Phytoecdysones and / or 20-hydroxyecdysone derivatives in combination with an active ingredient intended to restore SMN expression for use in the treatment of spinal muscular atrophy

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 capacity to increase the production of functional SMN protein, for their use in combination therapy in the treatment of spinal muscular atrophy in mammals.

Description

Title of the invention: Phytoecdysones and / or 20-hydroxyecdysone derivatives in combination with an active ingredient intended to restore SMN expression for use in the treatment of spinal muscular atrophy Technical field of the invention

[0001] The invention relates to the use of phytoecdysones and / or semi-synthetic 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. Previous technique

[0002] Neuromuscular diseases are characterized by impaired function of motor units, composed of motor neurons, neuromuscular junctions, and skeletal muscles. Regardless of the disease's origin—neurological, as in spinal muscular atrophy or amyotrophic lateral sclerosis, or muscular—all cause impaired motor function in patients, ranging from disability to premature death when vital muscles are affected.

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

[0004] Spinal muscular atrophies are 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 severity levels are described, based on the age of symptom onset and the progression of clinical damage, ranging from type 1, the most severe, to type 3, for which life expectancy can exceed 40 years. Patients with SMA present with symmetrical skeletal muscle weakness due to atrophy of isolated muscle fibers or fibers grouped into fascicles. Almost all SMAs are predominantly proximal, meaning they affect the muscles of the trunk and those close to the trunk. Progressively, the motor deficit extends, initially to the muscles of the lower limbs, and then, subsequently, 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 was named SMN for "survival of motor neurons" in Anglo-Saxon terminology (Lefebvre et al., 1995).

[0005] Although exhibiting significant clinical heterogeneity, genetic analyses have demonstrated that all forms of SMA are caused by a homozygous alteration of the telomeric gene SMN1 (Survival Of Motor Neurons), preventing the production of the Smn protein and leading to motor neuron degeneration, atrophy, and muscle weakness. In the human genome, there is an inverted centromeric copy of this gene, the SMN2 gene, which can be found in multiple copies (Lorson et al., 1998), but which only partially compensates for the loss of function of the SMN1 gene. Indeed, SMN2 has five nucleotide differences with SMN1, including one at exon 7, which favors 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 amount of functional SMN protein, either by modifying the maturation of SMN2 messenger RNA so that it reintegrates the missing exon 7 (Nusinersen, Risdiplam, Branaplam), or by delivering the SMN1 gene by gene therapy (Zolgensma®).

[0007] Others aim to slow the progression of the disease by protecting motor neurons or improving the function of neuromuscular junctions (Salbutamol, Pyridostigmine), or muscle performance (SRK-015, physical training). However, the majority of 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 modulating SMN2 splicing have also proven effective (Naryshkin et al., 2014; Palacino et al., 2015; Finkel et al., 2016; Finkel et al., 2017). These very positive results have led to regulatory approval of certain molecules.

[0009] Three treatments are currently available for SMA: The first treatment approved for SMA was Spinraza® (nusinersen). This treatment received marketing authorization in December 2016 in the United States and in June 2017 in Europe. It is an antisense oligonucleotide (ASO) developed to increase 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 regularly intrathecally, allowing for the re-expression of the SMN protein in motor neurons, with a real clinical benefit, although its magnitude varies depending on the type of SMA and the age at which treatment begins. Antisense oligonucleotide sequences capable of modifying the splicing of the SMN2 gene are given in applications WO2007 / 002390 and WO2018 / 014041. More recently, Zolgensma® (Onasemnogen abeparvovec or AVXS-101), a gene therapy product designed to deliver the SMN1 gene using a viral vector (AAV), was approved (marketing authorization in May 2019 in the United States and conditional marketing authorization in May 2020 in Europe). Its single administration is easier, as it is performed intravenously. Finally, Evrysdi® (Risdiplam or RO7034067) was authorized even more recently (Marketing Authorization 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, either orally or via a feeding tube.

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

[0011] Preclinical studies have shown that these post-treatment deficits were found in SMA mouse models, where treated animals exhibited reduced life expectancy, a deficit in body weight and muscle mass and function compared to healthy animals (Passini et al., 2010; Hua et al, 2010, 2011; Feng et al., 2016).

