A mixture of MONO-, di- and triglycerides of nonanoic acid for use in the treatment of muscle atrophy, sarcopenia and muscle-injury

Abstract

The present invention describes a mixture of free glycerol and mono-, di- and triglycerides of nonanoic acid for use in the treatment of sarcopenia muscle atrophy or muscle injury.

Description

[0001] INDUSTRIAL INVENTION PATENT APPLICATION TITLED:

[0002] A MIXTURE OF MONO- DI- AND TRIGLYCERIDES OF NONANOIC ACID FOR USE IN THE TREATMENT OF MUSCLE ATROPHY, SARCOPENIA AND MUSCLE-INJURY

[0003] FIELD OF THE INVENTION

[0004] The present invention refers to the field of substances for muscular disorders. STATE OF THE ART

[0005] Ageing, physical inactivity / disuse, nutritional deficiency, excessive exercise, and long-term use of immune suppressants such as glucocorticoids may lead to a decline in muscle mass or strength. Skeletal muscle atrophy and sarcopenia increase the risk of mortality in populations with chronic diseases, such as diabetes, heart failure, renal failure, and cancer. In addition, skeletal muscle atrophy can severely impact patients' quality of life and may lead to economic burden and psychological stress for their families. Despite its clinical relevance, no drug is currently approved for this indication. The use of hormonal drugs, such as dehydroepiandrosterone and testosterone, and anabolic steroids, has been studied as a potential treatment for sarcopenia. While these agents have shown some positive effects on muscle strength and mass, their use is limited owing to adverse effects, such as an increased risk of prostate cancer in men, virilization in women, and an overall high risk of cardiovascular events. As the population of adults aged over 60 years worldwide is predicted to expand to 2 billion by 2050, there is corresponding high need of developing preventive and / or therapeutic strategies against muscle atrophy and sarcopenia.

[0006] In contrast to chronic muscle diseases such as sarcopenia, muscle injuries inflict damage to localized muscle groups. Skeletal muscle injuries can stem from a variety of events, including surgery, direct trauma such as muscle lacerations and contusions or indirect insults such as muscle strains injuries (including muscle rupture and tearing, or tendon rupture) and are frequently observed in professional and amateur sport players.

[0007] Although injured muscle can usually regenerate spontaneously, the healing process is very slow and often incomplete or associated to the development of fibrosis. Common treatments include rest, ice, and use of nonsteroidal anti-inflammatory drugs to limit pain, however, therapeutic approaches have limited effectiveness and optimal strategies for such lesions are not known yet. Therefore, the development of new therapeutic strategies to faster muscle recovery after injury is necessary.

[0008] Aim of the present invention is therefore to provide a substance for use in the treatment of sarcopenia and muscle-injury.

[0009] SUMMARY OF THE INVENTION

[0010] Subject matter of the present invention is a mixture of mono-, di- and triglycerides of nonanoic acid for use in the treatment of sarcopenia, muscle atrophy and muscleinjury.

[0011] It was surprisingly found, in cell-based assays, that the mixture as above described increases the myogenic differentiation of C2C12 cells, exerts a hypertrophic effect as measured by enhanced myotubes width and length, protects against dexamethasone-induced muscle atrophy and exerts reparative effects against muscle strain injury.

[0012] Further subject matter of the present invention is a pharmaceutical composition comprising a mixture of mono-, di- and triglycerides of nonanoic acid and at least a pharmaceutically acceptable excipient.

[0013] DETAILED DESCRIPTION OF THE INVENTION

[0014] Preferably the mixture for use according to the invention is essentially consisting of mono-, di- and triglycerides of nonanoic acid and free glycerol (PC10).

[0015] Preferably the mixture for use according to the invention contains at least 10% of free glycerol, 40% of nonanoate monoglyceride and the remainder being nonanoate di- and triglyceride.

