Blood plasma fractions for use in muscle regeneration

Abstract

This invention describes methods and compositions for treating age-related diseases, as well as for muscle recovery, prevention of muscle degeneration, and maintenance of muscle mass. The compositions used in these methods include plasma and plasma-derived plasma components that have therapeutic and / or preventative effects against diseases.

Description

[Technical Field] Cross-citation of related applications Pursuant to Section 119(e) of Chapter 35 of the United States Code, this application claims priority over the filing dates of U.S. Provisional Patent Application No. 62 / 930,336, filed November 4, 2019; U.S. Provisional Patent Application No. 62 / 966,953, filed January 8, 2020; and U.S. Provisional Patent Application No. 63 / 062,735, filed August 7, 2020; the disclosures of these applications are incorporated herein by reference. This invention relates to the prevention and treatment of muscle diseases and injuries. It also relates to the use of blood products (such as plasma components) to treat and / or prevent age-related conditions, such as cognitive and psychodegenerative disorders. [Summary of the Invention] Skeletal muscle possesses a high regenerative capacity, which originates from satellite cells (myogenic stem cells). (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)). In adulthood, satellite cells are activated by muscle damage, but may be pathologically dysregulated in nutritional disorders. (Jang, ibid.) Skeletal muscle regeneration is considered to be coordinated through four processes: myofibril degeneration leading to cell necrosis; inflammation and the invasion of certain inflammatory cells into the muscle; regeneration through the activation of satellite cells, which subsequently differentiate into myoblasts, helping to support the formation of new myofibrils and repair existing surviving myofibrils; and, if the regenerated fibers mature, remodeling / repair and remodeling the extracellular matrix. (Ibid.) Muscle regeneration is also closely related to metabolism, which can alter these processes. (Ibid.) Sarcopenia is a progressive loss of skeletal muscle mass and strength due to aging. (Tabebordbar M et al., Annals of Pathology: Mechanisms of Disease, 8:441-75 (2013)). This is a growing global health problem, affecting approximately one-quarter of people over 70 years of age and 40% of people over 80 years of age. (Ibid.) This disease leads to decreased independence, loss of daily living activities, and a decline in quality of life. (Ibid.) With age, the regenerative capacity of skeletal muscle declines, at least in part due to a reduction in the number of muscle satellite cells and muscle nuclei in muscle fibers. (Ibid. and Brack AS et al., Science, 317:807-810 (2007)). Furthermore, the total number and size of muscle fibers also decrease / shrink with age. (Jang, Ibid.) In addition to age-related muscle loss and degeneration, muscle function can also be impaired by acute physical or chemical injuries, local ischemia / reperfusion (e.g., organ transplantation, stroke, hypovolemic shock), contractile injuries, inflammatory myopathy, and genetically related degenerative diseases. The latter can 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. (Ibid.) Currently, treatment options for muscular dystrophy are limited, and these options often focus on controlling symptoms through immune and inflammatory responses. (Ibid.) Therefore, novel methods are needed to reverse the effects of muscle damage and degeneration. Although it has been shown that serum from young mice treated with heterochronic xenobiotic techniques can reduce the rate of conversion of certain muscle cells from myogenic to fibrotic forms, more practical and standardized interventions are still needed. (Brack, Ibid.) This invention addresses these needs by providing specific components or products obtained through plasma fractionation. [Cited and incorporated] All publications and patents referenced in this specification are incorporated herein by reference, as each individual publication or patent is specifically and individually indicated to be incorporated by reference. [Simplified Explanation of the Diagram] [Figure 1] illustrates a method for separating components of mixed plasma. The mixed plasma is separated at low temperature into an effluent and a paste. The effluent is then separated into effluent I (component I effluent) and component I paste. This step can be repeated, for example, to