The present disclosure relates to the use of therapeutic agents, particularly to the use of a combination of an activator of neuronal nitric oxide synthase, and a nitric oxide precursor, which is convertible by said nitric oxide synthase to nitric oxide, for use in a method for preventing or treating muscular dystrophy. The pharmaceutical preparations and methods of the invention are useful for treating muscle diseases and conditions characterized by impaired nitric oxide (NO) signalling in muscle tissue.

Numerous diseases and conditions in which an altered NO metabolism is present, primarily and secondarily affect the muscles. Neuromuscular disorders (NMD) are a broad and heterogeneous group of diseases of the peripheral nerve system. NMD include hereditary and acquired disorders of the muscles, the neuromuscular junction, the peripheral nerves, and the second motor neurons within the spinal cord. Muscle diseases, also called myopathies, are primary disorders of skeletal muscle. Hereditary myopathies, which themself represent a large number of different disease entities, include muscle channelopathies, metabolic myopathies, congenital myopathies, and muscular dystrophies.

Muscular dystrophy refers to a group of hereditary, progressive, degenerative disorders characterized by progressive degeneration of muscle fibres and muscle tissue over time finally leading to progressive muscle weakness and premature death. Classical histological findings of muscular dystrophies consist of pathological fibre size variation, muscle cell degeneration (necrosis) and regeneration, and replacement of muscle by connective and adipose tissue.

Muscular dystrophies include congenital muscular dystrophies (CMD), dystrophinopathies such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), limb girdle muscular dystrophies (LGMD), distal myopathies, type I and type II myotonic dystrophies (DM 1 , DM2), facio-scapulo-peroneal muscular dystrophy (FSHD), autosomal and X-linked Emery-Dreifuss muscular dystrophy (EDMD), and oculopharyngeal muscular dystrophy (OPMD). Acquired myopathies include inflammatory myopathies, toxic and drug- induced myopathies, and muscle neoplasm. Disorders of the neuromuscular junction include hereditary (congenital myasthenic syndromes) and acquired immune-positive or antibody negative autoimmune myasthenia gravis.

Neurogenic secondary muscle dysfunction and altered NO metabolism is observed in many diseases, such as motor neuron diseases, spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), but also in peripheral nerve disorders. Examples of conditions that secondarily affect muscle include muscle atrophy and muscle wasting disorders, including sarcopenia, cachexia, ICU- and surgery-induced weakness, inducible nitric oxide synthase (iNOS) in muscle wasting syndrome, sarcopenia, and cachexia. Sarcopenia is the

degenerative loss of skeletal muscle mass and function associated with aging. Cachexia is a multifactorial syndrome resulting in a reduced life expectancy and accompanies many chronic disorders such as cancer, AIDS, sepsis, immune disorders, chronic heart failure, and chronic obstructive pulmonary disease.

Among primary muscle diseases, Duchenne muscular dystrophy (DMD) is the most common inherited muscular dystrophy, affecting almost one in 3500-6000 males, and is caused by mutations in the dystrophin gene. DMD is characterised by progressive muscular weakness affecting proximal muscles more than distal muscles. Onset is usually at five years of age. People with DMD become wheelchair dependent by the time they are 13 years old. All patients are affected by cardiomyopathy by the age of 18. Few survive beyond the third decade; most patients die because of respiratory complications and heart failure due to cardiomyopathy. Becker muscular dystrophy (BMD) is characterised by a later onset and a generally milder clinical course. About one in 20,000 boys is affected by BMD. There are a variety of animal models for DMD, including the mdx mouse, which has served as a standard animal model and which has been used extensively over the years in research.

Deficiency of dystrophin in DMD leads to increased membrane permeability and calcium (Ca ²⁺ ) entry. This results in Ca ²⁺ overload in mitochondria directly responsible for increased reactive oxygen species (ROS) generation, which in turn further contributes to membrane damage through peroxidation of sarcolemmal lipids. ROS generation stimulates further Ca ²⁺ entry into the muscle fiber, further contributing to mitochondrial dysfunction. Downstream of oxidative stress, ROS also induce activation of NF-κΒ. A key feature of chronic muscular dystrophies and other neuromuscular disorders is NF-κΒ activation. NF-kB blockade reduces skeletal muscle degeneration and enhances muscle function in mdx mice. NF-κΒ induces muscle wasting by stimulating the expression of iNOS, leading to increased nitric oxide (NO) concentrations. Nitric oxide synthases (NOS) are a family of enzymes converting L-arginine to L-citrulline, releasing NO in the process. There are three known NOS isoforms, two are constitutive (neuronal nNOS, endothelial eNOS) isoenzymes, while the third is inducible NOS (iNOS) and involved in inflammatory processes.

