阅读说明：本技术 用于治疗神经肌肉疾病的植物蜕皮素及其衍生物 (Phytoecdysone and its derivatives for the treatment of neuromuscular diseases ) 是由 玛蒂尔德·拉蒂伊 皮埃尔·迪尔达 勒内·拉丰 斯坦尼斯拉斯·韦耶 于 2020-03-12 设计创作，主要内容包括：本发明涉及20-羟基蜕皮素及其衍生物,其旨在用于治疗神经肌肉疾病例如脊髓性肌萎缩或肌萎缩侧索硬化,或者更特别地用于治疗在这些神经肌肉疾病的背景下发生的引起肌肉功能改变的特定运动神经元病症。(The present invention relates to 20-hydroxyecdysone and its derivatives intended for the treatment of neuromuscular diseases such as spinal muscular atrophy or amyotrophic lateral sclerosis, or more particularly for the treatment of specific motor neuron disorders causing alterations in muscle function that occur in the context of these neuromuscular diseases.)

1. A composition comprising at least 20-hydroxyecdysone and/or at least one 20-hydroxyecdysone semisynthetic derivative for use in the treatment of a specific motor neuron disorder, including an alteration in muscle function due to the specific motor neuron disorder, in a mammal suffering from a neuromuscular disease.

2. The composition for use according to claim 1, wherein 20-hydroxyecdysone is in the form of an extract of a plant selected from the group consisting of plants comprising at least 0.5% 20-hydroxyecdysone based on dry weight of the plant, or an extract of a plant part comprising at least 95% and preferably at least 97% 20-hydroxyecdysone.

3. Composition for use according to claim 2, notably comprising from 0% to 0.05% by dry weight of the extract of impurities capable of affecting the safety, availability or efficacy of pharmaceutical use of the extract.

4. The composition for use according to any one of claims 2 to 3, wherein the plant is selected from the group consisting of rhaponticum carthamoides (stemmacha carthamoides), Sclerotium arachnoideum (Cyanotis arachnoidea) and Cyanotis hybrida (Cyanotis vaga).

5. The composition for use according to any one of claims 1 to 4, wherein the alteration in muscle function results from an alteration in motor neuron function or degeneration thereof.

6. The composition for use according to any one of claims 1 to 5, wherein the motor neuron disorder is caused by a genetic alteration in a mammal suffering from a neuromuscular disease.

7. The composition for use according to any one of claims 1 to 6, wherein the altered muscle function is altered muscle function of striated skeletal muscle or altered muscle function of cardiac muscle.

8. The composition for use according to any one of claims 1 to 7, wherein the altered muscle function is associated with hypoplasia and/or atrophy.

9. The composition for use according to any one of claims 1 to 8, wherein the neuromuscular disease is infantile Spinal Muscular Atrophy (SMA) and/or amyotrophic lateral Sclerosis (SLA).

10. The composition for use according to claim 9, wherein 20-hydroxyecdysone and/or the at least one semi-synthetic derivative of 20-hydroxyecdysone is used in the treatment of at least one genetic alteration that leads to SMA or SLA.

11. The composition for use according to any one of claims 1 to 10, wherein the neuromuscular disease is caused by a mutation in at least one gene selected from the group consisting of: SMN1, SOD1, TARDBP, VCP, FUS/TLS and C9ORF 72.

12. The composition for use according to any one of claims 1 to 11, wherein the treatment of the specific motor neuron disorder comprises increasing motor neuron survival and/or accelerating the maturation of the neuromuscular junction.

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

14. The composition for use according to any one of claims 1 to 13, wherein 20-hydroxyecdysone and/or the at least one semi-synthetic derivative of 20-hydroxyecdysone is administered in one or more divided doses at a dose of 200 to 1000 mg/day in adults and 5 to 350 mg/day in human children or infants.

15. Composition for use according to any one of claims 1 to 14, comprising at least one compound of general formula (I):

[ solution 1]

Wherein:

V-U is a carbon-carbon single bond and Y is hydroxy or hydrogen, or V-U is a C ═ C olefinic bond;

x is oxygen, and X is oxygen,

q is a carbonyl group;

R¹selected from: (C)₁-C₆)W(C₁-C₆) A group; (C)₁-C₆)W(C₁-C₆)W(C₁-C₆) A group; (C)₁-C₆)W(C₁-C₆)CO₂(C₁-C₆) A group; (C)₁-C₆) A is optionally substituted by OH, OMe, (C)₁-C₆)、N(C₁-C₆)、CO₂(C₁-C₆) A heterocycle substituted with a group of the type; CH (CH)₂A Br group;

w is a heteroatom selected from N, O and S, preferably O and more preferably S.

16. The composition for use according to claim 15, wherein in said general formula (I):

y is hydroxy;

R¹selected from: (C)₁-C₆)W(C₁-C₆) A group; (C)₁-C₆)W(C₁-C₆)W(C₁-C₆) A group; (C)₁-C₆)W(C₁-C₆)CO₂(C₁-C₆) A group; (C)₁-C₆) A is optionally substituted by OH, OMe, (C)₁-C₆)、N(C₁-C₆)、CO₂(C₁-C₆) A heterocycle substituted with a group of the type;

w is a heteroatom selected from N, O and S, preferably O and more preferably S.

17. The composition for use according to any one of claims 15 to 16, wherein said at least one compound of general formula (I) is selected from:

-n ° 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;

-n ° 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;

-n ° 3: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2, 3, 14-trihydroxy-17- [2- (4-hydroxy-1-piperidinyl) acetyl ] -10, 13-dimethyl-2, 3, 4, 5, 9, 11, 12, 15, 16, 17-decahydro-1H-cyclopenta [ a ] phenanthren-6-one;

-n ° 4: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2, 3, 14-trihydroxy-17- [2- [4- (2-hydroxyethyl) -1-piperidinyl ] acetyl ] -10, 13-dimethyl-2, 3, 4, 5, 9, 11, 12, 15, 16, 17-decahydro-1H-cyclopenta [ a ] phenanthren-6-one;

-n ° 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;

-n ° 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 ] ethylsulfanyl acetate;

-n ° 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;

-n ° 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.

18. Composition for use according to any one of claims 1 to 17, comprising at least one compound of general formula (II):

[ solution 2]

Technical Field

The present invention relates to the use of phytoecdysone and semi-synthetic derivatives of phytoecdysone for the treatment of neuromuscular diseases, in particular infantile spinal muscular atrophy and amyotrophic lateral sclerosis.

Background

Neuromuscular diseases are characterized by altered function of the motor unit consisting of motor neurons, neuromuscular junctions and skeletal muscles. Regardless of the origin of the disease, nerves (as in infantile spinal muscular atrophy or amyotrophic lateral sclerosis) or muscles cause changes in the motor function of the patient, which can range from disability to premature death when important muscles are affected. The care of these neuromuscular diseases is still symptomatic to date and represents a significant economic cost in terms of the level of disability of the patients, the progressiveness of these pathological conditions and the manpower and material requirements they require. Research and development has led to a certain social and economic interest in treatments that can alleviate motor symptoms and dependencies in patients.

Of these neuromuscular diseases, two are described as affecting specifically motor neurons: infantile Spinal Muscular Atrophy (SMA), the symptoms of which occur in childhood, and amyotrophic lateral Sclerosis (SLA), the symptoms of which occur in adulthood. These two neurodegenerative diseases with different causes and clinical manifestations share a common progressive myodenervation, leading to muscle atrophy (Al-Chalabi et Hardiman, 2013; Crawford et Pardo, 1996).

