WO2023278684A1 - Compositions et méthodes de traitement de la dysferlinopathie - Google Patents

Compositions et méthodes de traitement de la dysferlinopathie Download PDF

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Publication number
WO2023278684A1
WO2023278684A1 PCT/US2022/035701 US2022035701W WO2023278684A1 WO 2023278684 A1 WO2023278684 A1 WO 2023278684A1 US 2022035701 W US2022035701 W US 2022035701W WO 2023278684 A1 WO2023278684 A1 WO 2023278684A1
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muscle
dysf
acid
pba
cells
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PCT/US2022/035701
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English (en)
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Leonard P. Guarente
Kana TOMINAGA
Naoomi TOMINAGA
Mohan VISWANATHAN
Eric WILLAMS
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Massachusetts Institute Of Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles

Definitions

  • Dysferlinopathy is an adult-onset, progressive, rare form of muscular dystrophy caused by recessive loss-of-function mutations in the gene encoding dysferlin, DYSF, and includes the clinical diagnoses of Limb-Girdle Muscular Dystrophy (LGMD) 2B/R2, Miyoshi Myopathy type 1, and distal anterior compartment myopathy.
  • LGMD Limb-Girdle Muscular Dystrophy
  • Miyoshi Myopathy type 1 Miyoshi Myopathy type 1
  • distal anterior compartment myopathy A diagnosis of dysferlinopathy is made when a patient is shown to have >80% reduction in DYSF protein by western, and is confirmed by sequencing of the DYSF gene to identify the causative mutation(s). Patients typically lose ambulation by age 40-45, and there are no current treatments. Thus, there is an urgent ongoing and unmet need for therapies which treat dysferlinopathy.
  • the present disclosure provides methods of treating dysferlinopathy, comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting and or a pharmaceutically acceptable salt thereof.
  • the present disclosure provides methods of treating a muscle injury, comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting and or a pharmaceutically acceptable salt thereof.
  • the present disclosure provides methods of treating or preventing muscle loss, comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting and ; or a pharmaceutically acceptable salt thereof.
  • the present disclosure provides methods of promoting muscle growth, comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting and or a pharmaceutically acceptable salt thereof.
  • FIG. 1A is a schematic outline of the 2A-assay.
  • Human DYSF isoform 8 cDNA was inserted into a bicistronic lentiviral vector expression vector regulated by a minimal cytomegalovirus (CMV) promoter and tetracycline response element (TRE).
  • CMV minimal cytomegalovirus
  • TRE tetracycline response element
  • Transfection of LV-TRE-DYSF-T2A-DsRed into HEK293T cells results in equimolar expression of DYSF- 2A, a fusion protein with a C-terminal 2A-peptide, and DsRed protein, which are separated by cleavage at the T2A peptide sequence during translation.
  • An a-2A peptide antibody recognizes the extracellular region of DYSF-T2A protein in live cells; binding of this antibody can be detected by flow cytometry enabling quantitation of PM-localized of DYSF.
  • FIG. IB depicts the validation of the 2A-assay which was performed using vector constructs expressing DYSFWT and three DYSFPMMs: DYSFV67D, D Y SFR555 Wand DYSFL1341Pknown pathogenic mutations found in dysferlinopathy patients, and DYSFA170E, a commonly occurring DYSF SNP which is predicted to be non-pathogenic.
  • HEK293T cells were transiently transfected, cultured, and processed for flow cytometry as described in materials and methods. PM localized expression of DYSFPMMs is determined relative to DYSFWT. ***p ⁇ 0.001 vs DYSFWT.
  • FIG. 1C shows immunofluorescence images localizing DYSFPMMs expressed in HEK293T cells. 48-hrs post transfection HEK293T cells expressing the listed DYSFPMMs were FACS sorted for DsRed, cultured, fixed, and stained with the Hamlet a-DYSF l°Ab (green) and the a-Na/K ATPase-Ab (red) to identify the plasma membrane (PM). Scale bar: 25 pm.
  • FIG. 2 depicts HEK293T cells that were transiently transfected with LV-TRE-DYSF- T2A-DsRed vectors bearing DYSFWT or one of 113 various DYSFPMMs.
  • DsRed positive cells were sorted and cultured on coverslips and subject to ICC to visually determine DYSF localization, red bars indicate no observable PM-localization, while blue bars indicate that PM-localization was observed for a given DYSFPMM.
  • FIG. 3A shows the restoration of DYSFL1341PPM-localization by 4-PBA and corr- 2b.
  • FIG. 3B depicts ICC DYSF localization in transfected (unsorted) HEK cells expressing DsRed and either DYSFWT or DYSFL1341Ptreated with either DMSO (0.1%), 4-PBA (1 mM), or corr-2b (25 pM) for 24 hrs.
  • Live cells were stained with a-2A peptide -Ab to identify PM-localized DYSF protein.
  • DAPI staining and a-Na/K ATPase-Ab hybridization was used to identify the nuclei and PM in all cells, respectively. Scale bar, 50 pm.
  • FIGS. 4A-4G show that treatment with 4-PBA restores membrane repair in GREG (DYSFL1341P) myotubes and MMex38 (DYSFL1360P) mouse myofibers.
