Use of bazedoxifene for increasing muscle survival

Abstract

The invention relates to the use of bazedoxifene as representing third-generation SERM, namely compounds comprising a 2-phenylindole group, for increasing muscle survival, in particular in the treatment of dysferlinopathies.

Description

[0001] USE OF BAZEDOXIFENE TO INCREASE MUSCLE SURVIVAL

[0002] TECHNICAL FIELD

[0003] The present invention aims to improve muscle function in humans or animals, by preserving the integrity and survival of muscle cells, including under adverse conditions.

[0004] Specifically, it advocates the use of bazedoxifene, a selective estrogen receptor modulator (SERM) to protect muscle function, particularly in pathological conditions such as muscular dystrophies.

[0005] PRIOR STATE OF THE ART

[0006] Neuromuscular diseases are a group of conditions that are generally associated with a transient or permanent loss of muscle strength. This loss of strength is most often accompanied by muscle wasting, also known as amyotrophy.

[0007] Among these muscle diseases, myopathies constitute an important group corresponding to damage to the muscle fiber itself. Among them, progressive muscular dystrophies are characterized by a decrease in muscle strength, generally with muscle atrophy, as well as by abnormalities in muscle biopsy revealing changes in the tissue. This group includes Duchenne muscular dystrophy (or DMD), Becker muscular dystrophy (or BMD), as well as limb-girdle muscular dystrophy.

[0008] For some of these pathologies, associated genetic anomalies have been identified. Thus, Duchenne or Becker muscular dystrophies are linked to alterations in the gene coding for dystrophin, limb-girdle dystrophy type 2A (LGMD 2A or RI or calpainopathy) to alterations in the calpain 3 gene, dysferlinopathy (LGMD 2B or R2) to alterations in the dysferlin gene, sarcoglycanopathies or limb-girdle myopathies of type LGMD 2C (or R5), LGMD 2D (or R3), LGMD 2E (or R4), LGMD 2F (or R6), to defects in the genes of y-, a-, P- and ô-sarcoglycans, respectively (McNally EM, Pytel P, Annu Rev Pathol. 2007; Vol 2:87-109).

[0009] In relation to dysferlinopathy, the L1341P mutation in the dysferlin gene has been shown to result in a misconfiguration of the resulting protein, associated with its aggregation in the endoplasmic reticulum. The absence of dysferlin in the sarcolemma then leads to membrane fragility, which ultimately leads to the death of muscle cells and therefore contributes to muscular dystrophy.

[0010] For these pathologies, different gene therapy strategies are being developed but remain difficult to implement.

[0011] However, and more generally in all cases of muscle weakness, there is a clear need to develop technical solutions to preserve muscle function, particularly using chemical compounds that are more easily controlled than gene therapy.

[0012] Thus, the present invention is based on the demonstration, by the inventors, of this property of bazedoxifene.

[0013] Document W02009 / 021750 describes the use of the compound (2-phenyl-1H-indol-3-yl)m ethanol, which is not a SERM, in connection with the treatment of Duchenne or Becker dystrophies and cachexia.

[0014] Document WO2015 / 108988 describes the use of a combination of a SERM (advantageously lasoxifene which does not include a 2-phenylindole group) and a 5a-reductase inhibitor to treat any pathology linked to a modification of sex hormone levels.

[0015] The paper XIA HUI et al. (PLOS ONE, 2017, Vol. 12(7)) describes the use of bazedoxifene for the specific treatment of rhabdomyosarcoma.

[0016] WANG JING et al. (FRONTIERS IN PHARMACOLOGY, 2021, Vol. 11, pages 1-13) describes the use of bazedoxifene for the treatment of a specific inflammatory cardiomyopathy, namely autoimmune myocarditis. DORCHIES et al. (JOURNAL OF NEUROMUSCULAR DISEASES, 2014, Vol. 1(1)) reports the beneficial effect of tamoxifen and other SERMs on ere or 2 eme generation on a mouse model of Duchenne disease.

[0017] The document WU BO et al. (THE AMERICAN JOURNAL OF PATHOLOGY, 2018, Vol. 188(4), pages 1069-80) reports the beneficial effect of oral administration of tamoxifen (SERM of the ere generation) and raloxifene (SERM of 2 eme generation) in a mouse model of dystroglycanopathy.

[0018] DETAILED DESCRIPTION OF THE INVENTION

[0019] The inventors have shown that bazedoxifene, a pharmacological compound primarily known in the prevention and treatment of postmenopausal osteoporosis due to its action as a selective estrogen receptor modulator (or SERM), is a promising candidate for treating dysferlinopathies. The present application reveals that it is effective for a broad spectrum of conditions associated with muscle cell fragility, particularly related to the integrity of their membranes.

[0020] Definitions

[0021] The definitions below give the meaning generally used within the scope of the invention and must be taken into account unless another definition is explicitly indicated.