Infantile spinal muscular atrophy represents the most common cause of death in children of genetic origin, with prevalence ranging from 1/6,000 to 1/10,000 birth (Crawford et Pardo, 1996). Three major severity categories from type 1 (most severe) to type 3 (which may have a lifespan greater than 40 years) are described, depending on the age at which symptoms appear and the progression of clinical effects. Patients with SMA have symmetric pain of skeletal muscle due to atrophy of isolated or fasciculated muscle fibers. Almost all SMAs have the proximal advantage of affecting the torso and muscles near the torso. Gradually, the motor deficit is extended in a first step to the muscles of the lower extremities and then in a second step to the muscles of the upper extremities by preferentially affecting the extensors. Despite great clinical heterogeneity, genetic analysis has demonstrated that all forms of SMA are caused by mutations in the gene SMN1 for survival of motor neurons located at the chromosomal region 5q 13.

In the human genome, there is an anti-centromeric copy of the gene, gene SMN2, which may be present in several copies (Lorson et al, 1998), but this makes it possible to only partially counteract the loss of function of gene SMN 1. In fact, SMN2 differs from SMN1 by 5 nucleotides, one of which is at exon 7, which favours the excision of 90% of the mRNA produced by the gene SMN2 by splicing. This alternative splicing results in the production of truncated and unstable SMNs^Δ7A protein. Thus, only 10% of the proteins produced by gene SMN2 are intact and functional (vite et al, 1997; Lefebvre et al, 1997). Thus, a link between the copy number of the gene SMN2, their expression level and the severity of the disease has been demonstrated. SMN proteins are small ubiquitous proteins located in discrete domains of the nucleus, gems called spirochete Gemini (Gemini of coded Body), and are in the cytoplasm of cells. Although the SMN protein has specific neuronal functions, such as axonal growth (McWhorter et al, 2003) and axonal transport (Akten et al, 2011; Peter et al, 2011), it is also involved in ubiquitous functions, such as biogenesis of ribonucleoproteins (Buhler et al, 1999; Liu et al, 1997; Zhang et al, 2008) or regulation of mRNA translation (Sanchez et al, 2013). Thus, neither expression of the SMN protein, nor its cellular function, would allow direct interpretation of specific degeneration of motor neurons.

However, the development of different animal models and the progress of basic studies have made it possible to demonstrate that only conditional deletion of the Smn gene at the neuron does not reproduce the complete symptoms of SMA nor motor neuron degeneration (Frugier et al, 2000). Other studies describe the role of SMN proteins in the normal functioning of astrocytes (riddt et al, 2015), schwann cells (Hunter et al, 2014), cardiac cells (Bevan et al, 2010; Heier et al, 2010; Shabadi et al, 2010; Biondi et al, 2010), hepatocytes (vite et al, 2004) and blood vessels (Somers et al, 2016), suggesting a role that energy metabolism plays in the progression and severity of the disease. Other further studies have focused on the motor units in SMA and revealed specific neuromuscular junction changes (Kariya et al, 2008; Kong et al, 2009; Muray et al, 2008; Biondi et al, 2008) which are important components of motor neuron survival. In fact, SMN proteins have been shown to participate in the normal functioning of neuromuscular junctions by acting on the neurotransmitter vesicle pool, synaptic activity (Torres-Benito et al, 2011), and maturation of the junction (a key step that makes it functional and maintains its function) (Kariya et al, 2008; Biondi et al, 2008). On the other hand, failure of the Smn gene in muscle cells alone can produce severe muscle changes (cifuents-Diaz et al, 2001; rajndra et al, 2007; Lee et al, 2011), such as severe dystrophy (cifuents-Diaz et al, 2001), dysorganization of the sarcomere structures that allow contraction (Walker et al, 2008), reduction of correct fusion of muscle stem cells (Nicole et al, 2003) and therefore their differentiation (Shafey et al, 2005). In addition, there are fewer factors such as CANP (Calcium-Activated Neutral Protease) necessary to establish neuromuscular contact in patients with SMA that have myogenic cells (Fidzianska et al, 1984; Vrbova et al, 1989). Finally, faster degeneration of motor neurons co-cultured with SMA myocytes has been observed in vitro (Vrbova et al, 1989).

To date, very different approaches have been considered for developing treatments for SMA. Current research strategies are mostly focused on gene therapy either directly or indirectly modifying the expression of SMN by increasing the expression of the SMN2 gene or modifying the transcription of spliced SMN2, or aiming to reintroduce the correct gene. Some studies have focused on methods independent of SMN by developing cellular therapies or by focusing on activating neuroprotection (orliosome) or on improving muscle function (troponin activators).

Despite the dozens of clinical trials in SMA, only two molecules have been approved by authorities to date.

The first is a troponin activator (CK-2127107) which is intended to increase the sensitivity of sarcomere to calcium in order to increase endurance and the manifestation of muscle function (Hwee et al, 2015). This molecule was recently designated by the U.S. regulatory agency as an orphan drug for SMA (5 months 2017).

The second is an antisense oligonucleotide (ASO) intended to facilitate the inclusion of exon 7 of the transcript produced by the SMN2 gene, so that the complete SMN protein can be overexpressed by spinal cord cells after intrathecal injection (Hache et al, 2016; Chiriboga et al, 2016). This molecule was developed by Biogen and named norcinagen (nusinessen), has been approved by the us and european authorities and constitutes the first molecule for therapeutic application to the market for the treatment of severe infantile spinal muscular atrophy type 1 and type 2. The first results obtained with ASO targeting intron 7, whether on animal models (Hua et al, 2010; passsini et al, 2011) or during phase II and III clinical trials in patients with severe SMA (Finkel et al, 2016), are very impressive and promising for this fatal disease. However, there are still several gray regions for the use of oligonucleotides in vivo, injected alone and/or chronically into the central nervous system. First, the scientific community is replete with a lack of long-term tolerance of patients to exogenous molecules of this type of targeted gene expression. In addition, other problems relate to the inherent risk in the massive and uncontrolled overexpression of SMN proteins whose function is linked to cell proliferation (Grice et al, 2011). In addition, the efficacy of norcinamide appears to be more important because of the early intervention in the progression of the disease at the beginning of the treatment, which is difficult to obtain in view of the time of diagnosis and the clinical course of the home. Finally, in SMA, it is important to note the importance of multiple tissue changes playing a role in disease progression. However, norcisianon is a molecule that does not cross the blood brain barrier and is treated by the intrathecal route so that it can target only neurons and glial cells and not other organs, such as muscle, liver, pancreas and the vascular system. Thus, their alteration may limit the therapeutic effect of norcinolone. Finally, for patients with type 3 SMA who have much higher expression levels of SMN protein and are rarely involved in important prognoses, no treatment authorization has been approved, making this severity therapeutically orphaned.

Therefore, it has been shown that there is a need to develop physiological and/or pharmacological approaches complementary to norcisianon to enhance its protective effect and to limit as far as possible the clinical course of the patient, its dependence and its clinical care.

Phytoecdysones represent a large family of polyhydroxysterols. These molecules are produced by a variety of plant species (ferns, gymnosperms, angiosperms) and are involved in the defense of these plants against pests. Most of the phytoecdysones in the kingdom Plantae are 20-hydroxyecdysones.

Patent FR 3021318 discloses that phytoecdysones and more particularly 20-hydroxyecdysone (20E) have been the subject of many pharmacological studies. These studies have emphasized the anti-diabetic and anabolic properties of this molecule. Its stimulatory effect on protein synthesis in muscle was observed both in rats in vivo (Syrov et al, 2000; Toth et al, 2008; Lawrence et al, 2012) and in vitro on murine myotubes C2C12 (Gorelick-Feldman et al, 2008). Some of the effects described above in animal models have been found in clinical studies, but are still rare. Thus, 20-hydroxyecdysone is beneficial for increasing muscle mass in young athletes (Simekin et al, 1988). Finally, french patent FR 3021318 also describes the use of 20-hydroxyecdysone and 20-hydroxyecdysone derivatives for the treatment and prevention of sarcopenia and sarcopenia obesity (Lafont et al, 2017).

Disclosure of Invention

The object of the present invention is to limit the loss of motor neurons associated with neuromuscular diseases and the consequences of such degeneration.

