USE OF CARNOSOL FOR INCREASING MUSCLE PROTEIN SYNTHESIS

Abstract

The present invention relates to the use of carnosol or of a composition comprising carnosol for increasing muscle protein synthesis and/or for reducing the muscle protein degradation in a subject.

Claims

1-16. (canceled)

17. A method for prevention and/or treatment of muscle atrophy and/or muscular dystrophy in a subject in need thereof, the method comprising administering carnosol or a composition comprising at least 0.1% w/w of carnosol to said subject.

18. The method according to claim 17, wherein the muscle atrophy is sarcopenia.

19. The method according to claim 17, wherein the subject is an elderly subject.

20. The method according to claim 18, wherein the subject is an elderly subject.

21. The method according to claim 17, wherein the muscle atrophy is a muscle atrophy associated with a disease selected from the group consisting of cancer, AIDS, congestive heart failure, chronic obstructive pulmonary disease (COPD), renal failure, trauma, sepsis, severe burns, mental disease, neuronal disease, cachexia, obesity, and drug-related iatrogenia.

22. The method according to claim 17, wherein the composition comprises at least 2.5% w/w of carnosol.

23. A non-therapeutic method for increasing muscle protein synthesis and/or for reducing muscle protein degradation in a subject in need thereof, the method comprising administering carnosol to said subject.

24. A non-therapeutic method for preventing and/or treating loss of muscle mass and/or for increasing muscle mass in a subject in need thereof, the method comprising administering carnosol or a composition comprising at least 0.1% w/w of carnosol to said subject.

25. The non-therapeutic method according to claim 23, wherein the subject is selected from the group consisting of a subject having a sedentary lifestyle, a subject on bed rest or having been on bedrest, an immobilized subject, an undernourished subject, a malnourished subject, and an astronaut.

26. The non-therapeutic method according to claim 24, wherein the subject is selected from the group consisting of a subject having a sedentary lifestyle, a subject on bed rest or having been on bedrest, an immobilized subject, an undernourished subject, a malnourished subject, and an astronaut.

27. The non-therapeutic method according to claim 23, wherein the subject is a sportsperson.

28. The non-therapeutic method according to claim 24, wherein the subject is a sportsperson.

29. The non-therapeutic method according to claim 24, wherein the method is for increasing muscle mass of a livestock animal and comprises administering carnosol or a composition comprising at least 0.1% w/w of carnosol to said animal.

30. The non-therapeutic method according to claim 24, wherein the composition comprises at least 2.5% w/w of carnosol.

31. The non-therapeutic method according to claim 29, wherein the composition comprises at least 2.5% w/w of carnosol.

Description

FIGURES

[0060] FIG. 1 shows the hypertrophic activity of hydroalcoholic extract of Rosemary leaves on myotubes from young human subjects. Area of myotubes in a culture medium which comprises only culture medium (CTRL), or dry extract of a hydroalcoholic extract of Rosemary leaves at a concentration of 20 μg/mL (RL) are shown.

[0061] FIG. 2 is a schematic diagram of the bioassay-guide fractionation of the extract of rosemary leaves (RL). The fractions having a hypertrophic activity are in grey.

[0062] FIG. 3 shows the hypertrophic activity of commercially available carnosol on myotubes from young human subjects. Area of myotubes in a culture medium which comprises only culture medium (CTRL), 5 μg/ml of fraction C (fraction C), 5 μg/ml of fraction M (fraction M), 5 μg/ml of commercially available carnosol (CO) is shown.

[0063] FIG. 4 shows the hypertrophic activity of carnosol (CO) on myotubes from young human subjects at various concentrations (0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2.5 μg/ml, 5 μg/ml).

[0064] FIG. 5 shows the hypertrophic activity of carnosol on myotubes of elderly subjects (subjects 1 and 2).

[0065] FIG. 6 shows the effect of the administration of carnosol (CO) on gastrocnemius muscle of mice compared to a control (CTRL).

