Plasma fractions for muscle regeneration

Abstract

Methods and compositions for treating aging-related diseases and muscle restoration, preventing muscle degeneration and maintaining muscle are described. The compositions used in the methods include plasma and plasma fractions derived from plasma that have therapeutic and / or disease-preventing effects.

Description

[0001] I. Cross-Reference to Related Applications

[0002] Pursuant to 35 USC §119(e), this application claims priority to U.S. Provisional Patent Application No. 62 / 930,336, filed on November 4, 2019; U.S. Provisional Patent Application No. 62 / 966,953, filed on January 28, 2020; and U.S. Provisional Patent Application No. 63 / 062,735, filed on August 7, 2020; the disclosures of which are incorporated herein by reference.

[0003] II. Technical field

[0004] The present invention relates to the prevention and treatment of muscle diseases and injuries.The present invention relates to the use of blood products, such as plasma fractions, for the treatment and / or prevention of conditions associated with aging, such as neurocognitive disorders and neurodegenerative disorders.

[0005] III. Summary of the Invention

[0006] Skeletal muscle has a high regenerative capacity, and satellite cells (myogenic stem cells) are the source of this capacity. (Kang JS et al., Curr Opin Clin Nutr Metab Care, 13(3):243-48 (2010) and Jang YC et al., Cold Spring Harb Symp Quant Biol. 76:101-11 (2011)). Satellite cells are activated in response to muscle damage during maturity, but may be pathologically dysregulated in malnutrition diseases (Jang, supra).

[0007] The regeneration of skeletal muscle is believed to be coordinated through four processes. The processes include: muscle fiber degeneration leading to necrosis; inflammation and invasion of certain inflammatory cells into the muscle; regeneration through activation of satellite cells and subsequent differentiation into myoblasts that help support the formation of new muscle fibers and repair existing surviving muscle fibers; and remodeling / repair, in which the regenerated fibers mature and the extracellular matrix is ​​remodeled (Ibid). The regenerative activity of muscle is also closely related to metabolism that can regulate the processes (Ibid).

[0008] Sarcopenia is the progressive loss of skeletal muscle mass and strength due to aging. (Tabebordbar M et al., Annu. Rev. Pathol. Mech. Dis., 8:441-75 (2013)). It is a growing health problem worldwide, affecting about a quarter of individuals over 70 years old and 40% of individuals over 80 years old (supra). It can lead to a decrease in independence, loss of ability to perform normal activities of daily living, and a decrease in quality of life (supra). With age, skeletal muscle regenerative capacity becomes defective, which is at least partially attributed to a decrease in the number of myonuclei in muscle satellite cells and myofibers (supra and Brack AS et al., Science, 317:807-810 (2007)). In addition, the total number and size of muscle fibers decrease / reduce with aging (Jang, supra).

[0009] In addition to muscle loss and degeneration associated with aging, muscle function may also be impaired by acute physical or chemical injury, ischemia / reperfusion (e.g., organ transplant surgery, stroke, hypovolemic shock), contraction-induced injury, inflammatory myopathies, and genetically related degenerative diseases. The latter may include, for example, Duchenne and Becker muscular dystrophy, myotonic dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy, and facioscapulohumeral muscular dystrophy (supra).

[0010] Currently, treatment options for muscle wasting diseases are limited and focus on managing symptoms, typically through management of immune and inflammatory responses (Ibid.). Therefore, new methods are needed to reverse the effects of muscle damage and degeneration. Although heterochronic parabiosis between young and old mice and serum from young mice have been shown to have some efficacy in reducing the myogenic to fibrogenic conversion of certain muscle cells, there remains a need for a more practical and standardized intervention (Brack, Ibid.). The present invention addresses these needs by providing specific fractions or products of plasma fractionation.

[0011] IV. Incorporation by Reference

[0012] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0013] V. Description of the Figures

[0014] Figure 1A method for fractionation of pooled plasma is described. Pooled plasma is cryo-separated into an effluent and a paste. The effluent is further separated into an effluent I (Fraction I effluent) and a Fraction I paste. This process can be repeated to obtain, for example, effluents and pastes of Fraction II+III, Fraction IV-1, Fraction IV-4, and Fraction V.

[0015] Figure 2 Depicted is a short-term treatment scheme for C2C12 cells differentiated prior to 5 days in 2% horse serum (HS), with glucose utilization assays initiated twenty-four hours after treatment.

[0016] Figure 3A The concentration of glucose remaining in the culture medium of C2C12 cells differentiated into myotubes for 5 days and then treated for twenty-four hours with various treatment conditions including: (1) untreated; (2) 1 mM metformin (Met) positive control; (3) 0.5 mM metformin; (4) 0.25 mM metformin; (5) vehicle (10%); (6) PPF1 (5 mg / mL); (7) HAS1 (5 mg / mL); and (8) recombinant human albumin (rhAlbumin 5 mg / mL).

[0017] Figure 3B For Figure 2 and 3A Still photos captured from the video of myotubes treated with PPF1 as described.

[0018] Figure 4 A long-term culture treatment design using either 0% or 2% horse serum is depicted, with glucose utilization assays starting six days after the start of treatment and 48 hours after the last application of treatment.

[0019] Figure 5 Shown according to Figure 4 Experimental design depicted in Figure 2. Micrographs of C2C12 cells cultured in 0% or 2% horse serum (HS). Treatment with PPF1 at 0% HS concentration resulted in a greater amount of myotube formation than in the untreated condition.

[0020] Figure 6 Shown are C2C12 cells treated with 0% horse serum and PPF1 that exhibit positive staining for the myogenic differentiation marker myosin heavy chain.

[0021] Figure 7 Reported in accordance with Figure 4 The experimental design depicted in Figure 2 was used to determine the remaining glucose utilization in the culture medium of C2C12 cells using 0% horse serum and the following treatments: untreated; vehicle; PPF1; HAS1; and rhAlbumin.

[0022] Figure 8 Reported in accordance with Figure 4 Experimental design depicted in Glucose utilization in the culture medium of C2C12 cells using 0% horse serum and the following treatments: untreated; PPF1; fraction IV-4 paste suspension and IV-4 effluent.

[0023] Fig. 9 Relative expression of glucose transporter type 4 (GLUT-4) in C2C12 myoblasts untreated or treated with control vehicle, PPF1 (5 mg / mL) or recombinant human albumin (rhAlbumin 5 mg / mL) is reported.

[0024] Fig.10 Reported Figure 8 The plasma fraction / fractionation product described in the present invention is used to measure the dose-response relationship between glucose utilization. Different treatment concentrations (0.15 mg / mL, 0.3 mg / mL, 0.6 mg / mL, 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL and 10 mg / mL in the culture medium) are added to the cells. After 6 days of treatment and 48 hours with the same culture medium, the amount of glucose remaining in the culture medium is analyzed by the glucose utilization assay described above. All three compositions show a dose-response relationship for glucose utilization.

[0025] Fig.11A Table 1 is a summary of several experiments performed on C57BL / 6 mice of different ages and young rats and tested muscle weight values ​​for the tibialis anterior, extensor digitorum longus, gastrocnemius and soleus muscles. Each experiment also tested the effect of different lengths of time after the last dose of treatment with vehicle or PPF1 on muscle weight.

[0026] Fig. 11B Figure 1 is a diagram of an experimental protocol for studying muscle-related metrics in 22-month-old male C57B6 mice treated with PPF1 or controls. Pulse administration of PPF1 or control vehicle (150 μL per dose, iv) was performed on 26-month-old male C57B6 mice for 7 consecutive days. Ten (10) days after the last dose, the following skeletal muscle groups were harvested: tibialis anterior (TA), extensor digitorum longus (EDL), and soleus (SOL). Muscle to body weight (BW) ratios were obtained from each muscle group.

[0027] Fig. 11C Shown, from Fig. 11B The tibialis anterior muscle tissue of the regimen significantly increased in weight when treated with PPF1 compared with the control (mean ± SEM, **p < 0.01, Welch test).

[0028] Fig.11D Shown, from Fig. 11BThe extensor digitorum longus muscle tissue of the regimen significantly increased in weight when treated with PPF1 compared with the control (mean ± SEM, **p < 0.01, Welch's test).

[0029] Fig.11E Shown, from Fig. 11B The soleus muscle tissue of the regimen significantly increased in weight when treated with PPF1 compared with the control (mean ± SEM, *p < 0.05, Welch test).

[0030] Fig. 12A Shown is the role of PPF1 in inducing a slow-twitch myofiber gene (Myl2) in mouse tibialis anterior muscle.

[0031] Fig. 12B , Fig. 12C and Fig.12D Shown is the effect of PPF1 in reducing the expression of fast-twitch myofiber genes (Myh1 (2x), Myh2 (2a), and Myh4 (2b)), respectively, in mouse tibialis anterior muscle.

[0032] Fig.13A , Fig. 13B , Fig. 13C and Fig.13D All show C2C12 cells after 3 days of culture in 0% horse serum combined with various treatment conditions.

[0033] Fig.13A Shown are C2C12 cells under untreated conditions.

[0034] Fig. 13B Shown are C2C12 cells treated with 0.3% PPF1 for 3 days.

[0035] Fig. 13C and 13D Shown are C2C12 cells treated with 15 mg / mL of Fraction IV-1 paste suspension and 0.6 mg / mL of IV-1 paste suspension for 3 days, respectively.

[0036] Fig.14 The dose-response relationship between the plasma fraction / fractionation product from the C2C12 supernatant and normalized glucose utilization (%) 24 hours after the last medium change is reported. Cells were cultured for a total of six days in 0% horse serum. The graph depicts the effect of increasing doses of PPF1, Fraction IV-1 paste suspension, and Fraction IV-1 effluent on glucose utilization.

[0037] Fig.15The dose-response relationship between the plasma fraction / fractionation product from the C2C12 supernatant and normalized glucose utilization (%) 24 hours after the last medium change is reported. Cells were cultured for a total of six days in 2% horse serum. The graph depicts the effect of increasing doses of PPF1, Fraction IV-1 paste suspension, and Fraction IV-1 effluent on glucose utilization.

[0038] Fig.16A and Fig. 16B reported the effect of insulin-like growth factor-1 (IGF-1) on glucose utilization in C2C12 cells treated in 2% horse serum.

[0039] Fig.16A The dose-response relationship between IGF-1 treatment (x-axis) and glucose utilization is reported, revealing an EC of 17.43 ng / mL 50 .

[0040] Fig. 16B reported a dose-response relationship between PPF1 treatment and glucose utilization, revealing that an EC of 2.9 mg / mL (containing 0.87 ng / mL IGF1) 50 .

[0041] Fig.17A Schematic representation of the experimental protocol for studying muscle recovery using different fraction treatments for the barium chloride-induced injury model.