[0022] In the context of the invention, the articles "a" and "an" are used to designate one or more (i.e., at least one) of the grammatical objects of the article. For example, "an element" designates at least one element, i.e., one or more elements.

[0023] The terms "about," "roughly," "in the order of," or "approximately" used herein to refer to a measurable value such as a quantity, time, and the like, should be understood to encompass variations of ± 20% or ± 10%, preferably ± 5%, more preferably ± 1%, and even more preferably ± 0.1% from the specified value. Intervals / Ranges: Throughout this disclosure, various aspects of the invention may be presented in the form of an interval of values ​​(range format). It should be understood that the description of values ​​in the form of an interval is for convenience and brevity only and should not be construed as a limitation of the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range.For example, a description of a range such as "1 to 6" should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within that range, e.g., 1, 2, 2, 7, 3, 4, 5, 5, 3, and 6. This applies regardless of the extent of the range.

[0024] “Isolated” means extracted or removed from its natural environment or state. For example, an isolated nucleic acid or peptide is a nucleic acid or peptide that has been extracted from the natural environment in which it is usually found, whether in a living plant or animal, for example. A nucleic acid or peptide, for example, that is naturally present in a living animal is not an isolated nucleic acid or peptide for the purposes of the invention, whereas the same nucleic acid or peptide partially or completely separated from other components present in its natural environment is itself “isolated” for the purposes of the invention. An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment such as, for example, a host cell.

[0025] The term “abnormal,” when used in the context of organisms, tissues, cells, or components thereof, means organisms, tissues, cells, or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from organisms, tissues, cells, or components thereof that exhibit the respective “normal” (expected) characteristic. Characteristics that are normal or expected for one type of cell or tissue may be abnormal for another type of cell or tissue.

[0026] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cell thereof, in vitro or in situ, that can be subjected to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is an animal, preferably a mammal, more preferably a human, either female or male. It may also be a mouse, rat, pig, dog, or non-human primate (NHP), such as a macaque monkey.

[0027] For the purposes of the invention, a “disease” or “pathology” is a state of health of an animal in which its homeostasis is negatively affected and which, if the disease is not treated, continues to deteriorate. Conversely, for the purposes of the invention, a “disorder” or “dysfunction” is a state of health in which the animal is able to maintain its homeostasis but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. In the absence of treatment, a disorder does not necessarily lead to a deterioration in the animal’s state of health over time.

[0028] A disease or disorder is "attenuated" ("reduced") or "improved" if the severity of a symptom of the disease or disorder, the frequency with which that symptom is experienced by the individual, or both, are reduced. This also includes the cessation of disease progression, i.e., the cessation of progression of the disease or disorder. A disease or disorder is "cured" ("restored") if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by the individual, or both, are eliminated.

[0029] In the context of the invention, a “therapeutic” treatment is a treatment administered to a subject who presents the symptoms (signs) of a pathology, with the aim of reducing or eliminating these symptoms. In the context of the invention, “treatment of a disease or disorder” means the reduction of the frequency or severity of at least one sign or symptom of a disease or disorder experienced by the subject. A treatment is said to be prophylactic when it is administered to prevent the development, spread or worsening of a disease, in particular if the subject does not present or not yet presents the symptoms of the disease and / or for which the disease has not been diagnosed.

[0030] As used herein, “treating a disease or disorder” means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. Disease and disorder are used interchangeably in the context of the treatment of the invention. As used herein, an “effective amount” or “effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to whom the compound is administered. The term “therapeutically effective amount” refers to an amount that is sufficient or effective to prevent or treat (in other words, delay or prevent the development of, prevent the progression of, inhibit, reduce, diminish, or reverse) a disease or disorder, including alleviating the symptoms of that disease or disorder.

[0031] The present invention therefore relates to the use of a specific estrogen receptor modulator (SERM), advantageously of the third generation, i.e. comprising a 2-phenylindole group, even more advantageously bazedoxifene or its derivatives, to combat the fragility or even the death of muscle cells, and therefore to increase muscle survival.

[0032] Selective estrogen receptor modulators (or SERMs) are nonsteroidal molecules capable of binding to estrogen receptors. They possess both estrogen agonist and antagonist properties. Such molecules can be identified using well-known tests such as the estrogen response element (ERE) transactivation assay or as described by Nelson (2016, Methods Mol Biol: 431-443).

[0033] In practice, different compounds exhibiting such activity are available:

[0034] So-called first-generation SERMs are triphenylethylene derivatives.

