may then be further
processed to be used in the form of food supplements that contain
bioactive peptides, or other products. Naturally occurring
follistatin may be separated, isolated, and purified to be used as
a therapeutic biopharmaceutical agent for the treatment of muscle
wasting (cachexia) and other diseases.
[0015] In other embodiments, the amount of follistatin in the
composition is at least 1 ng/mg of composition, 2 ng/mg of
composition, 4 ng/mg of composition, 5 ng/mg of composition, 7
ng/mg of composition, 10 ng/mg of composition, 15 ng/mg of
composition, 20 ng/mg of composition, 35 ng/mg of composition, 50
ng/mg of composition, 75 ng/mg of composition, 100 ng/mg of
composition, 200 ng/mg of composition, 500 ng/mg of composition,
1000 ng/mg of composition, 2000 ng/mg of composition, 5000 ng/mg of
composition, 10,000 ng/mg of composition, 20,000 ng/mg of
composition, 50,000 ng/mg of composition, or 100,000 ng/mg of
composition.
[0016] In one aspect is a composition comprising follistatin and
avian egg yolk membrane, wherein the avian egg yolk membrane is
substantially dehydrated and free of viable pathogens. In one
embodiment, the avian egg yolk membrane is from a fertilized egg.
In another embodiment, the composition comprises avian egg yolk. In
a further embodiment, the avian egg yolk membrane is from a chicken
egg, duck egg, or ostrich egg. In a further embodiment, the
composition also comprises acceptable excipients.
[0017] In another aspect any of the aforementioned composition
further comprise a component selected from whey protein, egg
protein, casein, creatine, amino acids, BCAAs, carbohydrates,
lipids, insulin, insulin mimics, insulin resistance products,
pro-hormones, hormones, prosteroids, steroids, 7-keto DHEA, DHEA,
NO enhancers, estrogen inhibitors, caffeine, stimulants, ephedrine
alkaloids, vitamins, colustrum, recombinant antibodies, antibodies,
growth factors, growth hormone releasing peptides, IGF1, mechano
growth factor, mannitol, DMSO, or combinations thereof.
[0018] In another aspect are methods for increasing muscle mass,
comprising administering to a subject any of the aforementioned
compositions.
[0019] In another aspect is a process for making a
follistatin-containing composition, the method comprising
sterilizing an avian egg yolk or avian egg yolk membrane, wherein
(i) the avian egg yolk or avian egg yolk membrane is from a
fertilized avian egg; or (ii) the avian egg yolk or avian egg yolk
membrane is supplemented with a composition containing
follistatin.
[0020] In a further embodiment, the avian egg yolk or avian egg
yolk membrane is from a fertilized avian egg. In a further
embodiment, the avian egg yolk is dehydrated prior to the
sterilization.
[0021] In another aspect is a process for the preparation of avian
follistatin product. The avian follistatin product is a non-albumen
fecundated avian ovum decoction standardized to specific ng/ml
follistatin.
[0022] In one aspect is a method for the preparation of avian
follistatin product that includes: [0023] a) collecting fertilized
avian egg(s); [0024] b) cracking the collected fertilized avian
egg(s) and separating the egg yolk from egg white; [0025] c)
emulsifying the egg yolk; [0026] d) lyophilizing the emulsified egg
yolk to obtain a protein powder; and [0027] e) irradiating the
protein powder obtained post lyophilization with e-beam or gamma
radiation.
[0028] In a further embodiment, the process includes removal of a
portion of the lipids and the carbohydrate materials from the
protein powder.
[0029] In one embodiment, the method involves adding addition water
during step c).
[0030] In one embodiment, the avian follistatin product is not
pasteurized.
[0031] In one embodiment, the fertilized avian eggs are chicken
eggs.
[0032] In aspect is a composition, comprising an avian follistatin
product prepared by the method described herein. In one embodiment,
the composition further includes excipients, binders or carriers.
In one embodiment, the composition is in the form of a powder.
[0033] In one aspect, provided is a method for increasing muscle
mass, comprising administering to a patient an avian follistatin
product. In further embodiment, the administered dose (in reference
to any of the aforementioned compositions) is between 10 and 2000
grams of the raw form (containing lipids and/or carbohydrates),
between 20 and 1000 grams of the raw form, between, 50 and 900
grams of the raw form, between 100 and 800 grams of the raw form,
between 300 and 800 grams of the raw form, between 500 and 800
grams of the raw form, or about 750 grams of the raw form. In
further embodiment, the administered dose (in reference to any of
the aforementioned compositions) is between 1 and 200 grams of the
lipid and carbohydrate free form, between 2 and 90 grams of the
lipid and carbohydrate free form, between, 5 and 80 grams of the
lipid and carbohydrate free form, between 10 and 75 grams of the
lipid and carbohydrate free form, between 20 and 70 grams of the
lipid and carbohydrate free form, between 30 and 70 grams of the
lipid and carbohydrate free form, or about 60 grams of the lipid
and carbohydrate free form.
[0034] In another embodiment, provided is the use of the avian
follistatin product described herein for the formulation of a
nutritional supplement or a dietary supplement.
[0035] Articles of manufacture, comprising packaging material, an
avian follistatin product, which is effective for increasing muscle
mass or inhibiting the activity of myostatin, within the packaging
material, and a label that indicates that the product is used for
increasing muscle mass or for the inhibition of myostatin activity,
are provided.
[0036] In some embodiments, formulations described herein are
administered to a human.
[0037] In some embodiments, formulations described herein are
orally administered.
[0038] Other objects, features and advantages of the methods,
compounds, and compositions described herein will become apparent
from the following detailed description. It should be understood,
however, that the detailed description and the specific examples,
while indicating specific embodiments, are given by way of
illustration only, since various changes and modifications within
the spirit and scope of the disclosure will become apparent to
those skilled in the art from this detailed description. All
references cited herein, including patents, patent applications,
and publications, are hereby incorporated by reference in their
entirety.
INCORPORATION BY REFERENCE
[0039] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
DETAILED DESCRIPTION
[0040] Wasting of skeletal muscle is not only the hallmark of many
muscle disorders, but it also accompanies immobility and chronic
diseases such as kidney failure, tumor cachexia and in a subset of
patients with AIDS. Furthermore, decline in muscle mass is a
feature of normal ageing and precipitates health problems secondary
to a reduction in mobility.
[0041] Loss of muscle mass is a problem that accompanies many
disorders and diseases, such as wasting disorders, traumatic
disorders, inflammatory disorders, degenerative disorders, and the
like. The treatments of the various disorders where muscle mass is
lost usually do not improve and hasten muscle regeneration. Method
of improving muscle regeneration are lacking and needed.
[0042] Methods that can stimulate muscle growth and prevent muscle
loss are likely to benefit a significant proportion of the
population. It may be possible to treat muscle-wasting disorders
not by addressing the primary causative agent or insult, but simply
by enhancing the muscle development programme.
[0043] In some embodiments, muscle regeneration may be sought for
those individuals who do not suffer from muscle wasting or from
disorders, diseases or conditions that cause muscle wasting. In
some embodiments, muscle regeneration may be sought for performance
enhancement. In other embodiments, muscle regeneration may be
desirable for athletes for increasing performance. In other
embodiments, muscle regeneration may be desirable for those who
have low muscle mass.
[0044] One approach for muscle regeneration is through myostatin
inhibition (Patel et al. "The function of Myostatin and strategies
of Myostatin blockade--new hope for therapies aimed at promoting
growth of skeletal muscle" Neuromuscular Disorders 15 (2005)
117-126).
[0045] Myostatin is an endogenous, negative regulator of muscle
growth. Myostatin is a growth factor that limits muscle tissue
growth, i.e. higher concentrations of myostatin in the body cause
the individual to have less developed muscles. Myostatin,
originally named growth differentiation factor 8, was identified
through its homology to other members of the transforming growth
factor-b (TGF-b) superfamily.
[0046] Human myostatin consists of two identical subunits, each
consisting of 110 amino acid residues. Its total molecular weight
is 25.0 kDa. The myostatin protein is produced in the skeletal
muscle cells, circulates in the blood and acts on muscle tissue by
apparently slowing down the development of muscle stem cells. It is
expressed almost exclusively in developing and mature skeletal
muscle. The precise mechanism remains unknown. Myogenic cells
respond to Myostatin by down-regulating the expression of key
transcriptional regulators of muscle development such as Pax-3,
MyoD and Myf-5, which inhibit differentiation and further growth of
muscle.
[0047] Genetically engineered mice lacking myostatin have a
dramatic hypermuscular phenotype. Homozygous myostatin null mice
are approximately 30% larger than their littermates, due entirely
to diffusely increased muscle mass.
[0048] Other animal models with altered myostatin function confirm
the importance of myostatin in postnatal muscle growth and reveal
that the degree of muscle fiber hypertrophy and/or hyperplasia is
in part secondary to the timing and mode of myostatin inhibition
during development and postnatal life. These characteristics of
myostatin deficient animals indicate that myostatin normally
functions as a negative regulator of muscle growth.
[0049] Follistatin is a secreted glycoprotein that inhibits the
activity of a number of TGF-b family members. There is significant
data that follistatin is an in vivo inhibitor of myostatin (Armthor
et al., "Follistatin complexes Myostatin and antagonizes
Myostatin-mediated inhibition of myogenesis" Dev Biol. 2004,
270:19-30). Follistatin is a secreted protein that binds and
antagonizes the function of numerous members of the TGF-b family.
Deletion of the follistatin gene led to muscle loss (Matzuk M M, et
al., "Multiple defects and perinatal death in mice deficient in
follistatin." Nature 1995; 374:360-3), whereas over-expression
resulted in excessive muscle growth, (Lee S J, et al., "Regulation
of Myostatin activity and muscle growth." Proc Natl Acad Sci USA
2001; 98:9306-11). The ability of follistatin to antagonize
myostatin was shown by simultaneously over-expressing both
molecules in adult skeletal muscle, which prevented myostatin's
ability to induce muscle loss, and recently it has been shown that
follistatin and myostatin interact directly with high affinity
(Amthor et al. Follistatin complexes Myostatin and antagonizes
Myostatin-mediated inhibition of myogenesis. Dev Biol 2004;
270:19-30).