obtain effluents and pastes of components II+III, IV-1, IV-4, and V. [Figure 2] illustrates the design of short-term treatment of previously prepared C2C12 cells differentiated for 5 days in 2% horse serum (HS), with glucose utilization assays starting 24 hours after treatment. [Figure 3A] Reports the residual glucose concentration in the culture medium of C2C12 cells (differentiated into myotubes, continued differentiation for 5 days, and then treated for 24 hours under various treatment conditions), including: (1) no treatment; (2) 1 mM metformin (Met) positive control; (3) 0.5 mM metformin; (4) 0.25 mM metformin; (5) solvent (10%); (6) PPF1 (5 mg / mL); (7) HAS1 (5 mg / mL); (8) recombinant human albumin (rhAlbumin 5 mg / mL). [Figure 3B] is a static screenshot of the myotube-related video processed with PPF1 as described in Figures 2 and 3A. [Figure 4] depicts a design for long-term culture medium treatment using 0% or 2% horse serum, with glucose utilization assays performed six days after the start of treatment and 48 hours after the last treatment. [Figure 5] shows micrographs of C2C12 cells cultured in 0% or 2% horse serum (HS) according to the experimental design shown in Figure 4. Compared with the untreated case, treatment with PPF1 and 0% horse serum resulted in more myotube formation. [Figure 6] shows that C2C12 cells treated with 0% horse serum and PPF1 were positive for staining of myosin heavy chain, a marker of myogenic differentiation. [Figure 7] Reports the utilization of residual glucose in C2C12 cell culture medium after treatment with 0% horse serum and the following treatments according to the experimental design shown in Figure 4: no treatment; solvent; PPF1; HAS1; and rhAlbumin. [Figure 8] Reports on glucose utilization in C2C12 cell culture medium after treatment with 0% horse serum and the following treatments according to the experimental design shown in Figure 4: no treatment; PPF1; component IV-4 paste suspension and IV-4 effluent. [Figure 9] Reports the relative expression of glucose transporter type 4 (GLUT-4) in C2C12 myoblasts that were untreated or treated with control solvent PPF1 (5 mg / mL) or recombinant human albumin (rhAlbumin 5 mg / mL). [Figure 10] reports the dose-response relationship between plasma components / fractional separation products and glucose utilization as described in Figure 8. Different concentrations of treatment agents were added to cells (in culture medium, concentrations of 0.15, 0.3, 0.6, 1.25, 2.5, 5, and 10 mg / mL). After treatment with the same culture medium for 6 days and 48 hours, the amount of residual glucose in the culture medium was analyzed using the above-described glucose utilization assay. All three compositions showed a dose-response relationship with glucose utilization. [Figure 11A] is a summary table of several experiments conducted on C57BL / 6 mice of different ages and young rats to test the muscle weight values ​​of the tibialis anterior, extensor digitorum longus, gastrocnemius, and soleus muscles. Each experiment also tested the effect of muscle weight on different time lengths after the last administration with solvent or PPF1. [Figure 11B] is a schematic diagram of the experimental protocol used to study muscle-related parameters in 22-month-old male C57B6 mice that received PPF1 or a control. 26-month-old male C57B6 mice were pulsatilely administered PPF1 or a control solvent for 7 consecutive days (150 μL per dose, intravenously). Ten (10) days after the last administration, the following skeletal muscle groups were harvested: tibialis anterior (TA), extensor digitorum longus (EDL), and soleus (SOL). The muscle-to-body weight (BW) ratio was obtained for each muscle group. [Figure 11C] shows that, compared with the control group, the weight of the tibialis anterior muscle tissue was significantly increased after receiving PPF1 according to the regimen shown in Figure 11B (mean ± SEM, ** p < 0.01, Welch test). [Figure 11D] shows that, compared with the control group, the extensor digitorum longus tissue weight was significantly increased after receiving PPF1 according to the regimen shown in Figure 11B (mean ± SEM, ** p < 0.01, Welch test). [Figure 11E] shows that, compared with the control group, the weight of soleus muscle tissue was significantly increased after receiving PPF1 according to the regimen shown in Figure 11B (mean ± SEM, * p < 0.05, Welch test). [Figure 12A] illustrates the role of PPF1 in inducing the slow-twitch muscle fiber gene (Myl2) in the mouse tibialis