Patent Description:
Muscular dystrophies span approximately thirty inherited disorders characterized by weakness and wasting away of muscle tissue, with or without the breakdown of nerve tissue. There are nine main types of muscular dystrophy, each of which involve an eventual loss of strength, increasing disability, and possible physical deformity. Duchenne muscular dystrophy (DMD), is the most well-known type of muscular dystrophy, affecting approximately <NUM> in every <NUM>,<NUM> male births worldwide. DMD is caused by loss of sarcolemma adhesion to the extracellular matrix.

The development of therapies for DMD is gaining momentum with the recent accelerated approval of eteplirsen in <NUM> and the increased private sector funding of rare disease programs. However, the existing FDA approved drugs for DMD are not sufficient to substantially slow disease progression. While corticosteroids dampen inflammation and extend ambulation by several years, they do not address adhesion complex and membrane stability deficiencies. The antisense oligonucleotide exon skipping therapy eteplirsen increases truncated dystrophin protein production, but is only applicable to the approximately <NUM>% of DMD patients with mutations amenable to exon <NUM> skipping. There remains a need to identify more robust treatments for muscular dystrophy and other muscle wasting diseases.

Any embodiment not falling under the scope of the appended claims does not form part of the invention. Any references in the present description to methods of treatment or prevention refer to the compounds, pharmaceutical compositions, and medicaments of the present invention for use in a method for treatment or prevention of the human or animal body by therapy.

The invention relates to a compound that increases the expression of sarcospan for use in the treatment or prevention of a disease related to dysfunction of a dystrophin related complex, wherein said disease is muscular dystrophy, wherein the compound is selected from: <NUM>-[<NUM>-(<NUM>,<NUM>-Dihydro-<NUM>,<NUM>,<NUM>-trimethyl-<NUM>-indol-<NUM>-ylidene)-<NUM>-propenyl]-<NUM>-ethyl-benzothiazolium iodide (AC-<NUM>), N-[(<NUM>-butylpyrrolidin-<NUM>-yl)methyl]-<NUM>-cyano-<NUM>-methoxynaphthalene-<NUM>-carboxamide (nafadotride), <NUM>-amino-<NUM>-(<NUM>-hydroxyethoxymethyl)-<NUM>-purin-<NUM>-one (acyclovir), <NUM>-O-ethyl <NUM>-O-methyl <NUM>-(<NUM>,<NUM>-dichlorophenyl)-<NUM>,<NUM>-dimethyl-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (felodipine), <NUM>-benzo[g]pteridine-<NUM>,<NUM>-dione (alloxazine), <NUM>-O-methyl <NUM>-O-propan-<NUM>-yl <NUM>-(<NUM>,<NUM>,<NUM>-benzoxadiazol-<NUM>-yl)-<NUM>,<NUM>-dimethyl-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (isradipine), methyl N-[(E)-(<NUM>-hydroxy-<NUM>-oxidoquinoxalin-<NUM>-ium-<NUM>-ylidene)methyl]iminocarbamate (carbadox), diethyl <NUM>,<NUM>-dimethyl-<NUM>-[<NUM>-[(E)-<NUM>-[(<NUM>-methylpropan-<NUM>-yl)oxy]-<NUM>-oxoprop-<NUM>-enyl]phenyl]-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (lacidipine), <NUM>-[<NUM>-hydroxy-<NUM>-(propan-<NUM>-ylamino)ethyl]benzene-<NUM>,<NUM>-diol (isoproterenol), <NUM>-O-methyl <NUM>-O-propan-<NUM>-yl <NUM>-cyano-<NUM>-methyl-<NUM>-(<NUM>-nitrophenyl)-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (nilvadipine), (2R,3R,<NUM>,<NUM>)-<NUM>-(<NUM>-aminopurin-<NUM>-yl)-<NUM>-(methylsulfanylmethyl)oxolane-<NUM>,<NUM>-diol (methylthioadenosine), or a pharmaceutically acceptable salt or ester thereof.

In a preferred embodiment, the muscular dystrophy is Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD), Duchenne muscular dystrophy (DMD), distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, or oculopharyngeal muscular dystrophy.

In a further preferred embodiment, the muscular dystrophy is Duchenne muscular dystrophy (DMD).

In other or further preferred embodiments, sarcospan mRNA transcript is increased.

In alternative or even more preferred embodiments, sarcospan protein level is increased.

Also described in the present disclosure, yet not encompassed by the claimed invention, are methods for treating or preventing diseases related to dysfunction of a dystrophin-related complex in a subject in need thereof, comprising administering to the subject a compound that increases the expression of sarcospan, e.g., thereby ameliorating one or more symptoms of the disease.

Sarcospan is a transmembrane protein found in skeletal, smooth, and cardiac muscle, that is associated with several adhesion complexes including integrin, the dystrophin-glycoprotein complex, and the utrophin-glycoprotein complex. Overexpression of sarcospan has been shown to ameliorate disease symptoms and improve skeletal and cardiac muscle, as well as respiratory dysfunction in several relevant mouse models of DMD.

