A NON-THERAPEUTIC USE OF ADENOSYLCOBALAMIN TO ENLARGE MUSCLE FIBER SIZE

Description

FIELD OF THE INVENTION

[0001] The present invention relates to the non-therapeutic use of vitamin B12 for maintaining or increasing muscle function and muscle mass in an ageing subject over 30 years old, wherein the vitamin B12 is adenosylcobalamin and wherein muscle fiber size is increased.BACKGROUND TO THE INVENTION

[0002] Age-related loss of muscle function and mass occurs inevitably in all individuals, however its progression depends on a range of genetic and environmental factors, such as physical activity and nutritional intake.

[0003] In some subjects, the effect of ageing on muscle may progress to a state of morbidity, specific conditions of which include sarcopenia and frailty. Sarcopenia is defined as occurring at the point at which the age-related loss of muscle function and mass becomes debilitating and impacts on quality of life (Sayer, A.A. et al. (2013) Age Ageing 42: 145-150). In contrast, frailty is a classification of age-related muscle dysfunction which relies on muscle strength and functionality, but not muscle mass (Morley, J.E. et al. (2013) J. Am. Med. Dir. Assoc. 14: 392-397).

[0004] Sarcopenia and frailty are multi-factorial syndromes which associate with pathophysiological changes, such as impaired neuro-muscular transition, altered excitation / contraction coupling, impaired regenerative capacity linked to stem cell exhaustion, defects of mitochondrial and energy metabolism in myofibers, and marbling of skeletal muscle with fat and fibrosis (Ali, S. et al. (2014) Gerontology 60: 294-305). The aetiology of these syndromes is therefore complex and poorly understood, but low physical activity, hormonal decline in anabolic hormones (e.g. androgens and IGF-1), and malnutrition and / or nutritional deficiencies play an important role (Mithal, A. et al. (2013) Osteoporos. Int. 24: 1555-1566).

[0005] Sarcopenia is becoming a major health concern in developed countries, where lessened physical activity with age and increased longevity are particularly prevalent. In severe cases, sarcopenia may result in a person losing their ability to live independently. In addition, sarcopenia is a predictor of wider-ranging disability in population-based studies, and has been linked to poor balance, gait speed, prevalence of falls and fractures.

[0006] Reduced physical activity is thought to increase the likelihood of sarcopenia and therefore increased exercise will likely be beneficial in combatting the condition. Indeed, resistance exercise is associated with increased synthesis of proteins in skeletal muscle. However, exercise as a treatment often suffers from poor patient compliance.

[0007] There are currently no pharmacological agents approved for the treatment of sarcopenia. A number of growth hormones have been studied in this context, however these have shown little effect. In addition, anabolic steroids may increase muscle mass and strength, but are associated with a number of side effects, such as increased risk of prostate cancer. Moreover, existing pharmacological and nutritional approaches are mainly directed at targeting muscle anabolism and do not adequately address the neuromuscular defects associated with the condition.

[0008] Accordingly there remains a significant need for methods of maintaining or increasing muscle function and mass in ageing subjects. In particular, there is a need for methods of treating sarcopenia and frailty.SUMMARY OF THE INVENTION

[0009] The inventors have unexpectedly found that increasing levels of vitamin B12 in ageing subjects is clinically applicable to the treatment of age-related decline in muscle function and mass, and therefore provides a route to the treatment of age-related conditions such as sarcopenia and frailty.

[0010] In particular, the inventors have surprisingly found that different vitamin B12 isoforms have particular effects on different systems and pathways. Accordingly, particular vitamin B12 isoforms, or combinations of vitamin B12 isoforms, can be chosen to provide particularly beneficial effects in a subject.

[0011] The invention is defined in the claims and provides the non-therapeutic use of vitamin B12 for maintaining or increasing muscle function and mass in an ageing subject over 30 years old, wherein the vitamin B12 is adenosylcobalamin and wherein muscle fiber size is increased.