iNOS is upregulated in both DMD and mdx muscle samples. NO generated by inducible nitric oxide synthase (iNOS) induces muscle atrophy via regulation of several transcription factors. NO can react with superoxide anions (O ^2" ) to form the toxic molecule peroxynitrite (ONOO-), leading to further oxidative stress, and muscle fiber loss. Although the detailed mechanism of how NO-induced stress leads to muscle wasting is yet to be elucidated, the production of NO, and the subsequent formation of peroxynitrite, has been shown to decrease mRNA levels of MyoD - an important transcription factor involved in myogenesis and maintenance of skeletal muscle. On balance, there is strong evidence that stimulation of NO via iNOS contributes to chronic inflammatory changes and muscle atrophy in DMD and other muscular dystrophies. Long term supplementation with L-arginine for 17-months worsened muscle function, increased muscle fibrosis, and increased skeletal deformities in the mdx model suggesting a negative effect on disease progression, which was attributed to iNOS function, suggesting that the NO generated from iNOS is harmful to skeletal muscle (Wehling- Henricks, et al., PLoS One, 2010. 5(5): p. e10763).

nNOS function is commonly impaired in various neuromuscular disorders. A muscle-specific alternatively spliced isoform of nNOS, nNOSp, is the major source of NO in adult skeletal muscle. In muscle, nNOS localizes to the sarcolemma via direct binding to α-1-syntrophin, a member of the dystrophin-glycoprotein complex (DGC). Not surprisingly, several forms of muscular dystrophy with disruption of the DGC, including DMD and several limb girdle muscular dystrophies (LGMD 2C, 2D, and 2E), display loss of nNOS at the sarcolemma. nNOS activity and NO signalling have different roles in muscle as they modulate blood flow, glucose and fat metabolism, fatigue response, and mitochondrial function. At the level of vasculature, NO offers a prominent mechanism in increasing blood flow to exercising skeletal muscle. With regard to energy metabolism in muscle NO downregulates creatine kinase activity in striated muscle thereby negatively influencing the ATP synthesis and limiting skeletal muscle contractility. Importantly, NO is also capable of influencing oxidative energy metabolism via modulating the mitochondrial respiratory chain complex IV (cytochrome c oxidase / COX). NO competes with molecular oxygen towards binding to the active site of COX, thereby inhibiting its activity. Thus NO reduces oxygen consumption in isolated mitochondria to various extents. In intact cells, physiological levels of NO acutely stimulate uptake and oxidation of glucose and fatty acids by skeletal muscle, while inhibiting the synthesis of glucose, glycogen, and fat. Chronic effects of physiological NO elevation in vivo include stimulation of angiogenesis and mitochondrial biogenesis by increasing the concentration SIRT1 and PGC-1 a. Genetic enhancement of nNOS expression attenuates skeletal muscle inflammation and necrosis, improves exercise performance, and reduces cardiac fibrosis and contractile dysfunction in the mdx model.

A potential strategy to compensate the impaired nNOS function in DMD consists in supplementation of NO precursors. In the mdx model, a combination of ibuprofen and ISDN (isosorbide dinitrate, a NO precursor) reduced muscle necrotic damage and inflammation and improved free voluntary movement and resistance to exercise. The effects of ISDN and ibuprofen administered separately were transient and significantly lower than those induced by their combination. Furthermore, treatment of mdx mice with a NO donating NSAID (nonsteroidal anti-inflammatory drug) also led to muscle function improvement. However, no significant improvement was reported in human DMD patients using a combined approach supplementing a NO donating NSAID (D'Angelo et al., Pharmacol Res, 2012. 65(4): p. 472- 9).

A major pathway of nNOS-derived NO is to stimulate cGMP production. Thus, impaired nNOS function may be partially restored with phosphodiesterase 5 (PDE5) inhibitors such as sildenafil. Downstream targets of cGMP include protein kinase G, cyclic nucleotide-gated ion channels, and cGMP-activated PDEs. PDE5 degrades cytosolic cGMP, thereby attenuating NO signaling intensity. Thus, inhibiting PDE5 can raise cytosolic cGMP levels and indirectly amplify NO signaling. PDE5 inhibition in the mdx mice model showed improvement of force in some but not all muscles, reduced fibrosis suggesting positive effects on long term progression but no change of fatigue resistance (Percival et al., J Pathol, 2012. 228(1 ): p. 77- 87).

NO is synthesized from L-arginine by nNOS, indicating that L-arginine supplementation could ameliorate DMD. Supplementation of NO-precursing substances such as L-arginine has been investigated in animals. Some investigators found improvement of muscle function in mdx mice. Unfortunately, long term dietary supplementation with L-arginine for 17-months worsened muscle function, increased muscle fibrosis and increased skeletal deformities in the mdx model, suggesting that the application of NO-precursors such as L-arginine or L- citrulline could be harmful to human DMD patients.

Additionally, studies in humans did not show a positive effect of arginine on muscle protein synthesis or muscle function: arginine supplementation neither influences muscle protein synthesis (Tang et al., J. Nutr. 201 1 , 141 , 195-200) nor muscle strengths nor muscle function (Alvares et al., Appl. Physiol. Nutr. Metab. 2012, 37, 1 15-126).