  • White arrowheads show site of laser wounding in FIGS. 4A, 4C, and 4F with the associated time course repair kinetics of FM1-43 dye infiltration after injury in FIGS. 4B, 4D, and 4G, respectively; n-values are the total number of fibers tested in a total of two independent experiments.
  • FIG. 4E Immunofluorescent staining of fresh frozen histological cross sections of EDL muscle isolated from 3 month old male +/+ and MMex38 mice treated with vehicle or 4-PBA (2mg/ml) in drinking water for 48 hrs.
  • FIGS. 5A & 5B show the validation of a-2A Ab detection of PM-localized DYSF in 2A assay.
  • FIG. 5A shows Flow cytometry dot plots analyzing DYSF-2A detection in non- transfected HEK cells and HEK cells transiently transfected with DYSF WT .
  • Cells expressing DYSFWT were hybridized with a-2A 1° Ab and Alexa Fluor 647 conjugated-IgG 2°Ab as labelled.
  • the flow cytometer fluorescence wavelength detector was set for allophycocyanin (APC), which detects Alexa Fluor 647.
  • FIG. 5A shows Flow cytometry dot plots analyzing DYSF-2A detection in non- transfected HEK cells and HEK cells transiently transfected with DYSF WT .
  • Cells expressing DYSFWT were hybridized with a-2A 1° Ab and Alexa Fluor 647 conjugated-IgG 2°Ab
  • *** p ⁇ 0.001 indicates difference between 4 ng/ml and 0 or 1 ng/ml.
  • FIG. 6 shows immunofluorescence images localizing DYSF PMMs expressed in HEK293T cells. 48-hrs post transfection HEK293T cells expressing DYSF L1341P were FACS sorted for DsRed, cultured, fixed, and hybridized with the Hamlet a-DYSF 1°C Ab and the a- Calreticulin 1° Ab to identify the endoplasmic reticulum (ER). Arrows point to perinuclear areas where DYSF L1341P localization is coincident with ER. Scale bar: 25 microns.
  • FIG. 7 is a PCR stitching and Gibson cloning scheme for introduction of DYSF PMMs into LV-TREDYSF-T2A-DsRed.
  • a DYSFPMM-containing PCR product was produced by PCR stitching two separate PCR products, PCR1 and PCR2, each with overlapping regions of homology containing the PMM introduced in the PCR oligonucleotides. This product was then used for Gibson assembly into linearized LV-TRE-DYSF-T2A-DsRed using the appropriate restriction enzymes.
  • Example shown is for Gibson assembly of PCR products containing DYSF PMMs between BstBI sites, similar products were generated for other regions of DYSF.
  • FIG. 8 shows immunofluorescence images showing PM localization of DYSF in HEK cells expressing DYSF P134L , DYSF R1342W , DYSF e1335K , or DYSF C1815F .
  • HEK cells were transiently transfected with a DYSFPMM expression construct, cultured for 48-hrs, and sorted for DsRed positive cells. Following 24-hrs of culture, cells were processed for ICC with a- DYSF Ab, a-sodium potassium ATPase Ab PM and a-calreticulin Ab ERto aid in identification of these organelles, as well as DAPI nuclear marker.
  • RE Relative expression to WT. Scale bar: 25 microns.
  • FIGS. 10A-10D shows the evaluation of 4-PBA and corr-2b efficacy.
  • FIG. 10A shows Cell proliferation assays for HEK cells treated with 4-PBA or corr-2b compared to DMSO alone for 24-hrs demonstrates that 4-PBA and corr-2b prevent cell proliferation at concentrations greater than lmM and 25 mM, respectively.
  • FIG. 10B shows the Dose response of 4-PBA and corr-2b in 2A-assays with DYSF L1341P shows optimal effect of 4-PBA and corr- 2b are at lmM and 25uM, respectively. All values are normalized to cells expressing DYSFWT treated with DMSO.
  • FIG. 10A shows Cell proliferation assays for HEK cells treated with 4-PBA or corr-2b compared to DMSO alone for 24-hrs demonstrates that 4-PBA and corr-2b prevent cell proliferation at concentrations greater than lmM and 25 mM, respectively.
  • FIG. 10B shows the Dose response
  • FIG. 10D shows the effect of drug treatments on DYSF expression in HEK cells transiently transfected with DYSF WT or DYSF L1341P .
  • HEK cells expressing DYSFWT or DYSFL1341P were incubated with either DMSO (0.1%), 4-PBA (1 mM), corr-2b (25 pM) for 24-hrs and subject to western blot of total protein lysates from each cell line under each treatment. Nontransfected HEK cells were used as negative control.
  • Membranes were hybridized with a-DYSF(Hamlet) Ab, and a-DsRed 1° Ab and a-GAPDH 1° Ab as loading control. *p ⁇ 0.05, **p ⁇ 0,01, ***p ⁇ 0.001, relative to untreated or DMSO control.
  • FIGS. 11A & 11B show membrane repair in GREG cell derived myotubes.
