New therapeutic uses of type 2 iodothyronine deiodinase (D2) inhibitors

Abstract

The use of type 2 iodothyronine deiodinase (D2) inhibitor compounds in the therapeutic treatment of muscle wasting and / or muscle and / or skin regeneration therapy is described. Pharmaceutical compositions comprising type 2 iodothyronine deiodinase (D2) inhibitor compounds in a pharma- ceutical acceptable carrier, and in combination with optional excipients, diluents and adjuvants, are also described.

Description

[Technical field]

[0001] The present invention relates to the field of therapeutic treatment of muscle and skin disorders, in particular muscle wasting disorders and muscle or skin tissue injuries. [Background technology]

[0002] Skeletal muscle atrophy, also known as muscle wasting, is a debilitating condition that develops slowly with age (sarcopenia) and after prolonged inactivity or fasting, or appears rapidly in various pathologies such as cancer (cachexia), muscle denervation, chronic kidney disease, heart failure, chronic obstructive pulmonary disease, sepsis, diabetes, liver cirrhosis, cystic fibrosis, or dystrophies, which are a group of inherited diseases that lead to muscle wasting by limiting motor skills.

[0003] Muscle wasting diseases are characterized by a massive and progressive loss of muscle mass, resulting in loss of muscle strength and muscle function. In general, the loss of muscle mass leads to a deterioration in the patient's physical condition, ranging from postural instability to impaired respiratory and cardiac capacity. For example, approximately 80% of advanced cancer patients show severe loss of muscle mass, a condition called cancer cachexia. In these cancer patients, loss of muscle mass is associated with poor prognosis and poor response to treatment.

[0004] Because muscle function is essential for breathing, movement, chewing, and swallowing, reduced muscle mass and function is associated with greater morbidity and mortality, and reduced quality of life.

[0005] Despite the large number of people suffering from any kind of muscle wasting, especially those caused by cachexia and sarcopenia, muscle wasting and muscle remains an undertreated organ.To date, there are no effective drugs available to improve muscle mass and function that have been proven to be able to interfere with the pathophysiological mechanisms (i.e., systemic inflammation) that underlie the loss of muscle wasting in muscle wasting diseases.

[0006] Current treatments for cachexia-sarcopenia are based on three different types of drugs: ghrelin receptor agonists (anamorelin); monoclonal antibodies against inflammatory mediators responsible for cachexia and muscle wasting (e.g., antibodies against IL-1, IL-6, TNF-α, myostatin); and selective nonsteroidal androgen receptor modulators (SARMs, e.g., enobosarm). The above drugs have been tested in phase 2 and phase 3 clinical trials (Becker C. et al., Lancet Diabetes Endocrinol. 2015 3(12):948-57 2015; Temel et al., Lancet Oncol. 2016; 17: 519-531). All of them showed pharmacoprophylactic effects against muscle loss and weight gain. However, so far there is no clinical data showing the ability of the tested drugs to increase muscle strength and improve muscle physical-mechanical function.

[0007] In addition to cachexia and sarcopenia, physical injury can also lead to loss of muscle mass. Muscle injury is usually the result of external forces such as muscle strains and muscle tears. Large muscle injuries have limited healing capacity, and the repair process usually results in the formation of scar tissue. The presence of intramuscular scars alters the normal muscle contraction vector, reducing strength and increasing fatigue.

[0008] Mechanical or chemical trauma can also damage the skin. Skin damage is most commonly associated with burns, lacerations, fractures, and crush injuries. Typically, such skin disorders can result in the loss of epidermis, and often the loss of part of the dermis and even subcutaneous fat. The treatment of these injuries can be complex and can have a significant impact on the function and aesthetics of patients. In addition, impaired skin healing process can cause chronic wounds, which represent a large and growing disease burden.

[0009] As mentioned above, muscle and skin damage can cause functional impairment and severe disability, as well as cosmetic deformity. As a result, these injuries create ongoing reconstruction and regeneration challenges in clinical work to enable tissue regeneration and reduce transplant complications.

[0010] Different strategies have been developed during the last century, and especially during the last few decades, to promote muscle and skin repair and regeneration, including surgical techniques, physical therapy, biomaterials, muscle tissue engineering, and cell therapy. However, the inherent limitations of current approaches highlight the need for alternative strategies. Summary of the Invention

[0011] There is therefore a strong need to provide new and effective therapeutic strategies aimed at slowing or preventing the progression of diseases involving muscle wasting, in particular age-related muscle degeneration as well as muscle wasting conditions secondary to pathologies.

[0012] There is also a strong need to provide new pharmacological approaches aimed at ameliorating muscle and / or skin damage by promoting tissue repair and tissue function regeneration.

[0013] These and other needs are met by an inhibitor compound of type 2 iodothyronine deiodinase (D2) for therapeutic use as defined in the accompanying claim 1, and a pharmaceutical composition comprising a D2 inhibitor compound as defined in the accompanying claim 7. Preferred embodiments of the invention are defined in the dependent claims.

[0014] The accompanying independent and dependent claims form an integral part of this specification.

[0015] Type 2 iodothyronine deiodinase (D2) is an enzyme that plays a key role in maintaining circulating and tissue levels of thyroid hormones. As known in the art, intracellular thyroid hormone concentrations are regulated by three seleno-membrane proteins, namely type 1, type 2, and type 3 deiodinases (D1, D2, D3) (Gereben B. et al, Endocr Rev. 2008 29(7):898-938). In particular, D1 and D2 convert the prohormone thyroxine (T4) to the active hormone T3. Conversely, D3 converts T3 (triiodothyronine) to T2 (diiodothyronine) and T4 to rT3 (reversed triiodothyronine), both of which are inactive thyroid hormone metabolites.

[0016] As described in more detail in the Experimental section below, the inventors observed for the first time that type 2 deiodinase (D2) is expressed in mitotically arrested stem cells of both muscle and skin and showed that this enzyme plays an important role in maintaining the quiescent state of these cells.

[0017] As known in the art, stem cells are important for the regeneration and homeostasis of adult tissues. Further experiments carried out by the present inventors have revealed that upon muscle injury, especially in acute regeneration conditions such as cardiotoxin-induced muscle injury and wound healing, genetic in vivo depletion of D2 improves and promotes the regeneration of muscle and skin tissues compared to wild-type animals.

[0018] By further investigating the role of type 2 deiodinase in muscle and skin, the inventors surprisingly found that expression of this enzyme was dramatically increased in cachectic muscle in an animal model of muscle atrophy. Moreover, in a genetic D2-depleted mouse model, the absence of type 2 deiodinase activity suppressed experimentally induced atrophy of skeletal muscle.

[0019] To further evaluate the effect of D2 regulation in muscle and skin, and to exploit the role of this enzyme as a potential clinical target, we performed dedicated in vitro and in vivo pharmacological studies by transiently blocking D2 activity using various inhibitor compounds.As shown in Figures 9, 10 and 11, mice treated with D2 inhibitor compounds were protected from denervation and cancer cachexia-induced muscle loss, consistent with the results obtained in preclinical models of atrophy and included herein.Notably, a significant increase in survival rate was also observed in tumor-bearing mice (Figure 10I).

