EP4429668A1

EP4429668A1 - Phytoecdysones and/or 20-hydroxyecdysone derivatives in combination with an active ingredient for restoring smn expression, for use in the treatment of spinal muscular atrophy

Info

Publication number: EP4429668A1
Application number: EP22813970.5A
Authority: EP
Inventors: Mathilde Latil; Cynthia BEZIER; Pierre Dilda; René LAFONT; Stanislas Veillet; Frederic Charbonnier; Olivier BIONDI
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Sorbonne Universite; Biophytis SA; Universite Paris Cite
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Sorbonne Universite; Biophytis SA; Universite Paris Cite
Priority date: 2021-11-10
Filing date: 2022-11-04
Publication date: 2024-09-18
Also published as: FR3128873A1; CA3237013A1; WO2023083718A1

Abstract

The invention relates to at least one phytoecdysone and/or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient having the ability to increase the production of functional SMN protein, for use in a combination therapy in the treatment of spinal muscular atrophy in mammals.

Description

Title of the invention: Phytoecdysones and/or derivatives of 20-hydroxyecdysone in combination with an active principle aimed at restoring SMN expression for their use in the treatment of spinal muscular atrophy

Technical field of the invention

[0001] The invention relates to the use of phytoecdysones and/or hemisynthetic derivatives of 20-hydroxyecdysone, in combination with an active ingredient aimed at restoring the expression of the SMN protein, for the treatment of spinal muscular atrophy .

Prior technique

[0002] Neuromuscular diseases are characterized by an alteration in the functioning of motor units, made up of motoneurons, neuromuscular junctions and skeletal muscles. Whatever the origin of the disease, nervous as in spinal muscular atrophy or amyotrophic lateral sclerosis, or muscular, all cause an impairment of motor function in patients, ranging from disability to premature death when vital muscles are reached.

[0003] Among these neuromuscular diseases, two are described as specifically affecting motor neurons: infantile spinal muscular atrophy (or SMA), the symptoms of which appear in childhood, and amyotrophic lateral sclerosis (or ALS), the symptoms of which adulthood. These two neurodegenerative diseases, with different causes and clinical manifestations, have in common progressive muscle denervation, responsible for amyotrophy (Al-Chalabi and Hardiman, 2013; Crawford and Pardo, 1996).

[0004] Spinal muscular atrophy represents the most common cause of infant mortality of genetic origin with a prevalence of 1/6,000 to 1/10,000 births (Crawford and Pardo, 1996). Three main types of severity are described, depending on the age at onset of symptoms and the progression of clinical damage, ranging from type 1, the most severe, to type 3, whose life expectancy can be over 40 years. The patients Patients with SMA show symmetric damage to the skeletal muscles by atrophy of isolated muscle fibers or grouped into fascicles. Almost all SMAs are predominantly proximal, ie affecting the muscles of the trunk and close to the trunk. Gradually, the motor deficit first spreads to the muscles of the lower limbs, then later to the muscles of the upper limbs, preferentially affecting the extensor muscles. The gene responsible for SMA was identified in 1995 on chromosome 5 and named SMN for “survival of motor neurons” in Anglo-Saxon terminology (Lefebvre et al., 1995).

[0005]Although presenting great clinical heterogeneity, genetic analyzes have been able to demonstrate that all forms of SMA are caused by the homozygous alteration of the telomeric gene SMN1 (Survival Of Motor Neurons), preventing the production of the Smn protein, inducing motor neuron degeneration, muscle atrophy and weakness. In the human genome, there is an inverse centromeric copy of this gene, the SMN2 gene, which can be found in several copies (Lorson et al., 1998), but which only partially compensates for the loss of function of the SMN1 gene. Indeed, SMN2 presents 5 nucleotide differences with SMN1, including one at the level of exon 7, favoring its excision by splicing in 90% of the mRNAs produced by the SMN2 gene. This alternative splicing leads to the production of a truncated and unstable SMNA7 protein. Thus, only 10% of the proteins produced by the SMN2 gene are complete and functional (Lefebvre et al., 1997; Vitte et al., 2007). A link has been demonstrated between the number of copies of the SMN2 gene, their level of expression and the severity of the disease.

[0006] Several therapeutic strategies, at different stages of development, are being explored in proximal spinal muscular atrophy linked to SMN1. Some strategies studied aim to increase the quantity of functional SMN protein, either by modifying the maturation of the SMN2 messenger RNA so that it reintegrates the missing exon 7 (Nusinersen, Risdiplam, Branaplam), or by providing the SMN1 gene by gene therapy (Zolgensma®). [0007] Others aim to slow down the progression of the disease by protecting motor neurons or by improving the functioning of neuromuscular junctions (Salbutamol, Pyridostigmine), or muscle performance (SRK-015, physical training). Nevertheless, the majority of the therapeutic approaches tested in SMA aim to increase the expression levels of the SMN protein, either locally in the central nervous system and/or more generally in other organs, peripherally.

[0008] Gene therapy to deliver the SMN1 gene using adeno-associated viral (AAV) vectors has shown significant beneficial effects in preclinical studies in mice, and in clinical trials in humans (Mendell et al. 2017; Passini et al., 2010; Lowes et al., 2018). Other approaches focusing on the modulation of SMN2 splicing have also proven their effectiveness (Naryshkin et al., 2014; Palacino et al., 2015; Finkel et al., 2016; Finkel et al., 2017). These very positive results have allowed the regulatory authorization of certain molecules.

[0009] Three treatments are currently available in SMA:

- The first treatment to be authorized in SMA is Spinraza® (nusinersen). This treatment received marketing authorization (AMM) in December 2016 in the United States and in June 2017 in Europe. It is an antisense oligonucleotide (ASO) developed with the aim of increasing the production of functional SMN protein by acting on the maturation (splicing) of the SMN2 gene. Since antisense oligonucleotides do not cross the blood-brain barrier, nusinersen must be administered on a regular basis by the intrathecal route, allowing reexpression of the SMN protein in motor neurons, with a real clinical benefit, but of variable importance depending on the type of SMA and age at start of treatment. Sequences of antisense oligonucleotides having the capacity to modify the splicing of the SMN2 gene are given in applications W02007/002390 and WO2018/014041.

- More recently, Zolgensma® (Onasemnogene abeparvovec or AVXS-101), a gene therapy product aimed at bringing the gene SMN1 using a viral vector (AAV) has been authorized (MA in May 2019 in the United States and a conditional MA in May 2020 in Europe). Its single administration is easier, because it is carried out intravenously.