[0035] Such compounds may for example be chosen from: tamoxifen of formula:

[0036] - tamoxifen citrate clomiphene or enclomiphene or clomiphene citrate (CLOMID)

[0037] - toremifene or toremifene citrate afimoxifene (4-hydroxy tamoxifen) levormeloxifene or ormeloxifene droloxifene idoxifene

[0038] - fispemifene

[0039] - ospemifene ((deaminohydroxy)toremifene) of formula:

[0040] The so-called second-generation SERMs are derivatives of benzothiophene, for example; the formula: arzoxifene The so-called third-generation SERMs are 2-phenylindole derivatives of the formula: Such compounds may for example be chosen from: bazedoxifene (CAS number: 198481-32-2) of formula:

[0041] - bazedoxifene acetate of formula: zindoxifene of formula: pipendoxifene of formula:

[0042] Other SERMs of potential interest are:

[0043] - nafoxidine of formula:

[0044] - lasofoxifene of formula:

[0045] Preferably, the compound used in the context of the present invention is a SERM and / or comprises a 2-phenylindole group of formula: Advantageously, it is chosen from the group consisting of: bazedoxifene, bazedoxifene acetate, zindoxifene, pipendoxifene, their derivatives and / or their combinations. Alternatively, it may be ospemifene, endoxifene or raloxifene.

[0046] According to a particular embodiment, it is not tamoxifen, nor fispemifene nor even raloxifene.

[0047] Even more advantageously, it is bazedoxifene or bazedoxifene acetate.

[0048] Bazedoxifene has been approved by the FDA and ANSM for the prevention of postmenopausal osteoporosis. This medicine, marketed under the name Conbriza®, is presented in the form of tablets comprising 20 mg of bazedoxifene acetate (with a tablet core containing: Lactose monohydrate (142.8 mg) + Microcrystalline cellulose + Pregelatinized starch (corn) + Sodium starch glycolate + Sodium lauryl sulfate + Colloidal anhydrous silica + Magnesium stearate + Ascorbic acid and a film-coating containing: Hypromellose + Titanium dioxide (E171) + Macrogol 400), with a daily oral intake of 1 tablet.

[0049] Also covered by the present invention are derivatives of these compounds, in particular bazedoxifene, having the same biological activity, in particular that of SERM or that reported in the examples, for example on the survival of muscle cells under conditions of osmotic shock.

[0050] The term "derivatives" includes derivatives and metabolites, as well as pharmaceutically acceptable salts. A derivative is a compound derived from another (the precursor, typically with a similar chemical structure) after transformation of the latter. The derivative may differ by one or more atoms or functional groups. A metabolite is a stable intermediate compound or a compound resulting from the biochemical transformation of an initial molecule by metabolism.

[0051] By "pharmaceutically acceptable salts" is meant the addition salts of the compound, which can be obtained by reacting this compound with a mineral or organic acid according to a method known per se. Among the acids which can be used for this purpose, mention may be made of hydrochloric (HCl), hydrobromic, sulfuric, phosphoric, 4-toluene sulfonic, methane sulfonic, cyclohexyl sulfamic, oxalic, succinic, formic, fumaric, maleic, citric, aspartic, cinnamic, lactic, glutamic acids. N-acetyl-aspartic, N-acetyl-glutamic, ascorbic, malic, benzoic, nicotinic and acetic acids. For example, bazedoxifene acetate is commonly used in the pharmaceutical field.

[0052] Among the esters on the hydroxy function, we can cite carboxylic acid esters having from 1 to 6 carbon atoms.

[0053] Such compounds, including bazedoxifene, may be modified to increase their stability, bioavailability and / or ability to reach target tissues, including muscle tissues and cells.

[0054] As known to those skilled in the art, said compounds, in particular bazedoxifene, may be present in the composition in a naked (free) form or contained in delivery systems which increase stability, targeting and / or bioavailability, such as liposomes, or incorporated into supports such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, vectors or in combination with a cationic peptide.

[0055] The present invention also relates to pharmaceutical compositions containing as active ingredient at least one compound as defined above, as well as the use of this compound or this composition as a medicament or medicinal product. According to a particular embodiment, the compound according to the invention is the only therapeutic active ingredient present in the pharmaceutical composition.

[0056] Thus, the present invention relates to pharmaceutical compositions comprising a compound according to the invention. Advantageously, these compositions comprise a therapeutically effective amount of said compound, and a pharmaceutically acceptable carrier. In a particular embodiment, the term "pharmaceutically acceptable" means approved by a federal or state regulatory body or listed in the United States or European Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic product is administered. These pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and others.Saline solutions and aqueous solutions of dextrose and glycerol may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, and others.

[0057] The composition, if any, may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, sustained-release formulations, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. These compositions contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with an appropriate amount of carrier so as to provide the form for adequate administration to the subject.

[0058] In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition suitable, for example, for oral administration to humans. Typically, compositions for oral administration are in the form of tablets, optionally scored tablets or effervescent tablets, further containing excipients suitable for the solid dosage form and for administration in humans. For example, commercially available forms of bazedoxifene are tablets which further contain lactose monohydrate, microcrystalline cellulose, pregelatinized starch (corn), sodium starch glycolate, sodium lauryl sulfate, colloidal anhydrous silica, magnesium stearate and ascorbic acid. These tablets can be crushed and mixed with liquids.