[0050] Targeted over-expression of follistatin in skeletal muscle
increased the weight of individual muscle of transgenic mice by
327% (Lee S J, et al., Regulation of Myostatin activity and muscle
growth. Proc Natl Acad Sci USA 2001; 98:9306-11). Muscle
enlargement resulted through a combination of hyperplasia (66%
increase) and hypertrophy (27%). The increase in muscle mass was
far greater than that observed following the deletion of the
myostatin gene (McPherron A C, et al., Regulation of skeletal
muscle mass in mice by a new TGF-beta superfamily member. Nature
1997; 387:83-90). One possible explanation for these differences is
that follistatin is likely to antagonize molecules in addition to
myostatin that also act as muscle growth inhibitors.
[0051] Follistatin has been identified in the yolks of fertilized
avian eggs and not in the eggs of unfertilized eggs. Fertilized
avian eggs may be obtained in abundance and techniques are known in
the art for processing proteins from avian eggs. For example,
fertilized eggs may be obtained from a when, optionally stored in
refrigerator or cold room for up to three weeks, the eggs are
cracked, the yolks are separated and then processed by methods
known in the art, such as lyophilization or spray drying, to
provide a protein powder that is enriched with follistatin. The
follistatin enriched protein powder may be subjected to electron
beam or gamma irradiation for sterilization of the product.
[0052] Described herein are compositions that contain follistatin
derived from fertilized avian eggs. Compositions described herein
that contain follistatin are natural forms of follistatin and are
not synthetic. Compositions described herein that contain
follistatin may be formulated into a variety of formulations
depending on the intended mode of administration.
[0053] Described herein are methods for the preparation of
compositions that contain follistatin from avian eggs. Methods
described herein for the preparation of compositions that contain
follistatin are easily carried out and applicable on an industrial
scale.
Use of Follistatin Products
[0054] Follistatin containing products may be administered to those
individuals desiring or needing muscle regeneration or increases in
muscle mass. In some situations, follistatin containing products
may be consumed by athletes desiring an increase in muscle mass. In
other situations, follistatin products may be given to the elderly,
in whom muscle mass has been lost due to ageing. In other
situations, muscle mass may have been lost due to health
conditions, diseases or disorders and thus follistatin containing
products may be beneficial to these individuals. In some other
situations, follistatin containing products may be beneficial for
those individuals that satisfy a number of the aforementioned
conditions or situations.
Muscle Disorders
[0055] Follistatin compositions may be used in the treatment of
muscle disorders, where increases in muscle mass are desired.
Muscular Dystrophies
[0056] It has been shown that myostatin blockade stimulates growth
and decreases fibrosis of dystrophic muscle (K. Patel, et al.,
Neuromuscular Disorders 15 (2005) 117-126). Thus, follistatin may
be used to stimulate muscle growth in muscular dystrophies.
Disuse Muscle Atrophy
[0057] Skeletal muscle will undergo rapid atrophy when its normal
workload is reduced and interestingly, myostatin expression has
been reported to be elevated in the muscle of patients who were
bedridden for prolonged periods (Reardon K A, et al., Muscle Nerve
2001; 24:893-9). Muscle mass loss and atrophy also results from the
process of ageing. Thus, follistatin may be used in situations
where disuse muscle atrophy has occurred.
Steroid Myopathy
[0058] Glucocorticoids can induce muscle atrophy by inhibiting
protein synthesis and promoting protein degradation. Administration
of high doses of glucocorticoids that induce muscle atrophy have
resulted in an up-regulation of myostatin expression in skeletal
muscle [Ma K, et al., "Glucocorticoid-induced skeletal muscle
atrophy is associated with upregulation of Myostatin gene
expression" Am J Physiol Endocrinol Metab 2003; 285:E363-E71).
Thus, follistatin may be used in steroid myopathy.
AIDS Wasting Syndrome
[0059] Serum myostatin and local myostatin levels in skeletal
muscle of patients suffering from AIDS wasting syndrome have been
characterized (Gonzalez-Cadavid N F, et al., Proc Natl Acad Sci USA
1998; 95: 14938-43). Patients with less than 10% weight loss during
the past 6 month showed higher myostatin serum levels compared to
healthy individuals, and patients with more than 10% weight loss
showed higher levels compared to patients with less than 10% weight
loss. Additionally, muscle from patients suffering from AIDS
wasting syndrome contained higher levels of myostatin protein than
muscle from healthy individuals. These results pointed to a
correlation between muscle loss and myostatin levels in humans.
Thus, follistatin may be used in aids wasting syndrome
(cachexia).
[0060] Other conditions, disorders, diseases, and/or states where
increases in muscle mass are needed or desired, follistatin
products disclosed herein may be used.
[0061] Disclosed herein are nutritional and dietary supplements in
the form of a fertilized egg protein powder or an extract of
follistatin that has been isolated and purified from fertile
eggs.
[0062] Disclosed herein are methods for the preparation of
nutritional and dietary supplements that contain follistatin from
fertilized avian eggs.
Method for the Preparation of Fertilized Egg Protein Powder
[0063] Fertilized avian eggs are obtained from hens. The eggs are
collected and stored in a refrigerator or cold room (at or lower
than 4.degree. C. (40.degree. F.)) until they are ready to be
processed. The fertilized eggs are used within 3 weeks.
[0064] The following methods for the preparation of avian
follistatin protein powder may be carried out using whole egg yolk
or egg yolk membrane.
[0065] The processing of fertilized avian eggs is conducted using
equipment and facilities that meet United States Department of
Agriculture (USDA) standards for egg processing plants.
[0066] Large volumes of fertilized eggs may be processed by the
following techniques. Fertilized eggs are placed on conveyor belts,
washed, and then passed over a bright light source. This process is
called candling. Usually blood, if present, can be observed and the
egg is removed by attendants. Electronic sensing equipment may also
be used to detect blood spots. The clean eggs are then
automatically placed into an egg-cracking machine. Such a machine
consists of a series of individual egg holders that crack the egg,
separate the yolk and white, and dump each component into a
separate pipe. Such a machine can process thousands of eggs an
hour, and operators typically monitor the machine to detect eggs
which do not separate properly or contain blood spots. If such a
situation arises, the attendants may discard the eggs that do not
separate properly or contain blood spots.
[0067] The combined egg yolks, or egg yolk membranes, are then
homogenized or agitated strongly to form an emulsion. The agitation
may be carried out by means of a mechanical stirrer. If desired,
water may be added to the homogenizer. The emulsified egg yolks are
then transferred to a flask or other suitable container that has an
adapter for connecting to a high vacuum apparatus (such as a
mechanical oil pump). The yolk emulsion is frozen. A vacuum is
applied at any stage, before, during or after the yolks are
frozen.
[0068] The follistatin powder that is obtained after lyophilization
is complete is transferred to any suitable container.
[0069] The follistatin protein powder that is obtained is not
pasteurized.
[0070] The follistatin protein powder is then irradiated with Ebeam
or gamma irradiation at levels of at least 27.5 kilo grades. The
product, post lyophilization and irradiation, is pathogen tested to
CDER and CBER standards.
[0071] Quality assurance is then conducted to determine the
follistatin content of the protein powder. In some embodiments, the
protein powder is analyzed using mass spectrometric methods and/or
ELISA.
[0072] The follistatin protein powder is then processed by methods
known in the art or as described herein and stored. The final
formulation is in compliance with standards set forth by government
agencies, such as the Food and Drug Administration (FDA).
[0073] The final form of the product is capsulated, bottled, gel
packed and or otherwise contained/delivered to FDA standards.
Certain Terminology
[0074] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the claimed subject matter belongs. In
the event that there are a plurality of definitions for terms
herein, those in this section prevail. Where reference is made to a
URL or other such identifier or address, it is understood that such
identifiers can change and particular information on the internet
can come and go, but equivalent information can be found by
searching the internet. Reference thereto evidences the
availability and public dissemination of such information.
[0075] It is to be understood that the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of any subject matter
claimed. In this application, the use of the singular includes the
plural unless specifically stated otherwise. It must be noted that,
as used in the specification and the appended claims, the singular
forms "a," "an" and "the" include plural referents unless the
context clearly dictates otherwise. In this application, the use of
"or" means "and/or" unless stated otherwise. Furthermore, use of
the term "including" as well as other forms, such as "include",
"includes," and "included," is not limiting.
[0076] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
the application including, but not limited to, patents, patent
applications, articles, books, manuals, and treatises are hereby
expressly incorporated by reference in their entirety for any
purpose.
[0077] It is to be understood that the methods and compositions
described herein are not limited to the particular methodology
described herein and as such may vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope
of the methods, compositions described herein.
[0078] "Biologically-acceptable" refers to being safe for human
consumption.
[0079] "Avian" refers to the class of animals named "birds". All
birds lay amniotic eggs and thus eggs from any bird is contemplated
for use in the methods and compositions described herein. In some
embodiments, eggs are obtained from, but not limited to, domestic
chicken, geese, pheasants, turkeys, ducks, emus, ostriches,
pigeons, grouse, quails, doves, woodcocks, songbirds, finches. In
some embodiments, avian refers to, chicken, duck, goose, ostrich,
and/or turkey. In some embodiments, avian refers to chicken.
[0080] As used herein, amelioration of the symptoms of a particular
disease, disorder or condition by administration of a particular
composition (e.g. containing follistatin) refers to any lessening
of severity, delay in onset, slowing of progression, or shortening
of duration, whether permanent or temporary, lasting or transient
that can be attributed to or associated with administration of the
compound or composition.
[0081] The term "modulate," as used herein, means to interact with
a target either directly or indirectly so as to alter the activity
of the target, including, by way of example only, to inhibit the
activity of the target, to limit the activity of the target, or to
reduce the activity of the target. In some embodiments, the target
is myostatin.
[0082] As used herein, the term "target activity" refers to a
biological activity capable of being modulated. Certain exemplary
target activities include but are not limited to, muscle
regeneration.