- Finally, Evrysdi® (Risdiplam or R07034067) was authorized even more recently (MA in August 2020 in the United States and in March 2021 in Europe). It is a small molecule that acts on the maturation of the SMN2 gene. It is administered daily by mouth or by feeding tube.

[0010] Although restoration of the SMN gene resulted in unprecedented improvement in functional measures in patients as well as their motor function, significant deficits persist in patients with SMA after treatment, even if the intervention is early (Mercuri et al., 2018; Finkel et al., 2017; Baranello et al., 2018).

[001 1 ] Preclinical studies have shown that these post-treatment deficits were found in SMA mouse models, where treated animals exhibited reduced life expectancy, deficit in body weight and mass and function. muscles compared to healthy animals (Passini et al., 2010; Hua et al, 2010, 201 1; Feng et al., 2016).

[0012] In addition, there still remain other limitations and significant concerns regarding these treatments. For example, nusinersen must be administered by highly invasive intrathecal injection several times a year. This mode of administration is very difficult and sometimes impossible for patients who have undergone surgery for scoliosis, which excludes nusinersen as a therapeutic option for these patients. In addition, intrathecal administration allows specific distribution to the central nervous system, which means that not all symptoms can be fully covered. On this last aspect, onasemnogen has an advantage, because it is administered intravenously, and allows systemic distribution. Nevertheless, the question of the bioavailability of the AAV9 serotype is still open, as well as the fact that long-term transgene expression should be restricted to postmitotic cells like neurons (Chaytow et al., 2021). Although onasemnogen is advertised as a therapy requiring only one injection, it remains to be seen if the treatment will last for the life of the patients, or if boosters will be necessary.

[0013] Risdiplam, for its part, is a less invasive systemic therapy, by daily oral administration. However, because risdiplam targets the splicing machinery, it may also affect other transcriptions, leading to unknown and uncontrollable off-target side effects. Indeed, risdiplam has been described as appearing to have an effect on a regulator of cell division at high concentrations, raising concerns about oncogenic side effects (Ratni et al., 2018).

[0014] Phytoecdysones represent an important family of polyhydroxylated sterols. These molecules are produced by various species of plants (ferns, gymnosperms, angiosperms) and participate in their defense against insect pests. The major phytoecdysone in the plant kingdom is 20-hydroxyecdysone.

[0015] Patent FR 3 021 318 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 effects on protein synthesis in the muscles are observed in rats in vivo (Syrov et al., 2000; Tôth et al., 2008; Lawrence et al., 2012) and on murine C2C12 myotubes in vitro (Gorelick- Feldman et al., 2008). Some of the effects described above in animal models have been found in clinical studies, which are still few in number. Thus, 20E promotes an increase in muscle mass in young athletes (Simakin et al., 1988).

Finally, French patent FR 19 02726 further describes the use of phytoecdysones and semi-synthetic derivatives thereof for the treatment of neuromuscular diseases, in particular infantile spinal muscular atrophy and amyotrophic lateral sclerosis (Latil and al., 2020).

[0017] The above elements indicate that, even if the therapies which increase the expression of the SMN protein can have important effects on the progression of the disease and the quality of life of the patients, it remains useful to find complementary therapeutic approaches that would make it possible to reduce the administration doses of the treatments already authorized, or their frequency of administration or finally to improve the functional gains in order to further reduce the burden of the disease.

Presentation of the invention

The present invention aims to provide a treatment for spinal muscular atrophy, said treatment being improved compared to existing treatments.

[0019] The inventors have unexpectedly discovered that phytoecdysones (in particular 20E and semi-synthetic derivatives thereof) have a beneficial and synergistic effect when they are used in combination therapy with an active ingredient having the ability to to increase the production of functional SMN protein, in mammals suffering from spinal muscular atrophy. Indeed, the use of phytoecdysones and/or of at least one hemi-synthetic derivative of 20-hydroxyecdysone, in combination with a treatment using an active principle having the capacity to increase the production of functional SMN protein, improves by synergy of these elements motor and functional performance as well as survival and weight of animals with spinal muscular atrophy (SMA).

To this end, the present invention relates to at least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active principle having the ability to increase the production of functional SMN protein, for their use in combination therapy in the treatment of spinal muscular atrophy.

[0021] In particular embodiments of the present invention, the invention relates to at least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active ingredient having the ability to increase the production of functional SMN protein, for use in combination therapy in the treatment of motor neuron disease, said disease participating in SMA.

[0022] In the context of the present application, the term "combination therapy" designates the co-administration of at least two biologically active agents. In the case of the present invention, a first active agent is chosen from phytoecdysones or semi-synthetic derivatives of 20- hydroxyecdysone or is a composition comprising at least one phytoecdysones and/or at least one semi-synthetic derivative of 20-hydroxyecdysone, a second active agent being the active principle having the capacity to increase the production of functional SMN protein. Combination therapy may comprise a single formulation or multiple formulations. The co-administration can be carried out simultaneously or sequentially. The co-administration can be effected by the same route of administration or by different routes of administration. It is considered a combination therapy as long as the effects of the two (or more) agents overlap in the subject to achieve additional, additive or synergistic clinical effects.

In particular embodiments, the invention also responds to the following characteristics, implemented separately or in each of their technically effective combinations.

[0024] In particular embodiments, said at least one phytoecdysone and/or said at least one semi-synthetic derivative of 20-hydroxyecdysone, and said at least one active principle having the capacity to increase the production of functional SMN protein are co-administered.

[0025] In particular embodiments, said at least one phytoecdysone and/or said at least one hemi-synthetic derivative of 20-hydroxyecdysone, and said at least one active principle having the capacity to increase the production of functional SMN protein are co-administered within the same composition.

In particular embodiments, said at least one phytoecdysone and/or said at least one hemi-synthetic derivative of 20-hydroxyecdysone are administered orally or systemically to the mammal, and said at least one active principle having the ability to increase production of functional SMN protein is administered to the mammal orally and/or intrathecally and/or intravenously.

For oral administration, said at least one phytoecdysone and / or said at least one semi-synthetic derivative of 20-hydroxyecdysone and / or the active principle having the ability to increase the production of functional SMN protein, is preferably incorporated into an acceptable pharmaceutical formulation which can be administered orally.