[0012] The ageing subject is a subject over the age of 30 years old, or may, for example, be a human subject over the age of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 years old.

[0013] Preferably, the muscle is skeletal muscle.

[0014] The vitamin B12 maintains or increases muscle function and muscle mass in an ageing subject over 30 years old, wherein the vitamin B12 is adenosylcobalamin and wherein muscle fiber size is increased.

[0015] The vitamin B12 substantially prevents or reduces a reduction in muscle mass. The prevention or reduction in muscle mass may be in comparison to the reduction in muscle mass that would be expected in the absence of the vitamin B12 of the invention.

[0016] The vitamin B12 may be in a combined preparation for simultaneous, combined, sequential or separate administration to a subject.

[0017] The vitamin B12 is adenosylcobalamin.

[0018] The vitamin B12 may be administered by oral, parental, sub-lingual, sub-cutaneous, transdermal or intra-nasal administration.

[0019] The vitamin B12 may be administered as an oral vitamin B12 supplement or a probiotic supplement comprising vitamin B12 producing bacteria. The vitamin B12 may, for example, be in the form of a nutritional composition or supplement, or a diet product.

[0020] The subject may have previously been determined to be vitamin B12 deficient.

[0021] Preferably, the muscle is skeletal muscle.

[0022] The vitamin B12 may, for example, be in the form of a nutritional composition or supplement, or a diet product.

[0023] The diet product may be for use in a vitamin B12 deficient subject.

[0024] Vitamin B12; or diet product may be used in combination with an exercise regime to maintain or increase muscle function and mass and increase muscle fiber size.