[0059] Alternatively, the composition may be in a liquid form, advantageously an aqueous composition. Any other suitable solvent may be used.

[0060] The amount of the therapeutic agent of the invention, i.e., a compound as described above, that will be effective in the treatment of a disease can be determined by standard clinical techniques. In addition, in vivo and / or in vitro assays, such as those described in the examples below, may optionally be used to help predict optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, the weight and severity of the disease, and should be decided according to the judgment of the practitioner and the conditions of the individual patient.

[0061] According to a particular embodiment, the composition of the invention is in a solid form, advantageously a tablet, comprising 20 mg of the active compound, in particular bazedoxifene or bazedoxifene acetate, or even less. Preferably, the composition comprises an amount equal to or less than 20 mg, 15 mg, 10 mg, or even equal to or less than 5 mg.

[0062] According to another embodiment, the composition of the invention is in a liquid form and advantageously comprises less than 30 pM or 10 pM of the active compound, in particular bazedoxifene or bazedoxifene acetate, more advantageously between 30 nM and 10 pM, even more advantageously between 100 nM and 5 pM, or even between 300 nM and 2 pM.

[0063] An appropriate route of administration should allow delivery of a therapeutically effective amount of the therapeutic product to the target tissues, depending on the disease.

[0064] Available routes of administration include topical (local), enteral (systemic action, but delivered through the gastrointestinal (GI) tract), or parenteral (systemic action, but delivered through routes other than the GI tract). In the specific case of muscle diseases, the preferred route of administration of the compositions disclosed herein is generally enteral, which includes oral administration. In other embodiments, it may be parenteral, including intramuscular (i.e., into the muscle) or systemic (i.e., into the circulating system) administration. In this context, the term “injection” (or “infusion” or “infusion”) encompasses intravascular, especially intravenous (IV), and intramuscular (IM) administration. Injections are generally performed using syringes or catheters.

[0065] According to one embodiment, the composition is administered orally, intramuscularly, intraperitoneally, subcutaneously, topically, locally or intravascularly, advantageously orally. According to a preferred embodiment, the composition is intended for oral administration. As already mentioned, a composition according to the invention is preferably in a solid dosage form suitable for oral administration, advantageously in the form of one or more capsules or tablets. Thus, they can be taken with a little water before or during the main meal. According to a preferred embodiment, the composition according to the invention is administered daily, for example once a day. The treatment can last several weeks, several months, several years or even a lifetime.

[0066] As already indicated, the patient is advantageously a human, in particular a newborn, a young child, a child, an adolescent or an adult, regardless of their sex. The therapeutic tool according to the invention can however be adapted and useful for the treatment of other animals, in particular pigs, mice, dogs or macaque monkeys.

[0067] In general, the present invention aims to combat (or reduce or attenuate or preserve) the mortality and / or fragility of muscle cells, fibers or tissues, in other words to improve (or increase or preserve) survival and / or strength and / or muscle function. Indeed and as demonstrated in the present application, the compounds described above improve membrane resistance, in particular to stress, for example under osmotic shock conditions or after laser irradiation.

[0068] In the context of the invention, the terms "muscle" and "muscular" advantageously relate to striated muscles, even more advantageously to skeletal muscles. According to a particular embodiment, the cardiac muscle is not targeted.

[0069] Thus, and as already mentioned, the present invention relates to the treatment of diseases associated with muscle fragility, in particular instability of the muscle cell membrane. In other words, the present invention can contribute to improving muscle survival, and consequently muscle function, in particular muscle strength.

[0070] According to a particular embodiment, the skeletal muscles are predominantly or even exclusively affected. Thus, the cardiac muscle can be spared from the disease. As illustrated in the examples, muscle fragility, particularly of the muscle cell membranes, can be assessed using in vitro tests under osmotic shock conditions.

[0071] Thus and more precisely, the invention also aims at:

[0072] - a composition comprising a compound as described above, in particular bazedoxifene as a representative of third-generation SERMs, namely compounds comprising a 2-phenylindole group, for treating pathologies associated with muscle fragility; the use of a compound as described above, in particular bazedoxifene as a representative of third-generation SERMs, namely compounds comprising a 2-phenylindole group, for the preparation of a medicament intended to treat pathologies associated with muscle fragility;

[0073] - a method of treating pathologies associated with muscle fragility comprising the administration of a compound as described above, in particular bazedoxifene as a representative of third generation SERMs, namely compounds comprising a 2-phenylindole group;

[0074] - a composition comprising a compound as described above, in particular bazedoxifene as a representative of third generation SERMs, namely compounds comprising a 2-phenylindole group, for increasing muscle survival / function / strength; the use of a compound as described above, in particular bazedoxifene as a representative of third generation SERMs, namely compounds comprising a 2-phenylindole group, for increasing muscle survival / function / strength;

[0075] - a method for increasing muscle survival / function / strength comprising administering a compound as described above, in particular bazedoxifene as a representative of third generation SERMs, namely compounds comprising a 2-phenylindole group.