[0066] FIG. 7 shows the effect of administration of carnosol (CO) on MURF1 protein level compared to a control (CTRL) in mice.

[0067] FIG. 8 shows the hypertrophic activity on myotubes of a culture medium which comprises 2 μg/ml of carnosol, 2 μg/ml of carnosic acid, 2 μg/ml of rosmarinic acid, 2 μg/ml of ursolic acid and 50 μg/ml of vitamin C.

[0068] FIG. 9 shows the hypertrophic activity on myotubes of carnosol (CO) and carnosic acid (CA). Area of myotubes in a culture medium which comprises only culture medium (CTRL), 1 μg/ml of carnosic acid (CA 1 μg/ml), 5 μg/ml of carnosic acid (CA 5 μg/ml), 1 μg/ml of carnosol (CO 1 μg/ml), 5 μg/ml of carnosol (CO 5 μg/ml).

[0069] FIG. 10 shows the hypertrophic activity on myotubes of 1 μg/ml of carnosol (CO 1 μg) and 5 mM of leucine (leucine 5 mM).

[0070] FIG. 11 shows the hypertrophic activity on myotubes of 1 μg/ml of carnosol (CO 1 μg), 25 μM of β-hydroxy-8-methylbutyrate (HMB 25 μM) and 100 μM of β-hydroxy-8-methylbutyrate (HMB 100 μM).

[0071] FIG. 12 shows the hypertrophic activity on myotubes of 1 μg/ml of carnosol (CO 1 μg), and 10.sup.−7M of vitamin D (Vit D 10.sup.−7M).

[0072] FIG. 13 is a schematic diagram of the method used to determine if the protein synthesis is the pathway involved in the hypertrophic activity of carnosol. In FIG. 13 is also shown the western blot comparing the protein synthesis of myotubes exposed to carnosol compared to a control.

[0073] FIG. 14 shows the level of puromycine, p-S6K/S6K, p-S6/S6, p-4EBP1/4EPB1, Murf1 after administration of carnosol.

[0074] FIG. 15 shows the hypertrophic activity on myotubes of 1 μg/ml of carnosol (CO 1 μg), 1 μg/ml of carnosic acid (CA 1 μg/ml), and 50 μM of dimethylfumarate (DMF 50 μM).

EXAMPLES

Culture Cell Model

[0075] In order to assay the hypertrophic effect on muscle of compounds from vegetal extracts, the inventors have designed a culture cell model of human skeletal muscle satellite cells. The skeletal muscle satellite cells are able to proliferate and to differentiate ex vivo. When cultured in a growth factors rich medium, the skeletal muscle satellite cells proliferate in the form of myoblasts. At confluence, the myoblasts morphologically differentiate by fusing thereby producing long multinucleated cells (myotubes or myofibers) which express muscular protein. Myotubes are able to respond to hypertrophic and atrophic signals by modulating the balance between the protein synthesis (hypertrophy) or the protein degradation (atrophy) (El Haddad M. et al., Cell Mol Life Sci. 2017 May; 74(10):1923-1936).

[0076] The recovered skeletal muscle satellite cells used in the culture cell model by the inventors were recovered from a muscular biopsy of young and elderly subjects.