[0083] As used herein, the term "antagonist" or "antagonize" refers
to a compound, the presence of which results in a decrease in the
magnitude of a biological activity of a protein. In certain
embodiments, the presence of an antagonist results in complete
inhibition of a biological activity of a protein, such as, for
example, myostatin.
[0084] The term "acceptable" with respect to a formulation,
composition or ingredient, as used herein, means having no
persistent detrimental effect on the general health of the subject
being treated.
[0085] By "pharmaceutically acceptable," as used herein, refers a
material, such as a carrier or diluent, which does not abrogate the
biological activity or properties of the compound, and is
relatively nontoxic, i.e., the material may be administered to an
individual without causing undesirable biological effects or
interacting in a deleterious manner with any of the components of
the composition in which it is contained.
[0086] "Bioavailability" refers to the percentage of the weight of
follistatin disclosed herein, that is delivered into the general
circulation of the animal or human being studied. The total
exposure (AUC(0-.infin.)) of a drug when administered intravenously
is usually defined as 100% bioavailable (F %). "Oral
bioavailability" refers to the extent to follistatin is absorbed
into the general circulation when the formulation is taken orally
as compared to intravenous injection.
[0087] "Blood plasma concentration" refers to the concentration of
follistatin disclosed herein, in the plasma component of blood of a
subject.
[0088] A measurable serum concentration or measurable plasma
concentration describes the blood serum or blood plasma
concentration, typically measured in mg, .mu.g, or ng of
therapeutic agent per ml, dl, or 1 of blood serum, absorbed into
the bloodstream after administration. As used herein, measurable
plasma concentrations are typically measured in ng/ml or
.mu.g/ml.
[0089] "Pharmacodynamics" refers to the factors which determine the
biologic response observed relative to the concentration of drug at
a site of action.
[0090] "Pharmacokinetics" refers to the factors which determine the
attainment and maintenance of the appropriate concentration of drug
at a site of action.
[0091] "Absorption" refers to the process of movement of
follistatin from site of administration across a barrier into a
blood vessel or the site of action, e.g., follistatin moving from
the gastrointestinal tract into the portal vein or lymphatic
system.
[0092] The terms "effective amount" or "therapeutically effective
amount," as used herein, refer to a sufficient amount of an agent,
or composition being administered which will relieve to some extent
one or more of the symptoms of the disease, disorder or condition
being treated. For example, an "effective amount" for therapeutic
uses is the amount of the avian follistatin product described
herein required to provide a clinically significant increase in
muscle mass. An appropriate "effective" amount in any individual
case may be determined using techniques known in the art.
Examples of Compositions and Methods of Administration
[0093] The compositions described herein can be formulated for
administration to a subject via any conventional means including,
but not limited to, oral, or buccal. As used herein, the term
"subject" is used to mean an animal, preferably a mammal, including
a human or non-human. The terms patient and subject may be used
interchangeably.
[0094] Moreover, the follistatin containing compositions described
herein, can be formulated into any suitable dosage form, including
but not limited to, aqueous oral dispersions, liquids, gels,
syrups, elixirs, slurries, suspensions and the like, for oral
ingestion by an individual in need, solid oral dosage forms,
controlled release formulations, fast melt formulations,
effervescent formulations, lyophilized formulations, tablets,
powders, pills, dragees, capsules, delayed release formulations,
aqueous liquid dispersions, self-emulsifying dispersions, solid
solutions, liposomal dispersions, solid dosage forms, powders,
tablets, capsules, pills, delayed release formulations.
[0095] Formulations for oral use can be obtained by mixing one or
more solid excipient with one or more of the compounds described
herein, 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
include, for example, fillers such as sugars, including lactose,
sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methylcellulose, microcrystalline
cellulose, hydroxypropylmethylcellulose, sodium
carboxymethylcellulose; or others such as: polyvinylpyrrolidone
(PVP or povidone) or calcium phosphate. If desired, disintegrating
agents may be added, such as the cross-linked croscarmellose
sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0096] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol
gel, polyethylene glycol, and/or 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 compound doses.
[0097] Formulations 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 can contain the active ingredients in admixture
with filler such as lactose, binders such as starches, and/or
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active compounds 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 such administration.
[0098] In some embodiments, the solid dosage forms disclosed herein
may be in the form of a tablet, (including a suspension tablet, a
fast-melt tablet, a bite-disintegration tablet, a
rapid-disintegration tablet, an effervescent tablet, or a caplet),
a pill, a powder (including a sterile packaged powder, a
dispensable powder, or an effervescent powder) a capsule (including
both soft or hard capsules, e.g., capsules made from animal-derived
gelatin or plant-derived HPMC, or "sprinkle capsules"), solid
dispersion, solid solution, pellets, granules. In other
embodiments, the pharmaceutical formulation is in the form of a
powder. In still other embodiments, the pharmaceutical formulation
is in the form of a tablet. Additionally, formulations described
herein may be administered as a single capsule or in multiple
capsule dosage form. In some embodiments, the formulation is
administered in two, or three, or four, capsules or tablets.
[0099] Soft gel or soft gelatin capsules may be prepared, for
example, without limitation, by dispersing the formulation in an
appropriate vehicle (vegetable oils are commonly used) to form a
high viscosity mixture. This mixture is then encapsulated with a
gelatin based film using technology and machinery known to those in
the soft gel industry. The industrial units so formed are then
dried to constant weight.
[0100] In some embodiments, the formulations may include other
medicinal or pharmaceutical agents, carriers, diluents, dispersing
agents, suspending agents, thickening agents, adjuvants, such as
preserving, stabilizing, wetting or emulsifying agents, solution
promoters, and/or buffers. In addition, the formulations can also
contain other therapeutically valuable substances.
[0101] The formulations described herein can include follistatin
product and one or more pharmaceutically acceptable additives such
as a compatible carrier, binder, filling agent, suspending agent,
flavoring agent, sweetening agent, disintegrating agent, dispersing
agent, surfactant, lubricant, colorant, diluent, solubilizer,
moistening agent, plasticizer, stabilizer, penetration enhancer,
wetting agent, anti-foaming agent, antioxidant, preservative, or
one or more combination thereof. In still other aspects, using
standard coating procedures, a film coating is provided around the
formulation of the compound described herein. In one embodiment,
some or all of the particles of the compound described herein are
coated. In another embodiment, some or all of the particles of the
compound described herein are microencapsulated. In still another
embodiment, the particles of the compound described herein are not
microencapsulated and are uncoated.
[0102] In certain embodiments, compositions may also include one or
more pH adjusting agents or buffering agents, including acids such
as acetic, boric, citric, lactic, phosphoric and hydrochloric
acids; bases such as sodium hydroxide, sodium phosphate, sodium
borate, sodium citrate, sodium acetate, sodium lactate and
tris-hydroxymethylaminomethane; and buffers such as
citrate/dextrose, sodium bicarbonate and ammonium chloride. Such
acids, bases and buffers are included in an amount required to
maintain pH of the composition in an acceptable range.
[0103] In other embodiments, compositions may also include one or
more salts in an amount required to bring osmolality of the
composition into an acceptable range. Such salts include those
having sodium, potassium or ammonium cations and chloride, citrate,
ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or
bisulfite anions; suitable salts include sodium chloride, potassium
chloride, sodium thiosulfate, sodium bisulfite and ammonium
sulfate.
[0104] Formulations including avian follistatin product described
herein may be manufactured in a conventional manner, such as, by
way of example only, by means of conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping or compression processes.
[0105] "Antifoaming agents" reduce foaming during processing which
can result in coagulation of aqueous dispersions, bubbles in the
finished film, or generally impair processing. Exemplary
anti-foaming agents include silicon emulsions or sorbitan
sesquoleate.
[0106] "Antioxidants" include, for example, butylated
hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium
metabisulfite and tocopherol. In certain embodiments, antioxidants
enhance chemical stability where required.
[0107] In certain embodiments, compositions provided herein may
also include one or more preservatives to inhibit microbial
activity. Suitable preservatives include mercury-containing
substances such as merfen and thiomersal; stabilized chlorine
dioxide; and quaternary ammonium compounds such as benzalkonium
chloride, cetyltrimethylammonium bromide and cetylpyridinium
chloride.
[0108] Formulations described herein may benefit from antioxidants,
metal chelating agents, thiol containing compounds and other
general stabilizing agents. Examples of such stabilizing agents,
include, but are not limited to: (a) about 0.5% to about 2% w/v
glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1%
to about 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM
EDTA, (e) about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to
about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v.
polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k)
cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m)
divalent cations such as magnesium and zinc; or (n) combinations
thereof.
[0109] "Binders" impart cohesive qualities and include, e.g.,
alginic acid and salts thereof; cellulose derivatives such as
carboxymethylcellulose, methylcellulose (e.g., Methocel.RTM.),
hydroxypropylmethylcellulose, hydroxyethylcellulose,
hydroxypropylcellulose (e.g., Klucel.RTM.), ethylcellulose (e.g.,
Ethocel.degree.), and microcrystalline cellulose (e.g.,
Avicel.RTM.); microcrystalline dextrose; amylose; magnesium
aluminum silicate; polysaccharide acids; bentonites; gelatin;
polyvinylpyrrolidone/vinyl acetate copolymer; crosspovidone;
povidone; starch; pregelatinized starch; tragacanth, dextrin, a
sugar, such as sucrose (e.g., Dipac.RTM.), glucose, dextrose,
molasses, mannitol, sorbitol, xylitol (e.g., Xylitab.RTM.), and
lactose; a natural or synthetic gum such as acacia, tragacanth,
ghatti gum, mucilage of isapol husks, polyvinylpyrrolidone (e.g.,
Polyvidone.RTM. CL, Kollidon.RTM. CL, Polyplasdone.RTM. XL-10),
larch arabogalactan, Veegum.RTM., polyethylene glycol, waxes,
sodium alginate, and the like.
[0110] The term "carrier," as used herein, refers to relatively
nontoxic chemical compounds or agents that facilitate the
incorporation of a compound into cells or tissues, and include,
e.g., binders, suspending agents, disintegration agents, filling
agents, surfactants, solubilizers, stabilizers, lubricants, wetting
agents, diluents, and the like.