[0020] Furthermore, in animal models of muscle or skin injury, administration of D2 inhibitor compounds resulted in a significant increase in the number of muscle fibers and activated myoblasts in the injured muscle of treated mice compared to control animals (Figure 7), as well as a significantly more rapid and efficient wound healing process following skin injury (Figure 8).

[0021] As further validation, we observed that in a Drosophila model of muscular dystrophy, chronic administration of a D2 inhibitor compound to flies allowed functional restoration of skeletal muscle contractility (FIGS. 12 and 13).

[0022] Without wishing to be bound by any theory, the inventors believe that activation of thyroid hormone (TH) action in muscle tissue by type 2 deiodinase enzyme is important in inducing accelerated muscle catabolism that leads to muscle loss in several disease states. The inventors further propose that D2 expression marks quiescent stem cells in muscle and skin, and that acute D2 depletion in quiescent stem cells leads to G 0 From G Alert State, i.e., G 0 Phase and G 1 We believe that our findings induce their spontaneous transition into an intermediate state of the cell cycle between the mitotic and mitotic phases, in which stem cells enter the cell cycle more rapidly in response to injury.

[0023] Based on all the surprising findings presented above, attenuation of D2 enzyme activity at tissue level provides a novel and highly effective therapeutic means to protect muscles from atrophy and / or promote the regeneration of damaged muscle and / or skin tissue.

[0024] Thus, a first aspect of the present invention is an inhibitor compound of type 2 iodothyronine deiodinase (D2) for use in the therapeutic treatment of muscle wasting and / or in muscle and / or skin regeneration therapeutic methods.

[0025] As used herein, the term "inhibitor compound" refers to a substance capable of reducing and / or blocking the activity of an enzyme by binding to the protein and modifying the catalytic properties of the enzyme. Unless otherwise specified or made clear, the expression "inhibitor compound" is understood to encompass both selective and non-selective type 2 iodothyronine deiodinase inhibitors.

[0026] As used herein, unless otherwise specified, the term "therapeutic treatment" refers to administration of a type 2 iodothyronine deiodinase (D2) inhibitor compound according to the present invention after the onset of symptoms of a disease state, including muscle wasting, as well as prior to the onset of symptoms, particularly to subjects at risk of the disease.

[0027] The term "regenerative therapy," as used herein, refers to therapeutic treatments aimed at re-growing, repairing, or replacing damaged or diseased cells, tissues, or organs in a subject.

[0028] In one embodiment, inhibitor compounds of type 2 iodothyronine deiodinase (D2) suitable for use according to the present invention are selected from the group consisting of inverted triiodothyronine (rT3), amiodarone (AMIO), desethylamiodarone (DEA), 5-methyl-2-thiouracil (MTU), 6-benzyl-2-thiouracil (BTU), xanthohumol (XTH), genistein (GEN), 6-propyl-2-thiouracil (PTU), methimazole (MMI), iopanoic acid (IAc), dexamethasone, gold thioglucose (GTG), and any combination thereof.

[0029] Preferred D2 inhibitor compounds according to the present invention are disclosed in Table 1 together with the D2 sensitivity index for inhibition by these compounds and relevant references.

[0030] [Table 1]

[0031] Preferably, the inhibitor compound of type 2 iodothyronine deiodinase (D2) for use according to the present invention is selected from reversed triiodothyronine (rT3), amiodarone (AMIO) and desethylamiodarone (DEA), more preferably selected from reversed triiodothyronine (rT3) and amiodarone (AMIO).

[0032] Inverted triiodothyronine compound (rT3, 3,3',5'-triiodothyronine, CAS number: 5817-39-0) is a metabolically inactive form of thyroid hormone, produced from the thyroxine (T4) prohormone by removal of the iodine atom in the inner ring of T4 via the type 3 5'-deiodinase enzyme.

[0033] Amiodarone (AMIO) is an iodized benzofuran derivative known primarily for its approved uses in the treatment of cardiac disease. The drug is a potent class III antiarrhythmic agent capable of inducing prolongation of the action potential and refractory period in the heart.

[0034] Desethylamiodarone (DEA) is the major bioactive metabolite of amiodarone and is produced in an N-demethylation reaction catalyzed by cytochrome P450 3A4.

[0035] A preferred embodiment according to the present invention is inverted triiodothyronine (rT3) for use in the therapeutic treatment of disease states such as cachexia, sarcopenia or muscle wasting due to muscle denervation.

[0036] Another preferred embodiment according to the present invention is inverted triiodothyronine (rT3) for use in muscle and / or skin regeneration treatment methods.

[0037] Yet another preferred embodiment according to the present invention is amiodarone (AMIO) for use in the therapeutic treatment of disease states such as cachexia, sarcopenia or muscle wasting due to muscle denervation.

[0038] Yet another preferred embodiment according to the present invention is amiodarone (AMIO) for use in muscle and / or skin regeneration treatment methods.

[0039] According to the present invention, it is understood that any possible combination of inhibitor compounds of type 2 iodothyronine deiodinase (D2) as defined above is encompassed by the present invention.

[0040] For example, in one embodiment of the present invention, rT3 is used in combination with 6-propyl-2-thiouracil (PTU). Advantageously, in this embodiment, PTU blocks hepatic D1 activity in the liver, thus increasing the half-life of rT3.

[0041] As mentioned above, therapeutic approaches based on the administration of D2 inhibitor compounds are particularly suitable for the treatment of disease states involving loss of muscle mass and strength, such as, for example, secondary wasting pathologies (cachexia) or age-related muscle degeneration (sarcopenia).

[0042] In addition to protecting against muscle atrophy, pharmacological blocking of type 2 deiodinase activity results in stimulation of muscle and skin stem cell proliferation, thereby making tissue-specific modulation of the D2 enzyme a valid and innovative tool in the therapeutic context of regenerative medicine.

[0043] By way of example and not limitation, muscle wasting according to the present invention may result from a disease state selected from the group consisting of sarcopenia, cancer, sepsis, diabetes, chronic heart failure, chronic obstructive pulmonary disease, chronic renal failure, cirrhosis of the liver, cystic fibrosis, muscle denervation, fasting, and any combination thereof.

[0044] A preferred embodiment of the present invention relates to a therapeutic method aimed at controlling neoplastic cachexia.

[0045] With respect to muscle regeneration treatment methods, exemplary diseases that can be treated according to the present invention include, but are not limited to, delayed onset muscle soreness (DOMS), muscle strains, pulled muscles, muscle tears, cancer cachexia, muscle atrophy, muscular dystrophy, and any combination thereof.

[0046] In the context of skin regeneration therapeutic methods, examples may include, but are not limited to, therapeutic treatment of ulcers, dermatitis, skin wounds, burns, lacerations, and any combination thereof.

[0047] The type 2 iodothyronine deiodinase (D2) inhibitor compounds for use according to the present invention may also be effectively administered in the form of a pharmaceutical composition.

[0048] Thus, a second aspect of the present invention is a pharmaceutical composition for use in the therapeutic treatment of muscle wasting and / or in a muscle and / or skin regeneration treatment method, said pharmaceutical composition comprising a therapeutically effective amount of at least one inhibitor compound of type 2 iodothyronine deiodinase (D2) as defined above and a pharma- ceutical acceptable carrier, excipient and / or diluent.