[0042] Fig. 17B Reported in Fig.17A Results of contractile force measurements performed on days 0 and 17 of the protocol described in , where treatments included vehicle, recombinant human albumin, PPF1, or HAS1.

[0043] Fig.18A Schematic representation of the experimental protocol to study the effects of plasma fractions on serum mouse IGF1 levels.

[0044] Fig.18B Revealing that even in Fig.18A PPF1 treatment was also accompanied by a significant increase in mouse IGF-1 in serum 10 days after the last dose in the described regimen.

[0045] Fig.19A Schematic representation of the experimental protocol for investigating whether the plasma fraction can reduce heart weight in aged C57BL / 6 mice in a model of cardiac hypertrophy observed in aged mammals.

[0046] Fig.19B Heart weights in milligrams are shown for vehicle-treated and PPF1-treated mice.

[0047] Fig.19C Shows Fig.19B Heart weight to body weight ratios of the same mice as described in .

[0048] Fig. 20A , Fig. 20B and Fig. 20C Reported Fig.19A , 19B and expression of RNA levels of cardioprotective markers in the heart as described in 19C.

[0049] Fig. 20A It was shown that the RNA expression of sarcoendoplasmic reticulum calcium-ATPase (SERCA2a) was significantly increased by PPF1 treatment compared with the control.

[0050] Fig. 20B It was shown that the RNA expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1a) was significantly increased by PPF1 treatment compared with the control.

[0051] Fig. 20C It was shown that the RNA expression of α-myosin heavy chain (aMHC) was significantly increased with PPF1 treatment compared to the control.

[0052] Fig.21A The amount of lactate (myogenic differentiation factor) produced in C2C12 cells after three (3) hours of treatment with various factors is shown. These factors include vehicle, 2-DG (negative control), metformin (positive control), oligomycin, HAS1, recombinant human albumin (rhAlbumin), PPF1, Fraction IV-1 paste suspension, and three different concentrations of Fraction IV-1 paste suspension.

[0053] Fig.21B Shows the Fig.21A Effects of various factors described in on lactate production in C2C12 cells after five (5) hours of treatment.

[0054] VI. Specific Implementation Methods

[0055] A. Introduction

[0056] The present invention relates to the treatment of muscle, including skeletal muscle disorders or diseases. Plasma fractions including plasma fractionation products have been shown to have significant activity in muscle regeneration processes, such as increasing the utilization of glucose by myoblasts, increasing the differentiation of myoblasts to myotube formation, increasing contractility, and inducing slow-contracted muscle fiber-related genes. Compared with whole plasma serum, plasma fractions have several advantages because the plasma fractionation process can remove problematic coagulation factors and eliminate the need for cross-matching. In addition, in certain analyses, plasma fractions have shown unexpected improvements in efficacy compared to young plasma (see, e.g., U.S. Patent Application No. 15 / 499,694 and U.S. Patent Application No. 16 / 432,114; both are incorporated herein by reference in their entirety). Therefore, there is no reasonable predictability in predicting efficacy of plasma fractionation products from whole plasma serum.

[0057] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific method or composition described, and thus, of course, can vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be restrictive, because the scope of the present invention will be limited only by the appended claims.

[0058] The publications discussed herein are provided only for their disclosures prior to the date of filing of the present application. Any information herein should not be construed as an admission that the present invention cannot precede these publications because it was invented prior to the invention. In addition, the publication date provided may be different from the actual publication date, which may require independent confirmation.

[0059] In the case of providing a numerical range, it should be understood that each intermediate value between the upper and lower limits of the range is also explicitly disclosed, to ten times the lower limit unit, unless otherwise clearly specified herein. Each smaller range between any value or intermediate value in the range and any other value or intermediate value in the range is included in the present invention. The upper and lower limits of these smaller ranges can be independently included in the range or excluded from the range, and when one of the two limits, none or all are included in the smaller range, it is also included in the present invention, unless it is any clearly excluded limit value in the specified range. When the range includes one or both of the limits, the scope excluding one or both of these contained limits is also included in the present invention.

[0060] It should also be noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as an antecedent basis for use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0061] Those skilled in the art will appreciate upon reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the invention. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

[0062] B. Definition

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, some possible and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the publications cited. It should be understood that if there is a conflict, the present disclosure replaces any disclosure incorporated into the publication.

[0064] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, reference to "the peptide" includes reference to one or more peptides and their equivalents, such as polypeptides, known to those skilled in the art, and the like.

[0065] When describing the methods of the present invention, the terms "host", "subject", "individual" and "patient" are used interchangeably and refer to any mammal that needs to be treated according to the disclosed methods. Mammals include, for example, humans, sheep, bovines, equines, swine, canines, felines, non-human primates, mice and rats. In certain embodiments, the subject is a non-human mammal. In some embodiments, the subject is a farm animal. In other embodiments, the subject is a pet. In some embodiments, the subject is a mammal. In certain embodiments, the subject is a human. Other subjects may include domestic pets (e.g., dogs and cats), livestock (e.g., cattle, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs and rats, for example, as in animal models of diseases) and non-human primates (e.g., chimpanzees and monkeys). Thus, the subject of the present invention may include, but is not limited to, mammals, for example, humans and other primates, such as chimpanzees and other ape and monkey species, etc., wherein the subject is a human in certain embodiments. The term subject is also meant to include humans or organisms of any age, budget, or other physical characteristics, where the subject may be an adult, a child, an infant, or a newborn.

[0066] "Young person" or "young individual" refers to an individual who is 40 years old or less than 40 years old, such as 35 years old or less than 35 years old, including 30 years old or less than 30 years old, such as 25 years old or less than 25 years old, or 22 years old or less than 22 years old by chronological age. In some cases, the individual serving as the source of the blood product comprising young plasma is 10 years old or younger, such as 5 years old or younger, including 1 year old or younger individuals. In some cases, the object is a newborn, and the source of the plasma product is the umbilical cord, wherein the plasma product is harvested from the umbilical cord of the newborn. Therefore, "young" and "young individual" can refer to an object between 0 and 40 years old, such as 0 years old, 1 year old, 5 years old, 10 years old, 15 years old, 20 years old, 25 years old, 30 years old, 35 years old or 40 years old. In other cases, "young" and "young individual" can refer to biological (different from chronological) age, such as individuals who have not shown the inflammatory cytokine levels in the plasma shown in older individuals. In contrast, "young" and "young individuals" can refer to biological (as opposed to chronological) age, e.g., individuals who exhibit higher levels of anti-inflammatory cytokines compared to levels in older individuals. For example, but not limited to, the inflammatory cytokine is eotaxin, and the fold difference between the young subject or young individual and the elderly individual is at least 1.5-fold. Similarly, the fold difference of other inflammatory cytokines between elderly individuals and young individuals can be used to refer to biological age. (See U.S. Patent Application No. 13 / 575,437, which is incorporated herein by reference). Typically, the individual is healthy, e.g., the individual does not have a malignant hematologic disease or an autoimmune disease at the time of collection.

[0067] As used herein, "treatment" refers to any of (i) preventing a disease or condition or (ii) alleviating or eliminating the symptoms of a disease or condition. Treatment can be performed prophylactically (before the onset of the disease) or therapeutically (after the onset of the disease). The effect of completely or partially preventing a disease or its symptoms can be referred to as preventive, and / or the effect of partially or completely curing a disease and / or a side effect attributable to a disease can be referred to as therapeutic. Therefore, as used herein, the term "treatment" covers any treatment of a condition associated with muscle damage, muscle disease, muscle disorder or muscle condition in a mammal that can benefit from improved muscle generation, regeneration, healing, function or repair, and includes: (a) preventing the subject from developing a condition; (b) inhibiting the condition, i.e., preventing it from occurring; or (c) alleviating the condition, i.e., causing the condition to subside. Treatment can cause a variety of different physical manifestations, for example, regulation of gene expression, restoration of tissues or organs, reduction of inflammation, etc. The therapeutic agent can be administered before, during or after the onset of the condition. The target therapy can be administered during the symptomatic period of the condition, and in some cases, the target therapy is administered after the symptomatic period of the condition.

[0068] Blood products containing plasma components. When implementing the target method, a blood product comprising a plasma component is administered to an individual in need, such as an individual suffering from one or more of the following conditions: muscle damage, muscle disease, muscle disorder or muscle disorder that can benefit from improved muscle generation, regeneration, healing, function or repair. Therefore, the method according to an embodiment of the present invention includes administering a blood product comprising a plasma component from an individual ("donor individual" or "donor") to an individual suffering from one or more of muscle damage, muscle disease, muscle disorder or muscle disorder that can benefit from improved muscle generation, regeneration, healing, function or repair ("receiving individual" or "recipient"). "Blood product comprising a plasma component" means any product containing plasma derived from blood (e.g., whole blood, plasma or a fraction thereof). The term "plasma" is used in its conventional sense and refers to the straw-colored / light yellow liquid component of blood, which consists of about 92% water, 7% protein, such as albumin, gamma globulin, antihemophilic factor and other coagulation factors, and 1% mineral salts, sugars, fats, hormones and vitamins. Non-limiting examples of plasma-containing blood products suitable for the subject methods include whole blood treated with an anticoagulant (e.g., EDTA, citrate, oxalate, heparin, etc.), blood products produced by filtering whole blood to remove leukocytes ("leukocyte filtration"), plasma from plasmapheretically-derived or apheretically-derived plasma, fresh frozen plasma, blood products consisting essentially of purified plasma, and blood products consisting essentially of plasma fractions. In some cases, the plasma product used is a non-whole plasma product, meaning that the product is not whole blood, and therefore lacks one or more components found in whole blood, such as red blood cells, white blood cells, etc., at least to the extent that the components are present in whole blood. In some cases, the plasma product is substantially acellular, if not completely acellular, wherein in such cases, the cell content may be 5% or less by volume, such as 1% or less than 1%, including 0.5% or less than 0.5%, wherein in some cases the acellular plasma fraction is a composition that is completely devoid of cells, i.e., they do not include cells.

[0069] Collection of blood products containing plasma components . Embodiments of the methods described herein include administering a blood product comprising a plasma component that may be derived from a donor, including a human volunteer. The term "derived from a human" may refer to such products. Methods for collecting blood products comprising plasma from a donor are well known in the art. (See, e.g., AABB TECHNICAL MANUAL, (Mark A. Fung et al., eds., 18th ed., 2014), which is incorporated herein by reference).