  • Dysferlin deficient GREG myoblasts were transfected with DYSFWT or DYSFL1341P expression vectors, cultured, and subsequently sorted for DsRed positive myoblasts. Sorted cells, as well as non-transfected GREG cells, were plated in chambered cover glass in differentiation media and cultured into myotubes. Myotubes were laser (405nm) wounded in the presence of calcium and FM1-43 dye. Untransfected and DYSF L1341P transfected myotubes failed to repair laser induced membrane damage, while myotubes expressing DYSF WT rapidly repaired the breach.
  • FIG. 11A & 11B show membrane repair in GREG cell derived myotubes.
  • FIG. 11A shows three image frames. The first image is at the time of wounding, the second in the middle of repair, and the third at the end of repair. Arrowheads show the site of injury.
  • FIG. 12 shows that 4-PBA treatment restores membrane repair to GREG myotubes expressing DYSF R555W .
  • Data are means ⁇ S.E.M, ***p ⁇ 0.001.
  • FIG. 13 shows that corr-2b treatment of GREG myotubes prevents membrane repair in the presence of DYSF WT and sensitizes non-transfected GREG myotubes to damage.
  • N.S.; not significant. Plotted values are means ⁇ S.E.M.
  • FIG. 14 shows the histological analysis of EDL muscle cross-section from MMex38 DYSF L1360P mice show hallmarks of dysferlinopathy.
  • Brightfield and immunofluorescence images of EDL muscle fibers obtained from C57BL/6NJ (20-month, male) or MMex38 (20- month, male) mice using Romeo a-DYSF 1° Ab with DAPI nuclear staining shows that sarcolemma localization of DYSF seen in control animals is absent in MMex38, as well as central nuclei (arrows) within myofibers in MMex38 mice, indicative of dystrophic muscle. Scale bar: 50 microns.
  • FIG. 15 shows the ex vivo treatment of MMex38 mice EDL with 4-PBA rescues DYSF sarcolemma expression.
  • EDL muscle isolated from male C57BL6/NJ (+/+) and MMex38 mice and cultured in either vehicle (0.1% DMSO) or 4-PBA (1 mM) for 24-hrs. Fresh frozen cross- sections of the treated EDL muscles were subsequently prepared for histological analysis.
  • DAPI staining was performed for nuclear localization and DYSF staining was done using Hamlet a-DYSF 1° Ab and Alexa 647 a-mouse fluorescent 2° Ab. Images were all taken at the same exposure time and magnification. Scale bar: 50 microns.
  • Dysferlin is a member of the Ferlin protein family found throughout metazoans.
  • a tail-anchored type-2 integral plasma membrane (PM) protein, DYSF has a very short extracellular domain and a large intracellular domain containing seven calcium binding C2-domains, as well as, FerA and DysF domains that together mediate intracellular membrane fusion events, calcium homeostasis, and lipid metabolism.
  • DYSF is expressed in skeletal muscle where it mediates Ca2+-dependent vesicle fusion and repair of sarcolemma following the creation of membrane breaches by tensile forces during muscle contractions.
  • DYSFPMMs loss-of-function DYSF patient missense mutations
  • DYSFL1341P protein misfolding, aggregation in the endoplasmic reticulum (ER), and degradation by the proteasome. It was surmised that this and other DYSFPMMs may be a class of endomembrane trafficking defective mutants that could be rescued by chemical chaperones or correctors.
  • Phenylbutyrate (4-PBA) was first approved in 1996 to treat patients with urea cycle disorders. The metabolized drug complexes with toxic ammonia creating an alternate ammonia elimination pathway in these patients. 4-PBA has also been frequently described as a chemical chaperone based on its ability to strongly attenuate ER-stress and protein aggregate formation. More recently, 4-PBA combination therapy with taurursodiol showed positive findings in a clinical trial for ALS. Here, we found the effectiveness of 4-PBA on DYSFPMMs to be quite broad, restoring localization to 25 of 64 DYSFPMMs tested in our 2A-assay system.
  • the present disclosure provides methods of treating dysferlinopathy, comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting and ; or a pharmaceutically acceptable salt thereof.
  • the dysferlinopathy is limb girdle muscular dystrophy 2b, myoshi myopathy, or distal compartment myopathy.
  • the present disclosure provides methods of treating a muscle injury, comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting and or a pharmaceutically acceptable salt thereof.
  • the muscle injury is a muscle strain.
  • the muscle strain is a grade 1 strain, a grade 2 strain, or a grade 3 strain.
  • the subject is an athlete (e.g., a professional athlete).
  • the muscle injury occurred during a sport or in connection with a sport.
  • the muscle injury is acute. In certain embodiments, the muscle injury is chronic.
  • the present disclosure provides methods of treating or preventing muscle loss, comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting and ; or a pharmaceutically acceptable salt thereof.
  • the method treats muscle loss. In certain embodiments, the method prevents muscle loss.
  • the muscle loss is sarcopenia. In certain embodiments, the muscle loss is muscle atrophy arising from a lack of physical activity. In yet another aspect, the present disclosure provides methods of promoting muscle growth, comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting and ; or a pharmaceutically acceptable salt thereof.
  • the muscle is skeletal muscle. In certain embodiments of the methods disclosed herein, the method comprises administering pharmaceutically acceptable salt thereof.