[0076] In practice, there are a number of conditions in which muscle function is impaired.

[0077] These may firstly be pathological conditions, in particular in the case of neuromuscular diseases. According to one embodiment, the targeted disease is of genetic origin, in particular caused by one or more mutations in one or more genes responsible for said disease.

[0078] Thus, muscular dystrophies caused by mutations in the dysferlin gene (DYSF) such as dysferlinopathy (LGMD 2B or R2), Miyoshi-type distal myopathy and distal anterior compartment myopathy are particularly targeted pathologies but all forms of neuromuscular diseases, in particular muscular dystrophies, can be treated.

[0079] As demonstrated in the examples, dysferlinopathy (LGMD 2B or R2) linked to the L1341P, G299R or SI 173X mutation can thus be managed.

[0080] In certain embodiments, a compound as described or a pharmaceutical composition comprising it is intended to be used to treat muscle diseases (i.e., myopathies) or muscle injuries, in particular neuromuscular genetic disorders, without liver involvement or even without cardiac involvement, such as for example: muscular dystrophies, congenital muscular dystrophies, congenital myopathies, distal myopathies, other myopathies, myotonic syndromes, ion channel muscle diseases, malignant hyperthermia, metabolic myopathies and other neuromuscular disorders, advantageously muscular dystrophies, congenital muscular dystrophies, congenital myopathies, distal myopathies and other myopathies.

[0081] Muscular dystrophies that can be treated include:

[0082] - Dystrophinopathies, a spectrum of X-linked muscle diseases caused by pathogenic variants of the DMD gene, which codes for the dystrophin protein. Dystrophinopathies include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and DMD-associated dilated cardiomyopathy;

[0083] - Limb-girdle muscular dystrophies (LGMDs), which are a group of disorders clinically similar to DMD, but occur in both sexes due to autosomal recessive or autosomal dominant inheritance. Limb-girdle dystrophies are caused by mutations in genes that encode sarcoglycans and other proteins associated with the muscle cell membrane, which interact with dystrophin. The term LGMD1 refers to genetic types with dominant (autosomal dominant) inheritance, while the term LGMD2 refers to types with autosomal recessive inheritance. Pathogenic variants have been reported at more than 50 loci (LGMD1A to LGMD1H; LGMD2A to LGMD2Y);

[0084] - Calpainopathy (LGMD2A / R1) is caused by a mutation in the CAPN3 gene, with more than 450 pathogenic variants described.

[0085] A non-limiting list of such diseases includes: centronuclear myopathy, preferably X-linked myotubular myopathy (XLMTM) and Charcot-Mari e-Tooth disease, limb-girdle muscular dystrophy, preferably LGMD2A / R1, LGMD2B / R2, LGMD2D / R3 or LGMD2I / R9, LGMD1D / D1, LGMD2L / R12, congenital muscular dystrophy type IC (MDC1C), Walker-Warburg syndrome (WWS), muscle-eye-brain disease (MEB), Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD), selenoprotein N deficiency congenital muscular dystrophy, selenoprotein N deficiency congenital muscular dystrophy, primary merosin deficiency congenital muscular dystrophy, Ullrich congenital muscular dystrophy, central core congenital myopathy, Congenital multi-minicore myopathy, autosomal centronuclear myopathy, myopathy with fiber disproportion, nemaline myopathy, congenital myasthenic syndromes, distal Miyoshi myopathy, dysferlinopathies,dystroglycanopathies and sarcoglycanopathies. In certain embodiments, the pharmaceutical composition of the invention is intended to be used for treating Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD), congenital limb-girdle muscular dystrophy, advantageously LGMD2A / R1, LGMD2B / R2, LGMD2D / R3, LGMD2I / R9, LGMD1D / D1 or LGMD2L / R12.,

[0086] In addition, cachexia or marasmus is also a pathological condition covered by the present invention. This condition is characterized by extreme thinness, particularly in skeletal muscles, caused by a long illness or insufficient protein or calorie intake.

[0087] This pathological condition is particularly observed in cases of chronic diseases such as cancer or AIDS or in people suffering from either heart failure - there is atrophy of skeletal muscles in 68% of patients - or urinary incontinence. Without being considered pathological strictly speaking, certain situations are associated with a loss or deterioration of muscle function or strength: aging, prolonged immobilization, etc. There is therefore again an interest in improving muscle survival and thus increasing muscle function or strength.

[0088] Thus, the present invention is based on the demonstration of protective properties of bazedoxifene as a representative of SERMs, in particular of the third generation, namely compounds comprising a 2-phenylindole group, on the membranes of muscle cells in particular.