[0049] As used herein, the term "therapeutically effective amount" refers to an amount of a D2 inhibitor compound for use according to the invention sufficient to exhibit a detectable therapeutic effect. The precise effective amount for a subject will depend on the subject's size and health, and the nature and severity of the condition being treated.

[0050] The term "pharmacologically acceptable" refers to compounds and compositions which may be administered to a mammal without undue toxicity at concentrations consistent with effective activity of the active ingredient.

[0051] The selection of suitable carriers, excipients and / or diluents for the pharmaceutical composition of the present invention can be determined by the person skilled in the art by using their knowledge in the art.

[0052] Preferably, the at least one inhibitor compound of type 2 iodothyronine deiodinase in the pharmaceutical composition for use according to the invention is selected from inverted triiodothyronine (rT3), amiodarone (AMIO), desethylamiodarone (DEA), 5-methyl-2-thiouracil (MTU), 6-benzyl-2-thiouracil (BTU), xanthohumol (XTH), genistein (GEN), 6-propyl-2-thiouracil (PTU), methimazole (MMI), iopanoic acid (IAc), dexamethasone, gold thioglucose (GTG), and any combination thereof.

[0053] A preferred pharmaceutical composition according to the present invention comprises inverted triiodothyronine (rT3), which is suitable for use in the therapeutic treatment of muscle wasting due to disease states such as cachexia or sarcopenia.

[0054] Another preferred pharmaceutical composition according to the present invention comprises inverted triiodothyronine (rT3), which is suitable for use in muscle and / or skin regeneration treatment methods.

[0055] Yet another preferred pharmaceutical composition according to the present invention comprises amiodarone (AMIO), which is suitable for use in the therapeutic treatment of muscle wasting due to disease states such as cachexia and sarcopenia.

[0056] Yet another preferred pharmaceutical composition according to the present invention comprises amiodarone (AMIO) and is suitable for use in muscle and / or skin regeneration treatment methods.

[0057] It is also contemplated herein that any combination of the above type 2 iodothyronine deiodinase (D2) inhibitor compounds can be present in the pharmaceutical compositions of the present invention.

[0058] In one embodiment, a pharmaceutical composition for use according to the invention comprises rT3 in combination with any of amiodarone (AMIO), desethylamiodarone (DEA), 6-propyl-2-thiouracil (PTU), and iopanoic acid (IAc).

[0059] In another embodiment, pharmaceutical compositions for use according to the invention comprise amiodarone in combination with any of rT3, desethylamiodarone (DEA), 6-propyl-2-thiouracil (PTU), and iopanoic acid (IAc).

[0060] The pharmaceutical compositions for use according to the present invention are suitable to be administered as a therapeutic treatment for muscle-related and / or skin-related diseases and / or to repair muscle and / or skin tissue damage, all as defined above for the therapeutic uses of D2 inhibitory compounds.

[0061] Pharmaceutical compositions for use according to the invention may be formulated into any suitable dosage form, for example for administration by topical, oral, enteral or parenteral routes.

[0062] As used herein, parenteral administration includes, but is not limited to, administration of pharmaceutical compositions by subcutaneous and intramuscular injection, implantation of sustained release depots, and intravenous injection administration.

[0063] Pharmaceutical compositions for use according to the invention may be administered by topical application in a solution, cream, ointment, spray, gel or any topical application. The pharmaceutical compositions of the invention may also be administered transdermally by means of drug eluting devices such as gauzes, patches, pads, or sponges.

[0064] The pharmaceutical compositions of the present invention can also be formulated into dosage forms for oral administration, such as tablets, capsules, powders, granules, solutions, suspensions, syrups, and the like.

[0065] In a pharmaceutical composition, the amount of active ingredient administered can vary widely according to considerations such as the particular compound and dosage unit used, the mode and time of administration, the duration of treatment, the age, sex, and general condition of the subject being treated, the nature and extent of the condition being treated, the rate of drug metabolism and excretion, potential drug combinations and drug-drug interactions.

[0066] Pharmaceutical compositions for use according to the present invention may be administered as a single dose or multiple doses, and as frequently as needed and for as long as needed to achieve the desired therapeutic effect. Those skilled in the art can readily determine the appropriate course of treatment using pharmaceutical compositions for use according to the present invention.

[0067] According to embodiments in which the pharmaceutical composition comprises inverted T3, the treatment regimen is based on administering to the patient a daily dose of rT3 preferably comprised between 50 micrograms (μg) and 500 μg.

[0068] According to another embodiment, in which the pharmaceutical composition comprises amiodarone, preferably the treatment regimen is based on administering to the patient a daily dose of amiodarone comprised between 100 mg and 700 mg, more preferably a daily dose of amiodarone comprised between 200 mg and 600 mg.

[0069] A preferred treatment regimen according to this embodiment involves orally administering to the patient a regimen consisting of a daily dose of 600 mg amiodarone during the first two weeks of treatment, a daily dose of 400 mg amiodarone during the third week of treatment, and a daily dose of 200 mg amiodarone during the maintenance period of treatment.

[0070] As is known in the art, amiodarone therapy can potentially result in a variety of side effects. Advantageously, this treatment regimen reduces the occurrence of drug-related side effects without affecting the efficacy of amiodarone. [Brief description of the drawings]