Furthermore, nNOS is involved in the mechanism of denervation-induced atrophy. nNOS/NO mediates muscle atrophy via regulation of Foxo transcription factors suggesting that nNOS stimulation could be harmful to humans (Suzuki et al., J Clin Invest. 2007 Sep; 1 17(9):2468- 76.).

The objective of the present invention is to provide means and methods for preventing or treating neuromuscular disorders such as muscular dystrophies, particularly Duchenne muscular dystrophy or Becker muscular dystrophy. This objective is attained by the subject matter of the independent claims 1 , 13, 14, 15 and 16. Surprisingly it was found that, contrary to the above referenced investigations, muscular dystrophies can be treated by combining stimulation of nNOS and supplementation of nitric oxide precursors such as arginine or citrulline.

According to one aspect of the invention a combined pharmaceutical preparation or combination product, for use in a method for preventing or treating muscular dystrophy is provided. This preparation comprises an activator of neuronal nitric oxide synthase (CAS Nr 125978-95-2; UniProt P29475) and a nitric oxide precursor, and the nitric oxide precursor is convertible by the nitric oxide synthase to nitric oxide.

The term combined pharmaceutical preparation refers to a pharmaceutical combination product that comprises an activator of neuronal nitric oxide synthase and a nitric oxide precursor in pharmaceutical purity, either within one and the same administration or dosage form, or supplied as two distinct dosage forms, one comprising the activator of neuronal nitric oxide synthase and the other dosage form comprising the nitric oxide precursor.

In certain embodiments, the muscular dystrophy is a congenital muscular dystrophy (CMD), a dystrophinopathy such as Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD), a limb girdle muscular dystrophy (LGMD), a distal myopathy, type I and type II myotonic dystrophy (DM1 , DM2), facio-scapulo-peroneal muscular dystrophy (FSHD), autosomal and X-linked Emery-Dreifuss muscular dystrophy (EDMD), or a oculopharyngeal muscular dystrophy (OPMD).

In certain embodiments the combined pharmaceutical preparation of the invention is administered alone, in which case the active ingredients of the preparation are administered staggered (at an interval) or jointly in combination. In certain embodiments the combined pharmaceutical preparation of the invention is administered in combination with one or more other therapeutic agents. Possible combination therapies can take the form of fixed combinations of the combined pharmaceutical preparation of the invention with one or more other therapeutic agents known to be active in the prevention or treatment of muscular dystrophy. The administration can be staggered or the combined agents can be given independently of one another or in the form of a fixed combination.

In certain embodiments, the activator of the neuronal nitric oxide synthase is selected from an activator of AMPK (AMP-activated protein kinase, CAS Nr 172522-01-9), 5,6,7,8- tetrahydrobiopterin (BH4, CAS Nr 62989-33-7 and 69056-38-8), FAD (CAS Nr 146-14-5), FMP (CAS Nr 146-17-8), riboflavin (CAS Nr 83-88-5) and NADPH (CAS Nr 53-59-3).

In certain embodiments, the nitric oxide precursor is L-arginine and/or L-citrulline.

In certain embodiments, the activator of AMPK is selected from: /V,/V-dimethylimidodicarbonimidic diamide (metformin, CAS Nr 657-24-9 or 1 1 15-70- 4);

^./V^dimethyl-S-cyclohexyl-Z^-thiohydroxybiguanidie,

^^^dimethyl-S-phenyl-^-thiohydroxylbiguandine:

tert-butyl 4-[(3-( V,A/-dimethylcabamimidoyl)guanidine)methyl]phenyl carbamate:

4-{[3-(/V, \/-dimethylcabamimidoyl)guanidine]methyl}phenyl octanoate:

4-{[3-( V, V-dimethylcabamimidoyl)guanidine]methyl}phenyl diethylcarbamate:

4-[(3-( V, V-dimethylcabamimidoyl)guanidine]methyl]-3-hydroxyphenyl pivalate:

3-[3-( V, V-dimethylcabamimidoyl)guanidine]propyl acetate:

[( V', V'-dimethylguanidino)iminomethyl]carbamic acid benzyl ester:

[( V', V'-dimethylguanidino)iminomethyl]carbamic acid 2,2,2-trichloethyl ester:

N H N H ₂ O

; and

[( V', V'-dimethylcarbamimidoyl)guanidino]-4-phenyl-1 ,3,2-dioxaphosphoramidate:

In certain embodiments, the combined pharmaceutical preparation comprises metformin and a NO precursor selected from L-arginine and L-citrulline in a mass ratio of 1 :5 (metformin: NO precursor), 1 :10 (metformin: NO precursor), 1 :15 (metformin: NO precursor) or 1 :20 (metformin: NO precursor).

In certain embodiments, the combined pharmaceutical preparation according to the invention is provided for use in a method for preventing or treating Duchenne muscular dystrophy or Becker muscular dystrophy. In certain embodiments the activator of neuronal nitric oxide synthase and the nitric oxide precursor are present in the same form of administration.

