WO2019170930A1 - Treatment for spinal muscular atrophy - Google Patents

Abstract

The present invention relates to a method for treating spinal muscular atrophy (SMA) in the different stages of development thereof, where the method comprises administering suitable amounts of a calpain inhibitor to a subject who may or may not have developed the first symptoms of the disease. The invention also relates to pharmaceutical compositions comprising a calpain inhibitor which is administered in combination with another compound to enhance the effect, such as histone deacetylase inhibitors and autophagy modulators.

Description

 TREATMENT FOR SPINAL MUSCLE ATROPHY

TECHNICAL FIELD OF THE INVENTION

 The present invention relates to a treatment that improves the symptoms, prognosis and / or development of spinal muscular atrophy (SMA), in any of its stages of development. The invention can be used for the treatment of patients who have already manifested symptoms of the disease, or who have not yet manifested them.

BACKGROUND OF THE INVENTION

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder and the main genetic cause of mortality in children under one year. It is characterized by degeneration of the motor neurons (MN) of the spinal cord that causes muscle atrophy and weakness. AME disease is caused by the homozygous alteration of the Survival Motor Neuron 1 (SMN1) gene located on chromosome 5q3 (telomeric region), which is responsible for the production of motor neuron survival protein (SMN). In humans there is a duplication of the SMN gene in the centromeric region, SMN2, which mainly produces a truncated isoform of SMN that is unable to compensate for the loss of SMN1 in patients. In AME, the number of copies of SMN2 determines the intracellular levels of SMN that define the onset and severity of the disease.

 Currently, SMA patients are classified depending on the severity of the disease in patients with SMA type I, SMA type II, SMA type III or SMA type IV. The different types of SMA are established based on the severity of the disease, the age at which the symptoms begin and the maximum motor function reached by the patients. The type of SMA correlates with the amount of SMN2 protein that, in turn, depends on the number of copies of that gene in the patient's genome.

Different therapeutic approaches to SMA have shown that raising the SMN protein improves disease symptoms (Finkel et al., 2017, N Engl J Med. 2017, 377: 1723-1732). Increasing the levels of SMN can be achieved by several mechanisms such as the increase in the expression of SMN2 and the promotion of the stability of the SMN protein. The mechanisms involved in regulating the stability of the SMN protein include the ubiquitin proteasome system (UPS) (Kwon et al., 2011. Hum Mol Genet. 2011 20: 3667-77), oligomerization of SMN (Burnett et al. , 2009. Mol Cell Biol. 29: 1107-15), histone deacetylase inhibitors (Kernochan et al., 2005. Hum Mol Genet. 14: 1171-82) and autophagy (Garcera et al., 2013. Cell Death Dis. 4: e686; Periyakaruppiah et al., 2016. Exp Neurol. 283: 287-97). Some of the SMN stabilizers have been used to analyze their effect on the phenotype of AME mice. Recent results have shown that treatment of SMNA7 mice using proteasome inhibitors and / or histone deacetylase inhibitors improves survival and motor function (Butchbach et al. Exp Neurol. 2016. 279: 13-26; Foran et al., 2016 Neurobiol Dis. 88: 118-24).

 There are currently no effective therapies for the treatment of SMA, especially the most severe forms of the disease, so there is a need in the art of alternative therapies suitable for the treatment of SMA in the different stages of the disease.

SUMMARY OF THE INVENTION

In a first aspect the invention relates to the use of a calpain inhibitor for the treatment of spinal muscular atrophy (SMA).

 In a second aspect the invention relates to a pharmaceutical composition comprising a calpain inhibitor and a compound selected from the group consisting of:

 (i) a histone deacetylase inhibitor (HDAC) and

 (ii) an autophagy modulator.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Effect of the reduction of endogenous calpain on the level of SMN protein in MN of spinal cord in culture. The MNs were transduced with shCalp lentivirus constructs or empty vectors (EV) and were maintained in the presence of a mixture of neurotrophic factors (NTF) (acronyms included according to their initials in English). (TO) Protein extracts from transduced cultures 3, 6 and 9 days were subjected to immunoblot analysis and tested with anti-SMN or anti-calpain antibodies. The membranes were retested with an anti-a-tubulin antibody, used as a loading control. The graph represents the expression of Smn and corresponds to the quantification of three independent experiments ± SEM. (B) Representative confocal images of cells transduced with the empty vector (EV) and shCalp maintained 6 days in the presence of NTFs. The cells were fixed and immunofluorescence was performed with anti-SMN antibody.

 Asterisks indicate significant differences using the unique ANOVA test or Student's t-test.

 Figure 2. Calpain attenuation prevents the reduction of SMN caused by membrane depolarization.

 Figure 3. Calpeptin treatment increases the level of SMN protein in MN. Figure 4. Calpeptin treatment prevents degeneration of neurites in AME mutant MNs.

 Figure 5. Calpeptin administration extends the survival of severe AME and AME SMNA7 mice.

 Figure 6. Calpeptin treatment improves motor function in severe AME mice, but not in SMNA7 mice.

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have identified that treatment with a calpain inhibitor in in vitro cultures of MNs and in animal models of AME is able to improve survival, the most apparent symptoms and the clinical course of the disease by attenuating the loss of weight and improve motor behavior.

Calpain inhibitor for use in the treatment of SMA

The inventors of the present invention have found that inhibition of calpain in mice suffering from AME results in an increase in the survival of said mice and an improvement in motor function. Consequently, in a first Aspect The invention relates to a calpain inhibitor for use in the treatment of SMA. The invention also relates to a method for the treatment of SMA which comprises administering a calpain inhibitor.

 The term "calpain" as used herein is used to refer generically to a family of neutral non-lysosomal cysteine proteases whose activity is calcium dependent. Calpain 1 (m-calpain) and Calpain 2 (m-calpain) are the best characterized calpains in humans and are activated by micro and millimolar calcium levels, respectively. In humans, calpain 1 corresponds to the protein encoded by the CAPN1 gene with accession number in ETniprot P07384 (date December 20, 2017); Calpain 2 corresponds to the protein encoded by the CAPN2 gene with accession number in Uniprot P17655 (dated November 22, 2017).

 The term "calpain inhibitor" as used herein is understood as any substance or compound that is capable of specifically preventing or blocking the transcription and / or translation of a gene that encodes the calpain protein or that is capable of preventing specifically that the protein encoded by said gene perform its func- tion (that is, prevent or block activity). In a preferred embodiment, the calpain inhibitor acts on calpain 1 and 2. In another preferred embodiment, the calpain inhibitor acts on calpain 1 without significantly affecting the activity or expression of calpain 2. In another preferred embodiment, The inhibitor is specific for calpain 2 without significantly affecting the activity or expression of calpain 1.

 Non-limiting examples of calpain inhibitors that act by reducing calpain expression include, without limitation, a small interfering RNA (siRNA), a short bracketed RNA (hRNA), a microRNA (miRNA), an antisense oligonucleotide or a ribozyme.

The term "small interfering RNA"("siRNA") refers to duplex of small inhibitory RNAs that induce the RNA interference pathway. These molecules may vary in length (generally 18-30 base pairs) and contain varying degrees of complementarity to their target mRNAs in the antisense chain. Some siRNAs, but not all, have outstanding unpaired bases at the 5 'or 3' end of the sense strand and / or the antisense strand. The term "siRNA" includes duplexes of two separate chains. As used herein, siRNA molecules are not limited to RNA molecules but also encompass nucleic acids with one or more chemically modified nucleotides, such as morpholinos.

