New therapeutic uses of endothelin receptor antagonists

Abstract

The present invention relates to endothelin receptor antagonists for use in the treatment or prevention of muscle fibrosis.

Description

[Technical field]

[0001] The present invention relates to new therapeutic uses of endothelin receptor antagonists. [Background technology]

[0002] In adult skeletal muscle, the extracellular matrix (ECM) plays a crucial role for contracting myofibers. The ECM is a three-dimensional network consisting of extracellular macromolecules such as collagens and glycoproteins as well as soluble factors such as growth factors and cytokines, providing structural and biochemical support to the surrounding cells. The ECM regulates the bioavailability of biofactors and maintains myofiber structure during regeneration. Muscle regeneration is a complex, time-regulated process orchestrated by various important cellular actors, including mantle cells, myofibers, inflammatory, fibroadipogenic progenitors, and endothelial cells. During this process, the ECM undergoes enormous changes in terms of quantity and quality, and all these changes induce specific responses in the different cell types involved in muscle regeneration (Bentzinger et al., 2012). Fibrosis, characterized by excessive ECM accumulation, is the result of tissue inflammation or injury, which further leads to scarring and impaired function of the affected organ. Fibrosis can occur in many tissues of the body, including skeletal muscle, and is a common pathological feature of muscular dystrophies (Smith and Barton, 2018). Fibrosis is perhaps one of the most detrimental symptoms of muscular dystrophies that has yet to be elucidated in humans. Furthermore, muscle fibrosis can impair the efficiency of gene and cell-based therapies developed for muscular dystrophies. There is still no efficient treatment to reverse this process in skeletal muscle. Such treatment is not only essential to combat the pathological process, but also to improve the efficiency of other therapeutic strategies, such as gene or cell therapy, which are largely hindered by the presence of fibrosis. Conversely, the accumulation of extracellular matrix in muscle tissue also affects elderly subjects without any muscular dystrophies, such as elderly subjects suffering from achalasia, for which there is no similarly efficient drug.

[0003] In mice, fibrosis involves mechanical, humoral, cellular, as well as soluble factors, such as TGFβ, pro- and anti-inflammatory cytokines, and growth factors such as CTGF and PDGF (Serrano et al., 2010), which have been described as one of the key drivers of ECM remodeling (Abrigo et al., 2018). At the cellular level, fibrosis involves inflammatory cells, fibroadipogenic precursors (FAPs), and possibly mantle cells (Pessina et al., 2015). FAPs are the main collagen-producing cells within the stromal tissue microenvironment, and these cells are increasingly recognized for their role as an autocrine source of profibrotic stimuli associated with fibrosis in dystrophic conditions. However, the involvement of FAPs in the development of muscular dystrophies in humans is still not fully clear, highlighting the need to better understand the molecular and cellular actors in human fibrosis, which often exacerbates muscular dystrophies. There remains a need to address many aspects of the cellular crosstalk that contribute to the initiation and maintenance of fibrosis in human skeletal muscle.

[0004] To date, there are no effective drugs to combat muscle fibrosis. Therefore, it is necessary to provide an efficient strategy to treat muscle fibrosis occurring either in patients suffering from myopathy, particularly muscular dystrophies, or in elderly people. It is also desirable to develop efficient drugs for use in the treatment or prevention of muscle fibrosis, which can be further combined with gene therapy or cell therapy to improve the condition of muscle tissue and treat muscular dystrophies. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Suzuki et al., J Pharm Biomed Anal. August 5, 2017; 142:84~90 Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a need to provide new therapies to treat or prevent muscle fibrosis. [Means for solving the problem]

[0007] We observed that human FAPs derived from fibrotic muscles differed from healthy non-fibrotic control muscles, exhibiting significantly higher proliferative capacity, impaired ECM protein production, and the ability to inhibit myoblast fusion. To identify potential candidate proteins expressed by FAPs derived from fibrotic muscles that may affect muscle differentiation, we analyzed the transcriptome profile of 189 genes deregulated in control human cricopharyngeal fibrotic muscle (CPM) FAPs and identified 111 candidate proteins with predicted extracellular properties. After multiple successive screens, we identified endothelin receptor B as a particularly interesting candidate target for inhibiting fibrosis progression. Indeed, we observed upregulation of this receptor in both control fibrotic muscle FAPs and pathological / OPMD (oculopharyngeal muscular dystrophy) fibrotic muscle FAPs. The ligand of endothelin receptor B is endothelin 1 (ET1), a known profibrotic peptide, secreted by myotubes (Le Bihan et al., 2012). ET-1 is a widely distributed, multifunctional hormone that acts in both paracrine and autocrine ways. ET-1 is the most potent and long-lasting vasoconstrictor ever discovered in humans (Yanagisawa et al., 1988). Originally described as being secreted by endothelial cells and having effects on smooth muscle cells, ET-1 has been linked to the development of fibrosis in various organs, including lung, cardiac fibroblasts or hepatic stellate cells (Abraham et al., 1997; Katwa et al., 2003; Rockey et al., 1998). However, the effect of ET-1 on FAP is completely unknown in the prior art. Also, the endothelin receptor B expression profile in FAP has never been described in the prior art. We first showed that ET-1 stimulates ECM production in fibrotic skeletal muscle FAPs but not in nonfibrotic control skeletal muscle FAPs. Furthermore, we showed that addition of an endothelin receptor B antagonist to cells from skeletal fibrotic muscle could reduce ECM production by FAPs and partially restore impaired myotube fusion.Thus, blockade of the endothelin pathway via its receptors provides a new target for the treatment of muscle fibrosis.

[0008] Thus, one aspect of the present invention is to provide an endothelin receptor antagonist for use in the treatment or prevention of muscle fibrosis, particularly human muscle fibrosis.

