Method for improving muscle mass, muscle strength, or muscle function by a combination of testosterone and growth hormone

Abstract

The present disclosure relates to methods, uses, compositions, and kits for using testosterones or testosterone derivatives and growth hormone (GH) or GH derivatives to improve muscle mass, muscle strength, or muscle function in a subject, or to reduce fatigue, pain, or obesity. One aspect of the present disclosure relates to combination therapies of testosterones or testosterone derivatives and GH or GH derivatives for treating various disorders associated with muscle wasting or conditions associated with muscle weakness.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 369,867, filed on July 29, 2022. The content of this application is hereby incorporated by reference in its entirety into this specification.

[0002] Government Interests This invention was made with government support under grant NS095813 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Reference to a Sequence Listing This application is filed with a sequence listing in electronic form. The sequence listing is provided as a file named SeqList_161118 - 03601, sized 2,078 bytes, created on July 12, 2023. The information in the electronic form of the sequence listing is hereby incorporated by reference in its entirety into this specification.

[0004] This disclosure relates to methods or uses for improving muscle mass, muscle strength, and muscle function, or methods or uses for treating disorders associated with muscle wasting or muscle weakness, or methods or uses for reducing fatigue, pain, or obesity.

Background Art

[0005] Muscles, the largest tissue in the human body, are essential for various body functions such as movement, support, protection, heat generation, and blood circulation. Many disorders, injuries, and conditions affect muscle mass, muscle strength, and muscle function. Muscle wasting is the loss of muscle mass due to muscle weakness and shrinkage, while muscle weakness is characterized by a lack of strength in the muscles. There are various potential causes of muscle wasting and / or muscle weakness, including certain medical conditions such as muscular dystrophy and amyotrophic lateral sclerosis. Deficits in muscle function have a profound impact on the quality of life. There is a need to improve muscle mass, muscle strength, and muscle function and to treat disorders associated with muscle wasting or muscle weakness.

Summary of the Invention

[0006] The present disclosure addresses the aforementioned needs in a number of aspects.

[0007] In one aspect, the present disclosure provides a method for doing so in a subject in need of (i) improvement in muscle mass, muscle strength, or muscle function, (ii) treatment of a disorder associated with muscle wasting or muscle weakness, or (iii) reduction of fatigue, pain, or obesity. The method includes administering to the subject an effective amount of testosterone or a derivative thereof, and administering to the subject an effective amount of growth hormone (GH) or a derivative thereof.

[0008] The present disclosure also provides for the use of (A) an effective amount of testosterone or a derivative thereof, (B) an effective amount of growth hormone or a derivative thereof, or (C) both in the manufacture of a pharmaceutical for (i) improving muscle mass, muscle strength, or muscle function, (ii) treating a disorder associated with muscle wasting or muscle weakness, or (iii) reducing fatigue, pain, or obesity. Improving, treating, or reducing includes administering to a subject in need thereof an effective amount of testosterone or a derivative thereof, and administering to the subject an effective amount of growth hormone or a derivative thereof.

[0009] The subject can be a healthy subject, can have a disorder or condition associated with muscle wasting or muscle weakness, or can be at risk of developing a disorder or condition. Examples of disorders or conditions include facioscapulohumeral muscular dystrophy (FSHD), sarcopenia, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, Becker muscular dystrophy, Pompe disease, myotonic dystrophy type 1, myotonic dystrophy type 2, inclusion body myositis, polymyositis, dermatomyositis, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy, Charcot-Marie-Tooth disease, HIV myopathy, frailty in the elderly, deconditioning, nutritional deficiency, injury, cancer-related cachexia, muscle dysfunction, nerve dysfunction, neuromuscular junction dysfunction, and motor neuron diseases. In one example, the disorder is FSHD.

[0010] A variety of teststerone derivatives can be used. In one embodiment, the derivative is a teststerone ester. In some embodiments, the derivative has the structure of formula (II), TIFF2025524901000002.tif41170wherein R is alkyl, alkanediyl, alkenyl, alkenediyl, alknyl, aralkyl, aryl, heteroaryl, or acyl. Examples of teststerone derivatives include teststerone enanthate, teststerone propionate, teststerone cypionate, teststerone undecanoate, teststerone oleate, and teststerone palmitate. In one example, the derivative is teststerone enanthate. Teststerone or its derivative can be administered at about 0.1 mg to 30,000 mg (e.g., 1 to 10,000 mg, 10 to 1000 mg, 20 to 800 mg, 30 to 600 mg, 40 to 500 mg, and 50 to 200 mg). In some embodiments, teststerone or its derivative is administered once every two weeks at about 70 to 170 mg, or once every two weeks at about 110 to 150 mg. For example, teststerone or its derivative can be administered once every two weeks at about 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, or 150 mg. In one example, teststerone or its derivative is administered once every two weeks at about 140 mg. Preferably, the subject is administered teststerone enanthate at about 140 mg once every two weeks, or a teststerone derivative at an equivalent daily dose, or weekly dose, or every two weeks dose. Teststerone or its derivative can be administered to the subject via any suitable route such as intramuscular injection.

[0011] Growth hormone or its derivative can be administered at about 0.01 μg / kg / day to 250 μg / kg / day (e.g., 0.1 - 200 μg / kg / day, 0.5 - 100 μg / kg / day, 1.0 - 50 μg / kg / day, 1.5 - 20 μg / kg / day, 2.0 - 10 μg / kg / day). In one embodiment, growth hormone or its derivative can be administered at about 2.5 - 6.0 μg / kg / day, or about 4.0 - 5.5 μg / kg / day. In other cases, growth hormone or its derivative can be administered at about 4.0 μg / kg / day, 4.1 μg / kg / day, 4.2 μg / kg / day, 4.3 μg / kg / day, 4.4 μg / kg / day, 4.5 μg / kg / day, 4.6 μg / kg / day, 4.7 μg / kg / day, 4.8 μg / kg / day, 4.9 μg / kg / day, 5.0 μg / kg / day, 5.1 μg / kg / day, 5.2 μg / kg / day, 5.3 μg / kg / day, 5.4 μg / kg / day, or 5.5 μg / kg / day. Preferably, growth hormone or its derivative is administered at about 5.0 μg / kg / day. Growth hormone or its derivative can be administered via any suitable route including subcutaneous injection.

[0012] For the method or use described above, testosterone or its derivative, or growth hormone or its derivative can be administered for any suitable duration. In some embodiments, the duration is at least 1 week, e.g., 2 weeks to 1 year or more, 4 - 72 weeks, 8 - 36 weeks, 12 - 30 weeks, or about 24 weeks.

[0013] The method or use can further include identifying a subject or evaluating a subject using various tests. Examples of tests include Quantitative Muscle Testing (QMT), Manual Muscle Testing (MMT), Facioscapulohumeral Dystrophy Clinical Outcome Measure (FSHD-COM), FSHD-Health Index (FSHD-HI), Forced Vital Capacity (FVC), Epworth Sleepiness Scale, Fatigue Severity Scale, Dual Energy X-Ray Absorptiometry (DEXA) fat-free mass (total and regional), PROMIS-57, Individualized Neuromuscular Quality of Life Questionnaire (INQoL), Beck Depression Inventory (BDI), and Six Minute Walk Distance. One or more of these tests and related steps of identifying or evaluating can be performed before, during, or after treatment or use.

[0014] In another aspect, the disclosure also provides a method or use for performing it in a subject in need of treatment of one or more of the disorders or conditions described herein. In one example, the disorder is FSHD and the method includes administering to the subject an effective amount of testolactone or a derivative thereof as described above and administering to the subject an effective amount of growth hormone or a derivative thereof as described above.

[0015] The disclosure also provides a kit for (i) improving muscle mass, muscle strength, or muscle function, (ii) treating a disorder associated with muscle wasting or muscle weakness, or (iii) reducing fatigue, pain, or obesity. The kit includes (a) an effective amount of testolactone or a derivative thereof and (b) an effective amount of growth hormone or a derivative thereof.

[0016] Details of one or more embodiments of the disclosure are described in the following description. Other features, objects, and advantages of the disclosure will be apparent from this specification and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] [Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H] Show the long-term effects of testosterone and rHGH on patients with FSHD over a 36-week study period (24-week treatment period + 12-week washout period).

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 1G

Figure 1H

Figure 1I

[0018] The present disclosure relates to methods, uses, compositions, and kits for improving muscle mass, muscle strength, or muscle function in a subject using testosterone or a testosterone derivative and growth hormone (GH) or a GH derivative or variant. One aspect of the present disclosure relates to a combination therapy of testosterone or a testosterone derivative and GH or a GH derivative / variant for treating various disorders associated with muscle wasting or conditions associated with muscle weakness.

[0019] Certain aspects of the present disclosure are based, at least in part, on unexpected findings from clinical trials in which combinations of testosteronederivatives and recombinant human growth hormone (rHGH) were used as treatments for individuals having muscle wasting, muscle weakness, gait limitations, functional impairment, or reduced quality of life. As disclosed herein, participants in the trials were found to have increases in a number of strength and function measurements without experiencing any serious adverse events. Accordingly, testosterone or a testosterone derivative can be used in combination with GH or a GH derivative / variant to treat disorders associated with muscle wasting or weakness in a subject or to improve muscle mass, muscle strength, or muscle function.

[0020] In one example, a clinical study was conducted to evaluate the methods and therapies disclosed herein. More specifically, in a population of adult males with FSHD, testosterone enanthate (in oil) was administered by intramuscular injection every two weeks in combination with rHGH (GENOTROPIN®) by subcutaneous injection after each evening meal. Testosterone enanthate was given at a dose of 140 mg every two weeks, and GENOTROPIN® was given at a dose of 5.0 μg / kg / day (calculated using the patient's pre-entry weight). Participants were given this treatment for 24 weeks, followed by a 12-week washout period.

[0021] Nineteen participants completed the study, and no participants experienced any serious adverse events. At week 24, it was found that the mean six-minute walk distance of the participants increased by 37.3 meters (p = 0.0007), the fat-free mass improved by 2.2 kg (p < 0.0001), and the total disease burden (FSHD-HI) decreased by 19% (p = 0.04). Participants were also found to have increases in a number of strength measurements.

