Phytoecdysones and their derivatives for use in the treatment of neuromuscular diseases

Abstract

The present invention relates to 20-hydroxyecdysone and derivatives thereof for use in the treatment of neuromuscular diseases such as spinal muscular atrophy or amyotrophic lateral sclerosis, or more particularly in the treatment of certain disorders of motor neurons that cause altered muscle function that occur as part of these neuromuscular diseases.

Description

Technical Field

[0001] The present invention relates to the use of plant exons, and semi-synthetic derivatives of plant exons, for the treatment of neuromuscular diseases, particularly infantile spinal muscular atrophy and amyotrophic lateral sclerosis.

Background Art

[0002] Neuromuscular diseases are characterized by functional changes in the motor unit composed of motor neurons, neuromuscular junctions, and skeletal muscles. Whether the cause of the disease is neurological, as in infantile spinal muscular atrophy or amyotrophic lateral sclerosis, or muscular, all of these diseases cause changes in the patient's motor function, which can range from handicap to early death when important muscles are affected. Care for these neuromuscular diseases is still symptomatic and presents a considerable economic cost in terms of the level of the patient's handicap, the progression of these symptoms, and the human and material needs required by these medical conditions. Research and development of therapies that can alleviate the symptoms related to movement and the patient's dependence have gathered a certain amount of social and economic interest.

[0003] Among these neuromuscular diseases, two of them are described as particularly affecting motor neurons: infantile spinal muscular atrophy (i.e., SMA) that appears in childhood, and amyotrophic lateral sclerosis (or SLA) that appears in adulthood. In these two neurodegenerative diseases with different causes and clinical symptoms, progressive muscle denervation is common, which causes muscular atrophy (Non-Patent Documents 1 and 2).

[0004] Infantile spinal muscular atrophy is the most common cause of gene - derived pediatric death, with an incidence of 1 / 6,000 - 1 / 10,000 of live births (Non - Patent Document 2). Depending on the age of onset and the progression of clinical symptoms, three main severity types have been described, from the most severe type 1 to type 3, where the lifespan can exceed 40 years. Patients with SMA show symmetric damage to skeletal muscle due to atrophy of isolated or grouped muscle fibers into fascicles. Almost all SMA is proximal - muscle - predominant, that is, it affects the trunk and the muscles near the trunk. The motor impairment progresses gradually, spreading to the muscles of the lower limbs in the first stage and subsequently to the muscles of the upper limbs in the second stage, preferentially affecting extensor muscles. Genetic analysis has demonstrated that all forms of SMA, although having great clinical heterogeneity, are caused by mutations in the SMN1 gene, which is located in the chromosomal region 5q13 and is essential for the survival of motor neurons.

[0005] The human genome contains an inverted - centromere copy of this SMN2 gene, which can be found in multiple copies (Non - Patent Document 3), but can only partially compensate for the loss of function of the SMN1 gene. In fact, SMN2 has a five - nucleotide difference from SMN1, one of which is located in exon 7, and by splicing 90% of the mRNA produced by the SMN2 gene, it favors its cleavage. This alternative splicing results in truncated and unstable SMN Δ7It leads to the production of proteins. Therefore, out of the proteins produced by the gene SMN2, only 10% are complete and non-functional (Non-Patent Documents 4 and 5). Thus, a relationship has been shown between the number of copies of the gene SMN2, its expression level, and the severity of the disease. The SMN protein is a small, ubiquitously distributed protein that is present within individual domains of the nucleus, abbreviated as "Gemini of Coiled Bodies" and called "GEMs", and in the cytoplasm of cells. The SMN protein has roles in specific neurons such as axonal growth (Non-Patent Document 6) and axonal transport (Non-Patent Documents 7 and 8), but is also involved in ubiquitous functions such as the biogenesis of ribonucleoproteins in the nucleus (Non-Patent Documents 9 to 11) or the control of mRNA translation (Non-Patent Document 12). Therefore, neither the expression nor the cellular function of the SMN protein can directly explain the specific degeneration of motor neurons.

[0006] However, the development of various animal models and the progress of basic research have demonstrated that even when the Smn gene is conditionally deleted only in neurons, neither the complete symptoms of SMA nor the degeneration of motor neurons are reproduced (Non-Patent Document 13). Other studies have explained the role of SMN protein in the proper functions of astrocytes (Non-Patent Document 14), Schwann cells (Non-Patent Document 15), heart cells (Non-Patent Documents 16 to 19), hepatocytes (Non-Patent Document 20), and blood vessels (Non-Patent Document 21), suggesting the role of energy metabolism in disease progression and severity. Other further studies have focused on the motor unit in SMA and clarified specific changes in the neuromuscular junction, which is an essential element for the survival of motor neurons (Non-Patent Documents 22 to 25). In fact, SMN protein is involved in the proper functioning of the neuromuscular junction by acting on the pool of neurotransmitter vesicles, synaptic activity (Non-Patent Document 26), and the maturation of the junction (Non-Patent Documents 22, 25), which is an important step enabling the acquisition and maintenance of functionality. On the other hand, the invalidation of the Smn gene only in muscle cells results in profound muscle changes such as severe dystrophy (Non-Patent Document 27); insufficient organization of the sarcomere structure that can generate contraction (Non-Patent Document 28); a decrease in the correct fusion of muscle stem cells (Non-Patent Document 29); and a concomitant decrease in the differentiation of muscle stem cells (Non-Patent Document 30) (Non-Patent Documents 27, 31, 32). Furthermore, myoblasts from patients with SMA are deficient in specific factors such as CANP (Calcium-Activated Neutral Protease), which is essential for the establishment of neuromuscular contact (Non-Patent Documents 33, 34). Finally, it has been observed in vitro that motor neurons degenerate more rapidly in co-culture with SMA muscle cells (Non-Patent Document 34).

[0007] Currently, completely different approaches are being considered to develop therapies for SMA. Most current research strategies focus on directly or indirectly altering SMN expression through gene therapy aimed at increasing the expression of the SMN2 gene, or altering the splicing of SMN2 transcripts, or reintroducing the correct gene. Some research has focused on approaches independent of SMN by developing cell therapies or by focusing on the activation of neuroprotection (oleoxim) or the improvement of muscle function (troponin activator).

[0008] Despite numerous clinical trials on the progression of SMN, currently only two molecules have been approved by the authorities.

[0009] The first, troponin activator (CK-2127107), aims to increase the sensitivity of the sarcomere to calcium for the improvement of muscle function durability and performance (Non-Patent Document 35). This molecule was designated as an orphan drug for SMA by the US regulatory authorities in May 2017.

[0010] The second, an anti-sense oligonucleotide (ASO), aims to enable the overexpression of full-length SMN protein in spinal cord cells after intrathecal injection by promoting the inclusion of exon 7 in the transcripts produced by the SMN2 gene (Non-Patent Documents 36 and 37). This molecule, named nusinersen and developed by Biogen, has been approved by the authorities in the United States and Europe and constitutes the first therapeutic molecule marketed for the treatment of severe infantile spinal muscular atrophy types 1 and 2. Despite being during Phase II and III clinical trials for severe SMA patients (Non-Patent Document 38), the initial results obtained with ASOs targeting intron 7 are very impressive and promising for this fatal disease. However, there are still some gray areas regarding the use of in vivo injection of oligonucleotides once and / or over a long period into the central nervous system. First, the scientific community lacks a perspective on the long-term tolerance of patients to this type of exogenous molecule that targets gene expression. And further questions are related to the risks inherent in the uncontrolled overexpression of the SMN protein, and the function of the above protein is related to cell proliferation (Non-Patent Document 39). Furthermore, the effectiveness of nusinersen seems to be increasingly important. This is because the start of treatment is to intervene early regarding clinical situations that are difficult to obtain from the relationship between disease progression, diagnosis time, and the clinical course of the family. Finally, in SMA, it is important to note the importance of the role played by changes in multiple tissues in disease progression. However, nusinersen is a molecule that does not cross the blood-brain barrier and requires treatment via the intrathecal route, which can only target neurons and glial cells without targeting other organs such as muscle, liver, pancreas, and the vascular system. Therefore, these changes may limit the therapeutic effect of nusinersen. Finally, patients with type 3 SMA, in whom the expression level of the SMN protein is extremely high and rarely involved in life prognosis, have not yet received treatment approval, and this severity remains isolated regarding treatment.