The increase in the production of SMN protein can be obtained by gene therapy or by gene therapy. According to a mode of implementation of gene therapy, the active principle having the capacity to increase the production of protein SMN aims to provide the SMN1 gene. According to one mode of implementation of a gene therapy, the active ingredient having the capacity to increase the production of SMN protein is either a small molecule which acts on the maturation of the SMN2 gene, or an antisense oligonucleotide (ASO) developed in the aim of increasing the production of functional SMN protein by acting on the splicing (maturation) of the SMN2 gene. In the latter case, the ASO modulates the splicing of SMN2 which makes it possible to increase the inclusion of exon 7 in the mRNA of SMN2 and thus makes it possible to increase the number of generated SMN proteins having the acids amines corresponding to exon 7 and therefore corresponding to a functional SMN protein.

[0029] Thus, in particular embodiments of the present invention, the active principle has the capacity to increase the production of functional SMN protein by gene therapy or by gene therapy.

In particular embodiments of the present invention, the active ingredient has the ability to increase the production of functional SMN protein by supplying the SMN1 gene.

[0031] In particular embodiments of the present invention, the active ingredient has the ability to increase the production of functional SMN protein by acting on the maturation of the SMN2 gene.

[0032] In particular embodiments, the active principle having the capacity to increase the production of functional SMN protein is an antisense oligonucleotide (ASO). This ASO preferably has a sequence of 10 to 30 nucleotides, more preferably of 12 to 30 nucleotides, preferentially 12 to 25 nucleotides or even preferentially 15 to 20 nucleotides. According to preferred embodiments of the present invention, ASO has a sequence of 18 nucleotides. In particular embodiments of the present invention, ASO has the ability to induce the inclusion of exon 7 in the sequence of the SMN2 gene.

In particular embodiments of the present invention, the ASO is complementary to at least 50%, preferably at least 60%, preferably at least 70%, more preferably to at least 80%, preferably at least 90%, preferably at least 95%, preferably 98%, preferably 100%, of the sequence of the nucleic acid coding for the pre-mRNA of the human SMN2 gene.

In particular embodiments of the present invention, the ASO is complementary to at least 50%, preferably at least 60%, preferably at least 70%, more preferably to at least 80%, preferably to at least 90%, preferably at least 95%, preferably 98%, preferably 100%, of a sequence belonging to intron 6, to intron 7 or to a part of exon 7 and a part of an intron adjacent to exon 7, nucleic acid encoding the pre-mRNA of the human SMN2 gene. In this way, exon 7 is included in the mRNA of SMN2, which makes it possible to obtain a functional SMN protein.

The sequence of the nucleic acid coding for the pre-mRNA of the human SMN2 gene is available on Genbank under the reference NG_008728, version NG_008728.1.

In particular embodiments, the ASO is a sequence that is at least 50% identical or similar to the sequence SEQ ID NO: 1 given in the sequence listing filed with the present application (5'-TCACTTTCATAATGCTGG-3 '), preferably identical or similar to at least 60%, preferably at least 70%, more preferably to at least 80%, preferably to at least 90% and preferably identical or similar to 100%. The sequence SEQ ID NO: 1 is complementary to 18 bases of intron 7 (bases 10 to 27) of the SMN2 gene.

In particular embodiments, the ASO has a sequence chosen from the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO : 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29. These sequences allow ASO to target intron 7 of the SMN2 gene.

When the ASO sequence has a percentage identity or similarity of less than 100% with respect to one of the sequences listed above, it may have insertions, deletions and/or substitutions with respect to this sequence of reference.

The percentage identity between two ASO sequences is determined by comparing the two optimally aligned sequences, through a comparison window. The part of the sequence of an ASO in the comparison window can thus include additions or deletions with respect to the reference sequence so as to obtain an optimal alignment between the two sequences.

[0041] The percentage of identity is calculated by determining the number of positions for which a nucleic base is identical in the two compared sequences, then by dividing this number of positions by the total number of positions in the comparison window, the number obtained being multiplied by one hundred.

The ASO is preferably composed of a phosphodiester skeleton. It may possess various modifications in its skeleton and/or its chemical structure in order to increase its stability and/or its affinity for RNA and/or to present a significant advantage in terms of pharmacokinetics.

In particular embodiments, the ASO comprises at least one modification chosen from:

- a modification in the phosphate group such as a phosphorothioate, or a methylphosphanate, or a phosphoroamidate,

- a chemical modification in the 2' position of the ribose, such as a 2'0-methyl (2'OMe) or a 2'0-methoxyethyl (2'MOE) or a 2' Fluoro (2'F),

- at least one modification of nucleobases such as pyrimidine methylation of the 5' methylcytosine, 5-methyluridine/ribothymidine or "G-clamp" type, - at least one substantial change in the structure of the sugar, leading to a variety of molecules, such as morpholinos (PMO for “phosphoroamidate morpholino oligomer” in English terminology) or peptide nucleic acids (PNA for “Peptide nucleic acid” in English terminology) Anglo-Saxon) or constrained type oligonucleotides (LNA for "locked nucleic acid" in Anglo-Saxon terminology or cEt for "2'-4'-constrained ethyl" in Anglo-Saxon terminology or tc-DNA for tricyclo-DNA).

In particular embodiments, the ASO is of sequence identical or similar to at least 50% with the sequence SEQ ID NO: 23, preferably identical or similar to at least 60%, preferentially at least 70%, more preferably at least 80%, more preferably at least 90% and preferably the same or similar to 100%. The sequence SEQ ID NO: 23 corresponds to the sequence SEQ ID NO: 1 with 2'-O-methoxyethyl on the 2' carbon atom of the deoxyribose for each base, with a phosphorothioate skeleton, and with 5-methyl cytosines at instead of cytosines. The phosphothioate skeleton advantageously improves the stability of the ASO, the 5-methyl cytosines advantageously make it possible to make the ASO less sensitive to nucleases and the 2'-O-methoxyethyl on the 2' carbon atom of the deoxyribose advantageously allows to reduce the immune response induced by the administration of ASO.

In particular embodiments of the present invention, ASO is administered at a dose of between 0.01 and 10 mg per kilogram in humans. Preferably this dose is administered daily or weekly.

For their use in the present invention, phytoecdysones and semi-synthetic derivatives of 20-hydroxyecdysone are advantageously purified to pharmaceutical grade.

A phytoecdysone that can be used according to the invention is, for example, 20-hydroxyecdysone.