Phytoecdysones represent a large family of plant polyhydroxysterols, the structure of which is apparent to insect ecdysones. These molecules are produced by many plant species and participate in their defense against pests and insects. Most phytoecdysones are 20-hydroxyecdysones.

The present inventors have unexpectedly found that phytoecdysone and semi-synthetic derivatives of phytoecdysone significantly improve survival and weight change in mammals suffering from spinal muscular atrophy. In addition, phytoecdysone and its semisynthetic derivatives limit the muscular atrophy and hypoplasia present in this pathology and significantly limit the loss of motor neurons in mammals with spinal muscular atrophy.

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 a specific motor neuron disorder, including an alteration in muscle function due to said specific motor neuron disorder, in a mammal suffering from a neuromuscular disease.

In some embodiments, the invention is also responsive to features of each of the following implementations, taken alone or in technically effective combinations thereof.

20-hydroxyecdysone and its derivatives are advantageously purified to pharmaceutical grade.

The 20-hydroxyecdysone used is more preferably in the form of a plant extract rich in 20-hydroxyecdysone or a composition comprising 20-hydroxyecdysone as active agent. Plant extracts rich in 20-hydroxyecdysone are, for example, extracts of rhaponticum carthamoides (also known as Leuzea carthamoides), Echinacea arachnoidea (Cyanotis arachnoidea) and Echinacea pallida (Cyanotis vaga).

The extract obtained is more preferably purified to pharmaceutical grade.

In one embodiment, the 20-hydroxyecdysone is in the form of an extract of a plant selected from plants comprising at least 0.5% 20-hydroxyecdysone based on dry weight of the plant, or a portion of a plant comprising at least 95%, and preferably at least 97%, 20-hydroxyecdysone. The extract is more preferably purified to pharmaceutical grade.

Hereinafter the extract will be referred to as BIO 101. It is noteworthy that it comprises impurities, such as trace compounds, which can affect the safety, availability or efficacy of the pharmaceutical use of said extract, in an amount of from 0% to 0.05% by dry weight of the extract.

According to one embodiment of the invention, the impurity is a compound having 19 or 21 carbon atoms, such as rubrosterone (rubrosterone), dihydrorubrosterone (dihydrorubrosterone) or peristerone (posttrone).

The plant producing BIO101 is more preferably selected from the group consisting of rhaponticum carthamoides (also known as Leuzea carthamoides), Echinacea and Echinacea.

Derivatives of 20-hydroxyecdysone are obtained by semisynthesis and may in particular be obtained in the manner described in european patent application No. ep 15732785.9.

According to a preferred embodiment, the change in muscle function is due to a change in motor neuron function or degeneration thereof.

In one embodiment, the change in muscle function is a change in muscle function of striated skeletal muscle or a change in muscle function of cardiac muscle.

In one embodiment, the altered muscle function is associated with hypoplasia and/or atrophy.

In a specific embodiment, the motor neuron disorder is caused by a genetic alteration in a mammal having a neuromuscular disease.

The term "genetic alteration" means a mutation, such as a substitution or insertion of a nucleotide or a deletion of a nucleotide.

In a particular embodiment, the present invention is directed to a composition for use in the treatment of infantile Spinal Muscular Atrophy (SMA) or amyotrophic lateral Sclerosis (SLA) in a mammal.

In a particular embodiment, the invention is directed to a composition for use in the treatment of sporadic neuromuscular disease (associated with random mutations in a causal gene or one or more susceptibility genes) or a familial form in which there is a mutation at least one gene selected from the group consisting of: SMN1, SOD1, TARDBP encoding TAR DNA binding protein 43, VCP (valosin containing protein) FUS/TLS (fused to sarcoma/translocated liposarcoma) and C9ORF72 (chromosome 9 open reading frame 72) involved in the SMA framework intervened in the SMA framework.

In a particular embodiment, the treatment of a particular motor neuron disorder comprises increasing motor neuron survival and/or accelerating the maturation of the neuromuscular junction.

In a specific embodiment, the phytoecdysone is administered in a human at a dose of 3 to 15 mg/kg/day. The term "phytoecdysone" is intended herein to mean phytoecdysone and its derivatives in general, 20-hydroxyecdysone (particularly in the form of an extract) and its derivatives.

Preferably, the phytoecdysone is administered in one or more doses at a dose of 200 to 1,000 mg/day in an adult and in one or more doses at a dose of 5 to 350 mg/day in a human child or infant. The term "phytoecdysone" is intended herein to mean phytoecdysone and its derivatives in general, 20-hydroxyecdysone (particularly in the form of an extract) and its derivatives.

In some embodiments, the composition comprises at least one compound believed to be a phytoecdysone derivative, the at least one compound having the general formula (I):

[ solution 1]

Wherein:

V-U is a carbon-carbon single bond and Y is hydroxy or hydrogen, or V-U is a C ═ C olefinic bond;

x is oxygen, and X is oxygen,

q is a carbonyl group;

R¹selected from: (C)₁-C₆)W(C₁-C₆) A group; (C)₁-C₆)W(C₁-C₆)W(C₁-C₆) A group; (C)₁-C₆)W(C₁-C₆)CO₂(C₁-C₆) A group; (C)₁-C₆) A is optionally substituted by OH, OMe, (C)₁-C₆)、N(C₁-C₆)、CO₂(C₁-C₆) A heterocycle substituted with a group of the type; CH (CH)₂A Br group;

w is a heteroatom selected from N, O and S, preferably O and more preferably S.

In the framework of the present invention, "(C)₁-C₆) "means any alkyl group, straight or branched, of 1 to 6 carbon atoms, in particular methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl. Advantageously, it is methyl, ethyl, isopropyl or tert-butyl, in particular methyl or ethyl, more particularly methyl.

In a preferred embodiment, in formula (I):

y is hydroxy;

R¹selected from: (C)₁-C₆)W(C₁-C₆) A group; (C)₁-C₆)W(C₁-C₆)W(C₁-C₆) A group; (C)₁-C₆)W(C₁-C₆)CO₂(C₁-C₆) A group; (C)₁-C₆) A is optionally substituted by OH, OMe, (C)₁-C₆)、N(C₁-C₆)、CO₂(C₁-C₆) A heterocycle substituted with a group of the type;

w is a heteroatom selected from N, O and S, preferably O and more preferably S.

In some embodiments, the composition comprises at least one compound selected from the group consisting of:

n ° 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;

n ° 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;

n ° 3: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2, 3, 14-trihydroxy-17- [2- (4-hydroxy-1-piperidinyl) acetyl ] -10, 13-dimethyl-2, 3, 4, 5, 9, 11, 12, 15, 16, 17-decahydro-1H-cyclopenta [ a ] phenanthren-6-one;

n ° 4: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2, 3, 14-trihydroxy-17- [2- [4- (2-hydroxyethyl) -1-piperidinyl ] acetyl ] -10, 13-dimethyl-2, 3, 4, 5, 9, 11, 12, 15, 16, 17-decahydro-1H-cyclopenta [ a ] phenanthren-6-one;

n ° 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;

n ° 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 ] ethylsulfanyl acetate;

n ° 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;

n ° 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.

In some embodiments, the composition comprises at least one compound believed to be a phytoecdysone derivative, the at least one compound having the general formula (II):

[ solution 2]

The compound of formula (II) is hereinafter referred to as BIO 103.

In some embodiments, the composition is incorporated into an orally administrable, pharmaceutically acceptable formulation.

In the framework of the present invention, the term "pharmaceutically acceptable" means that it can be used for the preparation of pharmaceutical compositions which are generally safe, non-toxic and acceptable for veterinary as well as human pharmaceutical use.