anterior muscle. [Figure 12B], [Figure 12C] and [Figure 12D] demonstrate the effect of PPF1 in reducing the expression of fast muscle fiber genes (Myh1(2x), Myh2(2a) and Myh4(2b)) in the anterior tibialis muscle of mice. Figures 13A, 13B, 13C, and 13D all show the state of C2C12 cells after 3 days of culture in 0% horse serum and under various treatment conditions. [Figure 13A] shows the state of C2C12 cells under untreated conditions. [Figure 13B] shows the state of C2C12 cells after 3 days of treatment with 0.3% PPF1. [Figure 13C] and [Figure 13D] show the state of C2C12 cells after being treated with 15 mg / mL component IV-1 paste suspension and 0.6 mg / mL IV-1 paste suspension for 3 days, respectively. [Figure 14] Reports the dose-response relationship between plasma components / component separation products (listed in order of normalized glucose utilization (%)) derived from the C2C12 supernatant 24 hours after the last culture medium change. Cells were cultured in 0% horse serum for a total of six days. This figure illustrates the effect of increasing the dosage of PPF1, component IV-1 paste suspension, and component IV-1 effluent on glucose utilization. [Figure 15]5 reports the dose-response relationship between plasma components / component separation products (listed in order of normalized glucose utilization (%)) derived from the C2C12 supernatant 24 hours after the last culture medium change. Cells were cultured in 2% horse serum for a total of six days. This figure illustrates the effect of increasing the dosage of PPF1, component IV-1 paste suspension, and component IV-1 effluent on glucose utilization. [Figure 16A] and [Figure 16B] report the effect of insulin-like growth factor-1 (IGF-1) on glucose utilization in C2C12 cells treated with 2% horse serum. [Figure 16A] reports the dose-response relationship between IGF-1 treatment (x-axis) and glucose utilization, revealing an EC50 of 17.43 ng / mL. [Figure 16B] reports the dose-response relationship between PPF1 treatment and glucose utilization, revealing an EC50 of 2.9 mg / mL, which contains 0.87 ng / mL IGF1. [Figure 17A] is an illustration of an experimental protocol used to study muscle recovery after administration of different components in a barium chloride-induced injury model. [Figure 17B] Reports the twitching force measurements obtained on days 0 and 17 of the protocol described in Figure 17A, wherein the administration formulations included solvent, recombinant human albumin, PPF1 or HAS1. [Figure 18A] is a schematic diagram of an experimental protocol used to study the effects of plasma components on the level of mouse IGF1 in serum. [Figure 18B] reveals that even 10 days after the last administration as described in the protocol shown in Figure 18A, PPF1 administration was associated with a significant increase in serum mouse IGF-1. [Figure 19A] is a schematic diagram of an experimental protocol used to study whether plasma components can reduce the heart weight of aged C57BL / 6 mice in a model of myocardial hypertrophy observed in aged mammals. [Figure 19B] shows the heart weight (in milligrams) of mice that received the solvent and PPF1. [Figure 19C] shows the ratio of heart weight to body weight of the same mouse described in Figure 19B. Figures 20A, 20B, and 20C report the expression levels of cardiac protective marker RNA in the heart as described in Figures 19A, 19B, and 19C, respectively. [Figure 20A] shows that, compared with the control group, the RNA expression of sarcoplasmic reticulum-endoplasmic reticulum calcium ion transport ATPase (SERCA2a) was significantly increased after PPF1 treatment. [Figure 20B] shows that, compared with the control group, the RNA expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1a) was significantly increased after PPF1 treatment. [Figure 20C] shows that, compared with the control group, the RNA expression of α-myosin heavy chain (aMHC) was significantly increased after PPF1 treatment. [Figure 21A] shows the amount of lactate (myogenic differentiation factor) produced by C2C12 cells after three (3) hours of treatment with various factors. These factors include solvent, 2-DG (negative control), metformin (positive control), oligomycin, HAS1, recombinant human albumin (rhAlbumin), PPF1, component IV-1 paste suspension, and three different concentrations of component IV-1 paste suspension. [Figure 21B] shows the effect of treatment with the various factors described in Figure 21A for five (5) hours on lactate production in C2C12 cells.