  • the method comprises administering or a pharmaceutically acceptable salt thereof.
  • compositions and methods of the present invention may be utilized to treat an individual in need thereof.
  • the individual is a mammal such as a human, or a non-human mammal.
  • the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters.
  • the aqueous solution is pyrogen-free, or substantially pyrogen-free.
  • the excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs.
  • the pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like.
  • the composition can also be present in a transdermal delivery system, e.g., a skin patch.
  • the composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention.
  • physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent depends, for example, on the route of administration of the composition.
  • the preparation or pharmaceutical composition can be a selfemulsifying drug delivery system or a selfmicroemulsifying drug delivery system.
  • the pharmaceutical composition also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention.
  • Liposomes for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier 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 patient.
  • materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide;
  • a pharmaceutical composition can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin).
  • the compound may also be formulated for inhalation.
  • a compound may be simply dissolved or suspended in sterile water.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
  • Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients.
  • an active compound such as a compound of the invention
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • Compositions or compounds may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents,
  • pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose
  • the pharmaceutical compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions that can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art,
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body.
  • dosage forms can be made by dissolving or dispersing the active compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutan
  • Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.
  • active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Methods of introduction may also be provided by rechargeable or biodegradable devices.
  • Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals.
  • a variety of biocompatible polymers including hydrogels, including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient 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 therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound 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.
  • terapéuticaally effective amount is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).
  • a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.
  • the patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.
  • compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.
  • contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts.
  • contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, lH-imidazole, lithium, L- lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2- hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts.
  • contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, 1 -hydroxy -2-naphthoic acid, 2,2-dichloroacetic acid, 2- hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethan
  • the pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared.
  • the source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (
  • agent is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • Agents include, for example, agents whose structure is known, and those whose structure is not known. The ability of such agents to inhibit AR or promote AR degradation may render them suitable as “therapeutic agents” in the methods and compositions of this disclosure.
  • a “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).
  • Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results.
  • Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • preventing is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, 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.
  • a condition such as a local recurrence (e.g., pain)
  • a disease such as cancer
  • a syndrome complex such as heart failure or any other medical condition
  • prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.
  • administering or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art.
  • a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct).
  • a compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • a compound or an agent is administered orally, e.g., to a subject by ingestion.
  • the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.
  • the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents).
  • the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially.
  • an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.
  • a “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect.
  • the full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
  • a therapeutically effective amount may be administered in one or more administrations.
  • the precise effective amount needed for a subject will depend upon, for example, the subject’s size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.
  • the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not.
  • “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.
  • modulate includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity.
  • compositions, excipients, adjuvants, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable salt” or “salt” is used herein to refer to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.
  • pharmaceutically acceptable acid addition salt means any non-toxic organic or inorganic salt of any base compounds disclosed herein.
  • Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate.
  • Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form.
  • mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sul
  • the acid addition salts of compounds disclosed herein are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms.
  • the selection of the appropriate salt will be known to one skilled in the art.
  • Other non-pharmaceutically acceptable salts e.g., oxalates, may be used, for example, in the isolation of compounds disclosed herein for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
  • pharmaceutically acceptable basic addition salt means any non-toxic organic or inorganic base addition salt of any acid compounds represented disclosed herein, or any of their intermediates.
  • Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide.
  • Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.
  • stereogenic center in their structure.
  • This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30.
  • the disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01/062726.
  • Prodrug or “pharmaceutically acceptable prodrug” refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host after administration to form the compound of the present disclosure (e.g., compounds disclosed herein).
  • Typical examples of prodrugs include compounds that have biologically labile or cleavable (protecting) groups on a functional moiety of the active compound.
  • Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound.
  • prodrugs using ester or phosphoramidate as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference.
  • the prodrugs of this disclosure are metabolized to produce a compound disclosed herein.
  • the present disclosure includes within its scope, prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.
  • Log of solubility is used in the art to quantify the aqueous solubility of a compound.
  • the aqueous solubility of a compound significantly affects its absorption and distribution characteristics. A low solubility often goes along with a poor absorption.
  • LogS value is a unit stripped logarithm (base 10) of the solubility measured in mol/liter.
  • Example 1 Treatment of Dvsferlinopathies with Exemplary Compounds Development of a quantitative DYSF membrane localization assay
  • LV-TRE-DYSF-T2A-DsRed a bicistronic expression vector with an intervening T2A translational cleavage sequence was constructed with human DYSFWT upstream of the fluorescent reporter gene DsRed (FIG. 1A) allowing equimolar expression of both genes from a single mRNA.
  • DYSFWT-T2A a fusion protein with a small C-terminal 2A-epitope exposed on the cell surface (FIG. 1A) allowing unique detection (FIG. 3B) and quantitation of PM-resident DYSF (FIG. 3B) in live cells by flow cytometry using an anti- (a)-2A antibody (Ab).
  • This assay is termed the 2A-assay.
  • 113 DYSFPMMs were selected with assistance from the Jain Foundation Dysferlin Registry and engineered into the bicistronic vector system for 2A-assay analysis.
