Fat-forming compounds

Abstract

Provided are myogenic compounds identified in egg yolk and their synthetic derivatives, nutritional supplement compositions of one or more of these myogenic compounds and their synthetic derivatives, and the use of these compounds and nutritional supplement compositions in increasing muscle mass in mammals.

Description

[Technical Field]

[0001] This patent application claims the benefit of priority from U.S. Provisional Application No. 63 / 417,479, filed October 19, 2022, U.S. Provisional Application No. 63 / 433,821, filed December 20, 2022, and U.S. Provisional Application No. 63 / 503,027, filed May 18, 2023, the teachings of each of which are incorporated by reference herein in their entirety.

[0002] Field The present disclosure relates to myogenic compounds identified in isolated fractions of fertilized egg yolk and the use of these myogenic compounds and combinations thereof, or synthetic derivatives and combinations thereof, in increasing muscle mass or reducing muscle loss in mammals. [Background technology]

[0003] background Increasing muscle mass is of general interest not only among professional athletes but also among the general public. In addition, sarcopenia, or muscle wasting, is a problem in older adults, affecting 5-13% of those aged 60-70 and 15-50% of those over 80 (Shafiee et al. Journal of Diabetes & Metabolic Disorders, 2017 16(1)).

[0004] Previous studies have identified myogenic benefits from adding protein supplements derived from hen's egg yolk extract to the diet (Sharp et al. Journal of the American College of Nutrition, 2016 35(8):679-691; Evans et al. The Journals of Gerontology: Series A, 2020 76(1):108-114).

[0005] Fertilized egg yolk has been used as a health food in the past, particularly in Asian cultures, as a food called balut. Furthermore, a study in dogs showed that diet supplemented with FORTETROPIN, a fertilized egg yolk product, improved the outcome of tibial plateau leveling osteotomy by reducing muscle atrophy in the affected limb (White et al. PLoS ONE 2020 15(4):p.1-10).

[0006] Egg yolk is composed of 70% lipids and 30% protein by dry weight; the lipids are triglycerides, phospholipids, and cholesterol, and the proteins are low-density lipoproteins (LDL), high-density lipoproteins (HDL), phosvitin, livetin, and riboflavin-binding protein (Mann, K. & Mann, M. PROTEOMICS 2008 8(1):178-191).

[0007] Egg yolk, due to its widespread consumption, has been studied extensively. As early as 1949, Romanov et al. described the separation of the plasma and granular portions of the egg (The avian egg, 1949, New York: J. Wiley). These experiments, along with additional work by Burley and Vadehra in 1989 (The avian egg: chemistry and biology, 1989, New York: Wiley), described the structure, biology, chemistry, and development of the egg yolk. It was found that egg yolk can be easily separated into plasma and granular fractions using centrifugation (McBee, L. & Cotterill, O. Poultry Science, 1973, Oxford University Press, Great Clarendon St., Oxford OX2 6DP, England), with the plasma and granular fractions containing equal amounts of protein and 90%:10% lipids, respectively. Plasma contains 85% LDL and 15% livestock. The insoluble granules consist mainly of phosvitin and HDL linked by association with calcium molecules.

[0008] It is currently unknown what compounds are present in the fertilized egg yolk that produce the observed myogenic effects. Summary of the Invention

[0009] overview The present disclosure relates to myogenic compounds identified in isolated fractions of fertilized avian egg yolk.

[0010] One aspect of the present disclosure relates to compositions comprising one or more of these myogenic compounds or their synthetic derivatives.

[0011] In one non-limiting embodiment, the composition comprises one or more myogenic compounds identified in avian fertilized egg yolk subfractions F7.11, F7.18, F7.21, FS.10, FS.11, and / or FS.22, or synthetic derivatives thereof.

[0012] In one non-limiting embodiment, the composition comprises one or more myogenic compounds comprising one or more peptide sequences shown in Table 2, or myogenically active fragments thereof.

[0013] In one non-limiting embodiment, the myogenic compound is a protein selected from gelsolin, actin-depolymerizing factor, vimentin, SERPIN domain-containing protein also known as pigment epithelium-derived factor (PEDF), hepatocyte growth factor activator, chicken nucleoside diphosphate kinase, inter-alpha trypsin inhibitor heavy chain, keratin type II cytoskeleton cochlea, desmin, apolipoprotein AI, albumin, actin cytoplasmic type 5, actin cytoplasmic 1, vitellogenin-1, actin cytoplasmic 2, IF rod domain-containing protein, vitellogenin-3, glial fibrillary acidic protein, plasminogen, or type II alpha keratin IIA or a myogenically active fragment thereof.

[0014] In one non-limiting embodiment, the myogenic compound is a protein selected from albumin, ovalbumin, gelsolin, lysozyme C, an SMB domain-containing protein, transthyretin, an IG-like domain-containing protein, a fibrinogen C-terminal domain-containing protein, plasminogen, phosvitin (VTG2), a peptidase S1 domain-containing protein, or fibrinogen C, or a myogenically active fragment thereof.

[0015] In one non-limiting embodiment, the myogenic compound is a peptide sequence or protein that exhibits at least 70%, 80%, 90%, 95% or 99% sequence identity with a peptide sequence or protein identified herein and exhibits myogenic activity.

[0016] In one non-limiting embodiment, the composition comprises one or more synthetic derivatives of myogenic compounds identified in avian fertile egg yolk subfractions F7.11, F7.18, F7.21, FS.10, FS.11, and / or FS.22 or comprising one or more peptide sequences set forth in Table 2.

[0017] In one non-limiting embodiment, the myogenic compound is a protein or myogenically active peptide fragment thereof similar to those identified herein, but derived from a species alternative to chicken, such as, but not limited to, human, canine, feline, equine, bovine, ovine, porcine, and primate.

[0018] Another aspect of the present disclosure relates to nutraceutical and / or pharmaceutical compositions comprising one or more myogenic compounds identified in an isolated fraction of fertilized avian egg yolk, or synthetic derivatives thereof, and one or more nutraceutically and / or pharmaceutically acceptable excipients.

[0019] In one non-limiting embodiment, the nutritional supplement and / or pharmaceutical composition comprises one or more myogenic compounds or synthetic derivatives thereof identified in avian fertilized egg yolk subfractions F7.11, F7.18, F7.21, FS.10, FS.11, and / or FS.22.

[0020] In one non-limiting embodiment, the nutritional and / or pharmaceutical composition comprises one or more myogenic compounds comprising one or more peptide sequences set forth in Table 2, or myogenically active fragments thereof.

[0021] In one non-limiting embodiment, the myogenic compound is a protein selected from gelsolin, actin-depolymerizing factor, vimentin, SERPIN domain-containing protein also known as pigment epithelium-derived factor (PEDF), hepatocyte growth factor activator, chicken nucleoside diphosphate kinase, inter-alpha trypsin inhibitor heavy chain, keratin type II cytoskeleton cochlea, desmin, apolipoprotein AI, albumin, actin cytoplasmic type 5, actin cytoplasmic 1, vitellogenin-1, actin cytoplasmic 2, IF rod domain-containing protein, vitellogenin-3, glial fibrillary acidic protein, plasminogen, or type II alpha keratin IIA or a myogenically active fragment thereof.

[0022] In one non-limiting embodiment, the myogenic compound is a protein selected from albumin, ovalbumin, gelsolin, lysozyme C, an SMB domain-containing protein, transthyretin, an IG-like domain-containing protein, a fibrinogen C-terminal domain-containing protein, plasminogen, phosvitin (VTG2), a peptidase S1 domain-containing protein, or fibrinogen C, or a myogenically active fragment thereof.

[0023] In one non-limiting embodiment, the myogenic compound is a peptide sequence or protein that exhibits at least 70%, 80%, 90%, 95% or 99% sequence identity with a peptide sequence or protein identified herein and exhibits myogenic activity.

[0024] In one non-limiting embodiment, the composition comprises one or more synthetic derivatives of myogenic compounds identified in avian fertile egg yolk subfractions F7.11, F7.18, F7.21, FS.10, FS.11, and / or FS.22 or comprising one or more peptide sequences set forth in Table 2.

[0025] In one non-limiting embodiment, the myogenic compound is a protein or myogenically active peptide fragment thereof similar to those identified herein, but derived from a species alternative to chicken, such as, but not limited to, human, canine, feline, equine, bovine, ovine, porcine, and primate.

[0026] Another aspect of the present disclosure relates to a method for increasing muscle mass in a mammal, comprising administering to the mammal a composition comprising one or more myogenic compounds identified in a subfraction of fertilized avian egg yolk, or synthetic derivatives thereof.

[0027] In one non-limiting embodiment, the composition comprises one or more myogenic compounds identified in avian fertilized egg yolk subfractions F7.11, F7.18, F7.21, FS.10, FS.11, and / or FS.22, or synthetic derivatives thereof.

[0028] In one non-limiting embodiment, the composition comprises one or more myogenic compounds comprising one or more peptide sequences shown in Table 2, or myogenically active fragments thereof.