[0025] Vitamin B12; or diet product may be used in combination with other pharmaceutical compositions, including selective androgen receptor modulators (SARMs), such as ostarine or myostatin blockers (e.g. myostatin antibodies, activin receptor antibodies and activin receptor-Fc), such as LY2495655 or Bimagrumab, or beta2 receptor agonists such as formoterol, or ghrelin receptor agonists such as anamorelin, or anabolic catabolic transforming agents (ACTA), such as MT-102.DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Circulating levels of neuregulin-1 (NRG1) decrease with age in rats. Neuregulin-1 (NRG1) levels were measured in serum from rats aged 8 months, 18 months or 24 months using the slow off-rate DNA aptamer detection and quantification was performed after median-normalisation of Relative Fluorescence Units (RFU). 10 animals per group were analysed. ** = p-value < 0.01. Figure 2 Neuregulin-1 (NRG1) protects neuromuscular junctions from damage in vitro. Nerve and muscle co-cultures were grown in vitro until neuromuscular junctions were mature. Damage was induced using beta-amyloid (Ab) incubation (2.5 µM) and riluzole was used as a positive control for preserving neuromuscular junctions from Ab-induced damage. The effect of neuregulin-1 (NRG1) on Ab-induced damage was evaluated by measuring the neuromuscular junction (NMJ) size. All values are from 6 wells per group and are shown as a percent of the control condition (CTL). ** = p-value < 0.01; *** = p-value < 0.001. Figure 3 Neuregulin-1 (NRG1) protects skeletal muscle from age-induced atrophy. Pre-sarcopenic rats aged 16 months were treated for 5 months with either neuregulin-1 (NRG1) or saline. NRG1 was injected sub-cutaneously at 1 µg / kg body weight 3 times per week. Hind-limb skeletal muscle mass was then evaluated and compared to a group of adult healthy rats (8 months old at the start of the experiment) injected with saline control. * = p-value < 0.05 ; ** = p-value < 0.01. Figures 4A-C Vitamin B12 protects neuromuscular junctions from damage in vitro. Nerve and muscle co-cultures were grown in vitro until neuromuscular junctions were mature. Damage was induced using beta-amyloid (Ab) incubation (2.5 µM) and riluzole was used as a positive control for preserving neuromuscular junctions from Ab-induced damage. The effect of methylcobalamin (MeCbl) and adenosylcobalamin (AdenoCbl) on Ab-induced damage was evaluated by measuring: Figure 4A the neuromuscular junction (NMJ) size, Figure 4B the neuromuscular junction number and Figure 4C the neurite network length. All values are from 6 wells per group and are shown as a percent of the control condition (CTL). ** = p-value < 0.01; *** = p-value < 0.001. Figures 5A and 5B Neuregulin-1 (NRG1) and vitamin B12 have additive effects on neuromuscular junction protection in vitro. Nerve and muscle co-cultures were grown in vitro until neuromuscular junctions were mature. Damage was induced using beta-amyloid (Ab) incubation (10 µM) and riluzole was used as a positive control for preserving neuromuscular junctions from Ab-induced damage. The effect of neuregulin-1 (NRG1) and / or adenosylcobalamin (AdoCbl) on Ab-induced damage was evaluated by measuring the neuromuscular junction (NMJ) number (A) and size (B). All values are from 6 wells per group and are shown as a percent of the control condition (CTL). ** = p-value < 0.01; *** = p-value < 0.001. Figures 6A and 6B Adenosylcobalamin induces an increase in muscle fiber size in aged rats. Old sarcopenic rats were treated with methyl-cobalamin (MeCbl) in Figure 6A or adenosylcobalamin (AdoCbl) in Figure 6B for 5 months between 18 and 23 months of age, and compared to age matched sarcopenic controls (old CTL) or young adult healthy controls (adult). Tibialis anterior muscle was dissected out, sectioned and immunostained for fibers. Fiber size distribution for representative type 2A is shown. All values are from 9 animals per group. Figure 7 Methylcobalamin protects from muscle atrophy Human myoblasts were induced to form myotubes in the presence of the atrophic factor TNFa and different forms of vitamin B12 on a 2D+ micro-pattern inducing myotube alignment. IGF is used as a positive control to prevent TNFa-induced atrophy. Fusion index was calculated as the percentage of nuclei inside myotubes (i.e containing ≥ 2 nuclei) over the total number of nuclei. Values are from 3 wells per group and are shown as a percent of the control condition (CTL). ** = p-value < 0.01 and *** = p-value < 0.001 compared to CTL condition. ## = p-value <0.01 compared to TNFa condition. Figures 8A-C Adenosylcobalamin specifically reverses gene expression signatures associated with sarcopenia while methylcobalamin affects different gene expression signatures in skeletal muscle. Old sarcopenic rats were treated with adenoysyl-cobalamin (AdoCbl) or methyl-cobalamin (MeCbl) for 5 months between 18 and 23 months of age, and compared to age matched sarcopenic controls (old CTL) or young adult healthy controls (adult). Tibialis anterior muscle was dissected out for RNA extraction and micro-array analysis. GSEA gene enrichments for top regulated pathways in Figure 8A old controls vs adult, Figure 8B old treated with adenosylcobalamin vs old controls and Figure 8C old treated with methyl-cobalamin versus old controls are shown. DETAILED DESCRIPTION OF THE INVENTION

[0027] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

[0028] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press.Vitamin B12

[0029] In one aspect, the invention provides a non-therapeutic use of vitamin B12 for maintaining or increasing muscle function and mass in an ageing subject over 30 years old, wherein the vitamin B12 is adenosylcobalamin and wherein muscle fiber size is increased.

[0030] Vitamin B12 (also known as cobalamine) is a class of cobalt-containing hydrosoluble vitamins which cannot be synthesised by the human body and must therefore be acquired from food or synthesised by the gut microbiota.

[0031] The vitamin B12 class may refer to several chemical forms of vitamin B12, depending on the upper axial ligand of the cobalt ion. These are: Cyanocobalamin (R = -CN) Hydroxocobalamin (R = -OH) Methylcobalamin (R = -CH3), and Adenosylcobalamin (R = - 5'-deoxyadenosyl).

