WO2023078807A1 - Inhibiteur de hdac de classe iia et decitabine pour traiter la myopathie - Google Patents

Inhibiteur de hdac de classe iia et decitabine pour traiter la myopathie Download PDF

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WO2023078807A1
WO2023078807A1 PCT/EP2022/080283 EP2022080283W WO2023078807A1 WO 2023078807 A1 WO2023078807 A1 WO 2023078807A1 EP 2022080283 W EP2022080283 W EP 2022080283W WO 2023078807 A1 WO2023078807 A1 WO 2023078807A1
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myopathy
pharmaceutical composition
patient
aza
muscle
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Susan TREVES
Francesco Zorzato
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Universität Basel
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4245—Oxadiazoles
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system

Definitions

  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising 5-Aza-2- deoxycytadine, and/or a class Ila HDAC inhibitor characterised by a trifluoromethyloxadiazole group, to treat a myopathy patient characterised by reduced expression of RYR1, and/or overexpression of class II histone deacetylases (HDAC) and/or DNA methyltransferases (DNMT) in a muscle sample.
  • HDAC histone deacetylases
  • DNMT DNA methyltransferases
  • Myopathy refers to medical conditions characterised by defects in skeletal muscle performance which cause muscle weakness.
  • Congenital myopathies a subset of rare neuromuscular disorders, are caused in approximately 30% of the patients, by mutations in the RYR1 gene.
  • Patients can present a variety of symptoms and phenotypes depending on whether the mutations are dominantly or recessively inherited.
  • patients with recessive RYR1 mutations often display involvement of extraocular muscles leading to ophthalmoplegia and or ptosis as well as involvement of respiratory muscles, often requiring assisted ventilation (Jungbluth H. et al. Nat. Rev. Neurol. 14, 151-167 (2016); Treves S. et al. Curr. Opin. Pharmacol.
  • Skeletal muscle contraction is initiated by a massive release of Ca 2+ from the sarcoplasmic reticulum (SR) via the opening of the ryanodine receptor 1 (RyR1 ), a calcium release channel, which is localized in the sarcoplasmic reticulum (SR) terminal cisternae (Rios E., Pizarro G., Physiol. Rev. 71 , 849-908 (1991 ); Endo M. Physiol. Rev. 57, 71-108 (1977); Fleischer S. Annu. Rev. Biophys. Chem. 18, 333-364 (1989)).
  • the signal causing the opening of the RyR1 is the depolarization of the sarcolemmal membrane, which is sensed by voltage-dependent L-type Ca 2+ channels (dihydropyridine receptor, DHPR) located in invaginations of the sarcolemma referred to as transverse tubules (TTs).
  • Skeletal muscle relaxation is brought about by SR Ca 2+ uptake via the activity of the sarco(endo)plasmic reticulum CaATPAses (SERCA).
  • SERCA sarco(endo)plasmic reticulum CaATPAses
  • Dis- regulation of Ca 2+ signals due to defects in key proteins (RyR1 and DHPR) involved in excitation-contraction (EC) coupling is the underlying feature of several neuromuscular disorders.
  • RYR1 Mutations in RYR1, the gene encoding RyR1 , are causative of malignant hyperthermia (MH; MIM #145600), central core disease (CCD), specific forms of multi-minicore disease (MmD) (MacLennan D. H. et al. Science 256, 789-794 (1992)) and centronuclear myopathy (CNM) (Jungbluth H. Nat. Rev. Neurol; Treves S. Curr. Opin. Pharmacol.; Lawai T. A. et al. Skeletal Muscle). RYR1 mutations result mainly in four types of channel defects (Treves S. Curr. Opin. Pharmacol.).
  • One class of mutations causes the channels to become hypersensitive to activation by electrical and pharmacological stimuli (MacLennan D. H. Science).
  • the second class of RYR1 mutations results in leaky channels leading to depletion of Ca 2+ from SR stores (Jungbluth H. Nat. Rev. Neurol; Treves S. Curr. Opin. Pharmacol.).
  • a third class of RYR1 mutations also linked to CCD causes EC uncoupling, whereby activation of the voltage sensor Ca v 1.1 is unable to cause release of Ca 2+ from the SR (Avila G. J. Gen. Physiol. 121 , 277-286 (2003)).
  • the fourth class comprises recessive mutations, which are accompanied by a decreased content of mutant RyR1 channels on SR membranes (Wilmhurst J. M. et al. Ann. Neurol. 68, 717-726 (2010) ; Monnier N. et al. Hum. Mutat. 29, 670-678 (2008) ; Zhou H. et al. Brain 130, 2024-2036 (2007); Zhou H. et al. Hum. Mutat. 34, 986-996 (2013)).
  • muscles of patients with recessive RYR1 mutations exhibit striking epigenetic changes, including altered expression of microRNAs, an increased content of HDAC-4 and HDAC-5 and hypermethylation of more than 3600 CpG genomic sites (Zhou H. et al. Am. J. Hum. Genet. 79, 859-968 (2006); Rokach O. Hum. Mol. Genet. 24, 4636-4647 (2015); Bachmann C. Hum. Mutat. 40, 962-974 (2019)).
  • hypermethylation of one of the internal RYR1 CpG islands correlated with the increased levels of HDAC-4 and HDAC-5.
  • the objective of the present invention is to provide means and methods to provide a treatment for patients diagnosed with a form of myopathy, particularly myopathy characterised by germline gene mutations, reduced RYR1 gene expression, or reduced Ryr1 protein, for which no current treatments exist.
  • the inventors evaluated the efficacy of drugs targeting epigenetic enzymes in terms of muscle function in dHT mice, and found a combination medicament rescues muscle strength, increases RyR1 protein content, and improves muscle morphology, i.e. the treatment partially rescues Ca2+ release units (CRUs, the intracellular sites containing RyR1 ), and mitochondria.
  • CRUs Ca2+ release units
  • the present invention relates to pharmaceutical compositions comprising a selective class Ila HDAC inhibitor, in combination with the DNMT inhibitor 5-aza’- 2-deoxycytidine (CAS no. 2353-33-5), for use in treating patients diagnosed with a form of myopathy to improve the function of soleus muscles.
  • the HDAC inhibitor comprises a trifluoromethyl-oxadiazole group, and specifically acts on the class Ila subset of HDAC enzymes.
  • Examples of such a class Ila specific HDAC inhibitors include, TMP269 (CAS no. 1314890-29-3), N-((R)-1-(((R)-sec-butyl)(methyl)amino)propan-2-yl)-4-(5- (trifluoromethyl)-l ,2,4-oxadiazol-3-yl)benzamide (NVS-HD1 ), N-(pyridin-4-ylmethyl)-5-(5- (trifluoromethyl)-l ,2,4-oxadiazol-3-yl)pyridin-2-amine (NVS-HD2), and N-(2- (dimethylamino)ethyl)-4-(5-(trifluoromethyl)-1 ,2,4-oxadiazol-3-yl)benzamide (NVS-HD3).
  • the two drugs of the pharmaceutical compositions for use according to the invention may be administered together, or in separate formulations with a timeframe ensuring their biological effect is overlapping.
  • Such pharmaceutical compositions are provided for use in myopathy patients, characterised by reduced, or abrogated expression of the RYR1 gene, or Ryr1 protein, and/or by pathogenic high expression of one or more genes encoding class Ila HDAC and/or DNMT enzymes in muscle tissues.
  • Myopathy patients who may benefit from treatment with a pharmaceutical composition according to the invention include, but are not limited to, congenital myopathy patients diagnosed with multi-minicore disease, centronuclear myopathy, or congenital fibre-type disproportion, rigid spine muscular dystrophy or X-linked myotubular myopathy.
  • a first aspect of the invention relates to a pharmaceutical composition for use in treating myopathy comprising a selective class Ila HDAC inhibitor drug characterised by a trifluoromethyloxadiazole group, and 5-aza’-2-deoxycytidine.
  • Further aspects relate to pharmaceutical compositions comprising either the class Ila HDAC inhibitor, or 5-aza’-2- deoxycytidine, for use in patients being administered 5-aza’-2-deoxycytidine, or class Ila HDAC inhibitors, respectively, such that the patient receives both drugs within a medically relevant time window, within which the epigenetic changes induced by each drug are maintained.
  • the present invention particularly relates to pharmaceutical compositions comprising the DNMT inhibitor 5-aza’-2-deoxycytidine (CAS no. 2353-33-5), in combination with a selective class Ila HDAC inhibitor drug characterised by a trifluoromethyloxadiazole group, such as TMP269, NVS-HD1 , NVS-HD2, or NVS-HD3.
  • a selective class Ila HDAC inhibitor drug characterised by a trifluoromethyloxadiazole group, such as TMP269, NVS-HD1 , NVS-HD2, or NVS-HD3.
  • a course of monotherapy with the DNMT inhibitor 5-aza’-2-deoxycytidine alone was surprisingly able to improve the function of fast-twitch muscles in a mouse model of myopathy, and thus may be further be of use to maintains fast twitch muscle function, particularly in myopathy patients in which disease (and hence, fast-twitch muscle atrophy) is not yet established, such as paediatric myopathy patients.
  • compositions comprising the DNMT inhibitor 5-aza’-2-deoxycytidine, for use in treating patients diagnosed with a form of myopathy.
  • the patient is a paediatric patient.
  • the pharmaceutical composition comprising 5-aza’-2-deoxycytidine is characterised by the absence of a HDAC inhibitor, particularly an HDAC inhibitor which comprises a trifluoromethyl-oxadiazole group, and specifically acts on the class Ila subset of HDAC enzymes.
  • HDAC inhibitor particularly an HDAC inhibitor which comprises a trifluoromethyl-oxadiazole group
  • N-((R)-1-(((R)-sec- butyl)(methyl)amino)propan-2-yl)-4-(5-(trifluoromethyl)-1 ,2,4-oxadiazol-3-yl)benzamide N-((R)-1-(((R)-sec- butyl)(methyl)amino)propan-2-yl)-4-(5-(trifluoromethyl)-1 ,2,4-oxadiazol-3-yl)benzamide
  • NVS- HD1 N-(pyridin-4-ylmethyl)-5-(5-(trifluoromethyl)-1 ,2,4-oxadiazol-3-yl)pyridin-2-amine
  • NVS-HD3 N-(2-(dimethylamino)ethyl)-4-(5-(trifluoromethyl)-1 ,2,4-oxadiazol-3-yl)benzamide
  • a further aspect of the invention relates to a pharmaceutical composition for use in treating myopathy comprising the active agent 5-aza’-2-deoxycytidine alone, for use in a patient not additionally receiving treatment with a class Ila HDAC inhibitor drug within a medically relevant window.
  • the pharmaceutical compositions for use according to the invention is provided for use in myopathy patients, characterised by reduced, or abrogated expression of the RYR1 gene, or Ryr1 protein, and/or by pathogenic high expression of one or more genes encoding class Ila HDAC and/or DNMT enzymes in muscle tissues, particularly paediatric patients.
