Muscle-derived compounds

Abstract

The present invention provides muscle-derived compounds identified in yolk and synthetic derivatives thereof, nutritional compositions of one or more of these muscle-derived compounds and synthetic derivatives thereof, and the use of these compounds and nutritional compositions in increasing muscle mass in mammals.

Description

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

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

[0003] Professional athletes and the general public are generally interested in increasing muscle mass. In addition, sarcopenia or muscle atrophy is a problem for the elderly, affecting 5 - 13% of people aged 60 to 70 years and 15 - 50% of people over 80 years old (Shafiee et al., Journal of Diabetes & Metabolic Disorders, 2017, 16(1)).

[0004] Previous studies have found that adding protein supplements from fertilized chicken egg yolk extracts to the diet has myogenic benefits (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 yolks have been used as health foods in the past, particularly in Asian cultures, and are known as Balut foods. In addition, studies on canines have shown that diets supplemented with the fertilized egg yolk product FORTETROPIN improved the outcomes of tibial plateau leveling osteotomy by reducing muscle atrophy in the affected limb (White et al., PLoS ONE, 2015(4): p.1 - 10).

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

[0007] Due to the wide range of consumption of egg yolk, extensive research has been carried out on it. As early as 1949, Romanoff et al. described the separation of the serous part and the granular part of the egg (The avian egg, 1949, NewYork: J.Wiley). These experiments and other studies carried out 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 the egg yolk can be easily separated into a serous fraction and a granular fraction by centrifugation (McBee, L. &Cotterill, O., Poultry Science, 1973, Oxford Univ Press Gret Clarendon St.Oxford OX2 6DP, UK), where the serous part and the granular part contain equal amounts of protein and 90%:10% lipids respectively. The serous fluid contains 85% LDL and 15% lipovitellin. The insoluble particles are mainly composed of phosvitin and HDL bound to calcium molecules.

[0008] It is not yet clear which compounds present in the fertilized egg yolk produce the observed myogenic effects. Summary of the Invention

[0009] The present disclosure relates to myogenic compounds identified in separated fractions of fertilized avian egg yolks.

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

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

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

[0013] In a non-limiting embodiment, the myogenic compound is a protein selected from the group consisting of 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-II type cytoskeletal cochleal, desmin, apolipoprotein A-I, albumin, cytoplasmic actin 5, cytoplasmic actin 1, vitellogenin-1, cytoplasmic actin 2, IF rod domain-containing protein, vitellogenin-3, glial fibrillary acidic protein, plasminogen, or type II alpha-keratin IIA or a myogenic active peptide fragment thereof.

[0014] In a non-limiting embodiment, the myogenic compound is a protein selected from the group consisting of 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, or fibrinogen C or a myogenic active fragment thereof.

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

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

[0017] In a non-limiting embodiment, the myogenic compound is a protein or a myogenic active peptide fragment thereof that is similar to the proteins or their myogenic active peptide fragments identified herein but from a non-chicken species, such as but not limited to human, dog, cat, horse, cow, sheep, pig, and primates.

[0018] Another aspect of the present disclosure relates to a nutritional composition and / or a pharmaceutical composition comprising one or more myogenic compounds or their synthetic derivatives identified in isolated fractions of fertilized avian egg yolk and one or more nutritionally and / or pharmaceutically acceptable excipients.

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

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

[0021] In a non-limiting embodiment, the myogenic compound is a protein selected from the following: 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-α-trypsin inhibitor heavy chain, keratin-II cytoskeletal cochlea, desmin, apolipoprotein A-I, albumin, cytoplasmic actin 5, cytoplasmic actin 1, vitellogenin-1, cytoplasmic actin 2, IF rod domain-containing protein, vitellogenin-3, glial fibrillary acidic protein, plasminogen, or type II α-keratin IIA or myogenic active peptide fragments thereof.

[0022] In a non-limiting embodiment, the myogenic compound is a protein selected from the following: 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 SI domain-containing protein, or fibrinogen C or myogenic active fragments thereof.

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

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

[0025] In a non-limiting embodiment, the myogenic compound is a protein or a myogenic active peptide fragment thereof that is similar to the proteins or their myogenic active peptide fragments identified herein but from a non-avian species, such as but not limited to human, dog, cat, horse, cow, sheep, pig, and primates.

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

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

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

[0029] In a non-limiting embodiment, the myogenic compound is a protein selected from the following: 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-II cytoskeletal cochlea, desmin, apolipoprotein A-I, albumin, cytoplasmic actin 5, cytoplasmic actin 1, vitellogenin-1, cytoplasmic actin 2, IF rod domain-containing protein, vitellogenin-3, glial fibrillary acidic protein, plasminogen, or type II alpha-keratin IIA or their myogenic active peptide fragments.

[0030] In a non-limiting embodiment, the myogenic compound is a protein selected from the following: 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 SI domain-containing protein, or fibrinogen C or their myogenic active fragments.

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

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

[0033] In a non-limiting embodiment, the myogenic compound is a protein or a myogenic active peptide fragment thereof that is similar to the proteins or their myogenic active peptide fragments identified herein but from a non-chicken species, such as but not limited to humans, dogs, cats, horses, cows, sheep, pigs, and primates. Detailed Description

[0034] The present disclosure provides myogenic compounds identified in subfractions of fertilized avian egg yolk and their synthetic derivatives, nutritional compositions and / or pharmaceutical compositions comprising one or more of the myogenic compounds or their synthetic derivatives, and methods of using these compositions to increase muscle mass in mammals.

[0035] As used herein, "myogenic" or "myogenic active" refers to a compound that increases muscle growth and / or reduces muscle loss. In a non-limiting embodiment, the myogenic compound increases myogenic differentiation in a cell line having a reporter gene expressed under the promotion of a myogenic transcription factor. In a non-limiting embodiment, the myogenic compound increases the growth of muscle in mammals. The amount of increase is determined by comparison with differentiation and / or muscle growth in the absence of the myogenic compound and / or when a negative control is administered. In a non-limiting embodiment, the myogenic compounds of the present disclosure increase muscle growth in mammals similarly to powdered egg yolk. In a non-limiting embodiment, the myogenic compounds of the present disclosure promote muscle growth to a greater extent compared to powdered egg yolk. In a non-limiting specific embodiment, the myogenic compounds of the present disclosure reduce muscle loss in mammals. In a non-limiting embodiment, the myogenic compounds of the present disclosure reduce muscle loss in mammals similarly to powdered egg yolk. In a non-limiting embodiment, the myogenic compounds of the present disclosure reduce muscle loss to a greater extent compared to powdered egg yolk.

[0036] In a non-limiting embodiment, the myogenic compound upregulates the activity of the mTor pathway in mammals, downregulates the activity of the ubiquitin proteasome pathway, downregulates serum myostatin levels, and / or reduces ActRIIB expression.

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

[0038] The myogenic compounds of the present disclosure differ from natural egg yolk in that the myogenic compounds are either physically separated from other components in natural egg yolk, including but not limited to non-myogenic components and lipid components in egg yolk, by mechanical means or produced by synthetic means, e.g., peptide synthesis or recombinant protein production through a maturation protocol. In addition, as will be understood by those skilled in the art upon reading the present disclosure, although identified in fertilized avian egg yolk, other alternative biological sources are expected to contain the identified myogenic compounds, and the myogenic compounds of the present disclosure may be derived from these alternative biological sources.

[0039] Skeletal muscle is a composite tissue mainly composed of muscle fibers, which have four different forms according to the myosin heavy chain: type I, IIa, IIx, and IIb (Schiaffino, S. & Reggiani, C., Physiological Reviews 2011, 91 (4): 1447-1531). Slow muscle fibers are mainly composed of type I, while fast muscle fibers are composed of all forms of type II. Type I fibers are responsible for continuous use and perform postural functions, while type II fibers are involved in tasks such as walking or running. Although both fiber types are maintained and can be repaired, there is ample evidence that sarcopenia leads to the loss of type II fibers (Brunner et al., J Aging Phys Act, 2007 15(3): 336-48).

[0040] A cell line with a reporter gene expressed under the promotion of a myogenic transcription factor was used to identify myogenic differentiation caused by the respective fractions of egg yolk separated into lipid and protein components. Using the MLC2 promoter region, a reporter cell line was formed from C2C12 myoblasts, and its luciferase expression was related to the myogenic phenotype. C2C12 myoblasts were selected because they are related to muscle differentiation. The promoter region is known to be related to adult muscle growth. More specifically, the MLC1f promoter was selected to drive the expression of the luciferase transgene because it is only expressed in type IIb fibers of rats (Neville Dev Genet, 1996, 19(2): 157-62), and thus is a useful gene for identifying myoblast fusion and muscle fiber maturation because it is related to muscle differentiation.