[0111] "Dispersing agents," and/or "viscosity modulating agents"
include materials that control the diffusion and homogeneity of a
compound through liquid media or a granulation method or blend
method. In some embodiments, these agents also facilitate the
effectiveness of a coating or eroding matrix. Exemplary diffusion
facilitators/dispersing agents include, e.g., hydrophilic polymers,
electrolytes, Tween.RTM. 60 or 80, PEG, polyvinylpyrrolidone (PVP;
commercially known as Plasdone.RTM.), and the carbohydrate-based
dispersing agents such as, for example, hydroxypropyl celluloses
(e.g., HPC, HPC-SL, and HPC-L), hydroxypropyl methylcelluloses
(e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),
carboxymethylcellulose sodium, methylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose phthalate,
hydroxypropylmethylcellulose acetate stearate (HPMCAS),
noncrystalline cellulose, magnesium aluminum silicate,
triethanolamine, polyvinyl alcohol (PVA), vinyl pyrrolidone/vinyl
acetate copolymer (S630), 4-(1,1,3,3-tetramethylbutyl)-phenol
polymer with ethylene oxide and formaldehyde (also known as
tyloxapol), poloxamers (e.g., Pluronics F68.RTM., F88.RTM., and
F108.RTM., which are block copolymers of ethylene oxide and
propylene oxide); and poloxamines (e.g., Tetronic 908.RTM., also
known as Poloxamine 908.RTM., which is a tetrafunctional block
copolymer derived from sequential addition of propylene oxide and
ethylene oxide to ethylenediamine (BASF Corporation, Parsippany
N.J.)), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17,
polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30,
polyvinylpyrrolidone/vinyl acetate copolymer (S-630), polyethylene
glycol, e.g., the polyethylene glycol can have a molecular weight
of about 300 to about 6000, or about 3350 to about 4000, or about
7000 to about 5400, sodium carboxymethylcellulose, methylcellulose,
polysorbate-80, sodium alginate, gums, such as, e.g., gum
tragacanth and gum acacia, guar gum, xanthans, including xanthan
gum, sugars, cellulosics, such as, e.g., sodium
carboxymethylcellulose, methylcellulose, sodium
carboxymethylcellulose, polysorbate-80, sodium alginate,
polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan
monolaurate, povidone, carbomers, polyvinyl alcohol (PVA),
alginates, chitosans and combinations thereof. Plasticizcers such
as cellulose or triethyl cellulose can also be used as dispersing
agents. Dispersing agents particularly useful in liposomal
dispersions and self-emulsifying dispersions are dimyristoyl
phosphatidyl choline, natural phosphatidyl choline from eggs,
natural phosphatidyl glycerol from eggs, cholesterol and isopropyl
myristate.
[0112] Combinations of one or more erosion facilitator with one or
more diffusion facilitator can also be used in the present
compositions.
[0113] The term "diluent" refers to chemical compounds that are
used to dilute the compound of interest prior to delivery. Diluents
can also be used to stabilize compounds because they can provide a
more stable environment. Salts dissolved in buffered solutions
(which also can provide pH control or maintenance) are utilized as
diluents in the art, including, but not limited to a phosphate
buffered saline solution. In certain embodiments, diluents increase
bulk of the composition to facilitate compression or create
sufficient bulk for homogenous blend for capsule filling. Such
compounds include e.g., lactose, starch, mannitol, sorbitol,
dextrose, microcrystalline cellulose such as Avicel.RTM.; dibasic
calcium phosphate, dicalcium phosphate dihydrate; tricalcium
phosphate, calcium phosphate; anhydrous lactose, spray-dried
lactose; pregelatinized starch, compressible sugar, such as
Di-Pac.RTM. (Amstar); mannitol, hydroxypropylmethylcellulose,
hydroxypropylmethylcellulose acetate stearate, sucrose-based
diluents, confectioner's sugar; monobasic calcium sulfate
monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate,
dextrates; hydrolyzed cereal solids, amylose; powdered cellulose,
calcium carbonate; glycine, kaolin; mannitol, sodium chloride;
inositol, bentonite, and the like.
[0114] The term "non water-soluble diluent" represents compounds
typically used in the formulation of pharmaceuticals, such as
calcium phosphate, calcium sulfate, starches, modified starches and
microcrystalline cellulose, and microcellulose (e.g., having a
density of about 0.45 g/cm.sup.3, e.g. Avicel, powdered cellulose),
and talc.
[0115] An "enteric coating" is a substance that remains
substantially intact in the stomach but dissolves and releases the
drug in the small intestine or colon. Generally, the enteric
coating comprises a polymeric material that prevents release in the
low pH environment of the stomach but that ionizes at a higher pH,
typically a pH of 6 to 7, and thus dissolves sufficiently in the
small intestine or colon to release the active agent therein.
[0116] "Erosion facilitators" include materials that control the
erosion of a particular material in gastrointestinal fluid. Erosion
facilitators are generally known to those of ordinary skill in the
art. Exemplary erosion facilitators include, e.g., hydrophilic
polymers, electrolytes, proteins, peptides, and amino acids.
[0117] "Filling agents" include compounds such as lactose, calcium
carbonate, calcium phosphate, dibasic calcium phosphate, calcium
sulfate, microcrystalline cellulose, cellulose powder, dextrose,
dextrates, dextran, starches, pregelatinized starch, sucrose,
xylitol, lactitol, mannitol, sorbitol, sodium chloride,
polyethylene glycol, and the like.
[0118] Flavoring agents and/or sweeteners useful in the
formulations described herein, include, e.g., acacia syrup,
acesulfame K, alitame, anise, apple, aspartame, banana, Bavarian
cream, berry, black currant, butterscotch, calcium citrate,
camphor, caramel, cherry, cherry cream, chocolate, cinnamon, bubble
gum, citrus, citrus punch, citrus cream, cotton candy, cocoa, cola,
cool cherry, cool citrus, cyclamate, cylamate, dextrose,
eucalyptus, eugenol, fructose, fruit punch, ginger,
glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit,
honey, isomalt, lemon, lime, lemon cream, monoammonium
glyrrhizinate (MagnaSweet.RTM.), maltol, mannitol, maple,
marshmallow, menthol, mint cream, mixed berry, neohesperidine DC,
neotame, orange, pear, peach, peppermint, peppermint cream,
Prosweet.RTM. Powder, raspberry, root beer, rum, saccharin,
safrole, sorbitol, spearmint, spearmint cream, strawberry,
strawberry cream, stevia, sucralose, sucrose, sodium saccharin,
saccharin, aspartame, acesulfame potassium, mannitol, talin,
sylitol, sucralose, sorbitol, Swiss cream, tagatose, tangerine,
thaumatin, tutti fruitti, vanilla, walnut, watermelon, wild cherry,
wintergreen, xylitol, or any combination of these flavoring
ingredients, e.g., anise-menthol, cherry-anise, cinnamon-orange,
cherry-cinnamon, chocolate-mint, honey-lemon, lemon-lime,
lemon-mint, menthol-eucalyptus, orange-cream, vanilla-mint, and
mixtures thereof.
[0119] "Lubricants" and "glidants" are compounds that prevent,
reduce or inhibit adhesion or friction of materials. Exemplary
lubricants include, e.g., stearic acid, calcium hydroxide, talc,
sodium stearyl fumerate, a hydrocarbon such as mineral oil, or
hydrogenated vegetable oil such as hydrogenated soybean oil
(Sterotex.RTM.), higher fatty acids and their alkali-metal and
alkaline earth metal salts, such as aluminum, calcium, magnesium,
zinc, stearic acid, sodium stearates, glycerol, talc, waxes,
Stearowet.RTM., boric acid, sodium benzoate, sodium acetate, sodium
chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a
methoxypolyethylene glycol such as Carbowax.TM., sodium oleate,
sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium
or sodium lauryl sulfate, colloidal silica such as Syloid.TM.,
Cab-O-Sil.RTM., a starch such as corn starch, silicone oil, a
surfactant, and the like.
[0120] "Plasticizers" are compounds used to soften the
microencapsulation material or film coatings to make them less
brittle. Suitable plasticizers include, e.g., polyethylene glycols
such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800,
stearic acid, propylene glycol, oleic acid, triethyl cellulose and
triacetin. In some embodiments, plasticizers can also function as
dispersing agents or wetting agents.
[0121] "Solubilizers" include compounds such as triacetin,
triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl
sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide,
N-methylpyrrolidone, N-hydroxyethylpyrrolidone,
polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl
cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol,
bile salts, polyethylene glycol 200-600, glycofurol, transcutol,
propylene glycol, and dimethyl isosorbide and the like.
[0122] "Stabilizers" include compounds such as any antioxidation
agents, buffers, acids, preservatives and the like.
[0123] Suitable suspending agents for use in the solid dosage forms
described here include, but are not limited to,
polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12,
polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or
polyvinylpyrrolidone K30, polyethylene glycol, e.g., the
polyethylene glycol can have a molecular weight of about 300 to
about 6000, or about 3350 to about 4000, or about 7000 to about
5400, vinyl pyrrolidone/vinyl acetate copolymer (S630), sodium
carboxymethylcellulose, methylcellulose,
hydroxy-propylmethylcellulose, polysorbate-80,
hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum
tragacanth and gum acacia, guar gum, xanthans, including xanthan
gum, sugars, cellulosics, such as, e.g., sodium
carboxymethylcellulose, methylcellulose, sodium
carboxymethylcellulose, hydroxypropylmethylcellulose,
hydroxyethylcellulose, polysorbate-80, sodium alginate,
polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan
monolaurate, povidone and the like.
[0124] "Surfactants" include compounds such as sodium lauryl
sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E
TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate,
polysorbates, polaxomers, bile salts, glyceryl monostearate,
copolymers of ethylene oxide and propylene oxide, e.g.,
Pluronic.RTM. (BASF), and the like. Some other surfactants include
polyoxyethylene fatty acid glycerides and vegetable oils, e.g.,
polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene
alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol
40. In some embodiments, surfactants may be included to enhance
physical stability or for other purposes.