In certain embodiments the combined pharmaceutical preparation of the invention is formulated for enteral administration, such as nasal, buccal, rectal (suppository), or oral administration, or as a transdermal or inhalation formulation. In certain embodiments, the NO precursor is administered orally and is provided in pulverized form in a sachet. In certain embodiments, the combined pharmaceutical preparation is formulated for parenteral administration, such as intravenous, intrahepatic, subcutaneous or intramuscular injection forms.

In certain embodiments of parenteral administration, use is made of solutions of a combined pharmaceutical preparation. Suspensions or dispersions are also considered. Particularly useful for parenteral administration are isotonic aqueous solutions, dispersions or suspensions which, for example, can be made up shortly before use. In certain embodiments, the combined pharmaceutical preparation is sterilized.

Transdermal/intraperitoneal and intravenous applications are also considered, for example using a transdermal patch, which allows administration over an extended period of time, e.g. from one to forty weeks.

In certain embodiments, the therapy of the invention is applied orally.

In certain embodiments the combined pharmaceutical preparation of the invention comprises 100-3000 mg metformin and 1 -10 g L-arginine, particularly 175-2000 mg metformin and 1 . 5 - 5 g L-arginine, and more particularly 250 mg metformin and 2.5 g L-arginine.

In certain embodiments the combined pharmaceutical preparation of the invention comprises 100-3000 mg metformin and 1 -10 g L-citrulline, particularly 175-2000 mg metformin and 1 . 5 -5 g L-citrulline, and more particularly 250 mg metformin and 2.5 g L-citrulline.

In certain embodiments, the pharmaceutical preparation of the invention comprises 100-3000 mg metformin and 1-10 g L-arginine within the same administration (dosage) form. In certain embodiments, the pharmaceutical preparation of the invention comprises 175-2000 mg metformin and 1. 5 -5 g L-arginine within the same administration (dosage) form.

In certain embodiments, the pharmaceutical preparation of the invention comprises 100-3000 mg metformin and 1-10 g L-citrulline within the same administration (dosage) form. In certain embodiments, the pharmaceutical preparation of the invention comprises 175-2000 mg metformin and 1. 5 -5 g L-citrulline within the same administration (dosage) form. In certain embodiments, the pharmaceutical preparation of the invention comprises approximately 250 mg metformin and approximately 2.5 g L-arginine within the same administration (dosage) form.

In certain embodiments, the pharmaceutical preparation of the invention comprises approximately 250 mg metformin and approximately 2.5 g L-citrulline within the same administration (dosage) form.

In certain embodiments, the pharmaceutical preparation of the invention comprises approximately 500 mg metformin and approximately 5 g L-arginine within the same administration (dosage) form.

In certain embodiments, the pharmaceutical preparation of the invention comprises approximately 500 mg metformin and approximately 5 g L-citrulline within the same administration (dosage) form.

In certain embodiments the combined pharmaceutical preparation of the invention comprises 175-2000 mg metformin and 2. 5 -7.5 g L-arginine and/or L-citrulline, and more specifically 500 mg metformin and 5 g L-arginine and/or L-citrulline.

In certain embodiments, the combined pharmaceutical preparation of the invention comprises 250 mg metformin and 2.5 g L-arginine. In certain embodiments, the combined pharmaceutical preparation of the invention comprises 500 mg metformin and 5 g L-citrulline.

In certain embodiments the combined pharmaceutical preparation of the invention is provided for use in children and comprises 150 mg to 350 mg metformin and 1 -4 g L-arginine and/or L-citrulline, particularly 175-300 mg metformin and 1. 5 -3 g L-arginine and/or L- citrulline, and more particularly 250 mg metformin and 2.5 g L-arginine and/or L-citrulline. In certain embodiments the combined pharmaceutical preparation of the invention is provided for use in adults and comprises 300 mg to 700 mg metformin and 2-8 g L-arginine and/or L- citrulline, particularly 400-600 mg metformin and 3 - 6 g L-arginine and/or L-citrulline, and more particularly 500 mg metformin and 5 g L-arginine and/or L-citrulline.

In certain embodiments the activator of neuronal nitric oxide synthase is present in a first form of administration, and the nitric oxide precursor is present in a second form.

In certain embodiments the combined pharmaceutical preparation of the invention comprises 100-400 mg, particularly 175-325 mg, more specifically 250 mg metformin within the first form, and 1 -4 g, particularly 1.75 -3.25 g, more specifically 2.5 g L-arginine and/or L-citrulline within the second form.

In certain embodiments the combined pharmaceutical preparation of the invention comprises 250-750 mg, particularly 300-600 mg, more specifically 500 mg metformin within the first form, and 2.5-7.5 g, particularly 3 -6 g, more specifically 5 g L-arginine and/or L-citrulline within the second form. In certain embodiments the metformin and the nitric oxide precursor are formulated for oral administration.

In certain embodiments the combined pharmaceutical preparation includes an acceptable pharmaceutical excipient.

The term "excipient" in the context of the present specification particularly refers to a pharmacologically inactive substance that is formulated with the active ingredients.