Drawings

The invention will be better understood when reading the following description, given by way of non-limiting example, and with reference to the accompanying drawings, which show:

figure 1A shows a graph of the change in body weight of SMA mice treated with vehicle or with BIO101 from birth (P0) to 11 days after birth (P11). Here and in the rest of the description, P corresponds to the number of days after birth (after birth), n corresponds to the sample size, and P corresponds to the "P-value" used to quantify the statistical significance of the results;

FIG. 1B is a Kaplan-Meier representation of the pre-P11 survival curves of SMA mice treated with vehicle or with BIO101 from P0 to P11;

figure 2 is a histogram showing the total number of myofibers of the tibialis anterior (tibialis) of healthy control mice (vehicle control), SMA treated with vehicle from P0 to P11 (SMA vehicle), or SMA treated with BIO101 (SMA BIO 101);

FIG. 3A is an image showing a section of tibialis anterior muscle stained with hematoxylin and eosin of a healthy control mouse (control);

figure 3B is an image showing sections of tibialis anterior stained with hematoxylin and eosin of SMA mice treated with vehicle from P0 to P11 (SMA vehicle);

FIG. 3C is an image showing sections of tibialis anterior muscle stained with hematoxylin and eosin of SMA mice treated with BIO101 from P0 to P11 (SMA BIO 101);

fig. 3D is a histogram showing the myofiber cross-sectional surfaces of the tibialis anterior (tibialis) of healthy control mice (vehicle control), SMA treated with vehicle from P0 to P11 (SMA vehicle), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

fig. 3E is a histogram showing the muscle fiber cross-sectional surface of the plantar muscle of healthy control mice (vehicle control), SMA treated with vehicle from P0 to P11 (SMA vehicle), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

fig. 3F is a histogram showing the muscle fiber cross-sectional surface of soleus muscle for healthy control mice (vehicle control), SMA treated with vehicle from P0 to P11 (SMA vehicle), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

fig. 4A is a histogram showing the distribution of muscle fibers of tibialis anterior (tibialis) according to their cross-sectional surface in SMA mice treated with vehicle from P0 to P11 (SMA vehicle) or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

fig. 4B is a histogram showing the distribution of muscle fibers of the plantar muscle according to their cross-sectional surface in SMA mice treated with vehicle from P0 to P11 (SMA vehicle) or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

fig. 4C is a histogram showing the distribution of muscle fibers of soleus muscle according to their cross-sectional surfaces in SMA mice treated with vehicle from P0 to P11 (SMA vehicle) or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

figure 5A shows a Western blot showing plantar AKT phosphorylation of healthy control mice (CTL VH), SMA mice treated with vehicle from P0 to P11 (SMA VH), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

fig. 5B shows a histogram showing quantification of plantaris AKT phosphorylation by densitometry of healthy control mice (CTL VH), SMA mice treated with vehicle from P0 to P11 (SMA VH), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

figure 5C shows a Western blot showing plantaris ERK phosphorylation of healthy control mice (CTL VH), SMA mice treated with vehicle from P0 to P11 (SMA VH), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

fig. 5D shows a histogram showing quantification of plantaris ERK phosphorylation by densitometry of healthy control mice (CTL VH), SMA mice treated with vehicle from P0 to P11 (SMA VH), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

figure 5C shows a Western blot showing the expression levels of SMN protein from the plantar muscle of healthy control mice (CTL VH), SMA mice treated with vehicle from P0 to P11 (SMA VH), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

fig. 5F shows a histogram showing quantification of SMN protein by densitometry of the plantar muscle of healthy control mice (CTL VH), SMA mice treated with vehicle from P0 to P11 (SMA VH), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

figure 6A shows a Western blot showing AKT phosphorylation at the spinal cord of healthy control mice (CTL VH), SMA mice treated with vehicle from P0 to P11 (SMA VH), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

fig. 6B shows a histogram showing quantification of AKT phosphorylation at spinal cord by densitometry of healthy control mice (CTL VH), SMA mice treated with vehicle from P0 to P11 (SMA VH), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

figure 6C shows a Western blot showing ERK phosphorylation at the spinal cord of healthy control mice (CTL VH), SMA mice treated with vehicle from P0 to P11 (SMA VH), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

fig. 6D shows a histogram showing quantification of ERK phosphorylation at the spinal cord by densitometry of healthy control mice (CTL VH), SMA mice treated with vehicle from P0 to P11 (SMA VH), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

figure 6E shows a Western blot showing expression levels of SMN protein at the spinal cord of healthy control mice (CTL VH), SMA mice treated with vehicle from P0 to P11 (SMA VH), or SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

fig. 6F shows a histogram showing quantification of SMN protein at the spinal lumbar spinal segment of healthy control mice (CTL VH), SMA mice treated with vehicle from P0 to P11 (SMA VH), or SMA treated with BIO101 from P0 to P11 (SMA BIO101) by densitometry;

FIG. 7A is an image showing immunofluorescent labeling of motor neurons (anti-choline acetyltransferase) on a cross section of the lumbar spinal cord region of healthy control mice (vehicle control);

FIG. 7B is an image showing immunofluorescent labeling of motor neurons (anti-choline acetyltransferase) on spinal cord lumbar region cross-sections of SMA mice treated with vehicle from P0 to P11 (SMA vehicle);

FIG. 7C is an image showing immunofluorescent labeling of motor neurons (anti-choline acetyltransferase) on a cross section of the lumbar region of the spinal cord of SMA mice (SMA BIO101) treated with BIO101 from P0 to P11;

figure 7D shows histograms of total number of motoneurons/slice/hemiventral spinal cord for healthy control mice (vehicle control), SMA treated with vehicle from P0 to P11 (SMA vehicle), SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

figure 7E shows histograms of the number of lateral and distal motoneurons/slice/semiventral spinal cord for healthy control mice (vehicle control), SMA treated with vehicle from P0 to P11 (SMA vehicle), SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

fig. 7F is a histogram showing motor neuron distribution according to the surface of its soma by sectioning the hemiventral spinal cord of healthy control mice (vehicle control), SMA treated with vehicle from P0 to P11 (SMA vehicle), SMA treated with BIO101 from P0 to P11 (SMA BIO 101);

FIG. 8 is a histogram showing the number distribution of different types of motor neurons (slow, intermediate and fast) in groups of healthy control mice (vehicle control n.gtoreq.5), SMA treated with vehicle from P0 to P11 (SMA vehicle, n.gtoreq.5), or SMA treated with BIO101 from P0 to P11 (SMA BIO101, n.gtoreq.6); slow motor neurons are characterized by the expression of the ChAT + ERR β + marker, fast motor neurons are characterized by the expression of the ChAT + MMP9+ marker, and intermediate motor neurons are characterized by the expression of the ChAT + ERR β -MMP 9-marker. Significance was considered to be achieved when p < 0.05. ") indicates significant differences compared to vehicle control mouse groups. "#" indicates a significant difference compared to the SMA vehicle mouse group.

FIG. 9A shows photographs of immunofluorescent labeling of neuromuscular junctions with different markers pre-synaptic (nerve filament and SNAP25) and post-synaptic (face post-synaptic) (alpha-bungarotoxin). Staining with bisbenzimide allowed identification of the nucleus. The scale bar represents 10 μm.

Figure 9B is a histogram showing the percentage distribution of different types of neuromuscular junctions according to their maturation status (in the so-called "pretzel" shape: mature; porous: during maturation; or uniform: immature) in the plantar muscles of the group of healthy control mice (vehicle control, n-4), SMA treated with vehicle from P0 to P11 (SMA vehicle, n-4) or SMA treated with BIO101 from P0 to P11 (SMA BIO101, n-4). ") indicates significant differences compared to vehicle control mouse group (p < 0.05).

Figure 9C is a histogram showing the percentage distribution of different types of neuromuscular junctions according to their maturation status (in the shape of a so-called "pretzel": mature; porous: during maturation; or uniform: immature) in the soleus muscles of the group of healthy control mice (vehicle control, n-4), SMA treated with vehicle from P0 to P11 (SMA vehicle, n-4) or SMA treated with BIO101 from P0 to P11 (SMA BIO101, n-4). ") indicates significant differences compared to vehicle control mouse group (p < 0.05). "#" indicates a significant difference compared to the SMA vehicle mouse group.