[0125] "Viscosity enhancing agents" include, e.g., methyl
cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl
cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl
cellulose acetate stearate, hydroxypropylmethyl cellulose
phthalate, carbomer, polyvinyl alcohol, alginates, acacia,
chitosans and combinations thereof.
[0126] "Wetting agents" include compounds such as oleic acid,
glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate,
triethanolamine oleate, polyoxyethylene sorbitan monooleate,
polyoxyethylene sorbitan monolaurate, sodium docusate, sodium
oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween
80, vitamin E TPGS, ammonium salts and the like.
[0127] In some embodiments, solid dosage forms, e.g., tablets,
capsules, are prepared by mixing avian follistatin products
described herein, with one or more pharmaceutical excipients to
form a bulk blend composition. When referring to these bulk blend
compositions as homogeneous, it is meant that the particles of
avian follistatin products described herein, are dispersed evenly
throughout the composition so that the composition may be readily
subdivided into equally effective unit dosage forms, such as
tablets, pills, and capsules.
[0128] Conventional techniques include, e.g., one or a combination
of methods: (1) dry mixing, (2) direct compression, (3) milling,
(4) dry or non-aqueous granulation, (5) wet granulation, or (6)
fusion. See, e.g., Lachman et al., "The Theory and Practice of
Industrial Pharmacy" (1986).
[0129] Suitable carriers for use in the solid dosage forms
described herein include, but are not limited to, acacia, gelatin,
colloidal silicon dioxide, calcium glycerophosphate, calcium
lactate, maltodextrin, glycerine, magnesium silicate, sodium
caseinate, soy lecithin, sodium chloride, tricalcium phosphate,
dipotassium phosphate, sodium stearoyl lactylate, carrageenan,
monoglyceride, diglyceride, pregelatinized starch,
hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate
stearate, sucrose, microcrystalline cellulose, lactose, mannitol
and the like.
[0130] Suitable filling agents for use in the solid dosage forms
described herein include, but are not limited to, lactose, calcium
carbonate, calcium phosphate, dibasic calcium phosphate, calcium
sulfate, microcrystalline cellulose, cellulose powder, dextrose,
dextrates, dextran, starches, pregelatinized starch,
hydroxypropylmethycellulose (HPMC), hydroxypropylmethycellulose
phthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS),
sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride,
polyethylene glycol, and the like.
[0131] Binders impart cohesiveness to solid oral dosage form
formulations: for powder filled capsule formulation, they aid in
plug formation that can be filled into soft or hard shell capsules
and for tablet formulation, they ensure the tablet remaining intact
after compression and help assure blend uniformity prior to a
compression or fill step. Materials suitable for use as binders in
the solid dosage forms described herein include, but are not
limited to, carboxymethylcellulose, methylcellulose (e.g.,
Methocel.RTM.), hydroxypropylmethylcellulose (e.g. Hypromellose USP
Pharmacoat-603, hydroxypropylmethylcellulose acetate stearate
(Aqoate HS-LF and HS), hydroxyethylcellulose,
hydroxypropylcellulose (e.g., Klucel.RTM.), ethylcellulose (e.g.,
Ethocel.RTM.), and microcrystalline cellulose (e.g., Avicel.RTM.),
microcrystalline dextrose, amylose, magnesium aluminum silicate,
polysaccharide acids, bentonites, gelatin,
polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone,
povidone, starch, pregelatinized starch, tragacanth, dextrin, a
sugar, such as sucrose (e.g., Dipac.RTM.), glucose, dextrose,
molasses, mannitol, sorbitol, xylitol (e.g., Xylitab.RTM.),
lactose, a natural or synthetic gum such as acacia, tragacanth,
ghatti gum, mucilage of isapol husks, starch, polyvinylpyrrolidone
(e.g., Povidone.RTM. CL, Kollidon.RTM. CL, Polyplasdone.RTM. XL-10,
and Povidone.RTM. K-12), larch arabogalactan, Veegum.RTM.,
polyethylene glycol, waxes, sodium alginate, and the like.
[0132] In general, binder levels of 20-70% are used in
powder-filled gelatin capsule formulations. Binder usage level in
tablet formulations varies whether direct compression, wet
granulation, roller compaction, or usage of other excipients such
as fillers which itself can act as moderate binder. Formulators
skilled in art can determine the binder level for the formulations,
but binder usage level of up to 70% in tablet formulations is
common.
[0133] Suitable wetting agents for use in the solid dosage forms
described herein include, for example, oleic acid, glyceryl
monostearate, sorbitan monooleate, sorbitan monolaurate,
triethanolamine oleate, polyoxyethylene sorbitan monooleate,
polyoxyethylene sorbitan monolaurate, quaternary ammonium compounds
(e.g., Polyquat 10.RTM.), sodium oleate, sodium lauryl sulfate,
magnesium stearate, sodium docusate, triacetin, vitamin E TPGS and
the like.
[0134] It should be appreciated that there is considerable overlap
between additives used in the solid dosage forms described herein.
Thus, the above-listed additives should be taken as merely
exemplary, and not limiting, of the types of additives that can be
included.
[0135] A capsule may be prepared, for example, by placing the bulk
blend of the formulation of the compound described above, inside of
a capsule. In some embodiments, the formulations (non-aqueous
suspensions and solutions) are placed in a soft gelatin capsule. In
other embodiments, the formulations are placed in standard gelatin
capsules or non-gelatin capsules such as capsules comprising HPMC.
In other embodiments, the formulation is placed in a sprinkle
capsule, wherein the capsule may be swallowed whole or the capsule
may be opened and the contents sprinkled on food prior to eating.
In some embodiments, the therapeutic dose is split into multiple
(e.g., two, three, or four) capsules. In some embodiments, the
entire dose of the formulation is delivered in a capsule form.
[0136] In another aspect, dosage forms may include
microencapsulated formulations. In some embodiments, one or more
other compatible materials are present in the microencapsulation
material. Exemplary materials include, but are not limited to, pH
modifiers, erosion facilitators, anti-foaming agents, antioxidants,
flavoring agents, and carrier materials such as binders, suspending
agents, disintegration agents, filling agents, surfactants,
solubilizers, stabilizers, lubricants, wetting agents, and
diluents.
[0137] Materials useful for the microencapsulation described herein
include materials which sufficiently isolate the compound from
other non-compatible excipients. Materials compatible avian
follistatin products described herein are those that delay the
release of the follistatin product in vivo.
[0138] Exemplary microencapsulation materials useful for delaying
the release of the formulations described herein, include, but are
not limited to, hydroxypropyl cellulose ethers (HPC) such as
Klucel.RTM. or Nisso HPC, low-substituted hydroxypropyl cellulose
ethers (L-HPC), hydroxypropyl methyl cellulose ethers (HPMC) such
as Seppifilm-LC, Pharmacoat.RTM., Metolose SR, Methocel.RTM.-E,
Opadry YS, PrimaFlo, Benecel MP824, and Benecel MP843,
methylcellulose polymers such as Methocel.RTM.-A,
hydroxypropylmethylcellulose acetate stearate Aqoat (HF-LS, HF-LG,
HF-MS) and Metolose.RTM., Ethylcelluloses (EC) and mixtures thereof
such as E461, Ethocel.RTM., Aqualon.RTM.-EC, Surelease.RTM.,
Polyvinyl alcohol (PVA) such as Opadry AMB, hydroxyethylcelluloses
such as Natrosol.RTM., carboxymethylcelluloses and salts of
carboxymethylcelluloses (CMC) such as Aqualon.RTM.-CMC, polyvinyl
alcohol and polyethylene glycol co-polymers such as Kollicoat
IR.RTM., monoglycerides (Myverol), triglycerides (KLX),
polyethylene glycols, modified food starch, acrylic polymers and
mixtures of acrylic polymers with cellulose ethers such as
Eudragit.RTM. EPO, Eudragit.RTM. L30D-55, Eudragit.RTM. FS 30D
Eudragit.RTM. L100-55, Eudragit.RTM. L100, Eudragit.RTM. S100,
Eudragit.RTM. RD100, Eudragit.RTM. E100, Eudragit.RTM. L12.5,
Eudragit.RTM. S12.5, Eudragit.RTM. NE30D, and Eudragit.RTM. NE 40D,
cellulose acetate phthalate, sepifilms such as mixtures of HPMC and
stearic acid, cyclodextrins, and mixtures of these materials.
[0139] In other embodiments, a powder including the formulations
with an avian follistatin product described herein may be
formulated to include one or more excipients and flavors. Such a
powder may be prepared, for example, by mixing the formulation and
optional excipients to form a bulk blend composition. Additional
embodiments also include a suspending agent and/or a wetting agent.
This bulk blend is uniformly subdivided into unit dosage packaging
or multi-dosage packaging units.
[0140] In still other embodiments, effervescent powders are also
prepared in accordance with the present disclosure. Effervescent
salts have been used to disperse medicines in water for oral
administration. Effervescent salts are granules or coarse powders
containing a medicinal agent in a dry mixture, usually composed of
sodium bicarbonate, citric acid and/or tartaric acid. When such
salts are added to water, the acids and the base react to liberate
carbon dioxide gas, thereby causing "effervescence." Examples of
effervescent salts include, e.g., the following ingredients: sodium
bicarbonate or a mixture of sodium bicarbonate and sodium
carbonate, citric acid and/or tartaric acid. Any acid-base
combination that results in the liberation of carbon dioxide can be
used in place of the combination of sodium bicarbonate and citric
and tartaric acids, as long as the ingredients were suitable for
pharmaceutical use and result in a pH of about 6.0 or higher.
[0141] In other embodiments, the formulations described herein,
which include avian follistatin product described herein, are solid
dispersions. Methods of producing such solid dispersions are known
in the art and include, but are not limited to, for example, U.S.
Pat. Nos. 4,343,789, 5,340,591, 5,456,923, 5,700,485, 5,723,269,
and U.S. Pub. Appl 2004/0013734. In still other embodiments, the
formulations described herein are solid solutions. Solid solutions
incorporate a substance together with the active agent and other
excipients such that heating the mixture results in dissolution of
the drug and the resulting composition is then cooled to provide a
solid blend which can be further formulated or directly added to a
capsule or compressed into a tablet. Methods of producing such
solid solutions are known in the art and include, but are not
limited to, for example, U.S. Pat. Nos. 4,151,273, 5,281,420, and
6,083,518.