  • DYSFV67D DYSFR555W
  • DYSFL1341P that show little to no DYSF protein in patient muscle biopsies
  • the frequently occurring (1% allele frequency, dbSNP rs34999029) non-pathogenic DYSF SNP, DYSFA170E were examined by 2A-assay.
  • the ratio of PM-resident mutant DYSFPMM relative to DYSFWT among transfected DsRed positive cells was quantified by flow cytometry; while no significant difference was observed between DYSFWT and DYSFA170E, less than 14% of DYSFV67D, DYSFR555W, or DYSFL1341Pwere found at the cell surface.
  • Fluorescent immunocytochemical (ICC) analysis was performed on similarly transfected cells selected by FACS for DsRed. Cells were fixed and treated with DAPI, a-DYSF Ab, a-Na/K ATPase Ab, and an appropriate fluorescently conjugated secondary Ab in order to localize nuclei, DYSF, and HEK cell PM, respectively.
  • DYSFA170E was localized to the plasma membrane similar to DYSFWT, while the known pathogenic DYSFPMMs showed substantially reduced expression at the PM (FIG. 1C), supporting our 2A-assay results. In cells where discrete intracellular DYSF localization was detected it was often perinuclear, consistent with ER aggregation, as previously reported for DYSFL1341P and other DYSFPMMs.
  • DYSFPMMs which are found to be homozygous in 90 individuals from the Dysferlin Registry, 26 (72%) of these have 2A-expression levels less than 25% of DYSFWT and are absent from the PM by ICC indicating that this class holds predicative value for determining PMM pathogenicity. The remaining 10 PMMs are above the 25% 2A-assay threshold but less than DYSFWT. Of these, eight are visually localized to the PM by ICC, while R2042C and T252M are not, suggesting they are likely pathogenic (FIG. 2).
  • I1607T, R1331L, and K1526T are likely being benign - I1607T and R1331L are found in individuals with other homozygous PMMs with 2A-values ⁇ 0. 25, and K1526T is cis with another pathogenic DYSF variant.
  • a 96-well 2A-assay-based chemical screening platform was developed to identify compounds capable of restoring PM-localization to the 64 DYSFPMMs with 2A-assay values ⁇ 25% of DYSFWT. 10 compounds were tested that previously reported to rescue misfolding of other disease relevant proteins in the ER, such as the cystic fibrosis transmembrane conductance regulator (CFTR), and a-sarcoglycan.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the drug 4-phenylbutyric acid (4-PBA) was also tested as it was used for the treatment of urea cycle disorder diseases, which has also been proposed to act as a chemical chaperone for diseases involving misfolded missense mutants, such as the Ryanodine Receptor (RYR) and the CFTR D508 mutant, and an ER-stress reducer improving outcomes in ALS and myopathies.
  • urea cycle disorder diseases which has also been proposed to act as a chemical chaperone for diseases involving misfolded missense mutants, such as the Ryanodine Receptor (RYR) and the CFTR D508 mutant, and an ER-stress reducer improving outcomes in ALS and myopathies.
  • HEK cells transiently expressing DYSFWT or the 64 DYSFPMMs mutants below 25% DYSFWT and DsRed+ sorted were treated with compounds for 24hrs prior to the 2A-assay and scored for elevated expression of PM-localized DYSFPMMs above 25% (FIG. 3A).
  • 4- PBA boosted expression above the 25% threshold in 25/64 DYSFPMMs (FIG. 3A).
  • corr-2b rescued DYSFW992R, DYSFE1335G, DYSFL1341P, and DYSFF1867L (FIG 3A). These 4 DYSFPMMs were also rescued and to a greater degree by 4-PBA.
  • GREG cells are a naturally immortalized DYSF-deficient mouse myoblast cell line derived from A/J mice.
  • a functional assay was developed using in vitro differentiated GREG myotubes transfected with DYSF expression vectors.
  • DsRed positive GREG cells were isolated by FACS following transfection with either DYSFWT, DYSFL1341P, sorted for DsRed+cells and plated in media to promote myoblast fusion. The resulting myotubes were assayed for DYSF-mediated membrane repair capacity following laser damage in the presence of the membrane impermeant fluorescent dye FM1-43 and calcium.
  • DYSF-deficient GREG myotubes The repair deficiency of non- transfected DYSF-deficient GREG myotubes was evidenced by the rapid and continuous FM1- 43 dye influx following membrane laser wounding. In stark contrast, rapid membrane resealing was observed in GREG myotubes expressing DYSFWT. Membrane wounding of GREG myotubes expressing DYSFL1341P resulted in rapid and sustained dye influx indicating significant repair deficiency. GREG myotubes expressing DYSFL1341P did display some residual repair capacity compared to non-transfected myoblasts; high level expression of DYSFL1341P could result in some membrane localization of this mutant, which is known to be repair proficient when at the membrane.
  • MMex38 mice are homozygous for mDYSFL1360Pwhich is analogous to the PMM DYSFL1341P. Histologically, MMex38 mice have no PM DYSF expression in mouse skeletal-muscle and display age-progressive dystrophic histological phenotypes consistent to that seen in dysferlinopathy patients. Extensor digitorum longus (EDL) muscle from MMex38 and C57BL/6NJ (+/+) control animals were explanted and treated in culture with vehicle or ImM 4-PBA for 24 hrs.