[0089] According to a particular embodiment, the pathology treated within the framework of the present invention is not rhabdomyosarcoma. According to another particular embodiment, the pathology treated within the framework of the present invention is not a cardiomyopathy, in particular autoimmune myocarditis.

[0090] As already stated, a composition comprising a compound according to the invention, intended for the treatment of pathologies associated with muscular fragility or intended for the improvement of muscular survival, may also contain any acceptable compound or excipient, in particular pharmaceutical.

[0091] This treatment can also be combined with other treatments intended to treat the same pathology or condition.

[0092] To promote the engraftment of precursor cells or stem cells, it may be advantageous to combine the administration of bazedoxifene as a representative of third-generation SERMs, namely compounds comprising a 2-phenylindole group, with the engraftment of cells (myoblasts, stem cells, etc.). This administration can be done simultaneously or at different times.

[0093] It may also be advantageous to combine gene therapy, intended for the treatment of a neuromuscular disease, with the administration of a compound as described above, in particular bazedoxifene as a representative of third-generation SERMs, namely compounds comprising a 2-phenylindole group. Thus and according to a preferred embodiment, a therapeutic gene, such as the native dysferlin gene, is associated with the treatment with a compound as described above, in particular bazedoxifene as a representative of third-generation SERMs, namely compounds comprising a 2-phenylindole group. The administration of the two treatments may be carried out simultaneously or at different times.

[0094] This treatment can also be combined with treatments allowing an increase in muscle mass such as the administration of decorin (W02010 / 106295), lumican or fibromodulin (WO2013 / 072587).

[0095] The beneficial effects of bazedoxifene as representing compounds as described above, in particular third-generation SERMs, namely compounds comprising a 2-phenylindole group, result in an increase in muscle cell survival, particularly under osmotic shock conditions. These positive effects can be observed for different skeletal muscles, both in an organism suffering from a pathology associated with muscle fragility and in a healthy individual. A priori, no side effects or immunological reactions are to be deplored.

[0096] Another possible application of the compounds as described above, in particular bazedoxifene as a representative of third-generation SERMs, namely compounds comprising a 2-phenylindole group, is the in vitro or ex vivo culture of myoblasts, myotubes or muscle fibers (or myofibers), for example as described by Tominaga et al. (i Science; 2021 Dec 20;25(1):103667). These cells or tissues can originate from healthy organisms as well as from organisms affected by the aforementioned pathologies.

[0097] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, known to those skilled in the art. These techniques are explained in detail in the literature, in particular in “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of Animal Cells” (Freshney, 2010); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1997); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Short Protocols in Molecular Biology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles, Applications and Troubleshooting” (Babar, 2011); “Current Protocols in Immunology” (Coligan, 2002).Techniques particularly useful for particular embodiments will be discussed in the following sections. The disclosures of each patent, patent application, and publication cited herein are incorporated by reference in their entirety.

[0098] Without further description, it is believed that one skilled in the art can, using the following description and illustrative examples, make and use the compounds of the present invention and practice the claimed methods.

[0099] EXAMPLES OF ACHIEVEMENT

[0100] The invention and the advantages arising therefrom will become more apparent from the following examples of embodiment, supported by the appended figures. However, these are not intended to be limiting.

[0101] The invention is further illustrated using bazedoxifene, noted Bazedoxifene, or bazedoxifene acetate, noted Bazedoxifene acetate, tested on immortalized myoblasts.

[0102] FIGURE CAPTIONS

[0103] Effect of Bazedoxifene on the quantity and localization of dysferlin

[0104] L1341P

[0105] (A) Quantification of dysferlin-positive cell number (•) and viability (■) after treatment of immortalized L1341P myoblasts with increasing concentrations of Bazedoxifene. Values ​​are normalized by treatment with 0.1% DMSO and each point represents the mean ± standard deviation of four replicates of a representative experiment (N=3).

[0106] (B) Immunofluorescence analysis of dysferlin localization after treatment with 0.1% DMSO or the maximum effective dose (5 pM) of Bazedoxifene. Endoplasmic reticulum, Golgi apparatus, lysosomes, and autophagosomes are also identified with KDEL, GM 130, Lampl, and LC3B. Nuclei are labeled with Hoechst (blue). Scale bar = 10 pM. 2. Measurement of Bazedoxifene protection against osmotic shock

[0107] (A) Visualization and (B) quantification of osmotic shock-induced mortality of immortalized L1341P myoblasts after treatment with increasing concentrations of Bazedoxifene or 0.03% DMSO.

[0108] (C) Determination of the duration (hours) of osmotic shock required to induce 50% cell mortality.

[0109] Each point or bar represents the mean ± standard deviation of four replicates of a representative experiment.

[0110] BZA = Bazedoxifene.

[0111] Mutation-independent protective effect of Bazedoxifene.