  • Myopathy patients who may benefit from treatment with a pharmaceutical composition according to the invention include, but are not limited to, congenital myopathy patients diagnosed with multi-minicore disease, centronuclear myopathy, or congenital fibretype disproportion, rigid spine muscular dystrophy or X-linked myotubular myopathy.
  • the present invention further relates to pharmaceutical compositions comprising at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient.
  • an article “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items.
  • an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components.
  • references to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”
  • gene refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.
  • ORF open reading frame
  • a polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.
  • gene expression or expression may refer to either of, or both of, the processes - and products thereof - of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products.
  • the term gene expression may also be applied to the transcription and processing of a RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and/or protein product.
  • myopathy in the context of the present specification refers to diseases or medical conditions characterised by defects in skeletal muscle performance, translating to muscle weakness or other impacts on voluntary movement such as motor delay, or in severe cases, respiratory impairment. Diagnosis of myopathy made by combination of clinical assessment of muscle performance, electromyography, imaging, genetic tests, and/or pathology assessment of muscle biopsies. Muscle fibres of patients diagnosed with myopathy are characterised by irregular, or necrotic muscle fibres.
  • congenital myopathy in the context of the present specification relates to the spectrum of genetic disorders characterised by symptoms including muscle weakness and atrophy, join contractures, and spinal deformities, sometimes associated with cardiorespiratory defects.
  • Congenital myopathies are caused by mutations in one or more genes, one of the most common being mutations which disturb the expression or function of the protein encoded by the RYR1 gene. Mutations in genes such as SEPN1, or MTM1 may also result in congenital myopathy according to the invention.
  • mutant in the context of the genes present specification relates to one, or in many cases two or three mutations in the RYRI, SEPN1, and/or MTM1 genes associated with congenital myopathy diseases targeted by treatment with the pharmaceutical composition according to the invention.
  • Representative mutations characteristic of congenital myopathy patients according to the invention may be found, but are not limited to, those listed in Tables 7 to 9.
  • RYR1 in the context of the present specification relates to the human gene encoding the ryanodine receptor 1 (Ryr1) protein (Ensembl: ENSG00000196218), also sometimes referred to as the skeletal muscle ryanodine receptor, or the sarcoplasmic reticulum calcium release channel (SKRR).
  • RyR1 functions as a calcium release channel for the sarcoplasmic reticulum and transverse tubule and is subject to muscle-specific gene regulation which contributes to the aetiology of common congenital myopathies.
  • SEPN1 also sometimes referred to as MDRS1, RSMDIm RSS, SELN, or SELENON
  • MDRS1 RSMDIm RSS
  • SELN SELENON
  • MTM1 in the context of the present specification relates to the human gene encoding the putative tyrosine phosphatase myotubularin (Ensembl: ENSG00000171100).
  • histone deacetylase HDAC
  • histone deacetylase enzyme in the context of the present specification relate to human enzymes that remove an acetyl group from lysine amino acid groups a function which allows DNA to assume a more compressed form when applied to lysine residues on histone proteins.
  • the HDAC enzyme family comprises several classes assigned according to their sequence homology to yeast homologues.
  • HDAC4 Ensembl: ENSG00000068024
  • HDAC5 Ensembl: ENSG00000108840
  • HDAC7 Ensembl:ENSG00000061273
  • HDAC9 Ensembl: ENSG00000048052
  • DNA methyltransferase, DNMT, or DNMT enzyme in the context of the present specification relate to enzymes which transfer methyl groups to the CpG residues nucleotides of DNA, and/or RNA, an important component of epigenetic regulation of gene expression.
  • DNMT enzyme gene transcripts demonstrated to be expressed at higher levels in muscle samples from myopathy patients that may benefit from treatment with the pharmaceutical composition according to the invention include, but are not limited to, DNMT1 (Ensembl: ENSG00000130816), DNMT3A (Ensembl: ENSG00000119772) and tRNA aspartic acid methyltransferase 1 , or TRDMT1 (Ensembl: ENSG00000107614).
  • the term pharmaceutical composition refers to a compound of the invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier.
  • the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration.
  • class Ila HDAC inhibitor or selective class Ila HDAC inhibitor refer to compounds characterised by a trifluoromethyloxadiazole group which specifically inhibit the enzymatic activity of class Ila HDAC enzymes HDAC4, HDAC5, HDAC7, and HDAC9, without significant concomitant inhibition class I, and class lib HDAC enzymes.
  • the IC50 for inhibition of a class Ila HDAC by a class Ila HDAC inhibitor is below 500 nM, particularly below 200 nM, while the IC50 required for inhibition of a class I, or class lib HDAC is equal to, or above 1 uM.
  • class Ila HDAC inhibitors according to the invention include TMP269 (see below), and those described in (Luo L. et al. 2019, Cell Rep. 29(3):4749):
  • NVS-HD1 N-((R)-1-(((R)-sec-butyl)(methyl)amino)propan-2-yl)-4-(5-(trifluoromethyl)-1 ,2,4- oxadiazol-3-yl)benzamide;
  • NVS-HD2 N-(pyridin-4-ylmethyl)-5-(5-(trifluoromethyl)-1 ,2,4-oxadiazol-3-yl)pyridin-2-amine;
  • NVS-HD3 N-(2-(dimethylamino)ethyl)-4-(5-(trifluoromethyl)-1 ,2,4-oxadiazol-3-yl)benzamide.
  • TMP269 in the context of the present specification relates to the selective class Ila HDAC inhibitor with the formula C 25 H 2 IF 3 N4O3S (I) (CAS no. 1314890-29-3). TMP269 is characterised by the functional metal chelating trifluoromethyloxadiazole group (CF3) capable of inhibiting the activity of HDAC9, HDAC7, HDAC5, and HDAC4.
  • CF3 trifluoromethyloxadiazole group
  • 5-aza’-2-deoxycytidine refers to the cytosine analogue with the formula C (CAS no. 2353-33-5), capable of acting as a DNMT inhibitor.
  • This drug is also known as Decitabine, sold under the brand name Dacogen.
  • NVS-HD1 or N-((R)-1-(((R)- sec-butyl)(methyl)amino)propan-2-yl)-4-(5- (trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide refers to the class Ila HDAC inhibitor of formula (III), synthesised as in Luo et al. 2019.
  • NVS-HD2 or N-(pyridin-4- ylmethyl)-5-(5-(trifluoromethyl)-1 ,2,4-oxadiazol-3- yl)pyridin-2-amine refers to the class Ila HDAC inhibitor of formula (IV).
  • NVS-HD2 may be obtained as described in W02013080120A1 , Example 31.
  • NVS-HD3, or N-(2- (dimethylamino)ethyl)-4-(5-(trifluoromethyl)-1 ,2, 4- oxadiazol-3-yl)benzamide refers to the class Ila HDAC inhibitor of formula (V) synthesised as in Luo et al. 2019.
  • the term pharmaceutically acceptable earner includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavouring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624).
  • paediatric patient refers to a myopathy patient under the age of 18 years.
  • treating or treatment of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (e.g., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof, particularly muscle weakness).
  • treating or treatment refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient, such as increasing Ryr1 protein content in muscle.
  • treating or treatment refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom, such as arresting muscle atrophy), physiologically, (e.g., stabilization of a physical parameter), or both.
  • compositions for use in the treatment of myopathy are provided.
  • the invention relates to pharmaceutical compositions for use in treating a patient diagnosed with myopathy.
  • the composition is for use treating congenital myopathy, a genetic disorder characterised by muscle weakness and atrophy, join contractures, spinal deformities, and/or cardio-respiratory defects.
  • Myopathy patients who will benefit from treatment with the pharmaceutical composition according to the invention are those characterised by certain gene expression signature.
  • the myopathy gene expression signature according to the invention is characterised by above average expression of epigenetic regulatory class II a HDAC enzymes, and/or DNMT enzymes in muscle tissue, or below average expression of RYR1 or the encoded protein (see Myopathy patients and Methods of identifying myopathy patients sections below). In some cases, this gene signature is caused by epigenetic modifications derived from congenital mutations in genes including, but not limited to RYR1, SEPN1, or MTM1, which produce a dysregulated gene expression pattern in muscle tissue.
  • a first aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a combination of two drugs for use in treating a patient diagnosed with a form myopathy, a selective class Ila HDAC inhibitor characterised by a trifluoromethyloxadiazole group, and the DNMT inhibitor 5-aza’-2-deoxycytidine (CAS no. 2353-33-5) of the formula C8H12N4O4 (II).
  • the selective class Ila HDAC inhibitor is NVS-HD1 , NVS-HD2, NVS- HD3 or the compound TMP269 (CAS no. 1314890-29-3) with the formula C25H21F3N4O3S (I).
  • said patient is characterised by increased expression in muscle tissue (increased above the average observed in muscle of healthy controls), of genes encoding class Ila HDAC enzymes.
  • the patient is characterised by above average expression of DNMT enzymes in muscle tissue.
  • a myopathy patient according to the invention is characterised by reduced expression (below the average expression in healthy muscle), of the RYR1 gene or encoded protein. Subsets of myopathy patients predicted to respond favourably to pharmaceutical compositions according to the invention are defined below at length in the sections entitled Myopathy patients and Methods of identifying myopathy patients.
  • compositions comprising a combination of two drugs for use in treating a patient diagnosed with a form myopathy, the selective class Ila HDAC inhibitor TMP269, and the DNMT inhibitor 5-aza’-2- deoxycytidine, wherein said patient is characterised by increased expression in muscle tissue (increased above the average observed in muscle of healthy controls), of genes encoding class Ila HDAC enzymes.
  • compositions comprising a combination of two drugs for use in treating a patient diagnosed with a form myopathy, the selective class Ila HDAC inhibitor TMP269, and the DNMT inhibitor 5-aza’-2- deoxycytidine, wherein the patient is characterised by above average expression of DNMT enzymes in muscle tissue.
  • pharmaceutical compositions comprising a combination of two drugs for use in treating a patient diagnosed with a form myopathy, the selective class Ila HDAC inhibitor TMP269, and the DNMT inhibitor 5-aza’-2- deoxycytidine, wherein the myopathy patient is characterised by reduced expression (below the average expression in healthy muscle), of the RYR1 gene.
  • compositions comprising a combination of the selective class Ila HDAC inhibitor TMP269, and the DNMT inhibitor 5-aza’-2-deoxycytidine, for use in treating a myopathy patient characterised by reduced expression (below the average expression in healthy muscle), of the RYR1 protein.
  • compositions comprising a combination of the selective class Ila HDAC inhibitor TMP269, and the DNMT inhibitor 5-aza’-2-deoxycytidine, for use in treating a myopathy patient characterised by reduced expression (below the average expression in healthy muscle), of the genes encoding both the RYR1 gene and a gene encoding a DNMT enzyme.