[0022] This is notable because patients with FSHD experience a progressive decline in walking, strength, and function over time. There has long been a felt unmet medical need for the treatment of FSHD and other forms of muscular dystrophy. Currently, there are no therapies effective or approved for patients with FSHD, and few for any muscular dystrophy. Any treatment strategy that can not only reduce but also produce an increase in function in muscular dystrophy patients would be novel and transformative. Furthermore, the combination therapy described above is safe, well-tolerated, and improves function, muscle mass, and disease burden in men with FSHD. As a non-disease-specific therapeutic approach, this combination therapy can also be used as a treatment modality for both men and women with all different types of muscular dystrophy and other medical disorders that result in physical disability or muscle weakness. Prior to the inventors' research, testosterone paired with rHGH had not been studied in any muscular dystrophy population. To the inventors' knowledge, these specific formulations and dosages have been continuously tested using rigorous safety and efficacy modeling in any human population for the first time. Accordingly, the present disclosure addresses the long-felt unmet medical needs described above.

[0023] Disorders and conditions associated with muscle wasting or muscle weakness The combination therapy disclosed herein can be used to treat a variety of disorders associated with muscle wasting or muscle weakness.

[0024] As used herein, the terms "disorders associated with muscle wasting" and "muscle wasting-related disorders" are used interchangeably to refer to any condition associated with loss of muscle strength, muscle function, or muscle mass. Examples of these conditions include sarcopenia, cachexia, AIDS wasting syndrome, muscular dystrophy (including Duchenne muscular dystrophy syndrome, Becker muscular dystrophy syndrome, facioscapulohumeral muscular dystrophy, myotonic dystrophy (types 1 and 2), limb-girdle muscular dystrophy, Pompe disease), spinal muscular atrophy, neuromuscular disease, anorexia nervosa, motor neuron disease, diseases of the neuromuscular junction, inflammatory myopathy (e.g., inclusion body myositis, polymyositis, dermatomyositis), diseases associated with neuropathy (e.g., Charcot-Marie-Tooth disease and spinal nerve root disorder), nutritional deficiency, cancer-induced wasting, other conditions or diseases associated with decreased muscle mass, and other related diseases, but are not limited thereto. These disorders include chronic or acute "deconditioning" that can result from illness and injury, or immobilization or inactivity associated with, for example, rigid immobilization such as during air travel and space travel. Muscle wasting, including muscle atrophy, can also result from denervation, injury, joint immobilization, forced bed rest (disuse atrophy), glucocorticoid treatment, sepsis, non-weight bearing, cancer, and aging. Jagoe et al. 2001 Curr. Opin. Clin. Nutr. Metab. Care 4:183. In addition, there are various rare forms of myopathy that result in severe pain, debilitation, fatigue, and impairment (disorders of carbohydrate metabolism, disorders of lipid metabolism, lysosomal myopathy, inclusion body myopathy, distal myopathy, autoimmune inflammatory myopathy, etc.). The combination therapies disclosed herein are particularly suitable for treating facioscapulohumeral muscular dystrophy.

[0025] The terms "conditions associated with muscle weakness" and "muscle weakness-related conditions" are used interchangeably to refer to any condition associated with the absence of strength or function of any of the muscles in the body. Examples of this condition include the aforementioned disorders, as well as muscle weakness, fatigue, obesity, or pain in healthy subjects or subjects having none of the disorders listed above.

[0026] Facioscapulohumeral muscular dystrophy Facioscapulohumeral muscular dystrophy (FSHD) is the second most common form of adult muscular dystrophy, with a prevalence of 1 in 15,000 to 1 in 20,000 (Padberg GW. Facioscapulohmeral disease (dissertation). The Netherlands: The University of Leiden, 1982; Flanigan KM FAU-Coffeen, C M, Coffeen CM FAU-Sexton, L, FAU SL, FAU SD, FAU BS, MF L. Genetic characterization of a large, historically significant Utah kindred with facioscapulohumeral dystrophy). Clinical manifestations of FSHD include progressive and substantial weakness of the muscles of the face, shoulders, arms, and pelvic girdle, as well as life-altering fatigue, gait disturbances, respiratory decline, social limitations, and activity limitations related to muscle weakness.

[0027] In 2012, a cross-sectional study of 328 patients with FSHD was completed, identifying the most important symptoms for this population. Six symptomatic themes were identified as having a prevalence of over 90%. These FSHD themes included the following: (1) problems with the shoulders or arms, (2) inability to perform activities, (3) fatigue, (4) weakness of the back, chest, and abdomen, (5) limitations in mobility or gait, and (6) changes in body image due to the disease. In addition to being very widely recognized in FSHD, these themes were also identified as having the greatest impact on the daily lives of FSHD patients.

[0028] In a recent paper on functional impairment in FSHD, patients were reported to have a loss of strength of 1% to 4% per year and a 24% likelihood of developing a need for a wheelchair over a 6-year period. See Statland JM, et al., Muscle Nerve 2014;49:520-7. Currently, there are no known disease-modifying therapies for FSHD that can improve or limit the functional decline of gait.

[0029] As disclosed herein, a combination therapy of (i) a test steroid or a test steroid derivative and (ii) GH or a GH derivative can be used to treat FSHD, or to improve or remit at least one physical parameter of FSHD. For example, the combination therapy can be used for the treatment of gait disorders in FSHD. The combination therapy can be a treatment for frailty in FSHD. The combination therapy can be a treatment for dysfunction in FSHD. The combination therapy can be a treatment for disease burden in FSHD. The combination therapy can be a treatment for muscle atrophy in FSHD. The combination therapy can be used to improve the quality of life in FSHD.

[0030] In some embodiments, the combination therapy can be used for the treatment of patients with other muscular dystrophies, including but not limited to: Duchenne muscular dystrophy, limb-girdle muscular dystrophy, Becker muscular dystrophy, Pompe disease, myotonic dystrophy type 1, myotonic dystrophy type 2, and others. The combination therapy described above can also be a treatment for patients with functional limitations related to those muscles or nerves, including but not limited to: inclusion body myositis, polymyositis, dermatomyositis, ALS, spinal muscular atrophy, Charcot-Marie-Tooth disease, and others. In some embodiments, the combination therapy can be used as a treatment for patients with sarcopenia due to any cause, such as HIV myopathy, frailty in the elderly, deconditioning, nutritional deficiency, injury, or cancer-related cachexia. The combination therapy can also be beneficial in a population of women, including a population of women having the conditions or disorders described above.

[0031] Testosterone and Testosterone Derivatives Testosterone is a naturally occurring androgen produced in both men and women. Testosterone promotes protein synthesis and has an anabolic effect on both muscle and bone (Shahidi NT. Clin Ther 2001;23:1355-90). It is commonly used in men with hypogonadism and conditions associated with low or absent endogenous testosterone (Bhasin S, et al., Best Pract Res Clin Endocrinol Metab 2011;25:251-70). It is also recommended in men to improve libido and erectile dysfunction (Bhasin S, et al. J Clin Endocrinol Metab 2010;95:2536-59). In women, testosterone replacement is used for acquired immunodeficiency syndrome, inoperable metastatic breast cancer, low libido, sexual dysfunction, muscle atrophy associated with muscle wasting, and as postmenopausal therapy. See, for example, Margo K, et al., Am Fam Physician 2006;73:1591-8, Blackman MR, et al. JAMA 2002;288:2282-92, Choi HH, et al. J Clin Endocrinol Metab 2005;90:1531-41, Dolan Looby SE, et al. AIDS 2009;23:951-9, Dolan S, et al. Arch Intern Med 2004;164:897-904, Miller K, et al. J Clin Endocrinol Metab 1998;83:2717-25, and Nachtigall L, et al. Gynecol Endocrinol 2011;27:39-48.

[0032] The Endocrine Society currently recommends testosterone alone for (1) increasing muscle strength and fat-free mass in patients with HIV and (2) improving bone mineral density in patients receiving high-dose glucocorticoids (Bhasin S, et al. J Clin Endocrinol Metab 2010;95:2536-59). In previous studies, testosterone alone has been shown to be safe and to increase fat-free mass, reduce body fat, and improve basal metabolic rate in a heterogeneous group of adult dystrophy populations (including FSHD participants) with normal baseline testosterone levels. In this study, the results were seen 3 months after treatment and were equivalent to those demonstrated in a group of normal men receiving the same therapy (Welle S, et al., J Clin Endocrinol Metab 1992;74:332-5). A second study of 40 patients with myotonic dystrophy found that testosterone alone was safe and tolerable and could improve both creatinine excretion and fat-free mass while not statistically improving overall strength (Griggs RC, et al. Neurology 1989;:219-22.).

[0033] Without wishing to be bound by theory, testosterone is generally known to those skilled in the art and is represented by a compound having formula (I). TIFF2025524901000003.tif53170

[0034] Testosterone, also known by the chemical name 17-β-hydroxyandrost-4-en-3-one (or 4-androstene 17β-ol-3-one), can be obtained in various ways and can be isolated and purified from nature or produced synthetically in any manner. As disclosed herein, any of testosterone or a testosterone derivative or a testosterone analog can be used.

[0035] As used herein, the terms "derivative," "variant," and "analog" are used interchangeably to refer to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein, e.g., testosterone or GH), the structure of which is sufficiently similar to that disclosed herein such that, based on that similarity, it is expected by one of ordinary skill in the art to exhibit the same or similar activity and utility as, or to induce the same or similar activity and utility as, the claimed compound, either as an active agent or as a precursor.

[0036] Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of the parent compound. Examples are shown in formula (II), wherein R is alkyl, alkanediyl, alkenyl, alkenediyl, alknyl, aralkyl, aryl, heteroaryl, acyl. Formula (II) Testosterone derivative TIFF2025524901000004.tif46170

[0037] A testosterone derivative or analog can be a prodrug, ester, salt, or metabolite of testosterone. These include any useful metabolite or precursor of testosterone, such as the metabolite dihydrotestosterone. In some embodiments, a testosterone analog can be, for example, a testosterone ester such as testosterone cypionate, enanthate, or propionate, or a combination thereof, a prodrug of testosterone or a fatty acid ester, a fatty acid ester of testosterone having a long chain (i.e., 14 or more carbons), methyltestosterone (where the methyl group is covalently bonded to the testosterone nucleus at the C17 position and inhibits liver metabolism), a testosterone alkyl ester, a testosterone undecanoate, or a composition such as those disclosed in US2020 / 0174026 and US2011 / 0251167, which are incorporated herein by reference.

[0038] As used in this application, "testosterone ester" is a derivative of testosterone that includes substitution of at least an acyl functional group or an acyl functional group substituted as defined below on the hydroxyl group on the cyclopentyl ring of the steroid core. When a carbon limitation is assigned to a testosterone ester, the carbon limitation is relative only to the carbon atoms on the acyl substitution.