[0012] Moreover, other limitations and significant concerns remain regarding these treatments. For example, nusinersen must be administered by highly invasive intrathecal injection several times a year. This method of administration is very difficult and sometimes impossible for patients who have undergone surgery for Scoliosis, which excludes nusinersen as a treatment option for these patients. Furthermore, intrathecal administration allows for specific distribution within the central nervous system, meaning that not all symptoms can be fully addressed. On this latter point, onasemnogen has an advantage, as it is administered intravenously, allowing for systemic distribution. Nevertheless, the bioavailability of the AAV9 serotype remains an open question, as does the fact that long-term transgenic expression should be limited to post-mitotic cells such as neurons (Chaytow et al., 2021). Although onasemnogen is advertised as a therapy requiring only a single injection, it remains to be seen whether the treatment will last the lives of patients or if booster doses will be necessary.

[0013] Risdiplam, on the other hand, is a less invasive systemic therapy administered orally daily. However, since risdiplam targets the splicing machinery, it can also affect other transcriptions, leading to unknown and uncontrollable off-target side effects. Indeed, risdiplam has been described as having 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 plant species (ferns, gymnosperms, angiosperms) and contribute to their defense against insect pests. The major phytoecdysone in the plant kingdom is 20-hydroxyecdysone.

[0015] French patent FR 3 021 318 discloses that phytoecdysones, and more specifically 20-hydroxyecdysone (20E), have been the subject of numerous pharmacological studies. These studies have highlighted the antidiabetic and anabolic properties of this molecule. Its stimulatory effects on protein synthesis in muscles have been 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, although these are still limited in number. For example, 20E promotes an increase in muscle mass in young athletes (Simakin et al., 1988).

[0016] 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 et al., 2020).

[0017] The above indicates that, even though therapies that increase SMN protein expression can have significant effects on disease progression and patients' quality of life, it remains useful to find therapeutic approaches complementary treatments that would allow for a reduction in the doses of already authorized treatments, or their frequency of administration, or finally to improve functional gains in order to further reduce the burden of the disease. Presentation of the invention

[0018] The present invention aims to provide a treatment for spinal muscular atrophy, said treatment being improved compared to existing treatments.

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

[0020] 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 ingredient having the capacity 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 semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient having the capacity to increase the production of functional SMN protein, for their use in combination therapy in the treatment of a motor neuron disorder, said disorder participating in SMA.

[0022] In the context of this application, the term "combination therapy" refers to the co-administration of at least two biologically active agents. In the case of the present invention, a first active agent is selected from phytoecdysones or semi-synthetic derivatives of 20-hydroxyecdysone, or is a composition comprising at least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, a second active agent being the active ingredient capable of increasing the production of functional SMN protein. The combination therapy may comprise a single formulation or several formulations. Co-administration may be carried out simultaneously or sequentially. Co-administration may be carried out by the same route of administration or by different routes of administration. It is considered 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.

[0023] In particular embodiments, the invention further complies with the following characteristics, implemented separately or in each of their technically operative 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 ingredient 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 semi-synthetic derivative of 20-hydroxyecdysone, and said at least one active ingredient having the capacity to increase the production of functional SMN protein are co-administered within the same composition.

[0026] In particular embodiments, said at least one phytoecdysone and / or said at least one semi-synthetic derivative of 20-hydroxyecdysone are administered orally or systemically to the mammal, and said at least one active ingredient having the capacity to increase the production of functional SMN protein is administered to the mammal orally and / or intrathecally and / or intravenously.

[0027] For oral administration, said at least one phytoecdysone and / or said at least one semi-synthetic derivative of 20-hydroxyecdysone and / or the active ingredient having the capacity to increase the production of functional SMN protein, is preferably incorporated into an acceptable pharmaceutical formulation that can be administered orally.

[0028] Increased SMN protein production can be achieved through gene therapy or gene therapy. In a gene therapy implementation, the active ingredient capable of increasing SMN protein production is targeted at delivering the SMN1 gene. In a gene therapy implementation, the active ingredient capable of increasing SMN protein production is either a small molecule that acts on the maturation of the SMN2 gene, or an antisense oligonucleotide (ASO) developed to increase the production of functional SMN protein by acting on the splicing (maturation) of the SMN2 gene. In the latter case, the ASO modulates SMN2 splicing, thereby increasing the inclusion of exon 7 in the SMN2 mRNA and thus increasing the number of SMN proteins generated with the amino acids corresponding to exon 7, and therefore corresponding to a functional SMN protein.

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

[0030] In particular embodiments of the present invention, the active ingredient has the capacity to increase the production of functional SMN protein by supplying the SMN1 gene.