In some aspects, methods of treating or preventing a disease related to dysfunction of a dystrophin-related complex in a subject in need thereof include administering to the subject a compound that increases the expression of sarcospan. The disease related to dysfunction of a dystrophin-related complex is muscular dystrophy (e.g., Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD), Duchenne muscular dystrophy (DMD), distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular dystrophy). The compound, in some embodiments, is <NUM>-[<NUM>-(<NUM>,<NUM>-Dihydro-<NUM>,<NUM>,<NUM>-trimethyl-<NUM>-indol-<NUM>-ylidene)-<NUM>-propenyl]-<NUM>-ethylbenzothiazolium iodide (AC-<NUM>); <NUM>-Azabicyclo[<NUM>. <NUM>]oct-<NUM>-yl acetate (aceclidine); <NUM>-amino-<NUM>-(<NUM>-hydroxyethoxymethyl)-<NUM>-purin-<NUM>-one (acyclovir); <NUM>-benzo[g]pteridine-<NUM>,<NUM>-dione (alloxazine); methyl N-[(E)-(<NUM>-hydroxy-<NUM>-oxidoquinoxalin-<NUM>-ium-<NUM>-ylidene)methyl]iminocarbamate (carbadox); ); <NUM>-[<NUM>-hydroxy-<NUM>-(propan-<NUM>-ylamino)ethyl]benzene-<NUM>,<NUM>-diol (isoproterenol); (2R,3R,<NUM>,<NUM>)-<NUM>-(<NUM>-aminopurin-<NUM>-yl)-<NUM>-(methylsulfanylmethyl)oxolane-<NUM>,<NUM>-diol (methylthioadenosine); N-[(<NUM>-butylpyrrolidin-<NUM>-yl)methyl]-<NUM>-cyano-<NUM>-methoxynaphthalene-<NUM>-carboxamide (nafadotride); <NUM>-O-ethyl <NUM>-O-methyl <NUM>-(<NUM>,<NUM>-dichlorophenyl)-<NUM>,<NUM>-dimethyl-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (felodipine); <NUM>-O-methyl <NUM>-O-propan-<NUM>-yl <NUM>-(<NUM>,<NUM>,<NUM>-benzoxadiazol-<NUM>-yl)-<NUM>,<NUM>-dimethyl-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (isradipine); diethyl <NUM>,<NUM>-dimethyl-<NUM>-[<NUM>-[(E)-<NUM>-[(<NUM>-methylpropan-<NUM>-yl)oxy]-<NUM>-oxoprop-<NUM>-enyl]phenyl]-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (lacidipine); <NUM>-O-methyl <NUM>-O-propan-<NUM>-yl <NUM>-cyano-<NUM>-methyl-<NUM>-(<NUM>-nitrophenyl)-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (nilvadipine); or a pharmaceutically acceptable salt or ester thereof. In some embodiments, the sarcospan mRNA transcript, sarcospan protein level, or both the sarcospan mRNA transcript and sarcospan protein level are increased. The subject can be a human.

A compound that increases the expression of sarcospan (or a composition including such a compound) for use in treatment or prevention of a disease related to dysfunction of a dystrophin-related complex in a subject in need thereof is disclosed. Also disclosed herein, yet not covered by the claimed invention, is the use of a compound that increases the expression of sarcospan in the manufacture of a medicament for treatment or prevention of a disease related to dysfunction of a dystrophin-related complex.

The heterogeneity of mutations and difficulty of delivery to muscle are major challenges to the development of therapies to treat DMD. There is an urgent need for improved therapies that can overcome these challenges. Sarcospan (SSPN) reduces the pathology of muscular dystrophy in the DMD murine model by increasing membrane localization of the utrophin-glycoprotein complex (UGC) and α7β1D-integrin adhesion complexes, effectively increasing laminin binding to compensate for the loss of dystrophin.

Development of small molecule therapies that increase SSPN expression may lead to standalone or combinatorial therapies to treat DMD and other forms of muscular dystrophy caused by deficits in membrane proteins. Small molecule therapies are ideal due to their ability to bypass the limitations of delivery and immune responses seen with viral and cell-based methods.

Also provided herein, yet not as such covered by the claimed invention are methods for treating or preventing a disease related to dysfunction of a dystrophin-related complex in a subject in need thereof, comprising administering to the subject a compound that increases the expression of sarcospan, whereby symptoms of the disease are reduced. In accordance with the claimed invention, the disease related to dysfunction of a dystrophin-related complex is a muscular dystrophy. The muscular dystrophy may be Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD), Duchenne muscular dystrophy (DMD), distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, or oculopharyngeal muscular dystrophy. In certain embodiments, the muscular dystrophy is Duchenne muscular dystrophy.

In some embodiments of the claimed invention, the compound is <NUM>-[<NUM>-(<NUM>,<NUM>-Dihydro-<NUM>,<NUM>,<NUM>-trimethyl-<NUM>-indol-<NUM>-ylidene)-<NUM>-propenyl]-<NUM>-ethyl-benzothiazolium iodide (AC-<NUM>), or a pharmaceutically acceptable salt or ester thereof. In other, yet unclaimed implementations, the compound may be a muscarinic receptor agonist, for example, <NUM>-Azabicyclo[<NUM>. <NUM>]oct-<NUM>-yl acetate (aceclidine), or a pharmaceutically acceptable salt or ester thereof. In other embodiments encompassed by the claimed invention, the compound is <NUM>-amino-<NUM>-(<NUM>-hydroxyethoxymethyl)-<NUM>-purin-<NUM>-one (acyclovir), or a pharmaceutically acceptable salt or ester thereof.