To this end, according to particular embodiments of the present invention, said at least one phytoecdysone is 20-hydroxyecdysone. 20-Hydroxyecdysone and its semi-synthetic derivatives are advantageously purified to pharmaceutical grade.

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

The extracts obtained are preferably purified to pharmaceutical grade.

In one embodiment, the 20-hydroxyecdysone is in the form of a plant extract or of a plant part, said extract comprising at least 95%, and preferably at least 97%, of 20-hydroxyecdysone. Said plant is preferably chosen from plants containing at least 0.5% of 20-hydroxyecdysone by dry weight of said plant. Said extract is preferably purified to pharmaceutical grade.

Said extract is subsequently called BIO101. It remarkably contains between 0 and 0.5%, by dry weight of the extract, of impurities, such as minor compounds, likely to affect the safety, availability or effectiveness of a pharmaceutical application of said extract.

The plant from which BIO101 is produced is preferably chosen from Stemmacantha carthamoides, Cyanotis arachnoidea and Cyanotis vaga.

The semi-synthetic derivatives of 20-hydroxyecdysone are obtained by semi-synthesis and can in particular be obtained in the manner described in European patent application No. EP 15732785.9.

[0056] In a particular embodiment, the phytoecdysones are administered at a dose of between 3 and 15 milligrams per kilogram per day in humans. Phytoecdysones are understood here to mean both phytoecdysones in general and 20-hydroxyecdysone (in particular in the form of an extract) and its semisynthetic derivatives.

Preferably, the phytoecdysones are administered at a dose of 200 to 1000 mg/day, in one or more doses, in an adult human, and a dose of 5 to 350 mg/day, in one or more doses, in the human child or the infant. Phytoecdysone is understood here to mean both phytoecdysone in general and 20-hydroxyecdysone (in particular in the form of an extract) and its semi-synthetic derivatives.

In particular embodiments of the present invention, said at least one semi-synthetic derivative of 20-hydroxyecdysone is chosen from: - a compound of general formula (I):

[Chem. 1 ] in which :

R ¹ is chosen from: a (Ci-C6)W(Ci-Ce) group; a (Ci-C6)W(Ci-C6)W(Ci-C6) group; a (Ci-C6)W(Ci-Ce)CO2(Ci-Ce) group; a (Ci-Ce)A group, A representing a heterocycle optionally substituted by a group of OH, OMe, (Ci-Ce), N(Ci-Ce), CO2(Ci-Ce) type; a CH2Br group;

W being a heteroatom chosen from N, O and S, preferably O and even more preferably S; And,

- a compound being of formula (II):

[Chem. 2]

In the context of the present invention, the term "(Ci-Ce)" means any alkyl group of 1 to 6 carbon atoms, linear or branched, in particular, methyl, ethyl, n-propyl, iso -propyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl. Advantageously it is a methyl, ethyl, iso-propyl or t-butyl group, in particular a methyl or ethyl group, more particularly a methyl group.

In the context of the present invention, the term “heterocycle” preferably means a cycle comprising 5 or 6 atoms including one or two heteroatoms (O, S or N), the remaining atoms being carbon atoms.

In a preferred embodiment of the present invention, in the general formula (I):

R ¹ is chosen from: a (Ci-C6)W(Ci-Ce) group; a (Ci-C6)W(Ci-C6)W(Ci-C6) group; a (Ci-C6)W(Ci-C6)CO2(Ci-C6) group; a (Ci-Ce)A group, A representing a heterocycle optionally substituted by a group of OH, OMe, (Ci-Ce), N(Ci-Ce), CO2(Ci-C ₆ ) type;

W being a heteroatom chosen from N, O and S, preferably O and more preferably S.

In particular embodiments of the present invention, said at least one semi-synthetic derivative of 20-hydroxyecdysone is a compound chosen 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 - pi peridyl)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, ethyl 3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-17-yl]ethyl]sulfanylacetate; 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.

According to another aspect, the present invention relates to a composition comprising:

- at least one phytoecdysone and/or at least one semi-synthetic derivative of 20-hydroxyecdysone,

- at least one active principle having the capacity to increase the production of functional SMN protein, for its use in the treatment of a neuromuscular disease in mammals, in particular spinal muscular atrophy.

This use of the composition can meet one or more of the characteristics described above with reference to the use in therapy combination of said at least one phytoecdysone and/or of at least one semi-synthetic derivative of 20-hydroxyecdysone, and of said at least one active principle having the capacity to increase the production of functional SMN protein.

[0065] In embodiments, the composition is incorporated into an acceptable pharmaceutical formulation that can be administered orally or intrathecally or systemically.

[0066] In the context of the present invention, the term "pharmaceutically acceptable" means that which is useful in the preparation of a pharmaceutical composition, which is generally safe, non-toxic and which is acceptable for veterinary as well as pharmaceutical use. human.

Brief description of figures

The invention will be better understood on reading the following description, given by way of non-limiting example, and made with reference to the figures which represent:

[0068] [Fig. 1] Figure 1 represents the curve of the weight change of SMA mice (Smn ^A7/A7 ; huSMN2 ^+/_ ) treated with BIO101 alone or with a mismatch ASO or with an ASO 10-27 alone, or with an ASO combination 10-27 + BIO101, from birth (PO) until death of mice. Here and in the rest of the description, P corresponds to the number of days after birth (post natal);

[0069] [Fig. 2] Figure 2 is a Kaplan-Meier representation of the survival curves of SMA mice (Smn ^A7/A7 ; huSMN2 ^+/_ ) treated with BIO101 alone or with a mismatch ASO or with an ASO 10-27 alone, or with an ASO 10-27 + BIO101 combination, from birth (PO);

[0070] [Fig. 3] figure 3 represents the motor performances (movement capacities) evaluated by the open field test of the SMA mice (Smn ^A7/A7 ; huSMN2 ^+/_ ) treated with BIO101 alone or with a mismatch ASO or with an ASO 10-27 alone, or with an ASO 10-27 + BIO101 combination, from birth (PO);

[0071] [Fig. 4] figure 4 represents the motor performance (muscle fatigability) evaluated by the grip test of SMA mice (Smn ^A7/A7 ; huSMN2 ^+/_ ) treated with BIO101 alone or with an ASO mismatch or with an ASO 10-27 alone, or with an ASO 10-27 + BIO101 combination, from birth (PO).

For each of the figures, the results are presented as mean ± SEM with *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

In the remainder of the description, n corresponds to the size of the sample and p corresponds to the “p-value” used to quantify the statistical significance of a result. Moreover, p=ns indicates the non-significance of the p-value.