  • compositions comprising a combination of the selective class Ila HDAC inhibitor TMP269, and the DNMT inhibitor 5-aza’-2-deoxycytidine, for use in treating a myopathy patient characterised by reduced expression (below the average expression in healthy muscle), of the genes encoding both the RYR1 gene and a gene encoding a class Ila HDAC enzyme.
  • Fig. 3 of the Examples demonstrates that daily administration of a formulation comprising both a selective class Ila HDAC inhibitor TMP269, and 5-aza’-2-deoxycytidine, delivers an unexpected synergistic improvement in muscle performance compared to either agent alone, in a mouse model of myopathy.
  • Table 10 shows doses of NVS-HD1 , NVS-HD2, NVS-HD3 predicted to have a similar effect as TMP269 in combination with 5-aza’-2-deoxycytidine.
  • selective class Ila HDAC inhibitors, such as TMP269, and 5-aza’-2-deoxycytidine have strong therapeutic advantages for improving symptoms and generating improvements in quality of life for congenital myopathy patients, both drugs have been associated with significant side effects.
  • a next aspect of the invention thus relates to a pharmaceutical composition comprising a selective class Ila HDAC inhibitor characterised by a trifluoromethyloxadiazole group alone as an active agent, for use in treating a myopathy patient as described in the Myopathy patients and Methods of identifying myopathy patients sections below.
  • a pharmaceutical composition comprising a selective class Ila HDAC inhibitor is administered to a patient who is currently receiving separate administration of the drug 5-aza’-2- deoxycytidine, as exposure to both drugs is required in order to achieve a synergistic amelioration of symptoms.
  • the two agents are administered within one month of each other.
  • the two agents are administered within one week of each other.
  • the class Ila HDAC inhibitor is administered to a myopathy patient who has received a dose of 5-aza’-2-deoxycytidine on the same day.
  • another aspect of the invention relates to a pharmaceutical composition comprising 5-aza’-2-deoxycytidine alone as an active agent, for use in a myopathy patient as described in the Myopathy patients and Methods of selecting myopathy patients sections below.
  • a pharmaceutical composition comprising 5-aza’-2- deoxycytidine is administered to a patient who is currently receiving separate administration of a class Ila HDAC inhibitor drug compound characterised by a trifluoromethyloxadiazole group, in order to achieve a synergistic amelioration of symptoms.
  • 5-aza’-2- deoxycytidine is administered to a myopathy patient who in the same month receives a dose of the class Ila HDAC inhibitor.
  • the two agents are administered within one month of each other. In particular embodiments, the two agents are administered within one week of each other. In more particular embodiments, the 5-aza’-2-deoxycytidine is administered to a myopathy patient who has received a dose of the class Ila HDAC inhibitor on the same day.
  • a next aspect of the invention thus relates to a pharmaceutical composition comprising TMP269 alone as an active agent, for use in treating a myopathy patient as described in the “ Myopathy patients and Methods of identifying myopathy patients” sections below.
  • a pharmaceutical composition comprising TMP269 is administered to a patient who is currently receiving separate administration of the drug 5-aza’-2-deoxycytidine, as exposure to both drugs is required in order to achieve a synergistic amelioration of symptoms.
  • the two agents are administered within one month of each other.
  • the two agents are administered within one week of each other.
  • the TMP269 is administered to a myopathy patient who has received a dose of 5-aza’-2-deoxycytidine on the same day.
  • a further aspect of the invention relates to a pharmaceutical composition comprising 5-aza’-2- deoxycytidine alone as an active agent, for use in a myopathy patient as described in the “Myopathy patients and Methods of selecting myopathy patients” sections below.
  • the pharmaceutical composition comprising 5-aza’-2-deoxycytidine is administered to a patient who is currently receiving separate administration of the drug TMP269, in order to achieve a synergistic amelioration of symptoms.
  • 5-aza’-2-deoxycytidine is administered to a myopathy patient who in the same month receives a dose of TMP269.
  • the two agents are administered within one month of each other.
  • the two agents are administered within one week of each other.
  • the 5-aza’-2-deoxycytidine is administered to a myopathy patient who has received a dose of TMP269 on the same day.
  • another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the DNMT inhibitor 5-aza’-2-deoxycytidine of the formula C8H12N4O4 (II) for use in treating a patient diagnosed with a myopathy.
  • the pharmaceutical composition comprising 5-aza’-2-deoxycytidine does not further comprise an HDAC inhibitor compound, as this combination was shown not to alleviate disease progression with regard to fast twitch EDL muscles.
  • the pharmaceutical composition according to the invention is provided for use in a patient not being administered a second composition comprising an HDAC inhibitor compound within a medically relevant time window of 5-aza’-2-deoxycytidine administration.
  • the composition is administered to a patient who is not currently receiving an HDAC inhibitor compound, and who has not recently received, or is not scheduled too shortly be administered an HDAC inhibitor compound.
  • the patient is characterised by not having received an HDAC inhibitor compound within 1 week before or after 5-aza’-2-deoxycytidine administration.
  • the patient is characterised by not having received an HDAC inhibitor compound either within 1 month before or after 5-aza’-2-deoxycytidine administration.
  • the patient has not received, and is not scheduled to receive, a selective class Ila HDAC inhibitor characterised by a trifluoromethyloxadiazole group, and such as NVS-HD1 , NVS-HD2, NVS- HD3 or the compound TMP269 (CAS no. 1314890-29-3) with the formula C25H21F3N4O3S (I).
  • a selective class Ila HDAC inhibitor characterised by a trifluoromethyloxadiazole group, and such as NVS-HD1 , NVS-HD2, NVS- HD3 or the compound TMP269 (CAS no. 1314890-29-3) with the formula C25H21F3N4O3S (I).
  • said pharmaceutical composition comprising only 5-aza’-2- deoxycytidine as an active ingredient, if for use in a patient with a muscle tissue sample characterised by increased expression (increased above the average observed in muscle of healthy controls), of genes encoding DNMT enzymes in muscle tissue.
  • a myopathy patient according to the invention is characterised by reduced expression (below the average expression in healthy muscle), of the RYR1 gene.
  • a myopathy patient according to the invention is characterised by reduced expression (below the average expression in healthy muscle), of the RYR1 protein. Subsets of myopathy patients predicted to respond favourably to pharmaceutical compositions according to the invention are defined below at length in the sections entitled Myopathy patients and Methods of identifying myopathy patients.
  • the pharmaceutical combinations according to the current invention increase the level of Ryr1 protein in muscle, and are provided for use in patients diagnosed with a form of myopathy, characterised by defects in skeletal muscle leading to muscle weakness.
  • Muscle weakness may be measured directly, for example in a grip force test, or assessed indirectly, for example by imaging analysis to assess muscle atrophy.
  • the pharmaceutical combination of a class Ila HDAC enzyme inhibitor, and 5-aza’-2-deoxycytidine is an adult myopathy patient characterised by established disease i.e clinical measures of muscle weakness.
  • compositions comprising 5-aza’-2-deoxycytidine as the only pharmacologically active ingredient according to the invention improve the function of fast twitch muscle, and are also provided for use in patients diagnosed with a form of myopathy characterised by defects in skeletal muscle leading to muscle weakness.
  • the pharmaceutical composition comprising only 5-aza’-2-deoxycytidine is provided for use a paediatric patient, as the inventors propose that the treatment may preserve fast twitch muscle function in such patients not yet characterised by fast twitch muscle atrophy observed in established myopathy disease.
  • the pharmaceutical composition comprising only 5-aza’-2-deoxycytidine is provided for use a paediatric patient less than 10 years old; in certain particular embodiments, the composition is provided to a patient less than five years old, in even more particular embodiments, the patient is less than 36 months of age.
  • the pharmaceutical composition according to the invention is for use in treating a myopathy disease characterised by upregulation expression, or overexpression, specifically in patient muscle tissue, of at least one gene encoding a class Ila HDAC enzyme selected from HDAC4, HDAC5, HDAC7, or HDAC9.
  • a myopathy disease characterised by upregulation expression, or overexpression, specifically in patient muscle tissue, of at least one gene encoding a class Ila HDAC enzyme selected from HDAC4, HDAC5, HDAC7, or HDAC9.
  • the myopathy is characterised by overexpression of two or more genes selected from HDAC4, HDAC5, HDAC7, and/or HDAC9 in patient muscle tissue.
  • the pharmaceutical composition according to the invention is for use in a myopathy patient characterised by upregulation expression, or overexpression, specifically in muscle tissue, of at least one gene encoding a DNMT.
  • said patient is characterised by overexpression of one or more genes selected from DNMT1, DNMT3A, and/or TRDMT1.
  • said patient is characterised by overexpression of two or more genes selected from DNMT1, DNMT3A, and/or TRDMT1, specifically in muscle tissue.
  • said patient is characterised by upregulated expression of all three genes DNMT1, DNMT3A, and TRDMT1, for example, a patient diagnosed with severe multi-minicore disease such as those analysed in in Fig. 8.
  • the myopathy patient according to the invention is characterised by upregulated expression in muscle tissue of a combination of genes encoding class Ila HDAC and DNMT according to the two paragraphs above.
  • composition according to the invention is provided for use in a myopathy patient characterised by hypermethylation of CpG sites within target genes of a HDAC, or a DNMT according to the invention.
  • the composition is provided for use in treating a patient diagnosed with a congenital myopathy, driven by an inherited gene mutation.
  • the pharmaceutical composition according to the invention is provided for use in a patient who has been diagnosed with multi-minicore disease.
  • the pharmaceutical composition according to the invention is provided for use in a patient who has been diagnosed with severe multi-minicore disease.
  • the pharmaceutical composition according to the invention is provided for use in a patient who has been diagnosed with multi-minicore disease with external ophthalmoplegia. In certain embodiments, the pharmaceutical composition according to the invention is provided for use in a patient who has been diagnosed with centronuclear myopathy.
  • the pharmaceutical composition according to the invention is provided for use in a patient who has been diagnosed with congenital fibre-type disproportion.
  • the pharmaceutical composition according to the invention is provided for use in a patient who has been diagnosed with X-linked myotubular myopathy.
  • the pharmaceutical composition according to the invention is provided for use in a myopathy patient characterised by downregulation of RYR1 expression in muscle tissue, in other words below the average expression in healthy human muscle.
  • the inventors demonstrate that below average expression of RYR1 in muscle is observed not only in patients characterised by RYR1 mutations, but also patients characterised by congenital myopathies associated with SEPN1, and MTM1 mutations (Fig. 8 -10).
  • the pharmaceutical composition according to the invention is provided for use in a congenital myopathy patient characterised by at least one mutation in each copy of the RYR1 gene, in other words, a double heterozygote, or compound heterozygous mutation, where one copy of the RYR1 gene harbours a loss-of-function genetic defect.
  • the patient carries two missense RYR1 mutations leading to a decrease of RYR1 transcript and protein in muscle tissue.