[0032] The vitamin B12 pool in the human body is composed of several forms: cyanocobalamin, which is inactive and requires conversion for activity, and methylcobalamin and adenosylcobalamin, which are the metabolically active forms of vitamin B12.

[0033] Two enzymes are known to rely on vitamin B12 as a cofactor: methionine synthase and methylmalonylCoA mutase. Methionine synthase is a cytoplasmic enzyme relying on methyl-cobalamine to convert homocysteine to methionine. It thereby plays a critical role in providing S-adenosylmethionine (SAM) as a methylation donor and preventing the toxic accumulation of homocysteine. Low SAM levels and high homocysteine levels observed upon severe vitamin B12 deficiency impair myelination of peripheral nerves and the spinal cord. Methionine synthase also catalyses the activation of 5-methyl-tetrahydrofolate into the bioactive tetrahydrofolate, which is required for 1-carbon metabolism and DNA synthesis, and thus for efficient red blood cell proliferation. MethylmalonylCoA mutase is a mitochondrial enzyme relying on adenosyl-cobalamine to convert methyl-malonylCoA to succinylCoA, which subsequently enters the TCA cycle. It is implicated in the degradation of branched-chain amino acids and odd-chain length fatty acids, and is essential during embryonic life to control neurological development, but is not vital in adult life

[0034] The vitamin B12 may or may not be administered in combination.

[0035] The vitamin B12 of the invention is in the form of adenosylcobalamin.

[0036] The adenosylcobalamin maintains or increases muscle mass and muscle function in an ageing subject over 30 years old, wherein the adenosylcobalamin increases muscle fibre size.

[0037] The present invention provides the non-therapeutic use of adenosylcobalamin for increasing maintaining or increasing muscle mass and muscle function in an ageing subject over 30 years old, wherein muscle fiber size is increased.

[0038] The adenosylcobalamin maintains or increases muscle size by maintaining or increasing muscle fiber size.

[0039] As used herein, muscle wasting may be synonymous with 'muscle atrophy' and is used to refer to a decrease in the mass of muscle. Reducing muscle atrophy may therefore be synonymous with maintaining muscle mass.

[0040] The vitamin B12 of the invention may be administered to a subject by any suitable route, for example orally, intranasally, intravenously, parentally, sub-linguially, sub-cutaneously, transdermally or intramuscularly.Vitamin B12 deficiency

[0041] The subject may be vitamin B12 deficient.

[0042] The Recommended dietary allowance (RDA) of US adults was set at 2.4 µg per day by the Institute of Medicine, based on an average absorption from food of ~50% (National Academy of Sciences, Institute of Medicine (2000); Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin and Choline, Chapter 9, pp306-56). It was noted that the daily requirement varies with body size.

[0043] The likelihood of vitamin B12 deficiency in humans may be defined according to the serum vitamin B12 level as follows: <148 picomols / L (<200 picograms / mL) indicates probable deficiency, 148 to 258 picomols / L (201 to 350 picograms / mL) indicates possible deficiency and >258 picomols / L (>350 picograms / mL) indicates that deficiency is unlikely (BMJ, Best Practice, http: / / bestpractice.bmj.com / best-practice / monograph / 822 / basics.html). However, because of the lack of a gold standard for determining vitamin B12 levels and related complications regarding active and inactive vitamin B12, assays of serum vitamin B12 are often combined with further biochemical assays or clinical assessment based on presenting symptoms, in order to diagnose vitamin B12 deficiency.

[0044] Additional assays which may be performed to give a further indication of a vitamin B12 deficiency include determining the level of, holotranscobalamine, methylmalonic acid and / or homocysteine in a sample isolated from the subject.

[0045] Holotranscobalamin refers to vitamin B12 bound to its bioactive serum transporter transcobalamine II. Holotranscobalamin levels may be determined using commercial available assays (e.g. ELISA assays). Low levels of holotranscobalamin are associated with a potential vitamin B12 deficiency.

