Methods for Alleviating, Inhibiting or Reversing Muscle Disuse

Atrophy in Mammals

This patent application claims the benefit of priority from U.S. Provisional Application Serial No. 62/790,612 filed January 10, 2019, U.S. Provisional Application Serial No.

62/756,279 filed November 6, 2018, and U.S. Provisional

Application Serial No. 62/724,263, filed August 29, 2018, the teachings of each of which are herein incorporated by

reference in their entireties.

Background

Muscle loss due to muscle disuse atrophy is a common problem that mammals experience following many common

orthopedic surgical procedures.

Skeletal muscle atrophy is characterized by a decrease in protein content, fiber diameter, force production and

resistance to fatigue. In muscle disuse atrophy, a decreased rate of protein synthesis along with an increase in protein degradation, also referred to as proteolysis, have been shown to be important contributing factors (Jackman, R W. and

Kandarian, S.C. American Journal of Physiology-Cell Physiology

2004 287 (4) :C834-C843) . In this 2004 review article, Jackman and Kandarian discuss the interaction of molecular pathways including ubiquitin-proteasome, lysosomal and calpain in muscle proteolysis during muscle atrophy (American Journal of

Physiology-Cell Physiology 2004 287 ( 4 ) : C834-C843 ) . Under specific conditions, TNF-a, glucocorticoids, myostatin and reactive oxygen species can induce muscle atrophy. In muscle disuse atrophy, the transcription factor NF-kB plays a

particularly important role. The key physiological stimulus in muscle disuse atrophy is when a muscle no longer bears weight (Goldspink et al. Pflugers Archiv 1986 407 (3) : 333-340) . The removal of weight bearing initiates a cascade of molecular signaling events.

Total hip arthroplasty (THA) is a common orthopedic surgery procedure that is performed to relieve pain and improve functional performance in humans with end-stage hip osteoarthritis. THA is typically followed by out-patient physical rehabilitation. However, it has been widely reported that many patients experience muscle atrophy and reduced muscle strength and functional performance at various time- points following their THA surgical procedure (Holm et al.

PloS One 2013 8(4) :e62109; Holstege et al. Archives of

Physical Medicine and Rehabilitation 2011 92 (2 ) : 236-241 ;

Jensen et al . Osteoarthritis and Cartilage 2011 19(9) :1108— 1116) . According to Freeman et al . (Clinical Biomechanics 2013 28 (2 ) : 171-177 ) , the failure of the central nervous system (CNS ) to activate muscles close to the operated hip joint as a result of intra-articular swelling may play a role.

A similar form of muscle atrophy referred to as

arthrogenic muscle inhibition is commonly observed after total knee arthroplasty (TKA) (Mizneret al . The Journal of Bone and Joint Surgery 2005 87(5) : 1047; Rice, D. A. and McNair P.J.

Seminars in Arthritis and Rheumatism. Vol . 40. No. 3. WB

Saunders, 2010) .

The anterior cruciate ligament (ACL) is a ligament that connects the tibia to the femur. An injury of this ligament typically involves the ACL becoming stretched, partially torn or completely torn. In order to repair the ACL, a surgical procedure known as ACL reconstruction surgery is performed and the surgery involves replacing the torn ACL with a graft prepared from a tendon that is taken from another source such as the patellar tendon, hamstring tendon or quadriceps tendon. The graft serves as a scaffolding, upon which a new ligament tissue will grow. Researchers have reported a statistically significant loss of muscle mass and muscle strength in the quadriceps six months following surgery.

Similar muscle loss is observed in dogs following orthopedic surgery.

For example, Tibial-Plateau-Leveling Osteotomy (TPLO) surgery is a veterinary orthopedic surgical procedure that is commonly performed on dogs in order to stabilize their stifle joint after a rupture of the cranial cruciate ligament (CCL) . The CCL is analogous to the anterior cruciate ligament (ACL) in humans . In dogs that undergo TPLO surgery, the CCL typically ruptures due to long-term degeneration as the fibers that comprise the ligament undergo degradation over a long period of time. Although the exact cause of the degeneration of the CCL is not fully understood, genome wide association studies (GWAS) have provided some insight into the genetic predisposition for CCL degeneration with the identification of single nucleotide polymorphisms (SNPs) identified in the

SORCS2 and SEMA5B genes that are thought to play a role (Baird et al. Animal Genetics 2014 45 (4) : 542-549) . Certain breeds of dogs such as Labradors, Rottweilers, Boxers, West Highland White Terriers and Newfoundlands are believed to be

genetically predisposed to CCL rupture. In addition to

genetics, factors such as obesity (Adams et al. Journal of Small Animal Practice 2011 52 (7 ) : 347-352 ) , individual

conformation (Guerrero et al . American Journal of Veterinary Research 2007 68 ( 12 ) : 1332-1337 ) , hormonal imbalance (Light et al . American Journal of Veterinary Research 2012 73(8) :1186- 1193) and inflammatory conditions (Lemburg et al.