  • said mutation is a compound of two heterozygous RYR1 mutations.
  • said mutation is a homozygous mutation, where the same mutation is present on both copies of the gene.
  • the composition is provided for use in a patient who expresses a decreased amount of RyR1 protein on SR membranes in a muscle tissue.
  • said condition is characterised by a RYR1 mutation listed in Table 7.
  • the pharmaceutical composition according to the invention is provided for use in a patient characterised as compound heterozygous for both the Q1970fsX16, and A4329D mutations in the RYR1 gene, as modelled by the double knock out mice utilised in Example 1.
  • the pharmaceutical composition according to the invention is provided for use in a myopathy patient characterised by 80% or less RYR1 gene expression in a muscle tissue sample obtained from said patient, in comparison to healthy controls.
  • a patient muscle tissue sample is characterised by 70% of the level of RYR1 mRNA in comparison to a healthy muscle tissue sample (a 30% reduction).
  • a myopathy patient can be identified by a 20%, or particularly a 30% reduction in Ryr1 protein levels.
  • Such comparisons can be made with a healthy tissue sample, or the average RYR1 gene expression level obtained from analysing a cohort of healthy muscle tissue samples, for example, a cohort of at least 20 healthy control samples.
  • Figure 6 of the examples demonstrates how a pharmaceutical composition according to the invention can restore mRNA, and protein levels of RyR1 to within the healthy, normal range associated with improved muscle cell function.
  • a pharmaceutical composition according to the invention can rescue reduced Ryr1 protein content to improve muscle function.
  • the composition is provided to improve muscle performance in patients with myopathies characterised by muscle tissue exhibiting a loss of at least 20%, or even 30% of Ryr1 protein content, compared to healthy muscle tissue.
  • Ryr1 protein content can be measured directly, by measurements such as western blot, or immunohistochemistry.
  • Ryr1 protein content is assessed indirectly, for example Ryr1 dysfunction can be measured by methods such as measuring Ca 2+ transients, or measuring single channel properties in reconstituted bilayers
  • the composition is provided for use in a patient diagnosed with a form of congenital myopathy attributed to compound heterozygous mutations in the RYR1 gene, such as the autosomal recessive conditions multi-minicore disease (MmD), MmD with external ophthalmoplegia, central core disease (CCD) or congenital fibre type disease, or other congenital myopathies which are accompanied by a decrease in RYR1 transcript and protein equal to or greater than 30% (such as X-linked myotubular myopathy, multi-minicore disease caused by SEPN1 (SELENON) mutations and nemaline myopathy).
  • MmD multi-minicore disease
  • CCD central core disease
  • congenital fibre type disease or other congenital myopathies which are accompanied by a decrease in RYR1 transcript and protein equal to or greater than 30% (such as X-linked myotubular myopathy, multi-minicore disease caused by SEPN1 (SELENON) mutations and nemaline myopathy).
  • the composition is provided for use in a patient diagnosed with a congenital myopathy with a similar aetiology to autosomal recessive RYR1 conditions described in the paragraph above.
  • diseases include severe multi-minicore disease or rigid spin muscular dystrophy associated with compound mutations in the SEPN1 gene, and X-linked myotubular myopathy associated with mutations in the MTM1 gene.
  • the composition is provided for use in a congenital myopathy patient characterised by at least one mutation in each copy of the SEPN1 gene.
  • the composition is provided for use in a congenital myopathy patient characterised by at least one combination of mutations in the SEPN1 gene listed in Table 8 of the examples.
  • the composition is provided for use in a congenital myopathy patient characterised by a mutation in the MTM1 gene.
  • the composition is provided for use in a congenital myopathy patient characterised by a mutation listed in Table 9 of the examples.
  • the composition is provided for use in a myopathy patient where a muscle tissue sample is characterised by at least a 1.5, particularly a 2-fold upregulation (compared to the average expression of a cohort of muscle samples obtained from human healthy controls) of at least one gene encoding a class Ila HDAC.
  • a muscle sample obtained from the myopathy patient is characterised by at least 1.5-fold upregulation of HDAC4.
  • a muscle sample obtained from the myopathy patient is characterised by at least 2.5-fold upregulation of HDAC4. In certain embodiments, a muscle sample obtained from the myopathy patient is characterised by at least 1.5-fold upregulation of HDAC5. In particular embodiments, a muscle sample obtained from the myopathy patient is characterised by at least 2.5-fold upregulation of HDAC5. In certain embodiments, a muscle sample obtained from the myopathy patient is characterised by at least 1.5-fold upregulation of HDAC9. In particular embodiments, a muscle sample obtained from the myopathy patient is characterised by at least 2.5-fold upregulation of HDAC9. In certain embodiments, a muscle sample obtained from the myopathy patient is characterised by at least 1.5-fold upregulation of HDAC7.
  • a muscle sample obtained from the myopathy patient is characterised by at least 2.5-fold upregulation of HDAC7.
  • a muscle sample obtained from the myopathy patient is characterised by at least 1 .5-fold upregulation of HDAC5 and HDAC9, as in the patients characterised in Fig. 8 and 9.
  • a muscle sample obtained from the myopathy patient is characterised by at least 2.5-fold upregulation of HDAC9.
  • Figure 6 of the examples demonstrates how a combination medicament according to the invention restores pathogenically upregulated HDAC4 mRNA levels to the range of healthy controls.
  • the composition is provided for use in a myopathy patient characterised by upregulation, or overexpression, specifically in muscle tissue (compared to the average expression of a cohort of muscle samples obtained from human healthy controls) of at least one gene encoding a DNMT.
  • a muscle sample obtained from the myopathy patient is characterised by at least 1 .5-fold upregulation of DNMT1.
  • a muscle sample obtained from the myopathy patient is characterised by at least 2.5-fold upregulation of DNMT1.
  • a muscle sample obtained from the myopathy patient is characterised by at least 1.5-fold upregulation of DNMT3A.
  • a muscle sample obtained from the myopathy patient is characterised by at least 2.5-fold upregulation of DNMT3A.
  • a muscle sample obtained from the myopathy patient is characterised by at least 1.5-fold upregulation of TRDMT1.
  • a muscle sample obtained from the myopathy patient is characterised by at least 2.5-fold upregulation of TRDMT1.
  • a muscle sample obtained from the myopathy patient is characterised by at least 1.5-fold upregulation of DNMT1, DNMT3A and TRDMT1, as in the patients characterised in Fig. 8 and 9.
  • a mutation in RYR1, SEPN1, or MNMT1 characterising a congenital myopathy patient who will benefit from treatment with one of the pharmaceutical compositions according to the invention may be identified by various genotyping assays known in the art.
  • various methodologies may be used such as Sanger sequencing, capillary electrophoresis and fragment analysis, or next generation sequencing, directed at specific genes, or the whole genome.
  • a dysregulated gene expression signature characterising a myopathy patient is identified by analysing gene expression levels, for example HDAC, or DNMT genes in a muscle tissue sample obtained from said patient.
  • Means for measuring gene expression include, but are not limited to, mRNA sequencing, nucleic acid arrays, microarrays, or standard or fluidics-based quantitative real time polymerase chain reaction (PCR). All of the above involve the steps of isolating nucleic acids from a tissue sample, and contacting said nucleic acids with one or more labelled molecular probes specific for a gene, or a gene product, followed by amplification, and detection appropriate to said labelled molecule probe.
  • the expression of a specified gene is measured using a quantitative polymerase chain reaction (qPCR).
  • the level of HDAC, RyR1, or DNMT gene expression is a qPCR cycle threshold.
  • cycle threshold or CT in the context of the present specification relates to a quantitative nucleic acid measurement, for example a measurement made with qPCR. This method involves repeated cycles of nucleic acid amplification using nucleic acid probes, or primers, which hybridise the target biomarker, to generate a product emitting a fluorescent signal, which can be measured to determine the amount of starting genetic material.
  • the cycle threshold may be an average value, or the average value of a number of replicate samples. Other quantitative measurements may substitute the cycle threshold, such as a crossing point, or an adjusted inflexion point.
  • the results of a real time PCR gene expression assay are expressed as relative gene expression normalized to musclespecific housekeeping gene, for example, Desmin (DES).
  • DES Desmin
  • expression levels are shown as fold-change compared to healthy control samples that were set to 1 .
  • DES may be substituted by genes selected from, but not limited to, GAPDH, ACTB, B2M, PPIA, or HMBS.
  • the expression level of the biomarker is compared to a baseline, or reference sample.
  • a negative control, or negative reference is a sample of healthy muscle tissue.
  • An example of a positive control, or positive reference sample may be a previously analysed obtained from muscle tissue of one or more previously patients diagnosed with a myopathy according to the invention.
  • patient samples may be compared to a range of pre-determined calibration samples, or standards, to provide appropriate technical and biological controls.
  • the combination medicament according to the invention comprising a class Ila HDAC inhibitor characterised by a trifluoromethyloxadiazole group, and/or 5-aza-2-deoxycytidine is provided as a pharmaceutical composition, or pharmaceutical administration form.
  • pharmaceutical compositions, or pharmaceutical administration forms comprising TMP269, and 5-aza-2-deoxycytidine.
  • Another aspect the invention relates to the compound 5-aza-2-deoxycytidine alone, provided as a pharmaceutical composition, or pharmaceutical administration form, for use in a patient not receiving treatment with a class Ila HDAC inhibitor compound.
  • the dosage regimen for the compounds of the present invention, class Ila HDAC inhibitors characterised by a trifluoromethyloxadiazole group, and/or 5-aza-2-deoxycytidine will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired.
  • the compounds of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.
  • the therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated.
  • a physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.
  • the class Ila HDAC inhibitor characterised by a trifluoromethyloxadiazole group is a not able to enter the central nervous system.
  • the class Ila HDAC inhibitor characterised by a trifluoromethyloxadiazole group is TMP269.
  • the drug TMP269 is administered at a dose of in the range of 0.5 to 50 mg/kg.
  • the dosage of TMP260 is in the range of about 10 to 40 mg/kg. In other particular embodiments, the dosage of TMP260 in the range of about 20 to 25 mg/Kg.
  • the class Ila HDAC inhibitor is NVS-HD1 : (N-((R)-1-(((R)-sec-butyl) (methyl) amino) propan-2-yl)-4-(5-(trifluoromethyl)-1 ,2,4-oxadiazol-3-yl) benzamide).
  • the NVS-HD1 is administered at a dose in the range of about 0.002 to 0.2 mg/Kg.
  • NVS-HD1 is administered in the range of about 0.01 to 0.1 mg/Kg.
  • the dose of NVS-HD1 is about 0.1 mg/Kg.
  • the class Ila HDAC inhibitor is NVS-HD2 (N-(pyridin-4-ylmethyl)-5-(5-(trifluoromethyl)-1 ,2,4- oxadiazol-3-yl) pyridin-2-amine).