[0016] Preferably the mixture of the invention has the following composition: free glycerol 10-20 % monoglyceride of nonanoic acid 40-60 % diglyceride of nonanoic acid 20-40 % triglyceride of nonanoic acid 1 -5 %

[0017] More preferably the mixture for use according to the invention has the following composition: free glycerol 12-18 % monoglyceride of nonanoic acid 45-55 % diglyceride of nonanoic acid 25-35 % triglyceride of nonanoic acid 2-5 %

[0018] Even more preferably the mixture for use according to the invention has the following composition: free glycerol 14-16 % monoglyceride of nonanoic acid 47-53 % diglyceride of nonanoic acid 27-33 % triglyceride of nonanoic acid 3-4 %

[0019] According to the present invention sarcopenia and muscle atrophy include any condition characterized by atrophy and / or muscle wasting and / or loss of muscle function for example due to malignancy, some autoimmune disorders, aging, malnutrition, obesity, physical inactivity, immobility and chronic corticosteroids therapy

[0020] According to the present invention muscle injury incudes traumatic muscle injury resulting from high stresses and strains to skeletal muscle tissue (often due to muscle activation while the muscle is lengthening), resulting in indirect and noncontact muscle injuries (strains or ruptures), or from external impact, resulting in direct muscle injuries (contusion or laceration). According to the present invention muscle injury include also that due by surgery.

[0021] The pharmaceutical composition according to the invention can be administered orally, topically, transdermally. Suitable excipients according to pharmacopeia can be used to properly formulate the pharmaceutical composition for each of these routes of administration. The pharmaceutical composition according to the invention is preferably for use in the treatment of sarcopenia and muscle injury as above.

[0022] The present invention will be better understood considering the following experimental section.

[0023] BRIEF DESCRIPTION OF THE FIGURES

[0024] Figure 1. Effect of PC10 on the cell viability of C2C12 myoblasts. Cells were treated with PC10, 100 pM for 48 h and compared to untreated control cells. Cell viability was analyzed using the MTS assay. Data are expressed as mean ± SE of three independent experiments. Figure 2. Representing images of haematoxylin-eosin staining for myotubes evaluation: A) C2C12 control, untreated cells; B) C2C12 treated with 2% HS for 6 days; C) C2C12 treated with 2% HS in the presence of PC10 100 pM for 6 days. Final magnification 200x. Scale bar 50 pm.

[0025] Figure 3. Representing images of C2C12 immuno-stained for a-actinin: A) C2C12 control, untreated cells; B) C2C12 treated with 2% HS for 6 day; C) C2C12 treated with 2% HS for 6 days in the presence of PC10 100 pM. Red staining highlights a- actinin positive myotubes formation. Final magnification 200x. Scale bar 50 pm.

[0026] Figure 4. Quantitative image analysis of myotube width (panel A) and length (panel B) in C2C12 control, untreated cells, in C2C12 treated with 2% HS for 6 day and in C2C12 treated with 2% HS for 6 days in the presence of PC10 100 pM. The data are expressed as mean ± SE. *P < 0.05 and ****P<0.0001 vs HS; §§§§ vs CTRL by one-way ANOVA and Dunnett's multiple comparisons test.

[0027] Figure 5. Effect of PC10 on width (panel A) and length (panel B) of C2C12 myotubes expressed as the ratio of the average length of myotubes in the HS+ dexamethasone (HS+DEXA) group. Data are expressed as the mean ±SE. **p<0.01 and ****p<0.0001 vs HS+DEXA; §p<0.05 and §§§p<0.001 vs CTRL by or Kruskal- Wallis and Dunn's multiple comparisons test.

[0028] Figure 6. Representing images of C2C12: A) C2C12 control, untreated cells after 6 hours from injury B) C2C12 treated with PC10 100 pM after 6 hours from injury. The scratch perimeter was drawn with a solid black line to better highlight its mean diameter. Final magnification 200x. Scale bar 50 pm.

[0029] Figure 7. Measurement of the length of the simulated muscle tear 6 hours after the damage, in the presence or absence of PC10 100pM. Values are means±SE. ***p<0.001 , unpaired t test.