  • EDL Extensor digitorum longus
  • DYSFL1360P was completely absent in histological cross-sections of vehicle-treated EDL muscle, however, DYSFL1360Pexpression and localization to muscle sarcolemma was partially restored after EDL muscle explant after 24 hour treatment with ImM 4-PBA. Dramatically, while myofibers from untreated MMex38 EDL explants were membrane repair deficient following laser injury, 24hr treatment with 4-PBA fully restored membrane resealing function, similar to EDL myofibers from either treated or untreated age-matched wild type animals (FIGs. 4C, 4D).
  • 4-PBA (2 mg/mL) was administered for 48 hours in the drinking water of MMex38 and control animals and assayed DYSF localization and membrane repair in EDL muscle explants. Histological cross sections of EDL muscle from wild type animals (+/+) showed clear sarcolemma localization of DYSFWT, which was enhanced by 24hr 4-PBA treatment (FIG. 4E), similar to HEK cells expressing DYSFWT treated with 4-PBA, above.
  • Muscle fiber repair activity was robust in both 4-PBA and untreated wild type mice (FIGS 4F & 4G).
  • EDL muscle fibers from untreated MMex38 mutant mice were devoid of EDL myofiber repair activity (FIGs. 4F & 4G).
  • two days of 4-PBA administration fully restored membrane repair activity to mutant myofibers (FIGs. 4F & 4G), resulting in membrane repair kinetics similar to wild type animals. Therefore the oral administration of 4-PBA is remarkably effective in rescuing the defects of DYSFL1360P (human DYSFLL1341P) and likely can do the same for other DYSFPMMs.
  • 4-Phenyl butyric acid (4-PBA) was purchased from Sigma.
  • N-phenyl-4-(4- vinylphenyl)thiazol-2-amine (corr-2b) was purchased from Exclusive Chemistry, both were dissolved in dimethyl sulfoxide (DMSO; Sigma).
  • Control experiments contained the solvent DMSO added at the same concentration as that used with the stimulatory agents.
  • sodium phenylbutyrate United States Pharmacopeia (USP) Reference Standard the organic sodium salt of 4-PBA, was purchased from Sigma.
  • HEK293T were purchased from the American Type Culture Collection. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Genesee Scientific) containing 10 % Fetal Bovine Serum (FBS, Avantor Seradigm), 100 units/mL penicillin, and 100 pg/mL streptomycin (Thermo Fisher Scientific); culture media was changed every 2 days.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS Fetal Bovine Serum
  • penicillin 100 units/mL
  • streptomycin Thermo Fisher Scientific
  • GREG cells a DYSF -deficient myogenic cell line established from the A/J mouse (30), were cultured in DMEM/Glutamax containing 20 % FBS, 1 mM sodium pyruvate (Sigma), 0.5 % chick embryo extract (Thermo Fisher Scientific), and 100 pg/mL Primocin (InvivoGen). Cells were washed with PBS and cultured in DMEM + 5 % horse serum, 1 mM sodium pyruvate (Sigma), and 100 pg/mL Primocin (InvivoGen) to induce cell fusion and myotube differentiation; media was changed daily.
  • HEK cells were cultured at 37 °C in 5 % CO2 and passaged following phosphate- buffered saline (PBS, pH 7.4, Genesee Scientific) rinse and trypsinization with PBS + 0.25 % trypsin-EDTA in Hanks' Balanced Salt Solution (Corning) for 5 min at 37°C.
  • HEK cells were grown to sub confluence and passaged at a ratio of 1 :4 - 1:8.
  • GREG cells were grown to 50% confluence and passaged at a ratio of 1 :4.
  • HEK cells were seeded in 96-well plates at 5,000 cells/well, after 24 hours.
  • Cell media was supplemented with compounds or DMSO (Sigma) vehicle control and incubated for 24 hours before cell viability was measured using Cell Titer 96TM AQueous Assay Kit (Promega) according to the manufacturers specifications.
  • LV-TRE-DYSF-T2A-DsRed was constructed by Gibson Assembly using plasmids LV- TRE-VP64 human MyoD-T2A-dsRedExpress2, a gift from Charles Gersbach (Addgene plasmid #60629) (15) and pCI FLAG-hDysferlin (gift from Dr. Robert Brown, University of Massachusetts, Worcester), containing human dysferlin cDNA (isoform 8, NCBI accession NM_003494) with an N-terminal 18 amino acid flag-tag-containing sequence.
  • LV-TRE-DYSFPMM-T2A-DsRed expression vectors were subsequently generated.
  • PMMs were introduced into each of two products via the PCRIPMMReverse primer or PCR2PMMForward primer used for PCR synthesis using LV-TRE-DYSF-T2A-DsRed as the PCR DNA template.
  • the PCRIPMMForward primer and PCRIPMMReverse primers contain vector homology encompassing each restriction site necessary for Gibson assembly.
  • PCRIPMMand PCR2PMM were generated in 25 pL reactions with 5ng pCI with 10 PCR cycles (95°C 15 sec, 60°C 30 sec, 72°C T) using primer pair and Kapa HiFi HotStart PCR Master Mix (Roche).