The compound may be <NUM>-benzo[g]pteridine-<NUM>,<NUM>-dione (alloxazine), or a pharmaceutically acceptable salt or ester thereof. In some embodiments, the compound may be methyl N-[(E)-(<NUM>-hydroxy-<NUM>-oxidoquinoxalin-<NUM>-ium-<NUM>-ylidene)methyl]iminocarbamate (carbadox), or a pharmaceutically acceptable salt or ester thereof.

In some embodiments, the compound may be <NUM>-O-ethyl <NUM>-O-methyl <NUM>-(<NUM>,<NUM>-dichlorophenyl)-<NUM>,<NUM>-dimethyl-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (felodipine), <NUM>-O-methyl <NUM>-O-propan-<NUM>-yl <NUM>-(<NUM>,<NUM>,<NUM>-benzoxadiazol-<NUM>-yl)-<NUM>,<NUM>-dimethyl-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (isradipine), diethyl <NUM>,<NUM>-dimethyl-<NUM>-[<NUM>-[(E)-<NUM>-[(<NUM>-methylpropan-<NUM>-yl)oxy]-<NUM>-oxoprop-<NUM>-enyl]phenyl]-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (lacidipine), <NUM>-O-methyl <NUM>-O-propan-<NUM>-yl <NUM>-cyano-<NUM>-methyl-<NUM>-(<NUM>-nitrophenyl)-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (nilvadipine), or pharmaceutically acceptable salts or esters thereof.

In other, yet unclaimed implementations, the compound may be a cRaf1 kinase inhibitor, such as (3Z)-<NUM>-[(<NUM>,<NUM>-dibromo-<NUM>-hydroxyphenyl)methylidene]-<NUM>-iodo-<NUM>-indol-<NUM>-one (GW5074), or a pharmaceutically acceptable salt or ester thereof. In accordance with the claimed invention, the compound may be <NUM>-[<NUM>-hydroxy-<NUM>-(propan-<NUM>-ylamino)ethyl]benzene-<NUM>,<NUM>-diol (isoproterenol), or a pharmaceutically acceptable salt or ester thereof. The compound may be (2R,3R,<NUM>,<NUM>)-<NUM>-(<NUM>-aminopurin-<NUM>-yl)-<NUM>-(methylsulfanylmethyl)oxolane-<NUM>,<NUM>-diol (methylthioadenosine), or a pharmaceutically acceptable salt or ester thereof.

The compound may be a N-[(<NUM>-butylpyrrolidin-<NUM>-yl)methyl]-<NUM>-cyano-<NUM>-methoxynaphthalene-<NUM>-carboxamide (nafadotride), or a pharmaceutically acceptable salt or ester thereof. In other, yet unclaimed implementations, the compound may be an anabolic androgenic steroid,for example, (8R,<NUM>,10R,<NUM>,<NUM>,<NUM>)-<NUM>-hydroxy-<NUM>-methyl-<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>-dodecahydro-<NUM>-cyclopenta[a]phenanthren-<NUM>-one (nandrolone), or a pharmaceutically acceptable salt or ester thereof.

In some embodiments, sarcospan mRNA transcript is increased. Additionally or alternatively, the sarcospan protein level is increased.

Also provided herein, yet not as such as encompassed by the claimed invention are methods for treating or preventing muscular dystrophy in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a compound that increases the expression of sarcospan.

As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein, "another" may mean at least a second or more.

As used herein, the term "biomarker" is anything that can be used as an indicator of a particular physiological state of an organism. For example, a biomarker can be the level(s) of a particular by-product, metabolite, mRNA or protein associated with a particular physiological state.

The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (<NUM>) sugars, such as lactose, glucose and sucrose; (<NUM>) starches, such as corn starch and potato starch; (<NUM>) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (<NUM>) powdered tragacanth; (<NUM>) malt; (<NUM>) gelatin; (<NUM>) talc; (<NUM>) excipients, such as cocoa butter and suppository waxes; (<NUM>) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (<NUM>) glycols, such as propylene glycol; (<NUM>) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (<NUM>) esters, such as ethyl oleate and ethyl laurate; (<NUM>) agar; (<NUM>) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (<NUM>) alginic acid; (<NUM>) pyrogen-free water; (<NUM>) isotonic saline; (<NUM>) Ringer's solution; (<NUM>) ethyl alcohol; (<NUM>) phosphate buffer solutions; and (<NUM>) other non-toxic compatible substances employed in pharmaceutical formulations.

The term "preventing" is art-recognized, and when used in relation to a condition, such as a local recurrence, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of a disease related to dysfunction of a dystrophin-related complex includes, for example, reducing problems in the joints and spine while increasing muscle strength in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the progression of the disease in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.

The term "prophylactic" or "therapeutic" treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal), then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof.

The term "subject" refers to a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

As used herein, the term "treating" or "treatment" includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject's condition.