[0009] Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of at least one endothelin receptor antagonist and a pharma- ceutically acceptable carrier for use in the treatment or prevention of muscle fibrosis.

[0010] Another aspect of the invention relates to a kit of parts for simultaneous, separate or sequential use for the treatment or prevention of muscle fibrosis in patients suffering from myopathy, in particular muscular dystrophies, comprising at least one endothelin receptor antagonist and means for performing gene therapy or cell therapy or drug therapy for use in the treatment of myopathy, in particular muscular dystrophies.

[0011] The present invention also provides an in vitro method for diagnosing myofibrosis in a human subject suspected of having myofibrosis, the method comprising: - determining the circulating level of endothelin in a biological sample obtained from the human subject; and - comparing said circulating endothelin level to a reference level and correlating said circulating endothelin level with the presence or absence of muscle fibrosis in said human subject. The present invention provides a method comprising:

[0012] The present invention also provides a method for assessing the effectiveness of a treatment for muscle fibrosis, the method comprising: - determining circulating endothelin levels in a biological sample obtained from a human subject undergoing treatment for muscle fibrosis; and - comparing said circulating endothelin level to a first level to assess the effectiveness of said treatment for muscle fibrosis. Includes. [Brief description of the drawings]

[0013] [Figure 1] Figure 1A shows quantification of ET-1 protein in non-concentrated conditioned medium secreted from differentiated human myoblasts at 24, 48 and 72 hours of differentiation. Figure 1B shows RT-qPCR quantification of EDNR b gene expression normalized to RPLP0 expression in FAPs from control skeletal muscle (MCT), control fibrotic muscle (FibMCT) and pathological / OPMD fibrotic muscle (FibMOP) (**P<0.01). Figure 1C shows RT-qPCR quantification of human EDNR b gene expression normalized to hB2M expression in mice Ta injected with FAPs from MCT, FibMCT and FibMOP (***P<0.001****P<0.0001). [Diagram 2] Figure 2A shows immunofluorescence images of FAPs derived from MCT, FibMCT and FibMOP cultured with DMSO, ET-1 (40 nM) + / - bosentan (10 μM), phalloidin (red), Hoechst (blue) and collagen 7a1 (green). Figure 2B shows quantification of the percentage of COL7A1 positive cells after treatment with DMSO or ET-1 (40 nM) or ET-1 and bosentan (10 μM). COL7A1 is used as a readout for ECM production. Figure 2C shows evaluation of proliferation (percentage of Edu positive cells) after treatment with DMSO or ET-1 (40 nM) or ET-1 and bosentan (10 μM). [Diagram 3] Figure 3A shows the fusion index of myoblasts alone or cultured with FibMOP-derived FAPs in the presence or absence of bosentan (10 μM) (*P<0.05, **P<0.01, ****P<0.0001). Figure 3B shows desmin (green) and Hoechst (blue) immunostaining of myoblasts alone or cultured with FibMOP-derived FAPs in the presence or absence of bosentan (10 μM). [Figure 4]Figure 4A shows the quantification of the percentage of COL7A1 positive cells after treatment with DMSO or ET-1 (40 nM) or ET-1 and bosentan (10 μM) or ET-1 and BQ788 (10 nM) or ET-1 and BQ123 (10 nM). COL7A1 is used as a readout for ECM production. Figure 4B shows the assessment of proliferation (percentage of Edu positive cells) after treatment with DMSO or ET-1 (40 nM) or ET-1 and bosentan (10 μM) or ET-1 and BQ788 (10 nM). [Diagram 5] Figure 5A shows RT-qPCR quantification of EDNR b gene expression normalized to RPLP0 expression in FAPs from control skeletal muscle (MCT), control fibrotic muscle (FibMCT), pathological / OPMD fibrotic muscle (FibMOP) and pathological / IBM fibrotic muscle (FibMIBM) (**P<0.01, ****P<0.0001). Figure 5B shows RT-qPCR quantification of EDNR a gene expression normalized to RPLP0 expression in FAPs from control skeletal muscle (MCT), control cricopharyngeal fibrotic muscle (FibMCT), pathological / OPMD fibrotic muscle (FibMOP) and pathological / IBM fibrotic muscle (FibMIBM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present inventors have carried out experiments on FAP derived from skeletal muscle to identify key regulators of skeletal muscle fibrosis. The present inventors have surprisingly shown that endothelin receptors are such key regulators. Thus, a first aspect of the present invention relates to an endothelin receptor antagonist for use in the treatment or prevention of muscle fibrosis.

[0015] The term "endothelin receptor" as used herein refers to all endothelin receptors, particularly endothelin receptor type A (ET A ), endothelin receptor type B (ET B), or any endothelin receptor-like G protein-coupled receptor that can bind endothelin, particularly isoforms of human endothelin, more specifically endothelin-1, endothelin-2, or endothelin-3.

[0016] Within the scope of the present invention, "endothelin receptor type A," "endothelin type A receptor," "endothelin receptor A," "ET A The terms "receptor" and "ENDRA" are interchangeable.

[0017] Within the scope of the present invention, "endothelin receptor type B," "endothelin type B receptor," "endothelin receptor B," "ET B The terms "ENDRB receptor" and "ENDRB" are interchangeable.

[0018] The term "endothelin receptor antagonist" as used herein refers to any substance capable of blocking or reducing the binding between an endothelin and its endothelin receptor, thereby abolishing or reducing the in vivo activation of the endothelin receptor.

[0019] In certain embodiments, the endothelin receptor antagonist is an antagonist of a human endothelin receptor.