[0041] The murine (mus musculus) C2C12 myoblast cell line, which can be induced to differentiate into myotubes, was selected as the cell line. These cells were derived from normal CH3 mice that had undergone crush injury 2 days prior to isolation to generate satellite cells (Yaffe, D. & Saxel, O.R.A., Nature (London), 1977, 270 (5639): 725-727). These cells were further amplified, and subclones that were diploid and reliably formed myotubes were selected (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 plasmid composed of three parts: the MLC1f promoter (Neville et al., Dev Genet, 1996, 19(2): 157-62), the blasticidin resistance gene, and the GLuc gene (New England BioLabs, Ipswich MA), which expresses Gaussia luciferase, an enzyme that is not expressed in mammalian cells and reacts with coelenterazine to produce luminescence.

[0042] The C2C12 cell line was transfected with this lentiviral vector and then selected using medium supplemented with blasticidin. After expansion of the blasticidin-resistant cell clones, the efficacy of the reporter gene was confirmed by detecting cell differentiation. Then, three groups were used to test the cell line: 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 the Biolux Gaussia luciferase assay kit and correlated with gene expression and image analysis of fluorescently stained cells.

[0043] The yolk was then separated into a protein fraction and a lipid fraction. Two different methods were used to separate the proteins and lipids in the yolk. The first method involved centrifugation and alginate to produce three different fractions containing different concentrations of protein, fat, and cholesterol. The second method was to remove the lipids in the yolk with an organic solvent.

[0044] The fertilized eggs were processed to remove the eggshell and vitelline membrane, leaving the raw yolk. The raw yolk was diluted with ddH2O in a ratio of 2:3 (v / v, yolk:ddH20), and the pH was adjusted to 7. The yolk was placed on a stirring plate at low speed (60 rpm) in a cold room (4 °C) overnight. Next, the yolk was centrifuged at 10,000 g and 4 °C for 45 minutes to produce pellets of the particulate fraction and a supernatant containing soluble proteins and lipids. Then, a 1% (w / v) stock solution of sodium alginate was added to the supernatant in a ratio of 1:9 to produce 0.1% w / v sodium alginate in the final solution. The resulting solution was then centrifuged at 20 °C and 10,000 g for 15 minutes to obtain an aqueous fraction and an alginate-lipid slurry (lipids). These fractions: particulate, lipid slurry, and aqueous solution were then analyzed to determine their general composition. These fractions were also used as media supplements in cell studies to determine their effects on myogenic differentiation of myoblasts in vitro.

[0045] SDS-PAGE was used to determine the composition of each fraction according to protein size. Samples were taken 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 into the lanes using a precast polyacrylamide gel mini-protean gtx. The gel was run at 200 v for approximately 30 minutes. The run of the gel was stopped when the dye front reached the black reference line on the gel. The results were compared with those of unfertilized eggs previously published by Laca et al. (Food Hydrocolloids - FOODHYDROCOLLOID, 2010, 24:434-443) and showed similar results, indicating that fertilization of the yolk does not result in significant changes in the yolk composition and that myogenic compounds can also be derived from unfertilized eggs.

[0046] For cell studies, luciferase assays were performed on cell culture media collected at different time points for differentiation measurements. The metabolic activity of each fraction group at each time point was also measured using the PrestoBlue assay to provide information on the biocompatibility of the fractions added to the media and to ensure that the addition of the fractions did not have a negative impact on cell growth and confluence. At days 0, 3, 7, and 10, media were removed from each fraction group, frozen for subsequent analysis, and then replaced with media containing 1:10% v / v PrestoBlue reagent in differentiation media and incubated at 37 °C for 1 hour. After incubation, the supernatant was collected and the fluorescence intensity was analyzed by excitation at 560 and emission at 590 to measure the conversion rate of non-fluorescent resazurin to fluorescent resorufin through metabolic pathways in the cells. For the luciferase assay, the cell culture media collected and frozen throughout the study were thawed at room temperature. 5 μl of the sample was added to 12.5 μl of luciferase assay buffer and the samples in each well were assayed in duplicate. Then coelenterazine reagent was placed in each well and allowed to react for 10 minutes, after which the luminescence value of each sample was read with an integration time of 10 milliseconds.

[0047] The Presto Blue assay results showed that the activity was similar among all fraction groups, but the activity of the dexamethasone group was significantly lower compared to all groups, while the activity of the high-concentration aqueous group was significantly higher compared to the basal cell culture media control. Dexamethasone is an inhibitor of muscle differentiation, which is reflected in the lower luciferase expression levels in cells treated with this media; if myoblasts are not allowed to differentiate, their activity decreases under serum starvation conditions, leading to a decrease in myoblast activity.

[0048] The results of the luciferase assay showed that myoblast MLC expression increased significantly in most fraction groups compared to the media control and dexamethasone-supplemented media (negative control). The two exceptions were the low-concentration alginate and the low-concentration aqueous group, which were only significantly higher than the dexamethasone group and not higher than the media control.

[0049] Therefore, as shown by these experiments, the particulate fractions of egg yolk have a positive effect on myoblast differentiation, indicating that one or more bioactive compounds in fertilized egg yolk can improve lean muscle growth.

[0050] To identify the specific bioactive compounds in egg yolk responsible for muscle growth, the particulate fractions were further fractionated and the differentiation ability of the sub-fractions thus obtained was tested.

[0051] The particles are insoluble in aqueous solution and form aggregates sized 1 - 8 μm. In a solution containing 0.3 M NaCl, the particles start to dissolve and turn into micelles on the order of 100 - 200 nm as sodium breaks the calcium bonds that lead to the formation of insoluble aggregates. Using phosphate - buffered saline (PBS) as the solvent, the particulate fraction is dissolved from the aggregate form in solution and forms micelles. These micelles are then separated by centrifugal force, and the in vitro biological effects of these sub - fractions are evaluated in a reporter cell line. Additionally, the biological effects of the unfertilized egg fraction are evaluated in comparison to the fertilized egg fraction.

[0052] Using centrifugation, three fractions consisting of particles of different sizes were formed. The first fraction, the heaviest fraction (referred to herein as F5), was separated by centrifuging the dissolved particles at 5000 relative centrifugal force (rcf) for 15 minutes and consists of the pellet obtained through this process. The next fraction (referred to herein as F7) was obtained by centrifuging the supernatant from the previous step at 7500 rcf for 15 minutes and then resuspending the pellet. The last fraction contains the lightest components (referred to herein as FS) and consists of the supernatant obtained from the previous step.

[0053] All fractions were assayed for total protein content by the bicinchoninic acid (BCA) assay and their size ranges were verified by gel. For these analyses, ddH2O at a concentration that could produce a homogeneous mixture was added. Samples were then taken from these mixtures and the aqueous fractions according to the manufacturer's instructions for BCA detection. Each sample was assayed at multiple concentrations of 1x, 10x, 100x dilutions. Samples were assayed in triplicate and reacted with BCA buffer at 37 °C for 30 minutes. After the reaction, the absorbance at 562 nm was read and compared to a BSA standard control of known concentration assayed simultaneously to obtain an estimate of the protein concentration in each sample. After the BCA assay, the samples were assayed by SDS - PAGE through the gel to determine the content of each fraction. Where possible, samples were diluted to each lane to provide 5 μg of protein or the highest available concentration. The gel was then run at 200 V for 35 minutes or until the dye reached the top reference line. After running, the gel was fixed in methanol - acetone - acetic acid and then 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 line disclosed herein were also examined.

[0055] Based on these results, size-exclusion chromatography was further used to separate the proteins in the F7 and FS fractions into smaller sub-fractions. More specifically, FPLC was performed using a Superdex 200 increase 10 / 300 column to separate the F7 and FS fractions into sub-fractions based on their native conformation in PBS. Samples with a volume of 1 mL were collected into microcentrifuge tubes, then quickly frozen and stored at -80 °C for subsequent analysis and cell culture. The samples were named according to their elution volume and precursor fraction. For example, F7.7 corresponds to the 7th mL of the eluate collected from running the F7 fraction through the column. Then, the samples containing the eluate with the peak absorbance at 280 nm were run through the gel column. Most of the fractions in the F7 fraction had different size ranges and different compositions for each fraction. For example, F7.4 showed a band at 250 kDa and then a band at 75 kDa, while the next observed fraction, F7.6, showed a band at 200 kDa and a faint band at 75 kDa. The next fraction, F7.7, again showed a band at 200 kDa. The later eluted gels in the F7 sub-fractions showed that most of the columns below the gel were lightly stained, indicating a trend of lighter proteins in these fractions. For the supernatant gels, an even smaller correlation between size and the number of fractions was observed.

[0056] The concentrations of the samples collected from chromatography were also measured using the BCA assay, and the total protein concentration data was used to calculate the appropriate amount of each sample to be added to the cell culture medium to test the effect of each fraction on differentiation when applied to myoblasts as described herein. The data from the assay is shown in Tables 1.1 and 1.2.