[0029] In one non-limiting embodiment, the myogenic compound is a protein selected from gelsolin, actin-depolymerizing factor, vimentin, SERPIN domain-containing protein also known as pigment epithelium-derived factor (PEDF), hepatocyte growth factor activator, chicken nucleoside diphosphate kinase, inter-alpha trypsin inhibitor heavy chain, keratin type II cytoskeleton cochlea, desmin, apolipoprotein AI, albumin, actin cytoplasmic type 5, actin cytoplasmic 1, vitellogenin-1, actin cytoplasmic 2, IF rod domain-containing protein, vitellogenin-3, glial fibrillary acidic protein, plasminogen, or type II alpha keratin IIA or a myogenically active fragment thereof.

[0030] In one non-limiting embodiment, the myogenic compound is a protein selected from albumin, ovalbumin, gelsolin, lysozyme C, an SMB domain-containing protein, transthyretin, an IG-like domain-containing protein, a fibrinogen C-terminal domain-containing protein, plasminogen, phosvitin (VTG2), a peptidase S1 domain-containing protein, or fibrinogen C, or a myogenically active fragment thereof.

[0031] In one non-limiting embodiment, the myogenic compound is a peptide sequence or protein that exhibits at least 70%, 80%, 90%, 95% or 99% sequence identity with a peptide sequence or protein identified herein and exhibits myogenic activity.

[0032] In one non-limiting embodiment, the composition comprises one or more synthetic derivatives of myogenic compounds identified in avian fertile egg yolk subfractions F7.11, F7.18, F7.21, FS.10, FS.11, and / or FS.22 or comprising one or more peptide sequences set forth in Table 2.

[0033] In one non-limiting embodiment, the myogenic compound is a protein or myogenically active peptide fragment thereof similar to those identified herein, but derived from a species alternative to chicken, such as, but not limited to, human, canine, feline, equine, bovine, ovine, porcine, and primate. DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description The present disclosure provides myogenic compounds and synthetic derivatives thereof identified in subfractions of fertilized avian egg yolk, as well as nutritional and / or pharmaceutical compositions comprising one or more of the myogenic compounds or synthetic derivatives thereof, and methods for using these compositions in increasing muscle mass in mammals.

[0035] As used herein, "myogenic" or "myogenic activity" refers to a compound that increases muscle growth and / or reduces muscle loss. In one non-limiting embodiment, a myogenic compound increases muscle differentiation in a cell line with a reporter gene expressed under the promotion of a myogenic transcription factor. In one non-limiting embodiment, a myogenic compound increases muscle growth in a mammal. The increase is determined by comparing differentiation and / or muscle growth in the absence of the myogenic compound and / or upon administration of a negative control. In one non-limiting embodiment, a myogenic compound of the present disclosure increases muscle growth in a mammal similar to powdered egg yolk. In one non-limiting embodiment, a myogenic compound of the present disclosure significantly increases muscle growth compared to powdered egg yolk. In one non-limiting embodiment, a myogenic compound of the present disclosure reduces muscle loss in a mammal. In one non-limiting embodiment, a myogenic compound of the present disclosure reduces muscle loss in a mammal similar to powdered egg yolk. In one non-limiting embodiment, a myogenic compound of the present disclosure significantly reduces muscle loss compared to powdered egg yolk.

[0036] In one non-limiting embodiment, the myogenic compound upregulates mTOR pathway activity, downregulates ubiquitin proteasome pathway activity, downregulates serum myostatin levels, and / or decreases ActRIB expression in a mammal.

[0037] As used herein, "mammal" is intended to include, but is not limited to, humans, dogs, cats, horses, cattle, sheep, pigs, and primates.

[0038] The myogenic compounds of the present disclosure are distinguishable from natural egg yolk in that the myogenic compounds are either physically separated by mechanical means from other components in the natural egg yolk, including, but not limited to, non-myogenic components of the egg yolk and lipid components of the egg yolk, or are produced via synthetic means, such as, for example, peptide synthesis or recombinant protein production according to well-established protocols. Furthermore, as will be understood by those of skill in the art upon reading this disclosure, myogenic compounds identified in fertilized avian egg yolk are expected to include, and the myogenic compounds of the present disclosure may be derived from, other alternative biological sources.

[0039] Skeletal muscle is a complex tissue primarily composed of muscle fibers classified into four distinct types based on their myosin heavy chains: type I, type IIa, type IIx, and type IIb (Schiaffino, S. & Reggiani, C. Physiological Reviews 2011 91(4):1447-1531). Slow-twitch muscle fibers are primarily composed of type I, while fast-twitch muscle fibers are composed of all types of type II. Type I fibers are responsible for continuous use and postural function, while type II fibers are involved in movements such as walking and running. While both types of fibers are constantly maintained and can be repaired, sarcopenia is well documented to result in a loss of type II fibers (Brunner et al. J Aging Phys Act 2007 15(3):336-48).

[0040] We used cell lines carrying reporter genes driven by myogenic transcription factors to identify myogenic differentiation arising from various fractions of egg yolk separated into lipid and protein components. Using the MLC2 promoter region, we developed reporter cell lines from C2C12 myoblasts, in which luciferase expression correlated with the myogenic phenotype. C2C12 myoblasts were selected because they are associated with muscle differentiation. The promoter region is known to be associated with adult muscle growth. More specifically, the MLC1f promoter was selected to drive expression of a luciferase transgene because it is expressed only in type IIb fibers in rats (Neville Dev Genet 1996 19(2):157-62). Therefore, it is a useful gene for identifying myoblast fusion and myofiber maturation, as it is associated with muscle differentiation.

[0041] The cell line selected was the mouse C2C12 myoblast cell line, which can be induced to differentiate and form myotubes. These cells were derived from normal CH3 mice that had undergone a compression injury two days prior to isolation to produce satellite cells (Yaffe, D. & Saxel, ORA Nature (London), 1977. 270(5639):725-727). These cells were further expanded, and a subclone was selected that was diploid and capable of reliably forming myotubes (Blau et al., Science (American Association for the Advancement of Science), 1985. 230(4727):758-766). These cells were transfected with a lentivirus containing a three-part plasmid consisting of the MLC1f promoter (Neville et al. Dev Genet, 1996. 19(2):157-62), a blasticidin resistance gene, and the GLuc gene (New England BioLabs, Ipswich MA), which expresses Gaussia luciferase, an enzyme not expressed in mammalian cells that reacts with coelenterazine to produce light.

[0042] The C2C12 cell line was transfected with this lentiviral vector and then selected using blasticidin-supplemented medium. After clonal expansion of blasticidin-resistant cells, the reporter's efficacy was confirmed by assessing cell differentiation. The cell lines were then tested using three groups: a positive control containing 50 ng / ml insulin-like growth factor (IGF) 1, a negative control containing 40 μg / ml dexamethasone, and a medium control. Luciferase expression was measured using a Biolux Gaussia luciferase assay kit and correlated with gene expression and image analysis of fluorescently stained cells.

[0043] The egg yolk was then separated into protein and lipid fractions. Two different methods were used to separate the proteins and lipids in the egg yolk. The first method used centrifugation and alginate to create three different fractions with different concentrations of protein, fat, and cholesterol. The second method removed lipids from the egg yolk using organic solvents.

[0044] Fertilized eggs were processed to remove the shell and vitelline membrane, leaving the raw yolk. The raw yolk was then diluted with ddH2O at a 2:3 ratio (v / v, egg yolk:ddH2O) and the pH was adjusted to 7. The yolk was stored overnight in a cold room (4°C) on a stirring plate set at low speed (60 rpm). The yolk was then centrifuged at 10,000g for 45 minutes at 4°C to produce a pellet of the granular fraction and a supernatant containing soluble proteins and lipids. A 1% (w / v) sodium alginate stock solution was then added to the supernatant at a 1:9 ratio to produce 0.1% w / v sodium alginate in the final solution. The resulting solution was then centrifuged at 10,000g for 15 minutes at 20°C to produce an aqueous fraction and an alginate-lipid paste (lipid). These fractions: granules, lipid paste, and aqueous were then analyzed to determine their general composition. Cell studies were performed using the fractions as media supplements to determine their effect on myogenic differentiation of myoblasts in vitro.

[0045] The composition of each fraction was determined based on protein size using SDS-PAGE. Samples were obtained from each fraction and mixed with 2x Laemmli buffer containing 5% (v / v) β-mercaptoethanol. The samples were then heated to 70°C for 15 minutes using a water bath and loaded in duplicate onto lanes using a pre-cast polyacrylamide gel, MiniProtean GTX. The gel was run at 200V for approximately 30 minutes. The gel was stopped when the dye tip reached the black reference line on the gel. The results were compared with those previously published by Laca et al. for unfertilized eggs (Food Hydrocolloids-FOOD HYDROCOLLOID 2010 24:434-443), showing similar results, thus demonstrating that fertilization of the egg yolk does not significantly alter the composition of the yolk and that myogenic compounds can be obtained from unfertilized eggs.

[0046] In cell studies, differentiation was measured using a luciferase assay on cell culture media harvested at various time points throughout the assay. To provide information on the biocompatibility of the fractions added to the culture media and ensure that their addition did not adversely affect cell proliferation and fusion, the metabolic activity of each fraction group was measured using a PrestoBlue assay at each time point. On days 0, 3, 7, and 10, the media was removed from each fraction group, frozen for later analysis, and then replaced with media containing 1:10% v / v PrestoBlue reagent in differentiation medium and incubated at 37°C for 1 hour. After incubation, the supernatant was harvested and analyzed by excitation at 560 and emission at 590. Fluorescence intensity was analyzed throughout the study to measure the conversion of non-fluorescent resazurin to fluorescent resorufin via reduction by the cellular metabolic pathway. To perform the luciferase assay, frozen cell culture media collected throughout the study were thawed at room temperature. Samples from each well were assayed in duplicate by adding 5 μl of sample to 12.5 μl of luciferase assay buffer. Coelenterazine reagent was then added to each well and allowed to react for 10 minutes, after which the luminescence of each sample was read using a 10-millisecond integration time.