[0020] In another particular embodiment, the endothelin receptor antagonist is ET A Receptor antagonists, especially human ET A Receptor antagonists, or ET B Receptor antagonists, especially human ET B It is a receptor antagonist.

[0021] In certain embodiments, the endothelin receptor antagonist is a substance capable of blocking or reducing the binding between endothelin-1 and a human endothelin receptor, in particular human endothelin receptor B (ENDRB) or human endothelin receptor A (ENDRA).

[0022] The endothelin receptor antagonist may be a chemical molecule, a protein, a fragment of a protein, a peptide, or an aptamer that antagonizes any isoform of endothelin, particularly an isoform of human endothelin, particularly endothelin-1, for binding to an endothelin receptor.

[0023] According to embodiments of the invention, the endothelin receptor antagonist may be a dual endothelin receptor antagonist, an antagonist that selectively binds to the endothelin type B receptor, or an antagonist that selectively binds to the endothelin type A receptor.

[0024] As used herein, the term "dual endothelin receptor antagonist" refers to an ET A and E.T. B Both receptors, especially human ET A and E.T. B Dual endothelin receptor antagonists refer to antagonists that block the receptor. Examples of such dual endothelin receptor antagonists include bosentan, macitentan, aprocitentan, or tezosentan.

[0025] As used herein, the term "antagonist that selectively binds to the endothelin type A receptor" refers to an antagonist that has greater affinity for the endothelin type A receptor than for the endothelin type B receptor, particularly an antagonist that selectively binds to the endothelin type A receptor, particularly an antagonist that selectively binds to ... B Human ET rather than receptor A This refers to an antagonist that has a greater affinity for the receptor. Examples of such antagonists include BQ123, sitaxsentan, atrasentan, avosentan, ambrisentan, ET A Antibodies to the receptor, ET A Antibody mimetics for receptors and ET A It is an aptamer for a receptor.

[0026] As used herein, the term "antagonist that selectively binds to the endothelin type B receptor" refers to an antagonist that has greater affinity for the endothelin type B receptor than for the endothelin type A receptor, particularly an antagonist that selectively binds to the endothelin type B receptor, and is particularly useful for antagonists that selectively bind to the endothelin type B receptor. A Human ET rather than receptor BThis refers to an antagonist that has a greater affinity for the receptor. Examples of such antagonists include IRL2500, K-8794, RES7011, Ro 46-8443, A192621, BQ788, ET B Antibodies to the receptor, ET B Antibody mimetics for receptors and ET B It is an aptamer for a receptor.

[0027] Antagonists of endothelin receptors for use according to the present invention may be antibodies against the receptor or antibody mimetics that specifically bind to the endothelin receptor. A or E.T. B Receptors, especially human ET A or E.T. B Antibodies for use in the present invention as receptor antagonists include, but are not limited to, monoclonal or polyclonal antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, and nanobodies, Fab, Fab' or F(ab') antibodies. 2 The antibody mimetics for use in the present invention include full length or fragments thereof, including ET, scFV or diabody. A / ET B The antibodies or antibody mimetics for use in the present invention may be peptides designed by any conventional method, in particular with the aid of routinely used bioinformatics software, following the analysis of the binding domain between the receptor and its neutralizing antibody. The antibodies or antibody mimetics for use in the present invention compete with endothelin, in particular ET-1, to bind to the target endothelin receptor.

[0028] The antagonist of the endothelin receptor for use according to the present invention can also be an aptamer against said receptor. The aptamer can be selected by standard methods, for example through the SELEX (enrichment of exogenous endothelin) process from a large oligonucleotide library. The aptamer used in the present invention competes with endothelin, particularly ET-1, to bind to the target endothelin receptor.

[0029] According to a particular embodiment, the endothelin receptor antagonist used in the present invention is a human ETB It is an antagonist that binds to the receptor. Human ET B Exemplary endothelin receptor antagonists include dual endothelin receptor antagonists, human ET B A selective receptor antagonist, human ET B Antibodies or antibody mimetics against the receptor and human ET B Examples include aptamers against receptors.

[0030] According to another specific embodiment, the endothelin receptor antagonist used in the present invention is a human ET A It is an antagonist that binds to the receptor. Human ET A Exemplary endothelin receptor antagonists include dual endothelin receptor antagonists, antagonists that selectively bind to the endothelin type A receptor, and antagonists that selectively bind to the human ET A Antibodies or antibody mimetics against the receptor and human ET A Examples include aptamers against receptors.

[0031] In a preferred embodiment, the endothelin receptor antagonist is selected from bosentan, TAK044, SB209670, A192621, BQ788, IRL2500, atrasentan, K-8794, RES7011, Ro 46-8443, macitentan, aprocitentan, ambrisentan, BQ123, sitaxsentan, antibodies, antibody mimetics or aptamers against the human endothelin type A receptor and antibodies, antibody mimetics or aptamers against the human endothelin type B receptor.

[0032] In a more preferred embodiment, the endothelin receptor antagonist is bosentan, macitentan, aprocitentan, or tezosentan, IRL2500, K-8794, RES7011, Ro 46-8443, A192621, BQ788, ET B Antibodies to the receptor, ET B Antibody mimetics for receptors and ET B The aptamer is selected from an aptamer against a receptor.

[0033] In another preferred embodiment, the endothelin receptor antagonist is selected from BQ123, bosentan or BQ788, more particularly bosentan or BQ788, even more particularly bosentan.

[0034] In the context of the present invention, the above-mentioned endothelin receptor antagonist is used in the treatment or prevention of muscle fibrosis.Indeed, as shown in the examples, the present inventors have demonstrated that skeletal muscle fibrosis can be controlled by endothelin receptor antagonists thanks to the experiments carried out on FAP originating from skeletal muscle.