Implementation Method

[099] 2. Example 2 Long-term treatment with different concentrations of horse serum Figure 4 illustrates the experimental setup for testing the effects of different concentrations of horse serum on C2C12 cells in culture. It was hypothesized that lower horse serum concentrations would better mitigate the effects of PPF1. On day -2, two days prior to the experiment (d-2), C2C12 myoblasts (Sigma Aldrich 91031101-1VI) were seeded at a density of 8,000 cells / well in C2C12 medium (DMEM + GlutaMAX (ThermoFisher Scientific) + 4.5 g / L glucose, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P / S)) in 96-well plates. Two days later, all culture media were replaced with treatment agents using C2C12 medium (DMEM + GlutaMAX + 1 g / L glucose, 0% or 2% horse serum (Gibco), 1% P / S) with or without 2% horse serum: (1) Untreated; (2) Solvent (10% in the medium); (3) PPF1 (5 mg / mL in the medium); (4) HAS1 (5 mg / mL in the medium); (5) Recombinant human albumin (rhAlbumin, 5 mg / mL in the medium). This day was designated as day zero (d0). On day 2 (d2), half of the culture medium was removed and replenished with the same concentration of treatment agent (5 mg / mL). On day 4 (d4), half of the culture medium was removed again and replenished with the same concentration of treatment agent (5 mg / mL). On day 6 (d6), glucose utilization was measured, and cells were fixed and stained. Figure 5 shows the state of C2C12 cells cultured in 0% and 2% horse serum. For each horse serum concentration, the cell states of untreated and PPF1-treated cells are shown. Treatment with PPF1 at both serum concentrations resulted in the formation of numerous myotubes, leading to myotube differentiation of C2C12 cells. The untreated C2C12 cells showed the lowest degree of myotube differentiation compared to those treated with 0% horse serum. Figure 6 shows that C2C12 cells treated with 0% horse serum and PPF1 were positive for staining of myosin heavy chain, a marker of myogenic differentiation. Figure 7 reports the residual glucose utilization (expressed as concentration (% OD)) in C2C12 cell culture medium after long-term treatment with 0% horse serum. Although horse serum was absent, cell-related results from PPF1 treatment showed a significant increase in glucose utilization compared to the solvent and other plasma components (e.g., HAS1). The researchers also observed that PPF1 tended to enhance myotube contraction to a greater extent compared to HAS1 or rhAlbumin. Therefore, PPF1 can enhance cellular metabolism in a manner different from HAS1 and rhAlbumin. Data are correlations from n=4 wells in three independent experiments ± SEM. Figure 8 reports the residual glucose utilization in C2C12 cell culture medium after long-term treatment with 0% horse serum and different plasma components and plasma component separation products (expressed as concentration (% OD)). The treatment protocols were implemented as shown in Figure 4 above, and the treatment methods were as follows: (1) No treatment; (2) PPF1 (5 mg / mL in culture medium); (3) filtrate IV-4 (5 mg / mL in culture medium); (4) a paste suspension of component IV-4 corresponding to the Cohn component separation process (5 mg / mL in culture medium; concentrated dialysate obtained by dialysis of IV-1 suspension with 0.9% NaCl / 10 mM HEPES at pH 7.3). The results showed that the two plasma components / component separation products exhibited similar effects to PPF1 in the glucose utilization assay of C2C12 cells. Figure 9 shows the relative expression of glucose transporter type 4 (GLUT-4), a protein that plays a key role in the regulation of systemic glucose homeostasis, in C2C12 myoblasts treated with untreated or controlled solvent PPF1 (5 mg / mL in culture medium) or 10% recombinant human albumin (rhAlbumin, 5 mg / mL in culture medium) solution (w / v / , 50 g / L). Data are from a single independent experiment with n=2 wells ±, ***p<0.001. Figure 10 reports the dose-response relationship between plasma components / component separations and glucose utilization as described in Figure 8. C2C12 myoblasts were differentiated into myotubes by in vitro culture in 0% horse serum for 6 days. Different concentrations of treatment agents were added to the cells (0.15, 0.3, 0.6, 1.25, 2.5, 5, and 10 mg / mL in the culture medium). After treatment with the same culture medium for 6 days and 48 hours, the amount of residual glucose in the culture medium was analyzed using the above-described glucose utilization assay. All three compositions showed a dose-response relationship with glucose utilization, with the component IV-4 paste suspension exhibiting the highest median potency (EC50). 3. Example 3 Short-term in vivo administration of PPF1 can increase muscle mass and induce slow-twitch muscle fiber-related genes. Figure 11A is a summary table of several experiments conducted on C57BL / 6 mice of different ages and young rats to test muscle weight values ​​for the tibialis anterior, extensor digitorum longus, gastrocnemius, and soleus muscles. Each experiment also tested the effect of muscle weight on different time lengths following the last administration with either solvent or PPF1. The table shows that a significant increase in muscle weight was associated with PPF1 administration, with a persistent effect observed even long after the most recent administration. Figure 11B is a schematic diagram of the experimental protocol used to study muscle-related parameters in 22-month-old male C57B6 mice administered PPF1 or a control. 