[0057] Table 1.1: Summary of statistical analysis of the 7500 g fraction

[0058]

[0059] n.s. (not significant); + (higher in the experimental group), - (lower in the experimental group); Tukey post hoc test p < 0.05

[0060] Table 1.2: Summary of statistical analysis of the supernatant fraction

[0061]

[0062] n.s. (not significant); + (higher in the experimental group), - (lower in the experimental group); Tukey post hoc test p < 0.05

[0063] From these tables, several fractions that induced higher MLC expression than the positive IGF control on day 10 were identified; these fractions were F7.11, F7.18, and F7.21 as well as FS.10, FS.11, and FS.22. These fractions, along with two control fractions, F7.7 and FS.8, that 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 subjected to 3 MS runs, resulting in a total of 24 raw data files from the 8 fractions.

[0064] More specifically, the samples were analyzed using LC-MS, which utilized a NanoLC-MS / MS (Dionex Ultimate 3000 RLSCnanoSystem, Thermofisher) interfaced with an Eclipse (ThermoFisher). 3 μl of the 12.5 μl in-gel digestion sample was loaded onto a fused silica capture column (Acclaim PepMap 100, 75umx2cm, ThermoFisher). After washing with 0.1% TFA at a rate of 5 μl / min for 5 minutes, the capture column was connected in series with the analytical column of the LC-MS / MS (Nanoease MZ peptide BEH C18, 130A, 1.7um, 75umx250mm, Waters). Peptides were fractionated at 300 nL / min using the following segmented linear gradient: 4 - 15% solution B (where solution A contained 0.2% formic acid and solution B contained 0.16% formic acid, 80% acetonitrile) for 30 minutes, 15 - 25% solution B for 40 minutes, 25 - 50% solution B for 44 minutes, 50 - 90% solution B for 11 minutes. Then the solution B was returned to 4% concentration for 5 minutes for the next run. The scan sequence started with an MSI spectrum (Orbitrap analysis, resolution 120,000, scan range M / Z 375 - 1500, automatic gain control (AGC) target 8E5, maximum injection time 100 ms). The top S (3 seconds) duty cycle scheme was used to determine the number of MSMS performed per cycle. Parent ions with a charge of 2 - 7 were selected for MSMS, and a 60-second dynamic exclusion was employed to avoid repeated sampling. The parent mass was isolated in the quadrupole with an isolation window of 1.2 m / z, an automatic gain control (AGC) target of 1E5, and fragmented by high-energy collision dissociation with a normalized collision energy of 30%. These fragments were scanned in the Orbitrap at a resolution of 15000. The MSMS scan range was determined by the charge state of the parent ion, but the lower limit was set to 110 amu.

[0065] Proteins identified as having relatively high concentrations in the initial analysis included albumin, ovalbumin, gelsolin, and lysozyme C. When the spectral counts for each protein were divided by the total spectral counts in each sample to account for variations in sample concentration, several other proteins were found to be differentially expressed compared to the undifferentiated fraction, including: proteins containing the SMB domain, transthyretin, proteins containing the IG-like domain, proteins containing the fibrinogen C-terminal domain, plasminogen, vitellogenin (VTG2), proteins containing the peptidase S1 domain, and fibrinogen C.

[0066] In addition, 15 peptide sequences were identified in at least 5 of the 6 active fractions by spectral counting and intensive peptide identification searches, but were not detected by this process in the 2 inactive fractions.

[0067] Table 2 shows these active peptides. Note: By definition, the 2 inactive fractions show 0.

[0068] Table 2:

[0069]

[0070] The symbols "^" and "$" in the peptide sequences of Table 2 represent the N-terminus and C-terminus, respectively. The numbers in parentheses represent the mass of the assumed chemical modification to the preceding residue or terminus. For example, "^(42)" represents N-terminal acetylation resulting in an additional mass of approximately 42 amu, and C(57) represents the (+57.021465) atomic mass unit resulting from carbamidomethyl modification of cysteine (C), and S(80), T(80), and Y(80) represent the (+79.96633) amu resulting from phosphorylation of their amino acid residues.

[0071] Accordingly, the present disclosure provides myogenic compounds and compositions thereof identified in the isolated fractions of fertilized avian egg yolk. As described above, the myogenic compounds and compositions of the present disclosure differ from natural egg yolk in that the myogenic compounds are either physically separated from other components in natural egg yolk, including but not limited to non-myogenic components of the egg yolk and lipid components of the egg yolk, by mechanical means, or are produced by synthetic means, e.g., by peptide synthesis or recombinant protein production via a maturation protocol. In addition, as will be understood by those skilled in the art upon reading the present disclosure, although identified in avian egg yolk, it is expected that other alternative biological sources also contain the identified myogenic compounds, and the myogenic compounds of the present disclosure may be derived from these alternative biological sources.

[0072] In one non-limiting embodiment, the composition comprises one or more myogenic compounds or synthetic derivatives thereof identified in subfractions F7.11, F7.18, F7.21, FS.10, FS.11, and / or FS.22 of fertilized avian egg yolk.

[0073] "Subfraction F7.11" refers to the fraction that is obtained by resuspending the pellet obtained by centrifuging the supernatant of dissolved yolk granules at 5000 rcf for 15 minutes and then centrifuging the resulting supernatant at 7500 rcf for 15 minutes (referred to herein as the F7 fraction) and has been further fractionated by size exclusion chromatography and corresponds to the 11th ml of liquid collected by running the F7 fraction through a size exclusion column.

[0074] "Subfraction F7.18" refers to the fraction that is obtained by resuspending the pellet obtained by centrifuging the supernatant of dissolved yolk granules at 5000 rcf for 15 minutes and then centrifuging the resulting supernatant at 7500 rcf for 15 minutes (referred to herein as the F7 fraction) and has been further fractionated by size exclusion chromatography and corresponds to the 18th ml of liquid collected by running the F7 fraction through a size exclusion column.

[0075] "Subfraction F7.21" refers to the fraction that is obtained by resuspending the pellet obtained by centrifuging the supernatant of dissolved yolk granules at 5000 rcf for 15 minutes and then centrifuging the resulting supernatant at 7500 rcf for 15 minutes (referred to herein as the F7 fraction) and has been further fractionated by size exclusion chromatography and corresponds to the 21st ml of liquid collected by running the F7 fraction through a size exclusion column.

[0076] "Subfraction FS.10" refers to the fraction that includes the supernatant obtained by centrifuging the supernatant of dissolved yolk granules at 5000 rcf for 15 minutes and then centrifuging the resulting supernatant at 7500 rcf for 15 minutes (referred to herein as the FS fraction) and has been further fractionated by size exclusion chromatography and corresponds to the 10th ml of liquid collected by running the FS fraction through a size exclusion column.

[0077] "Subfraction FS.11" refers to the fraction that includes the supernatant obtained by centrifuging the supernatant of dissolved yolk granules at 5000 rcf for 15 minutes and then centrifuging the resulting supernatant at 7500 rcf for 15 minutes (referred to herein as the FS fraction) and has been further fractionated by size exclusion chromatography and corresponds to the 11th ml of liquid collected by running the FS fraction through a size exclusion column.

[0078] "Subfraction FS.22" refers to the fraction that includes the supernatant obtained by centrifuging the supernatant of dissolved yolk granules at 5000 rcf for 15 minutes and then centrifuging the resulting supernatant at 7500 rcf for 15 minutes (referred to herein as the FS fraction) and has been further fractionated by size exclusion chromatography and corresponds to the 21st ml of liquid collected by running the FS fraction through a size exclusion column.

[0079] In a non-limiting embodiment, the composition comprises a powder prepared by drying one or more of subfractions F7.11, F7.18, F7.21, FS.10, FS.11, and / or FS.22.

[0080] In a non-limiting embodiment, the composition comprises one or more myogenic compounds, the myogenic compounds comprising one or more peptide sequences shown in Table 2. In a non-limiting embodiment, the myogenic compound is a protein comprising the peptide sequence shown in Table 2 or a myogenic active fragment thereof. In a non-limiting embodiment, the myogenic compound is a protein selected from the following: 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-II type cytoskeletal cochlea, desmuslin, apolipoprotein A-I, albumin, cytoplasmic actin 5, cytoplasmic actin 1, vitellogenin-1, cytoplasmic actin 2, IF rod domain-containing protein, vitellogenin-3, glial fibrillary acidic protein, plasminogen, or type II alpha-keratin IIA or a myogenic active peptide fragment thereof.

[0081] In a non-limiting embodiment, the myogenic compound is a protein selected from the following: 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 SI domain-containing protein, or fibrinogen C or a myogenic active fragment thereof.