[0046] Methyl-malonic acid (MMA) accumulates with low activity of the vitamin B12-dependent enzyme methylmalonylCoA mutase. As such high levels of MMA are associated with vitamin B12 deficiency.

[0047] Homocysteine accumulates with low activity of the vitamin B12-dependent enzyme methionine synthase. Low High levels of homocysteine are associated with vitamin B12 deficiency. However assays of homocysteine levels can be confounded by folate deficiency.

[0048] Vitamin B12 may, for example, be provided in the form of a tablet, liquid (e.g. for ingestion, or use in a nasal spray or injection) or transdermal patch. For example, vitamin B12 is available as a nutritional supplement either on its own or in combination with other supplements.

[0049] Oral supplementation typically involves giving 250 µg to 1 mg of vitamin B12 daily.

[0050] Herein described is administering a probiotic supplement comprising vitamin B12 producing bacteria to a subject.

[0051] The probiotic supplement can include any probiotic microorganism(s) which beneficially affect the host subject by improving its intestinal microbial balance to enhance vitamin B12 uptake. The probiotic microorganism can be selected from the group comprising of Bifidobacterium, Lactobacillus, Streptococcus, Enterococcus and Saccharomyces or mixtures thereof.

[0052] Certain probiotic microorganisms which are native components of the gut microbiota are known to produce vitamin B12, for example, lactic acid producing bacteria such as Lactobacillus. delbrueckii subsp. bulgaricus (see Le Blanc et al.; J App. Micro.; 111(6); (2011)). Advantageously, the probiotic supplement can enhance existing microorganisms in the gut that produce vitamin B12 in situ.

[0053] The oral vitamin B12 supplementation may be in the form of a food or beverage product. The food or beverage product may comprise a probiotic supplement comprising vitamin B12 producing bacteria or other probiotics which can enhange existing microorganisms in the gut that produce vitamin B12 in situ.

[0054] Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient. The dosage is such that it is sufficient to provide required levels of active vitamin B12.

[0055] By "simultaneous", it is to be understood that the two agents are administered concurrently, whereas the term "combined" is used to mean they are administered, if not simultaneously, then "sequentially" within a time frame that they both are available to act therapeutically within the same time frame. Thus, administration "sequentially" may permit one agent to be administered within 5 minutes, 10 minutes or a matter of hours after the other provided the circulatory half-life of the first administered agent is such that they are both concurrently present in therapeutically effective amounts. The time delay between administration of the components will vary depending on the exact nature of the components, the interaction therebetween, and their respective half-lives.

[0056] In contrast to "combined" or "sequential", "separate" is to be understood as meaning that the gap between administering one agent and the other agent is significant, i.e. the first administered agent may no longer be present in the bloodstream in a therapeutically effective amount when the second agent is administered.Muscle function and mass

[0057] The compounds, compositions, uses and methods herein described provide for the maintenance of or increase in muscle function and mass in an ageing subject.

[0058] The term "muscle function" refers to the ability of a muscle to perform in a manner that does not negatively impact on the life of a subject, and encompasses parameters of muscle strength, muscle contraction, muscle endurance and / or muscle elasticity.

[0059] Suitable tests for assessing muscle function include grip strength assessment using a dynamometer; one repeat maximum on leg press, chest press or leg extension; gait speed; 6 min walk test; time up and go; short physical performance battery; Fried frailty criteria; and stair climbing time assessments.

[0060] Muscle mass (which may equate with muscle volume, muscle thickness or myofiber / muscle fiber size) may be measured by dual-energy X-ray absorptiometry (DXA) or bioimpedance tests. Similarly, MRI may be used for assessing muscle volume and ultra-sound may be used for assessing muscle thickness and pennation angle.