[0077] The satellite cells can be easily recovered from a muscle biopsy and cultured ex vivo in dishes where they proliferate as myoblasts and differentiate into myotubes. The biopsy is treated according to an experimental protocol described and validated by us for human skeletal muscle from quadriceps (Kitzmann et al., 2006; El Haddad et al 2017). The biopsy is cut into a fragment of 1 mm.sup.3 and placed in a culture dish treated with type 1 collagen. The explants are trapped inside in a thin layer of Matrigel (BD Matrigel Matrix, BD Biosciences) in 35 mm-collagen coated Petri dishes with growth media (DMEM/F12, supplemented with 10% fetal bovine serum, 0.1% Ultroser G, 1 ng/ml basic FGF, and 10 microg/mL gentamicin). After 6 to 8 days, when cells migrate out of the explants, they are enzymatically harvested using dispase (BD Biosciences) and subcultured in growth medium. Harvested cells are purified by immunomagnetic cell sorting using magnetic activated cell sorter (MACS) microbeads (MiltenyiBiotec) coupled to an antibody against CD56. Usually in protocols of muscle culture cells, muscle differentiation is induced by growing confluent myoblasts in differentiation medium depleted of growth factors. At confluence, myoblasts start to differentiate and differentiation can be evidenced 1) morphologically (after 2 to 4 days), as the fusion of myoblasts generates long giant multinucleated cells (named myotubes) and 2) biochemically, as myotubes express proteins required for muscle contraction. Differentiation is assessed by immunofluorescence using the antibodies against Troponin T and Myogenin, two markers of muscle differentiation. The differentiation status is also confirmed by precisely measuring the expression of three differentiation markers (Myogenin, Sarcomeric Actin and Caveolin by RT-qPCR). Expression of RPLP0 is also quantified as internal control.

[0078] Briefly, myoblasts are seeded at 10.sup.5 cells/dish onto 35 mm collagen-coated dishes and cultured in growth medium (DMEM/F12, supplemented with 10% fetal bovine serum, 0.1% Ultroser G, 1 ng/ml basic FGF, and 10 microg/ml gentamicin). Myogenic differentiation of confluent cells was induced after 3 days by changing to DMEM containing 5% FBS (differentiation medium). Cells were kept in differentiation medium for 3 to 4 days.

[0079] Then the cells are fixed and the expression of troponin T (a protein of the cytoskeleton which is exclusively expressed by myotubes) is analysed by immuno-fluorescence. Then the area of myotubes is measured using the Image J software. If the area increases the compound has a hypertrophic activity and if the area decreases the compound has an atrophic activity.

Hypertrophic Activity of the Rosemary Extract

[0080] The hypertrophic activity of hydroalcoholic extract of rosemary leaves was tested in the cell model disclosed above.

[0081] The skeletal muscle satellite cells came from young people (aged under 30).

[0082] As shown in FIG. 1, a hydroalcoholic extract of rosemary leaves exerts a hypertrophic activity on myotubes when it is at a concentration of 20 μg/ml in the culture medium.

Isolation of the Hypertrophic Compound from the Extract of Rosemary Leaves (RL)

[0083] To isolate the compound(s) responsible for the hypertrophic activity of the extract of rosemary leaves, a bioassay-guided fractionation approach was used.

[0084] Wild rosemary was harvested in north of Montpellier (France). The dried leaves were crushed and an extraction was carried out directly. 150 g of crushed rosemary leaves were put in obscurity at room temperature in a mix comprising 900 g of ethanol absolute and 450 g of distilled water. The mix was manually agitated every 24 hours. After 7 days maceration, the extract was filtrated. Evaporation was made on dry reduced pression. 69 g of hydroalcoholic extract of rosemary leaves called hereinafter RL was obtained.

[0085] After several purification steps as illustrated in FIG. 2, an active compound was isolated in the fraction C.

[0086] The fraction M also shown a hypertrophic activity but weak compared to fraction C.

[0087] A NMR analysis shown that fraction C consists of pure carnosol.

[0088] Thus, the hypertrophic compound from the extract of rosemary leaves is the carnosol. In order to confirm this result, the hypertrophic activity of commercially available carnosol was assayed (FIG. 3). The commercially available carnosol shows the same hypertrophic activity as the fraction C purified from rosemary leaves.

Carnosol Shows a Hypertrophic Activity from 0.5 μg/ml (1-2 μM)

[0089] The hypertrophic activity of carnosol was assayed at various concentrations in the culture medium (0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2.5 μg/ml and 5 μg/ml). The results are shown in FIG. 4. The carnosol exhibits a hypertrophic activity from 0.5 μg/ml (i. e. from 1 to 2 μM) in the cell culture model.