[0142] In some embodiments, the solid dosage forms described herein
can be formulated as enteric coated delayed release oral dosage
forms, i.e., as an oral dosage form of a pharmaceutical composition
as described herein which utilizes an enteric coating to affect
release in the small intestine of the gastrointestinal tract. The
enteric coated dosage form may be a compressed or molded or
extruded tablet/mold (coated or uncoated) containing granules,
powder, pellets, beads or particles of the active ingredient and/or
other composition components. The enteric coated oral dosage form
may also be a capsule (coated or uncoated) containing pellets,
beads or granules of the solid carrier or the composition.
[0143] The term "delayed release" as used herein refers to the
delivery so that the release can be accomplished at some generally
predictable location in the intestinal tract more distal to that
which would have been accomplished if there had been no delayed
release alterations. In some embodiments the method for delay of
release is coating. Any coatings should be applied to a sufficient
thickness such that the entire coating does not dissolve in the
gastrointestinal fluids at pH below about 5, but does dissolve at
pH about 5 and above. It is expected that any anionic polymer
exhibiting a pH-dependent solubility profile can be used as an
enteric coating for the methods and compositions described herein
to achieve delivery to the lower gastrointestinal tract.
[0144] In some embodiments, formulations are provided that include
particles of the avian follistatin product described herein and at
least one dispersing agent or suspending agent for oral
administration to a subject. The formulations may be a powder
and/or granules for suspension, and upon admixture with water, a
substantially uniform suspension is obtained.
[0145] Liquid formulation dosage forms for oral administration can
be aqueous suspensions selected from the group including, but not
limited to, pharmaceutically acceptable aqueous oral dispersions,
emulsions, solutions, elixirs, gels, and syrups. See, e.g., Singh
et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp.
754-757 (2002). In addition to the particles of the avian
follistatin product described herein, the liquid dosage forms may
include additives, such as: (a) disintegrating agents; (b)
dispersing agents; (c) wetting agents; (d) at least one
preservative, (e) viscosity enhancing agents, (f) at least one
sweetening agent, and (g) at least one flavoring agent. In some
embodiments, the aqueous dispersions can further include a
crystalline inhibitor.
[0146] The aqueous suspensions and dispersions described herein can
remain in a homogenous state, as defined in The USP Pharmacists'
Pharmacopeia (2005 edition, chapter 905), for at least 4 hours. The
homogeneity should be determined by a sampling method consistent
with regard to determining homogeneity of the entire composition.
In one embodiment, an aqueous suspension can be re-suspended into a
homogenous suspension by physical agitation lasting less than 1
minute. In another embodiment, an aqueous suspension can be
re-suspended into a homogenous suspension by physical agitation
lasting less than 45 seconds. In yet another embodiment, an aqueous
suspension can be re-suspended into a homogenous suspension by
physical agitation lasting less than 30 seconds. In still another
embodiment, no agitation is necessary to maintain a homogeneous
aqueous dispersion.
[0147] Examples of disintegrating agents for use in the aqueous
suspensions and dispersions include, but are not limited to, a
starch, e.g., a natural starch such as corn starch or potato
starch, a pregelatinized starch such as National 1551 or
Amijelt.RTM., or sodium starch glycolate such as Promogel.RTM. or
Explotab.RTM.; a cellulose such as a wood product,
methylcrystalline cellulose, e.g., Avicel.RTM., Avicelt.RTM. PH101,
Avicel.RTM. PH102, Avicel.RTM. PH105, Elcema.RTM. P100,
Emcocel.RTM., Vivacel.RTM., Ming Tia.RTM., and Solka-Floc.RTM.,
methylcellulose, croscarmellose, or a cross-linked cellulose, such
as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol.RTM.),
cross-linked carboxymethylcellulose, or cross-linked
croscarmellose; a cross-linked starch such as sodium starch
glycolate; a cross-linked polymer such as crospovidone; a
cross-linked polyvinylpyrrolidone; alginate such as alginic acid or
a salt of alginic acid such as sodium alginate; a clay such as
Veegum.RTM. HV (magnesium aluminum silicate); a gum such as agar,
guar, locust bean, Karaya, pectin, or tragacanth; sodium starch
glycolate; bentonite; a natural sponge; a surfactant; a resin such
as a cation-exchange resin; citrus pulp; sodium lauryl sulfate;
sodium lauryl sulfate in combination starch; and the like.
[0148] In some embodiments, the dispersing agents suitable for the
aqueous suspensions and dispersions described herein are known in
the art and include, for example, hydrophilic polymers,
electrolytes, Tween.RTM. 60 or 80, PEG, polyvinylpyrrolidone (PVP;
commercially known as Plasdone.RTM.), and the carbohydrate-based
dispersing agents such as, for example, hydroxypropylcellulose and
hydroxypropyl cellulose ethers (e.g., HPC, HPC-SL, and HPC-L),
hydroxypropyl methylcellulose and hydroxypropyl methylcellulose
ethers (e.g. HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),
carboxymethylcellulose sodium, methylcellulose,
hydroxyethylcellulose, hydroxypropylmethyl-cellulose phthalate,
hydroxypropylmethyl-cellulose acetate stearate, noncrystalline
cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl
alcohol (PVA), polyvinylpyrrolidone/vinyl acetate copolymer
(Plasdone.RTM., e.g., S-630), 4-(1,1,3,3-tetramethylbutyl)-phenol
polymer with ethylene oxide and formaldehyde (also known as
tyloxapol), poloxamers (e.g., Pluronics F68.RTM., F88.RTM., and
F108.RTM., which are block copolymers of ethylene oxide and
propylene oxide); and poloxamines (e.g., Tetronic 9080, also known
as Poloxamine 908.RTM., which is a tetrafunctional block copolymer
derived from sequential addition of propylene oxide and ethylene
oxide to ethylenediamine (BASF Corporation, Parsippany, N.J.)). In
other embodiments, the dispersing agent is selected from a group
not comprising one of the following agents: hydrophilic polymers;
electrolytes; Tween.RTM. 60 or 80; PEG; polyvinylpyrrolidone (PVP);
hydroxypropylcellulose and hydroxypropyl cellulose ethers (e.g.,
HPC, HPC-SL, and HPC-L); hydroxypropyl methylcellulose and
hydroxypropyl methylcellulose ethers (e.g. HPMC K100, HPMC K4M,
HPMC K15M, HPMC K100M, and Pharmacoat.RTM. USP 2910 (Shin-Etsu));
carboxymethylcellulose sodium; methylcellulose;
hydroxyethylcellulose; hydroxypropylmethyl-cellulose phthalate;
hydroxypropylmethyl-cellulose acetate stearate; non-crystalline
cellulose; magnesium aluminum silicate; triethanolamine; polyvinyl
alcohol (PVA); 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with
ethylene oxide and formaldehyde; poloxamers (e.g., Pluronics
F68.RTM., F88.RTM., and F108.RTM., which are block copolymers of
ethylene oxide and propylene oxide); or poloxamines (e.g., Tetronic
908.RTM., also known as Poloxamine 908.RTM.).
[0149] Wetting agents suitable for the aqueous suspensions and
dispersions described herein are known in the art and include, but
are not limited to, cetyl alcohol, glycerol monostearate,
polyoxyethylene sorbitan fatty acid esters (e.g., the commercially
available Tweens.RTM. such as e.g., Tween 20.RTM. and Tween 80.RTM.
(ICI Specialty Chemicals)), and polyethylene glycols (e.g.,
Carbowaxs 3350.RTM. and 1450.RTM., and Carbopol 934.RTM. (Union
Carbide)), oleic acid, glyceryl monostearate, sorbitan monooleate,
sorbitan monolaurate, triethanolamine oleate, polyoxyethylene
sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium
oleate, sodium lauryl sulfate, sodium docusate, triacetin, vitamin
E TPGS, sodium taurocholate, simethicone, phosphotidylcholine and
the like
[0150] Suitable preservatives for the aqueous suspensions or
dispersions described herein include, for example, potassium
sorbate, parabens (e.g., methylparaben and propylparaben), benzoic
acid and its salts, other esters of parahydroxybenzoic acid such as
butylparaben, alcohols such as ethyl alcohol or benzyl alcohol,
phenolic compounds such as phenol, or quaternary compounds such as
benzalkonium chloride. Preservatives, as used herein, are
incorporated into the dosage form at a concentration sufficient to
inhibit microbial growth.
[0151] Suitable viscosity enhancing agents for the aqueous
suspensions or dispersions described herein include, but are not
limited to, methyl cellulose, xanthan gum, carboxymethyl cellulose,
hydroxypropyl cellulose, hydroxypropylmethyl cellulose,
Plasdon.RTM. S-630, carbomer, polyvinyl alcohol, alginates, acacia,
chitosans and combinations thereof. The concentration of the
viscosity enhancing agent will depend upon the agent selected and
the viscosity desired.
[0152] Examples of sweetening agents suitable for the aqueous
suspensions or dispersions described herein include, for example,
acacia syrup, acesulfame K, alitame, anise, apple, aspartame,
banana, Bavarian cream, berry, black currant, butterscotch, calcium
citrate, camphor, caramel, cherry, cherry cream, chocolate,
cinnamon, bubble gum, citrus, citrus punch, citrus cream, cotton
candy, cocoa, cola, cool cherry, cool citrus, cyclamate, cylamate,
dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger,
glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit,
honey, isomalt, lemon, lime, lemon cream, monoammonium
glyrrhizinate (MagnaSweet.RTM.), maltol, mannitol, maple,
marshmallow, menthol, mint cream, mixed berry, neohesperidine DC,
neotame, orange, pear, peach, peppermint, peppermint cream,
Prosweet.RTM. Powder, raspberry, root beer, rum, saccharin,
safrole, sorbitol, spearmint, spearmint cream, strawberry,
strawberry cream, stevia, sucralose, sucrose, sodium saccharin,
saccharin, aspartame, acesulfame potassium, mannitol, talin,
sucralose, sorbitol, swiss cream, tagatose, tangerine, thaumatin,
tutti fruitti, vanilla, walnut, watermelon, wild cherry,
wintergreen, xylitol, or any combination of these flavoring
ingredients, e.g., anise-menthol, cherry-anise, cinnamon-orange,
cherry-cinnamon, chocolate-mint, honey-lemon, lemon-lime,
lemon-mint, menthol-eucalyptus, orange-cream, vanilla-mint, and
mixtures thereof. In one embodiment, the aqueous liquid dispersion
can comprise a sweetening agent or flavoring agent in a
concentration ranging from about 0.001% to about 1.0% the volume of
the aqueous dispersion.