[0011] Therefore, it seems important to develop physiological and / or pharmacological approaches that complement nusinersen in order to enhance its protective effect and limit as much as possible the patient's clinical course, the patient's dependency, and the patient's clinical management.

[0012] Phytoecdysones are an important family of polyhydroxylated sterols. These molecules are produced by various types of plants (ferns, gymnosperms, angiosperms) and are involved in the defense of these plants against pests. Most phytoecdysones in the plant kingdom are 20-hydroxyecdysone.

[0013] Patent Document 1 discloses that phytoecdysones, and more particularly 20-hydroxyecdysone (20E), have been the subject of numerous pharmacological studies. These studies have highlighted the antidiabetic and anabolic properties of this molecule. Its stimulating effect on protein synthesis in muscle has been observed in vivo in rats (Non-Patent Documents 40 to 42) and in vitro in mouse myotubes C2C12 (Non-Patent Document 43). Although some of the above effects in animal models have been discovered in clinical studies, the number is still small. For example, 20-hydroxyecdysone promotes an increase in muscle mass in young athletes (Non-Patent Document 44). Finally, Patent Document 1 further describes the use of 20-hydroxyecdysone and 20-hydroxyecdysone derivatives for the treatment and prevention of sarcopenia and sarcopenic obesity (Non-Patent Document 45).

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Non-Patent Documents

[0015]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Non-Patent Document 39

Non-Patent Document 40

Non-Patent Document 41

Non-Patent Document 42

Non-Patent Document 43

Non-Patent Document 44

Non-Patent Document 45

Summary of the Invention

Problems to be Solved by the Invention

[0016] An object of the present invention is to limit the loss of motor neurons associated with neuromuscular diseases and the consequences of this degeneration.

[0017] Phytoecdysones are an important family of plant polyhydroxylated sterols that are structurally related to insect molting hormones. These molecules are produced by a number of plant species and are involved in these defenses against pests. Most phytoecdysones are 20-hydroxyecdysone.

[0018] The present inventors unexpectedly discovered that phytoecdysones and semi-synthetic derivatives of phytoecdysones significantly improve the survival rate and body weight changes of mammals affected by spinal muscular atrophy. Furthermore, phytoecdysones and their semi-synthetic derivatives limit muscular atrophy and hypoplasia present in this pathology and significantly limit the loss of motor neurons in mammals affected by spinal muscular atrophy.

Means for Solving the Problems

[0019] For this purpose, the present invention relates to a composition comprising 20-hydroxyecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone for use in the treatment of specific disorders of motor neurons in mammals suffering from neuromuscular diseases, said disorders comprising changes in muscle function due to said specific disorders of motor neurons.

[0020] In certain embodiments, the present invention further meets the following characteristics, implemented separately or in each technically operable combination.

[0021] 20-Hydroxyecdysone and its derivatives are preferably purified to pharmaceutical grade.

[0022] The 20-hydroxyecdysone used is more preferably in the form of a plant extract rich in 20-hydroxyecdysone or a composition containing 20-hydroxyecdysone as an active agent. Plant extracts rich in 20-hydroxyecdysone are, for example, extracts of Stemmacantha carthamoides (also called Leuzea carthamoides), Cyanotis arachnoidea and Cyanotis vaga.

[0023] The obtained extract is more preferably purified to pharmaceutical grade.

[0024] In certain embodiments, 20-hydroxyecdysone is in the form of a plant extract or a part of a plant, said plant being selected from plants containing at least 0.5% by dry weight of 20-hydroxyecdysone, and said extract contains at least 95%, preferably at least 97% of 20-hydroxyecdysone. The extract is more preferably purified to pharmaceutical grade.

[0025] The above extract is hereinafter referred to as BIO101. Notably, it contains impurities such as trace compounds that may affect the safety, availability, or effectiveness of the above extract for pharmaceutical applications, in an amount of 0 to 0.05% of the dry weight of the above extract.

[0026] According to an embodiment of the present invention, the impurities are compounds containing 19 or 21 carbon atoms, such as rubrosterone, dihydrorubrosterone, or poststerone.

[0027] The above plant from which BIO101 is produced is more preferably selected from Stemmacantha carthamoides (also known as Leuzea carthamoides), Cyanotis arachnoidea, and Cyanotis vaga.

[0028] The above derivative of 20-hydroxyecdysone is obtained by semi-synthesis and can be obtained in particular in the manner described in European Patent Application No. 15732785.9.

[0029] According to a preferred embodiment, the above change in muscle function is due to a change or degeneration of motor neuron function.

[0030] In one embodiment, the changing muscle function is the function of striated muscle or cardiac muscle.

[0031] In one embodiment, the above change in muscle function is related to hypoplasia and / or atrophy.

[0032] In a particular embodiment, the disorder of motor neurons is caused by genetic changes in mammals suffering from neuromuscular diseases.

[0033] The term "genetic alteration" means a mutation such as substitution or insertion of one or more nucleotides, or deletion of one or more nucleotides.

[0034] In certain embodiments, the present invention is directed to a composition for use in the treatment of mammalian infantile spinal muscular atrophy (SMA) or amyotrophic lateral sclerosis (SLA).

[0035] In certain embodiments, the present invention is directed to a composition for use in the treatment of sporadic neuromuscular diseases (associated with random mutations in the causative gene or one or more susceptibility genes), or in the form of a family in which a mutation is found in at least one gene selected from SMN1, which intervenes in the framework of SMA; SOD1; TAR DNA-binding protein 43, encoding TARDBP; VCP (Valosin Containing Protein); FUS / TLS (Fused in sarcoma / translocated in liposarcoma); and C9ORF72 (chromosome 9 open reading frame 72), which is involved in the framework of SLA.

[0036] In certain embodiments, the treatment of specific disorders of motor neurons includes improving the survival rate of motor neurons and / or accelerating the maturation of neuromuscular junctions.

[0037] In certain embodiments, phytoecdysone is administered to humans at a dose of 3-15 mg / kg / day. The term "phytoecdysone" here means common phytoecdysone and its derivatives, 20-hydroxyecdysone and its derivatives (especially in the form of extracts).

[0038] Preferably, phytoecdysone is administered to adult humans in divided doses of 200-1,000 mg / day, and to human children or infants in divided doses of 5-350 mg / day. The term "phytoecdysone" here means common phytoecdysone and its derivatives, 20-hydroxyecdysone and its derivatives (especially in the form of extracts).

[0039] In an embodiment, the above composition contains at least one compound considered to be a derivative of a plant ecdysone, and the above at least one compound has the general formula (I):

[0040]

Chemical formula

[0041] wherein V-U is a carbon-carbon single bond, Y is a hydroxyl group or hydrogen, or V-U is an ethylenic C=C bond; X is oxygen; Q is a carbonyl group; R 1 is: (C 1 -C 6 )W(C 1 -C 6 ) group; (C 1 -C 6 )W(C 1 -C 6 )W(C 1 -C 6 ) group; (C 1 -C 6 )W(C 1 -C 6 )CO 2 (C 1 -C 6 ) group; (C 1 -C 6 )A group (where A represents a heterocyclic ring optionally substituted with a group of the OH, OMe, (C 1 -C 6 ), N(C 1 -C 6 ), CO 2 (C 1 -C 6 ) type); CH 2 Br group; and is selected from; W is a heteroatom selected from N, O, and S, preferably O, and more preferably S.

[0042] In the context of the present invention, "(C 1 -C 6)」 means a linear or branched alkyl group having 1 to 6 carbon atoms, in particular any of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, n-hexyl groups. Advantageously, this is a methyl, ethyl, isopropyl, or t-butyl group, in particular a methyl or ethyl group, more particularly a methyl group.

[0043] In a certain preferred embodiment, in the above formula (I): Y is a hydroxyl group; R 1 is: (C 1 -C 6 )W(C 1 -C 6 ) group; (C 1 -C 6 )W(C 1 -C 6 )W(C 1 -C 6 ) group; (C 1 -C 6 )W(C 1 -C 6 )CO 2 (C 1 -C 6 ) group; (C 1 -C 6 )A group, (where A represents a heterocyclic ring optionally substituted with a group of the type OH, OMe, (C 1 -C 6 ), N(C 1 -C 6 ), CO 2 (C 1 -C 6 )); and is selected from: W is a heteroatom selected from N, O, and S, preferably O, more preferably S.