Hypertrophic Activity on Myotube Cells from Elderly Subject

[0090] The culture cell model disclosed above was used with skeletal muscle satellite cells recovered from elderly subjects (subject 1 and subject 2) in order to assay the hypertrophic activity of carnosol on myotube cells from elderly subject.

[0091] As shown in FIG. 5, the carnosol has also a hypertrophic activity on myotube cells of elderly subjects (carnosol: 2 μg/ml).

Hypertrophic Activity of Carnosol In Vivo To investigate whether carnosol can alter skeletal muscle physiology, adult mice (13 weeks old, C57 BL6 J) were treated for 12 days with a daily oral dose of 80 mg/Kg. Sections were made on gastrocnemius muscle with cryostats followed by staining with an anti-dystrophin antibody, revealed by immunofluorescence. The determination of the fibre areas (CSA) was made using the Myovision software.

[0092] As shown in FIG. 6, treatment with carnosol significantly increases the diameter of the fibers on the gastrocnemius

[0093] Then, a western blot analysis on muscle extract was performed to determine the impact of a carnosol treatment on MURF1 protein. It was found that carnosol down-regulates MURF1 protein level in gastrocnemius from control mice, in good correlation with its impact on gastrocnemius fiber area (FIG. 7).

Comparison of the Hypertrophic Activity of Carnosol with Other Compounds.

[0094] The hypertrophic activity of carnosol was compared to other antioxidant compounds. As shown in FIG. 8, only carnosol has a significant hypertrophic activity. The hypertrophic activity of carnosol goes beyond its antioxidant activity (carnosol: 2 μg/ml; carnosic acid: 2 μg/ml; rosmarinic acid: 2 μg/ml; ursolic acid: 2 μg/ml; vitamin C: 50 μg/ml).

[0095] Then, the hypertrophic activity of carnosol and carnosic acid was compared. Whereas carnosic acid has a structure very close from the one of carnosol, the hypertrophic activity of carnosol is significantly higher than the one of carnosic acid. This suggests that the hypertrophic activity of carnosol is specific (FIG. 9).

[0096] The hypertrophic activity of carnosol was compared to leucine and its metabolite β-hydroxy-6-methylbutyrate known for their anabolic properties. Human myotube were treated for 48 hours with carnosol, leucine and β-hydroxy-β-methylbutyrate. As shown in FIGS. 10 and 11, only carnosol treatment induced myotube hypertrophy.

[0097] Low vitamin-D levels are associated with decreased muscle strength and poor physical function in elderly individuals. In addition, it has been proposed that vitamin-D plays an important role for obtaining optimal skeletal muscle function. Thus, the hypertrophic activity of carnosol has been compared to the one of vitamin D. Human myotubes were treated for 48 hours with carnosol or vitamin D in the culture medium at indicated concentrations. Only carnosol induced myotube hypertrophy (FIG. 12).

Pathway(s) Involved in the Hypertrophic Activity of Carnosol

[0098] The muscular hypertrophic activity of carnosol may result from a global increase of the synthesis of proteins. To study this hypothesis, the culture cell model disclosed above was stimulated with carnosol. Then, 30 minutes before recovering the protein from the cells, the cells were treated with puromycin. The puromycin is incorporated in the neo-synthetized proteins. Then, a western blot was carried out with anti-puromycin antibodies in order to show the translation rate of the mRNA in the living cells (Schmidt et al., SUnSET, a nonradioactive method to monitor protein synthesis. Nat Methods. 2009; 6:275-277) (see FIG. 13).