[0071] The following experimental section is provided for illustrative purposes only and is not intended to limit the scope of the invention as defined in the appended claims. In the following experimental section, reference is made to the accompanying drawings. [Figure 1] Figure 1 shows that type 2 iodothyronine deiodinase (D2) is highly expressed in quiescent stem cells of different tissues. Results are based on IF for Pax7-nGFP and D2-3xflag mice in muscle and skin stem cells (SCs). (A) Representative immunofluorescence (IF) staining of Pax7 (green) and D2-flag (red) expression in cryosections of uninjured tibialis anterior (TA) muscle (scale bar, 50 μm). (B) Representative IF staining of CD34 (red) and Flag-D2 (green) expression in epidermal hair follicles (scale bar). [Figure 2-1] Figure 2 shows that D2 expression in muscle is induced in different animal models of muscle wasting. D2 and D3 mRNA expression was determined in TA muscle from (A) cancer cachexia (C26-treated) animals sacrificed 6, 9, and 11 days after cancer cachexia induction, (B) denervated mice sacrificed 6, 12, and 15 days after denervation, and (C) mice fasted for 6, 12, and 24 hours. Expression of TRa, TRP (thyroid hormone receptor), MCT8, and MCT10 (thyroid hormone transporter) mRNA in TA muscle from (D) cachexia, (E) denervated, and (F) fasted mice. [Figure 2-2] MuRF-1 mRNA expression in TA muscles from (G) cachectic, (H) denervated, and (I) fasted mice. Atrogin-1 mRNA expression in TA muscles from (J) cachectic, (K) denervated, and (L) fasted mice. [Figure 3-1] Figure 3 shows that D2 depletion in quiescent stem cells accelerates muscle regeneration. (A) (Top) Schematic of the experimental design. (B) Representative photomicrographs (bottom) showing hematoxylin and eosin (H&E) staining of TA sections from wild-type (wt) and deiodinase-deficient (cD2KO) mice after tamoxifen induction (Scale bar, 100 μm). (B) Percentage of centrally located nuclei assessed in the sections shown in A. (C) Quantification of the cross-sectional area of ​​TA sections as shown in A. [Figure 3-2] (D) Quantification of the percentage of positive cells by double staining for Pax7 / MyoD and Pax7 / BrdU (wt n=5; cD2KO N=5 in three individual experiments). (E) Quantification of cross-sectional area analysis of TA sections from cD2KO and wt(Pax7creER-td / GFP) adult mice 60 days after CTX injection. [Figure 4] Figure 4 shows that D2 depletion in quiescent stem cells promotes skin regeneration. Wound healing experiments were performed in scD2KO and wt mice after tamoxifen-induced D2 depletion. (Top panel) Representative images of wound area 0, 1, 7 and 10 days after wound healing. (Bottom panel) Graph showing wound area calculated using Cell*F Olympus Imaging Software (scale bar indicates 1 cm). [Figure 5-1] Figure 5 shows that global D2 depletion in mice attenuates cancer (cachexia)-induced muscle wasting. Representative photomicrographs showing (A) H&E staining and (B) graphs showing frequency distribution of mean fiber diameter and myofiber cross-sectional area (CSA) of TA muscles from wt and D2KO mice sacrificed 12 days after C26 cell injection. [Figure 5-2](C) Time course of weight change in wt and D2KO mice monitored for 14 days after C26 cell injection. (D) Heart weight and viability of wt and D2KO mice sacrificed 15 days after C26 cell injection. (E) Atrogin-1 and (F) Murf-1 mRNA expression in TA muscle from animals sacrificed 6, 9, and 12 days after C26 cell injection. [Figure 6-1] Figure 6 shows that global D2 depletion in mice attenuates denervation-induced muscle wasting. (A) Graph showing time course of body weight change in wt and D2KO mice monitored for 12 days after hindlimb denervation. (B) Graph showing frequency distribution of myofiber CSA in TA muscles from wt and D2KO mice sacrificed 12 days after denervation. [Figure 6-2] (C) Atrogin-1 and (D) Murf-1 mRNA expression in TA muscle from animals sacrificed 6, 9, and 12 days after denervation. [Figure 7-1] Figure 7 shows the therapeutic ability of rT3 to activate stem cells during muscle regeneration by blocking D2. (A) Schematic of the experimental design. (B) Representative photomicrographs showing H&E staining of TA sections from rT3-treated and untreated mice (control, ctr) sacrificed 7 days after injury (scale bar, 50 μm). (C) Quantification of the cross-sectional area of ​​tissue sections shown in B. (D) Graph showing the percentage of Pax7 / EdU staining for TA sections from rT3-treated and ctr mice (in two individual experiments, Ctr=3; rT3=3 mice). [Figure 7-2] (E) Representative photomicrographs showing H&E staining of rT3-treated and Ctr mice sacrificed 21 days after injury (scale bar, 50 μm). (F) Quantification of the cross-sectional area of ​​the tissue sections shown in E. [Figure 8]Figure 8 shows the therapeutic ability of rT3 to activate stem cells during skin regeneration by blocking D2. (A) Schematic of the experimental design. (B) Representative images of skin wound healing in rT3 vs. control treated mice. (C) Graph showing quantification of wound closure, shown as the mean of percent of initial wound size (n=4 in each group of 4 independent experiments) (scale bar 1 cm). (D) Representative image of animals 28 days after injury. Animals were administered rT3 5 days before shaving and skin wounds show improved hair regrowth. Data are shown as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 using Mann-Whitney test and multiple T-test when comparing two conditions. [Figure 9-1] Figure 9 shows that blocking D2 activity with rT3 attenuates muscle atrophy in vitro. (A) Representative image of immunoblot of PARP in pp6 cells. ERK serves as a loading control. (B) Representative image of immunoblot of PARP measured in pp6 cells. Tubulin serves as a loading control. (C) Representative IF staining of p65 / NFKB in pp6 cells treated with or without rT3. [Figure 9-2] (D) Representative image of immunoblot of p65 / NFKB in pp6 cells. Tubulin and PARP serve as loading controls for the cytoplasmic and nuclear compartments, respectively. (E) Representative image of immunoblot of p65 / NFKB and caspase 8 in pp6 cells. Tubulin serves as a loading control. (F) Representative image of immunoblot of PARP in C2C12 cells. Tubulin serves as a loading control. (G) Representative image of immunoblot of PARP in pp6 cells. [Figure 10-1]Figure 10 shows that amiodarone treatment protects against muscle wasting in a cancer cachexia mouse model. (A) Schematic of the experimental design. (B) Time course of body weight change in Ctr and amiodarone treated mice monitored for 11 days after C26 cell injection. (CE) Graphs showing (C) heart weight, (D) TA, and (E) gastrocnemius (GC) weight of Ctr and amiodarone treated mice sacrificed 11 days after C26 cell injection. [Figure 10-2] (F) Graph showing tumor weight in ctr and amiodarone treated mice sacrificed 11 days after C26 cell injection. (G) MURF-1 and (H) Atrogin-1 mRNA expression in TA muscle from animals sacrificed 11 days after C26 cell injection. (I) Graph showing overall survival (%) of Ctr and amiodarone treated mice monitored after C26 cell injection. (J) Frequency distribution of myofiber CSA in TA muscle from Ctr and amiodarone treated mice sacrificed 11 days after cachexia. [Figure 11] Figure 11 shows that amiodarone protects against muscle wasting in a denervated mouse model. (AB) Graphs showing relative mRNA expression levels of (A) MURF-1 and (B) atrogin-1 in Ctr (contralateral gastrocnemius) and DEN (denervated gastrocnemius after sciatic nerve transection in mice) with or without amiodarone treatment. (C) Graph showing muscle mass in mg as the difference between gastrocnemius Ctr and gastrocnemius DEN (ΔGC WEIGHT, NN DEN / DEN). [Figure 12]Figure 12: Graphs (A, B) showing the time course of the climbing index of dystrophic D. melanogaster flies treated with increasing concentrations of amiodarone compared to untreated and wild-type flies. Results are expressed as the percentage of flies that climbed to the 15 cm mark of the vial after 60 seconds. To facilitate data visualization, in panel (B) only data obtained after flies were treated with 10 mM amiodarone are shown. (C) Graph showing the number of days after treatment at which 50% of the dystrophic D. melanogaster population lost its climbing ability after treatment with increasing concentrations of amiodarone compared to untreated and wild-type flies. Comparison of fittings: parameters of the quadratic polynomial (quadratic) curves are different for each data set (p<0.001, extra sum-of-squares F test). [Figure 13] Figure 13 shows the climbing ability of dystrophic D. melanogaster flies after treatment with increasing concentrations of amiodarone for 10 days (A), 15 days (B), and 20 days (C) compared to untreated and wild type flies. Graph (D) summarizes the data. Results are expressed as the percentage of flies that climbed to the 15 cm mark of the vial after 60 seconds. *p<0.01 and **p<0.001 vs. Dys+DMSO, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] Materials and Methods animal Animals were bred and maintained in the animal facility of the CEINGE Biotecnologie Avanzate, Naples, Italy. Experiments and animal care were performed in accordance with the institutional guidelines. Tg:Pax7-nGFP;Pax7CreER was provided by Shahragim Tajbakhsh (Pasteur Institute, Paris, France) (Rocheteau, 2012 Cell 148(1-2): 112-125). Dio2 flox / flox (Luongo, 2015 Endocrinology 156(2): 745-754), D2-3xFLAG (Castagna, 2017 J Clin Endocrinol Metab 102(5): 1623-1630), global-D2KO (Christoffolete, 2007 Endocrinology 148(3): 954-960) were used in this study. C57BL / 6 and Balb / c mice were purchased from Jackson Laboratory (Stock No: 000664 and 000651). All mice used in the experiments were 12-16 weeks of age. Both sexes were used in the experiments as indicated. Animals were genotyped by PCR using tail DNA.