[0044] In an embodiment, the above composition comprises at least one compound selected from the following compounds: No.1: (2S,3R,5R,10R,13R,14S,17S)‐2,3,14‐Trihydroxy‐10,13‐dimethyl‐17‐(2‐morpholinoacetyl)‐2,3,4,5,9,11,12,15,16,17‐decahydro‐1H‐cyclopenta[a]phenanthren‐6‐one; No.2: (2S,3R,5R,10R,13R,14S,17S)‐2,3,14‐Trihydroxy‐17‐[2‐(3‐hydroxypyrrolidin‐1‐yl)acetyl]‐10,13‐dimethyl‐2,3,4,5,9,11,12,15,16,17‐decahydro‐1H‐cyclopenta[a]phenanthren‐6‐one; No.3: (2S,3R,5R,10R,13R,14S,17S)‐2,3,14‐Trihydroxy‐17‐[2‐(4‐hydroxy‐1‐piperidyl)acetyl]‐10,13‐dimethyl‐2,3,4,5,9,11,12,15,16,17‐decahydro‐1H‐cyclopenta[a]phenanthren‐6‐one; No.4: (2S,3R,5R,10R,13R,14S,17S)‐2,3,14‐Trihydroxy‐17‐[2‐[4‐(2‐hydroxyethyl)‐1‐piperidyl]acetyl]‐10,13‐dimethyl‐2,3,4,5,9,11,12,15,16,17‐decahydro‐1H‐cyclopenta[a]phenanthren‐6‐one; No.5: (2S,3R,5R,10R,13R,14S,17S)‐17‐[2‐(3‐dimethylaminopropyl(methyl)amino)acetyl]‐2,3,14‐trihydroxy‐10,13‐dimethyl‐2,3,4,5,9,11,12,15,16,17‐decahydro‐1H‐cyclopenta[a]phenanthren‐6‐one; No.6: 2‐[2‐Oxo‐2‐[(2S,3R,5R,10R,13R,14S,17S)‐2,3,14‐trihydroxy‐10,13‐dimethyl‐6‐oxo‐2,3,4,5,9,11,12,15,16,17‐decahydro‐1H‐cyclopenta[a]phenanthren‐17‐yl]ethyl]ethylsulfanylacetate; No.7: (2S,3R,5R,10R,13R,14S,17S)-17-(2-Ethylsulfanylacetyl)-2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one; No.8: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(2-hydroxyethylsulfanyl)acetyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H cyclopenta[a]phenanthren-6-one.

[0045] In an embodiment, the composition comprises at least one compound that is considered a derivative of a phytoecdysone, and the at least one compound has the general formula (II):

[0046] [Chemical formula] wherein.

[0047] The compound of formula (II) is hereinafter referred to as BIO103.

[0048] In an embodiment, the composition is incorporated into a pharmaceutically acceptable formulation that is orally administrable.

[0049] In the context of the present invention, the term "pharmaceutically acceptable" generally means safe, non-toxic, and useful for the preparation of pharmaceutical compositions that are acceptable for veterinary and human pharmaceutical use.

[0050] The present invention will be better understood by reading the following description given as a non-limiting example with reference to the drawings. [Brief Description of the Drawings]

[0051]

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DETAILED DESCRIPTION OF THE INVENTION

[0052] Hereinafter, the present invention will be described in a specific context of some of its preferred non-limiting application fields.

[0053] 1. Purification method of BIO101 BIO101 is prepared from 20 - hydroxyecdysone with a purity of 90% according to the following steps: i) A step of thermally dissolving 20 - hydroxyecdysone with a purity of 90% in methanol, filtering, and partially concentrating; ii) A step of adding three times the amount of acetone; iii) A step of cooling with stirring to a temperature of 0 - 5°C; iv) A step of filtering the obtained precipitate; v) A step of successively rinsing with acetone and water; and vi) A step of drying.

[0054] This purification involves a recrystallization process that is suitable for this molecule and can be carried out on an industrial scale.

[0055] The filtration in step i) is carried out using a 0.2 μm particle filter.

[0056] The partial concentration in step i) is preferably carried out by vacuum distillation at a temperature of about 50°C in the presence of MeOH.

[0057] The drying step vi) is carried out under vacuum at a temperature of about 50°C.

[0058] 2. Synthesis method of BIO103 BIO103 is obtained by semi - synthesis from 20 - hydroxyecdysone followed by purification to pharmaceutical grade according to the following preparation method:

[0059]

Chemical formula

[0060] Synthesis diagram of BIO103 in three steps: I) Oxidative cleavage of the side chain of 20-hydroxyecdysone between carbons C20 and C22 to obtain poststerone (a protocol known to those skilled in the art); II) Introduction of a bromine atom at the C21 position; III) Reaction of the bromine compound thus obtained with ethanediol.

[0061] 3. Biological activities of BIO101 and BIO103 a. Phenotypic analysis of the effects of BIO101 A mouse model of severe SMA was used against the FVB / NRj gene pool. This is characterized by the inactivation of exon 7 of the mouse gene Smn and the expression of the human transgene SMN2 (Smn Δ7 / Δ7 ; huSMN2 + / + )(Hsieh et al., 2000). Mice resulting from these matings with the genotype "FVB / NRj-Smn Δ7 / Δ7 huSMN2 + / + 2 copies" are described as "SMA". These mice are characterized by progressive growth retardation, which begins to be observed 4 days after birth, with degeneration of approximately 50% of the motor neurons in the anterior horn of the spinal cord at the end of their lifespan and an average lifespan of approximately 12 days (Hsieh et al., 2000). Mice with the "FVB / NRj-Smn + / Δ7 huSMN2 + / + 2 copies" genotype have no specific phenotype and are used as so-called "control" mice. The above mice were treated by forced oral administration at a dose of 50 mg / kg per day with molecule BIO101 complexed with a vehicle (in this case cyclodextrin), or with the vehicle alone (VH). Body weight and survival rate were analyzed daily until P11. In the following description, n corresponds to the sample size and p corresponds to the "p-value" used to quantify the statistical significance of the results.

[0062] The results demonstrate that in daily oral treatment from the birth of the mice, BIO101 alone (n = 18 (n is the sample size)) can significantly limit the weight loss of animals from day 9 after birth compared to animals treated with the vehicle (n = 22) (p < 0.05) (Figure 1A), and can also significantly reduce the mortality rate of treated animals before P11 (p < 0.05) (Figure 1B).

[0063] b. Analysis of the trophic effect of BIO101 At 1 hour after the last forced oral administration at P11, the treated mice were anesthetized with 1% pentobarbital at 6 μL / g (mouse body weight), and then the soleus muscle, which is a mixed extensor muscle, the plantaris muscle, which is a fast extensor muscle, and the tibialis anterior muscle, which is a fast flexor muscle, were harvested for histological or molecular studies.

[0064] After harvesting, the soleus muscle, plantaris muscle, and tibialis anterior muscle were individually placed in storage medium and frozen in cold isopentane. For each muscle, transverse medial sections with a thickness of 10 μm were prepared. These sections were stained with hematoxylin-eosin, dehydrated, and placed in mounting medium. Images of these sections were taken with a microscope (magnification 200×). The differential interference contrast technique was used to obtain relief-like images. From these images, the number of muscle fibers in each muscle and the cross-sectional area of 20% of these fibers were counted using image processing software (Figure 2).

[0065] Histological analysis of the cross-sections of the harvested muscles stained with hematoxylin-eosin shows the beneficial effect of BIO101 on muscular dystrophy (number of muscle fibers). Indeed, in the tibialis anterior muscle, it was shown that the number of muscle fibers in SMA mice treated with BIO101 was significantly increased compared to the number of fibers in animals treated with the vehicle (the number of fibers was 2,358 and 2,069 (+14%) respectively, p < 0.05).

[0066] Interestingly, the muscular atrophy present in SMA mice is restricted by treatment, regardless of the nature and type of muscle. Indeed, treatment with BIO101 significantly restricts atrophy in three muscles (flexor or extensor muscles) of the study controls.