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

[0032] In particular embodiments, the active ingredient capable of increasing the production of functional SMN protein is an antisense oligonucleotide (ASO). This ASO preferably has a sequence of 10 to 30 nucleotides, more preferably 12 to 30 nucleotides, preferably 12 to 25 nucleotides, or even more preferably 15 to 20 nucleotides. According to preferred embodiments of the present invention, the ASO has a sequence of 18 nucleotides.

[0033] In particular embodiments of the present invention, ASO has the ability to induce the inclusion of exon 7 in the SMN2 gene sequence.

[0034] 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 at least 80%, preferably at least 90%, preferably at least 95%, preferably 98%, preferably 100%, of the nucleic acid sequence encoding the pre-mRNA of the human SMN2 gene.

[0035] 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 at least 80%, preferably at least 90%, preferably at least 95%, preferably 98%, preferably 100%, of a sequence belonging to intron 6, intron 7, or a portion of exon 7 and a portion of an intron adjacent to exon 7, of the nucleic acid encoding the pre-mRNA of the human SMN2 gene. In this way, exon 7 is included in the SMN2 mRNA, thereby enabling the production of a functional SMN protein.

[0036] The nucleic acid sequence encoding the pre-mRNA of the human SMN2 gene is available on Genbank under reference NG_008728, version NG_008728.1.

[0037] In particular embodiments, the ASO is a sequence that is at least 50% identical or similar to the SEQ ID NO: 1 sequence given in the sequence listing filed with this application (5'-TCACTTTCATAATGCTGG-3'), preferably 60% identical or similar, preferably at least 70%, more preferably at least 80%, preferably at least 90%, and preferably 100% identical or similar. The SEQ ID NO: 1 sequence is 18 bases complementary to intron 7 (bases 10 to 27) of the SMN2 gene.

[0038] In particular embodiments, the ASO has a sequence selected from the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQID NO: 7, SEQID 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 the ASO to target intron 7 of the SMN2 gene.

[0039] When the ASO sequence has a percentage of 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 reference sequence.

[0040] The percentage of identity between two ASO sequences is determined by comparing the two optimally aligned sequences through a comparison window. The portion of an ASO sequence within the comparison window may thus include additions or deletions relative to the reference sequence in order to achieve 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 dividing this number of positions by the total number of positions in the comparison window, the resulting number being multiplied by one hundred.

[0042] The ASO is preferably composed of a phosphodiester backbone. It may have various modifications to its backbone and / or chemical structure in order to increase its stability and / or its affinity for RNA and / or to provide a significant advantage in terms of pharmacokinetics.

[0043] In particular embodiments, the ASO includes at least one modification selected 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'O-methyl (2'OMe) or a 2'O-methoxyethyl (2'MOE) or a 2' Fluoro (2'F), - at least one modification of nucleobases such as pyrimidine methylation of 5' methylcytosine, 5-methyluridine / ribothymidine, or "G-clamp", - at least one substantial change in the sugar structure, leading to a variety of molecules, such as morpholinos (PMO for "phosphoraamidate morpholino oligomer" in Anglo-Saxon terminology) or peptide nucleic acids (PNA for "peptide nucleic acid" in Anglo-Saxon terminology) 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).

[0044] In particular embodiments, the ASO is sequence identical or at least 50% similar to the sequence SEQ ID NO: 23, preferably identical or at least 60% similar, preferably at least 70%, more preferably at least 80%, preferably at least 90%, and preferably identical or 100% similar. The sequence SEQ ID NO: 23 corresponds to the sequence SEQ ID NO: 1 with 2'-O-methoxyethyl on the 2' carbon atom of deoxyribose for each base, with a phosphorothioate backbone, and with 5-methyl cytosines in place of cytosines. The phosphothioate skeleton advantageously improves the stability of ASO, the 5-methyl cytosines advantageously make ASO less sensitive to nucleases, and the 2'-O-methoxyethyl on the 2' carbon atom of deoxyribose advantageously reduces the immune response induced by the administration of ASO.

[0045] 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.

[0046] For their use in the present invention, phytoecdysones and semi-synthetic derivatives of 20-hydroxyecdysone are advantageously purified to pharmaceutical grade.

[0047] A phytoecdysone usable according to the invention is, for example, 20-hydroxyecdysone.

[0048] For this purpose, according to particular embodiments of the present invention, said at least one phytoecdysone is 20-hydroxyecdysone.

[0049] 20-Hydroxyecdysone and its semi-synthetic derivatives are advantageously purified to pharmaceutical grade.