[0061] The term "maintains" refers to a particular parameter, such as muscle function and / or mass, remaining substantially unchanged over a period of time (e.g. 5, 10, 15, 20, 25, 30, 40, 50 or more years).

[0062] In one embodiment, muscle mass increases by at least 1%, 2%, 3%, 4%, 5%, 10%, 15% or 20%.

[0063] In another embodiment, muscle mass increases by 1-2.5%, 1-5%, 1-10% or 1-20%.

[0064] Preferably, the muscle is skeletal muscle.Method of treatment

[0065] It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment; references to preventing are more commonly associated with prophylactic treatment. Treatment may also include arresting progression in the severity of a disease.Subject

[0066] The non-therapeutic use for mammals, particularly humans, is preferred. However, both human and veterinary treatments are within the scope of the invention.

[0067] The ageing subject to be treated is a subject over the age of 30, and may, for example, be a human subject over the age of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 years old. For veterinary applications, the age of the animal would be scaled from the human situation using the average lifespan for calibration.

[0068] The vitamin B12 may administered to an ageing subject as a medicament in order to maintain or increase muscle function and muscle mass, wherein muscle fiber size is increased, wherein the subject has reduced muscle mass and / or muscle function which causes debilitation and / or a negative impact on the subject's quality of life.Sarcopenia and frailty

[0069] Herein described are means to address loss of muscle function and mass that occurs with age. Age-related loss of muscle function and mass occurs inevitably in all individuals, however its progression depends on a range of genetic and environmental factors, such as physical activity and nutritional intake.

[0070] The specific condition of sarcopenia is defined as occurring at the point at which the age-related loss of muscle mass and function becomes debilitating and impacts on quality of life (Sayer, A.A. et al. (2013) Age Ageing 42: 145-150). In contrast, frailty is a classification of age-related muscle dysfunction which relies on muscle strength and functionality, but not muscle mass (Morley, J.E. et al. (2013) J. Am. Med. Dir. Assoc. 14: 392-397).

[0071] Sarcopenia and frailty are multi-factorial syndromes which associate with pathophysiological changes, such as impaired neuro-muscular transition, altered excitation / contraction coupling, impaired regenerative capacity linked to stem cell exhaustion, defects of mitochondrial and energy metabolism in myofibers, and marbling of skeletal muscle with fat and fibrosis (Ali, S. et al. (2014) Gerontology 60: 294-305). The aetiology of these syndromes is therefore complex and poorly understood, but low physical activity, hormonal decline in anabolic hormones (e.g. androgens and IGF-1), and malnutrition and / or nutritional deficiencies play an important role (Mithal, A. et al. (2013) Osteoporos. Int. 24: 1555-1566).Dietary intervention and product

[0072] The term "dietary intervention" refers to an external factor applied to a subject which causes a change in the subject's diet. The dietary intervention is a high calorie diet. The dietary intervention is a high protein and / or carbohydrate diet. The dietary intervention is a diet supplemented with vitamins and minerals, in particular vitamin B12 and / or vitamin D.

[0073] The dietary intervention is a diet supplemented with vitamin B12, in particular adenosylcobalamin.

[0074] The diet may be one which is adjusted to the starting body weight of the subject.

[0075] The dietary intervention may comprise administration of at least one diet product. The diet product may be a meal replacement product or a supplement product which may, for example, increase the subject's appetite. The diet product may include food products, drinks, pet food products, food supplements, nutraceuticals, food additives or nutritional formulas. Example oral nutritional supplements include Nestlé Boost and Meritene products.EXAMPLES Example 1 Materials and methods

[0076] Variation of circulating levels of neuregulin-1 (NRG1) with age in rats.

[0077] Neuregulin-1 (NRG1, in particular the NRG1 beta EGF domain) levels were measured in serum from rats aged 8 months, 18 months or 24 months using slow off-rate DNA aptamer detection (Gold, L. et al. (2010) PLoS ONE 5: e15004). 10 animals per group were analysed.