[0047] The Presto Blue assay results showed similar viability in all fraction groups, except for the dexamethasone group, which had significantly lower viability compared to all other groups, and the aqueous high-concentration group, which had significantly higher viability than the base cell culture medium control. Dexamethasone is an inhibitor of muscle differentiation, as reflected by the low expression levels of luciferase in cells treated with this medium; if myoblasts are unable to differentiate, the low viability results from decreased activity in serum-starved conditions.

[0048] Results from the luciferase assay showed a significant increase in myoblast MLC expression in most fraction groups when compared to the medium control and dexamethasone-supplemented medium, the negative control. The two exceptions were the low-concentration alginate and low-concentration aqueous fractions, which were significantly higher than the dexamethasone group but not the medium control.

[0049] Thus, as shown by these experiments, the granular fraction of egg yolk has a positive effect on myoblast differentiation, indicating that a bioactive compound or compounds within fertilized egg yolk improves lean muscle growth.

[0050] To identify specific bioactive compounds contained in the egg yolk that are involved in muscle growth, the granular portion was further subfractionated and the subfractions were tested for their differentiation potential.

[0051] The granules are insoluble in aqueous solutions and form aggregates on the order of 1–8 μm in size. In solutions containing 0.3 M NaCl, the particles begin to dissolve, becoming micelles on the order of 100–200 nm in size, as sodium disrupts the calcium bonds that form the insoluble aggregates. The use of phosphate-buffered saline (PBS) as a solvent dissolved the granule fraction from its aggregated form, allowing the formation of micelles in solution. These micelles were then separated by centrifugation, and the biological effects of these subfractions were evaluated in vitro in reporter cell lines. Additionally, the biological effects of the unfertilized egg fraction compared with the fertilized egg fraction were evaluated.

[0052] Centrifugation was used to generate three fractions composed of granules of various sizes. The first fraction, Harvest, herein designated F5, was isolated by centrifuging the solubilized granules at 5000 relative centrifugal force (rcf) for 15 minutes and consisted of the resuspended pellet obtained by this process. The next fraction, herein designated F7, was obtained by centrifuging the supernatant from the previous step at 7500 rcf for 15 minutes and resuspending the pellet. The final fraction, containing the lightest components, herein designated FS, consisted of the supernatant from the previous step.

[0053] All fractions were assessed by bicinchoninic acid (BCA) assay to determine total protein content and run on a gel to confirm their size range. For these analyses, ddH2O was added at a level that produced a homogenous mixture. Samples were then taken from these mixtures and aqueous fractions, and BCA assays were performed according to the manufacturer's instructions. Each sample was assessed at multiple concentrations: 1x, 10x, and 100x dilutions. Samples were assayed in triplicate and reacted with BCA buffer for 30 minutes at 37°C. After the reaction, the plate was read for absorbance at 562 nm and compared with a BSA standard control of known concentration evaluated simultaneously to obtain an estimate of the protein concentration of each sample. Following the BCA assay, samples were run through the gel using SDS-PAGE to determine the content of each fraction. Samples were diluted to provide 5 μg of protein per lane, where possible, or the highest concentration available. The gel was then run at 200 V for 35 minutes, or until the dye reached the upper reference line. After electrophoresis, the gel was fixed with methanol-acetone-acetic acid and stained with Coomassie blue for 24 hours. After staining, the gel was washed with diH2O to remove excess stain and then imaged.

[0054] The myogenic effects of the F5, F7, and FS fractions on the reporter cell lines disclosed herein were also investigated.

[0055] Based on these results, size-exclusion chromatography was used to further separate the proteins in the F7 and FS fractions into smaller subfractions. More specifically, the F7 and FS fractions were separated into subfractions based on their native conformation in PBS using FPLC on a Superdex 200 increase 10 / 300 column. One-milliliter samples were collected in microcentrifuge tubes, flash-frozen, and stored at -80°C for later analysis and cell culture. Samples were named according to their elution volume and proportion of the precursor fraction; for example, F7.7 corresponds to the 7th ml of liquid collected through the column from the F7 fraction. Samples containing the eluate with a peak absorbance at 280 nm were then run on a gel. Most of the F7 fractions exhibited a range of sizes, indicating different compositions within each fraction. For example, F7.4 shows a 250 kDa band followed by a 75 kDa band, while the next fraction observed, F7.6, has a band beginning at 200 kDa and a faint 75 kDa band. The next fraction, F7.7, has a 200 kDa band again. Gels for later elutions in the F7 subfractions show lighter staining in most columns further down the gel, indicating a tendency for these fractions to contain more faint proteins. For the supernatant gels, a poor correlation between size and fraction number was observed.

[0056] Samples collected from the chromatography were also assayed for concentration using the BCA assay, and the total protein concentration data was used to calculate the appropriate volume of each sample, which was then added to cell culture medium to test the effect of each fraction on differentiation when applied to myoblasts as described herein. The data from this assay are shown in Table 1.1 and Table 1.2. [Table 1-1] [Table 1-2]

[0057] From these tables, several fractions were identified that induced MLC expression above that of the positive IGF control on day 10; these were F7.11, F7.18, and F7.21, FS.10, FS.11, and FS.22. These fractions, along with two control fractions, F7.7 and FS.8, which did not exhibit myogenic activity, were then analyzed by mass spectrometry (MS) to identify the compounds responsible for the observed myogenic activity. Each fraction was run through MS three times, resulting in a total of 24 raw data files from the eight fractions.

[0058] More specifically, samples were analyzed by LC-MS using a Nano LC-MS / MS (Dionex Ultimate 3000 RLSCnano System, Thermofisher) compatible with Eclipse (ThermoFisher). Three microliters of the 12.5 μl in-gel digested sample was loaded onto a fused silica trap column (Acclaim PepMap 100, 75 μm x 2 cm, ThermoFisher). After washing with 0.1% TFA at 5 μl / min for 5 minutes, the trap column was inlined with an analytical column (Nanoease MZ peptide BEH C18, 130A, 1.7 μm, 75 μm x 250 mm, Waters) for LC-MS / MS. Peptides were fractionated at 300 nL / min using a segmented linear gradient: 4–15% solution B for 30 min (where solution A contains 0.2% formic acid, solution B contains 0.16% formic acid, 80% acetonitrile), 15–25% solution B for 40 min, 25–50% solution B for 44 min, and 50–90% solution B for 11 min. Solution B was then returned to 4% for 5 min for the next run. The scan sequence started with MS1 spectrum (Orbitrap analysis, resolution 120,000, scan range M / Z 375–1500, automatic gain control (AGC) target 8E5, maximum injection time 100 ms). A duty cycle scheme with top S (3 s) was used to determine the number of MSMS runs performed in each cycle. Parent ions with charges between 2 and 7 were selected for MSMS, and a 60 s dynamic exclusion was used to avoid repeated sampling. The parent mass was isolated in the quadrupole using a 1.2 m / z isolation window and an automatic gain control (AGC) target of 1E5, and fragmented by high-energy collisional dissociation at a normalized collision energy of 30%. The fragments were scanned in the Orbitrap at a resolution of 15,000. The MSMS scan range was determined by the charge state of the parent ion, with the lower limit set at 110 amu.

[0059] Proteins identified as being in higher concentrations in the initial analysis include albumin, ovalbumin, gelsolin, and lysozyme C. When the spectral counts of each protein were divided by the total spectral counts of each sample to account for variations in sample concentrations, several additional proteins were identified that differed in terms of their expression compared to the undifferentiated fraction, including SMB domain-containing protein, transthyretin, IG-like domain-containing protein, fibrinogen C-terminal domain-containing protein, plasminogen, phosvitin (VTG2), peptidase S1 domain-containing protein, and fibrinogen C.

[0060] Furthermore, using both spectral counting and focused peptide identification searches, 15 peptide sequences were identified in at least five of the six active fractions but were not detected in the two inactive fractions via this process.

[0061] Table 2 shows these active peptides. Note that by definition, the two inactive fractions are designated 0. [Table 2]

[0062] The symbol " in the peptide sequence in Table 2 ∧ " and "$" indicate the N-terminus and C-terminus, respectively. The number in parentheses represents the mass of the putative chemical modification to the previous residue or terminus. For example, " ∧ "(42)" represents the N-terminal acetylation resulting in an additional mass of approximately 42 amu, C(57) refers to the (+57.021465) atomic mass units of the carbamidomethyl modification to cysteine ​​(C) and S(80), and T(80) and Y(80) refer to the (+79.96633) amu of the phosphorylation to those amino acid residues.

[0063] Thus, provided herein are myogenic compounds and compositions thereof identified in isolated fractions of fertilized avian egg yolk. As discussed above, the myogenic compounds and compositions thereof of the present disclosure are distinguishable from natural egg yolk in that the myogenic compounds are either physically separated by mechanical means from other components in the natural egg yolk, including, but not limited to, non-myogenic components of the egg yolk and lipid components of the egg yolk, or are produced by synthetic means, such as peptide synthesis or recombinant protein production according to well-established protocols. Furthermore, as will be understood by those skilled in the art upon reading this disclosure, it is anticipated that the myogenic compounds identified in avian egg yolk will also be contained in other alternative biological sources, and the myogenic compounds of the present disclosure may be derived from these alternative biological sources.