Figure 9D is a histogram showing the percentage distribution of different types of neuromuscular junctions in the tibialis anterior muscle in the groups of healthy control mice (vehicle control, n-4), SMA treated with vehicle from P0 to P11 (SMA vehicle, n-4) or SMA treated with BIO101 from P0 to P11 (SMA BIO101, n-4) according to their maturation state (in the so-called "pretzel" shape: mature; porous: during maturation; or uniform: immature). ") indicates significant differences compared to vehicle control mouse group (p < 0.05). "#" indicates a significant difference compared to the SMA vehicle mouse group.

Fig. 10A shows motor performance of healthy control mice (vehicle control, n-13), SMA treated with vehicle from P0 to P9 (SMA vehicle, n-11), or SMA treated with BIO101 from P0 to P9 (SMA BIO101, n-12) evaluated by the open field test on days P5, P7, and P9. The numbers of "═ indicates significant differences compared to the vehicle control mouse groups (═ p < 0.05, ═ p < 0.01, ═ p < 0.001, and ═ p < 0.0001).

Fig. 10B shows muscle fatigue of healthy control mice (vehicle control, n-17), SMA treated with vehicle from P0 to P9 (SMA vehicle, n-11), or SMA treated with BIO101 from P0 to P9 (SMA BIO101, n-10) evaluated by the grip test on days P5, P7, and P9. The numbers of "═ indicates significant differences compared to the vehicle control mouse groups (═ p < 0.05, ═ p < 0.01, and ═ p < 0.001).

Figure 11A shows the body weight curves of SMA mice treated from P0 to P11 with vehicle or with BIO 103;

FIG. 11B is a Kaplan-Meier representation of the pre-P11 survival curves of SMA mice treated with vehicle or with BIO101 from P0 to P11;

fig. 12A is a histogram showing the myofiber cross-sectional surfaces of the tibialis anterior (tibialis) of healthy control mice (vehicle control), SMA treated with vehicle from P0 to P11 (SMA vehicle), or SMA treated with BIO103 from P0 to P11 (SMA BIO 101);

fig. 12B is a histogram showing the muscle fiber cross-sectional surface of the plantar muscle of healthy control mice (vehicle control), SMA treated with vehicle from P0 to P11 (SMA vehicle), or SMA treated with BIO103 from P0 to P11 (SMA BIO 101);

fig. 12C is a histogram showing the muscle fiber cross-sectional surface of soleus muscle for healthy control mice (vehicle control), SMA treated with vehicle from P0 to P11 (SMA vehicle), or SMA treated with BIO103 from P0 to P11 (SMA BIO 101);

fig. 13A is a histogram showing the muscle fiber distribution according to cross-sectional surface of tibialis anterior (tibialis) of SMA mice treated with vehicle from P0 to P11 (SMA vehicle) or SMA treated with BIO103 from P0 to P11 (SMA BIO 103);

fig. 13B is a histogram showing the muscle fiber distribution as a function of cross-sectional surface distribution of the plantar muscles of SMA mice treated with vehicle from P0 to P11 (SMA vehicle) or SMA treated with BIO103 from P0 to P11 (SMA BIO 103);

fig. 13C is a histogram showing the muscle fiber distribution of soleus muscle from P0 to P11 SMA mice treated with vehicle (SMA vehicle) or from P0 to P11 SMA treated with BIO103 (SMA BIO103) according to cross-sectional surface distribution.

Detailed description of the preferred embodiments

The invention will be described below in the specific context of some preferred and non-limiting fields of application of the invention.

1.BIO101 purification method

BIO101 was prepared from 90% pure 20-hydroxyecdysone according to the following procedure:

i) dissolving 90% pure 20-hydroxyecdysone in methanol, filtering and partially concentrating,

ii) 3 volumes of acetone were added,

iii) cooling to a temperature of from 0 ℃ to 5 ℃ with stirring,

iv) filtering the obtained precipitate,

v) continuous rinsing with acetone and water, and

vi) drying.

This purification involves a recrystallization process suitable for the molecule and capable of production on an industrial scale.

The filtration of step i) was performed through a 0.2 μm particle filter.

The partial concentration of step i) is advantageously carried out by vacuum distillation in the presence of MeOH at a temperature of about 50 ℃.

The drying of step vi) is carried out in vacuo at a temperature of about 50 ℃.

2.Synthesis method of BIO103

BIO103 was obtained by semi-synthesis from 20-hydroxyecdysone followed by purification to pharmaceutical grade according to the following preparation:

[ solution 3]

3-step synthesis of BIO 103:

I) oxidative cleavage of the side chain between carbons C20 and C22 of 20-hydroxyecdysone to obtain a presterone (a protocol known to the person skilled in the art),

II) introduction of a bromine atom at the C21 position,

III) reacting the bromine compound thus obtained with ethanethiol.

3.Biological Activity of BIO101 and BIO103

Phenotypic analysis of the effects of BIO101

A mouse model of severe SMA was used on the FVB/NRj gene pool, characterized by the failure of exon 7 of the murine gene Smn and expression of 2 copies of the human transgenic SMN2 (Smn)^Δ7/Δ7；huSMN2^+/+) (Hsieh et al, 2000). These hybrids produce a polypeptide having the formula "FVB/NRj-Smn^Δ7/Δ7huSMN2^+/+Mice of the 2 copy "genotype are described as" SMA ". These mice are characterized by progressive growth failure observed starting 4 days after birth, with motor neuron degeneration (progressive muscular atrophy) at the ventral angle of the spinal cord of about 50% at the end of life and a mean lifespan of about 12 days (Hsieh et al, 2000). Having "FVB/NRj-Smn^+/Δ7huSMN2^+/+Mice of the 2 copy "genotype have no specific phenotype and are used as so-called" control "mice. Mice were treated by gavage with the molecule BIO101 complexed with a vehicle, in this case cyclodextrin, or with Vehicle (VH) alone at a dose of 50mg/kg per day. Body weight and survival were analyzed daily until P11. In the remainder of the description, n corresponds to the sample size and p corresponds to the "p value" used to quantify the statistical significance of the result.

The results show that in mice orally treated daily from birth, BIO101 alone (n-18, where n is the sample size) makes it possible to significantly limit (P < 0.05) the loss of animal body weight seen from 9 days after birth (fig. 1A) and significantly reduce (P < 0.05) the mortality rate before P11 of treated animals (fig. 1B) compared to vehicle treated animals (n-22).

Analysis of muscular trophism of BIO101

At P11, one hour after the mice's last gavage, the treated mice were anesthetized with 1% pentobarbital at 6 μ L/g mice, and then the extensor soleus muscle (with mixed typology), extensor metatarsus (with fast typology), and flexor tibialis muscle (with fast typology) were sampled for histological or molecular studies.

After sampling, soleus, metatarsus and tibialis were each contained in a preservation matrix and then frozen in chilled isopentane. For each muscle, a 10 μm thick medial cross-section was achieved. These sections were stained with hematoxylin-eosin, dehydrated and mounted in inclusion matrices. Images of these sections were taken with a microscope (magnification x 200). In order to obtain an image with a relief impression, a differential interference contrast technique is used. From these images, the number of muscle fibers of each muscle and a cross-sectional surface of 20% of these fibers were calculated using image processing software (fig. 2).

Histological analysis of sampled muscle sections stained with hematoxylin-eosin showed a beneficial effect of BIO101 on muscular hypoplasia (number of muscle fibers). In fact, in the tibialis muscle, the number of muscle fibers in SMA mice treated with BIO101 was significantly increased compared to the number of fibers in animals treated with vehicle (2358 and 2069 fibers (+ 14%), p < 0.05), respectively).