[0070] In one embodiment, the donation is obtained by venipuncture. In another embodiment, the venipuncture is only a single venipuncture. In another embodiment, saline volume replacement is not used. In a preferred embodiment, a plasmapheresis process is used to obtain a blood product containing plasma. Plasmapheresis can include removing a volume of plasma adjusted according to weight and returning cellular components to the donor. In a preferred embodiment, sodium citrate is used during the plasmapheresis process to prevent cell coagulation. After citrate administration, the volume of plasma collected from the donor is preferably 690mL to 880mL, and is preferably coordinated with the weight of the donor.

[0071] C. Plasma fraction

[0072] During World War II, there was a need for a stable plasma expander that could be used on the battlefield when soldiers lost a lot of blood. Therefore, methods for preparing freeze-dried plasma were developed. However, since sterile water is required for reconstitution, it is difficult to use freeze-dried plasma in combat situations. As an alternative, Dr. EJ Cohn suggested that albumin could be used and prepared a ready-to-use stable solution that could be immediately introduced for treating shock. (See Johan, Current Approaches to the Preparation of Plasma Fractions in (Biotechnology of Blood) 165 (Jack Goldstein ed., 1st ed., 1991)). Dr. Cohn's method for purifying plasma fractions utilizes the denaturing effect of cold ethanol and utilizes changes in pH and temperature to achieve separation.

[0073] Embodiments of the methods described herein include administering plasma fractions to a subject. Fractionation is a method by which certain protein subgroups are separated from plasma. Fractionation techniques are known in the art and rely on steps developed by Cohn et al. in the 1940s. (E. Cohn, Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. 68 J Am Chem Soc 459 (1946), which is incorporated herein by reference). Several steps are involved in the process, each of which involves a specific ethanol concentration that results in selective protein precipitation and changes in pH, temperature, and osmotic molarity. The precipitate is also separated by centrifugation or precipitation. The original "Cohn fractionation method" involves separating proteins into five fractions by precipitation, referred to as fraction I, fraction II+III, fraction IV-1, fraction IV-4, and fraction V. Albumin is the end point (fraction V) product initially identified in the process. According to embodiments of the present invention, each fraction, filtrate (or effluent or sometimes referred to as waste stream from a previous separation step) contains or potentially contains a therapeutically useful protein fraction.(See Thierry Burnouf, Modern Plasma Fractionation, 21(2) Transfusion Medicine Reviews 101 (2007); Adil Denizli, Plasma fractionation: conventional and chromatographic methods for albumin purification, 4 J. Biol. & Chem. 315, (2011); Gjessing EC, et al., J. Biol. & Chem. (174): 682-96 (1948); and T. Brodniewicz-Proba, Human Plasma Fractionation and the Impact of New Technologies on the Use and Quality of Plasma-derived Products, 5 Blood Reviews 245 (1991), and U.S. Pat. Nos. 3869431, 5110907, 5219995, 7531513, and 8772461, which are incorporated herein by this reference). The above experimental parameters can be adjusted to obtain specific protein fractions.

[0074] Recently, fractionation has reached further complexity and therefore constitutes another embodiment of the present invention. This recent increase in complexity has occurred by the introduction of chromatography, resulting in the separation of new proteins from existing fractions such as cryoprecipitate, cryo-poor plasma and Cohn fractions; by integrating chromatography and ethanol fractionation processes to improve the recovery of IgG; and virus reduction / inactivation / removal (same). In order to capture proteins at physiological pH and ionic strength, anion exchange chromatography can be used. This maintains the functional activity of proteins and / or protein fractions. Heparin and monoclonal antibodies are also used for affinity chromatography. In addition, fractionation by gel filtration, fractionation by salts and fractionation by polyethylene glycol are used. (Hosseini M Iran J Biotech, 14 (4): 213-20 (2016), which is incorporated herein by reference). One of ordinary skill in the art will recognize that the above parameters can be adjusted to obtain a specific desired protein-containing plasma fraction.

[0075] Plasma fractionation can also be based on ammonium sulfate. (See, e.g., Odunuga OO, Biochem Compounds, 1:3 (2013); Wingfield PT, Curr Protoc Protein Sci, Appx. 3 (2001), incorporated herein by reference). In addition to obtaining specific blood fractions, ammonium sulfate-based fractionation is also employed to reduce a large number of proteins in plasma. (Saha S et al., J. Proteomics Bioinform, 5(8) (2012), incorporated herein by reference).

[0076] In an embodiment of the present invention, plasma is fractionated in an industrial environment. The frozen plasma is thawed at 1°C to 4°C. The thawed plasma is subjected to continuous cold centrifugation, and the cryoprecipitate is separated out. The recovered cryoprecipitate is frozen and stored at a temperature of -30°C or lower than -30°C. The cryoprecipitate-depleted (“cryo-poor”) plasma is immediately processed to capture (e.g., by preliminary chromatography) labile coagulation factors such as factor IX complex and its components, as well as protease inhibitors such as antithrombin and C1 esterase inhibitor. Continuous centrifugation and precipitation separation can be used for subsequent steps. Such techniques are known to those of ordinary skill in the art and are described, for example, in U.S. Patent Nos. 4,624,780, 5,219,995, 5,288,853, and U.S. Patent Applications Nos. 20140343255 and 20150343025, the disclosures of which are incorporated herein by reference in their entirety).

[0077] In an embodiment of the present invention, the plasma fraction may include a plasma fraction containing a high concentration of albumin. In another embodiment of the present invention, the plasma fraction may include a plasma fraction containing a high concentration of IgG or intravenous immunoglobulin (IGIV) (e.g., ). In another embodiment of the present invention, the plasma fraction may include an IGIV plasma fraction, for example, one that has been substantially depleted of immunoglobulin (IgG) by a method known to those of ordinary skill in the art, such as protein A-mediated depletion (See Keshishian, H. et al., Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields Novel Candidates for Early Myocardial Injury, Molecular & Cellular Proteomics, 14 at 2375-93 (2015)). In another embodiment, the plasma fraction can be a plasma fraction in which substantially all coagulation factors are removed in order to retain the efficacy of the fraction and reduce the risk of thrombosis. For example, the plasma fraction can be a plasma fraction described in U.S. Pat. No. 62 / 376,529, filed on August 18, 2016 (the disclosure of which is incorporated herein by reference in its entirety).

[0078] D. Albumin products

[0079] For those of ordinary skill in the art, albumin plasma products ("APP") are divided into two major categories: plasma protein fraction ("PPF") and human albumin solution ("HAS"). PPF is derived from a process with a higher yield than HAS but a lower minimum albumin purity than HAS (>83% for PPF and >95% for HAS). (Production of human albumin solution: a continuously developing colloid, P. Matejtschuk et al., British J. of Anaesthesia 85(6):887-95, at888(2000)). In some cases, the albumin purity of PPF is 83% to 95%, or 83% to 96%. Albumin purity can be determined by electrophoresis or other quantitative determination methods, such as by spectroscopy. In addition, some people point out that PPF has disadvantages due to the presence of protein "contaminants" such as PKA (same). Therefore, PPF preparations are no longer popular as albumin plasma products, and have even been removed from the pharmacopoeia of some countries (same). Contrary to these concerns, the present invention advantageously utilizes these "contaminants." In addition to the alpha, beta and gamma globulins and the above-mentioned PKA, the methods of the present invention also utilize additional proteins or other factors among the "contaminants" that promote processes such as neurogenesis, neuronal cell survival, improved cognitive or motor function, and reduced neuroinflammation.

[0080] Those skilled in the art will recognize that there are or have been several commercial sources of PPF ("commercial PPF preparations"). These commercial sources include Plasma-PlexTM PPF (Armour Pharmaceutical Co., Tarrytown, NY), Plasmanate TM PPF (Grifols, Clayton, NC), Plasmatein TM (Alpha Therapeutics, Los Angeles, CA) and Protenate TM PPF (Baxter Labs, Inc. Deerfield, IL).

[0081] Those skilled in the art will also recognize that there are or have been several commercial sources of HSA ("commercial HSA preparations"). These commercial sources include Albuminar TM (CSL Behring), AlbuRx TM (CSL Behring), Albutein TM (Grifols,Clayton,NC),Buminate TM (Baxatla, Inc., Bannockburn, IL), Flexbumin TM (Baxatla, Inc., Bannockburn, IL) and Plasbumin TM (Grifols, Clayton, NC).

[0082] 1. Plasma protein fraction (human) (PPF)

[0083] According to the U.S. Food and Drug Administration ("FDA"), "Plasma Protein Fraction (Human)" or PPF is a proprietary name for a product defined as "a sterile solution of proteins consisting of albumin and globulins derived from human plasma." (Code of Federal Regulations "CFR" 21 CFR 640.90, which is incorporated herein by reference). The source material for PPF is plasma recovered from whole blood prepared in accordance with the provisions of 21 CFR 640.1-640.5 (incorporated herein by reference) or source plasma prepared in accordance with the provisions of 21 CFR 640.60-640.76 (incorporated herein by reference).

[0084] The PPF was tested to determine compliance with the following standards pursuant to 21 CFR 640.92 (incorporated herein by reference):

[0085] (a) the final product should be a 5.0 + / - 0.30% protein solution; and

[0086] (b) The total protein in the finished product shall consist of at least 83% albumin and not more than 17% globulin. Gamma-globulin shall not exceed 1% of the total protein. The protein composition shall be determined by a method approved for each manufacturer by the Director of the Center for Biologics Evaluation and Research of the Food and Drug Administration.

[0087] As used herein, "plasma protein fraction" or "PPF" refers to a sterile solution of proteins consisting of albumin and globulin derived from human plasma, wherein the albumin content is at least 83% and the globulins (including α1, α2, β and γ globulins) and other plasma proteins do not exceed 17%, and the γ globulin does not exceed 1%, as determined by electrophoresis. (Hink, JH, Jr. et al., Preparation and Properties of a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2 (174) (1957)). PPF may also refer to a solid form, which has a similar composition when suspended in a solvent. The total globulin fraction can be determined by subtracting albumin from the total protein. (Busher, J., Serum Albumin and Globulin, CLINICAL METHODS: THE HISTORY, PHYSICAL, AND LABORATORY EXAMINATIONS, Chapter 10, edited by Walker HK, Hall WD, Hurst JD (1990)).

[0088] 2. Albumin (human) (HAS)

[0089] According to the FDA, "albumin (human)" (also referred to herein as "HAS") is the proprietary name of a product defined as a "sterile solution of albumin derived from human plasma". (Code of Federal Regulations "CFR" 21CFR 640.80, which is incorporated herein by reference). The source material of albumin (human) is plasma recovered from whole blood prepared in accordance with the provisions of 21CFR 640.1-640.5 (incorporated herein by reference) or source plasma prepared in accordance with the provisions of 21CFR 640.60-640.76 (incorporated herein by reference). Other requirements for albumin (human) are listed in 21CFR 640.80-640.84 (incorporated herein by reference).