[0050] The 20-hydroxyecdysone used is preferably in the form of a plant extract rich in 20-hydroxyecdysone or a composition containing 20-hydroxyecdysone as an active agent. Plant extracts rich in 20-hydroxyecdysone include, for example, extracts of Stemmacantha carthamoides (also called Leuzea carthamoides), Cyanotis arachnoidea, and Cyanotis vaga.

[0051] The extracts obtained are preferably purified to pharmaceutical grade.

[0052] In one embodiment, 20-hydroxyecdysone is in the form of a plant extract or a part of a plant, said extract comprising at least 95%, and preferably at least 97%, of 20-hydroxyecdysone. Said plant is preferably selected from plants containing at least 0.5% of 20-hydroxyecdysone by dry weight of said plant. Said extract is preferably purified to pharmaceutical grade.

[0053] Said extract is hereinafter referred to as BIO101. It contains remarkably between 0 and 0.5%, by dry weight of the extract, of impurities, such as minor compounds, etc. likely to affect the safety, availability or efficacy of a pharmaceutical application of said extract.

[0054] The plant from which BIO101 is produced is preferably chosen from Stemmacantha carthamoides, Cyanotis arachnoidea and Cyanotis vaga.

[0055] 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, phytoecdysones are administered at a dose of between 3 and 15 milligrams per kilogram per day in humans. Hereinafter, phytoecdysones include phytoecdysones in general, as well as 20-hydroxyecdysone (particularly in extract form) and its semi-synthetic derivatives.

[0057] Preferably, phytoecdysones are administered at a dose of 200 to 1000 mg / day, in one or more doses, to adult humans, and at a dose of 5 to 350 mg / day, in one or more doses, to children or infants. The term phytoecdysone refers to phytoecdysones in general, as well as 20-hydroxyecdysone (particularly in extract form) and its semi-synthetic derivatives.

[0058] In particular embodiments of the present invention, said at least one semi-synthetic derivative of 20-hydroxyecdysone is selected from: - a compound of general formula (I): in which: R1 is chosen from: a (Ci-C6)W(Ci-C6) group; a (Ci-C6)W(Ci-C6)W(Ci-C6) group; a (Ci-C6)W(Ci-C6)CO2(Ci-C6) group; a (Ci-C6)A group, A representing a heterocycle possibly substituted by a group of the type OH, OMe, (Ci-C6), N(Ci-C6), CO2(Ci-C6); a CH2Br group; W being a heteroatom chosen from N, O and S, preferably O and even more preferably S; and, - a compound having formula (II):

[0059] In the context of the present invention, "(Ci-C6)" means any linear or branched alkyl group of 1 to 6 carbon atoms, in particular, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, and n-hexyl groups. Advantageously, it is a methyl, ethyl, isopropyl, or t-butyl group, in particular a methyl or ethyl group, more particularly a methyl group.

[0060] In the context of the present invention, heterocycle is preferably understood to mean a ring comprising 5 or 6 atoms of which one or two are heteroatoms (O, S or N), the remaining atoms being carbon atoms.

[0061] In a preferred embodiment of the present invention, in the general formula (I): R1 is chosen from: a (Ci-C6)W(Ci-C6) group; a (Ci-C6)W(Ci-C6)W(Ci-C6) group; a (Ci-C6)W(Ci-C6)CO2(Ci-C6) group; a (Ci-C6)A group, A representing a heterocycle possibly substituted by a group of type OH, OMe, (CrC6), N(Ci-C6), CO2(CrC6); W being a heteroatom chosen from N, O and S, preferably O and preferably even S.

[0062] In particular embodiments of the present invention, said at least one semi-synthetic derivative of 20-hydroxyecdysone is a compound selected from the following compounds: No. 1: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-10,13-dimethyl-17-(2-morpholinoacetyl)-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one, No. 2: (2S,3R,5R, 10R, 13R, 14S, 17S)-2,3,14-trihydroxy-17-[2-(3-hydroxypyrrolidin-l-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-l-piperidyl)acetyl]-10,13-dimethyl-2,3,4,5,9,ll,12,15,16,17-decahydro-lH-cyclopenta[a]phenanthren-6-o ne; 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-decahy dro-1 Hc y clopenta[a] phenanthren- 6-one; No. 6: 2-[2-oxo-2-[(2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-10,13-dimethyl-6-oxo-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-17-yl]ethyl]sulfanylac ethyl state; No. 7: (2S,3R,5R,10R,13R,14S,17S)-17-(2-ethylsulfanylacetyl)-2,3,14-trihydroxy-10,13-dim ethyl-2,3,4,5,9,ll,12,15,16,17-decahydro-lH-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 cy-clopenta[a]phenanthren-6-one.