[0153] In another embodiment, the aqueous liquid dispersion can
comprise a sweetening agent or flavoring agent in a concentration
ranging from about 0.005% to about 0.5% the volume of the aqueous
dispersion. In yet another embodiment, the aqueous liquid
dispersion can comprise a sweetening agent or flavoring agent in a
concentration ranging from about 0.01% to about 1.0% the volume of
the aqueous dispersion.
[0154] In addition to the additives listed above, the liquid
formulations can also include inert diluents commonly used in the
art, such as water or other solvents, solubilizing agents, and
emulsifiers. Exemplary emulsifiers are ethyl alcohol, isopropyl
alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl
benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide,
sodium lauryl sulfate, sodium doccusate, cholesterol, cholesterol
esters, taurocholic acid, phosphotidylcholine, oils, such as
cottonseed oil, groundnut oil, corn germ oil, olive oil, castor
oil, and sesame oil, glycerol, tetrahydrofurfuryl alcohol,
polyethylene glycols, fatty acid esters of sorbitan, or mixtures of
these substances, and the like.
[0155] In some embodiments, the formulations described herein can
be self-emulsifying drug delivery systems (SEDDS). Emulsions are
dispersions of one immiscible phase in another, usually in the form
of droplets. Generally, emulsions are created by vigorous
mechanical dispersion. SEDDS, as opposed to emulsions or
microemulsions, spontaneously form emulsions when added to an
excess of water without any external mechanical dispersion or
agitation. An advantage of SEDDS is that only gentle mixing is
required to distribute the droplets throughout the solution.
Additionally, water or the aqueous phase can be added just prior to
administration, which ensures stability of an unstable or
hydrophobic active ingredient. Thus, the SEDDS provides an
effective delivery system for oral and parenteral delivery of
hydrophobic active ingredients. SEDDS may provide improvements in
the bioavailability of hydrophobic active ingredients. Methods of
producing self-emulsifying dosage forms are known in the art and
include, but are not limited to, for example, U.S. Pat. Nos.
5,858,401, 6,667,048, and 6,960,563.
[0156] It is to be appreciated that there is overlap between the
above-listed additives used in the aqueous dispersions or
suspensions described herein, since a given additive is often
classified differently by different practitioners in the field, or
is commonly used for any of several different functions. Thus, the
above-listed additives should be taken as merely exemplary, and not
limiting, of the types of additives that can be included in
formulations described herein. The amounts of such additives can be
readily determined by one skilled in the art, according to the
particular properties desired.
[0157] Buccal formulations that include avian follistatin product
described herein may be administered using a variety of
formulations known in the art. For example, such formulations
include, but are not limited to, U.S. Pat. Nos. 4,229,447,
4,596,795, 4,755,386, and 5,739,136. In addition, the buccal dosage
forms described herein can further include a bioerodible
(hydrolysable) polymeric carrier that also serves to adhere the
dosage form to the buccal mucosa. The buccal dosage form is
fabricated so as to erode gradually over a predetermined time
period. Buccal drug delivery, as will be appreciated by those
skilled in the art, avoids the disadvantages encountered with oral
drug administration, e.g., slow absorption, degradation of the
active agent by fluids present in the gastrointestinal tract and/or
first-pass inactivation in the liver. With regard to the
bioerodible (hydrolysable) polymeric carrier, it will be
appreciated that virtually any such carrier can be used, so long as
the desired drug release profile is not compromised, and the
carrier is compatible with the follistatin product described
herein, and any other components that may be present in the buccal
dosage unit. Generally, the polymeric carrier comprises hydrophilic
(water-soluble and water-swellable) polymers that adhere to the wet
surface of the buccal mucosa. Examples of polymeric carriers useful
herein include acrylic acid polymers and co, e.g., those known as
"carbomers" (Carbopol.RTM., which may be obtained from B.F.
Goodrich, is one such polymer). Other components may also be
incorporated into the buccal dosage forms described herein include,
but are not limited to, disintegrants, diluents, binders,
lubricants, flavoring, colorants, preservatives, and the like. For
buccal or sublingual administration, the compositions may take the
form of tablets, lozenges, or gels formulated in a conventional
manner.
[0158] In certain embodiments, delivery systems for pharmaceutical
compounds may be employed, such as, for example, liposomes and
emulsions. In certain embodiments, compositions provided herein can
also include an mucoadhesive polymer, selected from among, for
example, carboxymethylcellulose, carbomer (acrylic acid polymer),
poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic
acid/butyl acrylate copolymer, sodium alginate and dextran.
Examples of Methods of Dosing and Treatment Regimens
[0159] The compositions containing the avian follistatin products
described herein can be administered for prophylactic and/or
therapeutic treatments. In therapeutic applications, the
compositions are administered to a patient already suffering from a
disease or condition, in an amount sufficient to improve muscle
mass. Amounts effective for this use will depend on the severity
and course of the disease or condition, previous therapy, the
patient's health status, weight, and the judgment of the treating
health care provider.
[0160] In prophylactic applications, compositions containing the
avian follistatin products described herein are administered to a
patient requiring or desiring increase in muscle mass. In this use,
the precise amounts also depend on the patient's state of health,
weight, and the like. It is considered well within the skill of the
art for one to determine such prophylactically effective amounts by
routine experimentation (e.g., a dose escalation clinical trial).
When used in a patient, effective amounts for this use will depend
on the severity and course of the disease, disorder or condition,
previous therapy, the patient's health status and response to the
drugs, and the judgment of the health care provider.
[0161] In the case wherein the patient's condition does not
improve, upon the health care provider's discretion the
administration of the compounds may be administered chronically,
that is, for an extended period of time, including throughout the
duration of the patient's life in order to ameliorate or otherwise
control or limit the symptoms of the patient's disease or
condition.
Kits/Articles of Manufacture
[0162] For use in the therapeutic applications described herein,
kits and articles of manufacture are also described herein. Such
kits can include a carrier, package, or container that is
compartmentalized to receive one or more containers such as vials,
tubes, and the like, each of the container(s) including one of the
separate elements to be used in a method described herein. Suitable
containers include, for example, bottles, vials, and test tubes.
The containers can be formed from a variety of materials such as
glass or plastic.
[0163] The articles of manufacture provided herein contain
packaging materials. Packaging materials for use in packaging
pharmaceutical products are well known to those of skill in the
art. See, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252.
Examples of pharmaceutical packaging materials include, but are not
limited to, blister packs, bottles, tubes, bags, containers,
bottles, and any packaging material suitable for a selected
formulation and intended mode of administration and treatment. A
wide array of formulations of the compounds and compositions
provided herein are contemplated as are a variety of treatments for
any disease, disorder, or condition that would benefit by
administration of follistatin and increases in muscle
regeneration.
[0164] Such kits or article of manufacture optionally include a
formulation as described herein with an identifying description or
label or instructions relating to its use in the methods described
herein.
[0165] A kit will typically may include one or more additional
containers, each with one or more of various materials (such as
reagents, optionally in concentrated form, and/or devices)
desirable from a commercial and user standpoint for use of a
compound described herein. Non-limiting examples of such materials
include, but not limited to, buffers, diluents, filters, carrier,
package, container, labels listing contents and/or instructions for
use, and package inserts with instructions for use. A set of
instructions will also typically be included.
[0166] A label can be on or associated with the container. A label
can be used to indicate that the contents are to be used for a
specific therapeutic application. The label can also indicate
directions for use of the contents, such as in the methods
described herein.
[0167] The pack or dispenser can also be accompanied with a notice
associated with the container in 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 drug for human or veterinary administration. Such notice, for
example, can be the labeling approved by the U.S. Food and Drug
Administration for prescription drugs, or the approved product
insert. Compositions containing the follistatin products described
herein formulated in a compatible carrier can also be prepared,
placed in an appropriate container, and labeled for treatment of an
indicated condition.
EXAMPLES
[0168] These examples are provided for illustrative purposes only
and not to limit the scope of the claims provided herein.
Example 1
Determination of Follistatin Levels in Chicken Eggs
[0169] To determine the follistatin levels in conventional
fertilized and non-fertilized conventional store bought chicken
eggs were obtained and follistatin levels in the eggs were
determined via immobilizing follistatin via binding to Activin in a
C18 HPLC column (ExpertTox, Inc., Deer Park, Tex.). The assay was
standardized with Recombinant Follistatin 288 and was assayed in an
LC/MS with a third degree fragmentation (D. S., Tanetta, et al.
1977). Results revealed all fertile eggs contained significant
concentrations of follistatin (0.11, 1.05. 0.67. and 0.81 mcg/mg)
whereas two unfertilized eggs that were also tested contained no
detectable follistatin (0 and 0 mcg/mg).
Example 2
Measurement of Follistatin Levels in Humans
[0170] The following assay was then conducted to determine whether
orally ingested naturally occurring follistatin is absorbed and is
pharmaco-kinetically active in a human model. A male subject was
chosen because the normal baseline male physiology does not
regularly contain any measurable concentration of follistatin.