[0090] Albumin (human) was tested to determine compliance with the following standards in accordance with 21 CFR 640.82:

[0091] (a) Protein concentration. The final preparation should conform to one of the following concentrations: 4.0 + / - 0.25%; 5.0 + / - 0.30%; 20.0 + / - 1.2%; and 25.0 + / - 1.5% protein solution.

[0092] (b) Protein composition. At least 96% of the total protein in the final product shall be albumin as determined by a method approved for each manufacturer by the Director, Center for Biologics Evaluation and Research, Food and Drug Administration.

[0093] As used herein, "albumin (human)" or "HAS" refers to a sterile solution of proteins consisting of albumin and globulin derived from human plasma, wherein the albumin content is at least 95% and globulins (including α1, α2, β and γ globulins) and other plasma proteins do not exceed 5%. HAS may also refer to a solid form, which has a similar composition when suspended in a solvent. The total globulin fraction can be determined by subtracting albumin from the total protein.

[0094] As can be appreciated by those of ordinary skill in the art, PPF and HAS fractions may also be freeze-dried or in other solid forms. Such formulations with appropriate additives may be used to prepare, for example, tablets, powders, granules or capsules. The solid may be formulated into a formulation for injection by dissolving, suspending or emulsifying the solid form in an aqueous or non-aqueous solvent, such as a vegetable oil or other similar oil, a synthetic fatty acid glyceride, a higher fatty acid ester or a propylene glycol ester; and, if necessary, including conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0095] E. Fractions with reduced coagulation factors

[0096] Another embodiment of the present invention uses a plasma fraction from which substantially all coagulation factors are removed to retain the efficacy of the fraction and to reduce the risk of thrombosis. Conveniently, the blood product can be derived from a collection of young donors or young donors, and it can be made free of IgM to provide a young blood product compatible with ABO. At present, the plasma of the transfusion is matched with the ABO blood type because the presence of naturally occurring antibodies to A and B antigens can cause transfusion reactions. IgM is presented to be responsible for transfusion reactions when the patient receives ABO-mismatched plasma. Removing IgM from the blood product or fraction helps to eliminate the transfusion reactions of the object of the blood product and plasma fraction of the present invention.

[0097] Therefore, in one embodiment, the present invention relates to a method for treating an object suffering from an undesirable condition / indication associated with any of the following: a muscle injury, muscle disease, muscle disorder or muscle condition that can benefit from improved muscle generation, regeneration, healing, function or repair. The method comprises: administering to the object a blood product or blood fraction derived from whole blood of an individual or a group of individuals, wherein the blood product or blood fraction is substantially free of (a) at least one coagulation factor and / or (b) IgM. In some embodiments, one or more than one individual from which the blood product or blood fraction is derived is a young individual. In some embodiments, the blood product is substantially free of at least one coagulation factor and IgM. In certain embodiments, the blood product is substantially free of fibrinogen (factor I). In other embodiments, the blood product is substantially free of red blood cells and / or white blood cells. In other embodiments, the blood product is substantially acellular. In other embodiments, the blood product is derived from plasma. U.S. Patent Application No. 62 / 376,529, filed on August 18, 2016, further supports such embodiments of the present invention, and the patent application is incorporated herein by reference in its entirety.

[0098] F. Treatment of protein-rich plasma products

[0099] Another embodiment of the present invention uses a plasma fraction with a reduced albumin concentration compared to PPF, but with an increased amount of globulin and other plasma proteins (referred to as "contaminants"). Like PPF, HAS, effluent I and effluent II / III, effluent IV-1, effluent IV-4, and effluent V, the embodiments are all effectively free of coagulation factors. Such plasma fractions are hereinafter referred to as "protein-rich plasma protein products". For example, an embodiment of the present invention can use a protein-rich plasma protein product, which is composed of 82% albumin and 18% α, β and γ globulin and other plasma proteins. Another embodiment of the present invention can use a protein-rich plasma protein product, which is composed of 81% albumin and 19% α, β and γ globulin and / or other plasma proteins. Another embodiment of the present invention can use a protein-rich plasma protein product, which is composed of 80% albumin and 20% α, β and γ globulin and / or other plasma proteins. Another embodiment of the present invention may use a protein-enriched plasma protein preparation consisting of 70%-79% albumin and corresponding 21%-30% alpha, beta and gamma globulins and other plasma proteins. Another embodiment of the present invention may use a protein-enriched plasma protein preparation consisting of 60%-69% albumin and corresponding 31%-40% alpha, beta and gamma globulins and other plasma proteins. Another embodiment of the present invention may use a protein-enriched plasma protein preparation consisting of 50%-59% albumin and corresponding 41%-50% alpha, beta and gamma globulins and other plasma proteins. Another embodiment of the present invention may use a protein-enriched plasma protein preparation consisting of 40%-49% albumin and corresponding 51%-60% alpha, beta and gamma globulins and other plasma proteins. Another embodiment of the present invention may use a protein-enriched plasma protein preparation consisting of 30%-39% albumin and corresponding 61%-70% alpha, beta and gamma globulins and other plasma proteins. Another embodiment of the present invention may use a protein-rich plasma protein preparation consisting of 20%-29% albumin and corresponding 71%-80% alpha, beta and gamma globulins and other plasma proteins. Another embodiment of the present invention may use a protein-rich plasma protein preparation consisting of 10%-19% albumin and corresponding 81%-90% alpha, beta and gamma globulins and other plasma proteins. Another embodiment of the present invention may use a protein-rich plasma protein preparation consisting of 1%-9% albumin and corresponding 91%-99% alpha, beta and gamma globulins and other plasma proteins. Another embodiment of the present invention may use a protein-rich plasma protein preparation consisting of 0% albumin and 100% alpha, beta and gamma globulins and other plasma proteins.

[0100] The above described embodiments of the invention may also have a total gamma globulin concentration of 1-5%.

[0101] The specific concentration of protein in the plasma fraction can be determined using techniques known to those of ordinary skill in the relevant art. Such techniques include, by way of example and not limitation, electrophoresis, mass spectrometry, ELISA analysis, and Western blot analysis.

[0102] G. Preparation of Plasma Fractions

[0103] Methods for preparing PPF and other plasma fractions are well known to those of ordinary skill in the art. Embodiments of the present invention allow blood for preparing human plasma protein fractions to be collected in flasks with citrate or anticoagulant citrate dextrose solution (or other anticoagulants) to inhibit coagulation, and further separated into fractions I, II+III, IV and PPF according to the method disclosed by Hink et al. (See Hink, JH, Jr. et al., Preparation and Properties of a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2 (174) (1957), incorporated herein by reference). According to this method, the mixture can be collected to 2–8°C. The plasma can then be removed by centrifugation at 7°C and stored at -20°C. The plasma can then be thawed and fractionated at 37°C, preferably within eight hours after being taken out of -20°C storage.

[0104] Plasma can be separated from fraction I using 8% ethanol at pH 7.2 and a temperature of -2 to -2.5°C, with a protein concentration of 5.1 to 5.6%. Cold 53.3% ethanol (176 mL / L plasma) and acetate buffer (with H2O) can be added at a rate of, for example, 450 mL / min using a nozzle while the plasma temperature is lowered to -2°C. 2 (200 mL 4M sodium acetate, 230 mL glacial acetic acid to 1 L). Fraction I can be separated by ultracentrifugation and removed from the effluent (effluent I). Fibrinogen can be obtained from fraction I according to methods known to those of ordinary skill in the art.

[0105] Fraction II+III is separated from effluent I by adjusting the effluent to pH 6.8, 21% ethanol at a temperature of -6°C and a protein concentration of 4.3%. During the process of lowering the temperature of effluent I to -6°C, cold 95% ethanol (176 mL / L effluent I) and 10 M acetic acid for pH adjustment can be added using a nozzle at a rate of, for example, 500 mL / min. The resulting precipitate (fraction II+III) can be removed by centrifugation at -6°C. Gamma globulin can be obtained from fraction II+III using methods known to those of ordinary skill in the art.

[0106] Fraction IV-1 was separated from effluent II+III ("effluent II / III") by adjusting the effluent to pH 5.2, 19% ethanol at -6°C and a protein concentration of 3%. H can be added using a nozzle. 2 O and 10M acetic acid for pH adjustment while maintaining effluent II / III at -6°C for 6 hours. The precipitated fraction IV-1 can be left standing for 6 hours at -6°C and then separated from the effluent by centrifugation at the same temperature. The stable plasma protein fraction is recovered from effluent IV-1 by adjusting the ethanol concentration to 30%, pH 4.65, temperature -7°C and a protein concentration of 2.5%. This can be achieved by adjusting the pH value of effluent IV-1 with cold acid-alcohol (two parts of 2M acetic acid and one part of 95% ethanol). While maintaining a temperature of -7°C, 170mL of cold ethanol (95%) is added to each liter of conditioned effluent IV-1. The precipitated protein can be left standing for 36 hours and then removed by centrifugation at -7°C. Fraction IV-4 paste / precipitate can also be obtained using the Cohn fractionation process and can be resuspended. Indeed, fraction IV-4 and methods for its manufacture have been previously described (Schopfer LM et al., PLoS ONE, 14(1):e0209795 (2018), incorporated herein by reference in its entirety) (Schopfer LM et al., PLoS ONE, 14(1):e0209795 (2018) and Bertolini J, Goss N, Curlin J eds., Production of Plasma Proteins for Therapeutic Use, 16.4:231:232 (2013), incorporated herein by reference in its entirety).

[0107] The recovered protein (stabilized plasma protein fraction) may be dried (e.g., freeze-dried) to remove ethanol and H 2 O. The resulting dry powder can be dissolved in sterile distilled water, for example, using 15 liters of water / kg of powder, and the pH of the solution can be adjusted to 7.0 with 1M NaOH. The final concentration of protein can be adjusted to 0.004M acetyltryptophan, 0.004M octanoic acid, and 0.112M sodium by adding sterile distilled water containing sodium acetyltryptophan, sodium octanoate, and NaCl to a final concentration of 5%. Finally, the solution can be filtered at 10°C to obtain a clear solution, and then heat treated at 60°C for at least 10 hours to inactivate pathogens.

[0108] One of ordinary skill in the art will recognize that each of the various fractions and effluents described above can be used with the methods of the present invention to treat conditions such as muscle damage, muscle disease, muscle disorder, or muscle condition that can benefit from improved muscle generation, regeneration, healing, function, or repair. For example, but not limited to, effluent I or effluent II / III can be used to treat conditions such as muscle damage, muscle disease, muscle disorder, or muscle condition that can benefit from improved muscle generation, regeneration, healing, function, or repair, and are embodiments of the present invention.