[0035] Muscle fibrosis is characterized by the excessive accumulation of extracellular matrix components, and occurs most easily in patients with myopathy, especially muscular dystrophy.However, muscle fibrosis can also be observed in patients without myopathy, such as elderly subjects.By the way, muscle trauma can also be the cause of muscle fibrosis.Clinically, muscle fibrosis can be diagnosed by histological analysis of biopsy and / or by non-invasive imaging techniques such as magnetic resonance imaging (MRI).

[0036] According to one embodiment, the present invention relates to an endothelin receptor antagonist as described above, in particular a human ET 1 endothelin receptor antagonist, for use in the treatment or prevention of muscle fibrosis occurring in patients suffering from a myopathy, in particular a muscular dystrophy such as oculopharyngeal muscular dystrophy, Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy, congenital muscular dystrophy or Emery-Dreifuss muscular dystrophy, or other myopathies such as inclusion body myositis. B It concerns antagonists that bind to the receptor.

[0037] According to another embodiment, the present invention relates to an endothelin receptor antagonist as described above, in particular a human ET 10 antagonist, for use in the treatment or prevention of muscle fibrosis occurring in patients without myopathy, such as muscle fibrosis occurring in patients without myopathy but suffering from dysphagia, or in elderly people without myopathy but suffering from achalasia. BIt concerns antagonists that bind to the receptor.

[0038] In certain embodiments, the present invention relates to endothelin receptor antagonists for use in the treatment or prevention of pharyngeal muscular fibrosis, such as that occurring in patients suffering from oculopharyngeal muscular dystrophy or inclusion body myositis, or in elderly individuals suffering from achalasia.

[0039] The term "treatment" or "treating" includes administering an effective amount of an endothelin receptor antagonist to a subject to inhibit, reduce, or reverse the progression of a disease, condition, or disorder, or ameliorate the clinical symptoms of the disease. Treating further refers to achieving one or more of the following: (a) reducing the severity of the disorder, (b) limiting the occurrence of symptoms characteristic of the disorder being treated, (c) limiting the worsening of symptoms characteristic of the disorder being treated, (d) limiting the recurrence of the disorder in patients who previously had the disorder, and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic for the disorder.

[0040] A treatment is considered effective if, after a defined period of time, a statistically significant difference is observed for any one of the above four criteria between the mean value calculated from treated and untreated patients, or between the state of said patients before and after treatment. The presence or absence of said difference can be determined by any medical analytical method routinely used by physicians to assess the progression of a disease, in particular by any conventional method for assessing the progression of muscle fibrosis, for example by histological and biochemical assays, or by NMR and ultrasound as described by Martin-Bach et al., 2021.

[0041] The terms "prevention" or "preventing" include administering to a subject an effective amount of an endothelin receptor antagonist to prevent the appearance of clinical symptoms characteristic of the disease.

[0042] Prevention is considered effective if, after a defined period of time, the onset or appearance of lesions characteristic of the disease is delayed in treated patients compared to the mean values ​​obtained from untreated patients.

[0043] In another particular embodiment, circulating endothelin levels measured in a biological sample, in particular a blood sample, can also be used to determine the effectiveness of an antagonist of an endothelin receptor for use according to the present invention. According to this embodiment, the circulating endothelin levels measured in a subject undergoing treatment with the antagonist are compared to those measured in the subject before treatment. A decrease in the circulating endothelin levels in the treated subject is indicative of the effectiveness of the antagonist for treating muscle fibrosis, and an equal or increased circulating endothelin levels in the treated subject is indicative of the ineffectiveness of the antagonist for treating muscle fibrosis.

[0044] In yet another specific embodiment, the efficacy of an endothelin receptor antagonist for use according to the present invention may also be determined by comparing circulating endothelin levels measured in a subject receiving treatment with the antagonist with reference levels established from healthy subjects.

[0045] Circulating endothelin levels, particularly circulating endothelin-1 levels, in biological samples can be measured by any conventional method, such as by immunoassay or UPLC-MS / MS (Suzuki et al., J Pharm Biomed Anal. 2017 Aug. 5; 142:84-90).

[0046] The present invention also provides pharmaceutical compositions comprising the aforementioned endothelin receptor antagonists for use in the treatment or prevention of muscle fibrosis, the compositions comprising a therapeutically effective amount of at least one endothelin receptor antagonist and a pharma- ceutical acceptable carrier.

[0047] The term "pharmaceutical acceptable carrier" generally means a safe, non-toxic, and not biologically or otherwise undesirable carrier useful in the preparation of a pharmaceutical composition or formulation, including carriers acceptable for pharmaceutical use in humans. A carrier may act as a vehicle, a medium, or for diluting an active ingredient. The formulation of the pharmaceutical composition of the present invention may be determined and carried out according to well-known conventional techniques for drug formulation. The carrier material may be an organic or inorganic inert carrier material, such as one suitable for oral administration or injection. Suitable carriers include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, animal or vegetable oils, polyalkylene glycols, glycerin, and yellow petrolatum. The composition may also contain additional additives such as flavorings, preservatives, stabilizers, wetting agents, emulsifiers, and / or salts and / or buffers for changing osmotic pressure.

[0048] The compositions may be formulated in any conventional form, including tablets, pills, powders, lozenges, sachets, wafer packets, elixirs, suspensions, emulsions, solutions, syrups, solid forms for oral administration such as soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0049] The compositions of the invention may also be administered to a patient in accordance with the invention by topical (including transdermal, buccal or sublingual) or parenteral (including intraperitoneal, subcutaneous, intravascular (i.e., intravenous or intraarterial), intradermal or intramuscular injection) routes. For example, the compositions of the invention are preferably administered for the treatment of oculopharyngeal muscular dystrophy (OPMD) by oral administration.