26-month-old male C57B6 mice were pulsatilely administered PPF1 or a control solvent for 7 consecutive days (150 μL per dose, intravenously). Ten (10) days after the last administration, the following skeletal muscle groups were harvested: tibialis anterior (TA), extensor digitorum longus (EDL), and soleus (SOL). The muscle-to-body weight (BW) ratio was obtained for each muscle group. Figure 11C shows that the weight of the tibialis anterior muscle tissue was significantly increased after PPF1 administration compared to the control group (mean ± SEM, ** p < 0.01, Welch test). Figure 11D shows that the weight of the extensor digitorum longus muscle tissue was significantly increased after PPF1 administration compared to the control group (mean ± SEM, ** p < 0.01, Welch test). Figure 11E shows that, compared with the control group, the weight of soleus muscle tissue was significantly increased after PPF1 administration (mean ± SEM, * p < 0.05, Welch test). Figure 12A shows that PPF1 induces the expression of slow-twitch muscle fiber genes (Myl2(2a)) in the tibialis anterior muscle. Conversely, in mice treated with PPF1, the expression of fast-twitch muscle fiber genes (Myh1(2x) and Myh2(2a)) showed a decreasing trend (see Figures 12B and 12C, respectively). Figure 12D shows a slight decreasing trend in the fast-twitch muscle fiber-related gene Myh4(2b). Increased slow-twitch muscle fibers are a hallmark of endurance phenotypes. During exercise training in mice or humans, slow-twitch muscle fibers increase while fast-twitch fibers decrease. Slow-twitch fibers are more fatigue-resistant than fast-twitch fibers and burn more fat. This also suggests a potential link 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 mimicry drugs such as metformin, AICAR, and resveratrol. a) 4. Example 4 i. Effects of PPF1 and component IV-1 paste suspension on myotube formation Figures 13A, 13B, 13C, and 13D show the state of C2C12 cells after 3 days of culture in 0% horse serum and under various treatment conditions. Figure 13A shows the state of C2C12 cells under untreated conditions. Figure 13B shows the state of C2C12 cells after 3 days of treatment with 0.3% PPF1. Figures 13C and 13D show the state of C2C12 cells after 3 days of treatment with 0.3% IV-1 paste suspension and 1.25% IV-1 paste suspension, respectively. A comparison between C2C12 cells treated with 0.3% PPF1 and 0.3% IV-1 paste suspensions showed that, after 3 days, at the same concentration, the IV-1 paste suspension induced more myotube formation compared to PPF1. Figure 13D also shows that the induction by the IV-1 paste suspension was dose-dependent, as the visually increased myotube formation appeared to indeed lead to an increase in myotube formation (at treatment concentrations of 1.25% and 0.3%). All three treatment conditions (0.3% PPF1, 0.3% IV-1 paste suspension, and 1.25% IV-1 paste suspension) showed visually greater myotube formation compared to the solvents alone. Figure 14 reports the dose-response relationships between plasma components / fractional isolates of C2C12 (listed in normalized glucose utilization rate (%)) after six days of culture in 0% horse serum. The X-axis shows the escalating doses of IV-1 paste suspension, PPF1, and IV-1 effluent. EC50 values ​​are also reported, with the IV-1 paste suspension showing the highest potency (0.1 mg / ml), followed by the IV-1 effluent (0.4 mg / ml), and PPF1 showing the lowest potency, but still very effective (1.4 mg / ml). Figures 15A, 16B, and 16C report the dose-response relationships between plasma components / fractional isolates of C2C12 (listed in normalized glucose utilization (%), expressed as concentration (%OD)) after six days of culture in 2% horse serum. The X-axis plots the increasing doses (measured concentrations: 5, 2.5, 1.25, 0.6, 0.3, 0.15, 0.075 mg / ml) for each figure. EC50 values ​​are also reported, with the IV-1 paste suspension showing the highest potency (0.4 mg / ml), followed by the IV-1 effluent (1.7 mg / ml), and PPF1 showing the lowest potency, but still very effective (3.8 mg / ml%). 5. Example 5 Effects of IGF1 on metabolic activity Figures 16A and 16B report the effect of insulin-like growth factor-1 (IGF-1) on glucose utilization in C2C12 cells treated with 2% horse serum. On day -2 (d-2), cells were seeded in DMEM containing 4.5 g / L glucose and 10% fetal bovine serum (FBS). On day 0 (d0), the medium was replaced with DMEM containing 1 g / L glucose and 2% horse serum. On day 5 (d5), various treatments were added, and glucose utilization was determined on day 6 (d6). Figure 16A reports the dose-response relationship between recombinant human IGF-1 treatment (x-axis) and glucose utilization, revealing an EC50 of 17:43 ng / mL. Recombinant human IGF-1 was purchased from R&D Systems (catalog number: 291-G1). Figure 16B reports the dose-response relationship between PPF1 treatment and glucose utilization, revealing an EC50 of 2.9 mg / mL containing 0.87 ng / mL IGF-1. IGF-1 is known to affect myotube metabolism, and the calculated IGF-1 content in PPF1 is approximately 14.88 ng / mL. However, the data provided here indicate that the potency of PPF1 is 20 times higher than that of IGF-1 alone; therefore, IGF-1 alone cannot explain the enhanced potency observed under PPF1 treatment. Thus, the role of PPF1 must involve other factors. 