Veterinary Immunology and I munopathology 2004 97(3-4) :231- 240) that impact the joint are believed to exacerbate the degeneration of the CCL. The CCL traverses from the cranial mid part of the tibial intercondylar eminence to the lateral condyle of the femur. Under regular circumstances, the CCL prevents backward movement of the femur with respect to the tibia. However, due to selective breeding of dogs, the tibial plateau slope has sloped too far backwards which places constant stress on the CCL. This constant stress over time eventually leads to a degenerative rupture of the CCL.

Rupture of the CCL is very painful for the dog and typically leads to chronic progressive arthritis in the stifle if not properly treated. During the TPLO surgical procedure, the portion of the tibia that adjoins the stifle known as the tibial plateau, is cut by the surgeon and rotated so that its slope changes to approximately 5 degrees with respect to the horizontal plane. The surgical procedure prevents the femur from sliding down the slope of the tibial plateau where the canine exerts its weight on its knee. After undergoing TPLO surgery, the majority of dogs regain an active lifestyle without suffering from many post-operative complications and requiring drugs for long-term pain management. However, muscle loss due to atrophy of immobilization is a problem that dogs typically encounter after a TPLO surgical procedure (Monk et al . American Journal of Veterinary Research 2006 67(3) :529- 536) .

There is a need for compositions and methods of

alleviating muscle disuse atrophy in mammals. Summary

The present invention relates to a method for inhibiting, reversing or alleviating muscle disuse atrophy in a mammal via administration of a composition comprising egg yolk powder to the mammal.

In one nonlimiting embodiment, the composition

administered is FORTETROPIN.

In one nonlimiting embodiment, the composition is

administered to the mammal following a surgical procedure. In one nonlimiting embodiment, the surgical procedure is an orthopedic surgical procedure.

In one nonlimiting embodiment, the composition is

administered to the mammal prior to and following a surgical procedure. In one nonlimiting embodiment, the surgical procedure is an orthopedic surgical procedure.

In one nonlimiting embodiment, the composition is

administered to the mammal following an injury causing

immobilization .

In the present invention, administration of the

composition after or before and after the surgical procedure or after the immobilizing injury can result in reduced muscle atrophy of disuse, lower reduction in muscle thickness and/or muscle mass, improved muscle function, increased muscle force generation, reduced inflammation, reduced recovery time and/or lower increases in myostatin in the mammal.

Detailed Description

The present invention provides methods and compositions for inhibiting and/or reversing and/or alleviating muscle disuse atrophy in a mammal. Compositions administered in accordance with the present invention comprise egg yolk powder or one or more proteins and/or lipids derived from egg yolk which are effective in growth and development of lean muscle tissue.

In one nonlimiting embodiment, the composition

administered is FORTETROPIN. FORTETROPIN is a fertilized egg yolk derived product used as a dietary and nutritional supplement (MYOS RENS TECHNOLOGY INC. CORPORATION Cedar

Knolls, NJ) . A method for production of FORTETROPIN is disclosed in U.S. Patent 8,815,320, teachings of which are herein incorporated by reference in their entirety.

In another nonlimiting embodiment, the composition comprises an avian follistatin such as described in U.S.

Published Patent Application No. 2007/0275036, the disclosure of which is incorporated herein by reference in its entirety and/or other proteins and/or lipids found in avian eggs and which are beneficial in growth and development of lean muscle tissue such as, but not limited to, those described in U.S. Provisional Application Serial No. 62/755,601, the disclosure of which is incorporated herein by reference in its entirety.

For purposes of the present invention, when the phrases "egg yolk powder" or "composition comprising egg yolk powder" are used, they are meant to be inclusive, but are not limited to, egg yolk powder, one or more proteins and/or lipids derived from egg yolk which are effective in growth and development of lean muscle tissue, FORTETROPIN and avian follistatin .

Disclosed herein are methods for use of compositions comprising egg yolk powder in the alleviation, reversal and/or inhibition of muscle disuse atrophy in mammals. In these methods, the composition is administered to a mammal in need thereof in an amount effective to alleviate one or more symptoms of muscle disuse atrophy and/or reverse muscle disuse atrophy and/or inhibit muscle disuse atrophy in the mammal.

By "mammal" or "mammals" it is meant to include, but is not limited to, humans, apes, monkeys, cows, sheep, goats, dogs, cats, mice, rats, and transgenic species thereof.