Provided herein, yet not as such covered by the claimed invention, are methods of treating or preventing a disease related to dysfunction of a dystrophin-related complex in a subject by administering to the subject a therapeutically effective amount of compound that increases the expression of sarcospan. In certain implementations, the methods relate to treating muscular dystrophy, specifically Duchenne muscular dystrophy. Also provided, yet not as such covered by the claimed invention, are methods for treating muscular dystrophy in a subject by administering to the subject a therapeutically effective amount of a pharmaceutical composition disclosed herein.

Also described, but not covered by the claimed invention is a pharmaceutical composition comprising a compound that increases the expression of sarcospan. The composition may comprise a pharmaceutically acceptable carrier. The pharmaceutical composition may be delivered by any suitable route of administration, including orally, buccally, sublingually, parenterally, and rectally, as by powders, ointments, drops, liquids, gels, tablets, capsules, pills, or creams. The pharmaceutical compositions may be delivered systemically (e.g., via oral administration). The compositions may be delivered intravenously.

Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a subject, composition, and mode of administration, without being toxic to the subject.

The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could prescribe and/or administer doses of the compounds employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

RNA from myotubes treated for <NUM> hours was extracted from cells using Trizol-based (Thermo Fisher Scientific) phase separation. RNA concentrations were determined using a NanoDrop <NUM> (Thermo Fisher Scientific) and <NUM> ng of RNA in a <NUM>µl reaction was reverse transcribed using iScript cDNA synthesis (Bio-Rad) with the following cycling conditions: <NUM> for <NUM> minutes, <NUM> for <NUM> minutes, <NUM> for <NUM> minutes. For quantitative PCR, SsoFast EvaGreen Supermix (Bio-Rad), <NUM> of each optimized forward and reverse primer (for primer descriptions see Table <NUM>), and cDNA corresponding to <NUM> ng RNA were used to amplify cDNA measured by Applied Biosystems <NUM> (Thermo Fisher Scientific) with the following reaction conditions: <NUM> for <NUM> minutes, <NUM> for <NUM> minutes, <NUM> cycles of <NUM> for <NUM> seconds and <NUM> for <NUM> seconds, and dissociation stage. Each sample was run in triplicate. Data was analyzed using the ddCT method and normalized to reference gene, GAPDH or β-actin, with vehicle-treated samples serving as the calibrator (relative expression of vehicle control = <NUM>).

Table <NUM> shows the primers used for gene expression analysis. Primers were optimized by standard curve method using cDNA corresponding to 75ng RNA, diluted <NUM>-fold. AE: amplification efficiency, calculated using the equation AE = [<NUM>(-<NUM>/slope)]/ - <NUM>. SSPN, sarcospan; DMD, dystrophin; UTRN, utrophin; DAG, dystroglycan, SCGA, α-sarcoglycan; SCGB, β-sarcoglycan; ITGA7, α7 integrin; ITGB1, β1D integrin; MYOG, myogenin; MYF5, myogenic factor <NUM>; ACTB, β-actin; GADPH, glyceraldehyde <NUM>-phosphate dehydrogenase.

The SSPN promoter region was predicted using publically available data on UCSC genome browser (http://genome. Using the GRCh37/hg19 assembly, gene regulatory elements of the cardiac and skeletal muscle-specific SSPN transcript variant <NUM> (NM_005086. <NUM>) of the human SSPN gene (NG_012011. <NUM>) were identified. H2K4me3 marks, DNase hypersensitivity regions, and ChIP-seq data showing transcription factor binding locations from human skeletal muscle cultures indicated the promoter region to be upstream of exon <NUM> and within exon <NUM>. A <NUM> kb region encompassing the human SSPN promoter was amplified from human genomic DNA (Bioline) using Phusion High Fidelity DNA Polymerase (New England Biolabs) with the primers indicated in Table 2a or 2b. The primers contained leader sequences and restriction sites for BglII (AGATCT) or HindIII (TTCGAA). The PCR products were purified using PureLink HiPure Plasmid DNA Purification kit (Life Technologies) and digested with BglII and HindIII in NEBuffer <NUM> (New England Biolabs). The <NUM> kb digested PCR products were electrophoresed on agarose gels, excised, and purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research). T4 DNA ligase (Invitrogen) was used to ligate PCR products with promoter-less reporter plasmids, pmEGFP-<NUM> (Addgene, plasmid #<NUM>) or pgl4. <NUM> (Promega), prepared by digestion with BglII and HindIII. The plasmid constructs were linearized with BglII, which digested the region upstream of the SSPN promoter. The linearized plasmids were purified and used to transform One Shot TOP10 chemically competent E. coli (Thermo Fisher) grown on agar containing the appropriate antibiotic. Individual colonies were confirmed by colony PCR to contain the SSPN promoter construct and inoculated in liquid culture overnight. The plasmids were purified using PureLink Quick Plasmid Miniprep (Life Technologies) and subjected to DNA sequencing (Laragen Inc. ) using the primers in Table 2a or 2b to confirm presence and accuracy of the SSPN promoter region. Select bacterial cultures were grown in large cultures and collected for plasmid purification using the Plasmid Maxi Kit (Qiagen).

Table 2b shows the primers used for reporter construct cloning and sequencing. Cloning was optimized for human sarcospan (SSPN) gene region and pmEGFP-<NUM> plasmid (EGFP).