6. Example 6 PPF1 can improve muscle recovery after injury. Figure 17A illustrates the experimental protocol used to study muscle recovery from injury using different therapeutic agents. To induce muscle injury in vivo, C57BL / 6 mice were anesthetized via isoflurane inhalation. On day 2 of test agent administration, 50 μL of BaCl2 solution (Sigma-Aldrich B0750, 1.2% in 0.9% sterile NaCl) was injected into the left tibialis anterior muscle along the entire tibia (lengthwise) using a 30-gauge insulin syringe. 50 μL of physiological saline was injected into the right tibialis anterior muscle as a contralateral, uninjured control. During a 10-day fixation period, the left hind limb was wrapped with two layers of medical tape and sports tape (Durapore 3M 1538-2 and Hampton Adams 8542028768), followed by a 10-day recovery period during which the tape was removed. An aversion spray (Grannick bitter apple, GB11A8T) was applied to the outer surface of the tape to prevent the mice from chewing and removing it. Blood circulation in the toes and tape integrity were monitored daily. The hindlimbs of each anesthetized mouse were processed to allow for torque measurements of the aforementioned ankle joints (Gerlinger-Romero F et al., Journal of Visual Experimental Manipulation (J.Vis.Exp), 58696 (2019), doi: 10.3791 / 58696)). Twitching and tetanic forces were recorded using the aforementioned setup (Ho ATV et al., PNAS, 114: 6675-84 (2017)). Data on the left and hindlimbs were recorded prior to administration for baseline measurements, and values ​​at the end of the study (day 17) were compared with initial readings (day 0). For systemic administration, animals were given 150-50 μL of the test therapeutic agent via intravenous injection for 7 consecutive days. The solvent, PPF1, HAS1, and recombinant human albumin (rhAlbumin) were administered to different groups. Figure 17B reports the twitching force measurements obtained on days 0 and 17. On day 0 (before administration), all four columns produced similar maximum torque values. However, on day 17, only the column receiving PPF1 showed a significant increase in maximum torque compared to the control solvent column. Neither the recombinant human albumin (rhAlbumin) nor the HAS1 columns showed a significant increase in maximum torque compared to the control solvent column. Data are presented as mean ± SEM, *p < 0.05, Welch t-test. 7. Example 7 PPF1 is associated with elevated serum IGF-1 levels. Figure 18A illustrates the experimental protocol used to investigate the effect of plasma components on serum mouse IGF-1 levels. Blood was collected from 22-month-old C57BL / 6 mice that received the drug (as shown in Example 9) 10 days after the last day of a 7-day pulsatile administration of PPF1. Serum was separated, and IGF-1 levels in the mice were measured. Figure 18B reveals that even 10 days after the last administration, PPF1 administration was associated with a significant increase in serum mouse IGF-1, suggesting that plasma components (e.g., PPF1) can potentially induce skeletal muscle recovery from injury through a mechanism similar to that observed in the BaCl2 injury-induced model. Data are mean ± SEM, *p < 0.05, Welch t-test. 8. Example 8 PPF1 can reduce the weight of an aging heart in the body. Figure 19A is a schematic diagram of the experimental protocol used to investigate whether plasma components could reduce heart weight in aged C57BL / 6 mice in a model of myocardial hypertrophy observed in aged mammals. (See Kiper C et al., PLoS ONE 8(8): e70512). 26-month-old mice that had received pulsed PPF1 for seven consecutive days were sacrificed on day 17, and heart weight was measured. Figure 19B shows the heart weight (in mg) of mice that received the solvent and PPF1. The heart weight of mice receiving PPF was significantly reduced compared to the control group, indicating that plasma components (e.g., PPF1) can alleviate age-related hypertrophy. Figure 19C shows the ratio of heart weight to body weight in the same mice, where this ratio was significantly lower in mice receiving PPF1 compared to the control group, also indicating an alleviation of age-related hypertrophy. Figures 20A, 20B, and 20C report the expression levels of cardiac protective markers RNA in the heart described in Figures 19A, 19B, and 19C, respectively. Figure 20A shows that PPF1 treatment significantly increased RNA expression of sarcoplasmic reticulum-endoplasmic reticulum calcium ion transporter ATPase (SERCA2a) compared to the control group. SERCA2a is a key regulator of contractility and lymph node calcium circulation proteins in the pathogenesis of heart failure. Its reduction is associated with heart failure, and recovery with gene