[0082] In a non-limiting embodiment, the myogenic compound is a protein having at least 70%, 80%, 90%, 95%, or 99% sequence identity with the peptide sequence or protein identified herein and having myogenic activity.

[0083] In a non-limiting embodiment, the composition comprises one or more synthetic derivatives of myogenic compounds identified in subfractions F7.11, F7.18, F7.21, FS.10, FS.11 and / or FS.22 of fertilized avian egg yolk or comprising one or more peptide sequences shown in Table 2. In one embodiment, the synthetic derivative is prepared by recombinant protein expression using established methods. In a non-limiting embodiment, the synthetic derivative is a recombinant protein comprising the peptide sequence shown in Table 2 or a myogenic active fragment thereof. In a non-limiting embodiment, the synthetic derivative is a recombinant protein or a myogenic active fragment thereof having at least 70%, 80%, 90%, 95% or 99% sequence identity with the peptide sequence or protein identified herein and having myogenic activity.

[0084] As used herein, "myogenic active fragment" or "myogenic active peptide fragment" refers to a peptide sequence that is shorter in amino acid sequence than the full-length protein but retains myogenic activity similar to the full-length protein.

[0085] In a non-limiting embodiment, the myogenic compound is a protein or a myogenic active peptide fragment thereof that is similar to the protein or its myogenic active peptide fragment identified herein but from a non-chicken species, such as but not limited to human, dog, cat, horse, cow, sheep, pig, and primate.

[0086] In a non-limiting embodiment, the myogenic compound is a recombinant human protein or a myogenic active peptide fragment thereof.

[0087] The myogenic effects of several individual compounds identified herein were studied, namely, the chicken SERPIN domain-containing protein (also known as pigment epithelium-derived factor (PEDF)) and chicken nucleoside diphosphate kinase and their human recombinant proteins, SERPINF1 and human recombinant nucleoside diphosphate kinase (called NME2), and futopepin, on murine C2C12 cell proliferation and activation of the MLC1f promoter (by secreted Gaussia luciferase).

[0088] The protein containing the chicken SERPIN domain obtained from the ELISA kit significantly increased GLuc activity in the C2C12 cell line by 124 - 145% after four days of treatment. The vector of the chicken nucleoside diphosphate kinase protein in the ELISA kit was toxic to C2C12 cells, so the myogenic activity of this protein could not be evaluated using the available materials. However, two of its human recombinant proteins, SERPINF1 and human recombinant nucleoside diphosphate kinase (referred to as NME2), stimulated statistically significant proliferation of the murine C2C12 cell line in the concentration range of 62.5 to 1000 ng / ml: 109% to 120% of the control for SERPINF1 and 107% to 118% of the control for NME2. In addition, the human recombinant SERPINF1 protein significantly activated the MLC1f promoter determined by secreted GLuc in a dose-dependent manner in the C2C12 cell line, on the third day of treatment (128% to 115% for 250 ng / ml and 125 ng / ml respectively) and on the seventh day of treatment (141% to 135% for 250 ng / ml to 15.6 ng / ml). The human recombinant NME2 protein also significantly activated the MLC1f promoter determined by secreted GLuc in a dose-dependent manner in the C2C12 cell line, on the third day of treatment (135.8% to 121.2% for 250 ng / ml and 125 ng / ml respectively) and on the seventh day of treatment (126.4% to 113% for 250 ng / ml to 15.6 ng / ml). At different pH values and different incubation times, the protein extract from Fortopin also significantly increased GLuc activity in the C2C12 cell line (133 - 162%) after 4 days of treatment.

[0089] In addition, the presence of the protein containing the chicken SERPIN domain (also known as pigment epithelium-derived factor (PEDF)) and chicken nucleoside diphosphate kinase in the myogenic activity fertilized egg yolk product Fortopin was confirmed by ELISA assay. The highest concentration of the protein containing the chicken SERPIN domain extracted from 1 g of Fortopin was 140 ng, and the highest concentration of chicken nucleoside diphosphate kinase extracted from 1 g of Fortopin was 1270 ng.

[0090] The myogenic compounds identified herein can be formulated into nutritional compositions and / or pharmaceutical compositions for increasing muscle mass in mammals. In one non-limiting embodiment, the nutritional composition and / or pharmaceutical composition comprises more than one of the myogenic compounds described herein. In one non-limiting embodiment, the nutritional composition and / or pharmaceutical composition further comprises powdered egg yolk. In one non-limiting embodiment, the nutritional composition and / or pharmaceutical composition further comprises Fortetropin. Fortetropin is a product derived from fertilized egg yolk and is used as a dietary and nutritional supplement (MYOS CORP., Cedar Knolls, NJ). U.S. Patent No. 8,815,320 discloses a method for producing Fortetropin, the entire content of which is incorporated herein by reference. In another embodiment, the nutritional composition and / or pharmaceutical composition further comprises spray-dried egg yolk powder, such as that described in U.S. Patent Application Serial No. 16 / 151,601, the entire content of which is incorporated herein by reference. In one non-limiting embodiment, the nutritional composition and / or pharmaceutical composition increases muscle growth in mammals similarly to powdered egg yolk. In one non-limiting embodiment, the nutritional composition and / or pharmaceutical composition increases muscle growth to a greater extent compared to powdered egg yolk.

[0091] In a composition comprising more than one of the myogenic compounds disclosed herein, the compounds can be additive in myogenic activity or can be synergistic in myogenic activity, meaning that there is more than an additive effect.

[0092] Those skilled in the art will understand, upon reading the present disclosure, that the compositions described herein can be formulated for administration to mammals by any conventional means, including but not limited to oral or buccal administration.

[0093] In addition, the compositions described herein can be formulated into any suitable dosage form, including but not limited to aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, etc. for oral ingestion by an individual in need, solid oral dosage forms, controlled release formulations, fast-dissolving formulations, effervescent formulations, freeze-dried 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.

[0094] Oral formulations can be obtained by mixing one or more solid excipients with one or more of the compounds described herein, optionally grinding the resulting mixture, and processing the granule mixture with suitable auxiliaries (if required) to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars including glucose, fructose, lactose, sucrose, mannitol, sorbitol, stevia extract or sucralose; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If required, disintegrants can be added such as cross-linked sodium carboxymethylcellulose, polyvinylpyrrolidone, agar, or alginic acid or its salts such as sodium alginate.

[0095] The cores of dragees are provided with suitable coatings. For this purpose, concentrated sugar solutions can be used which can optionally contain gum arabic, talc, polyvinylpyrrolidone, carbomer gels, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the tablet or dragee coating in order to identify or characterize different combinations of the active compound dosage.

[0096] Oral formulations include push-fit capsules made of gelatin, and soft-sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules can contain a mixture mixed with a filler such as lactose, a binder such as starch and / or a lubricant such as talc or magnesium stearate and optionally a stabilizer. In soft capsules, the active compound can be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin or liquid polyethylene glycol. In addition, stabilizers can also be added. All formulations for oral administration should have a dosage suitable for oral administration.

[0097] In some embodiments, the solid dosage forms disclosed herein can be in the form of tablets (including suspension tablets, fast-dissolving tablets, chewable disintegrating tablets, fast-disintegrating tablets, effervescent tablets or sachet tablets), pills, powders (including aseptically packaged powders, dispensable powders or effervescent powders), capsules (including soft capsules or hard capsules such as capsules made of animal-derived gelatin or plant-derived HPMC, or "sprinkle capsules"), solid dispersions, solid solutions, pellets, granules. In other embodiments, the pharmaceutical formulation is in powder form. In other embodiments, the pharmaceutical formulation is in tablet form. In addition, the formulations described herein can be administered in single-capsule or multi-capsule dosage forms. In some embodiments, the formulation is administered in the form of two, three or four capsules or tablets.

[0098] Soft gelatin capsules can be prepared, for example but not limited to, by forming a high-viscosity mixture by dispersing the formulation in a suitable carrier, usually a vegetable oil. Then, using techniques and machinery known in the soft gelatin industry, the mixture is encapsulated in a gelatin-based film. The formed industrial units are then dried to a constant weight.

[0099] In some embodiments, the formulation can include other drugs or medicaments, carriers, diluents, dispersants, suspending agents, thickening agents, adjuvants (such as preservatives, stabilizers, wetting agents or emulsifying agents), solubilizing agents, and / or buffering agents. Additionally, other therapeutically valuable substances can also be present in the formulation.

[0100] The formulations described herein can include one or more myogenic compounds and one or more nutritionally or pharmaceutically acceptable additives, such as compatible carriers, binders, fillers, suspending agents, flavoring agents, sweetening agents, disintegrants, dispersants, surfactants, lubricants, coloring agents, diluents, solubilizers, wetting agents, plasticizers, stabilizers, penetration enhancers, wetting agents, defoaming agents, antioxidants, preservatives, or one or more combinations thereof. In other aspects, using standard coating procedures, a film coating is provided around the formulations of the compounds described herein. 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 and are not coated.