 The term "hRNA" or "short bracketed RNA" as used herein, refers to an rRNA where the two chains are linked by an uninterrupted nucleotide chain between the 3 'end of one strand and the 5' end of the other. respective strand to form a duplex structure.

 The term "micro RNA" or "miRNA" refers to short single stranded RNA molecules, typically about 21-23 nucleotides in length capable of regulating gene expression. The miRNAs can be synthetic (i.e., recombinant) or natural.

 Lina "antisense sequence", as used herein, includes antisense or sense oligonucleotides comprising a single stranded nucleic acid (RNA or DNA) sequence capable of binding to target mRNA (sense) or DNA (antisense) sequences.

 As used herein, the term "ribozyme" or "RNA enzyme" or "catalytic RNA" refers to an RNA molecule that catalyzes a chemical reaction.

 The nucleic acid capable of inhibiting calpain expression may contain one or more modifications in the nucleobases, in the sugars and / or in the bonds between nucleotides.

Modifications to one or more nucleic acid skeleton residues may comprise one or more of the following: modifications of T-sugar such as 2'-0-methyl (2'-OMe), 2'-0-methoxyethyl (2 '-MOE), 2'-0-methoxyethoxy, T-fluoro (2'-F), 2'-allyl, 2'-0- [2- (methylamino) -2-oxoethyl], 2'-0- ( N-methylcarbamate); modifications of the sugar in 4 'including 4'-uncle, bridge 4'-CH ₂ -0-2', bridge 4- (CH ₂ ) ₂ -0-2 '; closed nucleic acid (LNA); nucleic acid peptide (APN); intercalating nucleic acid (INA); intercalating coiled nucleic acid (TINA); hexitol nucleic acids (HNA); arabinonucleic acid (ANA); nucleic cyclohexane acids (CNA); Cyclohexenylnucleic acid (CeNA); treosyl nucleic acid (TNA); morpholino oligonucleotides; Gap-meros; Mix-meros; incorporation of arginine rich peptides; addition of synthetic 5'-phosphate to RNA; RNA aptamers (Que-Gewirth NS, Gene Ther. 2007 Feb; l4 (4): 283-9l.); regulated RNA aptamers with antidotes in the subject of the specific RNA aptamer (ref. Oney S, Oligonucleotides. 2007 Fall; 17 (3): 265-74) or any combination thereof.

 Modifications to one or more nucleoside bonds of the nucleic acids may comprise one or more of the following: phosphorothioate, phosphoramidate, phosphorodiamidate, phosphorodithioate, phosphorus selenoate, phosphorodiselenoate, phosphoranilothioate and phosphoranilidate, or any combination thereof.

 A closed nucleic acid (LNA), often referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose group of an LNA nucleotide is modified with an extra bridge that joins the T and 4 ’carbons (02’, C4’-methylene bridge). The bridge "closes" the ribose in the 3’-endo structural conformation, which is often found in the A-form of DNA or RNA. LNA nucleotides can be mixed with DNA or RNA bases in the nucleic acid when desired. Such oligomers are commercially available.

 A nucleic acid peptide (APN) is an artificially synthesized polymer whose skeleton is composed of repeating units of N- (2-aminoethyl) -glycine linked by peptide bonds. The different purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds.

 An intercalating nucleic acid (INA) is a modified nucleic acid analog comprising normal deoxyribonucleotides covalently linked to hydrophobic insertions.

 Hexitol nucleic acids (HNA) are nucleotides constructed of natural nucleobases and a phosphorylated 1,5-anhydrohexitol skeleton. The molecular associations between HNA and RNA are more stable than between HNA and DNA and between natural nucleic acids (cDNA, dsRNA, DNA / RNA). Other synthetically modified oligonucleotides comprise ANA (arabinonucleic acid), CNA (nucleic cyclohexane acids), CeNA (cyclohenexylnucleic acid) and TNA (threosylnucleic acid).

Morpholinos are synthetic molecules that are the product of a redesign of the natural structure of the nucleic acid. Structurally, the difference between morpholinos and DNA or RNA is that while the morpholinos have standard nucleobases, these bases are attached to 6-member morpholine rings instead of deoxyribose / ribose rings and non-ionic phosphorodiamidate bonds between the subunits They replace anionic phosphodiester bonds. Morpholinos are sometimes referred to as PMO (morpholino phosphorodiamidate oligonucleotide). The 6-member morpholine ring has the chemical formula 0- (CH ₂ -CH ₂ ) ₂ -NH.

 Gapmeros or "oligomeric compounds with gaps" are chimeric RNA-DNA-RNA oligonucleotide probes, where DNA windows or 'gaps' are inserted into an otherwise normal or modified RNA oligonucleotide known as' wings " This modification increases the stability of the oligonucleotide in vivo and the avidity of the interaction of the probe with the target, so that shorter probes can be used effectively. Preferably, the wings are modified 2'-0-methyl (OMe) or 2'-0-methoxyethyl (MOE) oligonucleotides that protect the internal block from nuclease degradation. In addition, the nucleotides that form the hollow or wings can be linked by phosphodiester bonds or phosphorothioate bonds, which makes them thus resistant to degradation by RNase. In addition, the nucleotides that form the wings can also be modified by incorporation of bonded bases. by 3 'methylphosphonate links.

 Inhibitors suitable for use in the present invention and acting by silencing the expression of the gene or genes encoding the different calpain variants include all those that cause a reduction in mRNA levels and / or a reduction in levels. of the corresponding protein of at least 5%, of at least 10%, of at least 15%, of at least 20%, of at least 25%, of at least 30%, of at least 35%, of at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% with respect to a reference value, said reference value being the level of the mRNA or corresponding protein in the absence of inhibitor.

 Suitable methods to determine if a calpain inhibitor is capable of lowering calpain levels include, without limitation, standard assays to determine mRNA expression levels such as qPCR, RT-PCR, RNA protection analysis, Northern blot, RNA dot blot, in situ hybridization and the like.

Suitable methods for determining whether an inhibitor acts by lowering calpain levels include quantification by conventional methods, for example, using antibodies capable of specifically binding to calpain and the subsequent quantification of the resulting antibody-antigen complexes. There is a wide variety of well known assays that can be used in the present invention, which use unlabeled antibodies (primary antibody) and labeled antibodies (secondary antibodies); These techniques include Western blot or Western blotting, ELISA (enzyme-linked enzyme immunoassay), RIA (radioimmunoassay), competitive EIA (enzyme immunoassay), DAS-ELISA (double sandwich ELISA antibody), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips or protein microarrays that include specific antibodies or tests based on colloidal precipitation in formats such as dip rods. Other ways to detect and quantify protein levels of interest include affinity chromatography techniques, binding ligand assays, etc.

 In another preferred embodiment, the calpain inhibitor acts by inhibiting protease activity. Suitable inhibitors that act by inhibiting calpain activity and that can be used in the present invention include, without limitation:

 Calpeptin or Benzyloxycarbonyleucyl-norleucinal or Z-Leu norleucinal) (CAS number 117591-20-5).