[0073] Animal Testing Approval All animal studies were performed in accordance with the guidelines of the Ministero della Salute and approved by the Institutional Animal Care and Use Committee (IACUC: 167 / 2015-PR and 354 / 2019-PR).

[0074] Cell culture, transfection, and reagents Primary muscle cultures (pp6) were isolated from the indicated mouse strains as described (Qu et al., 1998 J Cell Biol 142(5): 1257-1267). Immortalized myoblasts (C2C12) were obtained from ATCC. Proliferating cells were cultured in Dulbecco's modified Eagle's medium with 20% fetal bovine serum containing penicillin / streptomycin at 37°C. Transient transfections were performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. Anti-MyoD (sc-304) and tubulin (sc-8035) antibodies were purchased from Santa Cruz Biotechnology. Anti-Pax7 antibody was obtained from the Developmental Studies Hybridoma Bank. Anti-PARP was obtained from Cell Signaling Technology.

[0075] muscle damage Animals were anesthetized with a ketamine xylazine cocktail and 12.5 μl of 60 μg / ml CTX (Naja mossambica mossambica, Sigma-Aldrich) was injected into the right tibialis anterior (TA) muscle along its length (Yan, 2003 J Biol Chem 278(10): 8826-8836) and 25 μl into the gastrocnemius muscle. CTX was injected on a different day than Tam.

[0076] Cross-sectional area (CSA) Tibialis anterior (TA) muscles were dissected, fixed in isopentane in liquid nitrogen, and sliced ​​into 7 μm sections. Cross-sectional areas were analyzed and quantified using CellF* Olympus Imaging Software.

[0077] Immunofluorescence and histology Dissected muscles were snap frozen in liquid nitrogen-cooled isopentane, sectioned (7 μm thick), and stained. For immunofluorescence staining, cells or sections were fixed in 4% formaldehyde (PFA), permeabilized in 0.1% Triton X-100, then blocked with 0.5% goat serum and incubated with primary antibodies. Alexa FluorTM 594 / 647-conjugated secondary antibodies were used. Images were acquired using an IX51 Olympus microscope and Cell*F software. For hematoxylin / eosin staining (H&E), sections were fixed for 15 min, placed in hematoxylin for 5 min, washed, and placed in eosin for 5 min.

[0078] Wound creation, macroscopic examination and histological analysis The fur on the backs of mice was shaved and the skin was washed with 70% alcohol. Two 8 mm full-thickness skin wounds were created along the midline using a sterile 8 mm circular biopsy punch by tensioning the dorsal skin using forceps and pressing through both layers of the skin tension. Skin wound healing was measured every 2-3 days by anesthetizing the animals and imaging the wound area.

[0079] Excisional full-thickness skin wounds were aseptically created on the dorsal skin by folding the skin up at the midline and punching through two layers of skin using a sterile disposable biopsy punch with a diameter of 8 mm. In this way, two wounds were created simultaneously on either side of the midline. Each wound site was digitally photographed at the indicated intervals using a Nikon FX-35A, and wound area was determined photographically using CellF*. Changes in wound area were expressed as a percentage of the initial wound area.

[0080] qPCR experiments Total RNA was extracted from sorted or cultured cells using the Qiagen RNeasy Micro kit according to the manufacturer's instructions (Qiagen, Hilden, Germany) and then reverse transcribed into cDNA by using VIFO reverse transcriptase (Invitrogen Fife technologies Ltd). For real-time PCR, SYBR Green Master mix (Biorad) was used. Cyclophilin A was used as an endogenous control to normalize expression.

[0081] Western blot analysis Total protein extracts from cells and tissues were run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to Immobilon-P transfer membranes (Millipore, Billerica, MA). Membranes were then blocked with 5% nonfat dry milk in phosphate-buffered saline, probed with primary antibodies, washed, incubated with horseradish peroxidase-conjugated donkey anti-rabbit or mouse immunoglobulin G secondary antibodies (1:3000), and detected by chemiluminescence (Millipore). After extensive washing, membranes were incubated with anti-tubulin-specific or ERK antibodies (1:10,000; sc-8035, Santa Cruz, Dallas, TX) as loading controls.

[0082] Muscle wasting mouse model Denervation experiment Animals were anesthetized with ketamine-xylazine cocktail and a small incision (<0.5 mm) was made in the left tight skin. The sciatic nerve was exposed with the help of a small surgical hook and then cut before the separation of the sciatic nerve branches. A small (2-5 mm) nerve segment was removed to prevent reinnervation. As a negative control, denervation was performed unilaterally using the contralateral hindlimb. Tissues were harvested 2, 6, 12 or 15 days after the denervation surgery.

[0083] For denervation + / - amiodarone experiments, mice were treated with amiodarone for 13 days and sacrificed 5 days after induced denervation (days −7 to +5 of denervation).

[0084] Muscle wasting mouse model Cachexia mouse model Mouse colon 26 adenocarcinoma cells (C26) were cultured in DMEM containing 10% fetal bovine serum, 100 U / μl penicillin, and 100 μg / ml streptomycin. For in vivo inoculation, 7 × 10 cells were cultured in 100 μl saline. 6 A single suspension of cells was subcutaneously injected into the right flank of BALB / c mice, and the same volume of saline was injected into the control group.

[0085] In the cachexia + / - amiodarone experiment, mice were treated with amiodarone for 18 days and sacrificed 7 days after induced cachexia. Amiodarone was administered from day -7 to day +7 (day 1 being the day of cell injection).

[0086] Muscle wasting mouse model Fasting experiment C57BL6 mice were fasted for 6, 12 and 24 hours with unlimited access to water.

[0087] rT3 administration In vivo: Mice (C56BL6 mice) were treated systemically with 100 μl intravenous injections of 30 ng / μl rT3 (Sigma Aldrich) or PBS carrier control for 4 consecutive days.

[0088] In vitro: Pp6 or C2C12 cells were treated with rT3 (30 nM).

[0089] Amiodarone administration Mice (C57BL6 mice) were treated with 0.45 mg / ml amiodarone (Sigma, A8423, St. Louis, MO, USA) in drinking water ad libitum starting 1 week prior to induced denervation or cachexia until sacrifice (Bagchi et al., Circulation Research 1987; 60:621-625; Lee et al., Oncotarget, 2015; 6(40): 42976-42987).

[0090] Preparation of amiodarone: 45 mg was dissolved in 1 ml water at 80° C. for 10 min.

[0091] TNFα treatment Pp6 or C2C12 cells were treated with TNFα (40 ng / μl) for 24 h.