[0101] In certain embodiments, the composition can further contain one or more pH regulators or buffering agents, 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 tris(hydroxymethyl)aminomethane; and buffering agents such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. The amounts of such acids, bases, and buffering agents should maintain the pH value of the composition within an acceptable range.

[0102] In other embodiments, the composition can further contain one or more salts in amounts such that the osmotic pressure of the composition reaches an acceptable range. Such salts include salts 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.

[0103] As described herein, formulations comprising one or more myogenic compounds can be manufactured in a conventional manner, for example, by way of illustration only, by conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding, or compressing processes.

[0104] In certain embodiments, the compositions provided herein may also include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as phenylmercuric borate and thimerosal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0105] The formulations described herein may benefit from antioxidants, metal chelators, thiol-containing compounds, and other common stabilizers. Examples of such stabilizers include, but are not limited to: (a) from about 0.5% to about 2% w / v glycerol, (b) from about 0.1% to about 1% w / v methionine, (c) from about 0.1% to about 2% w / v monothioglycerol, (d) from about 1 mN to about 10 mN EDTA, (e) from about 0.01% to about 2% w / v ascorbic acid, (f) from 0.003% to about 0.02% w / v polysorbate 80, (g) from 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparin-like compounds, (m) divalent cations such as magnesium and zinc ions; or (n) combinations thereof.

[0106] Binders that impart cohesiveness may also be used. Examples include, but are not limited to, alginic acid and its salts; cellulose derivatives such as carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, ethyl cellulose, and microcrystalline cellulose; microcrystalline glucose; amylose; magnesium aluminum silicate; polygalacturonic acid; bentonite; gelatin; polyvinylpyrrolidone / vinyl acetate copolymer; crospovidone; povidone; starch; pregelatinized starch; tragacanth, dextrin, sugars (such as sucrose, glucose, dextrose, molasses, mannitol, sorbitol, xylitol, and lactose); natural or synthetic gums such as gum arabic, tragacanth, ghatti gum, mucilage of isapol husk, polyvinylpyrrolidone, larch arabinogalactan, polyethylene glycol, wax, sodium alginate, and the like.

[0107] Generally, the binder content used in powder-filled gelatin capsule formulations is 20 - 70%. The level of binder use in tablet formulations varies, whether by direct compression, wet granulation, roller compaction, or the use of other excipients such as fillers that can act as moderate binders themselves.

[0108] Those skilled in the art of formulating can determine the binder level of the formulation, but the level of binder use in tablet formulations is commonly up to 70%.

[0109] The composition may also contain a relatively non-toxic compound or chemical reagent that is favorable for incorporating the compound into cells or tissues as a carrier. Non-limiting examples include binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, etc. Suitable carriers for the solid dosage forms described herein include, but are not limited to, gum arabic, gelatin, colloidal silica, calcium glycerophosphate, calcium lactate, maltodextrin, glycerol, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium hydrogen phosphate, sodium stearoyl lactylate, carrageenan, glycerol monoesters, glycerol diesters, pregelatinized starch, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose, microcrystalline cellulose, lactose, mannitol, etc.

[0110] Dispersants and / or viscosity regulators include materials that control the diffusion and uniformity of a compound through a liquid medium or by granulation or mixing methods. In some embodiments, these reagents also enhance the effectiveness of coating or erodible matrices. Non-limiting examples of diffusion promoters / dispersants 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 KI 00, HPMC K4M, HPMC K15M, and HPMC KI 00M), sodium carboxymethyl cellulose, methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate stearate (HPMCAS), microcrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone / vinyl acetate copolymer (S630), polymer of 4-(1,1,3,3-tetramethylbutyl)-phenol with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamer, a block copolymer of ethylene oxide and propylene oxide; and poloxamer, a tetrafunctional block copolymer obtained by sequential addition of propylene oxide and ethylene oxide to ethylenediamine, polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone / vinyl acetate copolymer (S-630), polyethylene glycol (e.g., the molecular weight of polyethylene glycol can be from about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethyl cellulose, methylcellulose, polysorbate-80, sodium alginate, gums (such as tragacanth gum and gum arabic), guar gum, xanthan gums (including xanthan gum), sugars, celluloses (such as sodium carboxymethyl cellulose, methylcellulose, sodium carboxymethyl cellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, carbomer, polyvinyl alcohol (PVA), alginates, chitosan, and combinations thereof. Plasticizers such as cellulose or triethyl cellulose can also be used as dispersants. Dispersants that are particularly useful in liposome dispersions and self-emulsifying dispersions are dimyristoyl phosphatidylcholine, natural phosphatidylcholine from eggs, natural phosphatidylglycerol from eggs, cholesterol, and isopropyl myristate.

[0111] In the compositions of the present invention, a combination of one or more erosion promoters and one or more diffusion promoters can also be used.

[0112] The composition of the present invention may also contain a diluent for diluting the target compound before delivery. Diluents can also be used to stabilize the compound as 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 solutions. In certain embodiments, the diluent increases the volume of the composition to facilitate compression or create sufficient volume for uniform mixing for capsule filling. Such compounds include, for example, lactose, starch, mannitol, sorbitol, glucose, microcrystalline cellulose; calcium hydrogen 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, confectioner's sugar; calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; sodium chloride; inositol, bentonite, etc.

[0113] The composition may also contain an enteric coating, which is a substance that remains substantially intact in the stomach but dissolves and releases the myogenic compound in the small intestine or colon. Generally, the enteric coating contains a polymeric material that prevents release in the low pH environment of the stomach but ionizes at a higher pH (usually pH 6 to 7), thus dissolving sufficiently in the small intestine or colon to release the active agent therein.

[0114] In addition, the composition may also contain an erosion promoter, which is a substance that controls the erosion of a specific substance in gastrointestinal fluids. Erosion promoters are generally well known to those skilled in the art. Exemplary erosion promoters include, for example, hydrophilic polymers, electrolytes, proteins, peptides, and amino acids.

[0115] Fillers include compounds such as lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, powdered cellulose, dextrin, dextrates, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc. Fillers suitable for the solid dosage forms described herein include but are not limited to lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, powdered cellulose, dextrin, 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.

[0116] In addition, flavoring agents and / or sweetening agents can be used in the composition and can include gum arabic syrup, acesulfame potassium, alitame, anise, apple, aspartame, banana, Bavarian cream, berries, blackcurrant, butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream, chocolate, cinnamon, bubblegum, citrus, citrus punch, citrus cream, cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate, cyclohexylsulfamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger, glycyrrhetate, licorice syrup, grape, grapefruit, honey, isomalt, lemon, lime, lemon cream, monoammonium glycyrrhizinate, maltol, mannitol, maple, marshmallow, menthol, mint cream, mixed berries, neohesperidin DC, neotame, orange, pear, peach, peppermint, mint cream, raspberry, root beer, rum, saccharin, safrole, sorbitol, spearmint, spearmint cream, strawberry, strawberry cream, stevia, sucralose, sucrose, sodium saccharin, saccharin, aspartame, acesulfame potassium, mannitol, talin, sylitol, sucralose, sorbitol, Swiss cream, tagatose, tangerine, thaumatin, Tutti Fruitti, vanilla, walnut, watermelon, wild cherry, wintergreen, xylitol, or any combination of these flavoring components, such as anise-menthol, cherry-anise, cinnamon-orange, cherry-cinnamon, chocolate-mint, honey-lemon, lemon-lime, lemon-mint, mint-eucalyptus, orange-cream, vanilla-mint and mixtures thereof.

[0117] The composition may further comprise a lubricant and / or a glidant that prevents, reduces or inhibits 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 (such as aluminum, calcium, magnesium, zinc) salts, stearic acid, sodium stearate, glycerol, talc powder, wax, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (such as PEG-4000) or methoxypolyethylene glycol, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium lauryl sulfate or sodium lauryl sulfate, colloidal silica, starch (such as corn starch), silicone oil, surfactants, etc.

[0118] The composition may also contain a plasticizer, i.e., a compound used to soften the microcapsule material or film coating to make it less brittle. Examples of suitable plasticizers include, but are not limited to, polyethylene glycols (such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800), stearic acid, propylene glycol, oleic acid, triethyl cellulose, and glyceryl triacetate. In some embodiments, the plasticizer can also act as a dispersant or wetting agent.

[0119] The composition may further contain solubilizers such as glyceryl triacetate, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropanol, cholesterol, bile salts, polyethylene glycol 200 - 600, tetrahydrofurfuryl polyethylene glycol ether, carbitol, propylene glycol, and dimethyl isosorbide, etc.