[0067] Atrophy of muscle fibers in the context of SMA (Figure 3A) compared to healthy control mice (Figure 3B) can actually be observed in histological sections of the tibialis anterior muscle, and quantification of the cross-sectional area of muscle fibers shows that this atrophy is 56.1% in the tibialis anterior muscle of SMA mice (n = 4) compared to control mice (n = 4) (Figure 3D). This atrophy is significantly reduced to 32.7% at P11 by treatment with BIO101 (n = 4) (p < 0.05) (Figures 3C, 3D). This is also true for the soleus muscle (a significant atrophy of 46% of muscle fibers is observed in SMA mice compared to healthy control mice (p < 0.05), and treatment with BIO101 significantly restricts this atrophy to 17.9% (p < 0.05) (Figure 3E)), and the platysma muscle (for SMA mice, 50.2% (p < 0.01) compared to the control, and 37.8% (p < 0.05) by treatment with BIO101) (Figure 3F).

[0068] To perform a more precise analysis of the effect of BIO101 on atrophy, the distribution of muscle fibers was evaluated for each category of cross-sectional area. Muscle fibers have different cross-sectional areas depending on their properties. Type I fibers, which are characterized by slow contraction, have a smaller size than type II fibers, which are characterized by fast contraction. The significant decrease in atrophy by the molecule BIO101 observed by treating SMA mice from P0 to P11 results in a significant effect of this treatment on the distribution of fibers by cross-sectional area in the three muscles studied in SMA mice treated with BIO101 compared to SMA mice treated with vehicle (Figures 4A, 4B, 4C). In the tibialis anterior muscle of SMA mice treated with BIO101 from P0 to P11, a significant increase in the ratio of fibers with a cross-sectional area of 400 - 800 μm 2 was observed compared to the group of SMA vehicle mice, while for fibers with a cross-sectional area of 100 - 200 μm 2The ratio of fibers with a cross-sectional area of 200 - 400 μm significantly decreased (p < 0.05) (Figure 4A). In the soleus and gastrocnemius muscles of SMA mice treated with BIO101 from P0 to P11, the increase in the ratio of fibers with a cross-sectional area of 2 was 0 - 100 μm for the soleus muscle 2 and 100 - 200 μm for the gastrocnemius muscle 2 with a cross-sectional area (Figure 4B and 4C).

[0069] c. Molecular analysis Previous studies have demonstrated that insufficient activation of the AKT / CREB pathway and overactivation of the ERK / Elk-1 pathway are associated with low expression of SMN protein in the anterior horn of the spinal cord of "FVB / NRj-Smn Δ7 / Δ7 huSMN2 + / + 2-copy" transgenic mice, which are used as a mouse model of severe type II spinal muscular atrophy, suggesting the role of these pathways in the disease (Branchu et al., 2013).

[0070] The frozen plantar fascia was homogenized in extraction buffer by mechanical grinding. The supernatant containing the protein extract was collected and subsequently the protein extract was assayed according to the Lowry method. Electrophoresis was performed on an SDS-PAGE gel and then the separated proteins were transferred onto a membrane. The primary antibodies used were as follows: mouse monoclonal anti-SMN (1:5,000), rabbit polyclonal anti-Ser 473 phosphorylated AKT (1:1000), rabbit polyclonal anti-AKT (1 / 100), rabbit monoclonal anti-phosphorylated ERK 1 / 2 (1:500), anti-MAP kinase 1 / 2 (ERK 1 / 2) (1:1000). After rinsing these membranes, they were incubated with anti-mouse (1:5,000) or anti-rabbit (1:5,000) secondary antibodies conjugated to peroxidase. After the use of the primary antibodies, the antibody-light source complex was disrupted by incubation in dissociation solution and then the membranes were incubated again with rabbit anti-AKT and anti-MAP kinase 1 / 2 (ERK 1 / 2) antibodies (1:1000). The antibody complex was visualized by chemiluminescence and imaged by a digital image acquisition device for samples of the gel, membrane, or film. The optical density of each specific band was quantified by image processing software by subtracting the background and normalizing by the optical density of the β-actin band. For the vehicle-treated controls, the values obtained were determined to be 1 and the values of the other groups were normalized against these controls and expressed as relative amounts. Independent experiments were performed and animals from each group were obtained by using different multiple membranes when comparing each group to the control. The quantitative values of pAKT in the plantar fascia represent 3 mice per group treated with vehicle (control or SMA) and 4 mice treated with BIO101. The quantitative values of pERK in the plantar fascia represent at least 4 mice per group. The quantitative value of SMN represents n = 2. For the quantitative values of pAKT in the spinal cord, these represent n = 2 mice per group for pAKT and pERK and n = 4 per group for the level of SMN.

[0071] Molecular analysis of the plantar fascia confirmed that, compared to vehicle control mice (CTL VH), the AKT pathway was actually underactivated in SMA mice (Figs. 5A, 5B), while it was beneficial for the ERK pathway (Figs. 5C, 5D) (Branchu et al., 2013). Treatment with BIO101 for 11 days can reverse this relationship by significantly decreasing the level of phosphorylation of ERK (pERK) (Fig. 5D) and extremely greatly increasing the phosphorylation of AKT (pAKT) (Fig. 5B) in the muscles of mice treated from P0 to P11. This beneficial situation has already been demonstrated to be involved in the overexpression of the SMN protein (Branchu et al., 2013). As expected, the SMN protein is hardly expressed in SMA animals compared to healthy control animals. However, very surprisingly, no change in the level of SMN expression was observed after treatment with BIO101, suggesting the original molecular regulation (Figs. 5E, 5F).

[0072] Such a balance in the signal transduction pathway between ERK and AKT is also seen in the spinal cord of SMA mice. In fact, compared to healthy control mice (CTL VH), an increase in the phosphorylation of ERK (pERK) (Figs. 6C, 6D) was observed, along with an obvious decrease in the phosphorylation of AKT (pAKT) in SMA mice (Figs. 6A, 6B). Daily treatment of SMA mice with BIO101 for 11 days partially restores the above balance in the spinal cord of the animals by increasing the level of pAKT and decreasing the level of pERK (Figs. 6B, 6D). As expected for this model, the SMN protein is not expressed in SMA mice. As observed in the plantar fascia, treatment with BIO101 cannot restore the level of SMN in SMA mice treated for 11 days (Figs. 6E, 6F).

[0073] d. Analysis of motor neurons A quantitative and qualitative study of the population of motor neurons in thick sections of the lumbar region (L1-L5) of the spinal cord of healthy control mice or SMA mice treated with vehicle or BIO101 for 11 days was performed by immunofluorescent labeling using choline acetyltransferase (ChAT) as described above (Biondi et al., 2008; Boyer et al., 2013). Next, the analysis was refined by studying various motor neuron subpopulations by characterizing their position within the spinal cord (outer or inner position) and the distribution of the size of their cell bodies.

[0074] PBS was injected intracardially into anesthetized mice. The spinal cord of the mice was removed, fixed, and rinsed. The lumbar region (L1-L5) of the spinal cord was coated with a 4% agarose solution. 50-μm sections were prepared using a vibratome over the entire length of the sample. Next, one out of every five sections of the spinal cord was used for immunohistochemical analysis. After saturation with 0.1 M glycine, the tissue was permeabilized, blocked, and marked with an anti-choline acetyltransferase (ChAT) (1 / 400 e ) goat polyclonal primary antibody. The sections were then washed and incubated with an anti-goat polyclonal secondary antibody conjugated to cyanine 3 (1:400 e ). After marking the nuclei with bisbenzimide (1 / 1,000 e ), the sections were washed again and then treated with a photobleaching inhibitor of the fluorescent dye. The specificity of the marking was verified by control marking performed in the absence of the primary antibody.

[0075] Images were obtained using a camera that was installed on a microscope with a magnification of 200× and connected to a microcomputer-type central processing unit equipped with particularly suitable software for image acquisition. All counting was performed using image processing software.

[0076] In the immunofluorescence method, motor neurons marked with anti-ChAT antibody can be identified as a faint gray in the anterior horn of the spinal cord in the drawings (Figs. 7A, 7B, 7C). The number of motor neurons per section through the ventral half of the spinal cord was determined for each of three groups of mice. As expected, the number of motor neurons in SMA mice (n = 5) was significantly reduced compared to the number of motor neurons seen in the group of healthy control mice (n = 5) (Fig. 7A) (Fig. 7B). Indeed, this motor neuron degeneration was 25% between these two groups (p < 0.05) (Fig. 7D). Most interestingly, after daily treatment with BIO101 for 11 days, a significantly higher number of motor neurons (p < 0.05) was observed in these SMA mice compared to vehicle-treated SMA mice (n = 5) (Fig. 7C). Thus, it is observed that treatment with BIO101 significantly limits pathological motor neuron degeneration and that this treatment exerts a significant neuroprotective effect, with motor neuron loss limited to 13% in the group treated with molecule BIO101 (Fig. 7D).