[0063] 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 ingredient capable of increasing the production of functional SMN protein, for its use in the treatment of a neuromuscular disease in mammals, in particular spinal muscular atrophy.

[0064] This use of the composition may meet one or more of the characteristics described above with reference to the use in combination therapy of said at least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and said at least one active ingredient having the ability 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, intrathecally, or systemically.

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

[0067] The invention will be better understood upon reading the following description, given by way of non-limiting example, and made with reference to the figures which represent:

[0068] [Fig-1] [Fig.1] represents the weight gain curve 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 a combination of ASO 10-27 + BIO101, from birth (PO) until death of the mice. Here and in the rest of the description, P corresponds to the number of days after birth (postnatal);

[0069] [Fig.2] [Fig.2] is a Kaplan-Meier representation of the survival curves of SMA mice (SmnA7 / A7; huSMN2+ / ) treated with BIO101 alone or with an ASO mismatch or with an ASO 10-27 alone, or with a combination of ASO 10-27 + BIO101, from birth (PO);

[0070] [Fig.3] [Fig.3] represents motor performance (movement capabilities) evaluated by the openfield test of SMA mice (SmnA7 / A7; huSMN2+ / ) treated with BIO101 alone or with an ASO mismatch or with an ASO 10-27 alone, or with a combination of ASO 10-27 + BIO101, from birth (PO);

[0071] [Fig.4] [Fig.4] represents motor performance (muscle fatigue) evaluated by the grip test of SMA mice (SmnA7 / A7; huSMN2+ / -) treated with BIO101 alone or with an ASO mismatch or with an ASO 10-27 alone, or with a combination of ASO 10-27 + BIO101, from birth (PO).

[0072] 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.

[0073] In the following description, n corresponds to the sample size and p corresponds to the "p-value" used to quantify the statistical significance of a result. Furthermore, p=ns indicates that the p-value is not significant. Description of the implementation methods

[0074] 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)

[0076] The antisense oligonucleotide (ASO) of sequence SEQ ID NO: 23 (5'-TCACTTTCATAATGCTGG-3'), reference ISIS 396443, also known as ISIS-SMNRx, and referred to hereafter as ASO 10-27, is complementary to 18 bases (bases 10 to 27) of intron 7 of the SMN2 gene (targeting a site on intron 7, called ISS-N1, which represses the inclusion of exon 7 of the pre-messenger RNA (pre-mRNA) of the SMN2 gene). It has 2'-O-methoxyethyl modified nucleotides on the 2' carbon atom of deoxyribose for each base, with a phos-phorothioate backbone and 5-methyl cytosines, and the control ASO (noted as mismatch) of sequence SEQ ID NO: 24 (5'-TTAGTTTAATCACGCTCG-3') (Singh et al. 2006; Williams et al. 2009), were synthesized and purified as previously described (Baker et al. 1997, Hua et al. 2008; Passini et al., 2011). The oligonucleotides are resuspended at a concentration of 8 pg / uL in 0.9% NaCl and stored at -20°C.

[0077] On the day of injection, the ASO stock solution is diluted to a concentration of 2 pg / 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 8 pg of ASO 10-27 or ASO mismatch on postnatal day 1 (PO1) by unilateral intracerebroventricular injection using a Hamilton syringe and a 32G needle. The injection site is located 1 mm from the sagittal suture, between the bregma and lambda, landmarks visible through the skin at PO1. The needle is positioned perpendicular to the injection site on the skin surface and inserted to a depth of approximately 3 mm to reach the lateral ventricle.

[0078] 2. Co-administration of BIO101

[0079] The BIO101 cyclodextrin-complexed supplementary treatment is administered daily to the pups from the first postnatal day (PO) orally to the dose of 50mg / kg using a pipette.

[0080] 3. Biological activity of BIO101 in combination with ASO

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

[0082] A severe SMA mouse model was used with an FVB / NRj genetic background, characterized by the inactivation of exon 7 of the murine Smn gene and expressing two copies of the human SMN2 transgene (SmnA77A7; huSMN2 / +) (Hsieh et al., 2000). Mice resulting from these crosses with the genotype "FVB / NRj-SmnA7 / A7 huSMN2 / + 2 copies" are described as "SMA" (Hsieh et al., 2000). SMA mice were treated either with BIO101 alone, an ASO mismatch, or ASO 10-27 alone, or with a combined treatment of ASO 10-27 + BIO101 from PO. Survival ([Fig. 1]) and weight ([Fig. 2]) of the mice were analyzed daily.