[0064] In one non-limiting embodiment, the composition comprises one or more myogenic compounds identified in avian fertilized egg yolk subfractions F7.11, F7.18, F7.21, FS.10, FS.11, and / or FS.22, or synthetic derivatives thereof.

[0065] "Subfraction F7.11" refers to a fraction obtained by resuspension of the pellet generated by centrifugation of the supernatant generated by centrifugation of solubilized egg yolk granules at 5000 rcf for 15 minutes at 7500 rcf for 15 minutes (designated F7 fraction), which was further subfractionated by size exclusion chromatography, corresponding to the 11 ml of fluid collected after passing the F7 fraction through a size exclusion column.

[0066] "Subfraction F7.18" refers to a fraction obtained by resuspension of the pellet generated by centrifugation of the supernatant generated by centrifugation of solubilized egg yolk granules at 5000 rcf for 15 minutes at 7500 rcf (designated F7 fraction), which was further subfractionated by size exclusion chromatography, corresponding to the 18 ml of fluid collected after passing the F7 fraction through a size exclusion column.

[0067] 「"Subfraction F7.21" refers to a fraction obtained by resuspension of the pellet generated by centrifugation of the supernatant generated by centrifugation of solubilized egg yolk granules at 5000 rcf for 15 minutes at 7500 rcf for 15 minutes (designated F7 fraction), which was further subfractionated by size exclusion chromatography, corresponding to the 21 ml of fluid collected after passing the F7 fraction through a size exclusion column.

[0068] "Subfraction FS.10" refers to a fraction containing the supernatant obtained by centrifugation of solubilized egg yolk granules at 5000 rcf for 15 minutes at 7500 rcf (referred to as the FS fraction), which was further subfractionated by size exclusion chromatography, corresponding to the 10 ml of fluid collected after passing the FS fraction through a size exclusion column.

[0069] "Subfraction FS.11" refers to a fraction containing the supernatant obtained from centrifugation of solubilized egg yolk granules at 5000 rcf for 15 minutes at 7500 rcf (referred to as the FS fraction), which was further subfractionated by size exclusion chromatography, corresponding to the 11 ml of fluid collected after passing the FS fraction through a size exclusion column.

[0070] "Subfraction FS.22" refers to a fraction containing the supernatant obtained from centrifugation of solubilized egg yolk granules at 5000 rcf for 15 minutes at 7500 rcf (referred to as the FS fraction), which was further subfractionated by size exclusion chromatography, corresponding to the 21st / 22nd ml of fluid collected by passing the FS fraction through a size exclusion column.

[0071] In one non-limiting embodiment, the composition comprises a powder prepared by drying one or more subfractions of F7.11, F7.18, F7.21, FS.10, FS.11, and / or FS.22.

[0072] In one non-limiting embodiment, the composition comprises one or more myogenic compounds comprising one or more peptide sequences set forth in Table 2. In one non-limiting embodiment, the myogenic compound is a protein or a myogenically active fragment thereof comprising a peptide sequence set forth in Table 2. In one non-limiting embodiment, the myogenic compound is a protein selected from gelsolin, actin-depolymerizing factor, vimentin, SERPIN domain-containing protein also known as pigment epithelium-derived factor (PEDF), hepatocyte growth factor activator, chicken nucleoside diphosphate kinase, inter-alpha trypsin inhibitor heavy chain, keratin type II cytoskeleton cochlea, desmin, apolipoprotein AI, albumin, actin cytoplasmic type 5, actin cytoplasmic 1, vitellogenin-1, actin cytoplasmic 2, IF rod domain-containing protein, vitellogenin-3, glial fibrillary acidic protein, plasminogen, or type II alpha keratin IIA, or a myogenically active fragment thereof.

[0073] In one non-limiting embodiment, the myogenic compound is a protein selected from albumin, ovalbumin, gelsolin, lysozyme C, an SMB domain-containing protein, transthyretin, an IG-like domain-containing protein, a fibrinogen C-terminal domain-containing protein, plasminogen, phosvitin (VTG2), a peptidase S1 domain-containing protein, or fibrinogen C, or a myogenically active fragment thereof.

[0074] In one non-limiting embodiment, the myogenic compound is a protein that exhibits at least 70%, 80%, 90%, 95% or 99% sequence identity to a peptide sequence or protein identified herein and exhibits myogenic activity.

[0075] In one non-limiting embodiment, the composition comprises one or more synthetic derivatives of myogenic compounds identified in avian fertile egg yolk subfractions F7.11, F7.18, F7.21, FS.10, FS.11, and / or FS.22, or comprising one or more peptide sequences set forth in Table 2. In one embodiment, the synthetic derivatives are prepared by recombinant protein expression using well-established methods. In one non-limiting embodiment, the synthetic derivative is a recombinant protein or myogenically active fragment thereof comprising a peptide sequence set forth in Table 2. In one non-limiting embodiment, the synthetic derivative is a recombinant protein or myogenically active fragment thereof that exhibits at least 70%, 80%, 90%, 95%, or 99% sequence identity to a peptide sequence or protein identified herein and exhibits myogenic activity.

[0076] As used herein, "myogenically active fragment" or "myogenically active peptide fragment" refers to a peptide sequence that has a shorter amino acid sequence than the full-length protein but maintains the same myogenic activity as the full-length protein.

[0077] In one non-limiting embodiment, the myogenic compound is a protein or myogenically active peptide fragment thereof similar to those identified herein, but derived from a species alternative to chicken, such as, but not limited to, human, canine, feline, equine, bovine, ovine, porcine, and primate.

[0078] In one non-limiting embodiment, the myogenic compound is a recombinant human protein or a myogenically active peptide fragment thereof.

[0079] We investigated the myogenic effects of several individual compounds identified herein, namely, chicken SERPIN domain-containing protein, also called pigment epithelium-derived factor (PEDF), and chicken nucleoside diphosphate kinase, as well as its human recombinant counterpart, SERPINF1, and human recombinant nucleoside diphosphate kinase, also called NME2, as well as FORTETROPIN, on the proliferation of mouse C2C12 cells and activation of the MLC1f promoter by secreted Gaussia luciferase.

[0080] The chicken SERPIN domain-containing protein obtained from the ELISA kit significantly increased GLucocyte activity in the C2C12 cell line by 124-145% after 4 days of treatment. The vehicle for the chicken nucleoside diphosphate kinase protein from the ELISA kit was toxic to C2C12 cells, so the myogenic activity of this protein could not be assessed with the available material. However, both its human recombinant protein, SERPINF1, and a human recombinant nucleoside diphosphate kinase designated NME2, stimulated statistically significant proliferation of the mouse C2C12 cell line at concentrations ranging from 62.5 to 1000 ng / ml, from 109% to 120% of control for SERPINF1 and from 107% to 118% of control for NME2, respectively. Furthermore, human recombinant SERPINF1 protein statistically significantly activated the MLC1f promoter via secreted GLuc in the C2C12 cell line after 3 days of treatment (128% to 115% at 250 ng / ml and 125 ng / ml, respectively) and after 7 days of treatment (141% to 135% at 250 ng / ml to 15.6 ng / ml). Human recombinant NME2 protein also statistically significantly activated the MLC1f promoter via secreted GLuc in the C2C12 cell line after 3 days of treatment (135.8% to 121.2% at 250 ng / ml and 125 ng / ml, respectively) and after 7 days of treatment (126.4% to 113% at 250 ng / ml to 15.6 ng / ml). Protein extracts from FORTETROPIN at different pH levels and different incubation times also significantly increased GLuc activity in the C2C12 cell line (133–162%) after 4 days of treatment.

[0081] Furthermore, the presence of chicken SERPIN domain-containing protein, also known as pigment epithelium-derived factor (PEDF), and chicken nucleoside diphosphate kinase in FORTETROPIN, a myogenically active fertilized egg yolk product, was confirmed by ELISA assays, with the highest concentration of chicken SERPIN domain-containing protein extracted from 1 gram of FORTETROPIN being 140 ng and the highest concentration of chicken nucleoside diphosphate kinase extracted from 1 gram of FORTETROPIN being 1270 ng.

[0082] The myogenic compounds identified herein can be formulated into nutritional and / or pharmaceutical compositions for use in increasing muscle mass in mammals. In one non-limiting embodiment, the nutritional and / or pharmaceutical compositions comprise a plurality of the myogenic compounds identified herein. In one non-limiting embodiment, the nutritional and / or pharmaceutical compositions further comprise powdered egg yolk. In one non-limiting embodiment, the nutritional and / or pharmaceutical compositions further comprise FORTETROPIN. FORTETROPIN is a product derived from fertilized egg yolk used as a dietary or nutritional supplement (MYOS ​​CORP., Cedar Knolls, NJ). Methods for the production of FORTETROPIN are disclosed in U.S. Pat. No. 8,815,320, the teachings of which are incorporated herein by reference in their entirety. In another embodiment, the nutritional and / or pharmaceutical compositions further comprise spray-dried egg yolk powder as described in U.S. Patent Application No. 16 / 151,601, the disclosure of which is incorporated herein by reference in its entirety. In one non-limiting embodiment, the nutritional supplement and / or pharmaceutical composition increases muscle growth in a mammal similar to powdered egg yolk. In one non-limiting embodiment, the nutritional supplement and / or pharmaceutical composition increases muscle growth to a greater extent than powdered egg yolk.