[0112] Measurement of the effect of Bazedoxifene and / or Bazedoxifene acetate at 2 pM on cell viability in response to osmotic shock in different immortalized myoblast lines carrying the (A) L1341P (missense 1), (B) G299R (missense 2), (C) S1173X (ko), or (D) no dysferlin mutation. Each point represents the mean ± standard deviation of four replicates of a representative experiment.

[0113] I) MATERIALS AND METHODS

[0114] Cell culture and pharmacological treatment

[0115] The immortalized myoblasts used in this study were derived from patients with LGMDR2.

[0116] The main cell line used (814) originates from a vastus lateralis muscle biopsy of a 57-year-old male patient carrying the following mutation: homozygous c.4022T>C; p.L1341P. The cells were isolated by the team of Dr. Simone Spuler (University of Medicine - Charité, Berlin) and then immortalized by the team of Dr. Vincent Mouly (Institute of Myology, Paris). For osmotic shock experiments, the immortalized myoblast lines 107 and AB320 used carry the following mutations: heterozygous c.855+ldelG, c.895G>A; mRNA decay, p.G299R and heterozygous c.342-lG>A, c.3516_3517delTT; / , p.Serl l73X.

[0117] Cells were cultured in muscle cell proliferation medium: 1 volume of medium 199 (Invitrogen, 41150020) to 4 volumes of DMEM (Invitrogen, 61965-025), supplemented with 20% FCS, 25 pg / ml Fetuin (Life Technologies, 10344026), 5 ng / ml hEGF (Life Technologies, PHG0311), 0.5 ng / ml bFGF (Life Technologies, PHG0026), 0.2 pg / ml Dexamethasone (Sigma, D4902) and 5 pg / ml Insulin (Sigma, 91077). Cells were seeded on plates pre-coated with 0.1% gelatin and maintained under a controlled atmosphere at 37°C and 5% CO2.

[0118] Seventy-two hours after seeding, cells were treated with the compound bazedoxifene HCl or bazedoxifene acetate (Selleckchem) or dimethyl sulfoxide (DMSO, VWR) as a negative control. Cells were analyzed after 24 hours of treatment.

[0119] Quantification of dysferlin expression and viability assay

[0120] After 24 hours of treatment, cells were fixed with 4% paraformaldehyde (10 minutes, room temperature). Immunofluorescence was performed in phosphate-buffered saline (PBS) supplemented with 0.1% Triton for permeabilization (5 minutes, room temperature) followed by 1% bovine serum albumin (BSA; Sigma) for blocking (1 hour, room temperature). For the first hybridization step, cells were incubated (overnight, 4°C) with rabbit anti-Dysferlin (abeam, ab 124684) and goat anti-Desmin (R&D, AF3844) antibodies. The labels were then revealed during the second hybridization step by incubation (1 hour, room temperature) with fluorochrome-conjugated secondary antibodies (Invitrogen, anti-rabbit green and anti-goat red) and the nuclei were labeled with Hoechst (Invitrogen, 33342). Dysferlin expression was analyzed by the Cellinsight CX7 imager (Cellomics Inc).The first channel is used for nuclei identification, the second for cell segmentation and the third for dysferlin aggregate identification and quantification. Images were acquired with a 20x high-resolution objective and analyzed with a colocalization bioapplication. Using thresholding on the dysferlin intensity level, the number of cells positive for dysferlin staining (minimum one dysferlin aggregate in the cell) could be calculated so that it was less than 10% with the negative control. The total cell number was determined by counting the Hoechst-stained cells per well, which allowed quantification of cell viability by normalizing with the negative control. Dysferlin localization.

[0121] After 24 hours of treatment with Bazedoxifene (EC50=5pM) the cells were fixed and labeled (overnight, 4°C) with the following primary antibodies: anti Dysferlin (Novocastra), Kdel (EnzoLife), GM130 (BD Bioscineces), LAMP (Abeam) and LC3 A / B (ThermoScientific). After washing, the labels were then revealed using secondary antibodies coupled to fluorochromes (1 hour, room temperature) and analyzed using a confocal microscope (LSM 880, Carl Zeiss). Images were acquired with a 63x oil objective.

[0122] Osmotic Shock

[0123] Cells were seeded in 96-well plates and treated with different concentrations of Bazedoxifene, as indicated, for 24 hours. Cells were then incubated for 3 hours with a red viability probe (1 / 3000, Incucyte® Nuclight Rapid Red Dye - Sartorius). Once the probe was incorporated by the nuclei of live cells (incuCyte verification), an osmotic shock composed of 25% PBS and 75% water was applied in the presence of a green mortality probe (1 / 3000, Incucyte® Caspase-3 / 7 Green Dye - Sartorius). Red and green staining were acquired every 90 minutes for 72 hours with an IncuCyte® S3 Live-Cell Analysis, thus allowing measurement of cell resistance to osmotic shock (green to red ratio) after pharmacological treatment in comparison to the negative control (DMSO).