[0030] EXPERIMENTAL SECTION

[0031] EXAMPLE 1 - General procedure for the preparation of the tested product PC10

[0032] The esterification reaction was conducted in 10,000 kg batches in a reactor equipped with a vertical reflux condenser.

[0033] Starting glycerol and fatty / organic acid were loaded into the reactor at room temperature as shown in Table 1 . Table 1 : Product Reactants

[0034] The mixture was heated up to 110°C by dropping the starting materials into the reactor via the vertical reflux condenser.

[0035] Once the temperature of 110 °C had been reached, the reaction mixture was heated to 150 °C, raising the temperature by 2 °C at a time, keeping the pressure under control so that it did not exceed 0.5 BAR. When the mixture reached 150°C, the temperature at the head of the vertical condenser was set to 110°C to allow evaporation of the water from the esterification reaction and the reactor temperature was raised up to 235°C (235°C are reached by raising the temperature 1 °C at a time, keeping the Pressure always < 0.5 BAR). Once the reaction mixture reached a temperature of 235 °C, it was thermo-stabilized until the free acidity value (determined by ISO 660:2009 method) was equal to or less than 2%. When this value was reached, once the temperature of the vertical condenser had been set at a temperature equal to 140 °C, the vacuum was attached which allowed the unreacted starting acid to be distilled and therefore to reach a free acidity value equal to or less than 0.1 %.

[0036] The product was then discharged into a refrigerant and cooled to room temperature.

[0037] EXAMPLE 2 - Characterization of the products obtained from example 1 .

[0038] The product obtained according to Example 1 was characterized and analyzed using the following analytical methods shown in Table 2:

[0039] Table 2: Analytical methods used for the chemical characterization of the product.

[0040] Example 2.1 - Determination of Free Glycerol: This method specifies a titration process for the determination of content of glycerol in products containing mono- and triglycerides of fatty / organic acids and glycerol. The method is applicable to both liquid and powdered products.

[0041] Principle of the method

[0042] The cold oxidation of glycerol by sodium metaperiodate in an acid medium produces formic acid according to the following reaction:

[0043] HOCH2-CH(OH)-CH2OH + 2 IO42 HCHO + HCOOH + 2 IO3- + H20

[0044] After removing the excess periodate with 1 ,2-ethanediol, the formic acid produced by the reaction is titrated with a standard volumetric solution of potassium hydroxide, using the bromothymol blue indicator.

[0045] Procedure

[0046] Weigh 0.30-0.40 g of the sample to be analyzed into a 600 ml beaker.

[0047] Add 50 ml of distilled water to the sample using a 50 ml graduated cylinder. After adding the water, add 0.15-0.20 g bromothymol blue indicator (0.4 % alcohol solution) using a Pasteur pipette and acidify with 0.01 N hydrochloric acid solution until the solution turns yellow-green (this acidification step should only be done if the solution is not already yellow).

[0048] Add 0.1 N potassium hydroxide drop by drop until the color of the solution turns blue without any green tinge.

[0049] Add 50 ml of sodium metaperiodate solution (60g / l), stir gently and cover the beaker with a watch glass. Allow to stand / react (30 minutes) in the dark.

[0050] After the reaction time, add 10 ml of ethylene glycol solution, stir gently, and cover the beaker with a watch glass during the reaction period (20 minutes) in the dark. After the reaction time has elapsed, make up to volume up to 300 ml with distilled water, add 0.15-0.20 g bromothymol blue indicator using a pasteur pipette and stir gently.

[0051] Titrate the solution with 0.1 N potassium hydroxide solution until the solution turns blue without green tinge.

[0052] At the same time as the above determination and under the same conditions, carry out a blank test without the sample, using the same quantities of reagents.