C2C12 immortalized murine myoblasts cultured in growth media consisting of DMEM (Gibco) with <NUM>% Fetal Bovine Serum (Sigma-Aldrich) at <NUM> with <NUM>% CO<NUM> were transfected with the hSSPN-EGFP or hSSPN-Luciferase linearized plasmids using Lipofectamine <NUM> Transfection Reagent (Thermo Fisher Scientific). Transfected cells were selected using 800µg/ml of G418 (Sigma-Aldrich) for <NUM> weeks to generate stable cell lines expressing reporter protein under control of the human SSPN promoter.

hSSPN-EGFP myoblasts were seeded at <NUM> cells per well in <NUM>µl of growth media in <NUM>-well black, clear bottom microplates (Greiner) using a Multidrop <NUM> (Thermo Fisher Scientific) and incubated for <NUM> days. Upon reaching confluency, the growth media was replaced with <NUM>µl of differentiation media consisting of DMEM with <NUM>% horse serum (Sigma-Aldrich) using an EL406 combination washer dispenser (Biotek). At day <NUM> of differentiation, the media on the cells was aspirated, left with a residual volume of <NUM>µl, and replaced with <NUM>µl of fresh differentiation media. <NUM>µl of small molecule in DMSO or DMSO alone (for vehicle and positive control wells) was added to each well using a Biomek Fx (Beckman). To ensure proper mixing of the DMSO, <NUM>µl of additional differentiation media was added to all wells except the positive control treated wells, which instead received <NUM>µl of media containing insulin transferrin selenium (ITS, Gibco) to reach a final concentration of <NUM>% ITS. The final concentration of drug in each treated well was <NUM> in <NUM>% DMSO and <NUM>% DMSO for vehicle and positive control treated wells. After <NUM> hours of incubation, the media was replaced with Fluorobrite DMEM (Gibco) and each plate was imaged using ImageXpress Micro Confocal High Content Imaging System (Molecular Devices). The fluorescent intensity of imaged cells was determined using MetaXpress Analysis software (Molecular Devices). Analysis setting were as follows: top hat (size: <NUM>, filter shape: circle), adaptive threshold (source: top hat, minimum width: <NUM>, maximum width: <NUM>, intensity above local background: <NUM>), filter mask (filter type: minimum area filter, minimum value: <NUM>).

hSSPN-Luciferase myoblasts were cultured as described above. After <NUM> hours of treatment, plates were allowed to equilibrate to room temperature before the media was aspirated using an EL406 combination washer dispenser at room temperature. Bright-Glo luciferase assay system reagent (Promega) and differentiation media were added to cells at a <NUM>:<NUM> dilution using a Multidrop <NUM>. After a <NUM>-minute incubation at room temperature, luminescence signal was quantified using an Envision plate reader (PerkinElmer). The relative luminescence units were analyzed to determine fold change of treated over vehicle treated cells.

C2C12 cells (American Type Culture Collection) were grown at <NUM> with <NUM>% CO<NUM> in growth media containing DMEM (Gibco) with <NUM>% FBS (Sigma-Aldrich). Upon reaching confluency, the media was replaced with differentiation media containing DMEM with <NUM>% horse serum (Sigma-Aldrich). Conditionally immortalized H2K mdx myoblast cells with a nonsense mutation in exon <NUM> of dystrophin were a gift from Terrance Partridge, Ph. (Children's National Medical Center, Washington, D. Cells were allowed to proliferate on <NUM>% gelatin (Sigma-Aldrich) coated plates at <NUM> with <NUM>% CO<NUM> with growth media containing DMEM, <NUM>% HI-FBS (Invitrogen), <NUM>% L-glutamine (Sigma-Aldrich), <NUM>% chicken embryo extract (Accurate Chemical), <NUM>% penicillin-streptomycin (Sigma-Aldrich), and 20U/ml of fresh interferon gamma (Gibco). For differentiation, H2K mdx myoblasts were seeded on plates coated with <NUM>/ml matrigel (Corning) diluted in DMEM and grown in proliferation conditions. Upon reaching confluency, cells were grown at <NUM> with <NUM>% CO<NUM> in differentiation media containing DMEM with <NUM>% horse serum (Sigma-Aldrich), <NUM>% L-glutamine, and <NUM>% penicillin-streptomycin using established protocols.

Cells were treated for <NUM> hours beginning at day <NUM> of differentiation with DMSO (vehicle control, ATCC), felodipine (Sigma), nilvadipine (Sigma), alloxazine (Sigma), GW5074 (Sigma), methylthioadenosine (Santa Cruz Biotechnology), or ezutromid (Cayman Chemicals) diluted in cell type specific differentiation media at doses listed in the figures. For gene expression studies, each treatment was performed in duplicate. For protein studies, each treatment was performed with single or double replicates.