In certain embodiments, such an excipient is used to bulk up the formulation, to increase the stability of the active pharmaceutical ingredient, to control the release of the active

pharmaceutical ingredient or to improve the manufacturing of the active pharmaceutical ingredient. In certain embodiments the selected excipient is an adherent, a binder, a filler, a disintegrant, a coating, a flavor, a lubricant, a dye or pigment, a sorbent, a gildant, a preservative, a stabilizer, a wetting agent and/or an emulsifier, a solubilizer, a viscosity- increasing agent, a salt for regulating osmotic pressure, a carrier or a buffer substance. Such excipients are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes.

In certain embodiments, the combined pharmaceutical preparation of the invention for oral administration comprises fillers such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, and/or binders such as starches, cellulose derivatives and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, flow conditioners and lubricants, for example stearic acid or salts thereof and/or polyethylene glycol. In certain embodiments, the combined pharmaceutical preparation of the invention for oral administration is comprised within a tablet, where tablet cores in particular can be provided with suitable, optionally enteric, coatings, and dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient. In certain embodiments, the combined pharmaceutical preparation of the invention for oral administration is comprised within hard capsules consisting of gelatin and/or soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. In certain embodiments, the capsules contain the active ingredient in the form of granules or dissolved or suspended in suitable liquid excipients, such as in oils.

Another aspect of the invention is a dosage regimen provided for preventing or treating muscular dystrophy, where the dosage regimen comprises one, two or three dosage forms comprising 250 mg metformin and 2.5 g arginine and/or or citrulline, or one, two or three dosage forms comprising 250 mg metformin and one, two or three dosage forms comprising 2.5 g arginine and/or citrulline every day. Another aspect of the invention relates to a dosing regime for children of less than 12 years of age that comprises two or three daily administrations of the combined pharmaceutical preparation of the invention. In certain embodiments, such dosing regime provides for three daily administrations each comprising 150 mg to 350 mg metformin and 1-4 g L-arginine and/or L-citrulline, particularly 175-300 mg metformin and 1. 5 -3 g L-arginine and/or L- citrulline, and more specifically 250 mg metformin and 2.5 g L-arginine and/or L-citrulline.

In one embodiment, the dosing regime for children provides for three administrations per day of a combined pharmaceutical preparation of 150 mg to 350 mg metformin and 1 -4 g L- citrulline, specifically 250 mg metformin and 2.5 g L-citrulline, in one combined oral administration form (metformin and L-citrullin being part of the same form).

Another aspect of the invention relates to a dosing regime for adults that comprises three daily administrations of the combined pharmaceutical preparation of the invention, each comprising 300 mg to 700 mg metformin and 2-8 g L-arginine and/or L-citrulline. In certain embodiments the dosage regimen for adults provides for three daily administrations of 400- 600 mg metformin and 3 - 6 g L-arginine and/or L-citrulline. In certain embodiments the dosage regimen for adults provides for three daily administrations of 500 mg metformin and 5 g L-arginine and/or L-citrulline.

In one embodiment, the dosing regime for adults provides for three administrations per day of a combined pharmaceutical preparation of 300 mg to 700 mg metformin and 2-8 g L- citrulline, particularly 500 mg metformin and 5 g L-citrulline, in one combined oral administration form (metformin and L-citrullin being part of the same form).

Another aspect of the invention is a form of packaging (kit-of-parts) comprising 7 to 100, particularly 21 , 42, 63 or 84, or 14, 28, 42 or 56 dosing forms (corresponding to one, two, three or four weeks of a thrice-daily or twice daily regime, respectively) providing a combined pharmaceutical preparation in accordance with the invention. In certain embodiments, the packaging provides 3, 6, 9, 12, 15, 18 or 21 dosing forms on one blister, thereby facilitating following a thrice-daily administration regime. In certain embodiments, the packaging provides 2, 4, 6, 8, 10, 12 or 14 dosing forms on one blister, thereby facilitating following a twice-daily administration regime.

In one embodiment, said blister comprises a joint administration form of the combined pharmaceutical preparation comprising 150 mg to 350 mg metformin and 1-4 g L-citrulline, specifically 250 mg metformin and 2.5 g L-citrulline, for use in children, or 300 mg to 700 mg metformin and 2-8 g L-citrulline, specifically 500 mg metformin and 5 g L-citrulline for use in adults. Another aspect of the invention is the provision of a method for the manufacture of a medicament for use in a method for preventing or treating muscular dystrophy, comprising the use of an activator of neuronal nitric oxide synthase and a nitric oxide precursor in combination in accordance with the invention. Medicaments relating to the invention are manufactured by methods known in the art, especially by conventional mixing, coating, granulating, dissolving or lyophilizing.

A further aspect of the invention is the provision of a method for preventing or treating muscular dystrophy, comprising the administration of a combined pharmaceutical preparation in accordance with the invention. According to the invention such treatment may be for prophylactic or therapeutic purposes.

Wherever alternatives for single separable features are laid out herein as "embodiments", it is to be understood that such alternatives may be combined freely with alternatives of other single separable features to form discrete embodiments of the invention disclosed herein.