[0053] Expression of the result

[0054] The titer of glycerol is given, as a weight percentage, by the formula: (Vi - Vo) - N ■ 9.209

[0055] Glycerol content = - m

[0056] Vi = volume (ml) of the potassium hydroxide solution used for the titration of the sample

[0057] Vo = volume (ml) of the potassium hydroxide solution used for the blank test

[0058] N = normality factor of the standard volumetric potassium hydroxide solution m = mass (g) of the sample taken for determination

[0059] Example 2.2 - Determination of the content of glycerides of organic acids

[0060] This calculation method is used to determine the total glyceride content in mixtures containing only free glycerol, water, glycerides, and free organic acids.

[0061] This method can only be applied after other parameters have been determined using the following methods:

[0062] - Water content: ISO 8534:2017

[0063] - Free glycerol: Method example 2.1

[0064] - Free acidity: ISO 660:2009

[0065] The glyceride content (GC) is calculated as shown below:

[0066] GC= 100 - (WC+FG+FFA) wherein:

[0067] GC = glyceride content of fatty / organic acids

[0068] WC = water content

[0069] FG = Free Glycerol

[0070] FFA = Free acidity

[0071] Example 2.3 - Calculation of acid content (% on 100 g of product)

[0072] (Saponification number) ■ PM

[0073] % of acid on 100 g product = - — — - 10

[0074] 56.1 where:

[0075] PM = Molecular weight of the organic acid

[0076] 56.1 = Molecular weight Potassium hydroxide (KOH 0.1 N)

[0077] Table 3 shows the chemical-physical characterization of the products obtained from example 1 .

[0078] Example 2.4 - Quantification of Mono-, di- and triglycerides of nonanoic acid

[0079] This method describes the procedure for the quantitative determination of glycerol monononanoate, glycerol dinonanoate, glycerol trinonanoate on both liquid and solid samples.

[0080] Principle of the method

[0081] The sample containing the mixture of constituents in different ratios is derivatized as trimethyl silyl ether (TMSE). Silanising agents are added to the sample and the reaction on free -OH groups of glycerol is carried out at room temperature, using pyridine as a catalyst. After dilution, the sample is injected into a Gas Chromatograph equipped with an on-column injector, non-polar capillary column and FID detector.

[0082] All quantitative measurements are carried out by comparing the results obtained by Gas Chromatography with the sample saponification number, expressed in mg KOH / g and determined according to the ISO 3657:2013 method.

[0083] Operating conditions Gas Chromatograph

[0084] Carrier gas flow (Helium): 1 .5 ml / min

[0085] Column temperature ramp: 50°C (1 min) — 120°C (20°C / min) — 230°C (7°C / min) — 360°C (10°C / min) + (5 minutes in isotherm)

[0086] FID detector: 300°C

[0087] Type of injector: on-column

[0088] Procedure Weigh approximately 5-10 mg of representative sample into a 10 ml screw-capped test tube (weighing accuracy ± 0.0001 g). Dissolve the sample in 50 pl of pyridine and add 50 pl of silanisation reagent (99 parts of Bis-trimethylsilyl-trifluoroacetamide + 1 part of trimethylchlorosilane). Carefully close the test tube and allow to stand at room temperature for 20 minutes.

[0089] After the reaction time has elapsed, dilute the sample with 6 ml of n-heptane or isooctane, Inject 1 pl into the Gas Chromatograph using the one microsyringe. Expression of the results

[0090] Several constants must be taken into account when calculating the results.

[0091] Table 6 shows the molecular weights of interest.

[0092] Table 6: Molecular weights of Components

[0093] The responses to the FID of the different esters have a strong impact on analytica results. Table 7 shows the experimentally calculated response factors for all constituents.

[0094] Table 7: Response factors

[0095] The corrected peak areas of each component are calculated as follows:

[0096] Glycerol monononanoate (MO): Glycerol monononanoate area I RRF

[0097] Glycerol dinonanoate (DI): Glycerol dinonanoate area I RRF

[0098] Glycerol trinonanoate (TRI): Glycerol trinonanoate area I RRF

[0099] The weight percentages of the single components are calculated as follows:

[0100] Glycerol monononanoate (% m / m): 100 * MO I (MO+DI+TRI)

[0101] Glycerol dinonanoate (% m / m): 100 * DI / (MO+DI+TRI) Glycerol trinonanoate (% m / m): 100 * TRI I (MO+DI+TRI)

[0102] Table 8 shows the amounts of Monoglycerides, Diglycerides and Triglycerides of the product of example 1 .