  • the two corresponding PCR products were combined and purified using a PCR purification kit (Qiagen) and eluted in 30pL.
  • DYSFPMMs was introduced into LV-TRE- DYSFWT-T2A-DsRed by Gibson assembly of the stitched and amplified PMM-containing PCR product with gel purified linear LV-TRE-DYSFWT-T2A-DsRed vector DNA digested with specific restriction enzymes for each PMM. Gibson assemblies were performed in 1 OpL lx Gibson Assembly Master Mix (NEB) with 100 ng of linearized vector and approximately 10-20ng of purified PCR product. Reactions were incubated at 50°C for 6 hours prior to drop dialysis using a 0.025mM type-VS membrane filter (Millipore, Inc. #VSWP 0250) and electroporation into NEB Stable E.
  • NEB OpL lx Gibson Assembly Master Mix
  • DNA for transfection was prepared by electroporation into NEB Stable cells, overnight growth at 30°C with carbenicillin (100 pg/pL), and DNA purification using HiSpeed Plasmid Midi Kit (Qiagen).
  • HEK cells were seeded at a density of 6 c 105cells/well and GREG cells at 3 c 105cells/well in 6-well plates and incubated for 24hrs.
  • HEK cells were transfected with plasmid DNA (1.5pg) using calcium dichloride (120 pmol/mL, Sigma) in HEPES buffer (Sigma) with media change after 4hr of incubation, and collection 48hr later for flow cytometry.
  • GREG cells were transfected with plasmid DNA (1.5pg) and ViaFect Transfection Reagent (E4981, Promega, USA) using the 3:1 low-volume manufacturer’s protocol with subsequent culture in 2 pg/mL doxycycline.
  • DYSFPMM Flowing Software 2 (Cell Imaging Core, Turku Centre for Biotechnology. 2A-expression values for any given DYSFPMM is the ratio of (the calculated geometric mean of Alexa-647 fluorescence signal relative to the geometric mean of DsRed signal from the same live (non-DAPI stained) transfected DsRed positive cells) compared to (the same set of ratios of geometric means obtained from cells transfected with DYSFWT).
  • transiently transfected HEK cells were plated a density of 5 x 104cells/well into 96-well plates. After incubation for 24hr, compounds were added at indicated concentration in DMEM with 2% FBS. After a further 24hr incubation with compound, cells were trypsinized for 1 min and Ab treatments were carried out in 96-well U- bottom plates prior to flow cytometry analysis.
  • Membranes were incubated in Block Ace (BioRad) at 4°C overnight and then with anti-NCL- Hamlet DYSF (mouse, 1:2000, Leica), anti-RFP (DsRED, rabbit, 1:1000, Thermo Scientific) and anti-GAPDH (rabbit, 1 : 5000, Abeam) as primary monoclonal antibodies.
  • Membranes were washed 3 times in PBS with 0.1% Tween-20, then with horseradish peroxidase (HRP)- conjugated anti-rabbit IgG (1:5000, Cell Signaling Technology) or anti-mouse IgG (1:10000, GE Healthcare Life Science) as secondary antibodies. All antibody incubations were performed at room temperature (RT) for 1 hr.
  • HRP horseradish peroxidase
  • DsRed positive transfected cells and EDL muscle sections were fixed in pre-chilled ethanol for 10 min at -20°C, washed in PBS containing 0.1% Triton X-100 for 10 min, blocked with 3% bovine serum albumin (BSA, Sigma) at RT for 60 min, and incubated at RT for 1 hr with primary antibodies, anti-NCL-Hamlet DYSF (1:200), anti-Romeo DYSF (1:200), anti sodium potassium ATPase (1 :250, EP1845Y, Abeam) for plasma membrane localization, anti- calreticulin (EPR3924, Abeam) for ER localization, and anti-2 A peptide.
  • BSA bovine serum albumin
  • Plasma membrane repair capacity of myotubes and myofibers can be measured by the kinetics of intracellular uptake of the normally membrane impermeant dye FM1-43 following membrane laser wounding.
  • the DYSF deficient GREG myoblast cell line was used to generate myotubes.
  • DsRed positive cells of transiently transfected GREG cells were sorted by FACS (Aria, BD Biosciences) and seeded in a chambered coverglass (Nunc Lab-Tek II 155409PK, Thermo Scientific, USA). After an hour, culture media was changed to differentiation medium and incubated for 48 hrs to obtain myotubes. Prior to assay, the culture media was removed and switched to PBS with ImM calcium dichloride, glucose (4 mg/mL), and FM1-43 (equivalent to FM®1-43FX, 5 ng/pL, Biotium).
  • Plasma membrane repair capacity of GREG myotubes and myofibers can be measured by the kinetics of intracellular uptake of the normally membrane impermeant dye FM1-43 following membrane laser wounding.
  • DsRed positive GREG myoblasts are sorted by flow cytometry (FACS Aria) and seeded in a chambered cover glass (Nunc Lab-Tek II 155409PK, Thermo Scientific, USA). After an hour, culture media is changed to differentiation medium and incubated for 48hrs to obtain myotubes. Prior to assay, the culture media is removed and switched to PBS with ImM calcium dichloride, glucose (4mg/mL), and FM1-43 (5ng/pL, Biotium).