 Acetyl calpastatin ((CAS number: 123714-50-1)

 Calpain I inhibitor (CAS Number: 110044-82-1)

 - PD 150606 (CAS number: 179528-45-1)

 Aureusimine B (CAS number: 170713-71-0)

 - ALLM (CAS Number: 136632-32-1),

 - ALLN (CAS Number: 110044-82-1)

 - Leupeptin (CAS Number: 103476-89-7),

 Quinoline carboxyamide as described in US5622967A,

- Leu-Abu-CONHEt (AK275), (N - ((Phenylmethoxy) carbonyl) -L-leucyl-N- ethyl-L-2-aminobutanamide)

 - Cbz-Val-Phe-H (MDL28170) (CAS number: 88191-84-8)

 - Compounds PD 150606 and PD151746, corresponding to a-mercaptoacrylic acid derivatives described in Wang et al. (Proc Nati Acad Sci U S A., 1996, 93: 6687-6692)

 - MG-132 [Z-Leu-Leu-Leu-CHO] (CAS number: 133407-82-6)

Aclacinomycin A (CAS number: 57576-44-0) - E64-d (CAS number: 88321-09-9)

 - E64 (CAS number: 66701-25-5)

 The term "medium inhibition concentration" (IC50) as used herein is understood as the concentration of an inhibitor necessary to achieve a 50% target inhibition. The specificity of a given inhibitor is defined as the ratio between the IC50 value of a given inhibitor for the target of interest with respect to another target. In a preferred embodiment, the inhibitor is a calpain inhibitor. It is possible to determine if a compound is able to inhibit calpain in a more potent way than other targets by comparing IC50 values. For example, a specific calpain inhibitor will have a lower IC50 value for calpain than for other proteases. Said IC50 value may be at least 2 times less, at least 4 times less, at least 6 times less, at least 8 times less, at least 10 times less, at least 50 times less, at least 100 times less, at least 1,000 times less, at least 10,000 times less than the IC50 value for other targets, which would include other proteases. In another example a specific calpain inhibitor will have an IC50 value for calpain lower than for other cysteine proteases. Said IC50 value may be at least 2 times less, at least 4 times less, at least 6 times less, at least 8 times less, at least 10 times less, at least 50 times less, at least 100 times less, at least 1,000 times less, at least 10,000 times less than the IC50 value for other targets, which would include other cysteine proteases. In one embodiment, the calpain inhibitor has an IC50 equal to or less than 200nM, equal to or less than 150hM, equal to or less than 100mM, equal to or less than 90nM, equal to or less than 80nM, equal to or less than 70nM, equal to or less at 60nM, equal to or less than 55nM, equal to or less than 50nM, equal to or less than 45nM, equal to or less than 40nM, equal to or less than 35nM, equal to or less than 30nM, equal to or less than 25nM, equal to or less than 20nM , equal to or less than 15hM, equal to or less than lOnM, equal to or less than 5nM.

In a preferred embodiment, the IC50 of the calpain inhibitor is between 10 nM and 100 nM. In a more preferred embodiment the IC50 is between 30 nM-55 nM. In an even more preferred embodiment the IC50 is 52 nM, 34 nM, 15hM, 10hM or 20nM. A specific inhibitor for use in the present invention can inhibit the activity of calpain by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least one 50%, at least 75%, at least 90% and all ranges between 5% and 100%.

 Suitable methods to determine if an inhibitor acts by decreasing calpain activity include quantification by conventional methods known in the art. For example, they include, without limitation, initial velocity tests, progress curve tests, transient kinetics tests and relaxation tests. Continuous enzymatic activity tests include, without limitation, spectrophotometric, fluorometric, calorimetric, chemiluminescent, light scattering and microscale thermophoresis assays. Discontinuous enzymatic activity tests include, without limitation, radiometric and chromatographic tests. As those skilled in the art understand, factors that can influence enzyme activity include factors such as salt concentration, temperature, pH and substrate concentration.

The term "treatment" as used herein refers to any type of therapy, which aims to terminate, improve or reduce the susceptibility to AME. Thus, "treatment", "treat" and its equivalent terms refer to obtaining a desired effect pharmacologically or physiologically, which covers any treatment of SMA in a mammal, including humans. The effect can be prophylactic in terms of providing total or partial prevention of a disorder and / or adverse effect attributable to it. That is, "treatment" includes (1) inhibiting the disease, for example by stopping its development, (2) interrupting or ending the disorder or at least the symptoms associated with it, so that the patient would no longer suffer the disease or its symptoms, for example causing the regression of the disease or its symptoms by restoring or repairing a lost, absent or defective function, or stimulating an inefficient process, or (3), reducing, alleviating or improving the disease, or the associated symptoms to it, where reducing is used in a broad sense to refer to at least a reduction in the magnitude of a parameter or symptom, such as inflammation, pain, respiratory distress or inability to move autonomously. The term "Spinal muscular atrophy" (SMA) as used herein is a term applied to a varied number of disorders that have in common a genetic etiology and that manifest as weakness due to motor neuron lesions. The term "motor neuron" or "motor neuron" (MN) refers, in vertebrates, to the central nervous system neuron that projects its axon towards a muscle or gland.

 In a non-limiting manner, the symptoms that characterize SMA include: poor muscle tone, muscle weakness, lack of motor development, facial weakness, tongue fasciculation, difficulty swallowing and feeding, postural tremor of the fingers, slight contractures ( often in the knees, occasionally in the elbows) absence of tendon reflexes, or respiratory distress.

 Preferably, AME originates as a result of a disruption in the SMN1 (Survival Motor Neuron 1) gene, also known as telomere copy encoding SMN. In a preferred embodiment, AME originates as a result of a homozygous disruption of the SMN1 gene.

 "SMN1" (or "TelSMN"), as used in the present invention, refers to the telomeric SMNJ gene, which encodes the SMN protein {Survival Motor Neuron Proteiri). In humans it has the sequence shown in the Uniprot database with access number Q16637 (date 01/31/2018).

In a preferred embodiment, the patient to be treated according to the present invention contains an amplification of the SMN2 gene also known as a non-coding centromeric copy of SMN. In preferred embodiments, at least one of the SMN2 gene loci contains at least one copy, at least two copies, at least three copies, at least four copies, at least five, at least six, at least seven copies or at minus eight copies of the SMN2 gene. In a more preferred embodiment, at least one of the SMN2 gene loci contains 1 or 2 copies of the gene. In another preferred embodiment, the two SMN2 gene loci contain at least one copy, at least two copies, at least three copies, at least four copies, at least five, at least six, at least seven copies or at least eight copies of the SMN2 gene. In an even more preferred embodiment, at least the two SMN2 gene loci contain 1 or 2 copies of the gene. "SMN2" (or "CenSMN"), as used in the present invention, refers to the centromeric SMN2 gene, which encodes a truncated form of the SMN protein unable to fully compensate for the loss of the SMN1 gene. In humans it has the sequence shown in the Uniprot database with access number I2E4S9 (date 05/10/2017).

 In a preferred embodiment, the patient being treated with the calpain inhibitor according to the present invention is a patient affected by AME type I, AME type II, AME type III or AME type IV. There are four types of SMA, depending on the levels of SMN2, which determine the severity of the disease, the age at which the symptoms begin and the maximum motor function reached by the patients, although the correlation between the levels of SMN2 and The type of SMA is not absolute, so that the number of copies of the SMN2 gene is not predictive of the type of SMA that a certain patient can eventually develop, nor is it limiting of the present invention. SMA type I is diagnosed between 1 and 4 months of age. It is characterized in that the number of copies of SMN2 is between 1 and 3, preferably being 2. Patients affected by SMA type I are unable to sit down and show: hypotonia, weakness (especially in the control of the head and neck, and muscle weakness), difficulty feeding and breathing. In a preferred embodiment, the patient of SMA type I is diagnosed as having at least one symptom of the disease. In an even more preferred embodiment, the patient of SMA type I is diagnosed before presenting any symptoms of the disease.