[0077] Next, the above quantitative analysis was refined by studying various motor neuron subpopulations by analysis of their position within the spinal cord (outer or inner position) and by study of the distribution of the area of their cell bodies.

[0078] It is observed that the number of outer motor neurons (innervating distal muscles) is higher in SMA mice treated with BIO101 compared to vehicle-administered SMA mice, while there is no significant effect on inner motor neurons (Fig. 7E).

[0079] Analysis of the area of the cell bodies of motor neurons showed, as expected, atrophy of the motor neurons of vehicle-treated SMA mice when compared to healthy control mice, where the number of motor neurons with a cell body area of less than 600 μm 2 was significantly increased (p < 0.05). In parallel, in SMA mice, the number of motor neurons with a cell body area of 900 μm 2Motor neuron loss was observed to be significant (p < 0.05) (Figure 7F). Treatment with BIO101 can limit such atrophy of motor neurons by significantly restricting the number of small motor neurons with a cell body area of less than 300 μm 2 2 (p < 0.05) (Figure 7F). It is important to note that in the context of SLA, it has been explained that multiple motor units are not uniformly affected by the pathological process. In fact, a pre-onset mouse model of this disease has revealed preferential degeneration of FF (Fast Fatiguable) type motor units involving motor neurons with large cell bodies (Pun et al., 2006). Similar differential degeneration has also been reported in patients (Dengler et al., 1990; Theys, Peeters and Robberecht, 1999). Therefore, preferentially targeting motor neurons with large cell bodies and reducing the proportion of motor neurons with small cell bodies may be an interesting approach in the context of pathologies such as SLA.

[0080] To complement the quantitative analysis of the number and cell body size of motor neurons, and the qualitative study regarding their position (medial or lateral within the ventral spinal cord), the inventors studied the effect of treatment with BIO101 on the protection of subpopulations of motor neurons (slow motor neurons, intermediate motor neurons, and fast motor neurons). This qualitative analysis of the subpopulations of motor neurons was performed on thick sections of the lumbar region (L1 - L5) of the spinal cord of control, or SMA mice treated or untreated with BIO101, using immunofluorescent labeling with choline acetyltransferase as described above (Biondi et al., 2008; Branchu et al., 2013); and analysis of the type of motor neurons by simultaneous immunofluorescent labeling with estrogen-related receptor β (ERRβ), a specific marker for slow motor neurons, or matrix metallopeptidase 9 (MMP9), a specific marker for fast motor neurons.

[0081] PBS was injected into the heart of anesthetized mice. The spinal cords of the mice were removed, fixed, and rinsed. The lumbar region of the spinal cord (L1-L5) was coated with a 4% agarose solution. Cross-sections of 50 μm were prepared using a vibratome over the entire length of the sample. Next, one out of every five sections of the spinal cord was used for immunohistochemical analysis. After saturation with 0.1 M glycine, the tissues were permeabilized, blocked, and marked with the following primary antibodies: goat anti-ChAT antibody (1 / 400 e ), mouse anti-ERRβ antibody (1 / 400 e ), rabbit anti-MMP9 antibody (1 / 600 e ). After rinsing three times, the following antibodies were incubated with the sections: donkey anti-goat Cy5 antibody (1 / 400 e ), donkey anti-mouse Alexa 488 antibody (1 / 400 e ), donkey anti-rabbit Cy3 antibody (1 / 400 e ). After marking the nuclei with bisbenzimide (1 / 1,000 e ), the sections were rinsed again and then treated with a photobleaching inhibitor for the fluorescent dye. The specificity of the marking was verified by control marking performed in the absence of the primary antibody.

[0082] Images were obtained using a camera that was installed on a microscope with a magnification of 200 times and connected to a microcomputer-type central processing unit equipped with particularly suitable software for image acquisition. All counting was performed using image processing software.

[0083] The number of slow - moving neurons (ChAT+ ERRβ+) did not change significantly in these groups, regardless of whether they were healthy mice treated with vehicle or SMA mice treated with vehicle or BIO101 (Figure 8). In the group of SMA animals treated with vehicle, a significant loss of the mean number of fast - moving neurons (ChAT+ MMP9+) was observed compared to the group of healthy control mice (11 and 16 respectively; p<0.05). This loss was to the benefit of medium - speed type motor neurons (ChAT+ ERRb - MMP9 -), the number of which was significantly higher in the group of SMA vehicle mice compared to the number present in healthy control mice (4 and 7; p<0.05).

[0084] Interestingly, treatment of SMA mice with BIO101 preferably promotes the survival of fast - type motor neurons (ChAT+ MMP9+). Indeed, in the group of SMA mice treated with BIO101, the number of fast - moving neurons was significantly higher than in the group of SMA mice administered vehicle (14 and 11; p<0.05).

[0085] As a result, treatment with BIO101 limits the loss of motor neurons observed in this severe SMA model, particularly by protecting the mice from the loss of fast - moving neurons.

[0086] e. Analysis of the neuromuscular junction SMA is characterized by specific changes at the neuromuscular junction induced by the lack of the SMN protein and denervation induced by the specific death of motor neurons (Kariya et al., 2008; Biondi et al., 2008; Chali et al., 2016). The inventors performed morphological studies of the neuromuscular junction to determine the degree of maturation and fragmentation of the so-called "pretzel" - shaped mature structures. For this purpose, the inventors performed specific labeling of the presynaptic face (synaptophysin and neurofilament) and the postsynaptic face (α - bungarotoxin) by immunofluorescence (Leroy et al., 2014) on the subdivided muscle fibers of the soleus, plantaris, and anterior tibial muscles of SMA mice treated or not treated with molecule BIO101.

[0087] Longitudinal sections with a thickness of 75 μm were prepared with a vibratome. Next, the above sections were saturated with 0.1 M glycine while gently stirring and then washed with PBS. Subsequently, the above sections were blocked and permeabilized with a solution of 4% PBS - BSA - 5% goat serum - 0.5% Triton. Anti - neurofilament (1 / 800 e ) and anti - synaptophysin (SNAP25; 1 / 200 e ) primary antibodies, which are used to identify the presynaptic face of the neuromuscular junction, were incubated for 48 hours, visualized with a secondary antibody (anti - rabbit AlexaFluor® 647; 1 / 400 e ) and then washed. Finally, the sections were incubated with anti - α - bungarotoxin directly conjugated with AlexaFluor® 555 (1 / 500 e ). The sections were washed, the nuclei were marked with bisbenzimide (1 / 1,000 e ) and placed between a coverslip and a glass slide with a photobleaching inhibitor of the fluorescent dye for observation by epifluorescence microscopy imaging (Figure 9A).

[0088] The neuromuscular junctions are defined and quantified according to three categories: uniform plates, perforations, or "pretzel" - shaped, from the most immature to the most mature.

[0089] In all the muscles under study (soleus, flounder muscle, and anterior tibial muscle), the percentage of uniform neuromuscular junctions in SMA animals is significantly increased compared to healthy control mice. Such a delay in the maturation of neuromuscular junctions has been anticipated and has already been described in the literature (Biondi et al., 2008). Indeed, in the soleus muscle, which is a fast extensor muscle, the percentage of immature neuromuscular junctions at P10 is 89.7% compared to 49.7% in healthy control mice (p<0.05) (Figure 9B). In the flounder muscle, which is a slow extensor muscle, the percentage of immature neuromuscular junctions is 84.3% compared to 45.7% in healthy control mice (p<0.05) (Figure 9C). In the anterior tibial muscle, which is a fast flexor muscle, the percentage of immature neuromuscular junctions is 69.3% compared to 28.7% in healthy control mice (p<0.05) (Figure 9D). When SMA mice are treated daily with BIO101 from birth to P10, further significant maturation is observed at the neuromuscular junctions of all the muscles tested, the percentage of immature plates decreases, and plates with perforations become dominant, which is evidence of accelerated maturation. Indeed, in the soleus muscle, 16.3% of the junctions are of the perforated type compared to 10.4% in vehicle-administered SMA animals (p=ns) (Figure 9B). This difference is significant in the flounder muscle (26.5% of the junctions are perforated in the SMA group compared to 15.7% in the group of vehicle-treated SMA mice; p<0.05, Figure 9C) and in the anterior tibial muscle (45.7% are perforated junctions in the treated SMA group compared to 30.7% in the group of vehicle-treated SMA mice; p<0.05, Figure 9D).