  • Membrane repair assays and imaging are performed at RT with the chambered cover glass mounted on a confocal microscope (Olympus FV1200, 63x, 0.9 NA, oil immersion objective with laser scanning at 488nm).
  • a region of interest (ROI) on the PM was irradiated with a 405 nm laser at 60-100% power (15 mW) using the photo-activation mode for 5-10 seconds, depending upon myotube treatment conditions.
  • Image analysis of dye uptake was done using Fiji using the method described in Humphrey GW et. al.
  • Fluorescence intensity was normalized by the pre-irradiation intensity and the normalized response (AF/F0).
  • mice All animal procedures were performed according to Massachusetts Institute of Technology Committee on Animal Care. Mice were fed standard rodent chow diet and housed in a facility with 12 hr light and dark cycles. MMex38 mice were obtained from Dr. Simone Spuler (Max Delbruk Center, Berlin). MMex38 animals were rederived by embryo implantation and housed in the MIT mouse facilities. Mouse genotyping was performed by Transnetyx genotyping service using primer pairs and genotyping protocol described for MMex38 mice. MMex38 mice were backcrossed once to their progenitor strain C57BL/6NJ obtained from Jackson Laboratories and homozygous DYSFL1360P/DYSFL1360Pand +/+ lines were established from sibling F2 progenies. Experiments were performed with age- matched siblings of the same gender for each genetic line.
  • EDL muscle was surgically isolated from euthanized MMex38 or C57BL/6NJ male mice of specified ages and placed in solution, treated with collagenase for 1.5 hours and then pipetted to disrupt the tissue, and then put into a culture dish with culture medium and 4-PBA (1 mM) or DMSO for 24 hr.
  • 4-PB A treatment in vivo Sodium phenyl butyrate (USP, Sigma) was dissolved in sterile drinking water at a concentration of 2 mg/mL. C57BL6/NJ (+/+) mice and MMex38 mice were treated ad-libitum for 48 hours either with drug or water alone. Following treatment, mice were sacrificed, EDL muscle isolated, and fresh frozen sections prepared for histological analysis. DYSF staining was done using Romeo a-DYSF-l°Ab and Alexa 488 a-rabbit fluorescent 2° Ab and DAPI staining was performed for nuclear localization. To perform the membrane repair assay of mouse muscle fibers, the assay medium contained FM1-43 dye (5 ng/pL, Biotium).

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Abstract

La divulgation concerne des méthodes de traitement de lésions musculaires ou de perte musculaire, ou de maladies associées à celles-ci, telles que la dysferlinopathie.
PCT/US2022/035701 2021-06-30 2022-06-30 Compositions et méthodes de traitement de la dysferlinopathie WO2023278684A1 (fr)

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Citations (3)

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WO2004050076A1 (fr) * 2002-11-28 2004-06-17 Centre National De La Recherche Scientifique Cnrs Utilisation d'un inhibiteur d'histone deacetylase pour le traitement des dystrophies musculaires
US9526742B2 (en) * 2012-07-19 2016-12-27 Genethon Use of epigenome-modifying compounds for the treatment of genetic muscular diseases linked to a protein-conformational disorder
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WO2004050076A1 (fr) * 2002-11-28 2004-06-17 Centre National De La Recherche Scientifique Cnrs Utilisation d'un inhibiteur d'histone deacetylase pour le traitement des dystrophies musculaires
US9526742B2 (en) * 2012-07-19 2016-12-27 Genethon Use of epigenome-modifying compounds for the treatment of genetic muscular diseases linked to a protein-conformational disorder
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EMERY, C. A.: "Does decreased muscle strength cause acute muscle strain injury in sport? A systematic review of the evidence", PHYSICAL THERAPY REVIEWS, vol. 4, no. 3, 1 September 1999 (1999-09-01), pages 141 - 151, XP009542382, ISSN: 1083-3196, DOI: 10.1179/ptr.1999.4.3.141 *
TOMINAGA KANA, TOMINAGA NAOOMI, WILLIAMS ERIC O., RUFIBACH LAURA, SCHÖWEL VERENA, SPULER SIMONE, VISWANATHAN MOHAN, GUARENTE LEONA: "4-Phenylbutyrate restores localization and membrane repair to human dysferlin mutations", ISCIENCE, CELL PRESS, US, vol. 25, no. 1, 21 January 2022 (2022-01-21), US , pages 103667 - 103667, XP093021493, ISSN: 2589-0042, DOI: 10.1016/j.isci.2021.103667 *
VIANELLO SARA, HUA YU, VINCENT VOISIN, HAFEDH HADDAD, XUN HE, ARTHUR S. FOUTZ, CATHERINE SEBRIÉ, BRIGITTE GILLET, MORGANE ROULOT, : "Arginine butyrate: a therapeutic candidate for Duchenne muscular dystrophy", THE FASEB JOURNAL, vol. 27, 21 February 2013 (2013-02-21), pages 2256 - 2269, XP093021491, DOI: 10.1096/fj.12-215723 *

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