 In general, not limitation, SMA type II is diagnosed between 7 and 18 months of age. It is characterized in that the number of copies of SMN2 is between 2 and 4, preferably being 3. Patients affected by SMA type II are able to get to sit without help and some get to stand up, although they are unable to walk from independent way. In a preferred embodiment, the AME type II patient is diagnosed as having at least one symptom of the disease. In an even more preferred embodiment, the AME type II patient is diagnosed before presenting any symptoms of the disease.

Generally non-limiting, type III SMA is diagnosed between 18 months and 10 years of age. When the symptoms appear before the age of 3, it is a question of SMA type Illa, when it is after 3 years it is a question of SMA type Illb. Is characterized because the number of copies of SMN2 is between 3 and 5, preferably being 3 or 4. Patients affected by type III SMA come to walk independently. In a preferred embodiment, the type III SMA patient is diagnosed as having at least one symptom of the disease. In an even more preferred embodiment, the type III SMA patient is diagnosed before presenting any symptoms of the disease.

 Generally non-limiting, type IV SMA is diagnosed after 35 years of age. It is characterized in that the number of copies of SMN2 is between 4 and 8, preferably 4 or 5. The most characteristic symptom of SMA type IV is that these are patients who are able to walk independently and develop very mild symptoms, although they suffer from a progressive motor degeneration from the age of 35.

 In a preferred embodiment, the calpain inhibitor is calpastatin. In an even more preferred embodiment, the calpain inhibitor is calpeptin.

 The calpain inhibitor for use according to the present invention may be part of a pharmaceutical composition containing a vehicle suitable for administration to a subject, so that the calpain inhibitor will be administered to a subject in a pharmaceutical administration form suitable for This will include at least one pharmaceutically acceptable vehicle. Therefore, in a particular embodiment, the calpain inhibitor and, preferably, calpeptin, will be found to be part of a pharmaceutical composition comprising, in addition to calpeptin as an active ingredient, at least one vehicle, preferably, a pharmaceutically acceptable carrier. The term "vehicle" generally includes any diluent or excipient with which an active ingredient is administered. Preferably, said vehicle is a pharmaceutically acceptable vehicle for administration to a subject, that is, it is a vehicle (eg, an excipient) approved by a regulatory agency, for example, the European Medicines Agency (EMA), the "Food & American Drug Administration ”(FDA), etc., or are included in a pharmacopoeia (eg, the European, American Pharmacopoeia, etc.) recognized in general for use in animals, and, more particularly, in humans.

The calpain inhibitor can be dissolved for administration in any suitable medium. Illustrative, non-limiting examples of means in which the Active ingredient can be dissolved, suspended or with which emulsions can be formed, include: DMSO, water, ethanol, water-ethanol or water-propylene glycol mixtures, etc., oils, including petroleum derived oils, animal oils, vegetable oils or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In a preferred embodiment, calpeptin is administered in the form of a pharmaceutical composition comprising an organic solvent. Non-limiting examples of organic solvents are: acetone, methyl alcohol, ethyl alcohol, ethylene glycol, propylene glycol, glycerin, diethyl ether, chloroform, benzene, toluene, xylene, ethylbenzene, pentane, hexane, cyclohexane, tetrahydrofuran, carbon tetrachloride, chloroform, chloride of methylene, trichlorethylene, perchlorethylene, dimethylsulfoxide (DMSO).

Also, preparations in solid form of the pharmaceutical composition are included to be converted, shortly before use, into preparations in liquid form for oral or parenteral administration. Liquid forms of this type include solutions, suspensions and emulsions. The review of the different pharmaceutical forms of administration of active ingredients, the vehicles to be used, and their manufacturing procedures can be found, for example, in the Galician Pharmacy Treaty, C. Faulí i Trillo, Luzán 5, SA de Ediciones 1993 and in Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 20 ^th edition, Williams & Wilkins PA, USA (2000).

 In one embodiment, the medium in which the calpain inhibitor can be dissolved contains an organic solvent. In a preferred embodiment, the medium contains DMSO. In a more preferred embodiment the calpain inhibitor is calpeptin. In an even more preferred embodiment, calpeptin is dissolved in DMSO at a concentration of 25mg / ml. In an even more preferred embodiment, calpeptin is administered at a concentration between 0.1 and 1 mg / kg. In another more preferred embodiment the calpain inhibitor is calpastatin.

Non-limitingly, among the administration routes of the calpain inhibitor are non-invasive pharmacological administration routes, such as oral, gastroenteric, nasal or sublingual routes, and invasive administration routes, such as the parenteral route. In a particular embodiment, the calpain inhibitor is administered in a pharmaceutical form of parenteral administration (eg, intradermally, intramuscular, intraperitoneal, intravenous, subcutaneous, intrathecal, etc ...). By "parenteral route of administration" is meant that route of administration consisting in administering the compounds of interest by injection, therefore requiring the use of syringe and needle. There are different types of parenteral puncture depending on the tissue the needle reaches: intramuscularly (the compound is injected into muscle tissue), intravenously (the compound is injected into a vein), subcutaneously (injected under the skin) and intradermally (injected between the layers of the skin). The intrathecal route is used for administration in the Central Nervous System of drugs that cross the blood-brain barrier poorly, so that the drug is administered in the space surrounding the spinal cord (intrathecal space). In a preferred embodiment, the administration is intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous or intrathecal. In a preferred embodiment, the route of administration is subcutaneous.

 In another preferred embodiment, administration is performed monthly, preferably, once every 4 months, once every 3 months, once every 2 months, once a month. In another preferred embodiment, administration is performed weekly, preferably, once every 4 weeks, once every 3 weeks, once every two weeks, once a week. In another preferred embodiment, administration is performed daily, preferably, once every 6 days, once every 5 days, once every 4 days, once every 3 days, once every 2 days. In another preferred embodiment, administration is performed daily, preferably, once a day, 2 times a day, 3 times a day or 4 times a day.

The term "subject" or "patient" as used herein includes human beings, male or female, of any age or race. In a preferred embodiment, the subject is diagnosed with SMA. In a more preferred embodiment the subject is diagnosed with SMA for having developed at least one symptom of the disease, at least 2, at least 3, at least 4, at least 5. In another more preferred embodiment the subject is diagnosed by means of a genetic test without symptomatic manifestation. In another preferred embodiment, the subject is a human being with at least 1 day, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 days of life. In another preferred embodiment, the subject is a newborn human being with at least 1 week, at least 2, at least 3, at least 4 weeks of life. In another preferred embodiment, the subject is a newborn human being with at least 1 month, at least 2, at least 3, at minus 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 months of life. In another preferred embodiment, the subject is a human being of at least 1 year, at least 2 years, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, At least 10 years old.

 In a preferred embodiment, the SMA is type I, II, III or IV. In a more preferred embodiment, SMA is type I. In an even more preferred embodiment, treatment is administered for the first time to a patient from the day after birth, 2 days later, 3 days later, 4 days later, 5 days later, 6 days later, 7 days later; preferably, it is administered from the day following its birth. In another preferred embodiment, the treatment is first administered to a patient from the week following his birth, 2 weeks later, 3 weeks later, 4 weeks later. In another preferred embodiment, the treatment is first administered to a patient from the month following his birth, 2 months later, 3 months later, 4 months later, 5 months later, 6 months later, 7 months later, 8 months later. , 9 10 months later, 11 months later, 12 months later. In another preferred embodiment, the treatment is administered for the first time to a patient from the year following his birth, 2 years later, 3 years later, 4 years later, 5 years later, 6 years later, 7 years later, 8 years later. , 9 years later, 10 years later.