[0090] Therefore, these results indicate that treatment with BIO101 accelerates the maturation of neuromuscular junctions.

[0091] f. Analysis of the motor ability of a mouse model of severe SMA Phenotypic analysis of mice with severe type 2 SMA, treated or untreated with BIO101 from P0, was performed. From P5 to P9, longitudinal tests on the motor ability of the mice were performed every two days. As described above, the inventors evaluated the spontaneous locomotor ability by the open field test and the muscle fatigue susceptibility by the grip strength test (Biondi et al., 2008; Branchu et al., 2013; Chali et al., 2016).

[0092] The device used for the open field test varies depending on the age of the mouse. For animals from P0 to P6, the device is a plastic box of 15×15×5 cm, with a grid pattern of a field divided into 25 squares of 3 cm×3 cm. For animals from P7 to P21, it is a plastic box of 28×28×5 cm, with a grid pattern of a field divided into 16 squares of 7 cm×7 cm. The mice were tested individually and the evaluation device was cleaned at the end of each session. Each mouse placed centrally in the field at first was allowed to move freely for 5 minutes. During these 5 minutes, the measured values of the behavior were recorded by the experimenter, and the total number of squares traversed was recorded.

[0093] As expected, at all time points tested (P5, P7, P9), SMA mice treated with vehicle showed significantly reduced motor performance compared to healthy control mice (Figure 10A). In fact, the number of squares that the mice could cross was 17 at P5, 12 at P7, and 24 at P9 in the group of healthy control mice, while it was 10 at P5, 6 at P7, and 8 at P9 (p < 0.01, p < 0.0001, p < 0.001, respectively). At P5, treatment of SMA mice with BIO101 did not improve the motor performance of SMA mice (11 squares) compared to SMA mice treated with vehicle (10 squares). At P7, the motility of mice treated with BIO101 (crossing 10 squares) was significantly increased compared to SMA mice administered vehicle (crossing 6 squares; p < 0.05). At P9, although this difference was not large, treatment with BIO101 tended to have a beneficial effect on the mice, with the SMA BIO101 group being able to cross 11 squares while the SMA vehicle group could cross 8 squares (p = ns).

[0094] To evaluate muscle fatigue susceptibility, the grip strength of the forelimbs of mice from P5 to P9 was tested (grip strength test). The mice were suspended horizontally in the air by their forelimbs from a thin metal rod. The time they hung was recorded. Each mouse received 5 consecutive trials with a 1-minute break between the two tests. Only the best test was saved for the evaluation of muscle function.

[0095] The muscle fatigue susceptibility tested by the above grip strength test indicates that, as expected, SMA mice treated with vehicle have significantly reduced muscle function compared to healthy control mice (Figure 10B). In fact, the time that mice can continue to hang from the metal rod is 3.3 seconds at P5, 11.4 seconds at P7, and 16.1 seconds in the group of healthy control mice, whereas it is 0.2 seconds at P5, 6.2 seconds at P7, and 5.6 seconds at P9 (p<0.01, p<0.01, p<0.001 respectively). At P5, treatment of SMA mice with BIO101 significantly improves muscle performance (2.3 seconds) compared to SMA mice treated with vehicle (0.2 seconds; p<0.05). At P7, treatment with BIO101 has a tendency to increase, although not significantly, the hanging time of SMA mice compared to SMA mice administered vehicle (12.2 seconds versus 6.2 seconds, p = ns). Finally, at P9, BIO101 very significantly improves this parameter in mice treated with BIO101 compared to SMA mice treated with vehicle (5.6 seconds, p<0.01) (20.9 seconds).

[0096] In conclusion, BIO101 has beneficial effects on the spontaneous motor function and muscle fatigue susceptibility of SMA animals.

[0097] g. Phenotypic analysis of the effects of BIO103 Using the same type II mouse model, the phenotypic effects of the molecule BIO103 in a model of severe SMA were characterized. Mice were treated by forced oral administration at a dose of 50 mg / kg daily with the molecule BIO103 complexed with vehicle (in this case cyclodextrin), or with vehicle alone (VH). Body weight and survival were analyzed daily until P11.

[0098] The results demonstrate that in daily oral treatment from the birth of the mice, although BIO103 (n = 12) tends to limit the loss of animal body weight, the difference when compared to the vehicle-treated animals (n = 22) does not reach the significance threshold (Figure 11A). On the other hand, BIO103 significantly (p < 0.05) improves the survival rate of treated animals before P11 compared to vehicle-administered mice (36.3% survival rate at P11), with a survival rate of 66.6% at P11 (Figure 11B).

[0099] h.Analysis of the myotrophic effect of BIO103 Histological analysis of hematoxylin-eosin stained cross-sections of the harvested muscle shows a beneficial effect of BIO103 on muscular atrophy (cross-sectional area of muscle fibers).

[0100] Similar to the molecule BIO101, muscular atrophy present in the body of SMA mice is restricted by treatment regardless of the nature and type of muscle. Indeed, treatment with BIO103 can significantly limit the atrophy of the three muscles studied.

[0101] Quantification of the cross-section of muscle fibers shows, as expected, that this atrophy is very significant, being 56.1% in the tibialis anterior muscle of SMA mice (n = 4) compared to control mice (n = 4). This atrophy is significantly reduced to 22.1% at P11 by treatment with BIO103 (n = 4) (p < 0.05) (Figure 12A). This also applies to the soleus muscle (a significant atrophy of 46% of muscle fibers was observed in SMA mice compared to healthy control mice (p < 0.05), and treatment with BIO103 significantly limits this atrophy to 15.3% (p < 0.05) (Figure 12B)), and the gastrocnemius muscle (for SMA mice, 50.2% compared to the control (p < 0.01), and 37.1% by treatment with BIO103 (p < 0.05)) (Figure 12C).

[0102] To perform a more precise analysis of the effect of BIO103 on atrophy, the distribution of muscle fibers was evaluated for each category of cross-sectional area. The substantial reduction in atrophy by the molecule BIO103 observed by treating SMA mice from P0 to P11 results in a significant effect of this treatment on the distribution of fibers by cross-sectional area of the three muscles studied in SMA mice treated with BIO103 compared to SMA mice treated with vehicle (Figures 13A, 13B, 13C). In the tibialis anterior muscle of SMA mice treated with BIO103 from P0 to P11, a significant increase in the ratio of fibers with a cross-sectional area greater than 400 μm 2 was observed, while the ratio of small fibers with a cross-sectional area less than 200 μm 2 significantly decreased (p < 0.05) (Figure 13A). In the soleus muscle (Figure 13B) and the gastrocnemius muscle (Figure 13C) of SMA mice treated with BIO103 from P0 to P11, an increase in the ratio of muscle fibers with a cross-sectional area greater than 300 μm 2 was observed, accompanied by a decrease in muscle fibers with a cross-sectional area less than 200 μm 2 .

[0103] 4. Conclusions Considering the properties of BIO101 and BIO103 on the hypoplasia, atrophy, and degeneration of mammalian motor neurons affected by infantile spinal muscular atrophy, the use of phytoecdysones, particularly BIO101 and BIO103, as a capture for treatment aimed at modifying the effects of genetic changes alone or in combination can be proposed, thereby protecting muscle tissue and motor neurons and thus delaying the progression of neuromuscular diseases that cause a decline in muscle function and / or loss of motor neurons. Neuromuscular diseases particularly include amyotrophic lateral sclerosis and spinal muscular atrophy.

[0104] More generally, it should be noted that the aspects for the implementation and practice of the present invention discussed above are described as non-limiting examples, and thus other variations are also conceivable.