  • NVS-HD2 is administered to said patient at a dose in the range of about 0.15 to 15 mg/Kg.
  • the dose of NVS-HD2 is in the range of about 1 to 10 mg/Kg.
  • the dose of NVS-HD2 is about 7.8 mg/Kg.
  • the class Ila HDAC inhibitor is NVS-HD3 (N-(2-(dimethylamino) ethyl)-4-(5-(trifluoromethyl)- 1 ,2,4-oxadiazol-3-yl) benzamide).
  • NVS-HD3 is administered at a dose in the range of about 0.25 to 25 mg/Kg, particularly in the range of about 2 to 15 mg/Kg. In still more particular embodiments, the dose of NVS-HD3 is about 12 mg/Kg.
  • 5-aza-2-deoxycytidine must be carefully formulated and handled to minimise chemical instability and degradation to agents with no pharmacological activity.
  • 5- aza-2-deoxycytidine is preferably stored at low temperatures.
  • 5- aza-2-deoxycytidine is formulated such that solutions can be freshly ( ⁇ 8hours) prepared from a solid and reconstituted, for example in 5 to 10mM potassium phosphate with a pH of 7 to 7.4.
  • An optimal dosage for the recipient should be selected to avoid known side effects of weight loss and leukopenia.
  • the drug 5-aza-2-deoxycytidine is formulated for administration at a dose in the range of 0.01 to 1 mg/Kg. In particular embodiments, the dosage is in the range of 0.05 to 0.2 mg/Kg. In other particular embodiments, the dosage of 5-aza-2-deoxycytidine is about 0.5 mg/Kg
  • the composition further comprises N-methyl-2-pyrrolidone (NMP).
  • NMP N-methyl-2-pyrrolidone
  • the pharmaceutical composition is formulated such that the NMP is administered at about 250 uL/Kg.
  • the pharmaceutical composition for treating myopathy comprises dipropylene glycol dimethyl ether.
  • the composition is administered by a parenteral route.
  • the composition is introduced by the intravenous, route, for example, by infusion, or by means of an implanted pump.
  • the composition is administered by intramuscular injection.
  • the pharmaceutical composition according to the invention is applied by a subcutaneous injection, or subcutaneous slow-release implant.
  • the composition is administered by intradermal injection or implant.
  • HDAC inhibitors such as the HDAC class Ila inhibitor TMP269, or the DNMT inhibitor 5-aza-2-deoxycytidine to the cell is cell cycle dependent, meaning that daily treatment, or infusion over several hours, or treatment in weekly cycles may be preferred in order to provide an opportunity for the drugs to enter skeletal muscle cells. Repeated maintenance therapy may be applied, to ensure that epigenetic changes induced by each drug can be maintained in order to relieve symptoms.
  • a pharmaceutical composition comprising HDAC class Ila inhibitor, and/or 5-aza-2-deoxycytidine is administered daily.
  • the composition comprising HDAC class Ila inhibitor, and/or 5-aza-2- deoxycytidine is administered in cycles, for example for 3 days, or 3 times in a week, followed by a period without treatment, ranging from one, two or even three weeks.
  • the HDAC class Ila inhibitor, and 5-aza-2-deoxycytidine are administered as separate formulations with different timing, for example each is administered on different days of the week, or in alternating weekly cycles.
  • a pharmaceutical composition comprising TMP269, and 5-aza-2- deoxycytidine is administered daily.
  • the composition comprising TMP269, and 5-aza-2-deoxycytidine is administered in cycles, for example for 3 days, or 3 times in a week, followed by a period without treatment, ranging from one, two or even three weeks.
  • TMP269, and 5-aza-2-deoxycytidine are administered as separate formulations with different timing, for example each is administered on different days of the week, or in alternating weekly cycles.
  • a pharmaceutical composition comprising 5-aza-2-deoxycytidine alone is administered daily.
  • the composition comprising 5-aza-2-deoxycytidine is administered in cycles, for example for 3 days, or 3 times in a week, followed by a period without treatment, ranging from one, two or even three weeks.
  • any specifically mentioned drug compound mentioned herein may be present as a pharmaceutically acceptable salt of said drug.
  • Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion.
  • Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate.
  • the HDAC class Ila inhibitor e.g. TMP269
  • 5-aza-2-deoxycytidine of the present invention are typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handled product.
  • the invention further encompasses a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.
  • compositions of the present invention relate to a dosage form for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository.
  • pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).
  • compositions of the present invention relate to a dosage form for parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms.
  • a pharmaceutically acceptable carrier and/or excipient may be present.
  • the pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).
  • the invention further encompasses, as an additional aspect, the use of a class II a selective HDAC inhibitor, such as TMP269, together with 5-aza-2-deoxycytidine as identified herein, or a pharmaceutically acceptable salt of a class II a selective HDAC inhibitor, or 5-aza-2- deoxycytidine, as specified in detail above, for use in a method of manufacture of a medicament for the treatment of myopathy.
  • a class II a selective HDAC inhibitor such as TMP269
  • the invention encompasses methods of treatment of a patient having been diagnosed with a form of myopathy (as defined in the section Myopathy patients).
  • Said method comprises administering to the patient an effective amount of a class II a selective HDAC inhibitor, and 5- aza-2-deoxycytidine according to the above description, either as a single formulation, or as separate formulations administered together within a medically relevant window.
  • the method of treating a patient according the invention comprises obtaining a muscle tissue sample from the patient, determining the expression level of Ryr1 protein, and administering a pharmaceutical composition comprising a selective HDAC class II inhibitor characterised by a trifluoromethyloxadiazole group, and 5-aza’-2-deoxycytidine, if the expression level of Ryr1 protein is greater than that present in a healthy tissue control sample.
  • the method of treating a patient according the invention comprises obtaining a muscle tissue sample from the patient, determining the expression level of the RYR1 gene, and administering a pharmaceutical composition comprising a selective HDAC class II inhibitor characterised by a trifluoromethyloxadiazole group, and 5-aza’-2- deoxycytidine, if the expression level of Ryr1 protein is greater than that present in a healthy tissue control sample.
  • the method of treating a patient according the invention comprises obtaining a muscle tissue sample from the patient, determining the expression level of at least one gene encoding a HDAC or DNMT enzyme, particularly HDAC4, HDAC5, HDAC9, DNMT1, DNMT3A, and/or TRDMT1, and administering a pharmaceutical composition comprising a selective HDAC class II inhibitor characterised by a trifluoromethyloxadiazole group, and 5-aza’-2-deoxycytidine, if the expression level of at least one of the gene encoding a HDAC or DNMT enzyme is greater than that present in a healthy tissue control sample.
  • the invention further encompasses, as an additional aspect, the use TMP269, together with 5-aza-2-deoxycytidine as identified herein, or a pharmaceutically acceptable salt of a TMP269, or 5-aza-2-deoxycytidine, for use in a method of manufacture of a medicament for the treatment of myopathy.
  • the invention encompasses methods of treatment of a patient having been diagnosed with a form of myopathy (as defined in the section Myopathy patients).
  • Said method comprises administering to the patient an effective amount of a TMP269, and 5-aza-2-deoxycytidine according to the above description, either as a single formulation, or as separate formulations administered together within a medically relevant window
  • the method of treating a patient according the invention comprises obtaining a muscle tissue sample from the patient, determining the expression level of Ryr1 protein, and administering a pharmaceutical composition comprising TMP269, and 5-aza’-2- deoxycytidine, if the expression level of Ryr1 protein is greater than that present in a healthy tissue control sample.
  • the method of treating a patient according the invention comprises obtaining a muscle tissue sample from the patient, determining the expression level of the RYR1 gene, and administering a pharmaceutical composition comprising TMP269, and 5-aza’-2-deoxycytidine, if the expression level of Ryr1 protein is greater than that present in a healthy tissue control sample.
  • the method of treating a patient according the invention comprises obtaining a muscle tissue sample from the patient, determining the expression level of at least one gene encoding a HDAC or DNMT enzyme, particularly HDAC4, HDAC5, HDAC9, DNMT1, DNMT3A, and/or TRDMT1, and administering a pharmaceutical composition comprising TMP269, and 5-aza’-2-deoxycytidine, if the expression level of at least one of the gene encoding a HDAC or DNMT enzyme is greater than that present in a healthy tissue control sample.
  • the invention further encompasses, as an additional aspect, the use of 5-aza-2-deoxycytidine as identified herein, or a pharmaceutically acceptable salt of 5-aza-2-deoxycytidine, as specified in detail above, for use in a method of manufacture of a medicament for the treatment of myopathy, particularly in a pediatric patient.
  • the invention encompasses methods of treatment of a patient, particularly a paediatric patient, having been diagnosed with a form of myopathy (as defined in the section Myopathy patients). Said method comprises administering to the patient an effective amount of the 5-aza-2-deoxycytidine according to the above description.
  • the method of treating a patient according the invention comprises obtaining a muscle tissue sample from the patient, determining the expression level of Ryr1 protein, and administering a pharmaceutical composition comprising only 5-aza’-2- deoxycytidine, if the expression level of Ryr1 protein is greater than that present in a healthy tissue control sample.
  • the method of treating a patient according the invention comprises obtaining a muscle tissue sample from the patient, determining the expression level of the RYR1 gene, and administering a pharmaceutical composition comprising only 5-aza’-2- deoxycytidine, if the expression level of Ryr1 protein is greater than that present in a healthy tissue control sample.
  • the method of treating a patient according the invention comprises obtaining a muscle tissue sample from the patient, determining the expression level of at least one gene encoding a HDAC or DNMT enzyme, particularly HDAC4, HDAC5, HDAC9, DNMT1, DNMT3A, and/or TRDMT1, and administering a pharmaceutical composition comprising only 5-aza’-2-deoxycytidine, if the expression level of at least one of the gene encoding a HDAC or DNMT enzyme is greater than that present in a healthy tissue control sample.
  • the method comprises performing a genotyping reaction, to confirm at least one mutation in each copy of the RYR1 gene is present in the patient.
  • the method comprising if the patient is compound heterozygous for both the Q1970fsX16, and A4329D RYR1 mutations.
  • the method to treat a myopathy patient comprises performing a genotyping reaction, to confirm at least one mutation is present in each copy of the SEPN1 gene.
  • the method to treat a myopathy patient comprises performing a genotyping reaction, to confirm at least one mutation is present in at least one copy of the MTM1 gene.
  • the invention further encompasses the use of real time PCT primers specific for the genes RYR1, HDAC4, HDAC5 and HDAC9, DNMT1, DNMT3A and/or TRDMT1 for use in the manufacture of a kit for the detection of myopathy patients who are likely to respond to a pharmaceutical composition according to the invention.
  • the invention further encompasses the use of sequencing primers specific for the genes RYR1, SEPN1, or MTM1 for use in the manufacture of a kit for the detection of myopathy patients who are likely to respond to a pharmaceutical composition according to the invention.