[0083] In compositions comprising multiple myogenic compounds as disclosed herein, the compounds may be additive in myogenic activity or synergistic in myogenic activity, meaning more than an additive effect.

[0084] As one of ordinary skill in the art will appreciate upon reading this disclosure, the compositions described herein can be formulated for administration to a mammal by any conventional means, including, but not limited to, oral or buccal.

[0085] Additionally, the compositions described herein may be formulated into any suitable dosage form, including, but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions and the like, solid oral dosage forms, sustained release formulations, fast dissolve 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, for oral ingestion by an individual in need thereof.

[0086] Oral preparations can be obtained by mixing one or more solid excipients with one or more compounds described herein, optionally grinding the resulting mixture to obtain tablets or dragee cores, and then processing the resulting granules with the addition of suitable excipients.Suitable excipients include, for example, fillers such as sugars including glucose, fructose, lactose, sucrose, mannitol, sorbitol, stevia extract, or sucralose; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate.If necessary, disintegrants such as cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, alginic acid, or salts thereof, such as sodium alginate, can be added.

[0087] The dragee cores are provided with a suitable coating. For this purpose, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and concentrated sugar solutions, which may contain suitable organic solvents or solvent mixtures, may be used. Dyes or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0088] Orally usable preparations include push-fit capsules made of gelatin, soft sealed capsules made of gelatin, and plasticizers such as glycerol or sorbitol. Push-fit capsules may contain active ingredients mixed with fillers such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. All preparations for oral administration should be in a dosage suitable for such administration.

[0089] In some embodiments, the solid dosage forms disclosed herein may be in the form of tablets (including suspension tablets, fast-dissolving tablets, bite-disintegration tablets, rapid-disintegration tablets, effervescent tablets, or caplets), pills, powders (including sterile-packaged powders, dispensable powders, or effervescent powders), capsules (including both soft and hard capsules, e.g., capsules made from animal-derived gelatin or plant-derived HPMC, or "sprinkle capsules"), solid dispersions, solid solutions, pellets, or granules. In other embodiments, the pharmaceutical formulation is in the form of a powder. In yet other embodiments, the pharmaceutical formulation is in the form of a tablet. Furthermore, the formulations described herein may be administered in the form of a single capsule or multiple capsules. In some embodiments, the formulation is administered in two, three, or four capsules or tablets.

[0090] Soft gel or soft gelatin capsules may be prepared, for example and without limitation, by dispersing the formulation in a suitable vehicle (vegetable oil is commonly used) to form a thick mixture. This mixture is then encapsulated with a gelatin-based film using techniques and machinery known in the soft gel industry. The industrial units thus formed are then dried to a constant weight.

[0091] In some embodiments, the formulation may include other medicinal or pharmaceutical agents, carriers, diluents, dispersing agents, suspending agents, thickening agents, adjuvants, such as preservatives, stabilizing agents, wetting or emulsifying agents, solution-enhancing agents, and / or buffers. In addition, the formulation may also include other therapeutically valuable substances.

[0092] The formulations described herein may include one or more myogenic compounds and one or more nutraceutical or pharmaceutically acceptable additives, such as compatible carriers, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrants, dispersants, surfactants, lubricants, coloring agents, diluents, solubilizers, humectants, plasticizers, stabilizers, penetration enhancers, wetting agents, antifoaming agents, antioxidants, preservatives, or one or more combinations thereof. In yet another aspect, a film coating is applied around the formulation of the compounds described herein using standard coating procedures. In one embodiment, some or all of the particles of the compounds described herein are coated. In another embodiment, some or all of the particles of the compounds described herein are microencapsulated. In yet another embodiment, the particles of the compounds described herein are not microencapsulated or coated.

[0093] In certain embodiments, the compositions may also contain one or more pH adjusters or buffers, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts required to maintain the pH of the composition within an acceptable range.

[0094] In other embodiments, the composition may also include one or more salts in an amount necessary to bring the osmolality of the composition within 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.

[0095] Formulations containing one or more myogenic compounds as described herein may be manufactured in a conventional manner, such as by, for example only, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or compressing processes.

[0096] In certain embodiments, the compositions provided herein may also contain one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as mercury-containing compounds such as mercury-containing compounds and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0097] The formulations described herein may benefit from antioxidants, metal chelators, thiol-containing compounds and other general stabilizing agents. Examples of such stabilizers 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.

[0098] Binders that impart cohesive properties may also be used. Examples include, but are not limited to, alginic acid and its salts; cellulose derivatives such as carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, ethylcellulose, and microcrystalline cellulose; microcrystalline dextrose; amylose; magnesium aluminum silicate; polysaccharide acids; bentonite; gelatin; polyvinylpyrrolidone / vinyl acetate copolymer; crospovidone; povidone; starch; pregelatinized starch; sugars such as tragacanth, dextrin, sucrose, glucose, dextrose, molasses, mannitol, sorbitol, xylitol, and lactose; acacia, tragacanth, ghatti gum, mucilage of isapol husks, polyvinylpyrrolidone, larch arabogalactan, polyethylene glycol, waxes, and natural or synthetic gums such as sodium alginate.

[0099] Generally, binder levels of 20-70% are used in powder-filled gelatin capsule formulations. Binder levels in tablet formulations vary depending on the process used: direct compression, wet granulation, roller compaction, or the use of other excipients such as fillers that can themselves act as moderate binders.

[0100] A skilled formulator can determine the binder level for a formulation, but binder usage levels of up to 70% are common in tablet formulations.

[0101] The composition may further comprise a relatively non-toxic chemical compound carrier or an agent that promotes the uptake of the compound into cells or tissues. Non-limiting examples include binders, suspending agents, disintegrating agents, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, etc. 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, glycerin, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose, microcrystalline cellulose, lactose, mannitol, etc.

[0102] Dispersing agents and / or viscosity modifiers include materials that control the diffusion and homogeneity of the compound in the liquid medium, or through granulation or blending processes. In some embodiments, these agents also enhance the effectiveness of the coating or eroding matrix. Non-limiting examples of diffusion enhancers / dispersing agents include hydrophilic polymers, electrolytes, Tween, PEG, polyvinylpyrrolidone, and carbohydrate-based dispersants such as hydroxypropyl cellulose (e.g., HPC, HPC-SL, and HPC-L), hydroxypropyl methylcellulose (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M), sodium carboxymethylcellulose, methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate stearate (HPMCAS), amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone / vinyl acetate copolymer (S630), 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamer, block copolymers of ethylene oxide and propylene oxide;and poloxamine, a tetrafunctional block copolymer derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine, polyvinylpyrrolidone KL2, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone / vinyl acetate copolymer (S-630), polyethylene glycol, for example, the polyethylene glycol may 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 Dispersants include sugars, polysorbate-80, sodium alginate (e.g., gums such as tragacanth gum, acacia gum, guar gum, and xanthan gum), sugars, celluloses such as sodium carboxymethylcellulose, methylcellulose, and sodium carboxymethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, carbomer, polyvinyl alcohol (PVA), alginate, chitosan, and combinations thereof. Plasticizers such as cellulose and triethylcellulose can also be used as dispersants. Particularly useful dispersants for liposomal and self-emulsifying dispersions are dimyristoyl phosphatidylcholine, natural egg-derived phosphatidylcholine, natural egg-derived phosphatidylglycerol, cholesterol, and isopropyl myristate.

[0103] A combination of one or more erosion facilitators and one or more diffusion facilitators may also be used in the present compositions.

[0104] The composition of the present invention may further comprise a diluent that is used to dilute the compound of interest before delivery.Diluents can also be used to stabilize the compound because they can provide a more stable environment.Salts dissolved in buffer solutions (which can also provide pH control or maintenance) are used as diluents in the art, including but not limited to phosphate buffered saline.In certain embodiments, the diluent increases the bulk of the composition to facilitate compression or creates sufficient bulk for a homogeneous blend for capsule filling. Such compounds include, for example, 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, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, powdered sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates; hydrolyzed grain solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; sodium chloride; inositol, bentonite, and the like.

[0105] The composition may further comprise an enteric coating, which is a material that remains substantially intact in the stomach but dissolves in the small intestine or colon to release the myogenic compound. Generally, enteric coatings comprise a polymeric material that prevents release in the low pH environment of the stomach but ionizes at a higher pH, typically pH 6-7, and then dissolves sufficiently in the small intestine or colon to release the active agent.

[0106] In addition, the composition may contain an erosion enhancer, which is a material that controls the erosion of a particular material in gastrointestinal fluids. Erosion enhancers are generally known to those skilled in the art. Exemplary erosion enhancers include, for example, hydrophilic polymers, electrolytes, proteins, peptides, and amino acids.

[0107] Fillers including compounds such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc. Suitable fillers 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, starch, pregelatinized starch, hydroxypropyl methylcellulose (HPMC), hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate stearate (HPMCAS), sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc.