therapy is associated with promising clinical outcomes in indication subjects. (Chaanine AH et al., Stem Cell and Gene Therapy for Cardiovascular Disease—Chapter 30—SERCA2a Gene Therapy for Heart Failure, 389-400 (2016)). Figure 20B shows that PPF1 treatment significantly increased RNA expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1a) compared to the control group. PGC1a inhibition is associated with heart failure. (Riehle C and Abel D, Trends Cardiovasc Med, 22(4): 98-105 (2012)). Figure 20C shows that PPF1 treatment significantly increased RNA expression of α-myosin heavy chain (aMHC) compared to the control group. Decreased aMHC is associated with cardiac hypertrophy and heart failure. (Hilfiker-Kleiner D et al., Cardiovascular Research, 53: 460-69 (2002)). The increase in these cardioprotective markers suggests that plasma components (e.g., PPF1) can reactivate genetic pathways that alleviate cardiac hypertrophy. Data are mean ± SEM, *p<0.05, Welch t-test. 9. Example 9 Lactic acid is known to promote myoblast differentiation and induce myotube hypertrophy (e.g., see Tsukamoto S et al., International Journal of Molecular Sciences, 19:3649 (2018)). Therefore, measuring lactate production in myoblasts may be an indicator of muscle differentiation and growth. C12C2 myoblasts were subjected to in vitro differentiation treatment in 2% horse serum (HS) differentiation medium for 5 days to differentiate into myotubes. On day 5, various treatment agents were added to the cells. Based on EC50 values ​​assessed by glucose utilization, plasma components were added to the medium at a concentration of 5 mg / mL, but IV-1 paste was added at three different concentrations (0.25, 2.5, and 5 mg / mL). Similarly, on day 5, metformin (1 mM) was added as a positive control. On day 6, cells were treated again with metformin and oligomycin (250 nM) (as a positive control) and 2-deoxy-D-glucose (2-DG, 100 nM) (as a negative control) for three (3) hours or five (5) hours (see Figures 21A and 21B, respectively). After 3 or 5 hours, the culture medium was deproteinized, and lactate production was then determined by enzymatic reaction. Figure 21A shows the amount of lactate (myogenic differentiation factor) produced by C2C12 cells after three (3) hours of treatment with various factors. These factors included solvent, 2-DG (negative control), metformin (positive control), oligomycin, HAS1, recombinant human albumin (rhAlbumin), PPF1, component IV-1 paste suspension, and three different concentrations of component IV-1 paste suspension. Data are obtained from two wells of each of the three independent experiments ± SEM. The data show that after 3 hours of treatment: compared with the untreated control group, lactate production in the HAS1 and rhAlbumin groups did not increase. Lactate production in the PPF1 group showed a slight increasing trend. Lactate production in the component IV-4 and IV-1 paste suspension groups increased significantly. Data are obtained from two wells of each of the three independent experiments ± SEM, ****p<0.0001, nested one-way ANOVA. Lactic acid production is not fully correlated with glucose utilization of plasma components, suggesting that plasma components induce different mechanisms in cells. Figure 21B shows the effects of treatment with the various factors described in Figure 21A for five (5) hours on lactate production in C2C12 cells. Data are obtained from two wells of each of two independent experiments ± SEM. The data show that after 5 hours of treatment: lactate production in the HAS1 and rhAlbumin groups was not increased compared with the untreated control group. Lactate production in the PPF1 group showed an increasing trend. Lactate production in the IV-4 and IV-1 paste suspension groups was significantly increased. Data are obtained from two wells of each of two independent experiments ± SEM, ****p<0.0001, nested one-way ANOVA. Although the invention has been described in detail by way of illustration and examples for the purpose of clear understanding, the teaching of the invention is still of interest to those skilled in the art, who may make some changes and modifications without departing from the spirit or scope of the appended claims. Therefore, the foregoing has only described the principles of the invention. Although not explicitly described herein, those skilled in the art should be able to devise different arrangements that embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditions listed herein are intended to help the reader understand the principles of the invention and the inventors' ideas for further development in the field, and should be interpreted as not being limited to such specific examples and conditions. Moreover, references to the principles, aspects, and embodiments of the invention, and their specific examples, are intended to cover their structural and functional equivalents. Additionally, such equivalents include those currently known and those developed in the future, such as the development of any element having the same function regardless of its structure. Furthermore, whether explicitly stated in the claims, nothing disclosed herein is intended to be offered to the public.