Description of embodiments

The invention will be described below in the particular context of one of its preferred non-limiting fields of application.

[0075] 1 . Description and injection of the antisense oligonucleotide (ASO)

The antisense oligonucleotide (ASO) of sequence SEQ ID NO: 23 (5′-TCACTTTCATAATGCTGG-3′), reference ISIS 396443, also known as ISIS-SMNRx, and called ASO in the remainder of the description 10-27, complementary to 18 bases (bases 10-27) of intron 7 of the SMN2 gene (targeting a site in intron 7, called ISS-N1, which represses inclusion of exon 7 of the Pre-messenger RNA (pre-mRNA) of the SMN2 gene) having 2'-0-methoxyethyl modified nucleotides on the 2' deoxyribose carbon atom for each base, with a phosphorothioate backbone and 5-methyl cytosines and the ASO control (noted mismatch) of sequence SEQ ID NO: 24 (5'-TTAGTTTAATCACGCTCG-3') (Singh et al. 2006; Williams et al. 2009), were synthesized and purified as described previously (Baker et al. 1997, Hua et al., 2008; Passini et al., 2011). The oligonucleotides are re-suspended at a concentration of 8 μg/μL in 0.9% NaCl and stored at -20°C.

On the day of the injection, the stock solution of ASO is diluted to a concentration of 2 μg/μl in 0.9% NaCl. 0.04% methylene blue is added to the solution as an injection control. Newborn SMA mice receive a single dose of ASO 10-27 or ASO mismatch of 8 pg on postnatal ^day 1 (PO) by unilateral intracerebroventricular injection using a Hamilton syringe and a 32G needle. The injection site is located 1 mm from the sagittal suture, between bregma and lambda, landmarks visible through the skin at PO. The needle is positioned perpendicular to the injection site on the surface of the skin and inserted approximately 3mm deep to reach the lateral ventricle.

2. Co-administration of BIO1 Q1

The complementary treatment BIO101 complexed with cyclodextrin is administered daily to young mice from the first postnatal day (PO) orally at a dose of 50 mg/kg using a pipette.

3. Biological activity of BIO1 Q1 in combination with ASO

[0081] a. Analysis of the effects of combined treatment ASO 10-27 + BIO101 on weight and survival

A severe SMA mouse model was used on an FVB/NRj genetic background, characterized by the invalidation of exon 7 of the murine Smn gene and expressing two copies of the human SMN2 transgene (Smn ^A7/A7 ; huSMN2- ^{/ +} ) (Hsieh et al., 2000). The mice resulting from these crosses having the genotype “FVB/NRj-Smn ^A7/A7 huSMN2 ⁷⁺ 2 copies” are described as “SMA” (Hsieh et al., 2000). SMA mice were treated either with BIO101 alone or a mismatch ASO or ASO 10-27 alone, or with a combined ASO 10-27 + BIO101 treatment from PO. Survival (Figure 1) and weight (Figure 2) of the mice were analyzed daily.

The average lifespan of the pups (FIG. 1) treated with BIO101 alone (11 days) is substantially identical to that of the pups treated with the ASO mismatch (13 days). As described in 201 1 by Passini et al., treatment with ASO 10-27 alone at a dose of 8 pg significantly increases the average lifespan of SMA pups, with a median survival of 18 days (improvement of 38 % animal survival), versus a median survival of 13 days in the ASO mismatch control group. When the BIO101 treatment is administered in combination with ASO 10-27, the median survival increases to 22.5 days, i.e. a 73% improvement in animal survival.

The weight of the pups (FIG. 2) treated with BIO101 alone is comparable to that of the pups treated with ASO mismatch throughout their life.

[0085] Treatment with ASO 10-27 alone does not increase the average weight of SMA mice compared to the control treatment with ASO mismatch at P7, but it increases it significantly by 9.9% at P8 and 13.9% at P9. The ASO 10-27 + BIO101 combination induces a synergistic effect on the weight of the SMA young mice with a significant increase of 11.3% at P7, 16.5% at P8 and 24.9% at P9.

At P10, treatment with ASO 10-27 alone significantly increases the average weight of SMA pups (+16%) compared to the control treatment with ASO mismatch. In combination with ASO 10-27, BIO101 induces a synergistic effect on the weight of SMA pups with a significant increase of +30.9% at P10 (p<0.05), i.e. almost twice as high as that observed with ASO 10-27. 'ASO 10-27 alone.

This increase in the body weight of young mice treated with ASO 10-27 + BIO101 compared to that of young mice treated with ASO 10-27 alone continues until their death, with in particular a significant increase of 33% at P15 (p<0.05), by 49.4% at P20 (p<0.05), by 57.6% at P25 (p<0.01).

[0088] b. Analysis of the functional effects of the combined treatment ASO 10-27 + BIO101

We performed phenotypic analyzes of type 2 severe SMA mice treated with BIO101 alone or an ASO mismatch or an ASO 10-27 alone, or with a combined treatment ASO 10-27+BIO101 from PO. We carried out a longitudinal follow-up of the motor abilities of the mice. On the one hand, we evaluated the movement capacities by the open-field test (Figure 3), as well as the muscle fatigability by the grip-test (Figure 4), as described previously (Biondi et al., 2008; Branchu et al., 2013; Chali et al., 2016).

The device used for the open-field test consists of a plastic box measuring 28 x 28 x 5 cm with a grid of the field divided into 16 squares measuring 7 cm x 7 cm. The mice were tested individually and the evaluation device was washed after each session. Each mouse initially placed in the center of the field was allowed to move freely for 5 minutes with tail pinch stimulation every 15 seconds. Behavioral measurements are recorded by the experimenter during these 5 minutes and the total number of cross tiles is recorded. [0091] The BIO101 treatment, whether administered alone or in combination with ASO 10-27, accelerates the acquisition of walking with a tendency to increase the movement of the mice at P15 (1 10 ± 18 squares i.e. +58% with BIO101 treatment alone, and 100±19 squares i.e. +44% with ASO 10-27 + BIO101 treatment) compared to the mice treated with ASO 10-27 alone (70±20 squares).

From P23, the movement capacities of the mice treated with ASO 10-27 + BIO101 tend to be increased compared to those of the mice treated with ASO 10-27 alone (+51.3%, p=ns), and are significantly increased at P25 (+38.4%, p<0.05), or at P27 (+44.9%, p<0.01) (Figure 3).