[0120] In addition, the composition may also contain stabilizers such as antioxidants, buffers, acids, preservatives, etc.

[0121] Suitable suspending agents for the solid dosage forms described herein include, but are not limited to, polyvinylpyrrolidone, such as polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyethylene glycol, for example, the molecular weight of polyethylene glycol can be from about 300 to about 6000, or from about 3350 to about 4000, or from about 7000 to about 5400, vinylpyrrolidone / vinyl acetate copolymer (S630), sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethyl cellulose, polysorbate - 80, hydroxyethyl cellulose, sodium alginate, gums such as tragacanth and gum arabic, guar gum, xanthan gums (including xanthan gum), sugars, celluloses such as sodium carboxymethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, polysorbate - 80, sodium alginate, polyethoxylated sorbitan monolaurate, polyvidone, etc.

[0122] Surfactants may also be included, including compounds such as sodium lauryl sulfate, sodium docusate, Tween, glyceryl triacetate, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, poloxamer, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, etc. Other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, for example, polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkyl phenyl ethers, such as octoxynol 10, octoxynol 40. In some embodiments, surfactants can be added to enhance physical stability or for other purposes.

[0123] It may also include viscosity enhancers, including, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hypromellose acetate succinate, hypromellose phthalate, carbomer, polyvinyl alcohol, alginate, gum arabic, chitosan, and combinations thereof.

[0124] In addition, wetting agents may also be included in these compositions, including oleic acid, glycerol monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, glyceryl triacetate, Tween-80, vitamin E TPGS, ammonium salts, and other compounds.

[0125] In some embodiments, solid dosage forms (e.g., tablets, capsules) are prepared by mixing the myogenic compounds described herein with one or more pharmaceutical excipients to form a bulk blend composition. When these bulk blend compositions are referred to as homogeneous, this means that the particles of the myogenic compound are uniformly dispersed throughout the composition such that the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules.

[0126] 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, for example, Lachman et al., “The Theory and Practice of Industrial Pharmacy” (1986).

[0127] It should be understood that there is a significant overlap among the additives used in the solid dosage forms described herein. Thus, the additives listed above should be considered only as examples and not as limitations on the types of additives that may be included.

[0128] For example, capsules can be prepared by placing a bulk mixture of the formulations of the above compounds within a capsule. In some embodiments, the formulations (non-aqueous suspensions and solutions) are placed in soft gelatin capsules. In other embodiments, the formulations are placed in standard gelatin capsules or non-gelatin capsules (e.g., capsules containing HPMC). In other embodiments, the formulations are placed in sprinkle capsules, where the capsule can be swallowed whole or the capsule can be opened and the contents sprinkled on food before ingestion. In some embodiments, the therapeutic dose is divided into multiple (e.g., two, three, or four) capsules. In some embodiments, the entire dose of the formulation is delivered in capsule form.

[0129] In another aspect, the dosage form can include a microcapsule preparation. In some embodiments, one or more other compatible materials are present in the microcapsule material. Exemplary materials include, but are not limited to, pH regulators, dissolution accelerators, defoaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0130] Materials that can be used for the microencapsulation described herein include materials that can sufficiently isolate the compound from other incompatible excipients. Materials compatible with the myogenic compound are those that can delay the release of the myogenic compound in vivo.

[0131] In other embodiments, the preparation containing the myogenic compound described herein is a solid dispersion. Methods for producing such solid dispersions are known in the art and include, but are not limited to, for example, U.S. Patents 4,343,789, 5,340,591, 5,456,923, 5,700,485, 5,723,269, and U.S. Application 2004 / 0013734.

[0132] In other embodiments, the preparation described herein is a solid solution. A solid solution combines a substance with an active agent and other excipients. Heating the mixture causes the drug to dissolve, and then cooling the resulting composition provides a solid mixture, which can be further formulated or directly added to a capsule or compressed into a tablet. Methods for producing such solid solutions are known in the art and include, but are not limited to, for example, U.S. Patents 4,151,273, 5,281,420, and 6,083,518.

[0133] In some embodiments, the solid dosage form described herein can be formulated as an enteric-coated delayed-release oral dosage form, i.e., an oral dosage form of the pharmaceutical composition described herein that utilizes an enteric coating to achieve release in the small intestine of the gastrointestinal tract. The enteric-coated dosage form can be a compressed, molded, or extruded tablet / molding agent (coated or uncoated) containing granules, powders, pellets, beads, or particles of the active ingredient and / or other components. The enteric-coated oral dosage form can also be a capsule (coated or uncoated) containing pellets, beads, or granules of a solid carrier or composition.

[0134] As used herein, the term "delayed release" refers to a mode of delivery where release can occur at a generally predictable location in the intestine, which is further along the intestine than would be the case without the modification to provide delayed release. In some embodiments, the method of delayed release is coating. Any coating should be applied in a thickness sufficient to prevent the entire coating from dissolving in gastrointestinal fluids at a pH below about 5, but which will dissolve at a pH of about 5 or above. Any anionic polymer exhibiting pH-dependent solubility characteristics is expected to be useful as an enteric coating for the methods and compositions described herein to achieve delivery to the lower gastrointestinal tract.

[0135] In some embodiments, there are provided formulations comprising particles of the myogenic compounds described herein and at least one dispersant or suspending agent for oral administration to a subject. The formulations can be powders and / or granules for suspension and, when mixed with water, can provide a substantially uniform suspension.

[0136] Liquid dosage forms for oral administration can be aqueous suspensions selected from, but not limited to, the group consisting of pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. See, for example, Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Edition, pages 754 - 757 (2002). In addition to the particles of the myogenic compounds, the liquid dosage forms can also include additives such as: (a) disintegrants; (b) dispersants; (c) wetting agents; (d) at least one preservative; (e) viscosity enhancers; (t) at least one sweetening agent; and (g) at least one flavoring agent. In some embodiments, the aqueous dispersion can also contain crystallization inhibitors.

[0137] The aqueous suspensions and dispersions described herein can remain uniform for at least 4 hours, as defined by the United States Pharmacopeia (2005 Edition, Chapter 905). Uniformity should be determined by sampling methods consistent with determining the uniformity of the entire composition. In one embodiment, the aqueous suspension can be resuspended into a uniform suspension by physical agitation for less than 1 minute. In another embodiment, the aqueous suspension can be resuspended into a uniform suspension by physical agitation for less than 45 seconds. In another embodiment, the aqueous suspension can be resuspended into a uniform suspension by physical agitation for less than 30 seconds. In another embodiment, no agitation is required to maintain a uniform aqueous dispersion.

[0138] Suitable preservatives for the aqueous suspensions or dispersions described herein include, for example, potassium sorbate, parabens (e.g., methyl paraben and propyl paraben), benzoic acid and its salts, other esters of p-hydroxybenzoic acid such as butyl p-hydroxybenzoate, alcohols such as ethanol or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride. The preservatives used herein are added to the dosage form in a concentration sufficient to inhibit microbial growth.

[0139] In one non-limiting embodiment, the aqueous liquid dispersion can contain a sweetening or flavoring agent in a concentration range of from about 0.005% to about 0.5% by volume of the aqueous dispersion. In yet another embodiment, the aqueous liquid dispersion can contain a sweetening or flavoring agent in a concentration range of from about 0.01% to about 1.0% by volume of the aqueous dispersion.

[0140] In addition to the additives listed above, the liquid formulations can also include inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers. Exemplary emulsifiers are ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, sodium lauryl sulfate, sodium dioctyl sulfosuccinate, 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, sorbitan fatty acid esters, or mixtures of these substances, etc.

[0141] In some embodiments, the formulations described herein can be self-emulsifying drug delivery systems (SEDDS). An emulsion is a dispersion of one immiscible phase in another immiscible phase, usually in the form of droplets. Generally, emulsions are produced by vigorous mechanical dispersion.

[0142] Unlike emulsions or microemulsions, SEDDS form an emulsion spontaneously upon addition of an excess of water, without any external mechanical dispersion or agitation. One advantage of SEDDS is that the droplets can be distributed throughout the solution with only gentle mixing. In addition, water or an aqueous phase can be added immediately prior to administration, which ensures the stability of labile or hydrophobic active ingredients. Thus, SEDDS provide an effective delivery system for the oral and parenteral delivery of hydrophobic active ingredients. SEDDS can improve the bioavailability of hydrophobic active ingredients. Methods for producing self-emulsifying dosage forms are known in the art and include, but are not limited to, for example, U.S. Patents 5,858,401, 6,667,048, and 6,960,563.