Pharmaceutical compositions comprising calpain inhibitors and their use in the treatment of SMA.

The inventors of the present invention have found that the calpain inhibitor can be administered as a combination therapy with other compounds useful for the treatment of SMA. In another aspect, the invention relates to a method for the treatment of SMA comprising administering a calpain inhibitor in combination with an additional compound. Therefore, in a second aspect, the invention relates to a composition comprising a calpain inhibitor and a different additional active ingredient capable of increasing the improvement of patients selected from the group of: a histone deacetylase inhibitor or a modulator of autophagy In a preferred embodiment, the pharmaceutical composition according to the present invention comprises a calpain inhibitor and a histone deacetylase or HDAC inhibitor. By "HDAC inhibitor", as used in the present invention, it refers to a compound that inhibits the activity of histone deacetylase (HDAC), an enzyme involved in the removal of acetyl groups from lysine residues of histones. . HDAC inhibitors also have effects on non-histone proteins that are related to the acetylation process, including HSP90.

 To know if a compound is an HDAC inhibitor, it is possible to use any assay known in the state of the art to analyze histone deacetylase activity, such as the commercial EpiQuick HDAC Activity / Inhibition Assay Kit from Epigentek, so that if the compound to be tested inhibits histone deacetylase activity, said compound is an HDAC inhibitor. Non-limiting examples of histone deacetylase inhibitors are:

 valproic acid (CAS number 99-66-1),

 - magnesium valproate (CAS number 62959-43-7),

 sodium phenylbutyrate (CAS number 1716-12-7),

 pivanex (CAS number 122110-53-6),

 vorinostat (CAS number 149647-78-9),

 panobinostat (CAS number 404950-80-7),

 belinostat (CAS number 414864-00-9),

 tefinostat (CAS number 914382-60-8),

 givinostat (CAS number 497833-27-9),

 mocetinostat (CAS number 726169-73-9) and,

 entinostat (CAS number 209783-80-2).

 In a more preferred embodiment, the calpain inhibitor is calpastatin. In an even more preferred embodiment, the calpain inhibitor is calpeptin.

In another preferred embodiment, the pharmaceutical composition according to the present invention comprises a calpain inhibitor and an autophagy modulator. Autophagy, as used in the present invention, is a cellular process by which a recycling of cellular components occurs by incorporation into liosomes through the formation of vesicles called autophagosomes. ETn "autophagy modulator" as used herein is therefore it refers to a compound that increases or decreases a catabolic cellular mechanism that involves the degradation of unnecessary or dysfunctional cellular components such as intracellular proteins, protein aggregates, cell organelles, cell membranes, membrane organelles and other cellular components, through action of lysosomes. Although closely linked to apoptosis, autophagy is primarily characterized as a catabolic mechanism by which homeostasis of cellular energy is maintained. To know if a compound is an autophagy modulator, it is possible to use any assay known in the state of the art to analyze autophagy, such as immunohistochemistry.

 Non-limiting examples of autophagy modulators are:

 Chloroquine (CAS number 54-05-7),

 3-methyladenine (CAS number 5142-23-4),

 bafilomycin Al (CAS number 88899-55-2),

 - trehalose (CAS number 99-20-7),

 rapamycin (CAS number 53123-88-9),

 resveratrol (CAS number 501-36-0) and

 curcumin (CAS number 458-37-7).

 In a more preferred embodiment, the calpain inhibitor is calpastatin. In an even more preferred embodiment, the calpain inhibitor is calpeptin.

 The terms and limitations described above in relation to the use of a calpain inhibitor of the invention are equally applicable to this aspect.

* * *

 The invention is described below by means of the following examples, merely illustrative and not limiting the scope of protection of the invention.

EXAMPLES

Materials and methods

Isolation and culture of spinal cord MN

Embryonic CD1 or AME mouse spinal cord MN cultures of 12.5 days (El2.5) or 13 days (El 3) embryonic were prepared essentially as described (Gou-Fabregas et al, 2009. J Neurochem. 110: 1842-54; Garcera et al., 2011. Neurobiol Dis. 42: 415-26). The isolated cells were pooled in a tube containing culture medium and plated. The isolated MNs were seeded in either four-well culture plates (Nunc, Thermo Fisher Scientific, Madrid, Spain) for survival experiments (15,000 cells / well), evaluation of neurite degeneration (10,000 cells / well), and Immunoblot analysis (50,000 cells / well) or in 15 mm glass coverslips placed in four-well culture plates for immunofluorescence. The wells and glass coverslips were coated with polyiorithin / laminin (Sigma) as described (Soler et al., 1998). The culture medium was Neurobasal medium (Gibco, Invitrogen, Paisley, UK) supplemented with B27 (2% v / v, Gibco), horse serum (2% v / v, Fisher Scientific), L-glutamine (0.5 mM, Gibco), and 2-mercaptoethanol (25 mM, Sigma) and a mixture of recombinant NTF (brain derived neurotrophic factor 1 ng / ml, neurotrophic factor derived from glial cell line 10 ng / ml, ciliary neurotrophic factor 10 ng / ml, cardiotrophin-l 10 ng / ml and hepatocyte growth factor 10 ng / ml; Gibco).

AME animals

The AME mice used are FVB Cg-Tg (SMN2) ^89Ahmb Smnl ^tmlMsd / J (rnutAME) and FVB Cg-Tg (SMN2 * delta7) 4299 Ahmb Tg (SMN2) ^89Ahmb Srnnl ^tmlMsd / J (SMNA7). Heterozygous animals were crossed to obtain homozygous mutAME or SMNA7 Young of the same wild type (WT) litter were used for control experiments. For the purification of the MN embryos were removed from El 2, 5 of the uterus and a piece of the head was cut for genotyping; for in vivo treatment the neonatal offspring were tattooed and a tail fragment was collected. The REDExtract-N-Amp Tissue PCR Kit (Sigma, St Louis, MO, USA) was used for genomic DNA extraction and PCR organization, with the following primers: WT direct 5'-CTCCGGGATATTGGGATTG-3 ', reverse AME 5'-GGTAACGCCAGGGTTTTCC-3 'and reverse WT 5'- TTTCTTCTGGCTGTGCCTTT-3'.

Plasmids and lentivirus particle production

For RNA interference experiments, constructs were generated in pSUPER. retro pure (Oligo-Engine, Seattle, WA, USA) using oligonucleotides specific (Invitrogen) that are directed to the sequence of calpain-1 (shCalp) indicated by capital letters as follows, direct: 5'- gatccccGCGCCAAGCAGGTAACTTAttcaagagaTAAGTTACCTGCTTGGCGCttttt-3 'and reverse: 5'- agctaaaaaaGCGTAGTGTGTGTGGGGGGGGGGGGGGGGGGGGGGGGGG pSPAX2 and pMD2G. Viruses at 4 x 10 ⁵ -1 x 10 ⁶ UT / ml were used for the experiments. The empty vector (EV) was used as a control. For lentivirus transduction, MNs were seeded in four-well plates. Media containing lentivirus (2 UT / cell) was added 3 h later, and then changed after 20 h. In each experiment, the green fluorescent protein (GFP) positive cells were counted directly to track the effectiveness of the infection. The frequency of infection amounted to 99%.

Reagents and treatments

 On day 6 after sowing, the cultured MNs were treated with calpeptin (Calbiochem®) which was dissolved in DMSO (Sigma) or with potassium chloride (KC1, Sigma). Calpeptin was dissolved at a final concentration of 25 mM and added to the culture. KC1 was dissolved with NBM at a final concentration of 30 mM. For vehicle control, the same amount of solvent (DMSO) was added to the culture medium.