Interestingly, regardless of the nature and typology of the muscle, the muscle atrophy present in SMA mice is limited by the treatment. In fact, treatment with BIO101 makes it possible to significantly limit the atrophy of the three muscle flexors or extensors studied.

Myofiber atrophy in SMA background (fig. 3A) was indeed visible on tibialis histological sections (fig. 3B) relative to healthy control mice, and quantification of the myofiber cross-sectional surface indicated that this atrophy was 56.1% in the tibialis of SMA mice (n-4) relative to control mice (n-4) (fig. 3D). This atrophy was significantly reduced (P < 0.05) to 32.7% at P11 due to treatment with BIO101(n ═ 4) (fig. 3C and 3D). The same applies to the plantar muscles, where 46% of significant muscle fiber atrophy (p < 0.05) was observed in SMA mice compared to healthy control mice and where treatment with BIO101 significantly limited this atrophy to 17.9% (p < 0.05) (fig. 3E); and is equally applicable to soleus muscle (SMA mice have 50.2% muscle fiber atrophy compared to the control (p < 0.01) compared to 37.8% with BIO101 treatment (p < 0.05)) (fig. 3F).

To perform a more detailed analysis of the effect of BIO101 on atrophy, the distribution of muscle fibers by cross-sectional surface class was evaluated. Muscle fibers have different cross-sectional surfaces depending on their properties. The size of type I fibers characterized by slow shrinkage is smaller than the size of type II fibers characterized by fast shrinkage. The significant reduction in atrophy caused by the molecule BIO101 observed with the treatment of SMA mice from P0 to P11 resulted in a significant effect on the distribution of the fibers according to their cross-sectional surface in the three muscles studied for the SMA mice treated with BIO101 compared to the SMA mice treated with vehicle (fig. 4A, 4B and 4C). Cross-sectional surfaces of 400 to 800 μm were observed in the tibialis of SMA mice treated with BIO101 from P0 to P11, compared to the SMA vehicle mouse group²With a cross-sectional surface of 100 to 200 μm²The fiber fraction of (A) was significantly reduced (p < 0.05) (FIG. 4A). In the plantar and soleus muscles of SMA treated with BIO101 from P0 to P11, a plantar muscle cross-sectional surface of 200 to 400 μm was observed²Is increased with a cross-sectional surface of the metatarsal muscle of 0 to 100 μm²The fiber ratio of (A) and (B) of (B) and a soleus muscle cross-sectional surface of 100 to 200 μm²The reduction of the fiber ratio of (2) (fig. 4C).

c. Molecular analysis

Previous studies have made it possible to demonstrate insufficient activation of the AKT/CREB pathway and over-activation of the ERK/Elk-1 pathway with "FVB/NRj-Smn^Δ7/Δ7huSMN2^+/+Low expression of SMN protein at the ventral spinal cord corner of 2-copy "transgenic mice used as a model of severe spinal muscular atrophy type II mice, indicating a role of these pathways in disease (branch et al, 2013).

The frozen plantar muscles were homogenized in extraction buffer by mechanical milling. The supernatant containing the protein extract was sampled and the protein extract was then quantified according to the Lowry protein assay. The electrophoresis was performed on SDS-PAGE gels, and then the separated proteins were transferred to a membrane. The primary antibody used was as follows: mouse monoclonal anti-SMN (1: 5000), rabbit polyclonal anti-Ser 473 phospho-AKT (1: 1000), rabbit polyclonal anti-AKT (1/100), rabbit monoclonal anti-phospho-ERK 1/2 (1: 500), anti-MAP kinase 1/2(ERK 1/2) (1: 1000). After rinsing, the membranes were incubated with either an anti-mouse (1: 5000) or an anti-rabbit (1: 5000) secondary antibody conjugated to peroxidase. After the primary antibody was used, the antibody-antigen complex was destroyed by incubation in the dissociation solution, and then the membrane was again incubated with rabbit anti-AKT and anti-MAP kinase 1/2(ERK 1/2) antibody (1: 1000). The antibody complexes are visualized by chemiluminescence and imaged by a digital image acquisition device of a gel, membrane or film sample. The optical density of each specific band was quantified with image processing software by subtracting background and by normalization with the optical density of the β -actin band. For the vehicle treated controls, the obtained values were determined to be 1 and the values of the other groups were normalized to these controls and expressed as relative amounts. Animals from each group were obtained by performing independent experiments and by using different membranes on which each group was compared to controls. Quantitative values for pAKT at the plantaris muscles represent 3 mice/group treated with vehicle (control or SMA) and 4 mice treated with BIO 101. Quantitative values for pERK at the plantaris muscle indicate at least 4 mice/group. The quantitative value of SMN means n-2. For quantification of pAKT at spinal cord, they represent n2 mice/group for pAKT and pERK, and n 4/group for levels of SMN.

Molecular analysis in the plantaris muscle determined that the AKT pathway was indeed poorly activated in SMA mice (fig. 5A and 5B), beneficial to the ERK pathway (fig. 5C and 5D) compared to vehicle control mice (CTL VH) (Branchu et al, 2013). Treatment with BIO101 for 11 days allowed to reverse this relationship in the muscle of P0 to P11 treated mice by very significantly increasing AKT phosphorylation (pAKT) (fig. 5B), while ERK phosphorylation (pERK) levels were significantly reduced (fig. 5D). This beneficial and proven beneficial situation is associated with overexpression of SMN proteins (Branchu et al, 2013). As expected, SMN protein was hardly expressed in SMA animals compared to healthy control animals. However, and entirely surprisingly, no change in SMN expression levels could be observed after treatment with BIO101, indicating the original molecular regulation (fig. 5E and 5F).

This balance of signaling pathways between ERK and AKT was also found at the spinal cord of SMA mice. In fact, a significant decrease in AKT phosphorylation (pAKT) was observed in SMA mice (fig. 6A and 6B), while ERK phosphorylation (pERK) was increased (fig. 6C and 6D) compared to healthy control mice (CTL VH). SMA mice were treated with BIO101 daily for 11 days, partially restoring this balance in the animal spinal cord by increasing pAKT levels and decreasing pERK levels (fig. 6B and 6D). It is expected that in this model, SMN protein is not expressed in SMA mice. As observed in the plantar muscle, treatment with BIO101 failed to restore SMN levels in SMA mice treated over 11 days (fig. 6E and 6F).

d. Analysis of motor neurons

Healthy control mice or motor neuron populations on thick sections of the lumbar spinal area (L1 to L5) of SMA treated with vehicle or BIO101 for 11 days were studied quantitatively and qualitatively by immunofluorescent labeling with choline acetyltransferase (ChAT), e.g., as described above (Biondi et al, 2008; Boyer et al, 2013). This analysis was then refined by studying different motor neuron subpopulations, by characterizing their location in the spinal cord (lateral or medial) and their distribution of cell size.

The anesthetized mice were intracardiac infused with PBS. The spinal cord of the mice was taken, fixed, and then washed. The lumbar region of the spinal cord was coated in 4% agarose solution (L1 to L5). A 50 μm section was prepared using a vibrating microtome over the entire length of the sample. One section of each 5 spinal cord sections was then used for immunohistochemical analysis. After saturation with 0.1M glycine, the tissues were then permeabilized, blocked, and then goat polyclonal primary anti-choline acetyltransferase (ChAT) (1/400)^e) And (4) marking. Sections were then washed and then treated with anti-goat polyclonal secondary antibody conjugated with cyanine 3 (1: 400)^e) And (4) incubating. Bis-benzimide (1/1000) was used^e) The nuclei were labeled and then the sections were washed again and then blocked with a photobleaching inhibitor of a fluorescent dye.Since the control labeling was performed in the absence of primary antibody, the specificity of the labeling was verified.

The images are obtained using a camera mounted on a microscope with a magnification of x 200 and coupled with a central processing unit, in particular of the microcomputer type, containing suitable software for image acquisition. All counts were performed using image processing software.