[0143] Transbuccal formulations containing myogenic compounds can be administered using a variety of formulations known in the art. For example, such formulations include, but are not limited to, U.S. Patents 4,229,447, 4,596,795, 4,755,386, and 5,739,136. In addition, the transbuccal dosage forms described herein may also include bioerodible (hydrolyzable) polymer carriers, which are also used to adhere the dosage form to the buccal mucosa. The transbuccal dosage forms are manufactured to erode gradually over a predetermined period of time. Those skilled in the art will recognize that transbuccal drug delivery avoids the disadvantages encountered with oral drug administration, such as slow absorption, degradation of the active agent by the fluids present in the gastrointestinal tract, and / or first-pass inactivation in the liver. With respect to bioerodible (hydrolyzable) polymer carriers, it should be understood that almost any such carrier can be used, provided that the desired drug release characteristics are not impaired and the carrier is compatible with the myogenic compound and any other components that may be present in the transbuccal dosage unit. Generally, the polymer carrier includes hydrophilic (water-soluble and water-swellable) polymers that adhere to the moist surface of the buccal mucosa. Other components may also be added to the transbuccal dosage forms described herein, and other components include, but are not limited to, disintegrants, diluents, binders, lubricants, flavoring agents, coloring agents, preservatives, etc. For transbuccal or sublingual administration, the composition can be in the form of tablets, lozenges, or gels formulated in a conventional manner.

[0144] In certain embodiments, delivery systems for pharmaceutical compounds, such as liposomes and emulsions, can be employed. In certain embodiments, the compositions provided herein may also include mucoadhesive polymers selected from, for example, carboxymethylcellulose, carbomer (acrylic polymer), polymethylmethacrylate, polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran.

[0145] In one non-limiting embodiment, the nutritional composition and / or pharmaceutical composition may further comprise powdered egg yolk. In one non-limiting embodiment, the prime nutritional composition and / or pharmaceutical composition may further comprise Fortopine.

[0146] The following non-limiting embodiments further illustrate the invention. Detailed Description

[0147] Examples

[0148] Example 1: Evaluation of the Effects of Chicken SERPINF1, Chicken NME2, and Proteins Extracted from Fortopine on Activating the MLC1f Promoter by Secreted Gauss Luciferase

[0149] Materials and Methods

[0150] Compounds

[0151] Test compounds included: Fotopine provided by MYOS; chicken nucleoside diphosphate kinase B (NME2) from ELISA kit standard (Catalog No. MBS7244605; MyBioSource); chicken SERPINF1 or chicken pigment epithelium-derived factor (PEDF) standard from ELISA kit (Catalog No. MBS264953; MyBioSource); recombinant murine IGF1 from Peprotech; and dexamethasone from TCI America.

[0152] Cell culture

[0153] For cell culture, the C2C12 mouse muscle myoblast cell line carrying the reporter gene Gauss luciferase (GLuc) under the muscle-specific promoter of fast myosin light chain MLCf1 was used. Cells were amplified in proliferation medium consisting of 4.5 g / L DMEM supplemented with 10% v / v fetal bovine serum and 1 U / ml penicillin-streptomycin. The C2C12 myoblast cell line was seeded at a density of 25,000 cells / cm 2 in 48-well plates and cultured in proliferation medium with the medium changed daily for the first 3 days. There were three wells for each condition. On day 3, the medium was changed to differentiation medium consisting of DMEM with 4.5 g / L glucose supplemented with 1% FBS and 1 U / ml penicillin-streptomycin. To confirm the suitability of the experimental conditions and the success of the experiment, the activation of the reporter gene GLuc in the cell line was tested under three different treatment conditions: vehicle / media control; positive control containing 50 ng / ml insulin-like growth factor (IGF1); and negative control containing 50 μg / ml dexamethasone.

[0154] Protein extraction from Fotopine powder

[0155] Proteins were extracted according to the established protocol by Chalamaiah et al. (Food Chem 2018 268:369-377) to ensure accurate and reliable results. Briefly, 0.2 g or 2 g of Fotopine powder was carefully reconstituted in 10 ml of phosphate buffered saline at pH 4, 7, and 10, respectively. The mixture was then incubated at room temperature for 3 hours or left for 48 hours. During the 3-hour incubation, periodic vortexing was performed every 30 minutes to facilitate efficient extraction.

[0156] After the incubation period, the solution was carefully centrifuged at 2000 RPM and then transferred to an Eppendorf tube. To further use the extracted protein, the pH of the solution was neutralized to pH 7 and then centrifuged a second time at 15,000 RPM. Subsequently, the extracted protein was used for ELISA analysis to determine the concentrations of SERPINF1 and NME2.

[0157] The fotopine extract was also used in cytotoxicity assays and MLC1f promoter reporter gene activation assays, allowing for a comprehensive assessment of its biological activity.

[0158] ELISA

[0159] ELISA assays were performed according to the manufacturer's protocol to determine the concentrations of chicken SERPINF1 / pigment epithelium-derived factor (PEDF) and chicken nucleoside diphosphate kinase B (NME2) in the fotopine extract.

[0160] The ELISA kits used were the chicken nucleoside diphosphate kinase B (NME2) ELISA kit (catalog number MBS7244605; MyBioSource) and the chicken pigment epithelium-derived factor (PEDF) ELISA kit (catalog number MBS264953; MyBioSource).

[0161] Cytotoxicity assay

[0162] Cytotoxicity assessment is a crucial step in drug development. Cytotoxicity refers to the ability of a drug to cause damage or death to cells. Without assessing cytotoxicity, using high concentrations of a drug in testing may kill the cells, making it difficult to determine whether the drug is toxic or has an anti-proliferative effect. According to the manufacturer's instructions, the cytotoxicity was evaluated using the Cell Titer96 Aqueous One (Promega, WI, USA) basic assay. Cells were seeded in 96-well plates at a density of 10,000 cells / well and cultured overnight. They were treated with different doses of the compound for 24 hours in triplicate. Colorimetric analysis was performed using absorbance at 490 nm. An activity inhibition of more than 20% over the control value was considered cytotoxic.

[0163] Activation of the MLC1f promoter was evaluated by secreted Gauss luciferase (GLuc)

[0164] To evaluate the activation of the MLCf1 promoter by secreted Gluc, first, the MLC1f reporter C2C12 myoblasts were seeded at 25,000 cells / cm 2Inoculated at a density into 48-well plates and cultured in proliferation medium (DMEM containing 4.5 g / L glucose, 10% FBS, and 1 U / mL penicillin-streptomycin) for the first 3 days, with the medium changed daily in a cell culture incubator at 37°C and 5% CO2.

[0165] On the 3rd day, the medium was switched to differentiation medium, which is DMEM containing 4.5 g / L glucose, 1% FBS, and 1 U / mL penicillin-streptomycin. 2% and 20% futopin protein extracts at different pHs and SERPINF1 protein standards from an ELISA kit were evaluated.

[0166] In addition, this study also included three control groups (n = 3): 1) cells supplemented with 50 ng / mL murine IGF1 (Peprotech) to promote myoblast differentiation, 2) cells supplemented with 50 μg / mL dexamethasone to inhibit myoblast differentiation, and 3) cells fed with basal differentiation medium without any additives as a vehicle control.

[0167] After incubation for 1 day, 4 days, and 7 days, cell supernatants were collected and stored at -80°C. GLuc assays were performed according to the manufacturer's protocol (Thermo Fisher Scientific; Pierce Gaussia Luciferase Glow Assay Kit catalog number 16161).

[0168] Data statistics

[0169] 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 and Dunnett's multiple comparison post hoc. *P < 0.05 and **P < 0.01.

[0170] Results

[0171] Cytotoxicity analysis

[0172] Dexamethasone at concentrations up to 100 μg / ml to 12.5 μg / ml had no cytotoxicity on the cultured C2C12 cell line.

[0173] Cytotoxic effects of 88.3% to 80% were observed when using diluted NME2 protein from an ELISA kit. However, even when the concentration of the protein from the ELISA kit was 0, there was still toxicity, indicating the presence of proprietary supplements required for ELISA rather than the NME2 protein being harmful to cell culture.

[0174] The lyophilized standard of SERPINF1 / Pigment Epithelium-Derived Factor (PEDF) from the ELISA kit is cytotoxic only at a concentration of 10 ng / ml.

[0175] The proteins extracted from ftorafloxacin at different pH values are cytotoxic when undiluted and diluted 1:2, but not cytotoxic when diluted 1:4.

[0176] Cytotoxicity experiments were used to determine the highest non-toxic concentration of the test substance. These concentrations were then selected to further evaluate their biological activity in the C2C12 cell line.

[0177] After extracting proteins from ftorafloxacin powder under different pH values and incubation times, ELISA was used to evaluate the protein concentrations of NME2 and SERPINF1.

[0178] According to the ELISA data, the maximum amount of NME2 was extracted after 48 hours of protein extraction from 2% ftorafloxacin at pH = 10 at room temperature. It is estimated that 1 gram of ftorafloxacin contains approximately 1,270 ng of NME2.