[0092] Drosophila melanogaster model of muscular dystrophy Drosophila strains Dys, a genetic loss-of-function homozygous Drosophila mutant of dystrophin (dys) E17 (hiips: / / flybase.org / reports / FBal0241311.html) was employed as a Duchenne muscular dystrophy model. It harbors a point mutation on chromosome 3 causing the nucleotide mutation C19590458T and thus the amino acid mutation Q2807term|Dys-PA (position: 92A10, 3R: 19,590,458..19,590,458), which is a stop codon that determines that the full-length dys protein is abnormally truncated. As a control, the wild-type Oregon R strain was used.

[0093] Adult wild-type animals and mutant Drosophila were treated with increasing concentrations of amiodarone supplemented in the food for 30 days.

[0094] Rearing: Flies were reared on standard cornmeal agar medium (pH 5.5) at 25°C with minor modifications. Fly diet was prepared as follows: 100g yellow cornmeal, 100g brewer's yeast, 8g agar and 75g sucrose (5% w / v) were mixed and dissolved by adding warm water to a final volume of 1.5l as a hydration source for the flies. The mixture was autoclaved and allowed to cool slowly. The broad spectrum fungicide nipagin (3g dissolved in 16ml absolute ethanol) was added when the temperature reached approximately 50°C and then the mixture was distributed into vials.

[0095] Mating: A population of young adult flies (3 days old) was placed in a vial for mating and egg laying. The vials were visually inspected to ensure mating was occurring. Within 5-30 min, typically all females had paired with males. After 24 h of mating in untreated adults, individual eggs were gently picked using 2% agar plates supplemented with apple juice. Eggs were then washed in PBS, counted, and separated under a stereomicroscope. Approximately 5 days after mating, 3rd instar larvae that emerged from the food were sampled. Of note, rearing populations of dystrophic Drosophila were significantly more efficient at producing Dys E17 In heterozygous individuals, the third chromosome allele is balanced with the TM6,Tb balancer chromosome. The balancer allows the differentiation of larvae (heterozygous vs. homozygous) based on a reduced length and tortuous tracheal trunk. Homozygous Dys E17 The expected proportion of offspring is approximately 30% of the total.

[0096] Amiodarone administration Amiodarone compounds were dissolved in DMSO as stock solutions and aliquoted at -20°C.

[0097] Hatched Oregon R and homozygous Dys E17 Drosophila (1-2 days old at maturity) were placed in vials (10-15 flies / vial) and maintained in 4-5 ml of either standard medium or medium supplemented with increasing doses of amiodarone (100 nM, 1 μM, 10 μM, 20 μM, and 50 μM). Food was changed every 5 days. Amiodarone-supplemented food was freshly prepared by mixing amiodarone with the appropriate amount of standard food immediately before administration to the animals. Within each experiment, at least three vials per group were made. Each experiment was repeated at least three times. In total, a minimum of 60 animals per experimental condition were analyzed.

[0098] Treatment group: · Oregon R receiving vehicle (DMSO 1:1000); Dys administered with vehicle (DMSO 1:1000)E17 Flies; Dys receiving 100 nM amiodarone E17 Flies; Dys receiving 1 μM amiodarone E17 Flies; Dys receiving 10 μM amiodarone E17 Flies; Dys receiving 20 μM amiodarone E17 Flies; Dys receiving 50 μM amiodarone E17 Fly. Flies were treated for 30 days (treatment was terminated when animals reached 30 days of adult age) and food was renewed every 5 days.

[0099] Climbing assay Geotaxis was assessed using a climbing assay (negative gravitaxis reflex against the Earth's gravity). A horizontal line was drawn 15 cm above the bottom of the vial. After a 10 min resting period, the flies were dropped to the bottom of the vial, forcing all flies to start climbing (vertical walking). The number of flies that climbed to the 15 cm mark after 60 s was recorded as a percentage of success. A camera recorded the flight behavior during the experiment. Each test was performed three times with 1 min intervals and the results were averaged. Climbing ability was assessed every 5 days starting from 1-2 days of adult age (immediately after molting) and ending on the 30th day.

[0100] statistical analysis Significant differences were determined using one-way ANOVA, MannWhitney, and Student's t-tests, with p<0.05 considered statistically significant. All statistics and graphics were performed using GraphPad Prism7 and are presented in more detail in SI Appendix S20. In all figures, error bars indicate SEM. Values ​​of p<0.05 were considered significant (*P<0.05; **P<0.01; ***P<0.001).

[0101] result Example 1: D2 is expressed in quiescent stem cells of muscle and skin We analyzed D2 expression by immunofluorescence in previously characterized 3xFLAG-D2 knock-in mice (Castagna, 2017 J Clin Endocrinol Metab 102(5): 1623-1630). In these experiments, we found that D2 colocalized with the muscle stem cell marker Pax7+ (Seale, 2000 Cell 102(6): 777-786) in quiescent tibialis anterior (TA) muscle (Figure 1A). Interestingly, D2 expression colocalized with the hair follicle stem cell marker CD34, which specifically marks the hair follicle bulge where quiescent stem cells are localized (Figure 1B).

[0102] Overall, these findings indicate that D2 marks quiescent stem cells.

[0103] Example 2: Type 2 deiodinase is induced in muscle in different models of muscle wasting Furthermore, we analyzed TH metabolism in skeletal muscle atrophy using three different models of muscle atrophy: denervation, cancer cachexia, and fasting. Denervation was induced in 2-month-old mice by unilateral sciatic nerve removal. The contralateral leg was used as an internal control. The mRNA levels of different TH modulators (D2, D3, TRα, TRβ, MCT8, and MCT10) were measured in denervated mice harvested at different time points after denervation. D2 mRNA and protein were rapidly upregulated at day 6 after denervation, and the expression was sustained until day 15 (Figure 2A,D). Similarly, 7 × 10 6 D2 expression was enhanced in cachexia in BalbC mice subcutaneously (sc) injected with colon 26 adenocarcinoma cells (C26) (Figure 2B). Fasting also induced a similar increase in D2 expression early in starvation (Figure 2C). These results suggest that induction of D2, and consequently local amplification of TH signaling, is a common feature of different models of muscle atrophy.

[0104] Example 3: D2 depletion enhances stem cell proliferation during muscle regeneration To determine the consequences of D2 depletion in satellite cells upon injury, we analyzed TA muscles from satellite cell-specific cD2KO mice harvested 21 days after cardiotoxin (CTX) injection (Figure 3A).

[0105] D2 depletion resulted in a greater number of centrally located fibers and was associated with smaller fiber diameters compared to controls (Figure 3B-C). Thus, the number of Pax7+ / MyoD+ cells and the number of equivalent Pax7+ / BrdU+ cells increased, whereas the numbers of Pax7+ / MyoD- and Pax7- / MyoD+ cell populations decreased (Figure 3D). This suggested that the absence of D2 increased the proliferative capacity of stem cells, while delaying the completion of the myogenic program.

[0106] Interestingly, 60 days after CTX injury, fiber diameter was larger in cD2KO mice compared to controls, suggesting that full maturation may be achieved over an extended period in the absence of D2 (time 3E).