To assess muscle fatigability, the grip strength of the mice's forepaws was tested. The mice are suspended by their forepaws from a thin metal rod suspended horizontally in the air. The time spent hanging on is recorded. Each mouse was subjected to five successive attempts with a rest period of one minute between two tests. Only the best trial is kept for the evaluation of muscle functions.

At P7, the fatigue resistance of the young mice treated with ASO 10-27 alone is not improved compared to that of the young mice treated with the ASO mismatch. The resistance to fatigue of young mice treated with BIO101 alone is improved by 19.3%. At P15, the resistance to fatigue of the pups treated with ASO 10-27 alone and with BIO 101 alone are both higher (+7.7%) than that of the pups treated with the ASO mismatch. Surprisingly and advantageously, a synergy operates between BIO101 and ASO 10-27 during the combined treatment so that the resistance to fatigue of the treated SMA animals increases by 26.3% at P7 and by 21.2% at P15. .

From P19, the resistance to fatigue of young mice treated with ASO 10-27+BIO101 tends to increase compared with that of young mice treated with ASO 10-27 alone. This difference becomes evident from P21 (+54% in the group treated with the ASO 10-27 + BIO101 combination compared to ASO 10-27 alone, p=ns), then at P23 (+159.2% , p<0.05), at P25 (+337.7%, p<0.05) or at P27 (+296.7%, p=0.07). These differences important are maintained up to P35. From P39, these differences are less marked but the group of mice treated in combination ASO 10-27 + BIO101 nevertheless retains a greater resistance to fatigue than that observed in the group treated with ASO 10-27 alone (Figure 4 ).

[0096] 4. Conclusion

These results demonstrate the advantage of using the BIO101 treatment, in combination with an active principle aimed at restoring the expression of the SMN protein, in particular thanks to a therapeutic approach using ASOs having the effect of restoring SMN protein expression.

[0098] Indeed, this combined therapy shows significant beneficial effects in a mouse model of SMA, in particular at the level of the weight of the animals, and even more importantly, at the level of the physical performance of these animals, whether it is whether the animal's ability to move or its resistance to fatigue.

The combination of ASO 10-27 + BIO101 treatment, beyond improving the performance obtained by the treatment of an ASO 10-27 in monotherapy, undoubtedly has a synergistic effect.

More generally, it should be noted that the embodiments and embodiments of the invention considered above have been described by way of non-limiting examples and that other variants are therefore possible. .

[00101] In particular, the invention has been described mainly considering ASO 10-27 of sequence SEQ ID NO: 23. However, nothing excludes, in other types of embodiment, considering other active principles having the ability to increase SMN protein production like other ASOs having the ability to increase SMN protein production. Such ASOs are for example the ASOs of a sequence chosen from: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29. [00102] These sequences are given in particular in table 1 below:

[Table 1]

[00103] Bibliography

[00104] Al-Chalabi, A., & Hardiman, O. (2013). The epidemiology of ALS: a conspiracy of genes, environment and time. Nature reviews. Neurology, 9(11), 617-628.

[00105] Baker, BF, Lot, SS, Condon, TP, Cheng-Flournoy, S., Lesnik, EA, Sasmor, HM, & Bennett, CF (1997). 2'-0-(2-Methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (ICAM-1 ) oligonucleotides selectively increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 translation initiation complex in human umbilical vein endothelial cells. J. Biol. Chem., 272(18), 1 1994-12000.

[00106] Baranello, G., Servais, L., Day, J., Deconinck, N., Mercuri, E., Klein, A., Darras, B„ Masson, R„ Kletzl, H„ Cleary, Y. and para. (2018) FIREFISH Part 1: early clinical results following a significant increase of SMN protein in SMA type 1 babies treated with RG7916. In the 23rd World Muscle Society Congress, Mendoza, Argentina. Neuromuscular Disorders, Elsevier, Amsterdam, The Netherlands, Vol. 28pp. S109.

[00107] Biondi, O., Grondard, C., Lécolle, S., Deforges, S., Pariset, C., Lopes, P., Cifuentes-Diaz, C., Li, H., della Gaspera, B. , Chanoine, C. & Charbonnier, F. (2008). Exercise-induced activation of NMDA receptor promotes motor unit development and survival in a type 2 spinal muscular atrophy model mouse. The Journal of neuroscience: the official journal of the Society for Neuroscience, 28(4), 953-962.

[00108] Branchu, J., Biondi, O., Chali, F., Collin, T., Leroy, F., Mamchaoui, K., Makoukji, J., Pariset, C., Lopes, P., Massaad, C., Chanoine, C. & Charbonnier, F. (2013). Shift from extracellular signal-regulated kinase to AKT/cAMP response element-binding protein pathway increases survivalmotor-neuron expression in spinal-muscular-atrophy-like mice and patient cells. The Journal of neuroscience: the official journal of the Society for Neuroscience, 33(10), 4280-4294.

[00109] Chali, F., Desseille, C., Houdebine, L., Benoit, E., Rouquet, T., Bariohay, B., Lopes, P., Branchu, J., Della Gaspera, B., Pariset , C., Chanoine, C., Charbonnier, F. & Biondi, O. (2016). Long-term exercise-specific neuroprotection in spinal muscular atrophy-like mice. The Journal of physiology, 594(7), 1931-1952.

[001 10] Chaytow, H., Faller, K., Huang, Y. & Gillingwater, T. (2021 ) Spinal muscular atrophy: From approved therapies to future therapeutic targets for personalized medicine. Cell Reports Medicine, 2(7), 100346.

[001 1 1 ] Crawford, T.O., & Pardo, C.A. (1996). The neurobiology of childhood spinal muscular atrophy. Neurobiology of disease, 3(2), 97-110.

[001 12] Feng, Z„ Ling, KK, Zhao, X., Zhou, C„ Karp, G„ Welch, EM, Naryshkin, N., Ratni, H., Chen, KS, Metzger, F. et al. (2016) Pharmacologically induced mouse model of adult spinal muscular atrophy to evaluate effectiveness of therapeutics after disease onset. Hmm. Mol. Genet., 25, 964-975.

[001 13] Finkel, R.S., Chiriboga, C.A., Vajsar, J., Day, J.W., Montes, J., DeVivo, D.C., Yamashita, M., Rigo, F., Hung, G., Schneider, E. and para. (2016). Treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase 2, openlabel, dose-escalation study. Lancet, 388, 3017-3026.