C2C12 murine myotubes treated for <NUM> hours were lysed using RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (<NUM>µg/ml pepstatin A, <NUM>µg/ml aprotinin, <NUM>µg/ml leupeptin, <NUM> PMSF, <NUM> benzamidine, <NUM> calpain I inhibitor, <NUM> calpeptin). Cell lysates in RIPA buffer were rocked for <NUM> hour at <NUM> and centrifuged at <NUM> RPM for <NUM> minutes at <NUM>. The supernatant was collected, quantified for protein concentration using the DC protein assay (Bio-Rad), and normalized to <NUM>/ml in water and Laemmli sample buffer with a final concentration of <NUM>% glycerol (Sigma-Aldrich), <NUM>% beta-mercaptoethanol (Sigma-Aldrich), <NUM>% sodium dodecyl sulfate (Sigma-Aldrich), and <NUM>% bromophenol blue (Sigma-Aldrich). For SDS-PAGE, samples were heated to <NUM> for <NUM> minutes before loading <NUM>µg to a <NUM>-<NUM>% tris-glycine gel (Novex), run for <NUM> hours at <NUM> volts at room temperature, and transferred to a nitrocellulose membrane for <NUM> hours at <NUM> volts at <NUM>. Ponceau S staining was performed to visualize protein loading. Membranes were blocked with <NUM>% blotto (<NUM>% non-fat dried milk) in tris-buffered saline with <NUM>% tween-<NUM> (TBST, Sigma-Aldrich) for <NUM> hour at room temperature and incubated on a rocker overnight at <NUM> with the following primary antibodies and dilutions in <NUM>% blotto: SSPN (sc-<NUM>, <NUM>:<NUM>, Santa Cruz Biotechnology), GAPDH (Mab374, <NUM>:<NUM>,<NUM>, Millipore). Following three <NUM>-minute TBST washes, the membranes were incubated in goat anti-mouse IgG HRP (Ab6789, <NUM>:<NUM> for SSPN, <NUM>:<NUM>,<NUM> for GAPDH, Abcam) diluted in <NUM>% milk for <NUM> hours at room temperature. The membranes were then washed three times for <NUM> minutes each with TBST, incubated in SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) for <NUM> minutes at room temperature on an orbital shaker, and exposed to autoradiography films (Agfa). Autoradiography films were developed using a SRX-101A tabletop processor (Konica Minolta), scanned to a digital file, and analyzed by densitometry of bands using ImageJ version <NUM>. Target protein bands were normalized to loading control GAPDH with vehicle-treated cells serving as the calibrator sample (relative protein levels of vehicle control = <NUM>).

All data was analyzed on Prism version <NUM> (GraphPad Software) for Mac OS X using the two-tailed Kolmogorov-Smirnov test. Data are reported as mean ± SEM. A p-value of <<NUM> was considered statistically significant. * P<<NUM>, ** P<<NUM>, *** P<<NUM>, **** P<<NUM>.

Immortalized C2C12 myoblasts were selected for the screen due to their ease of culture and ability to grow to large quantities. However, it was unclear whether to conduct the assay using immature myoblasts or mature, differentiated myotubes. In order to understand SSPN gene activity in myoblasts and myotubes, SSPN gene expression in C2C12 cells was directly interrogated at each day during differentiation for a total of seven days. Cells underwent fusion starting at day <NUM> and appeared to be fully differentiated at day <NUM>, as previously reported. SSPN mRNA abundance was relatively unchanged during the first three days following incubation of C2C12 cells in differentiation media (<FIG>, panel a). However, SSPN levels began to increase exponentially at day <NUM>, reaching ten-fold levels by day <NUM>. The increased SSPN gene expression occurred just after the first evidence of visible myotube fusion at day <NUM> (<FIG>).

The expression of SSPN associated proteins was also evaluated, including dystrophin (Dmd), utrophin (Utrn), dystroglycan (Dag), α7 integrin (Itga7), β1D integrin (Itgb <NUM>), α-sarcoglycan (Sgca), and β-sarcoglycan (Sgcb). Gene activity increased immediately after myoblasts were switched from proliferation to differentiation media (<FIG>, panels b-i, and <FIG>). Interestingly, while dystrophin was not expressed in myoblasts, dystrophin mRNA levels increased nearly <NUM>,<NUM>-fold during differentiation. Utrn, Itga, and Itgb1 expression increased by ten-fold, while the sarcoglycans and dystroglycan increased nearly one hundred-fold during the same time period (<FIG>). As a control, muscle-specific transcription factors, myogenin (MyoG) and myogenic factor <NUM> (Myf5), that are known to regulate specific steps during myoblast differentiation were evaluated. MyoG gene activity increased immediately upon exposure to differentiation conditions (<FIG>, panel i), while Myf5 gradually decreased (<FIG>, panel j). Based on these results, cells were treated at day <NUM> of differentiation and data was collected at day <NUM> of differentiation as this would allow for increases in gene expression while minimizing possible saturation of gene activation.