[0179] According to the ELISA data, the maximum amount of SERPINF1 was also extracted after 48 hours of protein extraction from 2% ftorafloxacin at pH = 10 at room temperature. It is estimated that 1 gram of ftorafloxacin contains approximately 140 ng of SERPINF1.

[0180] The effects of SERPINF1 and proteins extracted from ftorafloxacin on the activation of the MLC1f promoter were evaluated by secreted Gauss luciferase.

[0181] The activation of the MLC1f promoter by different treatments was evaluated by measuring secreted Gauss luciferase. Monitoring the level of secreted Gauss luciferase can quantify the promoter activity under various experimental conditions. The C2C12 cell line was cultured in a 48-well plate for 5 days, and the medium was changed daily. In the first two days, the medium was replaced with growth medium. In the last 3 days, the medium was replaced with differentiation medium. On the 5th day of the experiment, 50 ng / ml IGF1 was used as a positive control, 50 μg / ml dexamethasone was used as a negative control, and the medium was used as a vehicle control. Supernatant samples were collected on the 3rd, 4th, and 7th days for G-luciferase assay.

[0182] The luciferase activity was directly evaluated and compared as a measure of promoter activity without normalizing the protein concentration.

[0183] On the first day, no induction of luciferase activity was observed even with the positive control IGF1.

[0184] On the 4th day after treatment, it was observed that luciferase driven by the MLC1f promoter was significantly activated by 144.6 ± 5.1 under the action of the positive control IGF1.

[0185] Meanwhile, the vehicle control and the negative control dexamethasone had no effect, showing luciferase activities of 106.5 ± 12.4 and 102 ± 3.4 respectively. These results indicated that the experimental setup was correct and the experiment was running as expected, verifying the reliability of our findings.

[0186] After four days of treatment, the SERPINF1 protein in the ELISA kit significantly increased the GLuc activity in the C2C12 cell line by 124 - 145%. The Gluc reporter showed reverse dose-dependent activation, indicating interference by the proprietary compound in the ELISA kit. The activity increased after protein dilution, indicating reduced interference at lower concentrations.

[0187] At different pH values and different incubation times, the fotopin protein extract significantly increased the GLuc activity in the C2C12 cell line (133 - 162%) on the 4th day after treatment.

[0188] On the 7th day, the luciferase activity of most treated samples generally decreased. However, in cells treated with the positive control IGF1, at pH 10, even at lower concentrations of SERPINF1 and the protein extracted from fotopin, there was still a statistically significant difference between the treated and untreated cells.

[0189] Example 2: Evaluation of the effects of human recombinant proteins SERPINF1 and NME2 on the proliferation of murine C2C12 cells and the activation of the MLC1f promoter (through secreted Gauss luciferase)

[0190] Materials and Methods

[0191] Test Compounds

[0192] The human recombinant protein NME2 was obtained from MyBioSource (Catalog No. 206177). Human pigment epithelium-derived factor (PEDF), a secreted glycoprotein encoded by the SERPINF1 gene, was also obtained from MyBioSource (Catalog No. 143337). Recombinant murine IGF1 was obtained from Peprotech, and dexamethasone was obtained from TCI America.

[0193] Cell culture, cytotoxicity test, evaluation of the activation of the MLC1f promoter through secreted Gauss luciferase (GLuc), and data statistics

[0194] Cell culture, cytotoxicity assay, assessment of activation of the MLC1f promoter by secreted Gauss luciferase (GLuc), and data statistics were all performed according to the method of Example 1.

[0195] Results

[0196] Cytotoxicity analysis

[0197] Dexamethasone at concentrations up to 100 μg / ml to 12.5 μg / ml had no cytotoxicity to the cultured C2C12 cell line. In addition, human SERPINF1 / pigment epithelium-derived factor (PEDF) human recombinant NME2 protein did not exhibit cytotoxicity.

[0198] Human recombinant SERPINF1 stimulated a statistically significant proliferation of the murine C2C12 cell line, which was 120% to 109% of the control group in the concentration range of 1000 ng / ml to 62.5 ng / ml, respectively.

[0199] Human recombinant NME2 stimulated a statistically significant proliferation of the murine C2C12 cell line, which was 118% to 107% of the control group in the concentration range of 1000 ng / ml to 62.5 ng / ml, respectively.

[0200] After performing the cytotoxicity experiment, the highest non-toxic concentration of the test substance was determined, and then these concentrations were selected to further evaluate their biological activities in the C2C12 cell line.

[0201] Assessment of the effects of recombinant human SERPINF1 and NME2 proteins on activation of the MLC1f promoter by secreted Gauss luciferase

[0202] The activation of the MLC1f promoter by different treatments was evaluated by measuring secreted Gauss luciferase (GLuc). By monitoring the level of secreted GLuc, the activity of the promoter under various experimental conditions could be quantified.

[0203] The C2C12 cell line was cultured in a 48-well plate for 5 days, and the culture medium was changed daily. In the first two days, the culture medium was replaced with growth medium. In the last 3 days, the culture medium was replaced with differentiation medium. On the 5th day of the experiment, 50 ng / ml IGF1 was used as a positive control, 25 μg / ml dexamethasone was used as a negative control, and the culture medium was used as a vehicle control. Supernatant samples were collected on the 1st, 4th, and 7th days for GLuc determination. The luciferase activity was directly evaluated and compared as a measure of promoter activity without protein concentration normalization.

[0204] On the first day, no induction of luciferase activity was observed even with the positive control IGF1.

[0205] On the 4th day after treatment, it was observed that under the action of the positive control IGF1, the luciferase driven by the MLC1f promoter was significantly activated by 172.4 ± 4.8. At the same time, the negative control dexamethasone had no effect, showing a luciferase activity of 100.4 ± 1.4. These results indicate that the experimental setup was correct and the experiment ran as expected, verifying the reliability of our findings.

[0206] Mouse C2C12 cell lines were treated with different concentrations of human recombinant SERPINF1 protein, which activated the MLC1f promoter in a dose-dependent manner. Specifically, as determined by secreted GLuc in the C2C12 cell line, human recombinant SERPINF1 protein significantly activated the MLC1f promoter statistically at three days after treatment (115% to 128% for 125 ng / ml and 250 ng / ml respectively) and at seven days after treatment (135% to 141% for 15.6 ng / ml to 250 ng / ml).

[0207] Importantly, on the 7th day, the luciferase activity in the positive control IGF1 decreased (from 172% on the 3rd day to 162% on the 7th day), while the luciferase activity in the cells treated with SERPINF1 increased (in the 250 ng / ml treated sample, the activity increased from 128% on the 3rd day to 140% on the 7th day).

[0208] Similar to treating mouse C2C12 cell lines with different concentrations of human recombinant SERPINF1 protein, dose-dependent activation of the MLC1f promoter was also observed with NME2 treatment. Specifically, as determined by secreted GLuc in the C2C12 cell line, human recombinant NME2 protein significantly activated the MLC1f promoter statistically at three days after treatment (121.2% to 135.8% for 125 ng / ml and 250 ng / ml respectively) and at seven days after treatment (113% to 126.4% for 15.6 ng / ml to 250 ng / ml). On the 7th day, the luciferase activity in the positive control IGF1 decreased (from 172% on the 3rd day to 162% on the 7th day), while the cells treated with NME2 still showed increased activity.

Claims

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

2. The composition according to claim 1, wherein the egg yolk is avian egg yolk.

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

4. The composition according to claim 3, wherein the one or more myogenic compounds are identified in subfractions F7.11, F7.18, F7.21, FS.10, FS.11 and / or FS.22 of fertilized avian egg yolk.

5. The composition according to claim 3, wherein the one or more myogenic compounds are synthetic derivatives of myogenic compounds isolated from subfractions F7.11, F7.18, F7.21, FS.10, FS.11 and / or FS.22 of fertilized avian egg yolk.

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

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

8. The composition according to 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-II type cytoskeletal cochlea, desmuslin, apolipoprotein A-I, albumin, cytoplasmic actin 5, cytoplasmic actin 1, vitellogenin-1, cytoplasmic actin 2, IF rod domain-containing protein, vitellogenin-3, glial fibrillary acidic protein, plasminogen and / or type II alpha-keratin IIA or a myogenic active fragment thereof.

9. The composition according to 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 myogenic active fragment thereof.

10. The composition according to claim 1, wherein the synthetic derivative is from a non-chicken species.

11. The composition according to claim 10, wherein the non-chicken species is selected from human, dog, cat, horse, cow, sheep, pig or primate.

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

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

14. The nutritional composition according to claim 12 or the pharmaceutical composition according to claim 13, which further comprises powdered egg yolk.

15. The nutritional composition according to claim 12 or the pharmaceutical composition according to claim 13, further comprising fostriecin.

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

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