[001 14] Finkel, R.S., Mercuri, E., Darras, B.T., Connolly, A.M., Kuntz, N.L., Kirschner, J., Chiriboga, C.A., Saito, K., Servais, L., Tizzano, E. et al. . (2017). Nusinersen versus Sham Control in Infantile-Onset Spinal Muscular Atrophy. AT/. English J. Med., 377, 1723-1732.

[001 15] Gorelick-Feldman, J., Maclean, D., Hic, N., Poulev, A., Lila, M.A., Cheng, D. & Raskin, I. (2008). Phytoecdysteroids increase protein synthesis in skeletal muscle cells. Journal of agricultural and food chemistry, 56(10), 3532-3537.

[001 16] Hua, Y., Sahashi, K., Hung, G., Rigo, F., Passini, M.A., Bennett, C.F. & Krainer, A.R. (2010). Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model. Genes Dev., 24, 1634-1644.

[001 17] Hua, Y., Sahashi, K., Rigo, F., Hung, G., Horev, G., Bennett, C.F. & Krainer, A.R. (2011). Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model. Nature, 478, 123-126.

[001 18] Hua, Y., Vickers, T.A., Okunola, H.L., Bennett, C.F., & Krainer, A.R. (2008). Antisense masking of an hnRNP A1/A2 intronic splicing silencer correct SMN2 splicing in transgenic mice. American journal of human genetics, 82(4), 834-848.

[001 19] Hsieh-Li, H. M„ Chang, J. G„ Jong, Y. J., Wu, M. H., Wang, N. M„ Tsai, C. H. & Li, H. (2000). A mouse model for spinal muscular atrophy. Nature genetics, 24(1), 66-70.

[00120] Latil, M., Dilda, P., Lafont, R. & Vei Het, S. (2019). Phytoecdysones and their derivatives for use in the treatment of neuromuscular diseases. Patent Application W02020187678.

[00121 ] Lawrence, MM (2012) Ajuga turkestanica as a countermeasure against sarcopenia and dynapenia. MS thesis, Appalachian State University. [00122] Lefebvre, S., Bürglen, L., Reboullet, S., Clermont, O., Burlet, P., Viollet, L., Benichou, B., Cruaud, C., Millasseau, P. & Zeviani, M (1995). Identification and characterization of a spinal muscular atrophy-determining gene. Cell, 80(1), 155-165.

[00123] Lefebvre, S., Burlet, P., Liu, Q., Bertrandy, S., Clermont, O., Munnich, A., Dreyfuss, G. & Melki, J. (1997). Correlation between severity and SMN protein level in spinal muscular atrophy. Nature genetics, 16(3), 265-269.

[00124] Lorson, C.L., Strasswimmer, J., Yao, J.M., Baleja, J.D., Hahnen, E., Wirth, B., Le, T., Burghes, A.H. & Androphy, E.J. (1998). SMN oligomerization defect correlates with spinal muscular atrophy severity. Nature genetics, 19(1), 63-66.

[00125] Lowes, L., Alfano, L., lammarino, M., Miller, N., Menier, M., Cardenas, J., Sproule, D„ Nagendran, S„ Al-Zaidy, S. & Mendell, J. (2018) AVXS-101 trial experience: CHOP-INTEND effectively quantifies early, rapid, and sustained improvements that precede subsequent milestone achievement but is not sensitive to continued advances in motor function in infants with SMA type 1. In the 23rd World Muscle Society Congress, Mendoza, Argentina. Neuromuscular Disorders, Elsevier, Amsterdam, The Netherlands, Vol. 28 p. S82.

[00126] Mendell, J.R., Al-Zaidy, S„ Shell, R„ Arnold, W.D., Rodino-Klapac, L.R., Prior, T.W., Lowes, L., Alfano, L., Berry, K., Church, K. et al. (2017) Singledose gene replacement therapy for spinal muscular atrophy. N.Engl. J. Med, 377, 1713-1722.

[00127] Mercuri, E., Darras, B.T., Chiriboga, C.A., Day, J.W., Campbell, C., Connolly, A.M., lannaccone, S.T., Kirschner, J., Kuntz, N.L., Saito, K. et al. (2018) Nusinersen versus Sham Control in later-onset spinal muscular atrophy. N.Engl. J. Med., 378, 625-635.

[00128] Naryshkin, N.A., Weetail, M., Dakka, A., Narasimhan, J., Zhao, X., Feng, Z., Ling, K.K., Karp, G.M., Qi, H., Woll, M.G. et al . (2014) Motor neuron disease. SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy. Science, 345, 688-693.

[00129] Palacino, J., Swalley, SE, Song, C., Cheung, AK, Shu, L., Zhang, X., Van Hoosear, M., Shin, Y., Chin, DN, Keller, CG and para. (2015) SMN2 splice modulators enhance U1-premRNA association and rescue SMA mice. Nat. Chem. Biol., 11, 511-517.

[00130] Passini, M. A., Bu, J., Richards, A. M., Kinnecom, C., Sardi, S. P., Stanek, L. M., Hua, Y., Rigo, F., Matson, J., Hung, G., Kaye , E. M., Shihabuddin, L. S., Krainer, A. R., Bennett, C. F., & Cheng, S. H. (2011 ). Antisense oligonucleotides delivered to the mouse CNS ameliorate symptoms of severe spinal muscular atrophy. Science translational medicine, 3(72), 72ra18.

[00131 ] Passini, M. A., Bu, J., Roskelley, E.M., Richards, A.M., Sardi, S.P., O'Riordan, C.R., Klinger, K.W., Shihabuddin, L.S. & Cheng, S.H. (2010) CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy. J. Clin. Invest., 120, 1253-1264.

[00132] Ratni, H., Ebeling, M., Baird, J., Bendels, S., Bylund, J., Chen, K.S., Denk, N., Feng, Z., Green, L., Guerard, M ., et al. (2018). Discovery of Risdiplam, a Selective Survival of Motor Neuron-2 (SMN2) Gene Splicing Modifier for the T reatment of Spinal Muscular Atrophy (SMA). J.Med. Chem. 61, 6501 - 6517.

[00133] Simakin, S. Yu., Panyushkin, V. V., Portugalov, S.N., Kostina, L. V, & Martisorov, E. G. (1998) Combined application of preparation Ecdysten. ScienceBulletin. No. 2, 29-31.