[0171] Follistatin-rich fertile egg yolk powder properly processed
to preserve active follistatin (Folstaxan.TM.) was obtained
(Celldyne Biopharma, San Antonio, Tex.). After initial blood draw
and subsequent oral Folstaxan.TM. dosing, serum follistatin levels
were qualitatively and quantitatively measured as an indicator of
absorption. Because follistatin is a negative modulator of
myostatin, serum myostatin levels were qualitatively and
quantitatively measured as an indicator hormonal influence and thus
true pharmacokinetic activity. Testing utilized a purchased
follistatin and myostatin standard for verification. Confirmations
were run by ELISA and quantitations by Liquid Chromatography Tandem
Mass Spectrometer with third degree fragmentation (Expertox, Deer
Park, Tex.).
[0172] Results showed a predicted zero level of follistatin at
baseline with a myostatin level of 46 pg/ml. Within twelve hours
after Folstaxan.TM. dosing, serum follistatin measured 57.1 pg/ml
with a decline of myostatin to 34 pg/ml. Twenty four hours after
the initial dosing, follistatin levels began to predictably drop
from the time of initial dosing to 11.4 pg/ml, yet myostatin
continued to decline slightly with a twenty four hour level of 31
pg/ml. These results indicate that a fertile egg yolk powder
properly processed to preserve active follistatin, when orally
ingested, results in detectable serum follistatin. Furthermore,
this resultant follistatin presence has significant pharmacokinetic
activity as shown by the hormonal down-regulation of serum
myostatin.
Example 3
Effect of Pasteurization of Fertilized Egg Yolks on Follistatin
[0173] Fertile egg yolks contain significant concentrations of
follistatin. Egg products are usually pasteurized in order to
minimize danger of microbe ingestion. In an effort to identify
whether pasteurization denatures or affects this source of
naturally occurring follistatin, this study was undertaken.
[0174] Fertile chicken eggs were cracked and separated using
commercial breakers and separators. The separated yolk was then
pasteurized a HTST at 192 F (89C) for 1.75 minutes. The egg yolks
were tested for quantity and quality of active follistatin. Testing
utilized purchased follistatin standardized for verification.
Confirmations were run by ELISA and quantitations by Liquid
Chromatography Tandem Mass Spectrometer with third degree
fragmentation (Expertox, Deer Park, Tex.).
[0175] Results showed no measurable active or viable follistatin.
This compares with previous results follistatin levels of 0.11 to
1.05 mcg/mg in non-pasteurized fertile egg yolk. These results
indicate that a fertile egg yolk cannot be pasteurized in order to
preserve active follistatin.
Example 4
Effect of Spray/Heat Drying of Fertilized Egg Yolks on
Follistatin
[0176] Fertile egg yolks contain significant concentrations of
follistatin. In an effort to identify whether Spray/Heat Drying
denatures or affects this source of naturally occurring
follistatin, this study was undertaken.
[0177] Fertile chicken eggs yolks were separated with commercial
separators then heat/spray dried with a commercial aerosolizer and
heat kept under 110.degree. Fahrenheit.
[0178] The resulting powder was tested for quantity and quality of
active follistatin. Testing utilized purchased follistatin
standardized for verification. Confirmations were run by ELISA and
quantitations by Liquid Chromatography Tandem Mass Spectrometer
with third degree fragmentation (Expertox, Deer Park, Tex.).
[0179] Results showed no measurable active or viable follistatin.
This compares with previous results follistatin levels of 0.11 to
1.05 mcg/mg in non-spray/heat dried fertile egg yolk. These results
indicate that a fertile egg yolk cannot be spray/heat dried in
order to preserve active follistatin.
Example 5
Effect of Lyophilization of Fertilized Egg Yolks on Follistatin
[0180] Fertile egg yolks contain significant concentrations of
follistatin. In an effort to identify whether lyophilization
denatures or affects this source of naturally occurring
follistatin, this study was undertaken.
[0181] Thirty dozen fertile chicken eggs yolks and forty-five dozen
unfertilized table eggs were freeze dried in a 18 sq. ft. research
dryer for 48 hours at -30.degree. Celsius. The resulting powder was
tested for quantity and quality of active follistatin. Testing
utilized purchased follistatin standardized for verification.
Confirmations were run by ELISA and quantitations by Liquid
Chromatography Tandem Mass Spectrometer with third degree
fragmentation (Expertox, Deer Park, Tex.).
[0182] Results showed good levels of active or viable follistatin,
comparable with previous results (follistatin levels of 0.11 to
1.05 mcg/mg in non-lyophilized fertile egg yolk). These results
indicate that a fertile egg yolk can be lyophilized in order to
preserve active follistatin.
Example 6
Effect of Gamma Irradiation of Fertilized Egg Yolks on
Follistatin
[0183] Fertile egg yolks contain significant concentrations of
follistatin. Egg products are usually pasteurized in order to
minimize danger of microbe ingestion. Gamma irradiation is a viable
alternative to pasteurization. In an effort to identify whether
gamma irradiation denatures or affects this source of naturally
occurring follistatin, this study was undertaken.
[0184] Fertile chicken eggs were gamma irradiated (FTSI, Mulberry,
Fla.) with the most common source of gamma rays for food
processing, the radioisotope cobalt 60. The eggs were treated
cobalt 60 gamma rays in a facility known as an irradiator. The egg
yolks were tested for quantity and quality of active follistatin.
Testing utilized purchased follistatin standardized for
verification. Confirmations were run by ELISA and quantitations by
Liquid Chromatography Tandem Mass Spectrometer with third degree
fragmentation (Expertox, Deer Park, Tex.).
[0185] Results showed no measurable levels of active or viable
follistatin. This compares with previous results follistatin levels
of 0.11 to 1.05 mcg/mg in non-irradiated fertile egg yolk. These
results indicate that a fertile egg yolk cannot be gamma irradiated
in order to preserve active follistatin.
Example 7
Comparison of Whole Fertilized Egg Yolks Versus Whole Fertilized
Egg Yolk Blastodisc/Membrane in Content of Active Follistatin
[0186] Fertile egg yolks contain significant concentrations of
follistatin. It is hypothesized that the blastodisc and egg yolk
membrane have significantly higher concentrations of follistatin
than whole egg yolk. In an effort to compare the follistatin levels
of these two egg products, this study was undertaken.
[0187] Fertile chicken eggs were cracked and separated by hand. The
separated yolk was then divided into two. The egg yolk membrane and
blastodiscs were extracted by hand using paper filters. The whole
egg yolks and the extraction were tested for quantity and quality
of active follistatin. Testing utilized purchased follistatin
standardized for verification. Confirmations were run by ELISA and
quantitations by Liquid Chromatography Tandem Mass Spectrometer
with third degree fragmentation (Expertox, Deer Park, Tex.).
[0188] Results showed, nearly a double level of follistatin in the
extraction versus the whole fertile egg yolk. The extraction
follistatin level was 0.84 mcg/mg compared with the whole yolk
follistatin level of 0.36 mcg/mg. These results indicate that
extracted blastodiscs and yolk membrane of fertile egg yolk is
higher in active follistatin than whole egg yolk.
Example 8
Protein Analysis of Lyophilized Fertilized Egg Yolks
[0189] Fertile egg yolk contains significant concentrations of
follistatin. In an effort to identify the major proteins and
corresponding levels contained in fertile lyophilized egg yolk,
this study was undertaken.
[0190] Analyses of fertilized lyophilized egg yolk were performed
using MALDI-TOF mass spectrometry analysis. There are two major
proteins detected by MALDI (35 kDa and 45 kDa) and a minor protein
(80 kDa) in most samples. This is consistent with SDS-PAGE analysis
of the same samples. No major degradation or aggregation, however
there are at least four major proteins detected by this method).
Performing in-gel digestion and Mass Spectrometer Peptide Mass
Fingerprinting determined that follistatin was one of the proteins
identified.
[0191] Results showed levels of active or viable follistatin
comparable with non-lyophilized levels. These results indicate that
a fertile egg yolk can be lyophilized and active follistatin will
be preserved along with three other proteins.
Example 9
Effect of Ebeam Irradiation of Fertilized Egg Yolks on
Follistatin
[0192] Fertile egg yolks contain significant concentrations of
follistatin. Egg products are usually pasteurized in order to
minimize danger of microbe ingestion. In an effort to identify
whether electron beam irradiation denatures or affects this source
of naturally occurring follistatin, this study was undertaken.
[0193] Six samples of lyophilized fertilized egg yolk were
irradiated using dual beam electron irradiation, shooting at
separate sample levels of: 0.0 kGy (control), 1.03 kGy, 3.00 kGy,
9.80 kGy, 28.58 kGy, and 51.65 kGy. The samples were irradiated
with a 10 MeV Electron Beam Linear Accelerator, Manufactured by
Varian Oncology--Papaytron Sled Assembly--Part # 100010432 REV. A
Varian Inc., 3120 Hansen Way, Palo Alto Calif. 94304-1030
[0194] The linear accelerators used were installed in a cell/system
designed by L3 Communications (formerly the Titan Corporation)
Pulse Sciences Division, located at 2700 Merced Street, San
Leandro, Calif. 94577.
[0195] The only visable change to the samples were change in
pigmentation of a bright yellow at 1 kGy to a pale/whitish yellow
at 51 kGy, with different shades of yellow corresponding to kGy
level.
[0196] Analyses of the six egg yolk samples were performed using
MALDI-TOF mass spectrometry analysis (control and 5 dosages). In
summary, the five radiation dosages do not seem to cause any
obvious degradation or aggregation of the sample.
[0197] There are two major proteins detected by MALDI (35 kDa and
45 kDa) and a minor protein (80 kDa) in most samples. This is
consistent with SDS-PAGE analysis of the same samples. No major
degradation or aggregation, however there are at least four major
proteins detected by this method (see earlier results). Performing
in-gel digestion and Mass Spectrometer Peptide Mass Fingerprinting
supported the identification of follistatin as one of the proteins
in the irradiated samples.
[0198] Results showed comparable levels of active or viable
follistatin regardless of radiation level. These results indicate
that a fertile egg yolk can be irradiated with an electron beam and
active follistatin will be preserved.
[0199] The examples and embodiments described herein are for
illustrative purposes only and various modifications or changes
suggested to persons skilled in the art are to be included within
the spirit and purview of disclosure and scope of the appended
claims. All publications, patents, and patent applications cited
herein are hereby incorporated by reference for all purposes.
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