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WO2019170930A1 - Traitement pour l'atrophie musculaire spinale - Google Patents

Traitement pour l'atrophie musculaire spinale Download PDF

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Publication number
WO2019170930A1
WO2019170930A1 PCT/ES2018/070177 ES2018070177W WO2019170930A1 WO 2019170930 A1 WO2019170930 A1 WO 2019170930A1 ES 2018070177 W ES2018070177 W ES 2018070177W WO 2019170930 A1 WO2019170930 A1 WO 2019170930A1
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Prior art keywords
calpain
sma
treatment
calpeptin
use according
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English (en)
Spanish (es)
Inventor
Rosa María SOLER TATCHÉ
Ana GARCERÁ TERUEL
Sandra DE LA FUENTE RUIZ
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Universitat de Lleida
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Universitat de Lleida
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • 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.
  • SMA Spinal muscular atrophy
  • MN motor neurons
  • 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).
  • STN1 Survival Motor Neuron 1
  • SMN2 motor Neuron survival protein
  • SMN2 motor neuron survival protein
  • 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.
  • SMN protein improves disease symptoms
  • 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.
  • UPS ubiquitin proteasome system
  • the invention relates to the use of a calpain inhibitor for the treatment of spinal muscular atrophy (SMA).
  • SMA spinal muscular atrophy
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a calpain inhibitor and a compound selected from the group consisting of:
  • HDAC histone deacetylase inhibitor
  • FIG. 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).
  • NTF neurotrophic factors
  • 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.
  • Calpeptin treatment increases the level of SMN protein in MN.
  • Calpeptin administration extends the survival of severe AME and AME SMNA7 mice.
  • 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.
  • 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).
  • 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).
  • the calpain inhibitor acts on calpain 1 and 2.
  • the calpain inhibitor acts on calpain 1 without significantly affecting the activity or expression of calpain 2.
  • 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.
  • siRNA small interfering RNA
  • hRNA short bracketed RNA
  • miRNA microRNA
  • an antisense oligonucleotide or a ribozyme include, without limitation, a small interfering RNA (siRNA), a short bracketed RNA (hRNA), a microRNA (miRNA), an antisense oligonucleotide or a ribozyme.
  • small interfering RNA 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.
  • siRNA includes duplexes of two separate chains.
  • siRNA molecules are not limited to RNA molecules but also encompass nucleic acids with one or more chemically modified nucleotides, such as morpholinos.
  • 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.
  • micro RNA 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.
  • antisense sequence 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.
  • 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); Cyclohex
  • 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 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 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 is a modified nucleic acid analog comprising normal deoxyribonucleotides covalently linked to hydrophobic insertions.
  • Hexitol nucleic acids 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.
  • the wings are modified 2'-0-methyl (OMe) or 2'-0-methoxyethyl (MOE) oligonucleotides that protect the internal block from nuclease degradation.
  • 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.
  • 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.
  • assays 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.
  • 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:
  • Acetyl calpastatin (CAS number: 123714-50-1)
  • Aclacinomycin A (CAS number: 57576-44-0) - E64-d (CAS number: 88321-09-9)
  • IC50 intermediate inhibition concentration
  • concentration of an inhibitor is defined as the ratio between the IC50 value of a given inhibitor for the target of interest with respect to another target.
  • 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.
  • 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.
  • 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.
  • 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.
  • factors that can influence enzyme activity include factors such as salt concentration, temperature, pH and substrate concentration.
  • treatment refers to any type of therapy, which aims to terminate, improve or reduce the susceptibility to AME.
  • treatment refers 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.
  • 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.
  • SMA Spinal muscular atrophy
  • MN motor neuron
  • 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.
  • AME originates as a result of a disruption in the SMN1 (Survival Motor Neuron 1) gene, also known as telomere copy encoding SMN.
  • SMN1 Sudvival Motor Neuron 1
  • AME originates as a result of a homozygous disruption of the SMN1 gene.
  • SMSN1 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).
  • 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.
  • 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.
  • at least one of the SMN2 gene loci contains 1 or 2 copies of the gene.
  • 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).
  • 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.
  • AME type I AME type II
  • AME type III AME type IV
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • vehicle generally includes any diluent or excipient with which an active ingredient is administered.
  • 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.
  • EMA European Medicines Agency
  • FDA Food & American Drug Administration
  • the calpain inhibitor can be dissolved for administration in any suitable medium.
  • suitable medium 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.
  • 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).
  • DMSO dimethylsulfoxide
  • 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 medium in which the calpain inhibitor can be dissolved contains an organic solvent.
  • the medium contains DMSO.
  • the calpain inhibitor is calpeptin.
  • calpeptin is dissolved in DMSO at a concentration of 25mg / ml.
  • calpeptin is administered at a concentration between 0.1 and 1 mg / kg.
  • the calpain inhibitor is calpastatin.