[0105] References Akten B, Kye MJ, Hao le T, Wertz MH, Singh S; et al. Interaction of survival of motor neuron (SMN) and HuD proteins with mRNA cpg15 rescues motor neuron axonal deficits. Proc. Natl. Acad. Sci. USA (2011); 108:10337-42. Al-Chalabi A & Hardiman O. The epidemiology of ALS: a conspiracy of genes, environment and time. Nat. Rev. Neurol. (2013); 9:617-28. Bevan AK, Hutchinson KR, Foust KD, Braun L, McGovern VL, et al. Early heart failure in the SMNDelta7 model of spinal muscular atrophy and correction by postnatal scAAV9-SMN delivery. Hum. Mol. Genet. (2010); 19:3895-905. Biondi O, Grondard C, Lecolle S, Deforges S, Pariset C, et al. Exercise-induced activation of NMDA receptor promotes motor unit development and survival in a type 2 spinal muscular atrophy model mouse. J. Neurosci. (2008); 28:953-62. Biondi O, Lopes P, Desseille C, Branchu J, Chali F, et al. Physical exercise reduces cardiac defects in type 2 spinal muscular atrophy-like mice. J. Physiol. (2012); 590:5907-25. Boyer JG, Murray LM, Scott K, De Repentigny Y, Renaud JM, Kothary R. Early onset muscle weakness and disruption of muscle proteins in mouse models of spinal muscular atrophy. Skelet. Muscle (2013); 3:24. Branchu J, Biondi O, Chali F, Collin T, Leroy F, et al. Shift from extracellular signal-regulated kinase to AKT / cAMP response element-binding protein pathway increases survival-motor-neuron expression in spinal-muscular-atrophy-like mice and patient cells. J. Neurosci. (2013); 33:4280-94. Buhler D, Raker V, Luhrmann R & Fischer U. Essential role for the tudor domain of SMN in spliceosomal U snRNP assembly: implications for spinal muscular atrophy. Hum. Mol. Genet. (1999); 8:2351-7. Chali, F. et al. Long-term exercise-specific neuroprotection in spinal muscular atrophy-like mice. J Physiol 594, 1931-1952 (2016). Chiriboga CA, Swoboda KJ, Darras BT, Iannaccone ST, Montes J, et al. Results from a phase 1 study of nusinersen (ISIS-SMN(Rx)) in children with spinal muscular atrophy. Neurology (2016); 86:890-7. Cifuentes-Diaz C, Frugier T, Tiziano FD, Lacene E, Robiot N, et al. Deletion of murine SMN exon 7 directed to skeletal muscle leads to severe muscular dystrophy. J. Cell Biol. (2001); 152:1107-14. Crawford TO & Pardo CA. The neurobiology of childhood spinal muscular atrophy. Neurobiol. Dis. (1996); 3:97-110. Dengler R, Konstanzer A, Kuther G, Hesse S, Wolf W, Struppler A. Amyotrophic lateral sclerosis: macro-EMG and twitch forces of single motor units. Muscle Nerve (1990); 13 (6):545-50. Fidzianska A, Rafalowska J, Glinka Z. Ultrastructural study of motoneurons in Werdnig-Hoffmann disease. Clin. Neuropathol. (1984); 3:260-5. Finkel RS, Chiriboga CA, Vaisar J, Day JW, Montes J, et al. Treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase 2, open-label, dose-escalation study. Lancet (2016); 388:3017-26. Frugier T, Tiziano FD, Cifuentes-Diaz C, Miniou P, Roblot N, et al. Nuclear targeting defect of SMN lacking the C-terminus in a mouse model of spinal muscular atrophy. Hum. Mol. Genet. (2000); 9:849-58. Gorelick-Feldman J, MacLean D, Ilic N, Poulev A, Lila MA, Cheng D, Raskin I. Phytoecdysteroids increase protein synthesis in skeletal muscle cells. J. Agric. Food Chem. (2008); 56:3532-37. Greensmith L & Vrbova G. Alterations of nerve-muscle interaction during postnatal development influence motoneurone survival in rats. Brain Res. Dev. Brain Res. (1992); 69(1):125-31 (1992). Grice SJ & Liu JL. Survival motor neuron protein regulates stem cell division, proliferation, and differentiation in Drosophila. PLoS Genetics (2011) 7:e1002030. Hache M, Swoboda KJ, Sethna N, Farrow-Gillespie A, Khandji A, et al. Intrathecal Injections in Children With Spinal Muscular Atrophy: Nusinersen Clinical Trial Experience. J. Child Neurol. (2016); 31:899-906. Heier CR, Satta R, Lutz C, DiDonato CJ. Arrhythmia and cardiac defects are a feature of spinal muscular atrophy model mice. Hum. Mol. Genet. (2010); 19:3906-18. Hsieh-Li HM, Chang JG, Jong YJ, Wu MH, Wang NM, et al. A mouse model for spinal muscular atrophy. Nat. Genet. (2000); 24:66-70. Hua Y, Sahashi K, Hung G, Rigo F, Passini MA, et al. Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model. Genes Dev. (2010); 24:1634-44. Hunter G, Aghamaleky Sarvestany A, Roche SL, Symes RC, Gillingwater TH. SMN-dependent intrinsic defects in Schwann cells in mouse models of spinal muscular atrophy. Hum. Mol. Genet. (2014); 23:2235-50. Hwee DT, Kennedy AR, Hartman JJ, Ryans J, Durham N, et al. The small-molecule fast skeletal troponin activator, CK-2127107, improves exercise tolerance in a rat model of heart failure. J. Pharmacol. Exp. Ther. (2015); 353:159-68. Kariya S, Park GH, Maeno-Hikichi Y, Leykekhman O, Lutz C, Arkovitz MS et al. Reduced SMN protein impairs maturation of the neuromuscular junctions in mouse models of spinal muscular atrophy. Hum. Mol. Genet. (2008); 17(16):2552-69. Kong L, Wang X, Choe DW, Polley M, Burnett BG, et al. Impaired synaptic vesicle release and immaturity of neuromuscular junctions in spinal muscular atrophy mice. J. Neurosci. (2009); 29:842-51. Lawrence MM. Ajuga turkestanica as a countermeasure against sarcopenia and dynapenia. MS thesis, Appalachian State University (2012). Lee YI, Mikesh M, Smith I, Rimer M, Thompson W. Muscles in a mouse model of spinal muscular atrophy show profound defects in neuromuscular development even in the absence of failure in neuromuscular transmission or loss of motor neurons. Dev. Biol. (2011); 356:432-44. Lefebvre S, Burlet P, Liu Q, Beltrandy S, Clermont O, et al. Correlation between severity and SMN protein level in spinal muscular atrophy. Nat. Genet. (1997); 16:265-9. Leroy, F. et al. Early intrinsic hyperexcitability does not contribute to motoneuron degeneration in amyotrophic lateral sclerosis eLife. 3, e04046 (2014). Liu Q, Fischer U, Wang F, Dreyfuss G. The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins. Cell (1997); 90:1013-21. Lorson CL, Strasswimmer J, Yao JM, Baleja JD, Hahnen E, et al. SMN oligomerization defect correlates with spinal muscular atrophy severity. Nat. Genet. (1998); 19:63-6. McWhorter ML, Monani UR, Burghes AH, Beattie CE. Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding. J. Cell Biol. (2003); 162:919-31. Murray LM, Comley LH, Thomson D, Parkinson N, Talbot K, Gillingwater TH.. Selective vulnerability of motor neurons and dissociation of pre- and post-synaptic pathology at the neuromuscular junction in mouse models of spinal muscular atrophy. Hum. Mol. Genet. (2008); 17:949-62. Nicole S, Desforges B, Millet G, Lesbordes J, Cifuentes-Diaz C, et al. Intact satellite cells lead to remarkable protection against Smn gene defect in differentiated skeletal muscle. J. Cell Biol. ‘2003); 161:571-82. Passini MA, Bu J, Richards AM, Kinnecom C, Sardi SP, et al. Antisense oligonucleotides delivered to the mouse CNS ameliorate symptoms of severe spinal muscular atrophy. Sci. Transl. Med. (2011); 3:72ra18. Peter CJ, Evans M, Thayanithy V, Taniguchi-Ishigaki N, Bach I, et al. The COPI vesicle complex binds and moves with survival motor neuron within axons. Hum. Mol. Genet. (2011); 20:1701-11. Pun S, Santos AF, Saxena S, Xu L, Caroni P. Selective Vulnerability and Pruning of Phasic Motoneuron Axons in Motoneuron Disease Alleviated by CNTF. Nature Neuroscience (2006); 9 (3):408-19. Rajendra TK, Gonsalvez GB, Walker MP, Shpargel KB, Salz HK, Matera AG. A Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle. J. Cell Biol. (2007); 176:831-41. Rindt H, Feng Z, Mazzasette C, Glascock JJ, Valdivia D, et al. Astrocytes influence the severity of spinal muscular atrophy. Hum. Mol. Genet. (2015); 24:4094-102. Sanchez G, Dury AY, Murray LM, Biondi O, Tadesse H, et al. A novel function for the survival motoneuron protein as a translational regulator. Hum. Mol. Genet. (2013); 22:668-84. Shababi M, Habibi J, Yang HT, Vale SM, Sewell WA, Lorson CL. Cardiac defects contribute to the pathology of spinal muscular atrophy models. Hum. Mol. Genet. (2010); 19:4059-71. Shafey D, Cote PD, Kothary R. Hypomorphic Smn knockdown C2C12 myoblasts reveal intrinsic defects in myoblast fusion and myotube morphology. Exp. Cell Res. (2005); 311:49-61. Simakin SYu, Panyushkin VV, Portugalov SN, Kostina LV, Martisorov EG. Combined application of preparation Ecdysten. Science Bulletin (1998); N°2, 29-31. Somers E, Lees RD, Hoban K, Steigh JN, Zhou H, et al. Vascular Defects and Spinal Cord Hypoxia in Spinal Muscular Atrophy. Ann. Neurol. (2016); 79:217-30. Syrov VN. Comparative experimental investigations of the anabolic activity of ecdysteroids and steranabols. Pharm. Chem. J. (2000); 34(4):193-197. Theys PA, Peeters E, Robberecht W. Evolution of Motor and Sensory Deficits in Amyotrophic Lateral Sclerosis Estimated by Neurophysiological Techniques. J. Neurol. (1999); 246 (6):438-42. 26 Torres-Benito L, Neher MF, Cano R, Ruiz R, Tabares L. SMN requirement for synaptic vesicle, active zone and microtubule postnatal organization in motor nerve terminals. PLoS One (2011); 6:e26164. Toth N, Szabo A, Kacsala P, Heger J, Zador E. 20-Hydroxyecdysone increases fiber size in a muscle-specific fashion in rat. Phytomedicine (2008); 15: 6918. Vitte J, Fassier C, Tiziano FD, Dalard C, Soave S, et al. Refined characterization of the expression and stability of the SMN gene products. Am J Pathol (2007); 171:1269-80. Vitte JM, Davoult B, Roblot N, Mayer M, Joshi V, et al. Deletion of murine Smn exon 7 directed to liver leads to severe defect of liver development associated with iron overload. Am. J. Pathol. (2004); 165:1731-41. Vrbova G, Fisher TJ. The Effect of Inhibiting the Calcium Activated Neutral Protease, on Motor Unit Size after Partial Denervation of the Rat Soleus Muscle. Eur. J. Neurosci. (1989); 1:616-25. Walker MP, Rajendra TK, Saieva L, Fuentes JL, Pellizzoni L, Matera AG. SMN complex localizes to the sarcomeric Z-disc and is a proteolytic target of calpain. Hum. Mol. Genet. (2008); 17:3399-410. Zhang Z, Lotti F, Dittmar K, Younis I, Wan L, et al. SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell (2008); 133:585-600.