[0108] Additionally, flavoring and / or sweetening agents may be used in the composition, such as acacia syrup, acesulfame K, alitame, anise, apple, aspartame, banana, bavarois cream, berry, blackcurrant, 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, scillamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger, glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey, isomalt, lemon, lime, lemon cream, monoammonium glycyrrhizinate, maltol, mannitol, maple, marshmallow, menthol, mint cream, mixed berry, neohesperidin DC, neotame, The flavoring ingredients may include 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, talc, cylitol, sucralose, sorbitol, Swiss cream, tagatose, tangerine, thaumatin, tutti frutti, vanilla, walnut, watermelon, wild cherry, wintergreen, xylitol, or any combination of these flavoring ingredients, such as 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.

[0109] The composition may further comprise a lubricant and / or glidant to prevent, reduce, or inhibit adhesion or friction of materials. Non-limiting examples of lubricants include stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, hydrocarbons such as mineral oil or hydrogenated vegetable oils such as hydrogenated soybean oil, higher fatty acids and their alkali metal and alkaline earth metal salts (aluminum, calcium, magnesium, zinc, etc.), stearic acid, sodium stearate, glycerol, talc, wax, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxypolyethylene glycol, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica, starch such as corn starch, silicone oil, surfactants, etc.

[0110] Plasticizers, compounds used to soften microencapsulated materials, or film coatings used to reduce brittleness, may also be included in the composition.Suitable examples of 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, triethylcellulose, and triacetin.In some embodiments, plasticizers can also function as dispersing agents or wetting agents.

[0111] The composition may further comprise a solubilizer such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, docusate sodium, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide.

[0112] In addition, the compositions may contain stabilizers such as antioxidants, buffers, acids, preservatives, and the like.

[0113] Suitable suspending agents for use in the solid dosage forms described herein 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 has a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, vinylpyrrolidone / vinyl acetate copolymer (S630), sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ... and the like. Examples of suitable celluloses include cellulose, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums such as tragacanth gum, acacia gum, guar gum, xanthans including xanthan gum, sugars, celluloses such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, and the like.

[0114] Surfactants may also be included, including compounds such as sodium lauryl sulfate, docusate sodium, Tween, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, poloxamer, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, etc. Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40, etc. In some embodiments, surfactants may be included to enhance physical stability or for other purposes.

[0115] Viscosity enhancers may also be included, including, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and mixtures thereof.

[0116] Additionally, wetting agents may be included in these compositions, including compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, Tween 80, Vitamin E TPGS, ammonium salts, and the like.

[0117] In some embodiments, solid dosage forms, e.g., tablets, capsules, are prepared by combining the myogenic compounds described herein with one or more pharmaceutical excipients to form a bulk blend composition. These bulk blend compositions are referred to as homogeneous, meaning that the particles of the myogenic compound are uniformly dispersed throughout the composition, allowing the composition to be easily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules.

[0118] Conventional techniques include, for example, a combination of one or more of the following methods: (1) dry blending, (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).

[0119] It should be understood that there is considerable overlap in the excipients used in the solid dosage forms described herein, and therefore the above excipients should be taken as merely exemplary of the types of excipients that may be included, and not as limiting.

[0120] Capsules may be prepared, for example, by placing a bulk blend of the formulation of the above-mentioned compound inside the capsule. In some embodiments, the formulation (non-aqueous suspension and solution) is placed in a soft gelatin capsule. In other embodiments, the formulation is placed in a non-gelatin capsule, such as a standard gelatin capsule or a capsule containing HPMC. In other embodiments, the formulation may be placed in a sprinkle capsule, and the capsule may be swallowed whole, or the capsule may be opened and the contents may be sprinkled on food before eating. In some embodiments, the therapeutic dose is divided into multiple (e.g., 2, 3, or 4) capsules. In some embodiments, the entire dose of the formulation is delivered in capsule form.

[0121] In another aspect, the dosage form may comprise a microencapsulated formulation. In some embodiments, one or more other compatible materials are present in the microencapsulated material. Exemplary materials include, but are not limited to, pH adjusters, erosion promoters, antifoaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegrating agents, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0122] Materials useful for microencapsulation as described herein include materials that sufficiently isolate the compound from other incompatible excipients. Materials that are compatible with the myogenic compound are those that delay the release of the myogenic compound in vivo.

[0123] In other embodiments, the formulations described herein that include a myogenic compound are solid dispersions. Methods for producing such solid dispersions are known in the art, including, but not limited to, U.S. Patent Nos. 4,343,789, 5,340,591, 5,456,923, 5,700,485, 5,723,269, and U.S. Application No. 2004 / 0013734.

[0124] In yet another embodiment, the formulations described herein are solid solutions. Solid solutions incorporate a substance along with an active agent and other excipients; the mixture is heated to dissolve the drug; the resulting composition is then cooled to yield a solid blend that can be further formulated, added directly to capsules, or compressed into tablets. Methods for producing such solid solutions are known in the art, including, but not limited to, U.S. Patent Nos. 4,151,273, 5,281,420, and 6,083,518.

[0125] In some embodiments, the solid dosage forms described herein can be formulated as enterically coated delayed-release oral dosage forms, i.e., oral dosage forms of the pharmaceutical compositions described herein that utilize an enteric coating to affect release in the small intestine of the gastrointestinal tract. The enteric coated dosage form can be a compressed, molded, or extruded tablet / mold (coated or uncoated) containing granules, powders, pellets, beads, or particles of the active ingredient and / or other composition components. The enteric coated oral dosage form can also be a capsule (coated or uncoated) containing pellets, beads, or granules of the solid carrier or composition.

[0126] The term "delayed release" as used herein refers to delivery that allows release to occur at a generally predictable location in the intestinal tract farther than would occur if the delayed release were not present. In some embodiments, the delayed release method is a coating. Any coating should be applied with a sufficient thickness so that the entire coating does not dissolve in gastrointestinal fluid at a pH below about 5, but dissolves at a pH above about 5. Any anionic polymer that exhibits a pH-dependent dissolution profile can be used as the enteric coating for the methods and compositions described herein, and is expected to achieve delivery to the lower gastrointestinal tract.

[0127] In some embodiments, a formulation is provided comprising particles of a myogenic compound described herein and at least one dispersing or suspending agent for oral administration to a subject. The formulation may be a powder and / or granules for suspension, which, when mixed with water, results in a substantially uniform suspension.

[0128] The liquid pharmaceutical dosage form for oral administration may be an aqueous suspension selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersion, emulsion, solution, elixir, gel, and syrup.For example, see Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp.754-757(2002).In addition to the particles of myogenic compound, the liquid dosage form may also contain additives such as (a) disintegrant; (b) dispersant; (c) wetting agent; (d) at least one preservative; (e) viscosity enhancer; (t) at least one sweetener, and (g) at least one flavoring agent.In some embodiments, the aqueous dispersion may further contain a crystallization inhibitor.

[0129] The aqueous suspensions and dispersions described herein can remain homogeneous for at least 4 hours as defined in the USP Pharmacists' Pharmacopeia (2005 edition, chapter 905). Homogeneity should be determined by a sampling method consistent with determining the homogeneity of the entire composition. In one embodiment, the aqueous suspension can be resuspended into a homogeneous suspension by physical agitation lasting less than 1 minute. In another embodiment, the aqueous suspension can be resuspended into a homogeneous suspension by physical agitation lasting less than 45 seconds. In yet another embodiment, the aqueous suspension can be resuspended into a homogeneous suspension by physical agitation lasting less than 30 seconds. In yet another embodiment, agitation is not required to maintain a homogeneous aqueous dispersion.

[0130] 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. The preservatives used herein are incorporated into the dosage form at concentrations sufficient to inhibit the growth of microorganisms.

[0131] In one non-limiting embodiment, the aqueous liquid dispersion can include a sweetener or flavoring agent at a concentration ranging from about 0.005% to about 0.5% by volume of the aqueous dispersion. In yet another embodiment, the aqueous liquid dispersion can include a sweetener or flavoring agent at a concentration ranging from about 0.01% to about 1.0% by volume of the aqueous dispersion.

[0132] In addition to the above-mentioned additives, liquid preparations can also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers.Exemplary emulsifiers include ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, sodium lauryl sulfate, docusate sodium, cholesterol, cholesterol esters, taurocholic acid, phosphatidylcholine, oils such as cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, fatty acid esters of sorbitan, or the mixture of these substances.

[0133] In some embodiments, the formulations described herein may be self-emulsifying drug delivery systems (SEDDS). An emulsion is a dispersion of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are produced by strong mechanical dispersion.

[0134] In contrast to emulsions and microemulsions, SEDDS spontaneously form emulsions when added to excess water without any external mechanical dispersion or agitation. The advantage of SEDDS is that only gentle mixing is required to disperse the droplets throughout the solution. Additionally, water or the aqueous phase can be added immediately before administration, ensuring the stability of unstable or hydrophobic active ingredients. Therefore, SEDDS provide an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. SEDDS may also improve the bioavailability of hydrophobic active ingredients. Methods for producing self-emulsifying dosage forms are known in the art, including, but not limited to, U.S. Patent Nos. 5,858,401, 6,667,048, and 6,960,563.