[0109] The aforementioned methods for preparing plasma fractions and plasma protein fractions (PPF) are exemplary only and relate only to embodiments of the present invention. One of ordinary skill in the art will recognize that these methods can be varied. For example, in different embodiments and methods of the present invention, pH, temperature, and ethanol concentration, among others, can be adjusted to produce different variations of plasma fractions and plasma protein fractions. In another example, additional embodiments of the present invention contemplate the use of nanofiltration to remove / inactivate pathogens from plasma fractions and plasma protein fractions.

[0110] Additional embodiments of the invention contemplate methods and compositions using and / or comprising additional plasma fractions. For example, the invention contemplates, among other things, that a particular concentration of albumin is not critical for treating a condition associated with a muscle injury, muscle disease, muscle disorder, or muscle condition that would benefit from improved muscle generation, regeneration, healing, function, or repair. Thus, the invention contemplates fractions having reduced albumin concentrations, such as fractions having less than 83% albumin.

[0111] H. Treatment

[0112] Aspects of the methods of the present invention described herein include treating subjects with blood products, such as plasma fractions, containing plasma, such as described above. Embodiments include treating human subjects with blood products containing plasma. Those skilled in the art will recognize that methods of treating subjects with blood products containing plasma are recognized in the art. As an example and not limitation, one embodiment of the methods of the present invention described herein includes administering fresh frozen plasma to a subject to treat a condition, such as muscle damage, muscle disease, muscle disorder, or muscle disorder that can benefit from improved muscle generation, regeneration, healing, function, or repair. In one embodiment, immediately, such as within about 12-48 hours of collection from a donor, a blood product containing plasma is administered to an individual suffering from muscle damage, muscle disease, muscle disorder, or muscle disorder that can benefit from improved muscle generation, regeneration, healing, function, or repair. In such cases, the product can be stored under refrigerated conditions, such as 0-10°C. In another embodiment, fresh frozen plasma is plasma that has been frozen and stored (cryopreserved) at or below -18°C. Prior to administration, fresh frozen plasma is thawed, and once thawed, administered to the subject 60-75 minutes after the thawing process begins. Each subject preferably receives a single unit of fresh frozen plasma (200mL-250mL), and the fresh frozen plasma is preferably derived from a donor in a predetermined age range. In one embodiment of the invention, fresh frozen plasma is donated from (derived from) a young individual. In another embodiment of the invention, fresh frozen plasma is donated from (derived from) a donor of the same sex. In another embodiment of the invention, fresh frozen plasma is donated from (derived from) a donor in the range of 18 to 22 years old.

[0113] In an embodiment of the present invention, after donation by blood type, blood products comprising plasma are screened. In another embodiment of the present invention, according to the requirements of 21CFR 640.33 and the suggestions included in the FDA guidance documents, blood products comprising plasma are screened for infectious pathogens such as HIV I & II, HBV, HCV, HTLV I & II, anti-HBc.

[0114] In another embodiment of the present invention, the subject is treated with a plasma fraction. In an embodiment of the present invention, the plasma fraction is a PPF, HAS, fraction IV-4 or fraction IV-4 paste suspension. In another embodiment of the present invention, the plasma fraction is a commercial PPF preparation in a commercial HAS preparation. In another embodiment of the present invention, the plasma fraction is a PPF, HAS, fraction IV-4 or fraction IV-4 paste suspension derived from a collection of individuals of a specific age range, such as young individuals, or a modified PPF, HAS, fraction IV-4 or fraction IV-4 paste suspension fraction that has been subjected to additional fractionation or processing, for example, a PPF, HAS, fraction IV-4 or fraction IV-4 paste suspension in which one or more than one specific protein has been partially or substantially removed. In another embodiment of the present invention, the plasma fraction is an IGIV plasma fraction that has been substantially depleted of immunoglobulin (IgG). A blood fraction that is "substantially depleted" or has a particular protein, such as IgG, "substantially depleted" refers to a blood fraction that contains less than about 50% of the amount present in a reference preparation or whole plasma, for example, less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, undetectable levels, or any integer between these values, as measured using standard assays well known in the art.

[0115] I. Application

[0116] Aspects of the methods of the present invention described herein include treating a subject with a blood product, such as plasma or a plasma fraction, containing plasma, such as described above. Embodiments include treating a human subject with a blood product containing plasma. Those skilled in the art will recognize that methods for treating a subject with a blood product containing plasma are well known in the art. As an example and not limitation, one embodiment of the methods of the present invention described herein includes administering fresh frozen plasma to a subject to treat muscle damage, muscle disease, muscle disorder, or muscle condition that can benefit from improved muscle generation, regeneration, healing, function, or repair. In one embodiment, immediately, such as within about 12-48 hours of collection from a donor, a blood product containing plasma is administered to an individual suffering from an undesirable condition, such as muscle damage, muscle disease, muscle disorder, or muscle condition that can benefit from improved muscle generation, regeneration, healing, function, or repair. In such cases, the product can be stored under refrigerated conditions, such as 0-10°C. In another embodiment, fresh frozen plasma is plasma that has been frozen and stored (cryopreserved) at or below -18°C. Before administration, fresh frozen plasma is thawed, and once thawed, 60-75 minutes after the thawing process begins, it is administered to the object. Each object preferably receives a single unit of fresh frozen plasma (200mL-250mL), and the fresh frozen plasma is preferably derived from a donor in a predetermined age range. In one embodiment of the invention, fresh frozen plasma is donated from (derived from) young individuals. In another embodiment of the invention, fresh frozen plasma is donated from (derived from) donors of the same sex. In another embodiment of the invention, fresh frozen plasma is donated from (derived from) donors in the range of 18 to 22 years old.

[0117] In an embodiment of the present invention, after donation by blood type, blood products comprising plasma are screened. In another embodiment of the present invention, according to the requirements of 21CFR 640.33 and the suggestions included in the FDA guidance documents, blood products comprising plasma are screened for infectious pathogens such as HIV I & II, HBV, HCV, HTLV I & II, anti-HBc.

[0118] In another embodiment of the present invention, the subject is treated with a plasma fraction. In an embodiment of the present invention, the plasma fraction is PPF or HAS. In another embodiment of the present invention, the plasma fraction is one of a commercial PPF preparation or a commercial HAS preparation. In another embodiment of the present invention, the plasma fraction is a PPF or HAS derived from a collection of individuals in a specific age range, such as young individuals, or is a modified PPF or HAS fraction that has been subjected to additional fractionation or processing, for example, a PPF or HAS in which one or more than one specific protein has been partially or substantially removed. In another embodiment of the present invention, the plasma fraction is an IGIV plasma fraction that has been substantially depleted of immunoglobulins (IgG). A blood fraction that is "substantially depleted" or has a particular protein, such as IgG, "substantially depleted" refers to a blood fraction that contains less than about 50% of the amount present in a reference preparation or whole plasma, for example, less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, undetectable levels, or any integer between these values, as measured using standard assays well known in the art.

[0119] Embodiments of the present invention include treating a subject with muscle damage, muscle disease, muscle disorder or muscle condition that can benefit from improved muscle generation, regeneration, healing, function or repair by administering an effective amount of plasma or plasma fractions to the subject. Another embodiment of the present invention includes administering an effective amount of plasma or plasma fractions, and then monitoring the improved function, wound healing, presence of markers, reduction of pain or reduction of inflammation of the subject. Another embodiment of the present invention includes treating a subject with a condition such as muscle damage, muscle disease, muscle disorder or muscle condition that can benefit from improved muscle generation, regeneration, healing, function or repair by administering an effective amount of plasma or plasma fractions to the subject, wherein the plasma or plasma fractions are administered in such a manner that, relative to the most recently administered dose, after the mean or median half-life of the plasma protein or plasma fraction protein has been reached, resulting in improved functional wound healing, marker presence, pain reduction or inflammation reduction (referred to herein as "pulse dosing" or "pulse dosing") (see U.S. Patent Application Nos. 15 / 499,697 and 62 / 701,411, which are incorporated herein by reference in their entirety). Another embodiment of the present invention includes applying plasma or plasma fractions by at least two consecutive days of dosing, and monitoring the improved function or HSC marker level of the object for at least 3 days after the date of the last application.Another embodiment of the present invention includes applying plasma or plasma fractions by at least 3 consecutive days of dosing, and monitoring the improved function, wound healing, the presence of markers, the reduction of pain or the reduction of inflammation of the object for at least 3 days after the date of the last application.Another embodiment of the present invention includes applying plasma or plasma fractions by at least two consecutive days of dosing, and monitoring the improved function, wound healing, the presence of markers, the reduction of pain or the reduction of inflammation when the average half-life of protein in plasma or plasma fractions has been reached after the date of the last application.Another embodiment of the present invention includes applying plasma or plasma fractions by a discontinuous 2-day to 14-day dosing, wherein each gap between administration can be 0-3 days respectively.

[0120] In some cases, according to the present invention, for example, pulse administration as described above includes the administration of a first group of doses, followed by a non-administration period, such as a "non-administration period", which is followed by the administration of another dose or another group of doses. The duration of the "non-administration period" can vary, but in some embodiments is 7 days or more than 7 days, such as 10 days or more than 10 days, including 14 days or more than 14 days, wherein in some cases, the non-administration period is 15 days to 365 days, such as 30 days to 90 days, including 30 days to 60 days. Thus, the embodiment of the method includes non-long-term (i.e., non-continuous) administration of plasma products, such as non-long-term administration. In some embodiments, the pulse administration and the subsequent non-administration period mode are repeated multiple times as needed, wherein in some cases, the mode is continued for 1 year or more than 1 year, such as 2 years or more than 2 years, up to and including the life of the object. Another embodiment of the present invention includes the administration of plasma or plasma fractions by the following dosing regimen: continuous 5 days of administration, 2 days-3 days of non-administration period, followed by continuous administration of 2 days-14 days.

[0121] Biochemically, an "effective amount" or "effective dose" of an active agent means an amount of an active agent that will inhibit, antagonize, reduce, decrease or eliminate by about 20% or more, such as 30% or more, 40% or more or 50% or more, in some cases 60% or more, 70% or more, 80% or more or 90% or more, in some cases about 100%, i.e., to a negligible amount, and in some cases, reverse an undesirable condition, such as a muscle injury, muscle disease, muscle disorder or muscle condition that would benefit from improved muscle generation, regeneration, healing, function or repair.

[0122] J. Plasma protein fraction

[0123] In practicing the methods of the invention, a plasma fraction is administered to a subject. In embodiments, the plasma fraction is a plasma protein fraction (PPF). In further embodiments, the PPF is selected from a commercial PPF preparation.