Survival of MN and neurite degeneration analysis

For the evaluation of the survival of the MN cells were seeded in complete medium containing a mixture of recombinant NTF or in basal medium without supplements or complete medium containing the experimental conditions. Three days after treatment, large bright phase neurons with long neuritic extensions present in photomicrographs of different microscopic areas of the culture plates were counted (4 central areas per well, 3 wells for each condition per experiment). The number of cells present in each plate on day 0 was considered the initial 100%. The counts were made every 3 days in the same microscopic areas as the initial count. Survival was expressed as the percentage of cells counted with respect to the initial value (100%). The morphometric analysis of neurite degeneration was performed as described (Press and Milbrandt, 2008. J. Neurosci. 28: 4861-71), with modifications. Briefly, the dissociated MNs were cultured and transduced as described above. Six, 9 and 12 days after transduction, phase contrast microscopy images were obtained with a 40x lens. A grid was created on each image with the NIH ImageJ software, using the grid complement (line area = 50,000). The cell count complement was used to score each neurite. Degenerating and healthy cells were counted in at least 10 large magnification fields per image (30-50 neurites) for each well. Three different wells were counted for each condition (with the observer without knowing the condition) and the experiments were repeated at least three different times. The neurite segments were considered degenerated if they showed evidence of swelling and / or blisters.

Administration of calpeptin in vivo

The mice were individually caged in propylene cages (33 cm ^c 18 cm x 14 cm) at an ambient temperature of 22 ± 2 ° C and a relative humidity of 40% ± 10%. Reproductive mice were given ad libitum water and rodent feed. The mice were kept in a light cycle: 12 h dark: 12 h (light period from 07:30 to 19:30). Young from the same mutAME and SMNA7 litters (mutants and WT) were randomly assigned to receive treatment or vehicle. Calpeptin (Calbiochem®, Merck, Madrid, Spain) was dissolved at a concentration of 50 mM in DMSO and injected at a dose of 0.006 mg per gram of weight in saline. The vehicle groups received equal volumes of saline solution with the same amount of DMSO. Administration was by subcutaneous injection (SC, interscapular region) once a day starting from P0 until death with a sterile polypropylene syringe (icogamma plus, 1 ml) and with a 30G needle (BD Microlance). WT animals received treatment or vehicle for up to 3-4 weeks. Birth was defined as postnatal day 0 (P0) for the experiments. The survival of the animals was analyzed, as well as body mass, size and behavioral tests (postural reflex and tube test).

Behavior analysis All tests were performed during the light period between 09:00 and 12:00 and before administration. For the postural reflex, each offspring turned on the spine on a flat surface and recorded the time it took to stand steadily on all four legs on the ground (time limit of 30 s). In the tube test, the animals were placed with the head down, hanging by the hind legs in a 50 ml plastic centrifuge tube with a mattress of a cotton ball at the bottom to protect the animals head after its fall The latency to fall from the edge of the tube was evaluated for a period of 30 s. Three repetitions were performed for each test with a minimum of 2 minutes rest between repetitions. Behavioral tests were performed daily starting at Pl through P10.

Immunoblot analysis

 To perform the immunoblotting, Total Cellular Used in polyacrylamide and SDS gels were separated and transferred to Immobilon-P polyvinylidene difluoride transfer membrane filters (Millipore, Billerica, MA, EE ETU) using a semi-dry Amersham Trans-Blot apparatus. Biosciences (Buckinghamshire, REÍ). The membranes were tested with anti-SMN antibody (1: 5000; BD Biosciences), anti-calpain-1 antibody (1: 1000; Biomol International Inc., Exeter, REÍ) and anti-clone AA6 clone (1: 4000; Biomol ). To control the specific protein content per lane, the membranes were retested with a monoclonal anti-tubulin antibody (1: 50,000; Sigma). The membranes were developed using Luminata ™ ForteWestem HRP Substrate (Millipore).

Immunofluorescence

Cultivated MNs were seeded on glass coverslips and treated with lentiviruses containing EV or shCalp. Six days after transduction with lentivirus the cells were fixed with 4% paraformaldehyde (Sigma) 10 minutes and with methanol (Sigma) for an additional 10 minutes. The MNs were permeabilized with 0.2% Triton X-100 and incubated for 1 h with 5% BSA in PBS. The primary antibody (antibody against SMN, 1: 100) was diluted in PBS and incubated overnight. After washing the secondary anti-mouse antibody ALEXA555 (Invitrogen), added at a 1: 400 dilution. Staining was performed with Hoechst (1: 400, Sigma) to identify the nuclear location in the soma of the MN. Samples were mounted using Mowiol mounting medium (Calbiochem®). Microscopy observations were performed on a FV10I Olympus confocal microscope (Tokyo, Japan).

Statistic analysis

 All experiments were performed at least three times. Values were expressed as mean ± E.E.M. Differences between groups were evaluated by univocal ANOVA of cultures transduced by lentiviruses at each time point; if significant, multiple post-hoc comparisons were made using the Bonferroni test; P values <0.05 were considered significant.

Results

 Calpain attenuation increases the level of SMN protein in MN of spinal cord in culture

To analyze the effect of calpain reduction on SMN in spinal cord MN, a lentivirus RNA interference method was used to decrease the level of calpain protein in these cells. Embryonic spinal cord MN (El2.5) were isolated and seeded in culture wells. Three hours later they were transduced with lentiviruses containing the empty vector (EV) or short bracketed RNA sequences directed to specific mouse calpain sites (shCalp) and maintained in the presence of a mixture of neurotrophic factors (NTF) (derived neurotrophic factor of brain 1 ng / ml, neurotrophic factor derived from glial cell line 10 ng / ml, ciliary neurotrophic factor 10 ng / ml, cardiotrophin-l 10 ng / ml and hepatocyte growth factor 10 ng / ml). At 3, 6 and 9 days after transduction, Cellular Used were collected and immunoblotted using an anti-SMN antibody. A significantly increased level of SMN protein was observed in shCalp cells after 3 (1.30 ± 0.09, / 0.0l), 6 (1.69 ± 0.19, / O, OI) and 9 (1, 75 ± 0.46, p <0.05) days of transduction, compared to the EV (Fig. 1A). Control transfer analysis using an anti-calpain antibody showed that the calpain protein is reduced in the shCalp condition compared to EV. To analyze whether this reduction in SMN is preferentially located in the soma of cells or neurites, SMN levels were measured by immunofluorescence using confocal microscopy. The MNs were seeded on glass coverslips and transduced using the EV or shCalp constructs. Six days after transduction, the cells were fixed and SMN immunostaining was performed with an anti-SMN antibody. The quantification of specific fluorescence levels showed that SMN increases both in the Soma of MN (115.8 ± 7.27, /><0.00l), and in neurites (12.29 ± 2.21, /><0.0005) in shCalp cells when purchased with the EV (85.43 ± 5.34 and 6.18 ± 0.56, respectively) (Fig. 1B).