In immunofluorescence, motor neurons labeled with anti-ChAT antibodies can indeed be identified in the figure in light gray at the ventral angle of the spinal cord (fig. 7A, 7B and 7C). The number of motor neurons per section of the hemiventral spinal cord was determined for each of the three mouse groups. It was expected that the number of motor neurons in SMA mice (n-5) was significantly reduced compared to the number of motor neurons found in the group of healthy control mice (n-5) (fig. 7A) (fig. 7B). In fact, this motor neuron degeneration was 25% (p < 0.05) between the two groups (FIG. 7D). In a highly interesting manner, it was observed that the number of motor neurons in these SMA mice (fig. 7C) was significantly higher (p < 0.05) than in SMA mice treated with vehicle (n ═ 5) after 11 days of daily treatment with BIO 101. Thus, it was observed that treatment with BIO101 significantly limited the degeneration of motor neurons due to pathology and that this treatment exerted a significant neuroprotective effect, wherein the loss of motor neurons in the group treated with molecular BIO101 was limited to 13% (fig. 7D).

The quantitative analysis is then refined by studying, on the one hand, the different motor neuron subpopulations, by analyzing their position in the spinal cord (lateral or medial position), and, on the other hand, by studying the distribution of their cell body surface.

Higher numbers of lateral motor neurons (innervating distal muscles) were observed in SMA mice treated with BIO101 compared to SMA mice that had received the vehicle, without significant effect on the medial motor neurons (fig. 7E).

As expected, analysis of the surface of the motor neuron cell showed that atrophy of motor neurons in vehicle-treated SMA mice was less than 600 μm on the surface of the motor neuron cell compared to healthy control mice²Is significantly increased (p < 0.05). At the same time, it was observed in SMA mice that the surface had a size greater than 900 μm²Loss of motor neurons from the soma (p < 0.05) (FIG. 7F). Treatment with BIO101 makes it possible to significantly limit the cell surface to less than 300 μm²The number of small motor neurons to limit this atrophy of motor neurons (p < 0.05) (fig. 7F). It is important to note that in the framework of an SLA it is described that the movement unit is also not affected by the pathological process. In fact, the pre-symptomatic murine model of the disease reveals a preferential degeneration of the FF (rapid fatigue) type motor unit, invoking motor neurons to function with the soma with a large surface (Pun et al, 2006). Similar differential denaturation has also been reported in patients (Dengler et al, 1990; Theys, Peeters et Robberecht, 1999). Thus, in the framework of pathologies such as SLA, it may be an interesting approach to reduce the proportion of motor neurons with small surfaces in favor of those with larger somal surfaces.

In a manner complementary to the quantitative analysis of the number of motor neurons and their size of the cell bodies, and to the qualitative study on their location (medial or lateral in the ventral spinal cord), we studied the effect of the treatment by BIO101 on the protection of subpopulations of motor neurons (slow motor neurons, intermediate motor neurons and fast motor neurons). This motor neuron subpopulation qualitative analysis was performed on thick sections of the lumbar spinal cord region (L1 to L5) of control or SMA mice treated with or without BIO101 by: immunofluorescent labeling with choline acetyltransferase, as described for example above (Biondi et al, 2008; Branchu et al, 2013), and analysis of the type of motor neuron by immunofluorescent co-labeling with Estrogen-related receptor-beta (ERR β) (a specific marker for slow motor neurons) or Matrix metallopeptidase 9 (MMP 9) (a specific marker for fast motor neurons).

The anesthetized mice were intracardiac infused with PBS. The spinal cord of the mice was taken, fixed, and then washed. The lumbar region of the spinal cord was coated in 4% agarose solution (L1 to L5). Over the entire length of the sampleA50 μm section was prepared using a vibrating microtome. One section of each 5 spinal cord sections was then used for immunohistochemical analysis. After saturation with 0.1M glycine, the tissue was then permeabilized, blocked, and then labeled with the following primary antibody: goat anti-ChAT antibody (1/400e), mouse anti-ERR beta antibody (1/400)^e) Rabbit anti-MMP 9 antibody (1/600)^e). After three washes, 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). Nuclei were labeled with bis-benzimide (1/1000e), and sections were washed again and then mounted with a photobleach inhibitor of a fluorescent dye. Since the control labeling was performed in the absence of primary antibody, the specificity of the labeling was verified.

The images are obtained using a camera mounted on a microscope with a magnification of x 200 and coupled with a central processing unit, in particular of the microcomputer type, containing suitable software for image acquisition. All counts were performed using image processing software.

The number of slow motor neurons (ChAT + ERR β +) did not change significantly in the group of either healthy mice treated with vehicle or SMA mice treated with vehicle or with BIO101 (fig. 8). A significant loss in the number of mean fastmoving neurons (ChAT + MMP9+) compared to healthy control mice was observed in the vehicle-treated group of SMA animals (11 and 16, respectively; p < 0.05). This loss occurred to benefit the intermediate type of motor neurons (ChAT + ERR β -MMP9-), the number of which was significantly higher in the SMA carrier mouse group compared to the number present in healthy control mice (7 vs 4; p < 0.05).

Interestingly, treatment of SMA mice with BIO101 preferentially facilitated survival of the fast type of motor neurons (ChAT + MMP9 +). In fact, the number of fastmoving neurons was significantly higher in the group of SMA mice treated with BIO101 than in the group of SMA mice that had received the vehicle (14 vs. 11; p < 0.05).

Thus, treatment with BIO101 limited the motor neuron loss observed in this severe SMA model, particularly by protecting mice from fast motor neuron loss.

e. Analysis of neuromuscular junctions

SMA is characterized by specific neuromuscular junction changes induced by the absence of SMN proteins and denervation induced by specific motor neuron death (Kariya et al, 2008; Biondi et al, 2008; Chali et al, 2016). We performed morphological studies of the neuromuscular junction to determine the degree of maturation and fragmentation of the so-called "pretzel" mature structure. To this end, we specifically labeled presynaptic (synaptophysin and neurofilaments) and postsynaptic (α -bungarotoxin) by immunofluorescence on torn muscle fibers of soleus, metatarsus and tibialis of SMA mice treated or not with the molecule BIO101 (Leroy et al, 2014).

Longitudinal sections of 75 μm thickness were achieved with a vibrating microtome. The sections were then saturated in 0.1M glycine with low agitation and then washed with PBS. It was then blocked and permeabilized with a solution of PBS-BSA 4% -goat serum 5% -triton 0.5%. Anti-neurofilament (1/800)^e) And anti-synaptophysin (SNAP 25; 1/200^e) Primary antibody (to determine presynaptic of neuromuscular junction) was incubated for 48 hours and secondary antibody (anti-lapin) was used647；1/400^e) Displayed, then washed. Finally, the slices are then combined555(1/500^e) Incubation with directly conjugated anti-alpha-bungarotoxin. Sections were washed and nuclei were labeled with bis-benzimide (1/1,000e) and then mounted between coverslips and slides with photobleach inhibitors of fluorescent dyes for observation in microscopic epifluorescence imaging (fig. 9A).

The neuromuscular junction is defined and quantified according to three categories (from least mature to most mature: uniform plate, perforated or "pretzel" shape).