To identify the human SSPN (hSSPN) promoter region, publically available data on UCSC genome browser was used. By assessing H2K4me3 marks, DNase hypersensitivity regions, and ChIP-seq data showing transcription factor binding, the promoter was predicted to be located directly upstream of exon <NUM> and within exon <NUM> (<FIG>). As gene activity can be easily visualized without the need for costly reagents, a fluorescence-based reporter for the primary screening assay was selected. A more reagent-consuming luminescence assay was used as the counterscreen to eliminate false positives from auto-fluorescent compounds. The immortalized C2C12 mouse myoblast cell line was used to generate stably transfected reporter cells containing an enhanced green fluorescent protein (EGFP) or luciferase reporter driven by the human sarcospan promoter (hSSPN) (<FIG>, panel a). The hSSPN-EGFP and hSSPN-Luciferase myoblasts expressed reporter protein at increasing levels throughout differentiation into early-stage myotubes, revealing that the reporters are reflective of endogenous SSPN gene activity (<FIG>, panel b). Insulin transferrin selenium (ITS) was selected as a positive control for the screening assays as it increases hSSPN-EGFP and hSSPN-Luciferase reporter levels by <NUM>-fold over vehicle after <NUM> hours of treatment (<FIG>), showing that the reporter cells are responsive to chemical stimulus. ITS increases protein synthesis and enhances the rate of myotube fusion, which increases SSPN expression as previously shown (<FIG>, panel a). Cells transfected with reporter plasmids lacking the SSPN promoter did not exhibit fluorescence or luminescence reporter activity.

Assay development is a challenging and laborious process that requires iterations of optimization. The obstacles that arise during assay development are confounded by the need to miniaturize assays and minimize the number of steps required, which both decrease handling time and directly increase throughput. To effectively screen large compound libraries, assays were scaled down to a <NUM>-well microplate format, which allowed for reduced reagent consumption and quick data collection. To optimize assay conditions for high-throughput screening in a <NUM>-well microplate format, numerous parameters for both hSSPN-EGFP and hSSPN-Luciferase reporter C2C12 cells were evaluated (<FIG>, panel a).

One limiting factor in high-throughput screens is the preparation of large quantities of cells. Subsequently, multiple seeding densities ranging from <NUM> to <NUM> cells per well were assessed. Seeding <NUM> cells per well followed by <NUM> days of incubation was sufficient for the myoblasts to reach confluency before differentiation. This also reduced the amount of pre-screen culture required, making it an optimal condition for scaling up to meet high-throughput requirements.

Next, the ability of the ITS positive control to increase reporter activity after <NUM> and <NUM> hours was tested. A detectable and significant change in reporter activity after <NUM> hours of treatment was observed (<FIG>). Screening core facilities typically store small molecules in <NUM>% DMSO, which can lead to cell toxicity from the DMSO vehicle alone. To assess DMSO toxicity in our cells, cells were dosed with <NUM>-<NUM>% DMSO. A decrease in cell viability at <NUM>% DMSO and higher after <NUM> hrs of treatment was observed. To ensure proper DMSO mixing to prevent regionally high concentrations of DMSO that can negatively impact cell viability, several mixing methods and conditions were tested. From these tests, it was determined that the addition of <NUM>µl of <NUM>% DMSO to an initial volume of <NUM>µl of media in each well, followed by <NUM>µl of additional media was sufficient to create a homogenous solution of DMSO, as detected by DMSO spiked with crystal violet dye. Image analysis using a custom analysis module in MetaXpress software identified myotubes based on specified dimensions and quantifies fluorescence pixel intensity of identified cells (<FIG>, panel b). Imaging two regions of each well at 10X magnification in low fluorescence media was sufficient for the custom analysis module to reliably detect a significant difference between vehicle and positive control treated cells (<FIG>, panel c).

To gain insight into the pathways involved in SSPN upregulation, libraries of well-characterized FDA approved compounds were screened. Using the hSSPN-EGFP cells, the Library of Pharmacologically Active Compounds (LOPAC), Prestwick Chemical, and NIHII small molecule libraries totaling <NUM>,<NUM> small molecules was screened. All images were analyzed for cellular fluorescence intensity and compared with values from vehicle treated cells. A hit cutoff of <NUM>-fold fluorescence intensity over vehicle was set and images with debris or small molecules that auto-fluoresce were eliminated. This led to <NUM> small molecules (<FIG>, panel b). Among the thirteen hits, six are L-type calcium channel antagonists (felodipine, isradipine, lacidipine, nifedipine, nilvadipine) with felodipine appearing twice from the LOPAC and NPW libraries, which indicated the robustness of the assay in duplicating results. In addition, at least five additional hits (isoproterenol, aceclidine, alloxazine, GW5074, and nandrolone) also affected intracellular calcium levels (an earlier assay also included AC-<NUM> and methylthioadenosine among the hits). These data strongly supported that endogenous SSPN expression is regulated in a calcium-dependent manner.

Table <NUM> provides information on plate quality control using robust strictly standardized mean difference. Robust strictly standardized mean difference (SSMD*) was used a measurement of quality control. Each of the <NUM> plates resulted in an SSMD* ≥ <NUM>, indicating a good quality difference between vehicle and positive control-treated cells.

Table <NUM> shows that validated hits from hSSPN-EGFP screen reveal an enrichment of calcium channel blockers. The screen resulted in <NUM> hits, which were further validated with the hSSPN-EGFP reporter cells. The validated hits included an overrepresented number of L-type calcium channel blockers.

Table <NUM> shows the validation of hits from screen on hSSPN-EGFP myotubes. Hits from the screen were retested on hSSPN-EGFP myotubes at <NUM> in <NUM> plates (n=<NUM> per plate). RFU, relative fluorescence units. *SSMD, robust strictly standardized mean difference.