To clarify the effect of calpain reduction on the viability of MN, survival experiments were performed. The cells were seeded at low density to avoid effects mediated by cell contact (7,890 cells / cm ² ) and cultured under various conditions: in the absence (NBM) or presence of a mixture of NTF and the NFT condition treated with EV or shCalp . Three, six and nine days after culture, the cells were counted in three wells of each condition. Considering 100% of the initial number of cells present in a well on day 0, survival was estimated as the percentage of cells remaining in the same well. As shown in Figure 1C, using the phase contrast microscope we observed that after 9 days without NTF, the MNs were largely degenerated (Press and Milbrandt 2008. J. Neurosci. 28: 4861-71) showing a percentage significantly lower surviving cells (day 3, NBM 3.38 ± 5.86, compared to NTF 64.38 ± 3.90, /><0.00l). However, the presence of EV or shCalp does not change the survival of the MN when compared to the NTF condition (day 9, 40.76 ± 2.63, 44.11 ± 5, 12 and 42.35 ± 2, 51 respectively). These results established that the reduction of endogenous calpain does not alter the normal viability of MN.

Calpain attenuation prevents the reduction of SMN caused by membrane depolarization

The addition of a high concentration of potassium (K ⁺ ) in the culture medium induces chronic depolarization of the neuronal plasma membrane, which in turn activates the voltage-regulated calcium channels (VGCC), which produces an entry of Ca ²⁺ and increased concentration of intracellular Ca ²⁺ . To clarify what is the effect of the increase in intracellular Ca ²⁺ at the level of SMN and the role of calpain in mediating this effect, spinal cord MNs were isolated from mice 2, 5 and a primary culture was established in the presence of NTF. The cells were transduced with the EV or shCalp constructs. Six days later, the cells were treated with 30 mM KC1 (30K) for 3 hours and protein extracts were obtained and immunoblotted using anti-SMN antibody. The results in Figure 2 show that the SMN protein was significantly reduced in cultures treated with 30K EV (1.81 fold reduction, p <0.0001) compared to untreated EV control. However, shCalp transduced cells showed no changes in SMN with treatment with KC1. Therefore, no significant differences in the level of SMN in 30K shCalp were observed when compared to the shCalp control condition. Consequently, treatment with a high concentration of potassium (K ⁺ ) in the culture medium induced reduction of the SMN protein through calpain activity in cultured spinal cord MN.

Calpeptin treatment increases the level of SMN protein in spinal cord MN in culture

 To further assess the role of calpain activity at the level of SMN protein in MN, the calpain cell permeable inhibitor, calpeptin, was used. First, the effect of calpeptin in basal culture conditions was analyzed. El2.5 MNs were isolated and cultured in the presence of NTF. Six days after sowing the culture medium was changed to new medium containing NTF or NTF plus 25 mM calpeptin. Total Cellular Uses were obtained at 3, 9, 16, 20 and 24 hours after treatment and subjected to immunoblot protein analysis using an anti-SMN antibody. The results shown in Figure 3A demonstrate that the level of SMN protein is significantly increased after 3, 9, 16 and 20 hours of treatment with calpeptin (3.34 ± 0.56, p <0.005, 4.40 ± 0, 58, p <0.005, 2.89 ± 0.61, p <0.05 and 2.06 ± 0.50, p <0.05 respectively). However, no changes in SMN were observed in cells treated 24 hours. These results together indicate that calpain inhibition increases SMN until 20/24 hours of treatment.

To study whether the effect of calpeptin treatment in MN with chronic depolarized membranes regulates the level of SMN protein, cultures were established. MN primary. Six days after seeding, the cells were treated with NTF (control) or NTF plus 30K or 25 mM calpeptin or 25 mM calpeptin + 30K. Three hours after treatment Cellular were obtained and subjected to immunoblotting using anti-a-fodrine or anti-SMN antibodies. Degradation of a-fodrin to specific fodrin degradation products of 150/145 kDa indicates activation of calpain. Product levels of 150/145 kDa in cells treated with NTF plus 30K (30K) were significantly increased compared to the NTF control condition (1.33 ± 0.13, /><0.0001). However, the addition of calpeptin to the control conditions NTF or 30K significantly reduced fodrine degradation products (0.66 ± 0.05,><0.001 and 0.63 ± 0.07, / K0, 001 , respectively) (Fig. 3B). This result suggests that calpain is activated after membrane depolarization in mouse MN. The level of SMN protein in cells treated with calpeptin + 30K (1.67 ± 0.26, / <0.05) was significantly increased compared to the 30K and control conditions (Fig. 3B). These results together indicate that SMN is reduced in cells treated with 30K and this effect can be prevented by treatment with calpeptin.

Calpain directly processes SMN in cultured MN

To identify whether SMN is directly processed by calpain, the N- and C-terminal degradation products were purified from MN of El2.3 of mouse CD1 and cultured for 6 days in the presence of NTF. Cells were treated for 3 hours with 30 or 50 mM KC1, or with 25 mM calpeptin and Cellular Used were analyzed by immunoblot using N-terminal / full length specific anti-SMN monoclonal antibodies (clone 8, BD Bioscience) and C-terminal (Acm 9F2) to detect degradation products. The quantification of full-length SMN showed that treatment with potassium significantly reduces protein levels while treatment with calpeptin increases levels of SMN. C-terminal fragments were detected in the membrane when the full length SMN band was overexposed. As shown in Figure 4 the degradation of SMN was inhibited by the addition of calpeptin. Calpeptin treatment increases the level of SMN protein and prevents degeneration of neurites in AME mutant MNs

 To determine whether treatment with calpeptin regulates SMN in MN of El2.5 embryos of the severe AME transgenic mouse model (FVB Cg-Tg (SMN2) 89AhmbSmnltmlMsd / J), mice were genotyped and the wild-type spinal cord dissected ( WT) and muiante (mutAME). MNs isolated from WT and mutAME were grown in the presence of NTF. Six days after seeding the cells were treated with 25 mM calpeptin for 3 hours. Protein extracts were collected and subjected to immunoblot analysis using an anti-SMN antibody. The results indicate that SMN increases in the WT condition treated with calpeptin ((2.43 ± 0.35, / K0.05) compared to the WT control (Figure 5A left graph) .In addition, in cultures of mutAME treatment with calpeptin also significantly increased the SMN protein compared to untreated cells (5, l8 ± 1.48, p <0.05) (Figure 5 A left and right).

Focal axonal swelling has been previously described in mouse tissues and MN with reduced SMN and in muscle biopsies of patients with severe SMA. To determine the presence of neurite degeneration in the primary culture model of MN, AME mouse model cells were isolated in 2.5 and cultured at a low density (5,200 cells / cm ² ). Neurite degeneration was assessed by quantifying the neurite segments in a given area of the plaque using phase contrast microscopy on days 6, 9 and 12. Neurites were considered degenerated if they showed evidence of swelling and / or blisters (see, Materials and methods). Nine and 12 days after sowing in the presence of NTF, mutAME MNs show a higher percentage of degeneration than WT MNs. To analyze the effect of calpain reduction on the reduction of neurite degeneration, mutAME and WT cells were transduced with the lentivirus that carries the shCalp or EV constructs. After 6 days of transduction, no significant differences were observed between groups. However, 9 days after transduction, a significant difference in neurite morphology was detected when shCalp mutAME cultures (16.64 ± 5.67% degenerated neurites) were compared with EV mutAME (28, l8 ± 6, 22% of degenerated neurites) (p <0.0l). After 12 days, the signs of degeneration increased to more than 36% of the neurites present in EV mutAME cultures, while shCalp mutAME cultures showed 26%. This percentage was not different from those observed in EV WT (26.29 ± 4.69) and shCalp WT (25.62 ± 3.6l). All these results together demonstrate that calpain regulates the level of SMN protein and neurite degeneration in MN with reduced SMN.