  • the invention further encompasses the use of sequencing primers specific for class Ila HDAC encoding genes or DNMT genes for use in the manufacture of a kit for the detection of myopathy patients who are likely to respond to a pharmaceutical composition according to the invention.
  • the invention further encompasses the following items:
  • a pharmaceutical composition for use in the treatment of myopathy comprising: i. a class Ila HDAC inhibitor characterised by a trifluoromethyloxadiazole group; and
  • 5-aza’-2-deoxycytidine (CAS no. 2353-33-5); wherein the pharmaceutical composition is administered to a patient characterised by: upregulation of at least one gene encoding a class Ila histone deacetylase (HDAC) and/or a DNA methyltransferase (DNMT), and/or
  • HDAC histone deacetylase
  • DNMT DNA methyltransferase
  • a pharmaceutical composition comprising 5-aza’-2-deoxycytidine for use in the treatment of myopathy, wherein the composition is administered to a patient characterised as specified in item A, and wherein said patient has recently received, is currently receiving, or is scheduled to receive administration of a class Ila HDAC inhibitor characterised by a trifluoromethyloxadiazole group.
  • a pharmaceutical composition comprising a class Ila HDAC inhibitor characterised by a trifluoromethyloxadiazole group for use in the treatment of myopathy, wherein the composition is administered to a patient characterised as specified in item A, and wherein said patient has recently received, is currently receiving, or is scheduled to receive administration of 5-aza’-2-deoxycytidine.
  • D The pharmaceutical composition for use according to any of items A to C, wherein the myopathy is selected from multi-minicore disease, centronuclear myopathy, congenital fibre-type disproportion, rigid spine muscular dystrophy, nemaline myopathy, or X-linked myotubular myopathy.
  • composition for use according to item E wherein the patient is characterised as being compound heterozygous for both the Q1970fsX16, and A4329D mutations in the RYR1 gene.
  • composition for use according to any one of the items A to D, wherein the patient is characterised by at least one mutation in the MTM1 gene.
  • composition for use according to any one of the items A to H wherein the class Ila HDAC inhibitor is N-(pyridin-4-ylmethyl)-5-(5-(trifluoromethyl)-1 ,2,4- oxadiazol-3-yl) pyridin-2-amine (NVS-HD2).
  • N The pharmaceutical composition for use according to item M, wherein the NVS-HD2 is administered at a dose in the range of about 0.15 to 15 mg/Kg, particularly in the range of about 1 to 10 mg/Kg, more particularly about 7.8 mg/Kg.
  • NVS-HD3 N-(2-(dimethylamino) ethyl)-4-(5-(trifluoromethyl)- 1 ,2,4-oxadiazol-3-yl) benzamide
  • P The pharmaceutical composition for use according to item O, wherein the NVS-HD3 is administered at a dose in the range of about 0.25 to 25 mg/Kg, particularly in the range of about 2 to 15 mg/Kg, more particularly about 12 mg/Kg.
  • composition for use according to any one of the items A to Q, wherein the composition further comprises N-methyl-2-pyrrolidone (NMP), particularly wherein the NMP is administered at a dose of about 250 uL/Kg.
  • NMP N-methyl-2-pyrrolidone
  • composition for use according to any one of the items A to R, wherein the composition is administered by a parenteral route.
  • T The pharmaceutical composition for use according to any one of the items A to S, wherein the composition is administered daily.
  • a muscle tissue sample obtained from the patient diagnosed with myopathy is characterised by at least a 1 .5, particularly a 2-fold upregulation of at least one gene encoding a class Ila HDAC and/or DNMT gene selected from HDAC4, HDAC5, HDAC9, DNMT1, DNMT3A, and/or TRDMT1.
  • AA.A pharmaceutical composition for use in the treatment of myopathy comprising:
  • TMP269 (CAS no. 1314890-29-3);
  • 5-aza’-2-deoxycytidine (CAS no. 2353-33-5); wherein the pharmaceutical composition is administered to a patient characterised by: upregulation of expression of at least one gene encoding a class Ila histone deacetylase (HDAC) and/or at least one gene encoding a DNA methyltransferase (DNMT), and/or downregulation of RYR1 expression.
  • HDAC histone deacetylase
  • DNMT DNA methyltransferase
  • a pharmaceutical composition comprising 5-aza’-2-deoxycytidine for use in the treatment of myopathy, wherein the composition is administered to a patient as specified in item AA, and wherein said patient has recently received, is currently receiving, or is scheduled to receive administration of TMP269.
  • a pharmaceutical composition comprising TMP269 for use in the treatment of myopathy, wherein the composition is administered to a patient as specified in item AA, and wherein said patient has recently received, is currently receiving, or is scheduled to receive administration of 5-aza’-2-deoxycytidine.
  • composition for use according to item EE wherein the patient is characterised as being compound heterozygous for both the Q1970fsX16, and A4329D mutations in the RYR1 gene.
  • composition for use according to any one of the items AA to DD, wherein the patient is characterised by at least one mutation in the MTM1 gene.
  • composition for use according to any one of the items AA to JJ, wherein the composition further comprises N-methyl-2-pyrrolidone (NMP), particularly wherein the NMP is administered at a dose of about 250 pL/Kg.
  • NMP N-methyl-2-pyrrolidone
  • composition for use according to any one of the items AA to KK, wherein the composition is administered by a parenteral route.
  • MM The pharmaceutical composition for use according to any one of the items AA to LL, wherein the composition is administered daily.
  • NN The pharmaceutical composition for use according to any one of claims items AA to MM, wherein in comparison to healthy muscle tissue sample, a muscle tissue sample obtained from the patient is characterised by at least a 1.5, particularly a 2-fold upregulation in the level of expression of at least one gene selected from the list consisting of HDAC4, HDAC5, HDAC9, DNMT1 , DNMT3A, and/or TRDMT1.
  • compositions for use according to any one of items AA to NN wherein in comparison to healthy muscle tissue sample, a muscle tissue sample obtained from the patient is characterised by at least a 30% reduction in the level of RYR1 protein, or a 30% reduction in the level of RYR1 mRNA.
  • the invention further encompasses the following numbered items:
  • a pharmaceutical composition comprising 5-aza’-2-deoxycytidine (CAS no. 2353-33- 5) for use in the treatment of myopathy, wherein the pharmaceutical composition does not further comprise a class Ila HDAC inhibitor.
  • a pharmaceutical composition for use according to item 1 for use in a patient who has not been administered a pharmaceutical composition comprising a class Ila HDAC inhibitor within a medically relevant window.
  • composition for use according to item 1 or 2 wherein the pharmaceutical composition is administered to a patient characterised by: upregulation of at least one gene encoding a class Ila histone deacetylase (HDAC) and/or a DNA methyltransferase (DNMT), and/or downregulation of RYR1 expression.
  • HDAC histone deacetylase
  • DNMT DNA methyltransferase
  • myopathy is selected from multi-minicore disease, centronuclear myopathy, congenital fibre-type disproportion, rigid spine muscular dystrophy, nemaline myopathy, or X-linked myotubular myopathy.
  • composition for use according to any one of the items 1 to 8, wherein the composition is administered by a parenteral route.
  • composition for use according to any one of the items 1 to 9, wherein the composition is administered daily.
  • a muscle tissue sample obtained from the myopathy patient is characterised by at least a 1 .5, particularly a 2- fold upregulation in the level of expression of at least one gene selected from HDAC4, HDAC5, HDAC9, DNMT1, DNMT3A, and/or TRDMT1.
  • a muscle tissue sample obtained from the myopathy patient is characterised by at least a 30% reduction in the level of RYR1 protein, or a 30% reduction in the level of RYR1 mRNA.
  • Fig. 1 shows pharmacokinetic profile of TMP269 following intraperitoneal injection in WT mice.
  • Fig. 2 shows daily i.p. injections with TMP269 and 5-Aza increase acetylation of Lys residues and of H3K9 in muscles from dHT mice.
  • Fig. 3 shows treatment of dHT mice with TMP269 and 5-Aza show improved in vivo muscle function as assessed using the grip strength test and voluntary running wheel.
  • Each symbol represents the average ( ⁇ S.D.) grip force value per mouse, calculated by averaging 5 measurements on the same mouse. Grip force (Force) values obtained on the first week were considered 100%.
  • Statistical analysis was conducted using the Mann-Whitney test. *p ⁇ 0.05.
  • B. Spontaneous locomotor (dark phase) activity (left panel) and total running speed (right panel) measured over 20 days in 21 -week-old dHT and WT littermates mice treated with vehicle or TMP269+5Aza. Data points are expressed as mean ( ⁇ S.D; n 4-5). *p ⁇ 0.05 (Mann-Whitney test).
  • Fig. 4 shows the mechanical properties of dHT mice treated with TMP269 and 5-Aza improve after 15 weeks of treatment.
  • Fig. 5 shows electrically evoked peak Ca 2+ transients in muscle fibres from treated dHT compound heterozygous (dHT) mice was rescued by TMP269 and 5-Aza administration.
  • Enzymatically dissociated FDB fibres were loaded with Mag- Fluo-4 and electrically stimulated by field stimulation.
  • B Whisker plots of peak twitch. Each symbol represents results obtained from a single FDB fibre.
  • C Representative Ca 2+ transient evoked by tetanic stimulation by a train of pulses delivered at 100 Hz for 300 msec.
  • Whisker plots of peak transient induced by tetanic stimulation FDB fibres were dissected from 4-5 mice per group; black line, vehicle treated WT, grey line, vehicle treated dHT, red line, vehicle TMP269 and 5-Aza treated dHT. Each symbol represents results obtained from a single FDB fibre. *p ⁇ 0.05 (ANOVA followed by the Bonferroni post hoc test).
  • Fig. 6 shows treatment with TMP269 and 5-Aza reverses RyR1 loss in soleus muscles from dHT mice.
  • Fig. 7 shows representative EM images at low magnification of Soleus fibres from vehicle treated (A) and drug treaded (B) dHT mice: small arrows point to dilated SR.
  • C and D Higher magnification images showing calcium release units (CRUs, black arrow) and autophagic material (empty arrow) from vehicle treated (C) and drug treated (D) dHT mice.
  • M mitochondria. Scale bars: A and B, 1 pm; C and D, 500 nm.
  • Fig. 8 shows qPCR measurements of the indicated epigenetic enzymes measured in muscle tissue biopsies from multi-minicore disease patients bearing SEPN1 gene mutations, and healthy controls. Gene expression is normalised to a housekeeping gene and expressed relative to the mean expression in the healthy control cohort.
  • Fig. 9 shows qPCR measurements of the indicated epigenetic enzymes measured in muscle tissue biopsies from X-linked myotubular myopathy patients bearing MTM1 gene mutations, and healthy controls. Gene expression is normalised to a housekeeping gene and expressed relative to the mean expression in the healthy control cohort.