Claims

1. A composition comprising at least 20-hydroxyecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone for its use in the treatment of a specific disorder of motor neurons in a mammal suffering from spinal muscular atrophy (SMA), wherein said disorder comprises a change in muscle function due to said specific disorder of motor neurons, and wherein at least one semi-synthetic derivative of 20-hydroxyecdysone is General formula (I): 【Chemical 1】 (wherein, V-U is a carbon-carbon single bond, Y is a hydroxyl group or hydrogen, or V-U is an ethylenic C═C bond; X is oxygen; Q is a carbonyl group; R １ is: (C １ -C ６ )W(C １ -C ６ ) group; (C １ -C ６ )W(C １ -C ６ )W(C １ -C ６ ) group; (C １ -C ６ )W(C １ -C ６ )CO ２ (C １ -C ６ ) group; (C １ -C ６ )A group, provided that A is a heterocyclic ring optionally substituted with a group selected from OH, OMe, (C １ -C ６ ), and CO ２ (C １ -C ６ ); CH ２ Br group; and a (2-hydroxyethylsulfanyl)methyl group; W is a heteroatom selected from O and S.) at least one compound of, or General formula (II): 【Chemical 2】 a compound of A composition comprising.

2. 20-Hydroxyecdysone is in the form of a plant extract or an extract of a part of a plant, said plant is selected from plants containing at least 0.5% of 20-hydroxyecdysone by dry weight of said plant, said extract contains at least 95% of 20-hydroxyecdysone, the composition for its use according to claim 1.

3. The composition for its use according to claim 2, comprising 0 to 0.05% by dry weight of the extract of impurities that may affect the safety, availability, or effectiveness of the application of said extract as a pharmaceutical.

4. Said plant is selected from Stemmacantha carthamoides, Cyanotis arachnoidea and Cyanotis vaga, the composition for its use according to claim 2 or 3.

5. Said change in muscle function is produced by a change in motor neuron function or its degeneration, the composition for its use according to any one of claims 1 to 4.

6. Said disorder of motor neurons is caused by a genetic change in a mammal suffering from SMA, the composition for its use according to any one of claims 1 to 5.

7. Said changing muscle function is the function of skeletal muscle or cardiac muscle, the composition for its use according to any one of claims 1 to 6.

8. Said change in muscle function is associated with hypoplasia and / or atrophy, the composition for its use according to any one of claims 1 to 7.

9. The composition for its use according to claim 8, wherein said at least one semi-synthetic derivative of 20-hydroxyecdysone and / or 20-hydroxyecdysone is used for treating at least one genetic change causing SMA.

10. The composition for its use according to any one of claims 1 to 9, wherein said SMA is caused by a mutation in the SMN1 gene.

11. The composition for its use according to any one of claims 1 to 10, wherein the treatment of said specific disorder of motor neurons includes improving the survival rate of motor neurons and / or accelerating the maturation of neuromuscular junctions.

12. The composition for its use according to any one of claims 1 to 11, wherein said at least one semi-synthetic derivative of 20-hydroxyecdysone and / or 20-hydroxyecdysone is administered to humans at a dose of 3 to 15 mg / kg / day.

13. The composition for its use according to any one of claims 1 to 12, wherein said at least one semi-synthetic derivative of 20-hydroxyecdysone and / or 20-hydroxyecdysone is administered to adult humans in divided doses of 200 to 1000 mg / day, and to human children or infants in divided doses of 5 to 350 mg / day.

14. In the general formula (I): Y is a hydroxyl group; R １ is: (C １ -C ６ )W(C １ -C ６ ) group; (C １ -C ６ )W(C １ -C ６ )W(C １ -C ６ ) group; (C １ -C ６ )W(C １ -C ６ )CO ２ (C １ -C ６ ) group; (C １ -C ６ ) A group, provided that A is a heterocyclic ring optionally substituted with a group selected from OH, OMe, (C １ -C ６ ), and CO ２ (C １ -C ６ ), and is selected from; W is a heteroatom selected from O and S, the composition for its use according to claim 1.

15. Said at least one compound of the general formula (I) is: - No. 1: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-10,13-dimethyl-17-(2-morpholinoacetyl)-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one; - No. 2: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(3-hydroxypyrrolidin-1-yl)acetyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one; - No. 3: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(4-hydroxy-1-piperidyl)acetyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one; - No. 4: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-[4-(2-hydroxyethyl)-1-piperidyl]acetyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one; - No. 6: 2-[2-oxo-2-[(2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-10,13-dimethyl-6-oxo-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-17-yl]ethyl]ethylsulfanylacetate; - No. 7: (2S,3R,5R,10R,13R,14S,17S)-17-(2-ethylsulfanylacetyl)-2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one; - No. 8: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(2-hydroxyethylsulfanyl)acetyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H cyclopenta[a]phenanthren-6-one The composition according to claim 1, selected from

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