[0050] The term "therapeutically effective amount" refers to an amount of said endothelin receptor antagonist in a pharmaceutical composition that is pharmacologically active and produces the desired therapeutic effect.

[0051] For example, the "therapeutically effective amount" of the aforementioned endothelin receptor antagonist for use according to the present invention includes, but is not limited to, 50 to 1000 mg / day, 50 to 900 mg / day, 50 to 800 mg / day, 50 to 700 mg / day, 50 to 600 mg / day, 50 to 500 mg / day, specifically 50 to 400 mg / day, 50 to 300 mg / day, 60 to 300 mg / day, 70 to 300 mg / day, 80 to 300 mg / day, 60 to 250 mg / day, and more specifically 80 to 250 mg / day.

[0052] Specifically, a "therapeutically effective amount" of bosentan for use according to the present invention includes, but is not limited to, an amount of 50-500 mg / day, specifically 50-400 mg / day, 50-300 mg / day, 60-250 mg / day, more specifically 80-250 mg / day.

[0053] The "therapeutically effective amount" of the aforementioned endothelin receptor antagonist for use according to the present invention can be determined by standard techniques, for example by in vivo and / or in vitro assays. The skilled artisan can also determine the optimal dosage range or adapt the dosage according to the route and frequency of administration, the age, weight, sex, health condition, severity of the disease of the patient, and / or the therapeutic purpose of the treatment or prevention.

[0054] The present invention also provides a method for treating or preventing muscle fibrosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an endothelin receptor antagonist as described above.

[0055] In another aspect of the present invention, the above-mentioned endothelin receptor antagonist can be used in combination with gene therapy or cell therapy to treat myopathy, particularly muscular dystrophy.In fact, muscle fibrosis can be an obstacle to the implementation and efficiency of such gene therapy or cell therapy.As shown by the present invention, endothelin receptor antagonist makes it possible to treat or prevent muscle fibrosis, which in turn allows gene therapy or cell therapy to reach target tissue or cell more efficiently, thus providing maximum efficiency of treatment.

[0056] Thus, the present invention also provides a kit of parts for simultaneous, separate or sequential use in the treatment or prevention of muscle fibrosis in patients suffering from myopathy, in particular muscular dystrophy. - at least one endothelin receptor antagonist, and - Means for carrying out gene therapy or cell therapy or drug therapy for use in the treatment of myopathies, in particular muscular dystrophies It comprises or consists of:

[0057] The gene therapy can be, for example, adeno-associated virus-based gene therapy for treating oculopharyngeal muscular dystrophy (Malerba et al., 2017).

[0058] Such cell therapy may, for example, be by way of autologous myoblast transplantation (Perie et al., 2014).

[0059] The medication can be, for example, an anti-aggregatory drug, specifically guanabenz for treating oculopharyngeal muscular dystrophy (Malerba et al., 2019).

[0060] The means for carrying out such gene therapy or cell therapy can be, for example, a viral vector, in particular an AAV vector, for carrying out gene therapy, or a cell suitable for carrying out cell therapy. The vector can harbor an antisense oligonucleotide, an shRNA, or an oligonucleotide encoding a functional human protein. The cell suitable for cell therapy can be, for example, an autologous or heterologous tissue cell, a pluripotent stem cell or a multipotent stem cell derived from the patient himself or a donor.

[0061] Treatment or prevention of muscle fibrosis with the above-mentioned endothelin receptor antagonists can be performed before, simultaneously with, or after gene therapy or cell therapy.

[0062] The endothelin receptor antagonists contained in the kit of parts of the present invention are any of the above-mentioned antagonists, particularly ET B Receptor antagonist or ET A Receptor antagonists, or ET B Receptor antagonists and ET A The combination may be with an antagonist of the receptor. The endothelin receptor antagonist may in particular be a dual endothelin receptor antagonist, an antagonist selectively binding to the endothelin type B receptor, or an antagonist selectively binding to the endothelin type A receptor, in particular an endothelin receptor antagonist selected from bosentan, TAK044, SB209670, A192621, BQ788, IRL2500, atrasentan, K-8794, RES7011, Ro 46-8443, macitentan, aprocitentan, ambrisentan, BQ123, sitaxsentan, an antibody, antibody mimetic or aptamer against the human endothelin type A receptor and an antibody, antibody mimetic or aptamer against the human endothelin type B receptor. In a more specific embodiment, the endothelin receptor antagonist is bosentan, BQ123 or BQ788.

[0063] Another aspect of the present invention is to provide an in vitro method for diagnosing muscle fibrosis in a human subject suspected of suffering from muscle fibrosis.

[0064] The method comprises: - determining the circulating level of endothelin in a biological sample obtained from the human subject; and - comparing said circulating endothelin level to a reference level and correlating said circulating endothelin level with the presence or absence of muscle fibrosis in said human subject. Includes.

[0065] The term "human subject suspected of having muscle fibrosis" refers to a human subject suffering from one of the diseases commonly known to cause muscle fibrosis, such as muscular dystrophy, or a subject who has experienced a traumatic injury to a muscle that may also cause muscle fibrosis. A human subject suspected of having muscle fibrosis is a person who is seriously suspected of having muscle fibrosis compared to any other medical condition that may also lead to changes in circulating endothelin levels.

[0066] The term "biological sample" refers to any biological sample that can be obtained from a human subject, in particular a sample of a body fluid such as urine or blood. In a preferred embodiment of the invention, the biological sample is a blood, plasma or serum sample.