Calpeptin administration extends the survival of mice with severe AME and AME SMNA7

 The evidence provided suggests that calpain directly regulates the level of SMN protein. It was decided to examine whether the administration of calpeptin has a positive effect on mice with AME. To further evaluate this hypothesis, a treatment protocol was started using two different AME mouse models: the FVB Cg-Tg (SMN2) 89AhmbSmnltmlMsd / J (mice with severe SMA, mutAME), and the FVB.Cg- Grm7Tg (SMN2) 89Ahmb SmnltmlMsd Tg (SMN2 * delta7) 4299Ahmb / J (or SMNA7). To determine if calpeptin affected the life span and body weight of these mice, animals were tattooed and genotyped on postnatal day 0 (P0). Young of the same WT litter and mutants were grouped into reference and treatment groups and subcutaneous injections began in Pl, with a daily dose of 0.006 mg of calpeptin per gram of weight. The results showed that the administration of calpeptin significantly increased the life span of mutAME calpeptin (average days 8, n = ll) compared to the reference mutAME (average days 4.5, h = 12, p <0.000l ) (Fig. 6A). Likewise, the life span of SMNA7 calpeptin mice (mean days 14, n = ll) was significantly increased compared to reference SMNA7 (mean days 11.5, n = 6, p <0.000l) (Fig. 6B ).

While the life span was extended, no increase in weight was observed in mutant animals treated with calpeptin. In contrast, the weight was slightly increased in the WT groups treated with calpeptin compared to the reference WT groups from day thirteen of treatment until the end of the experiment (MutSMA, day 15, WT treated 11.87 ± 0.23 gr , n = 2 vs. WT Sham 9.49 ± 0.33 gr, n = 8, p <0.000l) (SMNA7, day 15, WT treated 11.14 ± 0.59 gr, n = 2 vs. WT Sham 7.74 ± 0.66 gr, n = 5, p <0.00l) (Figure 6 C and D). Calpeptin treatment improves motor function in mice with SMA

 Since the mutAME and SMNA7 mouse models are characterized by severe impairment of motor function, it was analyzed whether the increase in life span observed in both models after treatment with calpeptin correlates with improved motor function. Two different behavioral tests were performed: the postural reflex test and the tube test.

 The postural reflex response is based on the ability of neonatal mice to return to their four legs after being placed supine. It can be measured in offspring as soon as P1-P2 and evaluated up to P9-P10. This test assesses overall body strength and coordination. Due to its simplicity, it allows the longitudinal study of the evolution of locomotor deterioration that is presented as an increase in the time to straighten. The test was performed on mice in the following groups: reference WT and treated with calpeptin, and reference mutant groups (mutAME and SMNA7) and calpeptin. The test was designed with a maximum time of 30 seconds and measurements were taken daily before the injection of calpeptin from Pl to P10. The test was done in triplicate for each animal with 5 minutes of rest time between tests. The results obtained in the WT groups of both mutAME and SMNA7 mice models show that the postural reflex time was consistently faster than in the mutant groups. In mutAME treated with calpeptin there were no significant differences from the start of treatment (Pl) to P7 compared to reference mutAME. However, from P7 until the end of the experiment the postural reflex time decreased progressively in mutAME treated with calpeptin (Fig. 7A). When SMNA7 mice were analyzed, a slight reduction in time was observed until the calpeptin-treated group was straightened from P7 until the end of the experiment (Fig. 7B).

The tube test is a non-invasive motor function test specifically designed for neonatal rodents. Evaluate the muscle strength of the hind legs, weakness and fatigue. The ideal age of the animal varies for this test from P2 to P8. In each test, the mouse is placed with the head down, hanging by the hind legs in a tube. Two parameters were evaluated in the present study: latency to fall off the edge of the tube (in seconds, time graphs) and the score of the hind legs (HLS graphs) that evaluates the placement of the legs and tail. The rats with motor weakness they show reduced time to fall and low HLS score. The test was performed on the same reference and treated groups of mice described. The test was designed with a maximum time of 30 seconds and triplicate measurements were made daily before the injection of calpeptin from Pl to P8. When the latency time was analyzed in the mutAME model, it was observed that from Pl to P5 all groups of mice did not show differences in time until falling. However, on days 6 and 7 after treatment (P6 and P7) mutAME mice treated with calpeptin showed significantly increased time to fall compared to the reference mutAME group (day 6, MutSMA treated 15.62 ± 10.66, n = 7 vs. Mut SMA Sham 3.85 ± 1.92, n = 3 p <0.05; and day 7, MutSMA treated 17.78 ± 10.62, vs. Mut SMA Sham 0.167 ± 0.41, p <0.00l) and without significant differences with respect to the WT groups (Fig. 7C). In the SMNA7 model, calpeptin treatment of mutants significantly increased the latency time compared to reference mutant controls from P3 until the end of the experiment (Fig. 7D). In both AME models, HLS measurements revealed that calpeptin treatment of mutant mice significantly increased the score compared to reference mutants (Fig. 7E and 7F). Together, the results obtained from the behavioral tests carried out suggest that treatment with calpeptin improves the motor function of AME mutant mice of severe models.

Claims

I- A calpain inhibitor for use in the treatment of spinal muscular atrophy (SMA).

2- The calpain inhibitor for use according to claim 1 wherein the AME is a consequence of a homozygous disruption in the SMN1 gene.

 3- The calpain inhibitor for use according to claims 1-2, wherein the patient suffering from AME is characterized by having a copy number equal to or less than 8 in the locus of the SMN2 gene.

4- The calpain inhibitor for use according to any of claims 1-3 wherein the SMA is type I, type II, type III or type IV.

 5- The calpain inhibitor for use according to claims 1-4 wherein the inhibitor is calpeptin or calpastatin

 6- The calpain inhibitor for use according to claim 5 wherein the calpeptin is administered in the form of a composition comprising an organic solvent

7- The calpain inhibitor for use according to claim 6 wherein the organic solvent is dimethylsulfoxide (DMSO).

 8- The calpain inhibitor for use according to claims 5-7 wherein the calpeptin is administered at a dose between 0.1 and 1 mg / kg.

9. The calpain inhibitor for use according to any of claims 1-10 wherein the treatment is administered to a patient who has developed at least one symptom of SMA.

 10-E1 calpain inhibitor for use according to any of claims 1-10 wherein the treatment is administered to a patient who has not developed any symptoms of SMA.

 I I-E1 calpain inhibitor for use according to any of claims 1-12 wherein the treatment is administered to a patient from the day after birth.

l2-Pharmaceutical composition comprising a calpain inhibitor and a compound selected from the group consisting of:

 (i) a histone deacetylase inhibitor (HDAC) and

(ii) an autophagy modulator. 13-Pharmaceutical composition according to claim 12 wherein the calpain inhibitor is calpeptin or calpastatin.

 14-Composition according to claims 12 or 13 for use in the treatment of SMA.

15. Composition for use according to claim 14 wherein the AME is a consequence of a homozygous disruption in the SMN1 gene.

 16- Composition for use according to claims 14 or 15 wherein the patient suffering from SMA is characterized by having a copy number equal to or less than 8 in the locus of the SMN2 gene.

17- Composition for use according to any of claims 14 to 16 wherein the

AME is type I, type II, type III or type IV.

 18- Composition for use according to any of claims 14 to 17 wherein the composition is administered to a patient who has developed at least one symptom of SMA.

19. Composition for use according to any of claims 14 to 18 wherein the composition is administered to a patient who has not developed any symptoms of SMA.

 20- Composition for use according to any of claims 12-19 wherein the treatment is administered to a patient from the day after birth.