[0099] Based on these results, the inventors studied if the carnosol is able to regulate various signaling pathways controlling the balance between the protein synthesis and the protein degradation. Two main pathways control the regeneration of contractile proteins: [0100] PI3K/Akt/mTOR which is the major pathway of muscle hypertrophy and [0101] pathway of the transcription factors of FOXO family which controls the expression of the genes involved in the proteasome degradation systems and the autophagy (respectively atrogenes and genes of the autophagy).

[0102] At the molecular level, it has been established that the activation of the PI3K/Akt/mTOR pathway stimulates the protein synthesis through their anabolizing targets (mTOR, protein S6 kinase 1 (S6K) and the eIF4E-binding proteins (4E-BP) and blocks the ubiquitine E3 of the family of atrogenes MuRF1 and MAFbx mediated proteolysis pathway (FOXO) (Egerman and Glass, Signaling pathways controlling skeletal muscle mass. Crit Rev Biochem Mol Biol. 2014 January-February; 49(1):59-68).

[0103] According to the available data, the expression of the MuRF1 and MAFbx mRNA is higher in conditions inducing skeletal muscle atrophy (inactivity, denervation, malnutrition, glucocorticoids treatment, oxidative stress, and inflammation). The experiments on mice wherein the expression of MuRF1 or MAFbx was inactivated suggest that MuRF1 would be a better candidate than MAFbx to develop targeted drugs in order to inhibit its expression and thus treat muscular atrophy. Indeed, deleting MuRF1 prevents muscular atrophy in more physiological or physiopathological conditions than the MAFbx deletion. Moreover, the muscular mass which is preserved in response to the MuRF1 deletion seems to be more functional with the strength proportional to the quantity of muscle (Bodine and Baehr, Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1; Am J Physiol Endocrinol Metab. 2014 Sep. 15; 307(6):E469-84).

[0104] The inventors evaluated with western blot the effects of carnosol on the various signaling pathways involved in the control of skeletal muscle hypertrophy or atrophy and found that carnosol stimulates the mTOR pathway (synthesis of proteins) by increasing the phosphorylation rate of P70 S6 kinase, PS6 and 4EBP1 (see FIG. 14). Moreover, carnosol inhibits strongly the MuRF1 expression (degradation of proteins) (see FIG. 14).

[0105] The inventors have also studied the NRF2 signaling pathway.

[0106] The regulation of Nrf2 signaling is believed to preserve redox homeostasis and protect the structure and function of skeletal muscle. Nrf2 is a transcription factor, inactivated by Keap-1 in the cytoplasm. Upon activation, Keap1 is degraded, and NRF2 mediates intracellular antioxidant response by binding to the antioxidant response element (ARE) in the promoter of its target genes and induces the expression of a set of antioxidant enzymes, called ‘phase 2 enzymes,’ including heme oxygenase-1 (HO-1). Recently it has been shown that Nrf2 deficiency caused accelerated aging and muscle loss during aging.

[0107] Since carnosic acid and carnosol are antioxidants molecules and can activate the NRF2 pathway in several type of cells, it was studied whether carnosic acid and carnosol are capable of inducing NRF2 pathway in skeletal muscle. The study has shown that carnosol (6 μM) and carnosic acid (6 μM) both induce NRF2 accumulation but only carnosol fully activates NRF2 pathway by inducing HO-1.

[0108] In order to know whether NRF2 pathway activation is sufficient to activate hypertrophy in skeletal muscle cells. An assay with DMF (dimethyl Fumarate) has been carried out. Given DMF is a NRF2 activator, if the NRF2 pathway activation is sufficient to activate hypertrophy in skeletal muscle cells then DMF should induce myotube hypertrophy.

[0109] The assay has shown that DMF induces the NRF2 pathway (NRF2 and HO-1) in skeletal muscle cells but DMF is a poor inductor of NRF2, efficient at doses superior to 20 μM whereas carnosol induces the NRF2 pathway from 3 μM.

[0110] Moreover, DMF treatment does not induce repression of MURF1 protein and myotube hypertrophy in contrast to carnosol treatment (FIG. 15).