[0067] The term "circulating endothelin" refers to a secreted isoform of endothelin. Specifically, the circulating endothelin is endothelin-1, endothelin-2, or endothelin-3. The circulating endothelin may be present in a biological sample, particularly in a body fluid, more particularly in blood, plasma, or serum. In a particular embodiment, the circulating endothelin is circulating endothelin-1 (ET1).

[0068] According to the diagnostic method of the present invention, circulating endothelin levels, in particular circulating endothelin-1 levels, in a biological sample may be measured by any conventional method, such as by immunoassay or UPLC-MS / MS (Suzuki et al., J Pharm Biomed Anal. 2017 Aug. 5; 142:84-90).

[0069] According to a particular embodiment, the reference level used in the diagnostic method of the invention is the average value of circulating endothelin levels calculated from a group of healthy human subjects. Further criteria such as age, sex, other health or medical conditions, or genetic background may also be taken into account for constructing said group.

[0070] In the context of the diagnostic method of the present invention, an increase in circulating endothelin levels measured in a human subject suspected of suffering from said muscle fibrosis compared to a reference level indicates the potential presence of muscle fibrosis.

[0071] In some embodiments, if the circulating endothelin level in a biological sample from a human subject suspected of suffering from muscle fibrosis is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% higher than the reference level, there is considered to be an increased circulating endothelin level in the human subject, and the human subject is considered to be likely suffering from muscle fibrosis.

[0072] According to one embodiment, the present invention also provides a method for monitoring the progression of muscle fibrosis in a human subject suspected of having muscle fibrosis, the method comprising: - determining the circulating level of endothelin in a biological sample obtained from the human subject; and - comparing said circulating endothelin levels with previous levels measured in biological samples obtained from the same human subject. Includes.

[0073] In the context of this method, "previous level" refers to the level measured in a biological sample obtained from the same human subject prior to a determined period of time. According to this method, an increase in the circulating endothelin level in the human subject compared to the previous level is indicative of progression of muscle fibrosis, and a decrease in the circulating endothelin level in the human subject compared to the previous level is indicative of a decrease in muscle fibrosis.

[0074] The present invention also provides a method for assessing the effectiveness of a treatment for muscle fibrosis, the method comprising: - determining circulating endothelin levels in a biological sample obtained from a human subject undergoing treatment for muscle fibrosis; and - comparing said circulating endothelin level to a first level to assess the effectiveness of said treatment for muscle fibrosis. Includes.

[0075] In the context of this method, "initial level" refers to the level measured in a biological sample obtained from the same human subject before treatment for muscle fibrosis. Thus, a decrease in the circulating endothelin level in the human subject compared to the initial level is indicative of the effectiveness of the treatment, and an increase or equal level of circulating endothelin in the human subject compared to the initial level is indicative of an ineffective or less effective treatment.

[0076] The present invention will now be described in more detail by the following examples. EXAMPLES

[0077] 1. Materials and Methods cell culture Human FAP cells were isolated from muscle biopsies obtained during surgical procedures, after informed consent in accordance with European recommendations and French legislation (authorisation AC-2019-3502), via Myobank, an affiliate of EuroBioBank. Human FAP, here isolated as a CD56 negative cell fraction (Perie, S. et al. 2006), were compared with control skeletal muscle: M CT , Control fibrotic cricopharyngeal muscle (CPM): FibM CT , fibrosis levels worsened OPMD fibrosis CPM: FibM OP , and fibrotic muscle from patients with inclusion body myositis: FibM IBMAll fibrotic muscles tested were skeletal fibrotic muscles. These cells primarily expressed PDGFRa, CD90, and CD105, known markers of fibro / adipogenic precursors (FAPs) in human skeletal muscle. The tissue type, cell type, age, pathology, and muscle fibrosis status of each biopsy sample are identified in the table below.

[0078] [Table 1]

[0079] The fibrosis status of the muscle of each biopsy sample is determined by histological analysis according to conventional methods.

[0080] Muscle biopsies were minced and explants were plated onto uncoated Petri dishes in a drop of fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) as previously described (Bigot et al., 2009). Cells were incubated at 37°C in 5% CO. 2The cells were cultured in a medium consisting of 1:4 ratio of 199 medium (Life technologies, Paisley, UK) and Dulbecco's modified Eagle's medium (DMEM, Life technologies) supplemented with 20% fetal bovine serum (FBS, Invitrogen), 25 μg / ml fetuin (Life technologies), 0.5 ng / ml bFGF (Life technologies), 5 ng / ml EGF (Life technologies), 5 μg / ml insulin (Sigma-Aldrich) and 50 μg / ml gentamicin (Life technologies) in a humid atmosphere containing 0.1% CO. Cells were labeled with CD56 antibody coupled to microbeads (130-050-401, MACS, Miltenyi Biotec, Paris, France) and then separated using an immunomagnetic cell sorting system (MACS) according to the manufacturer's instructions. The myogenic purity of both cell fractions (CD56+, myogenic cells and CD56−, non-myogenic cells) was monitored by immunocytochemistry using an antibody against desmin (clone D33, Dako, Trappes, France), which is exclusively expressed in myogenic cells.

[0081] animal Two- to three-month-old Rag2- / -Il2rb- / - immunodeficient mice were used as recipients for human cell transplantation. Mice were anesthetized by intraperitoneal injection of 80 mg / kg ketamine hydrochloride and 10 mg / kg xylazine (Sigma-Aldrich, St. Louis, MO). The study was performed in strict compliance with French legislation and in accordance with the ethical guidelines for animal experimentation of the European Union. The protocol was approved by the Committee on the Ethics of Animal Experiments Charles Darwin N°5 (protocol number 02704.01). All surgical procedures were performed under ketamine hydrochloride and xylazine anesthesia, and every effort was made to minimize suffering.