[0083] The mean lifespan of pups ([Fig. 1]) treated with BIO101 alone (11 days) is essentially the same as that of pups treated with ASO mismatch (13 days). As described in 2011 by Passini et al., treatment with ASO 10-27 alone at a dose of 8 pg significantly increases the mean lifespan of SMA pups, with a median survival of 18 days, compared to a median survival of 13 days in the ASO mismatch control group. When BIO101 is administered in combination with ASO 10-27, the median survival increases to 24 days.

[0084] The weight of the pups ([Fig.2]) treated with BIO101 alone is comparable to that of the pups treated with ASO mismatch throughout their lives.

[0085] At P10, treatment with ASO 10-27 alone significantly increased the mean weight of SMA pups (+11%) compared to the control treatment with ASO mismatch. In combination with ASO 10-27, BIO101 induced a synergistic effect on the weight of SMA pups with a significant increase of +32% at P10 (p<0.05), which is 3 times higher than that observed with ASO 10-27 alone.

[0086] This increase in body weight of mouse pups treated with ASO 10-27 + BIO101 compared to that of mouse pups treated with ASO 10-27 alone continues until their death, with in particular a significant increase of 32% at P15 (p<0.05), of 50% at P20 (p<0.05), of 54.6% at P25 (p<0.01).

[0087] b. Analysis of the functional effects of the combined ASO 10-27 + BIO101 treatment

[0088] We performed phenotypic analyses of type 2 severe SMA mice treated with BIO101 alone, an ASO mismatch, or ASO 10-27 alone, or with a combined ASO 10-27 + BIO101 treatment from PO. We performed longitudinal monitoring of the mice's motor abilities. We assessed locomotion abilities using the open-field test ([Fig. 3]), and muscle fatigue using the grip test ([Fig. 4]), as described previously (Biondi et al., 2008; Branchu et al., 2013; Chah et al., 2016).

[0089] The open-field testing device consists of a 28 x 28 x 5 cm plastic box with a grid divided into 16 squares of 7 cm x 7 cm. The mice were tested individually, and the testing device was washed after each session. Each mouse, initially placed in the center of the field, was allowed to move freely for 5 minutes, receiving tail-pinch stimulation every 15 seconds. Behavioral measures were recorded by the experimenter during these 5 minutes, and the total number of crossed squares was recorded.

[0090] 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 pups at P15 (110+18 squares or +58% with the BIO101 treatment alone, and 100+19 squares or +44% with the ASO 10-27 + BIO101 treatment) compared to the pups treated with ASO 10-27 alone (70+20 squares).

[0091] From P23 onwards, the movement capacities of mouse pups treated with PASO 10-27 + BIO101 tend to be increased compared to those of mouse pups treated with ASO 10-27 alone (+51.3%, p=ns), and are significantly increased at P25 (+38.4%, p<0.05), or even at P27 (+44.9%, p<0.01) ([Fig.3]).

[0092] To assess muscle fatigue, the grip strength of the mice's forepaws was tested. The mice were suspended by their forepaws from a thin metal rod suspended horizontally in the air. The time spent hanging was recorded. Each mouse underwent five successive attempts with a one-minute rest period between each test. Only the best attempt was retained for the evaluation of muscle function.

[0093] From P19 onwards, the fatigue resistance of pups treated with ASO 10-27 + BIO101 tends to increase compared to that of pups treated with PASO 10-27 alone. This difference becomes evident from P21 (+54% in the group treated with the combination of ASO 10-27 + BIO101 compared to PASO 10-27 alone, p=ns), then at P23 (+158.9%, p<0.05), at P25 (+320%, p<0.05) and again at P27 (+296.7%, p=0.07). These significant differences are maintained up to P35. From P39 onwards, these differences are less marked but the group of mice treated with ASO 10-27 + BIO101 combination nevertheless retains a resistance to fatigue greater than that observed in the group treated with FASO 10-27 alone ([Fig.4]).

[0094] 4. Conclusion

[0095] These results demonstrate the interest of using the BIO101 treatment, in combination with an active ingredient aimed at restoring the expression of the SMN protein, in particular through a therapeutic approach using ASOs which have the effect of restoring the expression of the SMN protein.