[0124] In another embodiment, the PPF is composed of 88% normal human albumin, 12% alpha and beta globulins, and no more than 1% gamma globulin as determined by electrophoresis. Additional embodiments for practicing the embodiments of the methods of the present invention include, for example, embodiments of a 5% solution of PPF buffered with sodium carbonate and stabilized with 0.004M sodium octanoate and 0.004M acetyltryptophan. Additional formulations may be used when practicing the methods of the present invention, including formulations that vary the percentage of PPF in the solution (e.g., from about 1% to about 10%, from about 10% to about 20%, from about 20% to 25%, from about 25% to 30%) and the concentrations of solvents and stabilizers.

[0125] K. Plasma fractions specific to donor age

[0126] Other embodiments of the present invention include administering plasma protein fractions derived from plasma of individuals of certain age ranges. Embodiments include administering PPF or HAS derived from plasma of young individuals. In another embodiment of the present invention, young individuals have a single specific age or a specific age range. In another embodiment, the average age of the donor is less than the age of the object or less than the average age of the object being treated.

[0127] Certain embodiments of the present invention include pooling blood or plasma from individuals within a specific age range, and fractionating the plasma as described above to obtain a plasma protein fraction preparation, such as PPF or HAS. In an alternative embodiment of the present invention, a plasma protein fraction or a specific plasma protein fraction is obtained from a specific individual that meets a specific age range.

[0128] L. Indications

[0129] One embodiment of the invention is the use of plasma fractions and plasma fractionation products for administration to subjects diagnosed with a disease, condition or disorder that would benefit from improved muscle generation, regeneration, healing, function or repair. Additional embodiments of the invention include treating the disease, condition or disorder when the disease or disorder is: muscle atrophy or weakness (for example and not limitation - deterioration or activity limitation due to exercise); sarcopenia; cachexia; McArdle disease; weakness associated with stroke; degeneration associated with amyotrophic lateral sclerosis; neuromuscular junction disorders; myasthenia gravis; toxic myopathy; inflammatory myopathy; lipid storage myopathy; acute physical or chemical injury; ischemia / reperfusion (e.g., organ transplant surgery, stroke, hypovolemic shock); injury caused by contraction; genetically related degenerative diseases; Duchenne and Becker muscular dystrophy; myotonic dystrophy; limb-girdle muscular dystrophy; Emory-Dreyfus muscular dystrophy; congenital muscular dystrophy; and facioscapulohumeral muscular dystrophy.

[0130] Other embodiments of the invention include when the disease, condition or disorder is: acute muscle injury from a single traumatic event such as sports, contact sports, or a traumatic accident such as from a collision (see Bahr, R., McCrory, P., LaPrade, RF, Meeuwisse, WH, & Engebretsen, L. (2012). The IOC manual of sports inyjuries: an illustrated guide to the management of injuries in physical activity. Wiley and Sons. 2012, which is incorporated herein by reference in its entirety); overuse injuries, such as from chronic use or use caused by exercise, in which repetitive microtrauma occurs to the muscle; muscle strains or sprains, including Grade I (mild - involving a small number of muscle fibers), Grade II (moderate - involving a large number of muscle fibers, pain recurs when the muscle contracts and the pain causes limited movement), and Grade III (severe - complete tear or rupture, in which the tendon separates from the muscle belly or the muscle belly is torn into 2 or more parts); muscle contusions or bruises; muscle cramps or spasms (sudden involuntary muscle contraction or excessive shortening); and muscle soreness, including delayed onset muscle soreness (DOMS), when treating the above diseases or conditions.

[0131] Other embodiments of the present invention include the use of plasma fractions and plasma fractionation products in combination with conventional treatments to treat muscle diseases, disorders or conditions. Embodiments of the present invention may include combined treatments using the principles of RICE (rest, ice, compression, elevation) or POLICE (protection, rest, ice, compression, elevation). Other embodiments may include treatments with plasma fractions / plasma fractionation products in combination with surgical intervention or physical therapy. Treatment with plasma fractions / plasma fractionation products in combination with more conventional treatments may be performed simultaneously, or conventional treatments may be performed before and / or after the administration of plasma fractions / plasma fractionation products.

[0132] Another embodiment of the present invention includes diseases, disorders or conditions of the myocardium, such as, but not limited to, reducing cardiac hypertrophy. Other examples of heart-related diseases, conditions or disorders include cardiomyopathy (heart enlargement), dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, congenital heart disease, heart attack and hypertension.

[0133] M. Reagents, Devices, and Kits

[0134] Also provided are reagents, devices and kits thereof for carrying out one or more than one of the above methods. The target reagents, devices and kits thereof can vary widely.

[0135] Reagents and devices of interest include those mentioned above with respect to the method of preparing a plasma-containing blood product for infusion into a subject in need thereof, such as anticoagulants, cryopreservatives, buffers, isotonic solutions, and the like.

[0136] The kit may also include blood collection bags, tubes, needles, centrifuge tubes, etc. In other embodiments, the kits described herein include two or more containers of plasma products, such as plasma protein fractions, such as three or more, four or more, five or more, including six or more containers of plasma products. In some cases, the number of different containers of plasma products in the kit may be 9 or more, 12 or more, 15 or more, 18 or more, 21 or more, 24 or more, 30 or more, including 36 or more, such as 48 or more. Each container may have associated therewith identification information, the identification information including a variety of data about the plasma product contained therein, which may include one or more of the age of the donor of the plasma product, processing details about the plasma product, such as whether the plasma product has been treated to remove proteins above the average molecular weight (such as described above), blood type details, etc. In some cases, each container in the test kit includes identification information about the plasma contained therein, and the identification information includes information about the donor age of the plasma product, such as identification information provides age-related data confirming the plasma product donor (wherein, the identification information can be the age of the donor when collecting). In some cases, each container of the test kit includes plasma products from donors of substantially the same age, that is, all containers include products from donors of substantially the same age (if not the same). Substantially the same age means that the different donors of the plasma products from which the test kit is obtained differ by 5 years or less than 5 years in some cases, such as 4 years or less than 4 years, such as 3 years or less than 3 years, including 2 years or less than 2 years, such as 1 year or less than 1 year, such as 9 months or less than 9 months, 6 months or less than 6 months, 3 months or less than 3 months, including 1 month or less than 1 month. The identification information can be present on any convenient part of the container, such as a label, an RFID chip, etc. As required, the identification information can be human-readable, computer-readable, etc. The container can have any convenient configuration. The volume of container can be changed, but in some cases, the volume is 10ml to 5000mL, for example 25mL to 2500mL, for example 50ml to 1000mL, including 100mL to 500mL.Container can be rigid or flexible, and can be made of any convenient material, for example polymeric material, including medical grade plastic material.In some cases, container has bag or pouch configuration.In addition to container, test kit can also include application device as described above.The assembly of test kit can be provided with any suitable packaging, for example, box or similar structure, it is configured to hold the assembly of container and other test kits.

[0137] In addition to the above-mentioned components, the target test kit may also include instructions for implementing the target method. Instructions may be present in the target test kit in various forms, one of which or more than one may be present in the test kit. A form in which instructions may exist is printed information on a suitable medium or substrate (e.g., one or more than one sheet of paper on which information is printed), in the packaging of the test kit, on a package insert, etc. Another way may be a computer-readable medium on which information is recorded, such as a floppy disk, a CD, a portable flash drive, etc. Another possible way is a website address, which can be used to access information at a remote site through the Internet. Any suitable device may be present in the test kit.

[0138] N. Experimental Examples

[0139] 1. Example 1

[0140] a) Short-term processing

[0141] On day minus two (d-2), C2C12 myoblasts (Sigma Aldrich 91031101-1VI) were seeded at 8,000 cells per well on 96-well plates in C2C12 medium (DMEM+GlutaMAX (ThermoFisher Scientific)+4.5 g / L glucose, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P / S)). Two days later, a complete medium replacement was performed with C2C12 differentiation medium (DMEM+GlutaMAX+1 g / L glucose, 2% horse serum (Gibco), 1% P / S). The addition of differentiation medium resulted in the fusion and differentiation of C2C12 myoblasts into myotubes. This day was designated as day zero (d0) (see Figure 2 Treatment assays were initiated on day 5 (d5) by completely removing the culture medium from each well and adding 150 μL of different culture medium plus treatments: (1) untreated; (2) 1 mM metformin (positive control - MedChem Express HY-17471A / C8-1851); (3) 0.5 mM metformin; (4) 0.25 mM metformin; (5) vehicle (10%, in culture medium); (6) PPF1 (5 mg / mL, in culture medium); (7) HAS1 (5 mg / mL, in culture medium); and (8) recombinant human albumin (rhAlbumin, 5 mg / mL, in culture medium).

[0142] PPF1 is a PPF with about 88% normal human albumin (relative to total protein), 12% alpha and beta globulins, and no more than 1% gamma globulin as determined by electrophoresis. Unless otherwise indicated, PPF1 is administered in the examples herein using a 5% solution (w / v, 50 g / L). HAS1 is a commercially available HAS, such as a 5% solution of the commercially available HAS preparation described above, and is stored at 4°C.

[0143] Twenty-four hours later, a glucose utilization assay was performed using a glucose assay kit (Abcam–ab65333). A preliminary test of glucose utilization was first performed to specify the appropriate dilution of the culture medium. Therefore, 100 μL of culture medium was removed from untreated cells and the supernatant was diluted in assay buffer (1:1 / 1:2 / 1:5 / 1:10, with a final volume of 50 μL / well). Reactants (46 μL) plus 2 μL of substrate and 2 μL of enzyme were then added to each well. After determining the correct dilution, the culture medium from all treatment groups and an additional untreated control was diluted accordingly, with a final volume of 50 μL / well.

[0144] Glucose standards from the Abcam kit were pipetted into the wells and the reaction mixture was added to the standards and treatment samples (46 μL assay buffer + 2 μL substrate + 2 μL enzyme per well). The reaction was incubated at 37°C for 30 minutes and then the absorbance was measured at 570 nm. The concentration of glucose was calculated based on the standards.

[0145] Figure 3A Glucose utilization is shown as the remaining concentration (% OD) in the culture medium. Compared with vehicle and other plasma fractions (e.g., HAS1), cells treated with PPF1 showed a significant increase in glucose utilization. It was also observed that PPF1 tended to promote the contraction of myotubes more than HAS1 or rhAlbumin. Therefore, unlike HAS1 and rhAlbumin, PPF1 enhances cell metabolism. Data are n=3 wells from three independent experiments, ± SEM.

[0146] Figure 3B For Figure 2 and 3A Still photograph capture of a video of myotubes treated with PPF1 as described in . Significantly increased contraction was observed compared to untreated and vehicle controls. This is associated with skeletal muscle recovery from conditions / indications such as aged muscle, weakness, and muscle recovery during and after surgery.