[0135] Buccal formulations containing myogenic compounds may be administered using various formulations known in the art. For example, such formulations include, but are not limited to, those described in U.S. Patent Nos. 4,229,447, 4,596,795, 4,755,386, and 5,739,136. Additionally, the buccal dosage forms described herein may further comprise a biodegradable (hydrolyzable) polymer carrier that also serves to adhere the dosage form to the buccal mucosa. The buccal dosage form is designed to gradually erode over a predetermined period of time. As will be appreciated by those skilled in the art, buccal drug delivery avoids the disadvantages encountered with oral drug administration, such as slow absorption, degradation of the active agent by fluids present in the gastrointestinal tract, and / or initial inactivation in the liver. It is understood that virtually any biodegradable (hydrolyzable) polymer carrier may be used, as long as the desired drug release profile is not compromised and the carrier is compatible with the myogenic compound and other components that may be present in the buccal dosage unit. Generally, the polymer carrier comprises a hydrophilic (water-soluble and water-swellable) polymer that adheres to the moist surface of the buccal mucosa.Other components, including but not limited to, disintegrants, diluents, binders, lubricants, flavorings, coloring agents, preservatives, etc., may also be incorporated into the buccal dosage forms described herein.For buccal or sublingual administration, the composition may take the form of a tablet, lozenge, or gel formulated in a conventional manner.

[0136] In certain embodiments, delivery systems for pharmaceutical compounds, such as, for example, liposomes and emulsions, may be employed. In certain embodiments, the compositions provided herein may also include a mucoadhesive polymer selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran.

[0137] In one non-limiting embodiment, the nutritional and / or pharmaceutical composition may further comprise powdered egg yolk. In one non-limiting embodiment, the nutritional and / or pharmaceutical composition may further comprise FORTETROPINE.

[0138] The following non-limiting examples further illustrate the present invention. [Example]

[0139] example Example 1: Evaluation of the effects of proteins extracted from chicken SERPINF1, chicken NME2, and FORTETROPIN on activation of the MLC1f promoter by secreted Gaussia luciferase material and method compound

[0140] The compounds tested included FORTETROPIN provided by MYOS Corp.; chicken nucleoside diphosphate kinase B (NME2) from an ELISA kit standard (cat#MBS7244605; MyBioSource); chicken SERPINF1 or chicken pigment epithelium-derived factor (PEDF) standard from an ELISA kit (cat#MBS264953; MyBioSource); recombinant mouse IGF1 from Peprotech; and dexamethasone from TCI America.

[0141] cell culture

[0142] For cell culture, we used the C2C12 mouse myoblast cell line, which carries the Gaussia luciferase (GLuC) reporter gene under the muscle-specific promoter of the fast myosin light chain MLCf1. Cells were grown in growth medium consisting of 4.5g / L DMEM supplemented with 10% v / v fetal bovine serum and 1U / ml penicillin-streptomycin. C2C12 myoblast cell line was plated at 25,000 cells / cm in 48-well plates. 2The cells were seeded at a density of 1000 kJ / well and cultured in growth medium with medium changes once daily for the first three days. Three wells were used for each condition. On day three, the medium was changed to differentiation medium consisting of DMEM 4.5 g / L glucose supplemented with 1% FBS and 1 U / ml penicillin-streptomycin. To confirm that the experimental conditions were appropriate and the experiment was successful, activation of the reporter gene GLuc in the cell lines was tested under three different treatment conditions: a vehicle / medium control; a positive control containing 50 ng / ml insulin-like growth factor (IGF1); and a negative control containing 50 μg / ml dexamethasone.

[0143] Protein extraction from FORTETROPIN powder

[0144] Protein extraction was performed according to the established protocol of Chalamaiah et al. (Food Chem 2018 268:369-377) to ensure accurate and reliable results. Briefly, 0.2 or 2 grams of powdered FORTETROPIN was carefully reconstituted in 10 ml of phosphate-buffered saline at various pH levels: 4, 7, and 10. The mixture was then incubated at room temperature for 3 hours or left for 48 hours. Periodic vortexing was performed every 30 minutes during the 3-hour incubation to promote effective extraction.

[0145] After the incubation period was completed, the solution was carefully centrifuged at 2,000 RPM and transferred to an Eppendorf tube. For further use of the extracted proteins, the pH of the solution was neutralized to pH 7, after which they were subjected to a second centrifugation step at 15,000 RPM. The extracted proteins were then used in ELISA analysis to measure the concentrations of SERPINF1 and NME2.

[0146] FORTETROPIN extracts were also used in cytotoxicity assays and activation of the MLC1f promoter reporter gene assay, allowing for a comprehensive evaluation of their biological activity.

[0147] ELISA

[0148] ELISA assays were performed according to the manufacturer's protocol to measure the concentrations of chicken SERPINF1 / pigment epithelium-derived factor (PEDF) and chicken nucleoside diphosphate kinase B (NME2) in FORTETROPIN extracts.

[0149] The ELISA kits used were a chicken nucleoside diphosphate kinase B (NME2) ELISA kit (cat#MBS7244605; MyBioSource) and a chicken pigment epithelium-derived factor (PEDF) ELISA kit (cat#MBS264953; MyBioSource).

[0150] Cytotoxicity assay

[0151] Cytotoxicity assessment is a crucial step in drug development. Cytotoxicity refers to a drug's ability to damage or kill cells. Using high drug concentrations without assessing cytotoxicity may result in cell death, making it difficult to determine whether a drug is toxic or has an antiproliferative effect. Cytotoxicity was assessed using a Cell Titer96 Aqueous One (Promega, WI, USA) assay according to the manufacturer's instructions. Cells were seeded at a density of 10,000 cells / well in 96-well plates and cultured overnight. Treatment was performed in triplicate with different compound doses for 24 hours. Colorimetric analysis was performed using absorbance at 490 nm. Inhibition of viability by more than 20% of the control value was considered cytotoxic.

[0152] Assessment of MLC1f promoter activation by secreted Gaussia luciferase (GLuc)

[0153] To assess activation of the MLCf1 promoter by secreted GLuc, MLC1f reporter C2C12 myoblasts were initially cultured at 25,000 cells / cm. 2The cells were seeded into 48-well plates at a density of 1000 and cultured in growth medium (DMEM 4.5 g / L glucose with 10% FBS and 1 μg / ml penicillin-streptomycin) for the first 3 days at 37°C in a cell culture incubator with 5% CO2, with daily medium changes.

[0154] On day 3, the medium was switched to differentiation medium containing DMEM 4.5g / L glucose with 1% FBS and 1u / ml penicillin-streptomycin. Protein extracts from 2% and 20% FORTETROPIN at different pH levels and SERPINF1 protein standard from the SELISA kit were evaluated.

[0155] Additionally, three control groups (n = 3) were included in the study: 1) cells supplemented with 50 ng / mL mouse IGF1 (Peprotech) to promote myoblast differentiation, 2) cells supplemented with 50 μg / mL dexamethasone to block myoblast differentiation, and 3) cells fed with basal differentiation medium without any additives for vehicle control.

[0156] After 1, 4, and 7 days of incubation, supernatants were collected from the cells and stored at −80° C. GLuc assays were performed according to the manufacturer's protocol (Thermo Fisher Scientific; Pierce Gaussia Luciferase Glow Assay Kit cat#16161).

[0157] statistics

[0158] The mean ± standard deviation of each group was calculated using GraphPad Prism 6.05 software (GraphPad Software, La Jolla, CA, USA), and statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison post-hoc test. *P<0.05 and **P<0.01.

[0159] result Cytotoxicity analysis

[0160] Dexamethasone at concentrations up to 100 μg / ml and 12.5 μg / ml was not cytotoxic to the cultured C2C12 cell line.

[0161] Using diluted NME2 protein from the ELISA kit, 88.3% to 80% cytotoxicity was observed. However, toxicity remained even at 0% concentration of the protein from the ELISA kit, suggesting that the presence of the proprietary supplements required for the ELISA, rather than the NME2 protein, was harmful to the cell culture.

[0162] The SERPINF1 / pigment epithelium-derived factor (PEDF) lyophilized standard from the ELISA kit is cytotoxic only at a concentration of 10 ng / ml.

[0163] Proteins extracted from FORTETROPIN at different pH levels were cytotoxic when undiluted and diluted 1:2, but not at a 1:4 dilution.

[0164] Cytotoxicity experiments were used to identify the highest non-toxic concentrations of the tested substances, and these concentrations were then selected for further evaluation of their biological activity in the C2C12 cell line.

[0165] Assessment of NME2 and SERPINF1 protein concentrations using ELISA after protein extraction from FORTETROPIN powder under various pH and incubation times

[0166] Based on the ELISA data, maximum NME2 was extracted from 2% FORTETROPIN at room temperature after 48 hours of protein extraction at pH 10. It is estimated that 1 gram of FORTETROPIN contains approximately 1,270 ng of NME2.

[0167] Based on the ELISA data, maximum SERPINF1 was extracted from 2% FORTETROPIN at room temperature after 48 h of protein extraction at pH = 10. It is estimated that 1 gram of FORTETROPIN contains approximately 140 ng of SERPINF1.

[0168] Evaluation of the effects of proteins extracted from SERPINF1 and FORTETROPIN on activation of the MLC1f promoter by secreted Gaussia luciferase

[0169] Activation of the MLC1f promoter was assessed by various treatments using secreted Gaussia luciferase assays. Monitoring the level of secreted Gaussia luciferase allows for quantification of promoter activity under various experimental conditions. C2C12 cell lines were cultured in 48-well plates for 5 days with daily medium changes. For the first 2 days, the medium was replaced with proliferation medium. For the last 3 days, the medium was replaced with differentiation medium. On day 5 of the experiment, 50 ng / ml IGF1 was used as a positive control, 50 μg / ml dexamethasone was used as a negative control, and medium was used as a vehicle control. Supernatant samples were collected on days 3, 4, and 7 for G-luciferase assays.