[0082] cell transplantation Cultures of human cells were washed with PBS, trypsinized, centrifuged, and resuspended in PBS. Cells were injected into both tibialis anterior (TA) muscles. Prior to injection, Ta of immunodeficient mice were subjected to three freeze-thaw cycles, each for 10 seconds, to damage muscle fibers and induce regeneration. Cultures of human cells were transplanted into recipient muscles immediately after freeze injury using a 25 μl Hamilton syringe as previously described (Negroni et al., 2009). For CD56- cells, 1.4x10 cells were transplanted in PBS immediately after freeze injury, then 4 and 8 days later. 5 15 μl of cell suspension containing 15 cells was injected. One month after the first injection, the mice were sacrificed and the Ta was collected and stored at −80° C. for analysis.

[0083] Co-culture experiments Non-myogenic (CD56-) and myogenic (CD56+) cells were seeded together at a final confluence of 21000 cells / cm2 in a 30% / 70% ratio. Once the cells had attached, the medium was replaced with differentiation medium composed of DMEM with 50 μg / mL gentamicin. To study the effect of bosentan on the fusion index in co-cultures, 10 μM bosentan (SML1265, Sigma Aldrich) was added to the wells on days 0 and 3. Cells were fixed on day 5 in 4% paraformaldehyde (PFA). The fusion index was calculated as the ratio between the number of nuclei per myotube identified by desmin staining (>2 nuclei) and the total number of desmin+ nuclei.

[0084] ET-1 experiment The method for quantification of ET-1 protein is described in Le Bihan et al., 2012. Treatment of FAPs to assess the effect of ET-1 was performed on 70-80% confluent cells rinsed twice with DMEM and treated for 3 days with growth medium of 1% FBS (instead of 20%) containing either DMSO or 40 nM ET-1 (E7764, Sigma-Aldrich) + / - 10 μM bosentan (SML1265, Sigma-Aldrich), 10 nM BQ788 (SML192621, Sigma-Aldrich), or 10 nM BQ123 (B150, Sigma-Aldrich). For proliferation experiments, Edu (10 μM) was added on day 2 and cells were fixed 24 h later. EdU labeling was performed using the Click-iT™ EdU Cell Proliferation Kit (C10338, Life Technologies) according to the manufacturer's instructions.

[0085] Immunofluorescence Immunostaining was performed on cells fixed with 4% PFA for 10 minutes (min) and incubated in blocking solution (PBS 2% FBS 0.2% Triton) for 30 minutes (min). Fixed cells were then incubated with primary antibodies (COL7A1 C6805 Sigma Aldrich 1 / 800, Desmin M0760 Dako 1 / 50) for 1 hour. Detection of immune complexes was performed using appropriate Alexa-Fluor secondary antibodies purchased from Life Technologies (Grand Island, NY) for 45 minutes. Nuclei and actin filaments were counterstained with Hoechst and phalloidin-Alexa 568 (Interchim 1 / 400), respectively.

[0086] RNA extraction and reverse transcription RNA was extracted from frozen muscle sections or cell pellets using TRIzol Reagent (Invitrogen, 15596026) according to the manufacturer's instructions. RNA concentration was determined using a NanoDrop® Spectrophotometer ND-1000. RNA was reverse transcribed using M-MLV (Invitrogen) according to the manufacturer's instructions.

[0087] quantitative PCR Quantitative polymerase chain reaction (qPCR) was performed using SYBR green mixed buffer (Roche Applied Science, Meylan, France) on a LightCycler 480 real-time PCR system (Roche Applied Science) as follows: 8 min at 95°C, followed by 50 cycles of 95°C for 15 s (s), 60°C for 15 s and 72°C for 15 s, with a program end of 95°C for 5 s and 65°C for 1 min. The specificity of the PCR products was checked by melting curve analysis using the following program: 65°C to 97°C ramp at 0.11°C / s. Gene expression levels were normalized to RPLP0 or hB2M expression and primer sequences are available upon request.

[0088] statistical analysis Data were expressed as mean ± SD. All statistical analyses were performed using GraphPad Prism (version 6.0d, GraphPad Software Inc., San Diego, CA). Statistical significance was assessed by one-way ANOVA test. Differences were considered significant when *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

[0089] Experimental Results Endothelin receptors as key regulators of muscle fibrosis To identify potential candidate proteins expressed by FAPs derived from fibrotic muscle that may affect muscle differentiation, we analyzed their transcriptome profiles. CT The 189 deregulated genes in FAP were subjected to rigorous computational filtering to predict their cellular distribution and identify proteins most likely to be in the extracellular space (Zhao et al., 2019). We identified 111 candidate proteins with predicted extracellular properties (signal peptide, transmembrane and specialized secretory pathways).

[0090] Of these 111, 66 were FibM CT It was upregulated in FAP. FibM OP The same analysis performed on FAP also showed that FibM OP It was possible to identify 63 candidate proteins that are upregulated in FAP. Combining these two lists yielded 19 candidate extracellular proteins.