[0147] 2. Example 2

[0148] a) Long-term treatment with different horse serum concentrations

[0149] Figure 4The diagram of the experiment of testing different horse serum concentrations on the influence of C2C12 cells in culture is shown. It is presumed that if the horse serum level is reduced, the effect of PPF1 can be resolved to a higher degree. C2C12 myoblasts (Sigma Aldrich 91031101-1VI) were seeded on 96-well plates in C2C12 culture medium (DMEM+GlutaMAX (ThermoFisher Scientific)+4.5g / L glucose, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P / S)) at -2 days (d-2) with 8000 cells per well. Two days later, a complete medium change was performed using C2C12 medium (DMEM+GlutaMAX+1g / L glucose, 0% or 2% horse serum (Gibco), 1% P / S) with or without 2% horse serum plus treatment agents: (1) untreated; (2) vehicle (10%, in the medium); (3) PPF1 (5mg / mL, in the medium); (4) HAS1 (5mg / mL, in the medium); and (5) recombinant human albumin (rhAlbumin, 5mg / mL, in the medium). This day was designated as day zero (d0). On day 2 (d2), half of the medium was removed and supplemented with the same concentration of treatment agent (5mg / mL). On day 4 (d4), half of the medium was removed again and supplemented with the same concentration of treatment agent (5mg / mL). On day 6 (d6), glucose utilization assays and cell fixation and staining were performed.

[0150] Figure 5 C2C12 cells cultured in 0% horse serum and 2% horse serum are shown. For each horse serum concentration, untreated and PPF1 treated cells are shown. Treatment with PPF1 in both serum concentrations resulted in a greater amount of myotube formation and thus differentiation of C2C12 cells into myotubes, with untreated C2C12 cells showing the least amount of myotube differentiation when using 0% horse serum. Figure 6 It is shown that C2C12 cells treated with 0% horse serum and PPF1 exhibited positive staining for the myogenic differentiation marker myosin heavy chain.

[0151] Figure 7 Glucose utilization is reported as the concentration (% OD) remaining in the culture medium of C2C12 cells treated for a long time in 0% horse serum. Despite the absence of horse serum, cells treated with PPF1 showed a significant increase in glucose utilization compared to vehicle and other plasma fractions (e.g., HAS1). It was also observed that PPF1 tended to promote the contraction of myotubes more than HAS1 or rhAlbumin. Therefore, unlike HAS1 and rhAlbumin, PPF1 enhances cell metabolism. Data are n=4 wells from three independent experiments, ± SEM.

[0152] Figure 8 Glucose utilization is reported as the remaining concentration (% OD) in the culture medium of C2C12 cells treated chronically with 0% horse serum and different plasma fractions and plasma fractionation products. The treatment scheme was as described above in Figure 4 The experiments were performed as described in , but the treatments used were: (1) untreated; (2) PPF1 (5 mg / mL in culture medium); (3) filtrate IV-4 (5 mg / mL in culture medium); and (4) a suspension of the paste of fraction IV-4 corresponding to the Cohn fractionation process (5 mg / mL in culture medium; concentrated dialysate of the IV-1 suspension dialyzed against 0.9% NaCl / 10 mM HEPES pH 7.3). The results showed that both plasma fractions / fractionation products showed similar effects as PPF1 on glucose utilization in C2C12 cells.

[0153] Fig. 9 The relative expression of type 4 glucose transporter (GLUT-4) in C2C12 myoblasts treated with control vehicle, PPF1 (5 mg / mL in culture medium) or recombinant human albumin (rhAlbumin, 5 mg / mL in culture medium) 10% solution (w / v, 50 g / L) is shown. GLUT-4 is a protein that plays a key role in regulating systemic glucose homeostasis. Data are n=2 wells from one experiment, ±***p<0.001.

[0154] Fig.10 Reported Figure 8 Dose-response relationship between plasma fractions / fractionation products and glucose utilization as described in . C2C12 myoblasts were differentiated into myotubes in vitro for 6 days in 0% horse serum differentiation medium. Different treatment concentrations (0.15 mg / mL, 0.3 mg / mL, 0.6 mg / mL, 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL and 10 mg / mL in the culture medium) were added to the cells. After 6 days of treatment and 48 hours with the same culture medium, the amount of glucose remaining in the culture medium was analyzed by the glucose utilization assay described above. All three compositions showed a dose-response relationship for glucose utilization, with the Fraction IV-4 paste suspension showing the strongest median efficacy (EC 50 ).

[0155] 3. Example 3

[0156] a) Short-term administration of PPF1 in vivo increases muscle mass and induces slow-twitch muscle fiber-related genes

[0157] Fig.11AThe summary table of several experiments carried out for C57BL / 6 mice of different ages and young rats and tested the muscle weight values ​​of tibialis anterior, extensor digitorum longus, gastrocnemius and soleus. Each experiment also tested the muscle weight influence of different time lengths after the last administration of vehicle or PPF1. The table shows that significant muscle weight increase is related to PPF1 processing, and even a long time after the most recent administration has been observed to last.

[0158] Fig. 11B Figure 1 is a diagram of the experimental protocol for studying muscle-related metrics in 22-month-old male C57B6 mice treated with PPF1 or control. Pulses of PPF1 or control vehicle (150 μL per dose, iv) were given to 26-month-old male C57B6 mice for 7 consecutive days. Ten (10) days after the last dose, the following skeletal muscle groups were harvested: tibialis anterior (TA), extensor digitorum longus (EDL), and soleus (SOL). Muscle to body weight (BW) ratios were obtained from each muscle group. Fig. 11C It is shown that the tibialis anterior muscle tissue significantly increased in weight when treated with PPF1 compared to the control (mean ± SEM, **p<0.01, Welch's test). Fig.11D It is shown that the extensor digitorum longus muscle tissue significantly increased in weight when treated with PPF1 compared to the control (mean ± SEM, **p<0.01, Welch's test). Fig.11E It is shown that soleus muscle tissue significantly increased in weight when treated with PPF1 compared to the control (mean ± SEM, *p<0.05, Welch's test).

[0159] Fig. 12A PPF1 induces a slow-twitch myofiber gene (Myl2(2a)) in the tibialis anterior muscle. In contrast, fast-twitch myofiber genes (Myh1(2x) and Myh2(2a)) tend to decrease in expression in mice treated with PPF1 (see Table 2, respectively). Fig. 12B and Fig. 12C ). Fig.12D The results showed that the fast-twitch muscle fiber-related gene Myh4 (2b) had a slight downward trend.

[0160] Increased slow-twitch muscle fibers are a hallmark of an endurance phenotype. When mice or humans undergo exercise training, slow-twitch muscle fibers increase and fast-twitch muscle fibers decrease. Slow-twitch muscle fibers are more resistant to fatigue than fast-twitch muscle fibers and burn more fat. This also suggests relevance to the treatment of obesity-related diseases, because if PPF1 promotes the formation of slow-twitch muscle fibers, its function would be very similar to other known exercise mimetics such as metformin, AICAR and resveratrol.

[0161] 4. Example 4

[0162] a) Effects of PPF1 and Fraction IV-1 paste suspension on myotube formation

[0163] Fig.13A , Fig. 13B , Fig. 13C and Fig.13D All show C2C12 cells after 3 days of culture in 0% horse serum combined with various treatment conditions. Fig.13A Shown are C2C12 cells under untreated conditions. Fig. 13B Shown are C2C12 cells treated with 0.3% PPF1 for 3 days. Fig. 13C and 13D Shown are C2C12 cells treated with 0.3% IV-1 paste suspension and 1.25% IV-1 paste suspension for 3 days, respectively.

[0164] Comparison between C2C12 cells treated with 0.3% PPF1 and 0.3% IV-1 paste suspension showed that after 3 days, IV-1 paste suspension induced more myotube formation than the same concentration of PPF1. Fig.13D It was also shown that the induction of IV-1 paste suspension was dose dependent, as the visual increase in myotube formation appeared to result in an increase in myotube formation (1.25% vs. 0.3% treatment concentration). All three treatment conditions (0.3% PPF1, 0.3% IV-1 paste suspension, and 1.25% IV-1 paste suspension) visually produced more myotube formation than vehicle alone.

[0165] Fig.14 The dose-response relationship between plasma fractions / fractionation products and normalized glucose utilization (%) of C2C12 cells grown in 0% horse serum for six days is reported. The x-axis depicts increasing doses of IV-1 paste suspension, PPF1, and IV-1 effluent. EC 50 Values, IV-1 paste suspension had the highest efficacy (0.1 mg / ml), IV-1 effluent had the second highest efficacy (0.4 mg / ml), and PPF1 had the lowest efficacy but was still highly effective (1.4 mg / ml).

[0166] Fig.15The dose-response relationship between the plasma fraction / fractionation product and the normalized glucose utilization (%) obtained from the residual glucose concentration (%OD) in the culture medium of C2C12 cells grown in 2% horse serum for six days is reported. For each graph, the x-axis depicts increasing doses of IV-1 paste suspension, PPF1 and IV-1 effluent, respectively (concentrations tested: 5 mg / ml, 2.5 mg / ml, 1.25 mg / ml, 0.6 mg / ml, 0.3 mg / ml, 0.15 mg / ml, 0.075 mg / ml). EC 50 Values, IV-1 paste suspension had the highest efficacy (0.4 mg / ml), IV-1 effluent had the second highest efficacy (1.7 mg / ml), and PPF1 had the lowest efficacy but was still highly effective (3.8 mg / ml%).

[0167] 5. Example 5

[0168] a) Effects of IGF1 on metabolic activity

[0169] Fig.16A and Fig. 16B The effects of insulin-like growth factor-1 (IGF-1) on glucose utilization of C2C12 cells treated in 2% horse serum are reported. Cells were seeded in DMEM plus 4.5 g / L glucose and 10% fetal bovine serum (FBS) on day -2 (d-2). The medium was replaced with DMEM plus 1 g / L glucose and 2% horse serum on day 0 (d0). Various treatments were added on day 5 (d5) and glucose utilization was measured on day 6 (d6). Fig.16A The dose-response relationship between recombinant human IGF-1 treatment (x-axis) and glucose utilization is reported, demonstrating an EC50 of 17.43 ng / mL. Recombinant human IGF1 was purchased from R&D Systems (Cat. No. 291-G1). Fig. 16B reported a dose-response relationship between PPF1 treatment and glucose utilization, revealing an EC of 2.9 mg / ml 50 , containing 0.87 ng / mL IGF1. IGF-1 is known to have an effect on the metabolism of myotubes, and its presence in PPF1 has been calculated to be approximately 14.88 ng / mL. However, the data presented here reveal that PPF1 is 20 times more effective than IGF-1 alone, so the presence of IGF-1 alone cannot explain the enhanced efficacy observed with PPF1. Therefore, other factors must be involved in the effects of PPF1.