[0039] The term "physiologically cleavable ester" refers to derivatives of the hydroxyls of formula (II) and acids or acid derivatives, and the products are cleaved in vivo to obtain the compound of formula (II) or an active metabolite. Such physiologically cleavable esters can be regarded as "prodrugs". Such "prodrugs" are valuable when such prodrugs increase the bioavailability of the corresponding hydroxyl compound when administered to a subject. For example, a "prodrug" administered intranasally can be more readily absorbed into the bloodstream, facilitate delivery of the parent compound to a biological compartment of the subject such as the brain or lymph, and also have pharmacokinetics for more favorable patient acceptance, safety profile, and / or specific modulation for use in the intended indication in the subject. General overviews of prodrugs are provided in (1) "Pro-drugs As Novel Delivery Systems," Vol. 14 of the ACS Symposium Series, by T. Higuchi and V. Stella, and (2) "Bioreversible Carriers in Drug Design," American Pharmaceutical Association, Pergamon Press, 1987, Edward B. Roche, Ed.

[0040] Testosterone has been esterified in various pharmaceutical preparations, and esters of propionate, enanthate (see formula (III)), cypionate, and undecanoate are sold as oral or injectable formulations for the treatment of hypogonadism. Formula (III) Testosterone enanthate: TIFF2025524901000005.tif60170

[0041] The "carbonyl group" of an ester that can be used as a derivative in accordance with the present disclosure, i.e., -C(O)-R, and the carboxylic acids that can form "prodrugs" include monocarboxylic acids derived from unsubstituted or substituted lower straight-chain or branched-chain alkyl, alkenyl, alkynyl, or arylalkyl entities. R is defined, for example, as follows. Naturally occurring carboxylic acids are generally a preferred class that can be acceptable cleavable esters of pharmaceutically active ingredients.

[0042] The term "lower alkyl" carboxylic acid refers to a monovalent saturated aliphatic hydrocarbon radical having 1 to 12 carbon atoms bonded to a carboxyl group. The alkyl can be a linear chain (i.e., straight chain), branched chain, or cyclic structure. Representative examples of lower alkyl radicals include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, isopentyl, amyl, sec-butyl, tert-butyl, tert-pentyl, cyclopropyl, cyclobutyl, cyclopentylethyl (cipionate), undecanoate, and the like.

[0043] As used herein, the term "saturated" means that a compound or group so modified does not have carbon-carbon double bonds and carbon-carbon triple bonds, except as shown below. In the case of substituted versions of saturated groups, one or more carbon-oxygen double bonds or carbon-nitrogen double bonds may be present. When such bonds are present, carbon-carbon double bonds that can occur as part of keto-enol tautomerism or imine / enamine tautomerism are not excluded.

[0044] The term "aliphatic", when used without the modifier "substituted", indicates that the so-modified compound / group is an acyclic or cyclic, but non-aromatic hydrocarbon compound or group. In an aliphatic compound / group, carbon atoms can be linked together in a linear chain, a branched chain, or a non-aromatic ring (alicyclic). Aliphatic compounds / groups can be saturated, i.e., linked by single bonds (alkanes / alkyls), or unsaturated and can have one or more double bonds (alkenes / alkenyls), or one or more triple bonds (alkynes / alkynyls).

[0045] The term "alkyl", when used without the modifier "substituted", refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, having a straight or branched acyclic structure, and having no atoms other than carbon and hydrogen. The groups --CH3 (Me), --CH2CH3 (Et), --CH2CH2CH3 (n-Pr or propyl), --CH(CH3)2 (i-Pr, i Pr, or isopropyl), --CH2CH2CH2CH3 (n-Bu), --CH(CH3)CH2CH3 (sec-butyl), --CH2CH(CH3)2 (isobutyl), --C(CH3)3 (tert-butyl, t-butyl, t-Bu, or t Bu), and --CH2C(CH3)3 (neo-pentyl) are non-limiting examples of alkyl groups.

[0046] The term "alkanediyl", when used without the modifier "substituted", refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as attachment points, having a straight-chain or branched acyclic structure, having no carbon-carbon double or triple bonds, and having no atoms other than carbon and hydrogen. The groups --CH2-- (methylene), --CH2CH2--, --CH2C(CH3)2CH2--, and --CH2CH2CH2-- are non-limiting examples of alkanediyl groups. "Alkane" refers to the compound H--R, where R is alkyl as defined above for this term. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by --OH, --F, --Cl, --Br, --I, --NH2, --NO2, --CO2H, --CO2CH3, --CN, --SH, --OCH3, --OCH2CH3, --C(O)CH3, --NHCH3, --NHCH2CH3, --N(CH3)2, --C(O)NH2, --OC(O)CH3, or S(O)2NH2. The following groups are non-limiting examples of substituted alkyl groups. --CH2OH, --CH2Cl, --CH3, --CH2CN, --CH2C(O)OH, --CH2C(O)CH3, --CH2C(O)NH2, --CH2C(O)CH3, --CH2OCH3, --CH2OC(O)CH3, --CH2NH2, --CH2N(CH3)2, and -CH2CH2Cl.

[0047] The term "alkenyl", when used without the modifier "substituted", refers to a monovalent unsaturated aliphatic group having a carbon atom as an attachment point, having a straight-chain or branched acyclic structure, having at least one non-aromatic carbon-carbon double bond, having no carbon-carbon triple bond, and having no atoms other than carbon and hydrogen. Non-limiting examples include the following. --CH=CH2 (vinyl), --CH=CHCH3, --CH=CHCH2CH3, --CH2CH=CH2 (allyl), --CH2CH=CHCH3, and --CH=CHCH=CH2.

[0048] The term "alkenediyl", when used without the modifier "substituted", refers to a divalent unsaturated aliphatic group having two carbon atoms as attachment points, having a linear or branched acyclic structure, having at least one non-aromatic carbon-carbon double bond, having no carbon-carbon triple bond, and having no atoms other than carbon and hydrogen. The groups -CH=CH--, --CH.dbd.C(CH3)CH2--, --CH=CHCH2--, and --CH2CH=CHCH2-- are non-limiting examples of alkenediyl groups. It should be noted that although the alkenediyl group is aliphatic, when connected at both ends, this group is not precluded from forming part of an aromatic structure. The terms "alkene" or "olefin" are synonymous and refer to a compound having the formula H-R, where R is an alkenyl as defined above. "Terminal alkene" refers to an alkene having only one carbon-carbon double bond, the bond of which forms a vinyl group at one end of the molecule. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by --OH, --F, --Cl, --Br, --I, --NH2, --NO2, --CO2H, --CO2CH3, --CN, --SH, --OCH3, --OCH2CH3, --C(O)CH3, --NHCH3, --NHCH2CH3, --N(CH3)2, --C(O)NH, --OC(O)CH3, or --S(O)2NH2. The groups -CH=CHF, -CH=CHCl, and -CFI=CHBr are non-limiting examples of substituted alkenyl groups.

[0049] The term "alkynyl", when used without the modifier "substituted", refers to a monovalent unsaturated aliphatic group having a carbon atom as a point of attachment, having a linear or branched acyclic structure, having at least one carbon-carbon triple bond, and having no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not exclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups --CCH, --CCCH3, and --CH2CCCH3 are non-limiting examples of alkynyl groups. "Alkyne" refers to a compound FI--R, where R is alkynyl.

[0050] When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by --OH, --F, --Cl, --Br, --I, --NH2, --NO2, --CO2H, --CO2CH3, --CN, --SH, --OCH3, --OCH2CH3, --C(O)CH3, --NHCH3, --NHCH2CH3, --N(CH3)2, --C(O)NH, --OC(O)CH3, or --S(O)2NH2.

[0051] The term "aryl", when used without the modifier "substituted", refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as a point of attachment, said carbon atom forming part of one or more six-membered aromatic ring structures, the ring atoms all being carbon, and the group not being composed of atoms other than carbon and hydrogen. Where two or more rings are present, the rings may or may not be fused. As used herein, this term does not exclude the presence of one or more alkyl or aralkyl groups (to the extent carbon number limitations permit) attached to the first aromatic ring or any additional aromatic rings present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, --C6H4CH2CH3 (ethylphenyl), naphthyl, and monovalent groups derived from biphenyl. The term "arenediyl", when used without the modifier "substituted", refers to a divalent aromatic group having two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more six-membered aromatic ring structures, the ring atoms all being carbon, and the monovalent groups not being composed of atoms other than carbon and hydrogen. As used herein, this term does not exclude the presence of one or more alkyl, aryl, or aralkyl groups (to the extent carbon number limitations permit) attached to the first aromatic ring or any additional aromatic rings present. Where two or more rings are present, the rings may or may not be fused. Non-fused rings may be connected via one or more of the following: a covalent bond, an alkanediyl, or an alkenediyl group (to the extent carbon number limitations permit). Non-limiting examples of arenediyl groups include the following.

[0052] "Arene" refers to the compound H--R, where R is an aryl as defined above for that term. Benzene and toluene are non-limiting examples of arenes.

[0053] When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by --OH, --F, --Cl, --Br, --I, --NH2, --NO2, --CO2H, --CO2CH3, --CN, --SH, --OCH3, --OCH2CH3, --C(O)CH3, --NHCH3, --NHCH2CH3, --N(CH3)2, --C(O)NH2, OC(O)CH3, or --S(O)2NH2.

[0054] The term "aralkyl", when used without the modifier "substituted", refers to the monovalent group --alkanediyl--aryl, where the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.

[0055] When the term aralkyl is used with the modifier "substituted", one or more hydrogen atoms from the alkanediyl and / or aryl group are independently replaced by --OH, --F, --Cl, --Br, --I, --NH2, --NO2, --CO2H, --CO2CH3, --CN, --SH, --OCH3, --OCH2CH3, --C(O)CH3, --NHCH3, --NHCH2CH3, --N(CH3)2, --C(O)NH2, OC(O)CH3, or --S(O)2NH2. Non-limiting examples of substituted aralkyl are the following. (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl.

[0056] The term "heteroaryl", when used without the modifier "substituted", refers to a monovalent aromatic group having an aromatic carbon atom or a nitrogen atom as a point of attachment, said carbon atom or nitrogen atom forming part of one or more aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen, or sulfur, and the heteroaryl group being composed of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. When two or more rings are present, the rings may or may not be fused. As used herein, this term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (to the extent carbon number limitations permit) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl, pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolinyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as a point of attachment. "Heteroarene" refers to the compound H--R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes. When these terms are used with the modifier "substituted", one or more hydrogen atoms are independently replaced by --OH, --F, --Cl, --Br, --I, --NH2, --NO2, --CO2H, --CO2CH3, -CN, --SH, --OCH3, ---CH2CH3, --C(O)CH3, --NHCH3, --NHCH2CH3, --N(CH3)2, --C(O)NH, --OC(O)CH3, or --S(O)2NH2.