[0170] Luciferase activity was assessed directly and compared as a measure of promoter activity without normalization to protein concentration.

[0171] On day 1, no induction of luciferase activity was observed even with the positive control IGF1.

[0172] Four days after treatment, significant activation of MLC1f promoter-driven luciferase by 144.6±5.1 was observed in response to the positive control IGF1.

[0173] In contrast, the vehicle control and negative control dexamethasone had no effect, demonstrating luciferase activity of 106.5 ± 12.4 and 102 ± 3.4, respectively. These results demonstrate that the experimental setup was correct and the experiment performed as expected, validating the reliability of our study.

[0174] The SERPINF1 protein from the ELISA kit significantly increased GLuc activity in the C2C12 cell line by 124-145% after 4 days of treatment. There was an inverse dose-dependent activation of the GLuc reporter, suggesting interference from the proprietary compound in the ELISA kit. Dilution of the protein showed increased activity, while lower concentrations showed decreased interference.

[0175] Protein extracts of FORTETROPIN at different pH levels and different incubation times significantly increased GLuc activity (133–162%) in the C2C12 cell line after 4 days of treatment.

[0176] By day 7, there was an overall decrease in luciferase activity in most treated samples. However, in the positive control IGF1-treated cells, even at low concentrations of SERPINF1 and protein extracted from FORTETROPIN at pH 10, statistically significant differences remained between treated and untreated cells.

[0177] Example 2: Evaluation of the effects of human recombinant proteins SERPINF1 and NME2 on proliferation of mouse C2C12 cells and activation of the MLC1f promoter by secreted Gaussia luciferase material and method Compounds tested

[0178] NME2 human recombinant protein was obtained from MyBioSource (cat#206177). Human pigment epithelium-derived factor (PEDF), a secreted glycoprotein encoded by the SERPINF1 gene, was also obtained from MyBioSource (cat#143337). Recombinant mouse IGF1 was obtained from Peprotech, and dexamethasone was obtained from TCI America.

[0179] Cell culture, cytotoxicity assay, evaluation and statistics of MLC1f promoter activation by secreted Gaussia luciferase (GLuc)

[0180] Cell culture, cytotoxicity assays, evaluation of mLC1f promoter activation by secreted GLuc, and statistics were performed as described in Example 1.

[0181] result Cytotoxicity analysis

[0182] Dexamethasone at concentrations up to 100 μg / ml and 12.5 μg / ml was not cytotoxic to cultured C2C12 cells. Furthermore, human SERPINF1 / pigment epithelium-derived factor (PEDF) and human recombinant NME2 protein were not cytotoxic.

[0183] Human recombinant SERPINF1 stimulated statistically significant proliferation of the murine C2C12 cell line from 120% to 109% of the control at concentrations ranging from 1000 ng / ml to 62.5 ng / ml, respectively.

[0184] Human recombinant NME2 stimulated statistically significant proliferation of the murine C2C12 cell line from 118% to 107% of control at concentrations ranging from 1000 ng / ml to 62.5 ng / ml, respectively.

[0185] After conducting cytotoxicity experiments, the highest non-toxic concentrations of the tested substances were identified, and then these concentrations, their biological activity in the C2C12 cell line, were selected for further evaluation.

[0186] Assessment of the effect of recombinant human SERPINF1 and NME2 proteins on activation of the MLC1f promoter by secreted Gaussia luciferase.

[0187] Activation of the MLC1f promoter by various treatments was assessed using secreted Gaussia luciferase (GLuc) assay. By monitoring the levels of secreted GLuc, promoter activity was quantified under various experimental conditions.

[0188] C2C12 cell lines were cultured in 48-well plates for 5 days with daily medium changes. For the first 2 days, the medium was replaced with proliferation medium. For the final 3 days, the medium was replaced with differentiation medium. On day 5 of the experiment, 50 ng / ml IGF1 was used as a positive control, 25 μg / ml dexamethasone was used as a negative control, and medium was used as a vehicle control. Supernatant samples were collected on days 1, 4, and 7 for GLuc assay. Luciferase activity was assessed directly and compared as a measure of promoter activity without normalization for protein concentration.

[0189] On day 1, no induction of luciferase activity was observed even with the positive control IGF1.

[0190] On day 4 of treatment, significant activation of MLC1f promoter-driven luciferase was observed in response to the positive control IGF1, with 172.4 ± 4.8 μM. In contrast, the negative control dexamethasone had no effect, demonstrating a luciferase activity of 100.4 ± 1.4 μM. These results demonstrate that the experimental setup was correct and the experiment functioned as expected, validating the reliability of our study.

[0191] Treatment of mouse C2C12 cell lines with various concentrations of human recombinant SERPINF1 protein activated the MLC1f promoter in a dose-dependent manner. Specifically, human recombinant SERPINF1 protein statistically significantly activated the MLC1f promoter, as measured by secreted GluC, in C2C12 cell lines after 3 days of treatment (115% to 128% at 125 ng / ml and 250 ng / ml, respectively) and 7 days of treatment (135% to 141% at 15.6 ng / ml and 250 ng / ml, respectively).

[0192] Importantly, luciferase activity of the positive control IGF1 decreased on day 7 (from 172% on day 3 to 162% on day 7), while luciferase activity in SERPINF1-treated cells increased (in the 250 ng / ml-treated sample, activity went from 128% on day 3 to 140% on day 7).

[0193] Similar to treatment of mouse C2C12 cell lines with different concentrations of human recombinant SERPINF1 protein, NME2 treatment also resulted in dose-dependent activation of the MLC1f promoter. Specifically, human recombinant NME2 protein statistically significantly activated the MLC1f promoter, as measured via secreted GLuc in C2C12 cell lines, after 3 days of treatment (121.2% to 135.8% at 125 ng / ml and 250 ng / ml, respectively) and after 7 days of treatment (113% to 126.4% at 15.6 ng / ml to 250 ng / ml). At day 7, when luciferase activity of the positive control IGF1 decreased (from 172% on day 3 to 162% on day 7), NME2-treated cells still showed increased activity.

Claims

1. A composition comprising one or more myogenic compounds identified in a fraction of egg yolk, or synthetic derivatives thereof.

2. 2. The composition of claim 1, wherein the egg yolk is an avian egg yolk.

3. The composition according to claim 2, wherein the egg yolk is a fertilized avian egg yolk.

4. 4. The composition of claim 3, wherein the one or more myogenic compounds are identified in avian fertile egg yolk subfractions F7.11, F7.18, F7.21, FS.10, FS.11, and / or FS.

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5. 4. The composition of claim 3, wherein the one or more myogenic compounds are synthetic derivatives of myogenic compounds isolated from avian fertilized egg yolk subfractions F7.11, F7.18, F7.21, FS.10, FS.11, and / or FS.

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6. 2. The composition of claim 1, wherein the one or more myogenic compounds comprise a peptide sequence shown in Table 2 or a peptide sequence having at least 70% sequence identity to a peptide sequence shown in Table 2.

7. The composition of claim 1 , wherein the myogenic compound is a protein or a myogenically active fragment thereof.

8. 8. The composition of claim 7, wherein the protein is selected from gelsolin, actin depolymerizing factor, vimentin, SERPIN domain-containing protein, pigment epithelium-derived factor, chicken nucleoside diphosphate kinase, hepatocyte growth factor activator, inter-alpha trypsin inhibitor heavy chain, keratin type II cytoskeleton cochlea, desmin, apolipoprotein A-I, albumin, actin cytoplasmic type 5, actin cytoplasmic 1, vitellogenin-1, actin cytoplasmic 2, IF-rod domain-containing protein, vitellogenin-3, glial fibrillary acidic protein, plasminogen and / or alpha keratin type II IIA or a myogenically active fragment thereof.

9. 8. The composition of claim 7, wherein the protein is selected from albumin, ovalbumin, gelsolin, lysozyme C, SMB domain-containing protein, transthyretin, IG-like domain-containing protein, fibrinogen C-terminal domain-containing protein, plasminogen, phosvitin (VTG2), peptidase S1 domain-containing protein, and / or fibrinogen C, or a myogenically active fragment thereof.

10. 10. The composition of claim 1, wherein the synthetic derivative is derived from an alternative species of chicken.

11. 11. The composition of claim 10, wherein the alternative species to chicken is selected from humans, dogs, cats, horses, cows, sheep, pigs, or primates.

12. A nutritional composition comprising the composition of any one of claims 1 to 11 and a nutritionally acceptable excipient.

13. A pharmaceutical composition comprising the composition of any one of claims 1 to 11 and a pharmaceutically acceptable excipient.

14. 14. The nutritional supplement composition of claim 12 or the pharmaceutical composition of claim 13, further comprising powdered egg yolk.

15. 14. The nutritional supplement composition of claim 12 or the pharmaceutical composition of claim 13 further comprising FORTETROPIN.

16. A method for increasing muscle mass in a mammal, said method comprising administering to said mammal a composition according to any one of claims 1 to 15.

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