[0170] 6. Example 6

[0171] a) PPF1 improves muscle recovery from injury

[0172] Fig.17A Schematic representation of the experimental protocol for studying muscle recovery from injury using different treatments. To induce muscle injury in vivo, C57BL / 6 mice were anesthetized by inhalation of isoflurane. On the second day of test article administration, 50 μL of BaCl was injected intramuscularly into the left tibialis anterior muscle over the entire length of the tibia using a 30-gauge insulin syringe. 2 Solution (Sigma-Aldrich B0750, 1.2% in sterile 0.9% NaCl). 50 μL of saline was injected intramuscularly into the right tibialis anterior muscle as a contralateral uninjured control.

[0173] The left hind limb was wrapped with 2 layers of surgical tape and sports tape (Durapore 3M 1538-2 and Hampton Adams 8542028768) for 10 days during the immobilization phase and unwrapped during the 10-day recovery phase. A disgusting spray (Grannick's bitter apple, GB11A8T) was applied to the outer surface of the tape to discourage mice from chewing and biting off the tape. The animals' toe circulation and tape integrity were monitored daily.

[0174] The hind limbs of each anesthetized mouse were prepared for torque measurements above the ankle as previously described (Gerlinger-Romero F et al., J. Vis. Exp. 58696 (2019), doi: 10.3791 / 58696). Contraction force and tetanic force were recorded using the setup as previously described. (Ho ATV et al., PNAS, 114: 6675-84 (2017)). Baseline measurements of the left limb and hind limb were recorded before dosing, and the values ​​at the end of the study (day 17) were compared with the initial readings (day 0).

[0175] For systemic treatment, animals were pulse dosed intravenously with 150 50 μL of the test treatment for 7 consecutive days. Vehicle, PPF1, HAS1 and recombinant human albumin (rhAlbumin) were administered to different groups.

[0176] Fig. 17B The results of the contractility measurements performed at day 0 and day 17 are reported. At day 0 (before administration), the contractility readings of all four groups produced similar maximum torque values. However, at day 17, only the PPF1 treatment group produced a significantly increased maximum torque compared to the control vehicle. Both recombinant human albumin (rhAlbumin) and HAS1 failed to produce a significantly increased maximum torque compared to the control vehicle. Data are mean ± SEM, *p < 0.05, Welch's t test.

[0177] 7. Example 7

[0178] a) PPF1 is associated with elevated IGF-1 serum levels

[0179] Fig.18A Schematic representation of the experimental protocol for studying the effect of plasma fractions on serum mouse IGF1 levels. Blood was collected for 10 days from 22 month old C57BL / 6 mice treated as described in Example 9 after the last day of a 7-day pulse treatment with PPF1. Serum was separated and the levels of mouse IGF-1 were determined. Fig.18B revealed that PPF1 treatment was accompanied by a significant increase in mouse IGF-1 in serum even 10 days after the last administration, suggesting one of several possible mechanisms whereby plasma fractions such as PPF1 could induce skeletal muscle to undergo apoptosis from the presence of BaCl 2 Recovery from injury induced in the injury-inducing model. Data are mean ± SEM, *p < 0.05, Welch's t test.

[0180] 8. Example 8

[0181] a) PPF1 reduces heart weight in the elderly in vivo

[0182] Fig.19A Schematic representation of the experimental protocol for investigating whether the plasma fraction can reduce heart weight in aged C57BL / 6 mice in a model of cardiac hypertrophy observed in aged mammals. (See Kiper et al., PLoS ONE 8(8):e70512). 26-month-old mice treated with pulse administration of PPF1 for seven consecutive days were sacrificed on day 17 and heart weights were measured. Fig.19B Heart weights of vehicle-treated mice and PPF1-treated mice are shown in milligrams. PPF-treated mice exhibited significantly reduced heart weights compared to controls, suggesting that age-related hypertrophy can be reduced with plasma fractions such as PPF1. Fig.19C Shown is the ratio of heart weight to body weight of the same mice, which was significantly reduced in PPF1 treated mice compared to controls, also indicating a reduction in age-related hypertrophy.

[0183] Fig. 20A , Fig. 20B and Fig. 20C Reported Fig.19A , 19B and expression of RNA levels of cardioprotective markers in the heart as described in 19C. Fig. 20AIt is shown that the RNA expression of the endoplasmic reticulum calcium-ATPase (SERCA2a) was significantly increased with PPF1 treatment compared with the control. SERCA2a is a key regulator of contractility and lymph node calcium cycling proteins and is the pathogenesis of heart failure. Its reduction is associated with heart failure, and gene therapy repair is accompanied by promising clinical results in subjects with indications. (Chaanine AH et al., Stem Cell and Gene Therapy for Cardiovascular Disease–Chapter 30–SERACA2a Gene Therapy for Heart Failure, 389-400 (2016)). Fig. 20B It was shown that the RNA expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1a) was significantly increased by PPF1 treatment compared with the control. PGC1a inhibition is associated with heart failure. (Riehle C and Abel D, Trends Cardiovasc Med, 22(4):98-105 (2012)). Fig. 20C It is shown that the RNA expression of alpha-myosin heavy chain (aMHC) was significantly increased with PPF1 treatment compared to controls. A decrease in aMHC is associated with cardiac hypertrophy and heart failure. (Hilfiker-Kleiner D et al., Cardiovasc. Res., 53:460-69 (2002)). The increase in these cardioprotective markers suggests that plasma fractions such as PPF1 can reactivate genetic pathways that reduce cardiac hypertrophy. Data are mean ± SEM, *p < 0.05, Welch's t test.

[0184] 9. Example 9

[0185] Lactate is known to promote myoblast differentiation and myotube hypertrophy (see, e.g., Tsukamoto S et al., Int. J. Molec. Sci. 19:3649 (2018)). Therefore, measuring lactate production in myoblasts can be used as an indicator of differentiation and growth in muscle. C2C12 myoblasts were differentiated into myotubes in vitro in 2% horse serum (HS) differentiation medium for 5 days. On day 5, various treatments were added to the cells. Based on EC assessed by glucose utilization 50The plasma fraction was added to the culture medium at 5 mg / mL, except that IV-1 paste was added at three different concentrations (0.25 mg / mL, 2.5 mg / mL, and 5 mg / mL). Also on day 5, a positive control, metformin (1 mM), was added. On day 6, cells were again treated with metformin and oligomycin (250 nM) as a positive control, and with 2-deoxy-D-glucose (2-DG, 100 nM) as a negative control for three (3) hours or five (5) hours (respectively, Fig.21A and 21B ). After 3 hours or 5 hours, the medium was deproteinized and then the amount of lactate produced was measured by enzymatic reaction.

[0186] Fig.21A The amount of lactate (myogenic differentiation factor) produced in C2C12 cells after three (3) hours of treatment with various factors is shown. These factors include vehicle, 2-DG (negative control), metformin (positive control), oligomycin, HAS1, recombinant human albumin (rhAlbumin), PPF1, fraction IV-1 paste suspension, and three different concentrations of fraction IV-1 paste suspension. The data are from two wells, each from three independent experiments, ± SEM. The data show that after 3 hours of treatment: HAS1 and rhAlbumin did not show an increase in lactate production compared to the untreated control; PPF1 showed a very slight trend to increase lactate production; fraction IV-4 and IV-1 paste suspensions showed a significant increase in lactate production. Data: n = 2 wells, from three independent experiments, ± SEM, ****p <0.0001, nested one-way ANOVA. The production of lactate is not completely correlated with glucose utilization when using plasma fractions, indicating that plasma fractions induce different mechanisms in cells.

[0187] Fig.21B Shows the Fig.21A Effects of various factors described in on lactate production in C2C12 cells after five (5) hours of treatment. Data are from two wells, each from two independent experiments, ± SEM. The data show that after 5 hours of treatment: HAS1 and rhAlbumin did not show an increase in lactate production compared to the untreated control; PPF1 showed a trend of increased lactate production; Fraction IV-4 and IV-1 paste suspensions showed a significant increase in lactate production. Data: n=2 wells, from two independent experiments, ± SEM, ****p<0.0001, nested one-way ANOVA.

[0188] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one skilled in the art that certain changes and modifications may be made thereto in light of the teachings of this invention without departing from the spirit or scope of the appended claims.

[0189] Therefore, the foregoing only illustrates the principles of the present invention.It should be understood that those skilled in the art will be able to design various arrangements, although not explicitly described or shown herein, such arrangements embody the principles of the present invention and are included in its spirit and scope.In addition, all examples and conditional languages ​​recorded herein are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventors to promote this area, and should be interpreted as not being limited to the examples and conditions specifically recorded in this way.Moreover, all statements herein that record the principles, aspects and embodiments of the present invention and its specific examples are intended to cover their structural and functional equivalents.In addition, such equivalents are intended to include currently known equivalents and equivalents developed in the future, that is, any elements of the same function developed, regardless of the structure.Moreover, any content disclosed herein is not intended to contribute to the public, regardless of whether such disclosure is clearly recorded in the claims.

Claims

1. Use of a plasma fraction in the preparation of a blood product for improving muscle regeneration in a subject diagnosed with a muscle disorder, thereby alleviating the symptoms of a muscle disorder including muscle degeneration, in, The plasma fractions are: i) Plasma protein fraction (PPF), comprising 83% to 95% albumin and not more than 17% globulins and other plasma proteins; ii) Fraction IV-4; or iii) Fraction IV-1, The muscle disorder is selected from the group consisting of: Duchenne and Becker muscular dystrophy; myotonic dystrophy; limb-girdle muscular dystrophy; Emery-Dreyfus muscular dystrophy; congenital muscular dystrophy; facioscapulohumeral muscular dystrophy; McArdle disease; muscle weakness associated with stroke; degeneration associated with amyotrophic lateral sclerosis; myasthenia gravis; Toxic myopathy; Inflammatory myopathy; Lipid storage myopathy; and muscle disorders caused by acute muscle damage.

2. The use according to claim 1, wherein the acute muscle damage is a contraction-induced damage.

3. The use according to claim 1, wherein the plasma fraction is PPF.

4. The use according to claim 3, wherein the PPF contains no more than 1% of gamma-globulin.

5. The use according to claim 1, wherein the plasma fraction is fraction IV-4. The use according to claim 1 , wherein the plasma fraction is fraction IV-1.

7. The use according to any one of claims 1 and 3-6, wherein the muscle disorder is a muscle disorder caused by acute muscle injury.

8. The use according to claim 7, wherein the acute muscle injury is caused by sports activity.

9. The use according to any one of claims 1 to 6, wherein reducing the symptoms of the muscle disorder comprises eliminating the symptoms of the muscle disorder.

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