[0057] The term "acyl", when used without the modifier "substituted", refers to the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl, or heteroaryl as those terms are defined above. Groups, --CHO, --C(O)CH3 (acetyl, Ac), --C(O)CHCH3, --C(O)CH2CHCH3, --C(O)CH(CH3)2, --C(O)CH(CH)2, --C(O)C6H5, --C(O)C6H4CH3, --C(O)CH2C6H5, --C(O)(imidazolyl) are non-limiting examples of acyl groups. "Thioacyl" is defined in a similar manner except that the oxygen atom of the group --C(O)R is replaced by a sulfur atom, --C(S)R. The term "aldehyde" corresponds to the alkanes defined above, with at least one of the hydrogen atoms replaced by a --CHO group. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms (including, if present, hydrogen atoms directly bonded to the carbon atom of the carbonyl or thiocarbonyl group) are independently replaced by --OH, --F, --Cl, --Br, --I, --NH2, --SH, --OCH3, --OCH2CH3, --NHCH3, --NHCH2CH3, --N(CH3)2, --OC(O)CH3, or --S(O)2NH2. Groups, --C(O)CHCH3, --CO2 (carboxyl), --CO2CH3 (methyl carboxyl), --CO2CH2CH3, --C(O)NH2 (carbamoyl), and --CON(CH3)2 are non-limiting examples of substituted acyl groups. The term lower "alkenyl" carboxylic acid refers to an aliphatic group having from 1 to 12 carbons, which can be linear chains, branched chains, and cyclic groups and having up to three double bonds, all of which can be optionally substituted in the same manner as alkyl groups. Representative examples of lower alkenyl radicals in carboxylic acids include vinyl (ethenyl), allyl (propene-3-yl), l-buten-4-yl, 2-buten-4-yl, l-penten-5-yl, and the like.

[0058] The term "pharmaceutically acceptable carboxylic acid" means that the carboxylic acid moiety useful in the formation of pharmaceutical formulations and compositions is also physiologically acceptable and generally non-toxic to the subject receiving that moiety.

[0059] A variety of compositions are available for the therapies described herein, e.g., testosterone patches or injections, intramuscular injections, implants, oral tablets of alkylated T (e.g., methyltestosterone), subcutaneous formulations, intranasal formulations, buccal formulations, transdermal formulations such as topical gels and solutions, or topical patches.

[0060] In some embodiments, the composition can be a solid dosage formulation (e.g., tablet, capsule, granule, powder, sachet, or chewable), solution, gel, suspension, emulsion, shampoo, conditioner, cream, foam, gel, lotion, ointment, transdermal patch, film, liniment, or paste. Further, methods and uses of the compositions described herein for treating a disease, preventing a disease, treating a condition, and / or preventing a condition are provided herein.

[0061] Formulations of testosterone or a testosterone derivative and its analogs or salts can provide a dosage of testosterone sufficient to maintain the serum total testosterone level of a male subject within the normal male range (in the range of approximately 300 - 1000 ng / dL) based on the measured value of serum total testosterone. The pharmaceutically effective amount of testosterone or a testosterone derivative and its analogs or salts present in the compositions disclosed herein can depend on the patient's starting serum total testosterone and the mode of administration.

[0062] For oral administration, the composition can be provided in the form of tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, and 100 milligrams of the active ingredient for symptomatic adjustment of the dosage to the subject being treated. An effective amount of the drug can usually be supplied at a dosage level of about 0.0002 mg / kg to about 50 mg / kg of body weight per day. More specifically, this range can be about 0.001 to 7 mg / kg of body weight per day. In particular, testosterone and testosterone derivatives, and analogs or salts thereof delivered by intramuscular injection can be provided by injections of 50 to 750 mg every 1 to 4 weeks. In one embodiment, testosterone and testosterone derivatives, and analogs or salts thereof are provided by intramuscular injection of 100 to 500 mg every 1 to 4 weeks. In one class of this embodiment, testosterone and testosterone derivatives, and analogs or salts thereof are provided by intramuscular injection of 50 to 250 mg every 1 to 4 weeks.

[0063] Testosterone and testosterone derivatives, and analogs or salts thereof can be provided in the form of a gel or cream at a dosage of 20 to 200 mg per day. In one embodiment, testosterone and testosterone derivatives, and analogs or salts thereof are provided in a gel at dosages of 50 to 100 mg / day, particularly 50 mg / day, 75 mg / day, and 100 mg / day.

[0064] Transdermal patches can be used to deliver 1 to 10 mg per day, particularly 4 to 6 mg / day of testosterone and testosterone derivatives, and analogs or salts thereof.

[0065] Testosterone, testosterone derivatives, and analogs or salts thereof can also be provided using a buccal gel at a dose of 10 mg / day to 100 mg / day. In one embodiment, the dose of testosterone or a testosterone derivative, and analogs or salts thereof, is a buccal gel and is 40 to 80 mg / day. In one class of this embodiment, the dose of testosterone or a testosterone derivative, and analogs or salts thereof, in the buccal gel is 60 mg / day.

[0066] Commercially available testosterone therapies are known in the art and include, for example, topical testosterone formulations (e.g., ANDROGEN, AXIRON, FIRST-TESTOSTERONE, FIRST-TESTOSTERONE MC, FORTESTA, and TESTIM), transdermal patch formulations (e.g., ANDRODERM), and buccal testosterone formulations (e.g., STRIANT).

[0067] A pharmaceutical composition containing testosterone, a testosterone derivative, a testosterone analog, or a salt thereof may further contain a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition can be formulated or administered in the same manner as (e.g., using the same excipients in the same ratios and / or including the same dosage strength as) commercially available testosterone, testosterone prodrug, or testosterone derivative products, including, but not limited to: ANDRODERM, ANDROGEL, ANDROID 10, ANDROID 25, ANDROID 5, AVEED, AXIRON, DELATESTRYL, DEPO - TESTADIOL, DEPO - TESTOSTERONE, DITATE - DS, FORTESTA, JATENZO, METANDREN, METHYLTESTOSTERONE, NATESTO, ORETON, ORETON METHYL, STRIANT, TESTIM, TESTODERM, TESTODERM TTS, TESTOPEL, testosterone, testosterone cypionate, testosterone cypionate - estradiol cypionate, testosterone enanthate, testosterone enanthate and estradiol valerate, testosterone propionate, testosterone undecanoate, TESTRED, VIRILON, VOGELXO, XYOSTED (autoinjector). The FDA approval labeling for each of these products available on the FDA website, including information regarding their formulations, dosages, and administrations.

[0068] GH, GH variants, and GH derivatives Growth hormone (GH), also known as somatotropin, is a peptide hormone that stimulates growth, cell reproduction, and cell regeneration in humans and other animals. Human growth hormone is produced in the pituitary gland of both males and females. This hormone is a single polypeptide chain of 191 amino acids and has a molecular weight of approximately 22 kDa. HGH exhibits a number of biological effects, including, among others, linear growth (somatogenesis), lactation, macrophage activation, and insulin-like and diabetogenic effects (Chawla, R., et al., Ann. Rev. Med. 34:519-547 (1983), Isaksson, O., et al., Ann. Rev. Physiol., 47:483-499 (1985), Hughes, J. and Friesen, H., Ann. Rev. Physiol., 47:469-482 (1985)). Shown below is the amino acid sequence of the GH polypeptide encoded by the human GH gene corresponding to Genbank accession number AIA66930.1 or AAF23136.1. FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQT SLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANS LVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNS HNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF (SEQ ID NO: 1)

[0069] The structure of HGH is well-known (Goeddel, D., et al., Nature 281:544-548 (1979)), and the three-dimensional structure of HGH has been elucidated by X-ray crystallography (de Vos, A., et al., Science 255:306-312 (1992)). This protein has a compact globular structure starting from the N-terminus and containing four amphipathic alpha-helix bundles called A-D, which are linked by loops. HGH also contains four cysteine residues involved in two intramolecular disulfide bonds, one forming a large loop within the molecule between Cys-53 and Cys-165, and the other forming a small loop near the C-terminus between Cys-182 and Cys-189. In solution, HGH exists mainly as a monomer and to a small extent as a dimer and high molecular weight oligomers. Under certain conditions, HGH can be induced to form larger amounts of dimers, trimers, and higher oligomers.

[0070] Numerous naturally occurring variants of HGH have been identified. These include HGH-V (Seeburg, DNA 1:239 (1982); U.S. Patent Nos. 4,446,235, 4,670,393, and 4,665,180, which are incorporated herein by reference), and 20 kDa HGH containing a deletion of residues 32-46 of HGH (Kostyo et al., Biochem. Biophys. Acta 925:314 (1987), Lewis, U., et al., J. Biol. Chem., 253:2679-2687 (1978)). In addition, numerous HGH variants resulting from post-transcriptional, post-translational, secretion, metabolic processes, and other physiological processes have been reported (Baumann, G., Endocrine Reviews 12:424 (1991)).

[0071] The biological effects of HGH are derived from its interaction with specific cell receptors. The interaction between HGH and the extracellular domain of its receptor is among the best understood hormone-receptor interactions. High-resolution X-ray crystallographic data (Cunningham, B., et al., Science, 254:821-825 (1991)) indicate that HGH has two receptor-binding sites and uses different sites on the molecule to sequentially bind to two receptor molecules. The two receptor-binding sites are designated as site I and site II. Site I includes the carboxy terminus of helix D, a portion of helix A, and the A-B loop, whereas site II encompasses the amino-terminal region of helix A and a part of helix C. The binding of GH to its receptor occurs first at site I, followed by site II. Site II then engages the second GH receptor, leading to receptor dimerization and activation of the intracellular signaling pathway that results in the cellular response to the hormone. An HGH variant in which the G120R substitution has been introduced into site II can bind to a single HGH receptor but cannot dimerize the two receptors. The variant acts as an HGH antagonist in vitro, presumably by occupying the receptor site without activating the intracellular signaling pathway (Fuh, G., et al., Science 256:1677-1680 (1992)).

[0072] Similar to testosterones, HGH can stimulate cell growth and regeneration (Giannoulis MG, et al., Endocr Rev 2012;33:314-77). In HGH deficiency states, patients experience exercise intolerance, low bone mineral density, changes in body composition, and worsening of cholesterol profiles (Toogood AA, et al., J Clin Endocrinol Metab 1999;84:131-6). HGH supplementation can relieve these disorders. Recombinant HGH (rHGH), a synthetic preparation of HGH, is used to treat children and adults with growth hormone deficiency, as well as muscle wasting, Turner syndrome, Prader-Willi syndrome, chronic renal insufficiency, and idiopathic short stature. See, for example, Giannoulis MG, et al., Endocr Rev 2012;33:314-77, Ross JL, et al. N Engl J Med 2011;364:1230-42, Mogul HR, et al. J Clin Endocrinol Metab 2008;93:1238-45, Youssef DM. Saudi J Kidney Dis Transpl 2012;23:755-64, and Kemp SF, et al. J Clin Endocrinol Metab 2005;90:5247-53.