  • Fig. 10 shows mutations in genes causing congenital myopathies such as RYR1, SEPN1 (SELENON) and MTM1, lead to, or are accompanied by a decrease in RYR1.
  • Heterozygous mutations in RYR1, SEPN1, or MTM1 (1 ) are associated with biochemical changes in muscles including hyper-methylation of >6000 genes (2), and increased content of class II HDACs and DNMT (epigenetic modifying enzymes).
  • class II HDACs particularly HDAC4 and 5 bind and sequester transcription factors belonging to the myocyte enhancer factor 2 (mef2) family (3).
  • HDAC-mediated sequestration of mef2 members leads to reduced transcription of muscle-specific mef2 target genes mediating excitation contraction such as RYR1 (4).
  • the inhibitory activity of gene transcription via chromatin modification is a result of the combined enzymatic activity of HDACs (5) and DNMT, therefore inhibiting the enzymatic activity of both group of enzymes results in the reversal of their effect on chromatin structure and thus availability to be transcribed.
  • Fig. 11 shows mechanical properties of EDL muscles from dHT mice treated with 0.05 mg/kg 5-Aza for 15 weeks are significantly improved.
  • Statistical analysis of force generated after twitch or tetanic force (50 and 100 Hz) in EDL muscles.
  • Table 1 List of hypomethylated protein-encoding genes in soleus muscles from dHT mice treated for 16 weeks with TMP269 and 5-Aza za drug versus vehicle treated dHT mice.
  • Table 4 Analysis of electrically evoked calcium transients in single FDB muscle fibres isolated from WT and dHT littermates, treated with vehicle or TMP269 and 5- Aza (25 mg/Kg) for 16 weeks.
  • Table 5 Quantitative analysis of CRUs in Soleus fibres from drug treated dHT mice, frequency of total CRUs and dyads (incomplete CRUs) are significantly rescued (columns A and B), suggesting a better preservation of CRUs and an improved structure of triads.
  • Table 10 Class II specific HDAC inhibitors IC50 and predicted dosing to improve muscle function in myopathy.
  • IC50 (nM) of inhibition of HDAC activity by class I Is-specific HDAC inhibitors.
  • IC50 of NVS compounds adapted from Supplementary Table 1 Luo et a/ 2019.
  • Predicted dosing for NVS compounds extrapolated from IC50 of HDAC4 inhibition compared to optimal TMP269 dosing demonstrated to increase grip force without side effects in dHT knockout mice.
  • Drug injection protocol The class II HDAC inhibitor TMP269 was purchased from Selleckchem (S7324), the DNA methyltransferase inhibitor 5-aza-2 deoxycytidine was from Sigma-Aldrich (A3656), Polyethylenglycol 300 (PEG300) was from Merck (8.17019), N-methyl-2-pyrrolidone (NMP) was from Sigma-Aldrich (328634).
  • Intraperitoneal injections IP, 30-gauge needle
  • IP Intraperitoneal injections started at the sixth week of age and continued daily for 10 - 15 weeks.
  • mice were sacrificed and their skeletal muscles were isolated, flash frozen and stored in liquid nitrogen.
  • muscle samples were homogenized using a tissue homogenizer (Mikro-Dismembrator S, Sartorius, Aubagne, France) for two sequences of 30 sec each.
  • TMP269 in blood and in skeletal muscle was quantified by high pressure liquid chromatography tandem mass spectrometry (LC-MS/MS) as described (Duthaler U. J. Pharm. Biomed. Anal. 172, 18-25 (2019)). Briefly, A Shimadzu LC system (Kyoto, Japan) was used, TMP269 and TMP195 (internal standard, IS) were analysed by positive electrospray ionization and multiple reaction monitoring (MRM). Methanol containing IS (5 nM TMP195) was used to extract TMP269 from blood and muscle homogenate samples (20 pL blood/muscle plus 175 pL methanol).
  • MRM positive electrospray ionization and multiple reaction monitoring
  • In Vivo Muscle Strength Assessment In vivo muscle performance was evaluated in male mice, by performing the following measurements: (i) forelimb grip strength and (ii) spontaneous locomotor activity. Forelimb grip strength was assessed once per week for a period of 10 weeks using a Grip Strength Meter from Columbus Instruments (Columbus, OH, USA), following the manufacturer’s recommendations. The grip force value obtained per mouse was calculated by averaging the average value of 5 measurements obtained on the same day on the same mouse. To avoid experimental bias during measurements of grip force, experimenters were blinded to the genotype and treatment of mice.
  • mice were individually housed in cages equipped with a running wheel carrying a magnet as previously described (Elbaz M. Hum. Mol. Genet. 28, 2987-2999 (2019Wheel revolutions were registered by reed sensors connected to an I-7053D Digital-Input module (Spectra), and the revolution counters were read by a standard laptop computer via an I-7520 RS-485-to-RS-232 interface converter (Spectra). Digitized signals were processed by the “mouse running” software developed at Santhera Pharmaceuticals. Total running distance (kilometer) and speed (Km/h) were evaluated.
  • EDL extensor digitorum longus
  • soleus muscles were dissected from 21 weeks old male WT and dHT mice, after 15 weeks of treatment with vehicle alone, or with the combination of TMP26915-Aza. Isolated EDL and soleus muscles were mounted onto a muscle force transducing setup (MyoTronic Heidelberg) as previously described (Mosca B. Nat. Commun. 4, 1541 (2013)). Muscle force was digitized at 4 kHz by using an AD Instrument converter and stimulated with 15 V pulses for 1.0 msec.
  • Tetanus was recorded in response to a train of pulses of 400 msec and 1100 msec duration delivered at 10/20/50/100/150 Hz, and 10/20/50/100/120 Hz, for EDL and soleus, respectively.
  • Specific force was normalized to the muscle cross-sectional area [CSA_wet weight (mg)/length (mm)_1 .06 (density mg/mm 3 )] (Mosca B. Nat. Commun. 4, 1541 (2013)). The experimenter performing the measurements was blinded with respect to the mouse genotype and treatment.
  • Flexor digitorum brevis (FDB) muscles were isolated and digested with 0.2% of Collagenase type I (Clostridium hystoliticum Type I, Sigma-Aldrich) and 0.2% of Collagenase type II (Clostridium hystoliticum Type II, Worthington) in Tyrode’s buffer (137 mM NaCI, 5.4 mM KCI, 0.5 mM MgCI 2 , 1 .8 mM CaCI 2 , 0.1 % glucose, 11 .8 mM HEPES, pH 7.4 NaOH) for 45 minutes at 37 °C as described (Brooks S. V. J. Physiol. 404, 71-82 (1988)).
  • Biochemical analysis of total muscle homogenates Total muscle homogenates were prepared from soleus from WT and dHT vehicle and drug treated mice. SDS-polyacrylamide electrophoresis and Western blots of total homogenates were carried out as previously described ((Elbaz M. Hum. Mol. Genet. 28, 2987-2999 (2019); Calderon J. C. J. Muscle Res. Cell Motil. 30, 125-137 (2000)). Western blots were stained with the primary antibodies, followed by peroxidase-conjugated Protein G (Sigma P8170; 1 :130’000) or peroxidase- conjugated anti-mouse IgG (Fab Specific) Ab (Sigma A2304; 1 :200’000). The immuno-positive bands were visualized by chemiluminescence using the WesternBright ECL- HRP Substrate (Witec AG). Densitometry of the immune-positive bands was carried out using the Fusion Solo S (Witec AG).
  • Sections were using the following primary antibodies mouse anti-mouse IgX MyHC I (1 :50), mouse anti-mouse IgY MyHC Ila (1 :200), mouse anti-mouse IgZ MyHC lib (1 :100) and rabbit anti-mouse laminin (1 :1500), followed by incubation with the following secondary antibodies: goat anti-mouse Alexa Fluor 568 IgG 1 (1 :1000), goat anti-mouse Alexa Fluor 488 IgM (1 :1000), goat anti-mouse Dylight 405 IgG (1 :400) and Donkey anti-rabbit Alexa Fluor 647 IgG (1 :2000). Images were obtained using an Eclipse Ti2 Nikon Fluorescence microscope with 10X air objective lens.
  • Muscles from 3 mice per group were evaluated. Images were analysed using Fiji plugins in order to obtain information on fibre types and minimal Feret's diameter (Briguet A. et al. Neuromuscul. Disord. 14, 675-682 (2004)), the closest possible distance between the two parallel tangents of an object, using a combination of cell segmentation and intensity thresholds as described.
  • Ultrathin sections ( ⁇ 40 nm) were cut in a Leica Ultracut R microtome (Leica Microsystem, Austria) using a Diatome diamond knife (DiatomeLtd. CH-2501 Biel, Switzerland) and examined at 60 kV after double-staining with uranyl acetate replacement and lead citrate, with a FP 505 Morgagni Series 268D electron microscope (FEI Company, Brno, Czech Republic), equipped with Megaview III digital camera (Munster, Germany) and Soft Imaging System (Germany).
  • Total muscle homogenates were prepared from soleus from WT and dHT vehicle and drug treated mice. SDS-polyacrylamide electrophoresis and Western blots of total homogenates were carried out as previously described ((Elbaz M. Hum. Mol. Genet. 28, 2987-2999 (2019); Briguet A. Neuromuscul. Disord. 14, 675-682 (2004); Calderon J. C. J. Muscle Res. Cell Motil. 30, 125-137 (2000)).
  • Western blots were stained with the primary antibodies listed in Supplementary Table S6, followed by peroxidase-conjugated Protein G (Sigma P8170; 1 :130’000) or peroxidase-conjugated antimouse IgG (Fab Specific) Ab (Sigma A2304; 1 :200’000).
  • the immuno-positive bands were visualized by chemiluminescence using the WesternBright ECL- HRP Substrate (Witec AG). Densitometry of the immune-positive bands was carried out using the Fusion Solo S (Witec AG).
  • Quantitative PCR Quantitative PCR: Quadriceps muscle biopsies from patients with genetically confirmed mutations and healthy non-affected individuals were used. Isolation of RNA from muscle biopsies was performed using TRIzolTM reagent (Thermo Fischer; 15596026) following the manufacturer’s protocol. For subsequent quantitative real-time polymerase chain reaction (qPCR), 1000 ng of RNA were reverse-transcribed to cDNA using the High-capacity cDNA Reverse Transcription Kit (Applied Biosystems; 4368814) on an Applied Biosystems 2720 Thermal Cycler.
  • qPCR quantitative real-time polymerase chain reaction
  • the cDNA was amplified using PowerllPTM SybrTM Green Master Mix (Applied Biosystems; A25742) for regular DNA quantification on an Applied Biosystems 7500 Fast Realtime PCR System running 7500 software version 2.3. Quantification was based on the comparative AACt method. Each reaction was performed in duplicate with validated qPCR primers, and results are expressed as relative gene expression normalized to muscle-specific housekeeping gene desmin (DES). Expression levels are shown as fold-change compared to healthy control samples that were set to 1 .