To validate the primary screen results, a select number of candidate drugs were counterscreened in both hSSPN-EGFP and hSSPN-Luciferase reporter cells in triplicate. The hSSPN-Luciferase cell line was used to exclude molecules that were detected based solely on auto-fluorescence. Felodipine, nilvadipine, methylthioadenosine (MTA), alloxazine, and GW5074 increased activity of both reporters at doses between <NUM> to <NUM> after <NUM> hours of treatment (<FIG>, panels a-b). To determine whether this increase in human SSPN promoter activity translated to an increase in mouse SSPN transcript levels, C2C12 myotubes were treated with <NUM>-<NUM> of felodipine and gene expression was assessed using qPCR. After <NUM> hours of felodipine treatment, SSPN expression increased over vehicle (<FIG>, panel c).

To assess the effect of felodipine in a dystrophin-deficient cell model, immortalized mdx myoblasts containing a nonsense mutation in exon <NUM> of the dystrophin gene were used. Mdx myotubes were treated with <NUM>-<NUM> of felodipine. An increase in SSPN gene expression with treatment was observed (<FIG>, panel d). Because increases in transcript levels do not necessarily correlate with changes in protein levels, SSPN protein levels were also assessed. Immunoblot analysis on C2C12 cells treated with <NUM> -<NUM> of felodipine revealed an increase in SSPN protein levels with treatments (<FIG>, panel e). In mdx cells, treatment with felodipine increased SSPN protein levels (<FIG>, panel f).

To assess the effect of SSPN-modulating candidate drugs identified in the screen on gene expression relative to drugs in clinical stage development targeting similar mechanisms, C2C12 cells were dosed with the known UTRN upregulator, ezutromid (SMT C1100). Ezutromid was identified in a similar cell-based, promoter-reporter screen and is currently in clinical trials. While ezutromid did not significantly increase UTRN levels in C2C12 myotubes, a nearly <NUM>-fold increase in UTRN gene expression was observed with <NUM> of felodipine treatment (<FIG>, panel a). Ezutromid did not significantly increase SSPN gene expression, while treatment with <NUM> of felodipine induced a <NUM>-fold increase in SSPN levels (<FIG>, panel b). To determine whether ezutromid affected SSPN gene expression in the dystrophin-deficient context, mdx myotubes were treated with ezutromid and UTRN and SSPN transcript levels were quantified. As expected from previously published findings, UTRN expression increased by up to <NUM>-fold (<FIG>, panel c). Interestingly, ezutromid treatment increased SSPN expression by <NUM>-fold, indicating that ezutromid may directly or indirectly increase SSPN levels (<FIG>, panel d).

Claim 1:
A compound that increases the expression of sarcospan for use in the treatment or prevention of a disease related to dysfunction of a dystrophin related complex, wherein said disease is muscular dystrophy, wherein the compound is selected from: <NUM>-[<NUM>-(<NUM>,<NUM>-Dihydro-<NUM>,<NUM>,<NUM>-trimethyl-<NUM>-indol-<NUM>-ylidene)-<NUM>-propenyl]-<NUM>-ethyl-benzothiazolium iodide (AC-<NUM>), N-[(<NUM>-butylpyrrolidin-<NUM>-yl)methyl]-<NUM>-cyano-<NUM>-methoxynaphthalene-<NUM>-carboxamide (nafadotride), <NUM>-amino-<NUM>-(<NUM>-hydroxyethoxymethyl)-<NUM>-purin-<NUM>-one (acyclovir), <NUM>-O-ethyl <NUM>-O-methyl <NUM>-(<NUM>,<NUM>-dichlorophenyl)-<NUM>,<NUM>-dimethyl-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (felodipine), <NUM>-benzo[g]pteridine-<NUM>,<NUM>-dione (alloxazine), <NUM>-O-methyl <NUM>-O-propan-<NUM>-yl <NUM>-(<NUM>,<NUM>,<NUM>-benzoxadiazol-<NUM>-yl)-<NUM>,<NUM>-dimethyl-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (isradipine), methyl N-[(E)-(<NUM>-hydroxy-<NUM>-oxidoquinoxalin-<NUM>-ium-<NUM>-ylidene)methyl]iminocarbamate (carbadox), diethyl <NUM>,<NUM>-dimethyl-<NUM>-[<NUM>-[(E)-<NUM>-[(<NUM>-methylpropan-<NUM>-yl)oxy]-<NUM>-oxoprop-<NUM>-enyl]phenyl]-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (lacidipine), <NUM>-[<NUM>-hydroxy-<NUM>-(propan-<NUM>-ylamino)ethyl]benzene-<NUM>,<NUM>-diol (isoproterenol), <NUM>-O-methyl <NUM>-O-propan-<NUM>-yl <NUM>-cyano-<NUM>-methyl-<NUM>-(<NUM>-nitrophenyl)-<NUM>,<NUM>-dihydropyridine-<NUM>,<NUM>-dicarboxylate (nilvadipine), (2R,3R,<NUM>,<NUM>)-<NUM>-(<NUM>-aminopurin-<NUM>-yl)-<NUM>-(methylsulfanylmethyl)oxolane-<NUM>,<NUM>-diol (methylthioadenosine), or a pharmaceutically acceptable salt or ester thereof.