[0073] As disclosed herein, it has unexpectedly been found that combination therapy of GH and testosterone / testosterone derivatives leads to improvement in muscle mass and strength and functional recovery in subjects having disorders associated with muscle wasting or weakness such as FSHD. Thus, GH and its derivatives or analogs can be used as therapeutic agents in the therapies disclosed herein. Examples of GH that can be used as a therapeutic agent include, but are not limited to, GH analogs, GH isoforms, GH mimetics, GH fragments, hybrid GH proteins, fusion proteins, oligomers and multimers thereof, homologs thereof including receptor agonists, glycosylation pattern variants thereof, and variants thereof, regardless of whether produced from cDNA or genomic DNA, recombinant vector expression, synthesis, transgenic, and gene activation methods, and regardless of their synthetic or manufacturing methods.

[0074] As used herein, "growth hormone" or "GH" includes, but is not limited to, growth hormone from any mammalian species, including human (HGH), recombinant human (rHGH), bovine (bGH), porcine, and chicken, and other domestic or farm animals, and regardless of its biological activity, further includes polypeptides and proteins having at least one biological activity of GH analogs, GH isoforms, GH mimetics, GH fragments, hybrid GH proteins, fusion proteins, oligomers and multimers, homologs, glycosylation pattern variants, variants, splice variants, and mutants thereof, regardless of whether produced from recombinant (cDNA, genomic DNA, synthetic DNA, or other forms of nucleic acid), in vitro, in vivo, microinjection of nucleic acid molecules, synthesis, transgenic, and gene activation methods, and regardless of their synthetic or manufacturing methods.

[0075] Accordingly, examples of GH include, but are not limited to, growth hormone proteins and their species and sequence variants, including the 191 single-chain amino acid sequence of human GH. GH can be a naturally occurring full-length protein or a cleavage fragment or sequence variant that retains at least a portion of the biological activity of the native protein. There are two known types of pituitary-derived human GH, one having a molecular weight of approximately 22,000 daltons (22kD HGH) and the other having a molecular weight of approximately 20,000 daltons (20kD HGH). 20kD HGH has an amino acid sequence corresponding to that of 22kD HGH consisting of 191 amino acids, except that it lacks 15 amino acid residues from positions 32 to 46 of 22kD HGH. In some reports, 20kD HGH has been found to exhibit lower risk and higher activity than 22kD HGH. The present disclosure contemplates the use of 22kD, 20kD HGH, and their species and sequence variants and cleavage fragments. The cloned gene for HGH has been expressed in secreted form in Escherichia coli (U.S. Patent No. 4,898,830, Chang, C.N., et al., Gene 55:189

[1987] ), and its DNA and amino acid sequences have been reported (Goeddel, et al. Nature, 281:544

[1979] , Gray, et al., Gene 39:247

[1985] ).

[0076] Examples of suitable GH variants herein include, for example, sequences having homology to the GH sequence, natural sequence fragments, and unnatural sequence variants having at least a portion of the biological activity or biological function of GH, from humans, non-human primates, mammals (including domestic animals), and that are useful for preventing, treating, mediating, or alleviating GH-related diseases, deficiencies, disorders, or conditions. GH sequences of non-mammals are well described in the literature. For example, the sequence alignment of carp GH can be found in Genetics and Molecular Biology 2003 26p.295-300. An analysis of the evolution of avian GH sequences is presented in Journal of Evolutionary Biology 2006 19p.844-854. Some exemplary sequences are also described in U.S. Patent Nos. 8143216, 7662393, and US2018 / 0161443, which are incorporated by reference. In addition, natural sequences homologous to human GH can be found by standard homology search techniques such as NCBI BLAST.

[0077] In one embodiment, GH comprises a GH polypeptide having a sequence corresponding to a protein found in nature. In another embodiment, GH is a sequence variant, fragment, homolog, or mimetic of a natural sequence that retains at least a portion of the biological activity of the corresponding natural GH. Any of these GH sequences or homologous derivatives (e.g., constructed by shuffling individual mutations between species or families) that retain at least a portion of the biological activity of natural GH may be useful in the therapies described herein. Such GH can exhibit at least about 60%, 70%, 75%, or 80% sequence identity, or preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the GH sequence (e.g., SEQ ID NO: 1).

[0078] Generally, a GH variant or analog or derivative exhibits binding specificity for a given target or another desired biological characteristic when used in vivo or utilized in an in vitro assay. For example, it may exhibit the ability to bind to a transmembrane receptor for growth hormone. In one embodiment, binding to the growth hormone receptor leads to receptor dimerization and leads to at least a portion of the activation of the intercellular signaling pathway compared to native growth hormone.

[0079] The term "GH polypeptide" encompasses GH polypeptides that include one or more amino acid substitutions (e.g., particularly conservative modifications or conservative substitutions), additions, or deletions. Exemplary substitutions include, for example, substitution of lysine at position 41 of native HGH or phenylalanine at position 176. In some cases, the substitution can be an isoleucine or arginine residue if the substitution is at position 41, or a tyrosine residue if the position is 176. Position F10 can be substituted with, for example, A, H, or I. Position M14 can be substituted with, for example, W, Q, or G. Other exemplary substitutions include, but are not limited to, any substitution or combination thereof: R167N, D171S, E174S, F176Y, I179T; R167E, D171S, E174S, F176Y; F10A, M14W, H18D, H21N; F10A, M14W, H18D, H21N, R167N, D171S, E174S, F176Y, I179T; F10A, M14W, H18D, H21N, R167N, D171A, E174S, F176Y, I179T; F10H, M14G, H18N, H21N; F10A, M14W, H18D, H21N, R167N, D171A, T175T, I179T; or F10I, M14Q, H18E, R167N, D171S, I179T.

[0080] For example, see U.S. Patent No. 6,143,523, which is incorporated herein by reference.

[0081] Exemplary substitutions (e.g., particularly conservative modifications or conservative substitutions) at various amino acid positions in naturally occurring HGH are described, including substitutions that increase agonist activity, increase protease resistance, convert the polypeptide to an antagonist, etc., and are encompassed by the term "HGH polypeptide."

[0082] Agonist GH, for example, as an HGH sequence, includes, for example, naturally occurring HGH sequences that include the following modifications: H18D, H21N, R167N, D171S, E174S, I179T. For example, see U.S. Patent No. 5,849,535, which is incorporated herein by reference. Additional agonist HGH sequences include H18D, Q22A, F25A, D26A, Q29A, E65A, K168A, E174S; H18A, Q22A, F25A, D26A, Q29A, E65A, K168A, E174S; or H18D, Q22A, F25A, D26A, Q29A, E65A, K168A, E174A. For example, see U.S. Patent No. 6,022,711, which is incorporated herein by reference. The HGH polypeptide containing substitutions at H18A, Q22A, F25A, D26A, Q29A, E65A, K168A, E174A enhances the affinity for the HGH receptor at site I. For example, see U.S. Patent No. 5,854,026, which is incorporated herein by reference. HGH sequences with increased protease resistance include, but are not limited to, HGH polypeptides that contain one or more amino acid substitutions within the C-D loop. In some embodiments, substitutions include, but are not limited to, R134D, T135P, K140A, and any combination thereof. For example, see Alam et al. (1998) J. Biotechnol. 65:183-190.

[0083] The term "GH polypeptide" also includes pharmaceutically acceptable salts and prodrugs of naturally occurring GH, as well as prodrugs of salts, polymorphs, hydrates, solvates, biologically active fragments, biologically active variants, and stereoisomers, and agonists and mimetic variants of naturally occurring HGH, and polypeptide fusions thereof. Fusions containing additional amino acids at the amino terminus, carboxyl terminus, or both are encompassed by the term "GH polypeptide". Exemplary fusions include, for example, methionyl growth hormone in which methionine is linked to the N-terminus of GH resulting from recombinant expression of the mature form of HGH lacking a secretion signal peptide or a portion thereof, fusions for purification purposes (including but not limited to polyhistidine or affinity epitopes), fusions with serum albumin-binding peptides, and fusions with serum proteins such as serum albumin, among others.

[0084] The term "GH polypeptide" includes polypeptides conjugated to polymers such as PEG and may be composed of one or more additional derivatizations of cysteine, lysine, or other residues. In addition, the GH polypeptide may contain a linker or polymer, and the amino acids to which the linker or polymer is conjugated may be non-natural amino acids or may be conjugated to naturally encoded amino acids using techniques known in the art such as coupling to lysine or cysteine. Polymer conjugation of GH polypeptides has been reported. See, for example, U.S. Patent Nos. 5,849,535, 6,136,563, and 6,608,183.

[0085] The term "GH polypeptide" also includes glycosylated GH, and without limitation thereto, polypeptides glycosylated at any amino acid position, N-linked or O-linked glycosylated forms of the polypeptide. Variants containing single nucleotide changes are also considered biologically active variants of the GH polypeptide. In addition, splice variants are included. The term "GH polypeptide" also includes any one or more GHs linked by chemical means or expressed as a fusion protein, e.g., a GH polypeptide or any other polypeptide, protein, carbohydrate, polymer, small molecule, linker, ligand, or any other type of biologically active molecule, e.g., a GH polypeptide heterodimer, homodimer, heteromultimer, or homomultimer, and polypeptide analogs containing, for example, specific deletions or other modifications but still maintaining biological activity.

[0086] In some embodiments, the GH, e.g., the HGH polypeptide, further includes additions, substitutions, or deletions that modulate the biological activity of the GH or HGH polypeptide. For example, the addition, substitution, or deletion may modulate one or more properties or activities of the GH, e.g., HGH. For example, the addition, substitution, or deletion may modulate the affinity for the GH, e.g., HGH polypeptide receptor, modulate receptor dimerization (including, but not limited to, increasing or decreasing), stabilize the receptor dimer, modulate the circulation half-life, therapeutic half-life, modulate the stability of the polypeptide, modulate cleavage by proteases, modulate the dosage, modulate release or bioavailability, facilitate purification, or improve or modify a particular route of administration. Similarly, the GH, e.g., the HGH polypeptide, may include protease cleavage sequences, reactive groups, antibody binding domains (including, but not limited to, FLAG or poly-His), or other affinity-based sequences (including, but not limited to, FLAG, poly-His, GST, etc.), or linked molecules (including, but not limited to, biotin) that improve the detection (including, but not limited to, GFP), purification, or other properties of the polypeptide.