[0091] Among the various cellular receptors that were upregulated, ENDRB was identified as a particularly interesting candidate because its ligand, the profibrotic peptide endothelin (ET1), is secreted by myotubes (Figure 1A). - / - Il2rb - / - When injected into immunodeficient mice, FibM CT and FibM OP Overexpression of EDNRB in FAP was confirmed by qPCR in vitro (Fig. 1B) and in vivo (Fig. 1C). The expression level of EDNRB mRNA was also significantly increased by FibM. IBM Similarly, M CT Compared with levels in IBM Overexpression of EDNRB mRNA can be observed in FAP (Figure 5A). CT , FibM CT , FibM OP and FibM IBM The expression level of EDNRA mRNA in FAP of the patients was further measured. A significant increase in EDNRA mRNA expression was also observed in M CT Compared with the expression level in OP and FibM IBM This was also observed in FAPs (Figure 5B). Using COL7A1 immunostaining as a readout for ECM production and Edu to assess proliferation, the addition of ET1 to the medium of FAPs increased FibM CT and FibM OP ECM production and proliferation were increased in FAP, whereas M CTIt was confirmed that the effect of ET1 was not increased in FAP (Figures 2A, 2B, and 2C). The addition of the ENDR antagonist bosentan (Clozel et al., 1994) partially abolished the effect of ET1 (Figures 2A, 2B, and 2C). Two other ENDR antagonists, BQ788 and BQ123, each selectively inhibited EDNR B Antagonists and EDNR A The addition of BQ788 and BQ123 also inhibited FibM CT and FibM OP This resulted in a decrease in ECM production in FAP (Figure 4A). In addition, FibM after treatment with BQ788 and bosentan CT and FibM OP A decrease in the proliferation of FAP can also be observed (FIG. 4B). These results illustrate that blockade of endothelin receptors can decrease the secretion and proliferation of FAP.

[0092] Co-culture of myoblasts with FAPs derived from fibrotic muscle at a 70% / 30% ratio impairs myoblast fusion. Blocking the endothelin receptor EDNR in the presence of bosentan partially restored the fusion index to control levels (Figure 3A,B). Overall, these data indicate an important role for endothelin in fibrosis by acting on FAPs in fibrotic tissues.

[0093] In conclusion, the data presented above demonstrate a key role for endothelin receptors in FAPs in human skeletal muscle fibrosis and identify them as potential drug targets to combat human muscle fibrosis.

[0094] [References] TIFF2024541034000002.tif207170TIFF2024541034000003.tif102170

Claims

1. An endothelin receptor antagonist for use in the treatment or prevention of muscle fibrosis.

2. 2. An endothelin receptor antagonist for use according to claim 1, wherein the antagonist is an antagonist of the endothelin type B receptor or an antagonist of the endothelin type A receptor, in particular an antagonist of the endothelin type B receptor.

3. An endothelin receptor antagonist for use according to claim 1 or 2, wherein the antagonist is a dual endothelin receptor antagonist, an antagonist that selectively binds to endothelin type B receptors, or an antagonist that selectively binds to endothelin type A receptors.

4. 2. The endothelin receptor antagonist for use according to claim 1, wherein the endothelin receptor antagonist is selected from bosentan, TAK044, SB209670, A192621, BQ788, IRL2500, atrasentan, K-8794, RES7011, Ro 46-8443, macitentan, aprocitentan, ambrisentan, BQ123, sitaxsentan, antibodies, antibody mimetics or aptamers against human endothelin type A receptors and antibodies, antibody mimetics or aptamers against human endothelin type B receptors.

5. 5. The endothelin receptor antagonist for use according to claim 4, wherein the endothelin receptor antagonist is selected from BQ123, bosentan or BQ788.

6. 10. An endothelin receptor antagonist for use according to claim 1 in the treatment of muscle fibrosis occurring in patients suffering from myopathy, in particular muscular dystrophies such as oculopharyngeal muscular dystrophy, Duchenne muscular dystrophy, inclusion body myositis, facioscapulohumeral muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, or in the treatment of muscle fibrosis not associated with myopathy, such as muscle fibrosis occurring in patients without muscular dystrophy but suffering from dysphagia or in elderly people without muscular dystrophy but suffering from achalasia.

7. An endothelin receptor antagonist for use as claimed in claim 1 for the treatment or prevention of muscle fibrosis occurring in patients suffering from pharyngeal muscle fibrosis, in particular muscle fibrosis occurring in patients suffering from oculopharyngeal muscular dystrophy or inclusion body myositis or in elderly people suffering from achalasia.

8. For simultaneous, separate or sequential use in the treatment or prevention of muscle fibrosis in patients suffering from myopathy, in particular muscular dystrophy; at least one endothelin receptor antagonist, and - Means for carrying out gene therapy or cell therapy or drug therapy for use in the treatment of myopathies, in particular muscular dystrophies A kit of parts comprising or consisting of:

9. 9. The kit for use according to claim 8, wherein the endothelin receptor antagonist is an antagonist of the endothelin type B receptor or an antagonist of the endothelin type A receptor, in particular a dual endothelin receptor antagonist, an antagonist that selectively binds to the endothelin type B receptor, or an antagonist that selectively binds to the endothelin type A receptor.

10. A pharmaceutical composition comprising a therapeutically effective amount of at least one endothelin receptor antagonist and a pharmaceutically acceptable carrier for use in the treatment or prevention of muscle fibrosis.

11. 11. The pharmaceutical composition for use according to claim 10, wherein the endothelin receptor antagonist is administered in an amount of 50-500 mg / day, particularly 50-400 mg / day, 50-300 mg / day, 60-250, more particularly 80-250 mg / day.

12. 1. An in vitro method for detecting muscle fibrosis in a human subject suspected of having muscle fibrosis, said method comprising: - determining the circulating level of endothelin in a biological sample obtained from said human subject; and - comparing said circulating endothelin level to a reference level and correlating said circulating endothelin level with the presence or absence of muscle fibrosis in said human subject. A method comprising:

13. 1. An in vitro method for assessing the effectiveness of an antifibrotic treatment, said method comprising: - determining circulating endothelin levels in a biological sample obtained from a human subject undergoing treatment for muscle fibrosis; and - comparing the circulating endothelin level to an initial level to assess the effectiveness of treating the muscle fibrosis. A method comprising:

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