[0107] Example 4: D2 depletion enhances stem cell proliferation during skin regeneration To characterize the effect of D2 depletion in vivo during tissue regeneration, a wound healing assay was performed by the inventors. The time course of epidermal regeneration showed that wound closure was significantly faster in D2-deficient epidermis than in control skin (Figure 4A). Overall, these data point to a critical role for D2 in protecting hair follicle stem cells from uncontrolled activation.

[0108] Example 5: Global D2 depletion in mice attenuates muscle wasting To evaluate the functional role of D2 induction in muscle atrophy, we performed a cancer cachexia study in D2KO mice. Matched pairs of D2KO and wild-type (WT) mice were injected with C26 cells and muscle wasting was monitored at days 12 and 15 after injection.

[0109] WT muscles underwent dramatic muscle wasting from days 8-9 to day 15 after injection, as shown by hematoxylin and eosin staining, fiber cross-sectional area (CSA), body weight, and heart weight (Figure 5A-D). Conversely, D2KO muscles were protected from massive muscle wasting, and up to day 15 after injection, the atrophy process was attenuated compared to WT mice (Figure 5A-D).

[0110] Importantly, while the mean survival time of WT mice was 12.7 days, D2KO mice nearly doubled their survival time (22.4 days, Fig. 5D).Interestingly, atrogen induction, a common sign of ongoing muscle atrophy, was highly reduced in D2KO mice (Fig. 5E and F).

[0111] Muscle denervation studies in D2KO mice showed a similar phenotype of reduced muscle wasting in D2KO mice compared to WT mice. On days 9 and 12 after denervation, gastrocnemius (GC) weights in WT animals were reduced by 38% and 34%, respectively, whereas muscle weight reductions in D2KO mice were 4% and 6% (Figure 6A). Consistently, D2KO mouse fiber diameters were significantly larger on day 12 after denervation when compared to WT mice (Figure 6B).

[0112] The ubiquitin ligases MuRF-1 and atrogin-1 were also strongly downregulated in denervated D2KO mice compared with denervated WT mice, indicating that UPS stimulation was dramatically reduced in the absence of D2 (Fig. 6C and D).

[0113] These data demonstrate the efficiency of D2 depletion and the resulting reduction in TH action for the dramatic attenuation of experimentally induced atrophy in vivo.

[0114] Example 6: Drug-induced D2 inhibition as a novel therapeutic tool to activate stem cells To therapeutically use the ability of D2 depletion to activate stem cells and promote muscle tissue repair, mice were treated with oral inverted triiodothyronine (rT3, a specific D2 inhibitor) for 7 days (-5 / +2 relative to CTX injury) (Figure 7A). Hematoxylin and eosin analysis 7 days after CTX showed the presence of an increased number of fibers, and fibers were also smaller in rT3-treated mice than controls (Figures 7B and 7C). In rT3-treated mice, Pax7+ / EdU+ cells were increased over controls (Figure 7D). Importantly, larger fibers were observed in rT3-treated mice than controls at 21 days after injury, consistent with the limited time frame (7 days) of D2 inhibition (Figures 7E and 7F).

[0115] In a similar setting, we also tested whether D2 inhibition positively affects wound regeneration in the skin. Indeed, rT3-treated mice repair wounds more quickly and efficiently than controls (Figure 8A-B). Interestingly, at later time points after skin wounding, it was observed that animals treated with rT3 had a more complete regrowth of hair in shaved areas (Figure 8D).

[0116] Taken together, these results demonstrated that D2 blocking with rT3 could promote regeneration in a variety of tissue contexts by "activating" stem cells and could be used therapeutically in vivo.

[0117] Example 7: TH regulates the NFKB / TNFα signaling pathway During muscle atrophy, most pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, are overexpressed along with enhanced nuclear factor kappa B (NFKB) activation (Bonaldo P. and Sandri M., Dis Model Mech. 2013 Jan;6(l):25-39).

[0118] We tested the effect of TH modulation on TNFα treatment of muscle cells. When cells were treated with TNFα in normal serum, cell death was observed at 24 h (Figure 9A-G). Conversely, cells cultured in the absence of TH (or when TH activation was prevented via D2 blocking with rT3) were protected from apoptosis, while the TNFα receptor further rescued the anti-apoptotic effect of D2 blocking (Figure 2A and B). TNF is one of the most potent physiological inducers of the nuclear transcription factor NF-κB, which mediates many of its biological effects, such as apoptosis, and here we demonstrated that NF-κB activation and, consequently, translocation of the p65 subunit to the nucleus were blocked by rT3 (Figure 9C and D).

[0119] We observed that TNFα induced caspase-8 activity in normal serum-grown cells without altering total p65 levels, and this effect was largely absent in the absence of TH (or when TH activation was prevented via D2 blocking with rT3, or when cells were cultured in charcoal-stripped medium) (Figure 9E). Consistent with this, overexpression of D3 in muscle cells protected them from TNF-induced apoptosis (Figures 9F and G), whereas addition of TNF receptors rescued the protective effect of D3 overexpression (Figure 9G).

[0120] Taken together, these data indicate a key role for TH in inducing key mediators of muscle atrophy and confirm the protective role of D2 inactivation against atrophy-induced muscle cell death.

[0121] Example 8: Drug-induced D2 inhibition as a novel therapeutic tool to attenuate muscle wasting To therapeutically use the ability of D2 blocking to protect mice from massive muscle wasting, we used amiodarone, a specific D2 inhibitor. In a tumor-induced cachexia mouse model, mice were treated with oral amiodarone for 18 days (-7 / +11 relative to C26 injection) (Figure 10A). Matched groups of amiodarone-treated and control mice were injected with C26 cells and muscle wasting was monitored up to different days after injection. Muscles of control mice underwent dramatic muscle wasting as indicated by fiber cross-sectional area (CSA), body, heart, TA (tibialis anterior) and GC (gastrocnemius) weights (Figures 10B-E and 10J). Conversely, muscles of amiodarone-treated mice were protected from massive muscle wasting (Figures 10B-E). Furthermore, there was no significant effect on tumor weight between amiodarone-treated and control mice, indicating that treatment with amiodarone did not inhibit tumor growth (Figure 10F).

[0122] The ubiquitin ligases MuRF-1 and atrogin-1 were also strongly downregulated in amiodarone-treated mice compared to controls (Fig. S10G and S10H). Importantly, while the survival percentage of control mice was about 12.7 days, amiodarone-treated mice nearly doubled their survival (27 days longer, Fig. S10I), similar to what was observed in tumor-induced cachexia in genetically D2-depleted mice.

[0123] In the denervation mouse model, mice were treated with oral amiodarone for 13 days (from day -7 to day +5 of denervation). Consistent with the tumor-induced cachexia model in D2KO mice, ubiquitin ligases MuRF-1 and atrogin-1 were also strongly downregulated in amiodarone-treated mice compared to control mice (Figures 11A and B). Furthermore, even after denervation, muscles of amiodarone-treated mice were protected from massive muscle wasting, as indicated by the preservation of gastrocnemius muscle mass (Figure 11C).

[0124] These data demonstrate the efficacy of D2 depletion to dramatically attenuate experimentally induced muscle atrophy in vivo.