[0087] In some embodiments, any recombinant HGH currently sold as a daily injectable product, including HUMATROPE (ELI LILLY & CO.), NUTROPIN (GENENTECH), NORDITROPIN (NOVO-NORDISK), GENOTROPIN (PFIZER), and SAIZEN / SEROSTIM (SERONO), can be used. In the United States, the following are available: GENOTROPIN, GENOTROPIN MINIQUICK, HUMATROPE, NORDITROPIN FLEXPRO, NUTROPIN AQ NUSPIN 10, NUTROPIN AQ NUSPIN 20, NUTROPIN AQ NUSPIN 5, OMNITROPE, SAIZEN, SAIZENPREP, SEROSTIM, ZOMACTON, ZOMACTON (FOR ZOMA-JET 10)[DSC], and ZORBTIVE. In Canada, the following are available: GENOTROPIN GOQUICK, GENOTROPIN MINIQUICK, HUMATROPE, NORDITROPIN NORDIFLEX PEN, NUTROPIN AQ NUSPIN 10, NUTROPIN AQ NUSPIN 20, NUTROPIN AQ NUSPIN 5, OMNITROPE, SAIZEN, and SEROSTIM.

[0088] Assessment and Evaluation The methods or uses disclosed herein can further include the step of identifying a subject in need of treatment or the step of evaluating a subject. One or more of these steps can be performed before or after administering the therapies disclosed herein. A variety of assessments and tests known in the art can be used for the identifying or evaluating step. Some examples are described below.

[0089] Efficacy Assessment - Neuromuscular Assessment Six-minute walk test: The six-minute walk test (6MWT) measurement has been widely studied and utilized in the neuromuscular population. See, for example, Kierkegaard M, et al. Neuromuscul Disord 2007;17:943-9, McDonald CM, et al. Muscle Nerve 2010;42:966-74, and McDonald CM, et al. Muscle Nerve 2010;41:500-10. It represents the patient's function and is one of the most widely recognized problems that impair the quality of life in FSHD. See, for example, Heatwole C, et al. Neurology 2012;78:S15.004.

[0090] Quantitative muscle testing (QMT): The maximum voluntary isometric contraction test of the limb muscles can be performed using a quantitative muscle assessment (QMA) system. This system uses an adjustable cuff to attach the patient's arm or leg to an inelastic strap connected to a force transducer with a load of 0.5 to 1,000 Newtons. The measurements resulting from this method of strength testing have been used in several neuromuscular diseases. See, for example, Kissel JT, et al. Neurology 2001;57:1434-40, Personius KE, et al. The FSH DY Group. Phys Ther 1994;74:253-63, Andres PL, Skerry LM, Munsat TL. Measurement of Strength in Neuromuscular Diseases. In: Munsat TL, ed. Quantification of neurologic deficit. Stoneham: Butterworths Publishers, 1989:87-100, and Muscle Study Group. A randomized, pilot trial of etanercept in dermatomyositis. Ann Neurol 2011;70:427-36.

[0091] Six selected muscle groups (biceps, triceps, quadriceps, hamstrings, grip, and external rotators of the shoulder) can be examined bilaterally. These specific muscles were selected because they demonstrate excellent test-retest reliability in neuromuscular patients and normal volunteers and because they reflect the affected muscle groups in FSHD, which are susceptible to therapeutic intervention (Kissel JT, et al. Neurology 2001;57:1434-40). The composite QMT score can be generated by expressing each muscle strength score as a percentage of normal predicted, taking into account the age, gender, and height of the subject, and averaging across all muscles. See, for example, Kissel JT, et al. Neurology 2001;57:1434-40, and Tawil R, et al. Neurology 1997;48:46-9. Additional analysis of QMT strength can include absolute strength analysis, as well as separate analysis of upper and lower extremity strength.

[0092] Manual muscle testing (MMT): Manual muscle testing can be performed on 30 muscle groups (bilateral shoulder abductors, external rotators of the shoulder, elbow flexors, wrist flexors, wrist extensors, hip flexors, hip abductors, knee extensors, hip extensors, knee flexors, hip adductors, elbow extensors, ankle dorsiflexors, and plantar flexors, as well as neck extensors and neck flexors). Measurements resulting from this method of strength testing have been utilized in previous clinical trials of musculoskeletal disorders. See, for example, Personius KE, et al. The FSH DY Group. Phys Ther 1994;74:253-63, Muscle Study Group. A randomized, pilot trial of etanercept in dermatomyositis. Ann Neurol 2011;70:427-36, Florence JM, et al. Phys Ther 1992;72:115-22, and Griggs RC, et al. Arch Neurol 1991;48:383-8.

[0093] FSHD Clinical Outcome Measure (FSHD-COM): FSHD-COM is an assessor-administered test that measures multiple aspects of physical function known to be impaired in FSHD. The tests included in FSHD-COM evaluate leg function, arm / shoulder function, trunk function, hand function, and balance. Leg function is evaluated using the time from sit to stand (Wagner KR, et al. Ann Neurol 2008;63:561-71), 6-minute walk test, self-selected walking speed (Bohannon RW. J Geriatr Phys Ther 2006;29:64-8, and Iosa M, et al. Clin Biomech (Bristol, Avon) 2007;22:1074-82), time to walk 30 feet, and time to climb stairs (Personius K, et al. Phys Ther 1994;74:253-63). Arm and shoulder function are measured using the range of motion determined at the shoulder and elbow (Anonymous A prospective, quantitative study of the natural history of facioscapulohumeral muscular dystrophy (FSHD): implications for therapeutic trials. The FSH-DY Group. Neurology 1997;48:38-46), and the time it takes the subject to put on and take off a coat (Brown M, et al., J Gerontol A Biol Sci Med Sci 2000;55:M350-5). Trunk function is measured using the time it takes the subject to pick up a penny from the floor (Brown M, et al., J Gerontol A Biol Sci Med Sci 2000;55:M350-5), the ability to rise up holding both feet, and the ability to move from supine to sitting position. Hand function is measured using determination of QMT grip strength. Finally, balance is estimated using the timed up and go test (Podsiadlo D, et al., J Am Geriatr Soc 1991;39:142-8).Each of the individual tests of FSHD-COM (and the FSHD-COM total score) can be analyzed.

[0094] Effectiveness Evaluation - Functional Evaluation Forced vital capacity: Respiratory function can be measured using forced vital capacity.

[0095] The Epworth Sleepiness Scale can be completed to estimate the subject's daytime sleepiness.

[0096] The Fatigue Severity Scale can be completed to estimate the subject's fatigue.

[0097] Dual-energy X-ray absorptiometry (DEXA). Lean body mass (LBM) can be measured via dual-energy X-ray absorptiometry (DEXA). DEXA provides a practical and effective approach for determining lean muscle mass and has been widely used in previous neuromuscular clinical trials. See, for example, Logigian EL, et al. Neurology 2010;74:1441-8, and Heatwole CR, et al. Arch Neurol 2011;68:37-44.

[0098] Both total and regional LBM can be measured (Skalsky AJ, et al. Neuromuscul Disord 2008;18:873-80). DEXA measurements have several advantages over more recently described and discussed MRI techniques. Specifically, compared to MRI, DEXA has a longer history of use, more standardized analysis, does not rely on perfect subject positioning, is more accessible, is more cost-effective, can be used in patients with metallic foreign bodies, aneurysm clips, pacemakers, claustrophobia, and metallic implants, and has a better correlation with changes in 6-minute walk distance compared to MRI thigh measurements. See, for example, Skalsky AJ, et al. Phys Med Rehabil Clin N Am 2012;23:67,73, x and Amato AA, et al. Neurology 2014;83:2239-46. Most importantly, the use of DEXA as a biomarker is supported by previous studies of combination therapies in which an increase in LBM (measured by DEXA) occurred prior to a subsequent observable improvement in patient strength and function (Schroeder ET, Eur J Appl Physiol 2012;112:1123-31).

[0099] Effectiveness assessment Self-reported assessment FSHD-Health Index (FSHD-HI): The FSHD-HI is a disease-specific patient-reported outcome measurement designed to evaluate patient-related effects during a therapeutic trial (Heatwole C, et al. Neurology 2012;78:S15.004). This instrument measures the patient's (1) mobility, (2) hand and arm function, (3) emotional problems, (4) cognitive impairment, (5) decreased satisfaction in social situations, (6) decreased performance in social situations, (7) specific activity impairments, (8) fatigue, (9) pain, (10) eating problems, (11) communication difficulties, (12) problems with the shoulder and arm, (13) weakness of the back, chest, or abdomen, and (14) patient's evaluation of body image, and is created to meet FDA recommendations for use in supporting drug labeling applications. The FSHD-HI is currently being validated in a long-term NIH-funded study of 40 FSHD patients at the University of Rochester.

[0100] PROMIS-57: The PROMIS-57 generates scores for depression, anxiety, fatigue, pain interference, pain intensity, physical function, sleep disturbance, and satisfaction with participation in social roles (Dunn-Lewis C, et al. Nutr J 2011;10:90).

[0101] Individualized Neuromuscular Quality of Life Questionnaire (INQoL): The INQoL is a quality of life instrument focused on muscle diseases (Vincent KA, et al. Neurology 2007;68:1051-7).

[0102] Beck Depression Inventory (BDI): The BDI is a widely used instrument for monitoring changes in depressive symptoms (BECK AT, et al. Arch Gen Psychiatry 1961;4:561-71).

[0103] Kit Kits (e.g., pharmaceutical packs) are also encompassed by the present disclosure. In certain embodiments, a kit includes one or more pharmaceutical compositions described herein and instructions for using the pharmaceutical composition.

[0104] In some embodiments, the kit includes two containers each containing a GH / GH derivative and a testosterone / testosterone derivative, respectively. In some embodiments, the kit includes one or more pharmaceutical compositions or compounds described herein and a first container (e.g., a vial, an ampoule, a bottle, a syringe, and / or a dispenser package, or other suitable container), the first container containing a first pharmaceutical composition (e.g., a GH / GH derivative or testosterone / testosterone). In certain embodiments, the kit may optionally further include a second container (e.g., a vial, an ampoule, a bottle, a syringe, and / or a dispenser package, or other suitable container). In some embodiments, the second container contains a pharmaceutical excipient for diluting or suspending the pharmaceutical composition or compound described herein. In certain embodiments, combining the pharmaceutical compositions or compounds described herein provided in the first container and the second container forms one unit dosage form. In some embodiments, the kit further includes instructions for use, such as instructions for combining the container components and / or instructions for administering the container components to a subject.