[0103] Table 8: Quantitative characterisation of Product of example 1

[0104] EXAMPLE 3 - Cell assays

[0105] Material and Methods

[0106] Cell culture

[0107] The C2C12 cell line, derived from mouse skeletal muscle myoblasts is a common cell model for skeletal muscle research. Murine C2C12 myoblasts were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). For the maintenance of the C2C12 myoblasts, the cells were cultured in growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, 100 U / ml penicillin and 100 pg / ml streptomycin in 5% CO2 at 37°C. The growth medium was changed every 2 days. All reagents were provided by Thermo Fisher Scientific (Milan, Italy).

[0108] Cytotoxicity assay

[0109] C2C12 myoblasts were cultured in 96-well plates (5x103 cells / well) overnight in 5% CO2 incubator at 37°C. Then over a period of 48 h in 5% CO2 incubator at 37°C myoblasts were treated with PC10 100 pM. The medium of each well was discarded after treatment and mitochondrial functionality and cell viability were assessed by the colorimetric method based on [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES) (Promega Corporation, Wl, USA) The optical density of the chromogenic product was measured at 490 nm (Cinci et al., Cancer Med. 2016 Jun;5(6):1279-91 ).

[0110] Induction of myogenic differentiation and treatment with PC 10 For the induction of myogenic differentiation, the C2C12 myoblasts at 80-90% confluence, were transferred to differentiation medium composed of DMEM supplemented with 2% horse serum (HS) to initiate the differentiation of the myoblasts into myotubes. The medium was changed with fresh differentiation medium and fresh PC10 every day. Myotube differentiation was induced for 6 days in presence or absence of PC10 100 pM.

[0111] Hematoxylin-eosin staining for myotubes evaluation

[0112] C2C12 cells were seeded, 105cells in glass dishes and treated as described above. At the end of treatments, specimens were fixed in 4% formaldehyde (freshly prepared from paraformaldehyde as normally used for experiments in optical microscopy) in 0.1 mol / L phosphate buffer, pH 7.4, for 10 min and stained with hematoxylin-eosin. Briefly: 1 ) specimens were incubated with hematoxylin for 2 minutes; 2) washed in tap water for 10 minutes; 3) incubated in 50% ethanol for 2 minute; 4) incubated in eosin for 1 minute; 5) washed in 96% and 100% ethanol; 6) incubated in xylene for 2 minute and then mounted. Images were captured with a Leitz microscope (Leica Microsystems MicrosystemsGmbH, Wetzlar, Germany) equipped with an image capture sys-tem (ProgRes CI Oplus, Carl Zeiss, Gottingen, Germany). At least 10 microscopic fields were randomly taken for each specimen at final magnification of 200X. Measurements of myotubes were carried out using the Imaged 1.33 free-share image analysis software.

[0113] Sarcomeric protein evaluation: o-actinin immunocytochemistry

[0114] C2C12 cells were seeded and treated as described above. At the end of treatments, specimens were fixed in 4% formaldehyde (freshly prepared from paraformaldehyde as normally used for experiments in optical microscopy) in 0.1 mol / L phosphate buffer, pH 7.4, for 10 min and then preincubated in 0.1 % (v / v) Triton (Sigma Aldrich) and 1 % (w / v) bovine serum albumin (Sigma Aldrich) in PBS for 15 min at room temperature. The cells were incubated with primary antibody anti-mouse a-Actinin (Cell Signaling Technology, Danvers, Massachusetts, USA) at final dilution of 1 :40 overnight at 4°C. Immunoreaction was revealed by using the secondary antibody Alexa Fluor 568 anti-rabbit (Invitrogen, San Diego, CA) 1 :200 for 2 h at room temperature. At least 10 microscopic fields were randomly taken for each specimen. a-Actinin presence and myotube measurements were carried out.