The invention is further characterized, without limitations, by the following example, from which further features, advantages or embodiments can be derived. The examples do not limit the invention but illustrate it.

Description of the figures

Fig. 1 shows the individual 2-minute walking distance (2MWD, A) and motor function

measure (MFM,B) values at baseline (left point of each set) and after treatment with L-arginine and metformin (squares) and predicted values (triangles) of all DMD patients. The y-axis corresponds to the 2-minute walking distance in metres (A) and total score in percent of the motor function measure (B), with the X-axis showing the age of the patient (in years) at baseline and after treatment.

Examples

The present disclosure relates to combinations of therapeutic agents and preparations to treat a skeletal muscle disease or condition characterized by impaired NO-dependent signaling. Among them there are primary muscle diseases such as Duchenne muscular dystrophy (DMD) which is caused by a loss of dystrophin and which is the most common inherited muscular dystrophy. Becker muscular dystrophy (BMD) is the milder variant of dystrophinopathy due to partial dystrophin expression. Embodiments also include the use of the preparation of the invention for therapy or prevention of other muscular dystrophies, such as congenital muscular dystrophies, limb girdle muscular dystrophies, distal myopathies, type I and type II myotonic dystrophies, facio-scapulo-peroneal muscular dystrophy, autosomal and X-linked Emery-Dreifuss muscular dystrophy, and oculopharyngeal muscular dystrophy. The disease or condition may be treated by administering a combination using a direct or indirect precursor of nitric oxide, such as L-arginine or L-citrulline co-administered with another therapeutic agent that activates the neuronal nitric oxide synthase (nNOS).

Secondary neurogenic muscle dysfunction with impaired NO function as observed in spinal muscular atrophy and amyotrophic lateral sclerosis and finally muscle atrophy and muscle wasting conditions, such as sarcopenia and cachexia, may also be treated by this combination.

Exemplary direct nNOS activators are NADPH, flavin adenine dinucleotide, flavin

mononucleotide, and tetrahydrobiopterin (BH4), which are cofactors of the nNOS. An indirect AMPK stimulator, and thereby indirect nNOS activator, is metformin. Metformin is known to elevate AMPK intracellular concentrations. Metformin indirectly stimulates AMPK activation by perturbing energy homeostasis. Specifically, it disrupts the oxidative phosphorylation through inhibition of complex I of the electron transport chain, thereby reducing the ATP resynthesis, thereby removing inhibition to AMPK activation. Thus, metformin can be used to stimulate AMPK, and thereby nNOS, indirectly.

A combined approach supplementing Duchenne muscular dystrophy patients with L-arginine (NO precursor) and metformin (indirect nNOS activator) for 16 weeks was used. Surprisingly this treatment seems to be superior compared to all other above-mentioned strategies used in DMD patients and in the mdx mice model for DMD, and also superior to the standard treatment of care in DMD (steroids). Important outcomes in muscular dystrophies and conditions with impaired muscle function are muscle force and function, muscle fatigue resistance, and reducing long term progression (slowing of the progressive replacement of muscle by fat and fibrosis).

No significant improvement has been reported for supplementation a NO releasing antiinflammatory NSAID in human DMD patients. This approach using NO precursors had been applied to the mdx model with very promising results. In contrast, when assessing muscle force and function using the motor function measure (MFM) scale, patients treated with L- arginine and metformin treated surprisingly showed an important MFM improvement. The mean total MFM (+3.5%) and the mean D1 MFM (standing and transfers) subscore (+6%) improved unexpectedly in the L-arginine and metformin treated patients more than the reported MFM and MFM D1 improvement (both improving less than 2%) after beginning the standard (steroid) treatment of care.

In the mdx mice model, supplementation of NO precursing substances such as L-arginine and PDE5 inhibition has been investigated. An improvement of muscle function was observed when supplementing L-arginine and deflazacort. Unfortunately, long term supplementation with L-arginine for 17-months worsened muscle function, increased muscle fibrosis, and increased skeletal deformities in the mdx model suggesting a negative effect on disease progression.

Quantitative muscle magnetic resonance imaging (MRI) showed that DMD patients treated with metformin and L-arginine had a muscle fat content (MFC) increase of quadriceps muscles of only 1.4% corresponding to an annual increase of 4.4%, while we observed that age-matched ambulant DMD patients older than 7 years (group 2 in table 2) receiving standard steroid treatment showed a median annual MFC increase of 10,2%.

This indicates that - in contrast to the observed L-arginine effect in the mdx model - L- arginine and metformin slow the disease progression in human Duchenne patients. As mentioned before, PDE5 inhibition in the mdx model showed improvement of force in some but not all muscles and a reduced fibrosis, suggesting positive effects on long term progression, but no change of fatigue resistance. Surprisingly, the approach of the present invention seems also to be superior to PDE5 inhibition. In addition to the observed MFM and quantitative muscle MRI data suggesting improvement of force and slowing of the disease progression, the mean two-minute walking distance of DMD patients treated with L-arginine and metformin also increased (+10 meters), suggesting an improved fatigue resistance as well, an effect not observed with PDE5 inhibition.