[0105] In certain embodiments, the instructions are for administering the pharmaceutical composition to a subject in need thereof. In certain embodiments, the instructions include information required by a regulatory agency such as the US Food and Drug Administration (FDA) or the European Medicines Agency (EMA). In certain embodiments, the instructions include prescription information.

[0106] Definitions As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein" and "polypeptide" refer to polymers of amino acids that include modified amino acids (e.g., phosphorylation, glycosylation, glycosylation, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are often used in relation to relatively large polypeptides, while the term "peptide" is often used in relation to small polypeptides, but the use of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include the aforementioned gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, as well as other equivalents, variants, fragments, and analogs.

[0107] A "recombinant" peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein produced by recombinant DNA technology, i.e., a peptide, polypeptide, or protein produced from a cell transformed with an exogenous DNA construct encoding the desired peptide. A "synthetic" peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein prepared by chemical synthesis. The term "recombinant," when used in connection with, for example, a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the modification of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Within the scope of the present invention are fusion proteins containing one or more of the above-described sequences and heterologous sequences. A heterologous polypeptide, nucleic acid, or gene is one that originates from a foreign species or, in the case of the same species, is substantially modified from its original form. Two fusion domains or sequences are heterologous to each other if they are not adjacent to each other in the naturally occurring protein or nucleic acid.

[0108] Conservative modifications or functional equivalents of the peptides, polypeptides, or proteins disclosed in the present invention refer to polypeptide derivatives of the peptides, polypeptides, or proteins, e.g., proteins having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. It substantially retains the activity of the parent peptide, polypeptide, or protein (such as those disclosed in the present invention). Generally, conservative modifications or functional equivalents are at least 60% identical to the parent (e.g., SEQ ID NO: 1) (e.g., any number from 60% to 100% (including both ends), e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%). Accordingly, within the scope of the present invention are hinge regions having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof.

[0109] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm as described in the following non-limiting examples. The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program of the GCG software package (available at www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0110] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the GH receptor binding characteristics of a GH containing the amino acid sequence. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art.

[0111] Amino acid substitutions can, in some cases, be made by selecting substitutions that do not significantly differ in their effects on maintaining (a) the structure of the peptide backbone in the substitution area, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. For example, naturally occurring residues can be grouped based on side chain properties: (1) hydrophobic amino acids (norleucine, methionine, alanine, valine, leucine, and isoleucine), (2) neutral hydrophilic amino acids (cysteine, serine, threonine, asparagine, and glutamine), (3) acidic amino acids (aspartic acid and glutamic acid), (4) basic amino acids (histidine, lysine, and arginine), (5) amino acids that affect chain orientation (glycine and proline), and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups can be considered conservative substitutions. Examples of substitutions include substitution of valine with alanine, lysine with arginine, glutamine with asparagine, glutamic acid with aspartic acid, serine with cysteine, asparagine with glutamine, aspartic acid with glutamic acid, proline with glycine, arginine with histidine, leucine with isoleucine, isoleucine with leucine, arginine with lysine, leucine with methionine, leucine with phenylalanine, glycine with proline, threonine with serine, serine with threonine, tyrosine with tryptophan, phenylalanine with tyrosine, and / or leucine with valine, but are not limited thereto. Exemplary substitutions are shown in the following table. Amino acid substitutions can be introduced into human erythropoietin, and the product is screened for retention of the biological activity of human erythropoietin. TIFF2025524901000006.tif104170

[0112] "Prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized in a host after administration, for example, by hydrolysis or oxidation, to form a compound of the present disclosure (e.g., a compound of formula (I), (II), or (III)). The present disclosure includes within its scope prodrugs of the compounds described herein. Such examples include, but are not limited to, choline ester derivatives, N-alkylmorpholine esters, etc. Other derivatives of the compounds described herein are active in both their acid and acid derivative forms, but in the acid-sensitive form, they often provide advantages in solubility, tissue compatibility, or delayed release in mammalian organisms (see Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well-known to those skilled in the art, such as esters prepared by reaction of a suitable alcohol with the parent acid, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides, etc. Simple aliphatic or aromatic esters, amides, and anhydrides derived from pendant acidic groups on the compounds described herein are specific prodrugs. In some cases, it is desirable to prepare double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkyl esters. C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, aryl, C7-C12 substituted aryl, and C7-C12 arylalkyl esters of the compounds described herein may be preferred in some cases. Examples of prodrugs of testosterone include, but are not limited to, testosterone undecanoate, testosterone cypionate, testosterone enanthate, testosterone propionate, and testosterone buciclate. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs" Ed. H. Bundgaard, Elsevier, 1985.As used herein, "prodrug" can also refer to a naturally occurring precursor of testosterone, such as androstenedione.

[0113] When a compound disclosed herein is an ester (e.g., testosterone acetate) or a prodrug (e.g., testosterone undecanoate) of a compound of formula (I), (II), or (III), it is understood that the ester or prodrug constituent (e.g., for testosterone undecanoate, the undecylenic acid ester) can include additional instances of hydrogen not shown in the drawings of formula (I), (II), or (III).

[0114] The term "biologically active metabolite" means a pharmacologically active product produced through the metabolism in vivo of a particular compound (e.g., a compound of formula (I), (II), or (III), or a salt thereof).

[0115] The term "pharmaceutically acceptable salts" refers to salts that are suitable for use in contact with the tissues of humans and lower animals within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, etc., and that are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or using organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from appropriate bases include alkali metals, alkaline earth metals, ammonium, and N + (C 1-4 alkyl)4 -Salts are included. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0116] As used herein, the term "effective amount" refers to the amount of testosterone or GH necessary, for example, to reduce at least one or more symptoms of a disease or disorder, and relates to a pharmacological composition in an amount sufficient to provide the desired effect. Thus, the term "therapeutically effective amount" refers to the amount of testosterone or GH, for example, that is sufficient to provide a beneficial effect when administered to a typical subject. The effective amount as used herein will also, in various contexts, include an amount sufficient to delay the onset of symptoms of a disease, modify the course of a symptomatic disease (e.g., without limitation, slow the progression of symptoms of the disease), or reverse the symptoms of a disease. Thus, it is generally not practical to specify an exact "effective amount". However, for any given case, the appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0117] An effective amount, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (lethal dose for 50% of the population) and the ED50 (therapeutically effective dose at 50% of the population). The dosage may vary depending on the dosage form used and the route of administration utilized. The dose ratio between the toxic and therapeutic effects is a therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods showing a large therapeutic index are preferred. A therapeutically effective dose can first be estimated from cell culture assays. Also, the dosage can be formulated in an animal model to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test sterol or GH that achieves half-maximal suppression of the symptoms) determined in cell cultures or in a suitable animal model. The levels in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored, inter alia, by suitable bioassays, for example, assays for free test sterol or GH. The dosage is determined by the physician and can be adjusted as needed to conform to the observed effects of the treatment.

[0118] The terms "decrease", "reduced", "reduction", and "inhibit" are all used herein to mean a decrease by a statistically significant amount. In some embodiments, "reduce", "reduction", or "decrease", or "inhibit" typically means a decrease by at least 10% compared to a reference level (e.g., in the absence of a given treatment), e.g., at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. The decrease can preferably be reduced to a level that is acceptable within the normal range for an individual without a given disorder.

[0119] The terms "improve", "increased", "increase", "enhance", or "activate" are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms "improve", "increased", "increase", "enhance", or "activate" mean an increase of at least 10% compared to a reference level, e.g., an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to the reference level, or an increase up to 100%, or any increase from 10 to 100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold compared to the reference level, or any increase from 2-fold to 10-fold or more. In the context of a marker or symptom, "increase" means a statistically significant increase in such levels.

[0120] As used herein, "subject" or "individual" means a human or an animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal, or game animal. Examples of primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of domestic animals and game animals include cows, horses, pigs, sheep, goats, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as salmon, catfish, and trout. In some embodiments, the subject is a mammal, such as a human or a non-human mammal. The mammal can be, but is not limited to, a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Non-human mammals can be advantageously used as subjects representing animal models of disorders. The terms "individual", "patient", and "subject" are used interchangeably herein. The subject can be male or female.

[0121] The subject can be a subject who has or has previously been diagnosed with or identified as having a condition or disorder that requires treatment (e.g., FSHD), or one or more complications associated with such a condition or disorder, and optionally, can be a subject who has already received treatment for such a condition or disorder, or one or more complications associated with the condition or disorder. Alternatively, the subject can also be a subject who has not previously been diagnosed as having a condition or disorder, or one or more complications associated with the condition or disorder. For example, the subject can be a subject who exhibits one or more risk factors for a condition or disorder, or one or more complications associated with the condition or disorder, or a subject who does not exhibit a risk factor.

[0122] A "subject in need of treatment" for a particular condition or disorder can be a subject who has the condition or disorder, has been diagnosed as having the condition or disorder, or is at risk of developing the condition or disorder.

[0123] As used herein, the term "administer" refers to placing a drug (e.g., GH or testosterone or a derivative thereof) as disclosed herein in a subject by a method or route that results in at least partial delivery of the drug to the desired site. A pharmaceutical composition containing a drug disclosed herein can be administered by any suitable route that provides an effective treatment in a subject. The terms "administer" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those of skill in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, intraocular administration, intratympanic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration including injection such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various embodiments, the preparation can be administered therapeutically, i.e., to treat an existing disease or condition. In further various embodiments, the preparation can be administered prophylactically, i.e., to prevent a disease or condition.

[0124] As used herein, the terms "treat," "treatment," "treating," or "remission" refer to therapeutic treatment, with the goal of reversing, reducing, alleviating, inhibiting, slowing, or halting the progression or severity of a disease or disorder, such as a condition associated with FSHD. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder associated therewith. Treatment is generally "effective" when one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" when the progression of the disease is reduced. That is, "treatment" includes not only improvement of symptoms or markers, but also slowing or arresting the progression or worsening of symptoms as compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of the degree of the disease, stabilization (i.e., not worsening) of the disease state, delay or slowing of the progression of the disease, remission or amelioration of the disease state, remission (whether partial or complete), and / or decrease in mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative treatment).

[0125] A "therapeutically effective amount" is an amount sufficient to ameliorate a disease state or symptom, particularly a condition or symptom associated with the disease state, or otherwise prevent, impede, delay, or reverse in any way the progression of a disease state or any other undesirable symptom associated with the disease or disorder.

[0126] A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, has the intended prophylactic effect, e.g., preventing or delaying the onset (or recurrence) of a disease state, or reducing the likelihood of the onset (or recurrence) of a disease state or associated symptoms. A complete therapeutic or prophylactic effect is not necessarily achieved by administration of a single dose and may occur only after administration of a series of doses. Accordingly, a therapeutically effective amount or a prophylactically effective amount may be administered in one or more administrations.