[0124] II) RESULTS

[0125] 1 / Identification of Bazedoxifene as an inhibitor of the degradation of the malformed dysferlin protein

[0126] As demonstrated in Figure 1A, the use of increasing doses of Bazedoxifene results in an increase in the number of immortalized L1341P mutant myoblasts expressing at least one dysferlin aggregate, without revealing significant toxicity, except at very high doses. This effect is confirmed by the histological analysis presented in Figure 1B: in the presence of the maximum effective dose of Bazedoxifene (5pM), the amount of dysferlin detected in immortalized L1341P myoblasts is increased compared to the DMSO control treatment. It has been shown that the L1341P mutation leads to the malconformation of dysferlin, its absence at the sarcolemma and its aggregation at the endoplasmic reticulum; thus leading to membrane fragility and ultimately muscle cell death.Analysis of dysferlin L1341P localization, by labeling different cellular compartments in Figure 1B, demonstrates the exit of the protein from the endoplasmic reticulum after Bazedoxifene treatment.

[0127] Based on these experiments, it can be hypothesized that Bazedoxifene prevents the degradation of the malformed protein by the ERAD system, thereby increasing the amount of functional dysferlin in muscle cells and potentially its membrane functionality and cell survival.

[0128] 2 / Bazedoxifene improves cell survival

[0129] To demonstrate the functionality of Bazedoxifene on muscle cell fragility, the survival of treated immortalized L1341P myoblasts was assessed after osmotic shock destabilizing cell membranes. Figures 2A and 2B demonstrate that at appropriate doses of Bazedoxifene, treatment of immortalized L1341P myoblasts results in an increase in cell viability under osmotic shock conditions. In addition, according to Figure 2C, muscle cell survival is significantly improved after treatment with a dose equivalent to FEC50 of Bazedoxifene, which notably results in an increase of more than 40% in the number of dysferlin-positive myoblasts (Figure 1).

[0130] Figure 3 confirms that the protective effect of Bazedoxifene (HCl or acetate) on cell survival under osmotic shock conditions, and therefore on muscle cell viability, results from a more general effect on cells and not only on aggregated dysferlin. Indeed, immortalized myoblasts resist osmotic shock in a similar way after treatment with Bazedoxifene whether they carry the L1341P (missense 1), G299R (missense 2), S1173X (ko) dysferlin mutations. Surprisingly, this same effect is observed in WT cells, i.e. immortalized myoblasts not mutated in the dysferlin gene.

[0131] This application thus reveals that Bazedoxifene is effective for a broad spectrum of conditions associated with muscle cell fragility, particularly related to the integrity of their membranes.

Claims

DEMANDS 1 / Compound comprising a 2-phenylindole group, which is a selective estrogen receptor modulator (SERM), for use in the treatment of a pathology associated with muscle fragility. 2 / Compound for its use according to claim 1, characterized in that the pathology is a neuromuscular disease or cachexia. 3 / Compound for its use according to claim 1 or 2, characterized in that the pathology is a genetic disease. 4 / Compound for its use according to one of the preceding claims, characterized in that the pathology affects skeletal muscles. 5 / Compound for its use according to one of the preceding claims, characterized in that the pathology is muscular dystrophy. 6 / Compound for its use according to claim 5, characterized in that muscular dystrophy is a dysferlinopathy. 7 / Non-therapeutic use of a compound comprising a 2-phenylindole group, which is a selective estrogen receptor modulator (SERM), for the enhancement of muscle survival, in particular resistance to stress. 8 / Use according to claim 7, characterized in that the improvement of muscle survival aims to compensate for deterioration resulting from immobilization or old age. 9 / Compound for use according to any one of claims 1 to 6 or use according to any one of claims 7 to 8, characterized in that the compound is bazedoxifene or bazedoxifene acetate. 10 / Compound for use according to any one of claims 1 to 6 and 9 or use according to any one of claims 7 to 9, characterized in that the compound is formulated in a composition. 11 / Compound for its use according to claim 10 or use according to claim 10, characterized in that the composition is in solid form, advantageously in the form of a tablet. 12 / Compound for its use according to claim 10 or 11 or use according to claim 10 or 11, characterized in that the composition, advantageously the tablet, comprises 20 mg of bazedoxifene or bazedoxifene acetate. 13 / Compound for use according to any one of claims 1 to 6 and 9 to 12 or use according to any one of claims 7 to 12, characterized in that the compound or composition is administered orally. 14 / Compound for use according to any one of claims 1 to 6 and 9 to 13 or use according to any one of claims 7 to 13, characterized in that the compound or composition is associated with other treatments, in particular gene therapy and cell transplantation. 15 / Method for ex vivo or in vitro culture of myoblasts, myotubes or muscle fibers carried out in the presence of a compound comprising a 2-phenylindole group, which is a selective estrogen receptor modulator (SERM), advantageously in the presence of bazedoxifene or bazedoxifene acetate.

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