[0078] Effect of neuregulin-1 (NRG1) on neuromuscular junctions in vitro.

[0079] Nerve and muscle co-cultures were grown in vitro until neuromuscular junctions were mature. Damage was induced using beta-amyloid (Ab) incubation (2.5 µM) and riluzole (5 µM) was used as a positive control for preserving neuromuscular junctions from Ab-induced damage. The effect of neuregulin-1 (NRG1; various concentrations, as specified in Figure 2) on Ab-induced damage was evaluated by measuring the neuromuscular junction (NMJ) size. 6 wells per group were analysed and compared to a control condition. The uninjured control condition is the neuromuscular junction size without Ab treatment. All other conditions are to be compared to the Ab treatment alone which represents the reference value for damaged neuromuscular junction size.

[0080] Effect of neuregulin-1 (NRG1) on age-induced atrophy of skeletal muscle.

[0081] Pre-sarcopenic rats aged 16 months were treated for 5 months with either neuregulin-1 (NRG1; NRG1 beta EGF domain from Reprokine (catalog number RKQ02297)) or saline. NRG1 was injected sub-cutaneously at 1 µg / kg body weight 3 time per week. Hind-limb skeletal muscle mass was then evaluated and compared to a group of adult healthy rats (8 months at start of experiment) injected with saline control. Hind-limb skeletal muscle mass was then evaluated and compared to an adult healthy group of rats.

[0082] Effect of vitamin B12 on neuromuscular junctions in vitro.

[0083] Nerve and muscle co-cultures were grown in vitro until neuromuscular junctions were mature. Damage was induced using beta-amyloid (Ab) incubation (2.5 µM) and riluzole (5 µM) was used as a positive control for preserving neuromuscular junctions from Ab-induced damage. The effect of methylcobalamin (MeCbl; 1 or 100 nM) and adenosylcobalamin (AdenoCbl; 1 or 100 nM) on Ab-induced damage was evaluated by measuring the neuromuscular junction (NMJ) number and size, and the neurite network. 6 wells per group were analysed and compared to a control condition (CTL). The uninjured control condition is the neuromuscular junction size without Ab treatment. All other conditions are to be compared to the Ab treatment alone which represents the reference value for damaged neuromuscular junction size.

[0084] Effect of vitamin B12 on skeletal muscle in vivo.

[0085] Pre-sarcopenic rats aged 16 months were treated for 5 months with either adenosylcobalamin (AdoCbl, C0884, Sigma aldrich), methylcobalamin (MeCbl, M9756, Sigma aldrich) or saline. AdoCbl and MeCbl were injected sub-cutaneously at 1mg / kg body weight 3 time per week. Tibialis Anterior (TA) was then dissected out and frozen for further analyses. For histology, TA was cryo-sectioned at 10µm and stained with laminin (L9393, Sigma aldrich) to delineate fibers. Specific fiber types 1, 2A and 2B were immunolabeled subsequently with appropriate antibodies (clones BAD5, BFF3, and SC71 respectively, DSHB). Images were acquired using a slide scanner (VS-120, Olympus) and analyzed using an in-house MetaXpress journal (Molecular Devices, Sunnyvale, USA). Images from old animals were compared to a group of adult healthy rats (8 months at start of experiment) injected with saline control. For gene expression analysis, total RNA was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instruction and RNA quality was checked using the Standard Sensitivity RNA Analysis Kit on a Fragment Analyzer (Advanced Analytical Technologies). Samples were then hybridized on Affymetrix Rat 230 PM 96-Array following standard Affymetrix protocol, based on the Eberwine T7 procedure. Statistical analysis was performed using LIMMA and exploited in GSEA to compare old control animals versus adult animals, and old animals treated with either AdoCbl or MeCbl versus old control animals.Effect of vitamin B12 on protection from muscle atrophy

[0086] HSMM human myoblasts were grown in vitro and induced to differentiate into mature myotubes. Myotube atrophy was induced by incubation with TNFa at 40ng / ml for 4 days. MeCbl (1nM) and AdoCbl (1nM) were incubated together with TNFa to test their effect on induced atrophy. IGF1 is used as a positive control at 15nM to prevent muscle atrophy. After 4 days of treatment, cells were stained for nuclei and myosin heavy chain to quantify the proportion of nuclei inside myotubes (i.e fusion index).