Of all muscles studied (metatarsals, soleus and tibialis), the percentage of uniform neuromuscular junctions in SMA animals was significantly increased relative to healthy control mice. This delayed maturation of the neuromuscular junction is expected and has been described in the literature (Biondi et al, 2008). In fact, the percentage of immature neuromuscular junctions at P10 was 89.7% in the extensor fastigii plantaris muscle relative to 49.7% (P < 0.05) in healthy control mice (fig. 9B), 84.3% in the extensor slowly soleus muscle relative to 45.7% (P < 0.05) in healthy control mice (fig. 9C), 69.3% in the flexor tibialis muscle relative to 28.7% (P < 0.05) in healthy control mice (fig. 9D). When SMA mice were treated daily with BIO101 from their birth to P10, more significant maturation was observed in the neuromuscular junction of all tested muscles, a reduction in the percentage of immature plates, benefiting the well plates, demonstrating the acceleration of maturation. In fact, 16.3% of the linkers were of the poromeric type in the metatarsal muscle, compared to 10.4% in SMA animals that had received the vehicle (p ═ ns) (fig. 9B). This difference was significant in soleus muscle (26.5% for the porous linker in the SMA group compared to 15.7% in the vehicle-treated SMA mouse group, p < 0.05, fig. 9C) and tibialis muscle (45.7% for the porous linker in the treated SMA group compared to 30.7% in the vehicle-treated SMA mouse group, p < 0.05, fig. 9D).

Thus, these results indicate that treatment with BIO101 accelerated the maturation of the neuromuscular junction.

f. Analysis of motor Capacity in Severe SMA mouse model

Phenotypic analysis was performed on type 2 severe SMA mice, with or without treatment with BIO101, starting from P0. Mice were tested for motor ability every two days longitudinally from P5 to P9. The inventors evaluated spontaneous displacement capacity by an open field test and muscle fatigue by a grip test, such as described above (Biondi et al, 2008; Branchu et al, 2013; Chali et al, 2016).

The devices used for the open field test are different according to the age of the mice. For animals P0 to P6, it consisted of a 15 x 5cm plastic box with a grid pattern divided into 25 fields of 3cm x 3cm squares. For animals P7 to P21, it consisted of a 28 x 5cm plastic box with a grid pattern divided into 16 fields of 7cm x 7cm squares. Mice were tested individually and the evaluation device was washed after each session. Each mouse initially placed at the center of the field was able to move freely for 5 minutes. The experimenter recorded the behavioral measurements during these 5 minutes and recorded the total number of squares crossed.

It is expected that SMA mice treated with vehicle showed a significant reduction in motor performance compared to healthy control mice at all test times (P5, P7, and P9) (fig. 10A). In fact, the number of squares they were able to cross was 10 at P5, 6 at P7 and 8 at P9, compared to 17 at P5, 12 at P7 and 24 at P9 in the healthy control group of mice (P < 0.01, P < 0.0001 and P < 0.001, respectively). At P5, treatment of SMA mice with BIO101 did not improve their motor performance (11 squares) compared to vehicle-treated SMA mice (10 squares). At P7, mobility was significantly improved in mice treated with BIO101 (P < 0.05 across 10 squares) compared to SMA mice that had received the vehicle (across 6 squares). This difference was not significant at P9, but treatment with BIO101 tended to have a beneficial effect, with mice in the SMA mouse BIO101 group being able to cross 11 squares compared to 8 squares (P ═ ns) in the SMA vehicle group.

To evaluate muscle fatigue, the grip force of the hind feet of the mice was tested from P5 to P9 (grip test). The mice were suspended by their hind legs on thin metal rods that were suspended horizontally in the air. The time spent hanging is recorded. Five consecutive trials were performed per mouse with a one minute rest period between the two tests. Only the best test was retained for evaluating muscle function.

Muscle fatigue tested by the grip test showed that SMA mice treated with vehicle were expected to have significantly reduced muscle function compared to healthy control mice (fig. 10B). In fact, they were able to remain suspended on the metal rod for 0.2 seconds at P5, 6.2 seconds at P7 and 5.6 seconds at P9, compared to 3.3 seconds at P5, 11.4 seconds at P7 and 16.1 seconds at P9 in the group of healthy control mice (P < 0.01, P < 0.01 and P < 0.001, respectively). At P5, SMA mice treated with BIO101 significantly improved their muscle performance (2.3 seconds; P < 0.05) compared to vehicle-treated SMA mice (0.2 seconds). At P7, the suspension time tended to increase, but not significantly (12.2 and 6.2 seconds, respectively, P ═ ns) for SMA mice treated with BIO101 compared to SMA mice that had received the vehicle. Finally, at P9, BIO101 improved this parameter very significantly in mice treated with BIO101 (20.9 seconds, P < 0.01) compared to SMA mice treated with vehicle (5.6 seconds).

In conclusion, BIO101 had a beneficial effect on the voluntary motility and muscle fatigue of SMA animals.

Phenotypic analysis of the effects of BIO103

In the severe SMA model, the same type II murine model was used to characterize the phenotypic effect of the molecule BIO 103. Mice were treated by gavage with the molecule BIO103 complexed with a vehicle, in this case a cyclodextrin, or with Vehicle (VH) alone at a dose of 50mg/kg per day. Body weight and survival were analyzed daily until P11.

The results show that BIO103 (n-12) tends to limit the weight loss of the animals in mice orally treated daily from birth compared to vehicle treated animals (n-22), although the difference did not reach the significance threshold (fig. 11A). On the other hand, BIO103 significantly (P < 0.05) increased survival before P11 (66.6% survival at P11) in treated animals compared to vehicle-received mice (36.3% survival at P11) (fig. 11B).

Analysis of muscular trophism of BIO103

Histological analysis of hematoxylin and eosin stained sections of sampled muscles showed a beneficial effect of BIO103 on muscle atrophy (surface of sections of muscle fibers).

As with the molecule BIO101, the muscle atrophy present in SMA mice is limited by this treatment regardless of the nature and typology of the muscle. In fact, treatment with BIO103 makes it possible to significantly limit the atrophy of the three muscles studied.

It is expected that quantification of the surface of the muscle fibers shows that this atrophy is very significant and 56.1% in tibialis of SMA mice (n-4) compared to control mice (n-4). This atrophy was significantly reduced (P < 0.05) to 22.1% at P11 due to treatment with BIO103(n ═ 4) (fig. 12A). The same applies to the plantar muscles, in SMA mice 46% of significant muscle fiber atrophy (p < 0.05) was observed compared to healthy control mice, and treatment with BIO103 significantly limited this atrophy to 15.3% (p < 0.05) (fig. 12B); and is equally applicable to soleus muscle (50.2% muscle fiber atrophy compared to control in SMA mice (p < 0.01) compared to 37.1% with BIO103 treatment (p < 0.05)) (fig. 12C).

To perform a more detailed analysis of the effect of BIO103 on atrophy, the distribution of muscle fibers by cross-sectional surface class was evaluated. The significant reduction in atrophy caused by the molecule BIO103 observed with SMA mice treated from P0 to P11 resulted in a significant effect on the distribution of the fibers according to their cross-sectional surface in the three muscles studied for the SMA mice treated with BIO103 compared to the SMA mice treated with vehicle (fig. 13A, 13B and 13C). Cross-sectional surfaces of greater than 400 μm were observed in the tibialis of SMA mice treated with BIO103 from P0 to P11, compared to the SMA vehicle mouse group²With a cross-sectional surface of less than 200 μm²The small fiber fraction of (2) was significantly reduced (p < 0.05) (FIG. 13A). In the plantar muscle (FIG. 13B) and soleus muscle (FIG. 13C) of SMA treated with BIO101 from P0 to P11, a cross-sectional surface greater than 300 μm was observed²With a cross-sectional surface of less than 200 μm²The ratio of muscle fibers is reduced.

4.Conclusion

Given the hypoplastic, dystrophic and degenerative nature of BIO101 and BIO103 in motor neurons of mammals suffering from infantile spinal muscular atrophy, it may be advisable to use phytoecdysones, and in particular BIO101 and BIO103 (alone or as a supplement to therapies aimed at correcting the effects of genetic alterations), to protect muscle tissue as well as motor neurons and thus slow down the changes in neuromuscular diseases with consequences of worsening muscle function and/or loss of motor neurons. Neuromuscular diseases include, in particular, amyotrophic lateral sclerosis and spinal muscular atrophy.

More generally, it should be noted that the modes considered above for carrying out and carrying out the invention are described by way of non-limiting examples, and that other alternatives are therefore conceivable.

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