[0125] Example 9: Drug-induced D2 inhibition as a novel therapeutic tool for restoring skeletal muscle function To further validate the therapeutic potential of D2 inhibitor compounds, we investigated the therapeutic potential of Dystrophic phenotype (Dys E17 We investigated the effects of amiodarone on skeletal muscle in a fly model characterized by Drosophila dystrophin. The Drosophila dystrophin gene is as complex as its mammalian counterpart, since it encodes three large isoforms of dystrophin-like protein (DLP1, DLP2, and DLP3) and three truncated products (Dp186, Dp205, and Dp117).

[0126] In the fruit fly, Drosophila melanogaster, complex questions of system function / dysfunction can be interrogated more rapidly than in other model organisms. Drosophila has significant strengths that allow specific expression experiments in the context of a powerful and well-established genetic framework. In this respect, the Drosophila system, representative and highly relevant for human diseases, has a key role in target identification, in vivo repurposing, and drug discovery via high-throughput compound screening, thus favoring the translation of lead compounds into human therapeutics. In the fly life cycle, fly larvae hatch from laid fertilized eggs, feed continuously, and only stop to molt twice after the first and second instar stages. At 5 / 6 days after egg laying, third instar larvae "migrate" as they leave food and prepare to undergo metamorphosis (9 / 10 days) into adult flies. For these reasons, D. melanogaster is a suitable organism for chronic drug delivery in vivo from fertilized eggs to the adult stage. Moreover, feeding experiments in Drosophila are important to support efficient activity and oral delivery of molecules in vivo as a step to evaluate biological effects in multicellular organisms.

[0127] For validation studies, adult wild-type and Dys E17The climbing ability and muscle tissue of the mutants were analyzed after dietary supplementation with increasing concentrations of amiodarone for 0-30 days (geotaxis assay). The rate of decline in climbing in dystrophic Drosophila mutants was significantly faster than in wild-type flies, suggesting that dystrophin-like proteins are required for normal muscle tissue. Thus, using different stocks, it has been previously demonstrated that Drosophila dys mutants begin adult life with normal mobility against gravity and then display a time-dependent muscle defect comparable to our model.

[0128] As shown in FIG. 12, Dys treated with only 1:1000 DMSO (the carrier used for 50 mM amiodarone) for 30 days E17 The climbing ability of the dystrophic flies was lower than that of wild-type flies. In particular, treatment of animals with increasing doses of amiodarone, from 100 nM to 10 µM, induced a dose-dependent beneficial effect on fly motility, with the most effective concentration being 10 µM. Higher concentrations of amiodarone, i.e., 20 to 50 µM, did not alter the reduced climbing of dystrophic animals.

[0129] Determining the number of treatment days when only 50% of the flies were able to climb (FIG. 12C) allowed us to show further amiodarone effects: Climbing reduction: Oregon - 24.5 days; Dys - 14.2 days; Dys + amiodarone 100 nM - 15.5 days; Dys + amiodarone 1 μM - 16.5 days; Dys + amiodarone 10 μM - 17.5 days; Dys + amiodarone 20 μM - 12.8 days; Dys + amiodarone 50 μM - 12.3 days.

[0130] Analysis of the climbing ability of Dys flies at different treatment days (Figure 13) showed that the amiodarone-induced increase in motility was evident after 15 and 20 days of treatment (and followed a dose-dependent trend), while no effect was observed at shorter times. Notably, the decline in climbing of dystrophic flies was almost halved after 15 days of animal treatment (lesioned animals 35% vs. 18%) and 20 days of animal treatment (lesioned animals 55% vs. 32%) with 10 μM. Notably, after 25-30 days, both untreated and amiodarone-treated Dys animals showed the same functional deficits (see results above). These results suggest that amiodarone may improve the climbing ability of dystrophic flies within a certain time window.

[0131] The above results of the experimental design show that chronic administration (30 days) of 10 mM amiodarone to adult dystrophic flies significantly improved the climbing efficiency of the animals. It is also noteworthy that a dose-dependent beneficial trend was observed in animals with concentrations of amiodarone below 10 mM, whereas no effect was seen at amiodarone concentrations ranging from 20 to 50 mM, suggesting a somewhat deleterious threshold. The highest beneficial effect of amiodarone was achieved after young ages of Drosophila, i.e. after 15-20 days of treatment, and disappeared in middle-aged-aged flies (after 25 days). Functionally restored muscles of dystrophic mutants of D. melanogaster were still injured at the structural level.

[0132] Taken together, these results indicate that amiodarone positively affects skeletal muscle contractility but has no effect on the disruption of dystrophic myofibrils.

Claims

1. Inhibitor compounds of type 2 iodothyronine deiodinase (D2) for use in the therapeutic treatment of muscle wasting and / or in muscle and / or skin regeneration therapeutic methods.

2. 2. The inhibitor compound of type 2 iodothyronine deiodinase (D2) for use according to claim 1, selected from the group consisting of inverted triiodothyronine (rT3), amiodarone (AMIO), desethylamiodarone (DEA), 5-methyl-2-thiouracil (MTU), 6-benzyl-2-thiouracil (BTU), xanthohumol (XTH), genistein (GEN), 6-propyl-2-thiouracil (PTU), methimazole (MMI), iopanoic acid (IAc), dexamethasone, gold thioglucose (GTG), and any combination thereof.

3. 3. The inhibitor compound of type 2 iodothyronine deiodinase (D2) for use according to claim 1 or 2, wherein the muscle wasting is due to a disease state selected from the group consisting of sarcopenia, cancer, sepsis, diabetes, chronic heart failure, chronic obstructive pulmonary disease, chronic renal failure, cirrhosis of the liver, cystic fibrosis, muscle denervation, fasting, and any combination thereof.

4. 4. The inhibitor compound of type 2 iodothyronine deiodinase (D2) for use according to claim 3, wherein said disease state is neoplastic cachexia.

5. 3. The inhibitor compound of type 2 iodothyronine deiodinase (D2) according to claim 1 or 2 for use in the therapeutic treatment of muscle diseases selected from the group consisting of delayed onset muscle soreness (DOMS), muscle strains, pulled muscles, muscle tears, cancer cachexia, muscle atrophy, muscular dystrophy, and any combination thereof.

6. 3. An inhibitor compound of type 2 iodothyronine deiodinase (D2) according to claim 1 or 2 for use in the therapeutic treatment of a skin disease selected from the group consisting of ulcers, dermatitis, skin wounds, burns, lacerations, and any combination thereof.

7. A pharmaceutical composition for use in the therapeutic treatment of muscle wasting and / or muscle and / or skin regeneration therapeutic method, comprising a therapeutically effective amount of at least one type 2 iodothyronine deiodinase (D2) inhibitor compound according to claim 1 and a pharma- ceutical acceptable carrier, excipient, and / or diluent.

8. 8. A pharmaceutical composition for use according to claim 7, which is in a pharmaceutical dosage form for topical, oral, enteral or parenteral administration.

9. 9. The composition of claim 8, wherein the pharmaceutical dosage form for topical administration is selected from the group consisting of a cream, an ointment, a spray, a gel, a gauze, a pad, a patch, a medicated patch, and a sponge.

10. 9. The composition of claim 8, wherein the pharmaceutical dosage form for oral administration is selected from the group consisting of tablets, capsules, powders, granules, solutions, suspensions, and syrups.

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