[0115] Dexamethasone -induced muscle atrophy of C2C12 cells

[0116] Dexamethasone (DEXA) was used to induce muscle atrophy in C2C12 myotubes. DEXA is a synthetic glucocorticoid that is often used as a representative inducer of muscle atrophy in in vitro and in vivo models (Li et al., Biomed Pharmacother. 2023 Nov; 167:115517). To induce muscle atrophy, fully differentiated C2C12 cells were treated with DEXA (1 pM) for 48 h in the presence or absence of PC 100 pM.

[0117] Muscle strain injury model.

[0118] Muscle injury was simulated by mechanical disruption of a culture of C2C12 myoblasts. C2C12 cells were seeded into glass-embedded plates and allowed to grow to confluence. Two hours before the experiment, the cells were starved in serum-free DMEM so that they synchronized. Muscle injury was then simulated by scraping the cells at confluence with a sterile 1000 pL pipette tip. After washing with PBS, the cells were incubated for 6 hours at 37°C with DMEM medium with 10% FBS in the presence or absence of 100 pM PC10.

[0119] Statistical Analysis

[0120] Data were analyzed using GraphPad Prism Software, version 6.0 (GraphPad Software, Inc., San Die-go, CA, United States). Results are expressed as mean ± SEM. Normality of the data was verified with the Kolmogorov-Smirnov and D'Agostino & Pearson test. Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons post hoc test for multiple comparisons or Kruskal-Wallis and Dunn's multiple comparisons test when data were not normally distributed. A p value < 0.05 was considered statistically significant.

[0121] Results

[0122] PC 10 is not cytotoxic to C2C12 cells.

[0123] Before testing PC10 in the process of muscle differentiation, first was determined the effects of PC10 on cell viability. As shown in Figure 1 , at concentration 100 pM, PC10 did not reduce cell viability compared to untreated control cells, demonstrating that PC 10 was not cytotoxic to C2C12 cells.

[0124] PC 10 increases the myogenic differentiation of C2C12 cells.

[0125] C2C12 cells are murine myoblasts derived from satellite cells, which can spontaneously differentiate into myotubes when moved from high-serum medium to low-horse serum medium. In the present study, C2C12 cells were therefore used to examine the effects of PC10 on myogenic differentiation. In particular, we examined the effects of PC10 on the morphological changes of C2C12 cells (such as the loss of their typical triangular morphology and the gradual change in cell shape with the acquisition of an elongated shape) that are associated with the differentiation process (Figure 2). On the 6th day of differentiation, myotubes formation was increased by HS treatment (Figure 2 panel B) compared to control cells (Figure 2 panel A). Interestingly, compared to cells treated with HS alone, PC10 further increased myotubes formation (Figure 2 panel C). These data suggest that PC10 promotes the myogenic differentiation of C2C12 cells.

[0126] PC 10 exerts a hypertrophic effect as measured by enhanced myotubes width and length.

[0127] The immune-cytological analysis was carried out to identify the a-actinin protein and to measure myotubes width and length. The differentiation of myoblasts in fact involves the exit from the cell cycle, the subsequent alignment of the myoblasts with each other and finally, the fusion between cells to form myotubes, multinucleated syncytia which present most of the characteristics of skeletal muscle fibers. When fusion between multiple myoblasts occurs, the resulting myotube undergoes a phenotypic change dependent on the activation of muscle-specific genes encoding for proteins such as actin, myosin, troponin and a-actinin. a-actinins constitute the cytoskeleton of the muscle fiber and have the function of maintaining the stability and integrity of the cell membrane during muscle contraction. Myotube width and length were determined at day 6th day of differentiation. As shown in Figure 4, PC10 100 pM treatment significantly increased muscle fiber length (panel B) and width (panel A) compared to the HS group (p < 0.0001 and p < 0.05, respectively. This effect was associated with the activation of muscle-specific genes, such as a-actinin (Figure 3). These data indicate that PC10 induces myogenic hypertrophy, which is the augmentation of skeletal muscle through an increase in size of myotubes.