[0096] Indeed, this combined therapy shows significant beneficial effects in a murine model of SMA, particularly with regard to the animals' weight, and in a even more important, in terms of the physical performance of these animals, whether it be the ability to move or the resistance to fatigue of the animal.

[0097] The combination of ASO 10-27 + BIO101 treatment, beyond improving the performance obtained by ASO 10-27 monotherapy treatment, undoubtedly has a synergistic effect.

[0098] More generally, it should be noted that the methods of implementation and realization of the invention considered above have been described as non-limiting examples and that other variants are therefore conceivable.

[0099] In particular, the invention has been described by considering mainly the ASO 10-27 of sequence SEQ ID NO: 23. Nothing excludes, however, in other types of embodiments, considering other active ingredients having the capacity to increase the production of SMN protein as other ASOs having the capacity to increase the production of SMN protein. Such ASOs are for example ASOs of 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.

[0100] These sequences are given in particular in Table 1 below: [Tables 1] Sequence name PHASE sequence Wheat residue per STEP in Fintron 7 of SMN2 SEQID NO : 1 TCACTTTCATAATGCTGG 10 to 27 SEQID NO : 2 ACTTTCATAATG CTGGCA 8to25 SEQID NO : QID : 3 to NO : 3 CACTGTT6AT4 TTCACTTTCATAATGCTG 11 to 28 SEQID NO : 5 AGTAAGATTCACTTT 21 to 35 SEQID NO: 6 GATTCACTTTCATAA 16 to 30 SEQID NO:7 ATTCACTTTCATAAT 15 to 29 SEQID NO:8 NO:9 SEQCACT1 toATCATA TCACTTTCATAATGC 13to27 SEQID NO : 10 CACTTTCATAATG CT 12 to 26 SEQID NO : 11 ACÏTTCATAATGCTG 11 to 25 SEQ ID NO : 12 CTTTCATAATGCTGG 10 to 24 SEQID3 NOGTTCA NO : 13 TG 14 TTCATAATGCTGGCA 8 to 22 SEQID NO : 15 TCATAATGCTGGCAG 7to21 SEQID NO : 16 CATAATGCTGGCAGA 6 to 20 SEQID NO : 17 TGCTGGCAGACTTAC 1 to 15 SEQID 1 NOQAC 198 : NOTT29 AT29 TTCACTTTCATA 17 to 28 SEQID NO : 20 TCACTTTCATAA 16 to 27 SEQID NO : 21 CACTTTCATAAT 15 to 26 SEQID NO : 22 ACTTTCATAATG 14 to 25 SEQID NO : 25 SEGQID TTCTTCA to2NOATGC 13 12 to 23 SEQID NO: 27TTCATAATGCTG 11 to 22 SEQID NO : 28 TCATAATGCTGG 10 to 21 SEQ ID NO : 29 ATTCACTTTC ATAATGCTG G 10 to 29

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Claims

Demands

1. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient capable of increasing the production of functional SMN protein, for use in the treatment of spinal muscular atrophy in mammals.

2. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient 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 motor neuron disorder responsible for spinal muscular atrophy.

3. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient having the capacity to increase the production of functional SMN protein for its use according to claim 2, wherein the motor neuron disorder is an alteration of motor neuron function or their degeneration.

4. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient 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 capacity to increase the production of functional SMN protein by gene therapy or gene therapy.

5. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient 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 capacity to increase the production of functional SMN protein by supplying the SMN1 gene or by acting on the maturation of the SMN2 gene.

6. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient 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 capacity to increase the production of Functional SMN protein is an antisense oligonucleotide.

7. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient having the capacity to increase the production of functional SMN protein for its use according to claim 6, wherein the antisense oligonucleotide comprises 10 to 30 nucleotides.

8. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient capable of increasing the production of functional SMN protein for use according to any one of claims 6 to 7, wherein the antisense oligonucleotide is complementary to at least 90%, preferably at least 95%, preferably at least 98%, preferably 100%, of the nucleic acid sequence encoding the pre-mRNA of the human SMN2 gene.

9. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient capable of increasing the production of functional SMN protein for use according to any one of claims 6 to 8, wherein the antisense oligonucleotide is complementary to at least 90%, preferably at least 95%, preferably 98%, preferably 100%, of a sequence belonging to intron 6, intron 7 or exon 7 of the nucleic acid encoding the pre-mRNA of the human SMN2 gene.

10. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient having the capacity to increase the production of functional SMN protein for its use according to any one of claims 6 to 9, wherein 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%, preferably at least 70%, more preferably at least 80%, preferably at least 90% and preferably identical or similar to 100%.

11. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient capable of increasing the production of functional SMN protein for its use according to any one of claims 6 to 9, wherein the ASO has a sequence selected 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.

12. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient capable of increasing the production of functional SMN protein for its use according to any one of claims 6 to 11, wherein the ASO comprises at least one modification selected from: - a modification at 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'O-methyl (2'OMe) or a 2'O-methoxyethyl (2'MOE) or a 2' Fluoro (2'F), - at least one modification of nucleobases such as pyrimidine methylation of the 5' methylcytosine type, 5-methyluridine / ribothymidine, or "G-clamp", - at least one substantial change in the structure of sugar, leading to a variety of molecules,such as morpholinos, peptide nucleic acids, or constrained-type oligonucleotides.

13. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient having the capacity to increase the production of functional SMN protein for its use according to any one of claims 6 to 9, wherein 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%, preferably at least 70%, more preferably at least 80%, preferably at least 90% and preferably identical or similar to 100%.

14. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient capable of increasing 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.

15. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient having the capacity to increase the production of functional SMN protein for its use according to claim 14, wherein the 20-hydroxyecdysone is in the form of a plant extract or a part of a plant, said extract comprising at least 95%, and preferably at least 97%, of 20-hydroxyecdysone.

16. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient capable of increasing the production of functional SMN protein for its use according to any one of claims 1 to 15, wherein said at least one semi-synthetic derivative of 20-hydroxyecdysone is selected from: - a compound with general formula (I): in which: R1 is chosen from: a (Ci-C6)W(Ci-C6) group; a (C1-C6)W(C1-C6)W(C1-C6) group; a (C1-C6)W(C1-C6)CO2(C1-C6) group; a (Ci-C6)A group, A representing a heterocycle possibly substituted by a group of the type OH, OMe, (Ci-C6), N(Ci-C6), CO2(Ci-C6); a CH2Br group; W being a heteroatom chosen from N, O and S, preferably O and even more preferentially S; and, - a compound having formula (II): [Chem. 2]

17. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient having the capacity to increase the production of functional SMN protein for its use according to claim 16, wherein in the general formula (I): R1 is selected from: a (Ci-C6)W(Ci-C6) group; a (C1-C6)W(C1-C6)W(C1-C6) group; a (C1-C6)W(C1-C6)CO2(C1-C6) group; a (Ci-C6)A group, A representing a heterocycle optionally substituted by a group of the type OH, OMe, (Ci-C6), N(CrC6), CO2(CrC6); W being a heteroatom chosen from N, O and S, preferably O and preferably even S.

18. At least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, and at least one active ingredient capable of increasing the production of functional SMN protein for use according to any one of claims 16 to 17, wherein at least one compound of general formula (I) is selected 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, No. 2: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(3-hydroxypyrro lidin-l-yl)acetyl]-10,13-dimethyl-2,3,4,5,9,ll,12,15,16,17-decahydro-l H-cyclopenta[a]phenanthren-6-one; No. 3: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(4-hydroxy-l-pi peridyl)acetyl]-10,13-dimethyl-2,3,4,5,9,ll,12,15,16,17-decahydro-lH-cyclopenta[a]phenanthren-6-one; No. 4: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-[4-(2-hydroxyethyl)-l-piperidyl]acetyl]-10,13-dimethyl-2,3,4,5,9,ll,12,15,16,17-decahy dro-lH-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-2,3,4,5,9,11,1 2,15,16,17-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-dim ethyl-6-oxo-2,3,4,5,9,ll,12,15,16,17-decahydro-lH-cyclopenta[a]phenanthren-17-yl]ethyl]ethyl sulfanylacetate; No. 7: (2S,3R,5R,10R,13R,14S,17S)-17-(2-ethylsulfanylacetyl)-2,3,14-trihydr oxy-10,13-dimethyl-2,3,4,5,9,ll,12,15,16,17-decahydro-lH-cyclopenta[ a]phenanthren-6-one; No. 8: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(2-hydroxyethyl sulfanyl)acetyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro- 1H cyclopenta[a]phenanthren-6-one.

19. Composition comprising: - at least one phytoecdysone and / or at least one semi-synthetic derivative of 20-hydroxyecdysone, - at least one active ingredient capable of increasing the production of functional SMN protein, for its use in the treatment of spinal muscular atrophy in mammals.

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