Claims

1. Use of a plasma component in the preparation of a medicament for improving muscle regeneration in an individual diagnosed with a muscle disease, thereby reducing symptoms of the muscle disease, wherein the muscle disease includes muscle degeneration, and wherein the plasma component is: i) a plasma protein component (PPF) comprising 83% to 95% albumin and not more than 17% globulin, wherein the PPF comprises not more than 1% gamma globulin; ii) component IV-4; or iii) component IV-1.

2. As per the purpose of claim 1, wherein the plasma component is the PPF.

3. As per the purpose of claim 1, wherein the plasma component is component IV-4.

4. As per the purpose of claim 1, wherein the plasma component is component IV-1.

5. The use of the drug according to any one of claims 1 to 4, wherein the drug is administered according to a pulse-dose dosing regimen.

6. As per the use of any of claims 1 to 4, wherein the muscle disease is caused by an acute injury.

7. As used in any of claims 1 to 4, wherein the muscle disease is malnutrition.

8. As claimed in claim 7, wherein the malnutrition is selected from the group consisting of: Duchenne and Becker muscular dystrophy; myotonic dystrophy; limb-girdle muscular dystrophy; Emery-Dreifuss muscular dystrophy; congenital muscular dystrophy; and facioscapulohumeral muscular dystrophy.

9. As claimed in any of claims 1 to 4, wherein the muscle disease is selected from the group consisting of: muscle atrophy, myasthenia gravis, McCardell's disease, stroke-related weakness, degeneration associated with amyotrophic lateral sclerosis, neuromuscular junction diseases, myasthenia gravis, toxic myopathy, inflammatory myopathy, lipid storage myopathy, and injury caused by contraction.

10. The use of any one of claims 1 to 4, wherein the muscle disease is an acute muscle injury caused by physical activity.

11. According to the use of any one of claims 1 to 4, wherein reducing the symptoms of the muscle disease includes eliminating the symptoms of the muscle disease.

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