  • PowerllPTM SybrTM Green Master Mix Applied Biosystems; A25742
  • Quantification was based on the comparative AACt method. Each reaction was performed in duplicate with validated qPCR primers, and results are expressed as relative gene expression normalized to muscle-specific housekeeping gene desmin (DES). Expression levels are shown as fold-change compared to healthy control samples that were set to 1 .
  • Example 1 A combined pharmaceutical composition for myopathy
  • TMP269 The pharmacokinetics and bio-distribution of the class ll-HDAC inhibitor TMP269 was first assessed. After intraperitoneal injection of 25 mg/kg body weight of TMP269 dissolved in Polyethylenglycol 300 (PEG300) (500 pl/Kg) and N-methyl-2-pyrrolidone (NMP) (250 uL/Kg), blood and/or skeletal muscles were collected at different time points and the content of TMP269 was quantified by mass spectrometry (Duthaler U. J. Pharm. Biomed. Anal. 172, 18- 25 (2019)). The peak plasma concentration of TMP269 was achieved approximately 1 hour after injection. The circulating levels of TMP269 decay within 12 hours (Fig. 1A).
  • the class ll-HDAC inhibitor diffuses into skeletal muscle (Fig. 1A), and as expected, its concentration profile in skeletal muscle follows that observed in blood. Although the level of TMP269 accumulating in skeletal muscle is lower compared to that present in blood, its concentration in muscle is adequate to induce an inhibitory effect on class Ila HDACs activity (Choi S. Y. etal. Biomed. Pharmacother. 101 , 145-154 (2016)).
  • An identical protocol was used to monitor the optimal dose of 5-Aza-2-deoxycytidine (5-Aza), an FDA approved DNA methyltransferase (DNMT) inhibitor (Kaminskas E. et al. Oncologist 10, 176-182 (2005)).
  • the administration of the DNMT inhibitor resulted in hypomethylation of 173 genes (Table 1 ). Based on these results, 6 weeks old WT and dHT mice with vehicle alone (PEG300+ NMP), with TMP269 alone (25 mg/kg), 5-Aza alone (0.05 mg/kg) or with the two drugs combined on a daily base, for 10 consecutive weeks. Administration of TMP269 +5-Aza for 15 weeks increases the acetylation of Lys residues (Fig. 2A) and of H3K9 (Fig. 2B and D) in total homogenates from FDB fibres isolated from WT and dHT mice, compared to that observed in fibres from vehicle treated WT and dHT mice.
  • the epigenetic modifying drugs may affect numerous genes, which in turn leads to an improvement of the in vivo muscle performance of the dHT mice; the latter effect may result from an improvement of the mechanical properties of skeletal muscles and/or by an influence of the drugs on the metabolic pathways of muscles.
  • EDL and soleus muscles isolated from mice treated for 15 weeks with vehicle or TMP269+5-Aza were stimulated with a single 15 V pulse of 1.0 msec duration a (Fig. 4 A, C, E and G) or by a train of pulses delivered at 150 Hz for 400 msec (EDL, Fig. 4B and D) or 120 Hz for 1100 msec (soleus, Fig. 4F and H) to obtain maximal tetanic contracture.
  • the peak force developed after twitch stimulation of soleus muscles from dHT mice injected with vehicle alone mice was approx.
  • treatment with TMP269+5-Aza causes no changes in the content of the fibre type composition of soleus muscles (Table 3).
  • the improved specific force cannot be attributed to a major shift of minimal Feret’s fibre diameter distribution.
  • the average peak intracellular Ca 2+ transient induced by a single action potential in FDB fibres from dHT mice injected with vehicle is approx.
  • the RyR1 protein content of soleus muscles from dHT mice injected with vehicle is 46% lower compared to that of WT mice (Fig. 6B).
  • the RyR1 protein content is partially re-established by treatment with TMP269+5- Aza.
  • Skeletal muscle fibres from adult wild type are usually characterized by a regular transverse pale-dark striation.
  • the SR-TT junctions containing RyR1 are uniformly distributed, and mostly placed at A-l band transition (when sarcomeres are relaxed), on both sides of the Z line.
  • CRUs are formed by two SR terminal cisternae closely opposed to a central TT-oriented transversally with respect to the longitudinal axis of the myofibrils. These CRUs are called triads. CRUs are often associated to a mitochondrion, to form functional couples.
  • CRUs present some abnormal features: the SR is often dilated (small arrows in Fig. 6A) and sometimes they are incomplete, meaning they are formed by only 2 elements (dyads) (Fig. 7B, black arrow). Quantitative EM analysis in shows that in fibres of vehicle treated dHT mice there are 40.3 ⁇ 2.4 CRUs/100 pm2, 14.2 ⁇ 3.6% of them being dyads (Table 4, columns A-B). In soleus muscle fibres of dHT vehicle treated mice There are also regions with accumulation of autophagic material (Fig. 7B, empty arrow). In fibres from drug treated dHT mice (Fig.
  • the epigenetic enzymes targeted by the combined TMP269+5-Aza treatment demonstrated to relieve symptoms in the dHT mouse model may also be upregulated in other myopathies with a similar aetiology.
  • a transcriptional analysis was performed on muscle tissue samples obtained from patients diagnosed with a range of congenital myopathies caused by (but not limited to) mutations in RYR1, MTM1, SEPN1 (SELENON).
  • SEPN1 mutations in patients with Multi-minicore disease are associated with upregulation of DNA methylation genes (DNMT1.3A and TRDMT1 ) (Fig. 8).
  • HDAC5 and HDAC9 are significantly unregulated is samples from patients diagnosed a very severe childhood muscle disease called X-linked myotubular myopathy caused by disabling MTM1 mutation (Fig. 9).
  • up-regulation of transcripts encoding epigenetic enzymes belonging to the class II HDAC and DNMT families in both groups of patients was associated with down-regulation of RYR1 transcript (Fig. 8 and 9).
  • the present study demonstrates that the combined pharmacological treatment with an FDA approved DNMT inhibitor and TMP269 improves muscle strength and performance in a mouse model for recessive RYR1 congenital myopathy, providing the proof of concept for the development for pharmacological treatment of patients with myopathies linked to reduced transcription or activity of the protein encoded by the excitation contraction gene RYR1, such as diseases caused by mutations in RYR1, MTM1, and SEPN1 (Fig. 10).
  • Example 3 Class Ila HDAC inhibitors for treatment of myopathy
  • Muscle denervation causes muscle atrophy, an event which has been associated with an upregulation of class II HDACs (including HDAC-4, -5 and -9).
  • HDAC-4 leads to the expression of ubiquitin ligase which ultimately drives the processes of muscle loss.
  • HDAC4 and HDAC5 blunts the effects of the processes leading to atrophy observed during denervation.
  • HDAC4 and HDAC5 knock-out mice the loss of muscle mass upon denervation is reduced by approximately 20 % compared to that observed in WT mice (Moresi V. et al. 2010 Cell 143, 35-45).
  • the physiological and molecular signatures of muscle denervation likely depend on the duration of the process.
  • Luo et al utilized a novel class II specific HDAC inhibitor (NVS-HD1 , Luo et al. Cell Rep 2019 29:749-763) to counteract pathogenic HDAC expression, and ameliorate the early symptoms of muscle denervation.
  • NVS-HD1 novel class II specific HDAC inhibitor
  • the similar mode of action of TM269 and NVS-HD1 on HDAC4 activity suggests these related compounds are strongly likely to provide an equivalent therapeutic effect in myopathy treatment.
  • NVS-HD1 , NVS-HD2 and NVS- HD3 WO2013008162, WO2013080120A1 are non-CNS penetrant trifluoromethyl-oxadiazole derivatives, and specifically inhibit Class II a HDAC activity.
  • NVS-HD1 Due to the similarities between the HDAC class Ila specific inhibition action of the NVS-HD1 , NVS-HD2, and NVS-HD2 with TMP269 (Table 10), the inventors propose these molecules may be used as an alternative to TMP269 to provide synergistic class II HDAC inhibition in a combination medicament further comprising the DNMT inhibitor 5- Aza, for use treating myopathy according to the invention.
  • NVS-HD1 , NVS-HD1 , or NVS-HD3 suitable for use treating myopathy in humans in combination with 5-Aza was calculated based on the published IC50 for NVS compounds with reference the relative IC50 of HDAC4 inhibition, as well as the specific dosing of TMP269 demonstrated to effectively inhibit HDAC4 and be safe and effective treatment in a model of myopathy in combination with 5-Aza (Table 10).
  • Example 1 demonstrates that the mechanical properties of soleus muscles isolated from adult dHT mice were significantly improved following 15 weeks of treatment with TMP269+5-Aza, whereas the mechanical properties of EDL muscles were not ameliorated.
  • Such combined treatment may be efficacious in congenital myopathy patients with established disease, as these patients are usually characterized by an increasing proportion of slow twitch muscle fibers as fast fibers atrophy.
  • the inventors further assessed fast twitch muscle function in dHT mice receiving only one drug, namely only 0.05 mg/kg 5-Aza (i.p. on a daily basis), approved by the FDS for treating pediatric patients with myelodysplastic syndrome.
  • the results show that 15 weeks of treatment with 5-Aza improves the mechanical properties of EDL muscles (Fig. 11 ). This new result implies that in young patients, treatment with 5-Aza alone may improve the strength of fast twitch muscles.
  • Muscles were stimulated with a single twitch or tetanic stimulation (EDL: 150Hz, 400 ms duration; soleus 120 Hz, 400ms duration). Data are shown as mean specific force (mN/mm2) *p ⁇ 0.05, p ⁇ 0.01 dHT vs WT; p ⁇ 0.05 dHT vehicle vs dHT TMP269+5-Aza ⁇ SEM. ANOVA and Bonferroni post hoc test. Table 3

Abstract

La présente invention concerne une composition pharmaceutique comprenant un inhibiteur de HDAC de classe IIa caractérisé par un groupe trifluorométhyloxadiazole et/ou une 5-aza-2-désoxycytidine pour une utilisation dans le traitement de la myopathie.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2013008162A1 (fr) 2011-07-08 2013-01-17 Novartis Ag Nouveaux dérivés de trifluorométhyl-oxadiazole et leur utilisation pour le traitement de maladies
WO2013080120A1 (fr) 2011-11-28 2013-06-06 Novartis Ag Nouveaux dérivés trifluorométhyl-oxadiazoles et leur utilisation dans le traitement de maladies

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WO2013008162A1 (fr) 2011-07-08 2013-01-17 Novartis Ag Nouveaux dérivés de trifluorométhyl-oxadiazole et leur utilisation pour le traitement de maladies
WO2013080120A1 (fr) 2011-11-28 2013-06-06 Novartis Ag Nouveaux dérivés trifluorométhyl-oxadiazoles et leur utilisation dans le traitement de maladies

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