[00134] Singh, N.K., Singh, N.N., Androphy, E.J., & Singh, R.N. (2006). Splicing of a critical exon of human Survival Motor Neuron is regulated by a unique silencer element located in the last intron. Molecular and cellular biology, 26(4), 1333-1346.

[00135] Syrov, V.N. (2000). Comparative experimental investigations of the anabolic activity of ecdysteroids and steranabols. Pharma. Chem. J., 34(4), 193-197.

[00136] Tôth, N., Szabo, A., Kacsala, P., Héger, J. & Zâdor, E. (2008). 20- Hydroxyecdysone increases fiber size in a muscle-specific fashion in rat. Phyto med ici ne: international journal of phytotherapy and phytopharmacology, 15(9), 691-698.

[00137] Vitte, J., Fassier, C., Tiziano, FD, Dalard, C., Soave, S., Roblot, N., Brahe, C., Saugier-Veber, P., Bonnefont, JP & Melki, J. (2007). Refined characterization of the expression and stability of the SMN gene products. The American journal of pathology, 171 (4), 1269-1280.

[00138] Williams, J.H., Schray, R.C., Patterson, C.A., Ayitey, S.O., Tallent, M.K. & Lutz, G.J. (2009). Oligonucleotide-mediated survival of motor neuron protein expression in CNS improves phenotype in a mouse model of spinal muscular atrophy. The Journal of neuroscience: the official journal of the Society for Neuroscience, 29(24), 7633-7638.

Claims

28/34 Claims Claim 1 . At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein, for their use in the treatment of spinal muscular atrophy in mammals . Claim 2. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to claim 1, for their use in the treatment of a condition of the motor neurons responsible for spinal muscular atrophy. Claim 3. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to claim 2, in which the Motor neuron disease is an impairment of the function of motor neurons or their degeneration. Claim 4. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to any one of claims 1 to 3, wherein the active principle has the capacity to increase the production of functional SMN protein by gene therapy or by gene therapy. Claim 5. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to any one of claims 1 to 3, in which the active principle has the capacity to increase the production of functional SMN protein by supplying the SMN1 gene or by acting on the maturation of the SMN2 gene. Claim 6. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to any one of claims 1 to 3, wherein the active ingredient has the ability to increase functional SMN protein production is an antisense oligonucleotide. Claim 7. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to claim 6, in which the antisense oligonucleotide contains 10 to 30 nucleotides. Claim 8. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to any one of claims 6 to 7, in which the antisense oligonucleotide is complementary to at least 90%, preferably at least 95%, preferably at least 98%, preferably 100%, of the sequence of the nucleic acid encoding the pre-mRNA of human SMN2 gene. Claim 9. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to any one of Claims 6 to 8, in which the antisense oligonucleotide is complementary to at least 90%, preferably at least 95%, preferably 98%, preferably 100%, of a sequence belonging to intron 6, to intron 7 or to exon 7 of the nucleic acid encoding the pre-mRNA of the human SMN2 gene. Claim 10. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to any one of claims 6 to 9, in which the antisense oligonucleotide is a sequence identical or similar to at least 50% with the sequence SEQ ID NO: 1, preferably identical or similar to at least 60%, preferentially at least 70%, more preferably to at least least 80%, preferably at least 90% and preferably identical or similar to 100%. Claim 1 1. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to any one of claims 6 to 9, in which the ASO has a sequence chosen from the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 , SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29. Claim 12. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active ingredient having the ability to increase the production of functional SMN protein for its use according to any one of claims 6 to 11, in which the ASO comprises at least one modification chosen from: - a modification at the level of the phosphate group such as a phosphorothioate , or a methylphosphanate, or a phosphoroamidate, - a chemical modification at the 2' position of the ribose, such as a 2'0-methyl (2'OMe) or a 2'0-methoxyethyl (2'MOE) or a 2' Fluoro (2' F), - at least one nucleobase modification such as 5' methylcytosine, 5-methyluridine/ribothymidine, or "G-clamp" type pyrimidine methylation, - at least one substantial change in sugar structure , leading to a variety of molecules, such as morpholinos or peptide nucleic acids or constrained type oligonucleotides. Claim 13. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to any one of Claims 6 to 9, in which the antisense oligonucleotide is a sequence identical or similar to at least 50% with the sequence SEQ ID NO: 23, preferably identical or similar to at least 60%, preferentially at least 70%, more preferably to at least least 80%, preferably at least 90% and preferably identical or similar to 100%. Claim 14. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to any one of claims 1 to 13, wherein said at least one phytoecdysone is 20-hydroxyecdysone. Claim 15. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to claim 14, in which the 20 -hydroxyecdysone is in the form of a plant extract or of a plant part, said extract comprising at least 95%, and preferably at least 97%, of 20-hydroxyecdysone. Claim 16. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to any one of claims 1 to 15, in which said at least one semi-synthetic derivative of 20-hydroxyecdysone is chosen from: - a compound of general formula (I):

[Chem. 1 ] in which :

R ¹ is chosen from: a (Ci-C6)W(Ci-Ce) group; a (Ci-C6)W(Ci-C6)W(Ci-C6) group; a (Ci-C6)W(Ci-C6)CO2(Ci-C6) group; a group (Ci-Ce)A, A representing a heterocycle optionally substituted with a group of the OH, OMe, (Ci-Ce), N(Ci-Ce), CO2(Ci-Ce) type; a CH2Br group;

- a compound being of formula (II):

[Chem. 2]

Claim 17. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to claim 16, in which in general formula (I): R ¹ is chosen from: a (Ci-C6)W(Ci-Ce) group; a (Ci-C6)W(Ci-C6)W(Ci-C6) group; a (Ci-C6)W(Ci-C6)CO2(Ci-C6) group; a (Ci-Ce)A group, A representing a heterocycle optionally substituted by a group of OH, OMe, (Ci-Ce), N(Ci-Ce), CO2(Ci-Ce) type;

Claim 18. At least one phytoecdysone and/or at least one hemisynthetic derivative of 20-hydroxyecdysone, and at least one active principle having the capacity to increase the production of functional SMN protein for its use according to any one of claims 16 to 17, in which at least one compound of general formula (I) is chosen from: 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, #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- 1 H-cyclopenta[a]phenanthren-6-one; No. 3: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(4-hydroxy-1 - pi peridyl)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, ethyl 3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-17-yl]ethyl]sulfanylacetate; 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.

Claim 19. Composition comprising:

- at least one active principle having the capacity to increase the production of functional SMN protein, for its use in the treatment of spinal muscular atrophy in mammals.