Example 1 : Pilot trial with L-arginine and metformin in five ambulant DMD patients A small pilot study was performed to examine if a 16-week treatment with 3 x 2.5 g/d of L- arginine (NO precursor) and 2 x 250 mg/d metformin (pharmacological AMPK activator) could serve as treatment for DMD. A total of five ambulant DMD patients aged between 7 and 10 years were enrolled and treated in the pilot study. No serious side effects of the study medication were observed. None of the patients dropped out. Laboratory testing revealed no significant changes in creatine kinase levels, markers of renal and liver function, glucose levels, and cholesterol levels (HDL / LDL) comparing baseline vs. post-treatment test results. DEXA scans revealed that the average whole body muscle content (70.6% baseline vs. 70.3% post-treatment) and fat content did not change significantly.

Indirect calorimetry assessing energy and muscle metabolism in vivo showed important changes: the relative carbohydrate contribution to the oxidation rate decreased in all patients (mean change -17.9%), while the relative fatty acid contribution to the oxidation rate increased (mean change 13.9%). Quantitative muscle MRI showed that metformin and L- arginine treated patients had an MFC increase of 1 .4% corresponding to an annual increase of 4.4%. The motor function measure is a validated assessment tool to measure motor function in both ambulant and non-ambulant patients with neuromuscular disorders. It includes items to evaluate three dimensions of motor performance, including specific motor functions, such as transfers and standing posture (D1 subscore). In a recent study on DMD the annual decrease of the total MFM score was 5.8%. In ambulant patients with DMD, D1 was the most informative dimension, with a mean decrease of 17.2% per year before loss of ambulation in patients aged 6 years and over. In contrast, four of the five treated patients showed a marked improvement of their clinical and functional abilities. Surprisingly, the mean total MFM

(+3.5%) and the MFM 1 subscore improved by more than +6% in the L-arginine and metformin treated DMD group (Fig. 1 B). Also, the mean two-minute walking distance improved by 9.6 meters indicating improved fatigue resistance (Fig. 1A). Relevant changes observed in the trial are shown in Table 1.

Category Variable mean median SD range 95% CI

Calorimetry REE, kcal/24 h -51.2 -56.5 37 -84 to -8 -54 to -48.5

Calorimetry carbohydrate oxidation, % -17.9 -1 1 .3 18.4 -44.8 to -4.4 -20.7 to -15.1

Calorimetry fatty acid oxidation, % 13.9 9.6 13.7 2.9 to 33.6 1 1 .1 to 16.7

Walking 2 min. walking distance / meters +9.58 +9.6 29.3 -40.3 to 30.6 6.8 to 12.4

Motor function MFM total score, % +3.54 +7.29 6.93 -8.33 to 8.33 0.969 to 6.1 1

MFM D1 subscore, % +6.15 +7.69 1 1.6 -12.8 to 15.4 3.58 to 8.72

MFM D2 subscore, % + 1 .67 +2.78 4.65 -5.56 to 5.56 -0.903 to 4.24

MFM D3 subscore, % + 1 .91 0 5.43 -4.76 to 9.53 -0.665 to 4.48

Table 1 : Descriptive statistics (mean, median, standard deviation, range, and 95% confidence interval) for selected variables of all five treated patients (change from baseline to post-treatment).

This treatment improves skeletal muscle metabolism and delays muscle degeneration.

Muscle biopsies were obtained from all five treated patients before and after the treatment to assess the effects of the metformin and L-arginine treatment. One biopsy (after treatment) failed to obtain a sufficient amount of muscle tissue, therefore only four out of five patients sample pairs could be analysed. Furthermore, four control muscle biopsies were used from age-matched patients who underwent muscle biopsies for diagnosis of neuromuscular symptoms but ultimately were deemed to be normal by means of combined clinical, serological, electrophysiological, and histological criteria.

Different markers of nitric oxide, oxidative phosphorylation, and reactive oxygen species pathways were assessed. For that purpose, we performed cGMP-, nitrotyrosin-, and carbonylated protein-ELISA, and an OXPHOS multiplex western blot. In total, each analysis was done three times for each muscle biopsy sample.

Mean cGMP concentrations were slightly lower in DMD muscle than in control muscle, while mean nitrotyrosin concentrations were 20 % higher in DMD than in controls. Mean OXPHOS complex II (+1 1 %) and V (-1 1 %) concentrations were comparable to those of controls, while complex III (-29 %) and III (-55%) concentrations were lower than in controls suggesting an impaired mitochondrial protein expression in DMD muscle. Mean carbonylated protein concentrations were four times higher than in controls demonstrating high levels of oxidative stress in DMD muscle. After treatment with L-arginine and metformin the indirect NO concentration markers cGMP and nitrotyrosin increased by 77% and 19% respectively indicating that L-arginine and metformin treatment increased NO generation in DMD muscle. Furthermore, mean OXPHOS complex II (+12%), III (62%) and III (+84%) and V (+28%) concentrations increased after treatment indicating an increased expression of mitochondrial proteins. Finally, mean carbonylated protein concentration were 19% lower after treatment suggesting that L-arginine and metformin decreased oxidative stress in DMD muscle. For more details please refer to Table 5

Example 2: Clinical trial with L-citrulline and metformin in sixteen DMD patients

To examine and compare the effects of the drug combination a second clinical study was performed on adult patients with Becker muscular dystrophy (BMD). L-citrulline was used instead of L-arginine. The hypothesis tested thereby was that L-citrulline might be able to raise the NO production to a greater extent than L-arginine, when given in combination with an activator whit neuronal NO synthase.