[0128] PC 10 protects against dexamethasone-induced muscle atrophy.

[0129] When muscle atrophy is induced, the number and diameter of muscle fibers decrease through the breakdown of muscle proteins, resulting in a decrease in total muscle mass. To evaluate the protective effect of PC10 on muscle atrophy, the length and width of muscle fibers were analyzed by hematoxylin-eosin staining.

[0130] In the HS+ dexamethasone (HS+DEXA) group, the length and width of the myotube were significantly reduced compared to the HS group (p<0.001 and p<0.05, respectively), confirming that DEXA-induced muscle atrophy was effectively induced. In contrast, PC10 100 pM significantly increased muscle fiber length and width compared to the HS+DEXA group (p< 0.0001 and p< 0.01 , respectively; Figure 5). These results clearly indicate that PC10 protects against DEXA-induced muscle atrophy.

[0131] PC 10 exerts reparative effects against muscle strain injury

[0132] A muscle tear is an injury to one or more bundles of muscle fibers generally caused by excessive stress on the muscles which exceeds the physiological limit of tension that the muscle can withstand. Following muscle damage, repair and regeneration processes of the muscle begin with the migration of myoblasts to the site of the injury. The aim of this part of the experiments was to simulate a muscle tear on a myoblast cell culture and test the repair activity of PC10.

[0133] The results shown in Figures 6 and 7 demonstrate that PC10 100 pM, after six hours from the muscle tear simulated in vitro, is able to promote muscle repair: in cells treated with PC10 100 pM a significant reduction in the width of the injury itself compared to untreated cells (p<0.001 ) was observed. This result suggests that PC10 promotes the migration of myoblasts to the site of muscle damage, favouring its repair.

Claims

CLAIMS1 . A mixture of mono-, di- and triglycerides of nonanoic acid for use in the treatment of muscle atrophy, sarcopenia or muscle-injury.

2. The mixture for use according to claim 1 wherein said mixture is essentially consisting of mono-, di- and triglycerides of nonanoic acid and free glycerol.

3. The mixture for use according to claim 2, wherein said mixture is containing at least 10% of free glycerol, 40% of monoglyceride of nonanoic acid and the remainder being di- and triglyceride of nonanoic acid.

4. The mixture for use according to claim 3 wherein said mixture has the following composition: free glycerol 10-20 % monoglyceride of nonanoic acid 40-60 % diglyceride of nonanoic acid 20-40 % triglyceride of nonanoic acid 1 -5 %5. The mixture for use according to claim 4 wherein said mixture has the following composition: free glycerol 12-18 % monoglyceride of nonanoic acid 45-55 % diglyceride of nonanoic acid 25-35 % triglyceride of nonanoic acid 2-5 %6. The mixture for use according to claim 5 wherein said mixture has the following composition: free glycerol 14-16 % monoglyceride of nonanoic acid 47-53 % diglyceride of nonanoic acid 27-33 % triglyceride of nonanoic acid 3-4 %7. The mixture for use according to any one of claims 1 -6 wherein said sarcopenia or muscle atrophy includes any condition characterized by loss of skeletal muscle mass and function for example due to malignancy, an autoimmune disorder, aging, obesity, physical inactivity, immobility, and chronic corticosteroids therapy.

8. The mixture for use according to any one of claims 1 -6 wherein said muscle injury incudes traumatic muscle injury resulting from high stresses and strains to skeletal muscle tissue, resulting in indirect and non-contact muscle injuries, or from external impact, resulting in direct muscle injuries or due by surgery.

9. A pharmaceutical composition comprising a mixture of mono-, di- and triglycerides of nonanoic acid as defined in any one of claim 1 -6 and at least a pharmaceutically acceptable excipient.

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