[0087] Effect of neuregulin-1 (NRG1) and vitamin B12 on neuromuscular junctions in vitro.

[0088] Nerve and muscle co-cultures were grown in vitro until neuromuscular junctions were mature. Damage was induced using beta-amyloid (Ab) incubation (10 µM) and riluzole (5 µM) was used as a positive control for preserving neuromuscular junctions from Ab-induced damage. The effect of neuregulin-1 (NRG1; 30 nM) and / or adenosylcobalamin (AdoCbl; 1 nM) on Ab-induced damage was evaluated by measuring the neuromuscular junction (NMJ) size or number. 6 wells per group were analysed and compared to a control condition (CTL) without Ab-induced damage.Results

[0089] Using an aptamer-based screen (Somalogic) on serum from rats aged 8 months, 18 months or 24 months, we found that neuregulin-1 (NRG1) circulating levels decrease with age (Figure 1). This decrease is concomitant with the progression of sarcopenia.

[0090] Considering that neuregulin-1 plays major roles both at the nerve and the neuromuscular junction levels, we then sought to investigate whether neuregulin-1 could protect the neuromuscular junction from the damage that occurs during ageing. For this purpose, we used an in vitro co-culture model that allows the formation and maintenance of neuromuscular junctions, and we induced damage with b-amyloid incubation.

[0091] As shown in Figure 2, neuregulin-1 was able to protect the neuromuscular system from damage at a dose of 30 nM. Collectively these results suggest that neuregulin-1 is beneficial for the maintenance of the neuromuscular system, and that the loss of this protein with age may be linked with the progression of sarcopenia.

[0092] To test whether a neuregulin-1 treatment could rescue the sarcopenia phenotype, we treated pre-sarcopenic rats for 5 months with neuregulin-1 and evaluated their skeletal muscle mass as compared with adult rats and pre-sarcopenic rats treated with saline as control.

[0093] As shown in Figure 3, we observed that hindlimb muscles from rats treated with neuregulin-1 have a significantly higher mass compared to controls, demonstrating that neuregulin-1 has prevented the age-induced skeletal muscle atrophy.

[0094] Taken together, our results suggest that neuregulin-1 protects the neuromuscular system from age-related dysfunction and could therefore be used to prevent sarcopenia.

[0095] Given the previously reported actions of vitamin B12 on neurons and the nervous system, we also tested the effect of the 2 active forms of vitamin B12 (methylcobalamin and adenosylcobalamin). Adenosylcobalamin, but not methylcobalamin, was also able to protect the system at both doses tested (Figure 4).

[0096] Interestingly when the system was subjected to a stronger damage (longer incubation of beta-amyloid at a higher concentration), neither neuregulin-1 nor adenosylcobalamin alone could rescue the system but a co-treatment provided a mild protection suggesting that the two may have synergic effects (Figure 5).

[0097] As shown in Figure 6, adenosylcobalamin, but not methylcobalamin, was also able to induce an increase in muscle fiber size in aged rats.

[0098] In contrast, methylcobalamin was able to protect from muscle atrophy (Figure 7).

[0099] This differential effect may be caused by the fact that adenosylcobalamin and methylcobalamin rescue different age-related gene expression signatures in skeletal muscle (Figure 8).

Claims

1. A non-therapeutic use of vitamin B12 for maintaining or increasing muscle function and muscle mass in an ageing subject over 30 years old, wherein the vitamin B12 is adenosylcobalamin and wherein muscle fiber size is increased.