Eight BMD patients were treated for six weeks with metformin (3x 500 mg/d) and another eight BMD patients were treated for six weeks with L-citrulline (3x 5g/d). After these first six weeks all sixteen patients were treated with both drugs for additional six weeks.

All three treatments improved the clinical abilities of the patients when using the motor function measure (MFM) total score, the D1 MFM subscore (assessing standing and walking abilities) and the distance walked in six minutes (6MWD) (table 2, 3 and 4). Importantly, the greatest improvements on all three clinical scores were observed after the combination therapy proving that the combination is a more powerful treatment than either single treatment (table 4).

After six week treatment with L-citrulline mean resting plasma concentrations of L-citrulline and its product L-arginine were markedly increased (table 2). Furthermore, the marker of in vivo NO generation (mean urinary cGMP/creatinine ratio) was significantly increased.

Skeletal muscle tissue is the main amino acid and protein storage in humans. Urinary amino acid loss (mean urinary amino acids /creatinine ratio) decreased significantly after L-citrulline treatment arguing for an improved protein balance and skeletal muscle protection. As muscle protein breakdown (MPB, mean urinary 3-methylhistidine/creatinine ratio) did not change, this improved protein balance must be attributed to an increased muscle protein synthesis (MPS). L-citrulline had no relevant effect on oxidative stress levels (mean urinary oxidated DNA / creatinine ratio).

After six week treatment with metformin (table 3) BMD patients had significantly decreased oxidative stress markers (mean urinary oxidated DNA/ creatinine ratio). As the major sources of elevated reactive oxygen species (ROS) are mitochondria, this finding is consistent with an improved mitochondrial function after metformin treatment. In consequence to the impaired mitochondrial function, a low fat utilization and replacement of normal muscle tissue by fatty tissue has been observed in dystrophinopathy. Metformin significantly reduced plasma concentrations of triglyceride, cholesterine, and LDL-cholesterine. Metformin treatment also reduced the muscle fat fraction (MFF) assessed by quantitative thigh muscle magnet resonance imaging (MRI). As the primary pathological process consists in the transformation of muscle tissue into fatty tissue, metformin seems to reverse this process. In accordance, metformin treatment reduced the markers for muscle fiber necrosis (mean serum creatine kinase), muscle protein breakdown (mean urinary 3-methylhistidine/creatinine ratio), and also the urinary amino acid loss (significantly reduced mean urinary amino acids /creatinine ratio).

In conclusion, L-citrulline treatment increases serum L-arginine concentrations and NO generation. Without wishing to be bound by theory, the inventors hypothesize that L-citrulline improves muscle protein balance most likely via stimulation of MPS and improves the clinical muscle function. Metformin reduces oxidative stress most likely via improved mitochondrial function, reduces muscle fiber necrosis, MPB, and amino acid loss. It improves the utilization of triglycerides, reduces the irreversible transformation of muscle tissue into fatty tissue, and the clinical muscle function. While both drugs improved the clinical parameters of muscle function, the combination of both drugs was superior to either treatment. Table 2: treatment with L-citrulline

Table 3: treatment with metformin

Table 4: treatment with L-citrulline and metformin

Table 5

DMD 01 Muscle biopsy analysis

Pathway Variable Controls DMD DMD

Pretreatment Postreatment Change / %

Nitric oxide cGMP umol / mg protein 0,0025 0,0021 0,0036 77%

Nitro tyros in nmol/ mg protein 5,66 7,10 8,48 19%

OXPHOS Complex II Succinat Oxidoreductase 30kDa/Actin 0,71 0,54 0,62 14%

Complex II Succinat Oxidoreductase 30kDa/GAPDH 1 ,30 1 ,69 1 ,86 10%

Complex III Cyt c Oxidoreductase 48kDa/Actin 4,56 2,90 4,38 51 %

Complex III Cyt c Oxidoreductase 48kDa/GAPDH 9,02 6,78 11,67 72%

Com plex IV Cyt c Oxidase 24kDa/Actin 4,06 1,74 3,00 72%

Com plex IV Cyt c Oxidase 24kDa/GAPDH 5,42 2,49 4,86 95%

Com plex V ATP Synthase 55kDa/Actin 4, 19 3,30 3,81 16%

Com plex V ATP Synthase 55kDa/GAPDH 8, 14 7,68 10,86 41 %

ROS Carbonylated proteins nmol/ mg protein 45,3 179, 1 145,3 -19%