Nonlimiting examples of mammals "in need" for purposes of the present invention are mammals that have undergone as well mammals that are scheduled to undergo a surgical procedure and mammals that have suffered an immobilizing injury. In one nonlimiting embodiment, the surgical procedure or immobilizing injury is expected to result in muscle disuse atrophy. In one nonlimiting embodiment, the surgical procedure is an

orthopedic surgical procedure. Nonlimiting examples of orthopedic surgical procedures in which the composition comprising egg yolk powder can be administered after or before and after the procedure include TPLO, TKA, THA and ACL

reconstruction surgery. Nonlimiting examples of immobilizing injuries in which the composition comprising egg yolk powder can be administered after include sports injuries, broken bones and sprained ankles.

By "alleviation and/or reversal and/or inhibition of muscle disuse atrophy" for purposes of this invention, it is meant to include, but is not limited to, reduced muscle atrophy of disuse, lower reduction in muscle thickness and/or muscle mass, improved muscle function, increased muscle force generation, reduced inflammation, reduced recovery time and/or lower increases in myostatin, a protein which can induce muscle atrophy, in the mammal.

In one nonlimiting embodiment, the composition

administered is FORTETROPIN. In one nonlimiting embodiment, the FORTETROPIN is produced in accordance with the process described in U.S. Patent 8,815,320.

In one nonlimiting embodiment, FORTETROPIN production is optimized to enhance potency as it relates to muscle disuse atrophy by modifying one or more egg yolk-related parameters such as, but not limited to, incubation time post-lay,

fertility status and breed of chicken.

In one nonlimiting embodiment, the composition comprising egg yolk powder is administered orally on a daily basis, one, two or three times a day.

In one nonlimiting embodiment, the composition comprising egg yolk powder is administered in an amount effective to upregulate mTor pathway activity, downregulate ubiquitin proteasome pathway activity, downregulate serum myostatin levels and/or reduce ActRIIB expression, a receptor for myostatin.

In one nonlimiting embodiment, the composition comprising egg yolk powder is FORTETROPIN administered orally on a daily basis in an amount ranging from about 5 to about 25 grams/day, about 6.6 to about 19.8 grams/day or about 50 to about 300 mg/kg/day, about 80 to about 250 mg/kg/day in humans. In canines, FORTETROPIN is administered orally on a daily basis in an amount ranging from about 200-1000 mg/kg/day, about 300 to about 900 mg/kg/day.

In one nonlimiting embodiment, administration of the composition comprising egg yolk powder is started about 1 to about 4 weeks before surgery.

In one nonlimiting embodiment, administration of the composition comprising egg yolk powder occurs after surgery and continues for about 2 to 12 weeks, more preferably 6 to 12 weeks. In one nonlimiting embodiment, administration of the composition comprising egg yolk powder is started before surgery and is continued after surgery. In one nonlimiting embodiment, administration of the composition comprising egg yolk powder is started about 1 to about 4 weeks before surgery and continues after surgery for at least 2 to 12 weeks.

In one nonlimiting embodiment, the composition comprising egg yolk powder is administered in combination with one or more additional agents which alleviate pain and/or

inflammation post surgery or post injuy. Nonlimiting examples of additional agents which can be administered in combination with a composition comprising egg yolk powder include, but are not limited to, curcumin, omega-3-fatty acids, non-steroidal anti-inflammtories (NSAIDs) and/or opioids.

By "administered in combination" it is meant to include simultaneous administration in a single formulation,

simultaneous administration in separate formulations,

sequential administration of the composition comprising egg yolk powder followed by the one or more additional agents and vice versa, and administration of separate formulations at separate times .

The compositions described herein can be formulated for administration to a subject via any conventional means including, but not limited to, oral, or buccal.

Moreover, the 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 .

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 glucose, fructose,

lactose, sucrose, mannitol, sorbitol, stevia extract, or sucralose; 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 .

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.

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 fillers 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.

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 . 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 .

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 .

The formulations described herein can include egg yolk powder 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 ( s ) 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.

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 .

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.

Formulations including egg yolk powder, as 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.

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 .

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 , (1) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof .

Binders imparting cohesive qualities may also be used.

Examples include, but are not limited to, alginic acid and salts thereof; cellulose derivatives such as

carboxymethylcellulose , methylcellulose ,

hydroxypropylmethylcellulose , hydroxyethylcellulose , hydroxypropylcellulose , ethylcellulose, and

microcrystalline cellulose; 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, glucose, dextrose, molasses, mannitol, sorbitol, xylitol, and lactose; a natural or synthetic gum such as acacia, tragacanth, ghatti gum, mucilage of isapol husks, polyvinylpyrrolidone, larch arabogalactan, polyethylene glycol, waxes, sodium alginate, and the like.

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.