  • the calpain inhibitor is administered in a pharmaceutical form of parenteral administration (eg, intradermally, intramuscular, intraperitoneal, intravenous, subcutaneous, intrathecal, etc ).
  • 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.
  • intramuscularly the compound is injected into muscle tissue
  • intravenously the compound is injected into a vein
  • subcutaneously injected under the skin
  • 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).
  • the administration is intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous or intrathecal.
  • the route of administration is subcutaneous.
  • administration is performed monthly, preferably, once every 4 months, once every 3 months, once every 2 months, once a month.
  • administration is performed weekly, preferably, once every 4 weeks, once every 3 weeks, once every two weeks, once a week.
  • 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.
  • 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.
  • the subject is diagnosed with SMA.
  • 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.
  • the subject is diagnosed by means of a genetic test without symptomatic manifestation.
  • 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.
  • the subject is a newborn human being with at least 1 week, at least 2, at least 3, at least 4 weeks of life.
  • 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.
  • 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.
  • the SMA is type I, II, III or IV. In a more preferred embodiment, SMA is type I.
  • 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.
  • the treatment is first administered to a patient from the week following his birth, 2 weeks later, 3 weeks later, 4 weeks later.
  • 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.
  • 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.
  • compositions comprising calpain inhibitors and their use in the treatment of SMA.
  • the calpain inhibitor can be administered as a combination therapy with other compounds useful for the treatment of SMA.
  • 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
  • the pharmaceutical composition according to the present invention comprises a calpain inhibitor and a histone deacetylase or HDAC inhibitor.
  • HDAC inhibitor 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.
  • HDAC histone deacetylase
  • HDAC inhibitor an HDAC inhibitor
  • 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.
  • histone deacetylase inhibitors are:
  • panobinostat (CAS number 404950-80-7),
  • the calpain inhibitor is calpastatin. In an even more preferred embodiment, the calpain inhibitor is calpeptin.
  • 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.
  • 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.
  • autophagy 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.
  • autophagy modulators are:
  • Chloroquine (CAS number 54-05-7),
  • bafilomycin Al (CAS number 88899-55-2)
  • curcumin (CAS number 458-37-7).
  • the calpain inhibitor is calpastatin. In an even more preferred embodiment, the calpain inhibitor is calpeptin.
  • 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).
  • 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).
  • SSN2 * delta7 FVB Cg-Tg
  • SN2 * delta7 4299 Ahmb Tg
  • SMNA7 89Ahmb Srnnl tmlMsd / J
  • 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'.
  • 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'- gatccccGCGCCAAGCAGGTAACTTAttcaagagaTAAGTTACCTGCTTGGCGCtttt-3 'and reverse: 5'- agctaaaaaGCGTAGTGTGTGTGGGGGGGGGGGGGGGGGGGGGGGGGGGG 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.
  • MNs were seeded in four-well plates. Media containing lentivirus (2 UT / cell) was added 3 h later, and then changed after 20 h.
  • the green fluorescent protein (GFP) positive cells were counted directly to track the effectiveness of the infection. The frequency of infection amounted to 99%.
  • the dissociated MNs were cultured and transduced as described above.
  • 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.
  • 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).
  • SC subcutaneous injection
  • 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).
  • 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.
  • ALEXA555 Invitrogen
  • Calpain attenuation increases the level of SMN protein in MN of spinal cord in culture
  • 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.
  • NTF neurotrophic factors
  • SMN protein 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.
  • SMN levels were measured by immunofluorescence using confocal microscopy. The MNs were seeded on glass coverslips and transduced using the EV or shCalp constructs.
  • VGCC voltage-regulated calcium channels
  • Calpeptin treatment increases the level of SMN protein in spinal cord MN in culture
  • calpain cell permeable inhibitor calpeptin
  • calpeptin the calpain cell permeable inhibitor in basal culture conditions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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%.
  • Calpeptin administration extends the survival of mice with severe AME and AME SMNA7
  • 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 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.
  • 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.

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Abstract

La présente invention concerne une méthode de traitement de l'atrophie musculaire spinale (AMS) à des différents stades de développement qui consiste à administrer des quantités appropriées d'un inhibiteur de calpaïne à un sujet qui peut avoir developpé ou non les premiers symptômes de la maladie. L'invention se rapporte également à des compositions pharmaceutiques qui comprennent un inhibiteur de calpaïne qui est administré en combinaison avec un autre composé pour potentialiser l'effet, tels que des inhibiteurs d'histone désacétylases et des modulateurs de l'autophagie.
PCT/ES2018/070177 2018-03-09 2018-03-09 Traitement pour l'atrophie musculaire spinale Ceased WO2019170930A1 (fr)

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Citations (3)

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US5622967A (en) 1993-04-26 1997-04-22 Sterling Winthrop, Inc. Quinolone carboxamide Calpain inhibitors
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Patent Citations (3)

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US5622967A (en) 1993-04-26 1997-04-22 Sterling Winthrop, Inc. Quinolone carboxamide Calpain inhibitors
CA2631071A1 (fr) * 2008-05-09 2009-11-09 Tong-Jun Lin Inhibition des calpaines reduisant l'inflammation allergique
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