Compositions may further comprise carriers of

relatively nontoxic chemical compounds or agents that facilitate the incorporation of a compound into cells or tissues. Nonlimiting examples include binders,

suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. Suitable carriers for use in 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. 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. Nonlimiting examples of diffusion facilitators/ dispersing agents include

hydrophilic polymers, electrolytes, a Tween, PEG,

polyvinylpyrrolidone, and carbohydrate-based dispersing agents such as 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 , hydroxypropylce1lulose ,

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, block copolymers of ethylene oxide and propylene oxide; and poloxamines, tetrafunctional block copolymers derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine , 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. Plasticizers such as cellulose or triethyl cellulose can also be used as dispersing agents. Dispersing agents that are 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.

Combinations of one or more erosion facilitator with one or more diffusion facilitator can also be used in the present compositions.

Compositions of the present invention may further comprise diluents 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; dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate;

anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar; 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; sodium chloride;

inositol, bentonite, and the like.

Compositions may further comprise an enteric coating, a substance that remains substantially intact in the stomach but dissolves and releases the egg yolk powder 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 .

In addition, the compositions may comprise an erosion facilitator, a material that controls 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 .

Filling agents including 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 can also be included in the compositions. 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.

In addition, flavoring agents and/or sweeteners can be used in the compositions and may include 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, maltol, mannitol, maple, marshmallow, menthol, mint cream, mixed berry, neohesperidine DC, neotame, orange, pear, peach, peppermint, peppermint cream, 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.

The compositions may further comprise lubricants and/or glidants that prevent, reduce or inhibit adhesion or

friction of materials . Nonlimiting examples of lubricants include stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil, 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, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a

methoxypolyethylene glycol, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica, a starch such as corn starch, silicone oil, a surfactant, and the like.

Plasticizers, compounds used to soften the

microencapsulation material or film coatings to make them less brittle may also be included in the compositions.

Examples of suitable plasticizers include, but are not limited to, 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. The compositions may further comprise solubilizers 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.

In addition, the compositions my comprise

stabilizers such as antioxidation agents, buffers, acids, preservatives and the like.

Suitable suspending agents for use in 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-propylmethy1cellulose ,

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.

Surfactants including compounds such as sodium lauryl sulfate, sodium docusate, Tweens, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates , polaxomers, bile salts,

glyceryl monostearate, copolymers of ethylene oxide and propylene oxide and the like may also be included.

Additional 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 .

Viscosity enhancing agents including, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose,

hydroxypropyl cellulose, hydroxypropyl ethyl cellulose, hydroxypropylmethyl cellulose acetate stearate,

hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof may also be included.

In addition, wetting agents including 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 may be included in these compositions .

In some embodiments, solid dosage forms, e.g., tablets, capsules, are prepared by mixing the egg yolk powder 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 egg yolk powder, 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.

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).

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.

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.

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 .

Materials useful for the microencapsulation described herein include materials which sufficiently isolate the compound from other non-compatible excipients. Materials compatible with the egg yolk powder are those that delay the release of the egg yolk powder in vivo.

In other embodiments, the formulations described

herein, which include the egg yolk powder, 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.

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.

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.

In some embodiments, formulations are provided that include particles of egg yolk powder 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.

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 egg yolk powder, 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, (t) at least one sweetening agent, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersions can further include a crystalline

inhibitor .

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 regards 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.

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.

In one nonlimiting 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.

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 , dimethyl formamide, 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.

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.

Buccal formulations that include egg yolk powder 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 egg yolk powder, 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. 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 .

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 a

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 . The invention is further illustrated by the following example, which should not be construed as further limiting. The contents of all references, pending patent applications, and published patents cited throughout this application are hereby expressly incorporated by reference.

EXAMPLE

100 dogs with cranial cruciate ligament rupture were enrolled in this study. Dogs were randomized to receive either FORTETROPIN or a placebo in a double-blind fashion.

Dogs were given 300 mg/kg/day of either FORTETROPIN or placebo starting a week prior to TPLO surgery and continuing for 12 weeks post-surgery. At pre-surgery and 6 and 12 weeks post ^¬ surgery, thigh circumference, thickness of coxofemoral extensors and flexors, thickness of epaxial muscle, were measured. In addition, serum myostatin and C-reactive protein levels were measured and stance analysis was performed on each dog at pre-surgery and again 12-weeks post-surgery.

Dogs that received FORTETROPIN experienced a smaller decrease in thigh circumference over 8 weeks relative to dogs that received a placebo.

Dogs that received FORTETROPIN also experienced a smaller increase in myostatin concentration over 8 weeks relative to dogs that received a placebo.

In addition, dogs that received FORTETROPIN experienced a greater increase in force that could be exerted due to weight on the affected hind limb after 8 weeks relative to placebo.