[0127] As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharmaceutically acceptable carrier, such as carriers commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" as used herein means, within the scope of sound medical judgment, a compound, material, composition, and / or dosage form that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems, or complications commensurate with a reasonable benefit / risk ratio.

[0128] As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to a carrier medium or excipient that does not interfere with the effectiveness of the biological activity of the active ingredient of the composition and is not unduly toxic to the host at the concentrations at which it is administered. In the context of the present invention, the pharmaceutically acceptable carrier or excipient is preferably suitable for topical formulations. This term includes, but is not limited to, solvents, stabilizers, solubilizers, isotonicity enhancers, structurants, suspending agents, dispersing agents, chelating agents, emulsifying agents, defoaming agents, ointment bases, emollients, skin protectants, gelling agents, thickening agents, pH adjusters, preservatives, penetration enhancers, complexing agents, lubricants, viscous agents, viscosity enhancers, bioadhesive polymers, or combinations thereof. The use of such agents for the formulation of pharmaceutically active substances is well known in the art (see, for example, "Remington’s Pharmaceutical Sciences", E.W. Martin, 18th Ed., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety).

[0129] As used herein, the terms "a", "an", "the" and similar terms used in the context of the present invention (in particular, in the context of the claims) shall be construed to cover both the singular and the plural forms, unless otherwise indicated herein or clearly contradicted by the context. The recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the invention and does not impose a limitation on the scope of the invention as otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0130] As disclosed herein, several ranges of values are provided. It is understood that, unless otherwise clearly indicated by the context, each intervening value between the upper and lower limits of that range to one tenth of the unit of the lower limit is also specifically disclosed. Each smaller range between any of the specifically recited values or intervening values within the recited range and any other specifically recited value or intervening value within the recited range is included within the invention. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range, and any range having either, neither, or both of its limits included within the smaller range, subject to any specifically excluded limits detailed within the recited range, is included within the invention. Where the recited range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0131] The term "about" refers to within 10%, preferably within 5%, and more preferably within 1% of a given value or range. Alternatively, the term "about" refers to within the average acceptable standard error, as considered by one of ordinary skill in the art.

[0132] As used herein, the terms "comprising" or "comprises" are used with respect to compositions, methods, and respective components thereof, which are open to the inclusion of unspecified elements, whether essential or not, to a method or composition. The term "consisting of" refers to the compositions, methods, and respective components thereof described herein, excluding any element not recited in the description of that embodiment. As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. This term permits the presence of elements that do not substantially affect the basic and novel or functional characteristics of that embodiment.

Examples

[0133] The example describes a 4-month, proof-of-concept, single-center, open-label study of daily human growth hormone (Genotropin®, 5.0 μg / kg via subcutaneous injection) and testosterone (testosterone enanthate, 140 mg via intramuscular injection every 2 weeks) in men with FSHD. A total of 20 subjects were enrolled in this study at the University of Rochester Medical Center in Rochester, NY.

[0134] Example 1 This example describes the study objectives and measures, the trial protocol, and subject selection and enrollment.

[0135] The primary objective of this study was to investigate the safety and tolerability of rHGH and testosterone in adult male patients with FSHD. Secondary objectives included (i) investigating the pharmacodynamic effects of rHGH and testosterone on serum levels of free and total testosterone, IGF-1, thyroid function, luteinizing hormone, and follicle-stimulating hormone, and (ii) investigating the effects of rHGH and testosterone on total and regional fat-free mass. Exploratory objectives included investigating the effects of rHGH and testosterone on multiple measures of clinical function, including walking, strength, lung function, and disease burden reported by the patient.

[0136] Safety outcome measures included (1) changes from baseline in laboratory test results, vital signs, and EKG results, (2) the occurrence of serious adverse events, and (3) the occurrence of non-serious adverse events. Exploratory efficacy outcome measures included the following. 1. Composite quantitative muscle testing (QMT) score 2. Composite manual muscle testing (MMT) score 3. FSHD clinical outcome measure (FSHD-COM), total and individual test scores 4. FSHD-health index (FSHD-HI), disease-specific patient-reported outcome measure 5. Forced vital capacity (FVC) 6. Epworth Sleepiness Scale 7. Fatigue Severity Scale 8. Dual-energy X-ray absorptiometry (DEXA) fat-free mass (total and regional) 9. PROMIS-57 10. Individualized neuromuscular quality of life questionnaire (INQoL) 11. Beck Depression Inventory (BDI) 12. Six-minute walk distance

[0137] Participants received the following interventions: (1) 5.0 μg / kg of rHGH per day (after dinner) by subcutaneous injection, and (2) 140 mg of testosterone enanthate administered intramuscularly every two weeks. The dosage of rHGH was calculated using the patient's pre-entry weight. All participants were instructed to self-administer the study drug.

[0138] The following table provides information regarding the probability of observing at least one such event during the trial, considering different values for the true incidence rate of adverse events in the target FSHD population receiving combination therapy and the precision with which the incidence rate of specific adverse events can be estimated. A sample size of 20 participants allows for obtaining a 95% upper confidence bound for an incidence rate that is within approximately 20% of the observed incidence rate. Also, it is highly likely that an adverse event with a true incidence rate of 15% or more can be observed in at least one subject during the trial. TIFF2025524901000007.tif42170

[0139] Regarding the exploratory measure of efficacy, changes from baseline to week 24 in the total distance walked in six minutes (6MWT) were considered. Using data on six-month changes in 6MWT from nine participants in a long-term study of FSHD progression, an estimated standard deviation of 42.3 meters was derived. Using data on 12-month changes in 6MWT from 71 participants in a long-term study of progression in participants with DM1, a similar estimated value of 38.5 meters was derived. Assuming a standard deviation of 40 meters for the primary outcome variable, a sample size of 18 participants would provide 85% power to detect an average change of 30 meters using a t-test and a 5% significance level (two-sided). The mean 6MWT distance at baseline in the FSHD cohort was approximately 350 meters. Thus, the selected effect size of 30 meters represents an improvement of approximately 8.6% in 6MWT, relatively speaking. Recent studies in chronic obstructive pulmonary disease, coronary artery disease, and Duchenne muscular dystrophy support distances of 25 - 35 meters as representing minimally clinically important changes. With a sample size of 20 participants, a 10% expected attrition rate can be tolerated.

[0140] Analysis of lean body mass: The lean body mass obtained using DEXA was an objective primary marker used to determine whether combination therapy showed sufficient promise to warrant further study in FSHD. Analysis of this outcome involved the use of a repeated measures analysis of variance model (i.e., so-called "mixed model repeated measures," or MMRM), treating time (handled as a categorical variable) as the factor of interest. The covariance matrix for within-subject observations was modeled using an unstructured pattern. Ninety-five percent confidence intervals for the mean change from baseline to each visit were calculated using this model, with the 24-week time point being the main concern. Tests for the significance of the mean change from baseline to 24 weeks were performed using this model with a 5% significance level (two-sided). Analyses were conducted according to the intention-to-treat principle, including all registered participants. The repeated measures analysis of variance model used in these analyses employed a direct likelihood approach to estimate the parameters of interest using all available data from all participants. An important assumption underlying this analysis was that missing data were "missing at random" (MAR), i.e., the probability that a response was missing for a subject depended only on the set of observed data for that subject and not on the specific missing value that was not obtained. For lean body mass, an estimated standard deviation of 1.6 kg was derived using data on 24-week changes from participants in the inventors' previous long-term study of FSHD progression (PI: Heatwole, U01AR065119). Assuming a standard deviation of 1.6 kg for this outcome variable, a sample size of 18 participants provided 85% power to detect a mean change of 1.2 kg using a t-test and a 5% significance level (two-sided).

[0141] An experienced principal investigator in clinical trials was responsible for the recruitment and follow-up of 20 subjects for this clinical trial. The participants were ambulatory male FSHD patients aged 18 - 65 years with moderate disease. All patients were symptomatic and had either genetically confirmed FSHD or clinical symptoms suggestive of FSHD and had a first-degree relative with genetically confirmed FSHD. The complete inclusion and exclusion eligibility criteria are listed below. To be eligible for enrollment in this study, subjects must meet all of the following eligibility criteria. Inclusion Criteria ■ Males aged 18 - 65 years ■ Ambulatory symptomatic FSHD of moderate severity (genetically confirmed or with clinical symptoms suggestive of FSHD and having a first-degree relative with genetically confirmed FSHD) ■ Hematocrit of 50% or less ■ Prostate-specific antigen of 4.0 ng / ml or less (or 3.0 ng / ml or less if the participant is at high risk for prostate cancer) ■ Fasting blood glucose of less than 126 mg / dl ■ Able to walk continuously for 6 minutes at a distance greater than 75 meters and less than 700 meters (cane, walker, orthotics are permitted). Exclusion Criteria ■ Diabetes, obesity (BMI > 35 kg / m2), cardiovascular disease (heart failure, coronary artery disease, uncontrolled hypertension, untreated hypercholesterolemia) ■ Untreated thyroid disease ■ Deep vein thrombosis ■ Untreated severe sleep apnea ■ Past pituitary disease ■ Liver disease ■ Mental illness ■ Kidney disease ■ Severe lower urinary tract symptoms (International Prostate Symptom Score (IPSS) > 19) ■ Cancer (other than basal cell skin cancer) ■ Active desire for pregnancy, heavy alcohol use (> 50 g / day) ■ Current use of testosterone or HGH ■ Current use of drugs that interfere with the growth hormone or gonadal endocrine axis.

[0142] Volunteers from diverse ethnic and racial backgrounds were recruited using local and national recruitment tools. These individuals were evaluated at the University of Rochester, which has a history of successfully recruiting patients for FSHD clinical trials.

[0143] Example 2 This example describes drug administration and assignment.

[0144] GENOTROPIN® was supplied by PFIZER INC. The generic form of testosterone enanthate was purchased by the investigational pharmacy from a local vendor. Both drugs were received and dispensed by the investigational drug service department at the University of Rochester.

[0145] Participants were given the study drug during their baseline visit and then received GENOTROPIN® for 5 weeks (lasting until the 4-week visit, allowing for a 1-week delay) to take home in a properly cooled container. Participants were also given testosterone dosages at 2 and 4 weeks. At the 4- and 8-week study visits, participants were given GENOTROPIN® again for 5 more weeks, sufficient to last from the inpatient visit, and testosterone dosages were given at 2 and 4 weeks later. Prior to the 12-week mark of the study, participants were mailed the appropriate amount of testosterone (given at weeks 12 and 14) and the exact amount of GENOTROPIN® to complete the study. The treatment packs were numbered for tracking. Compliance was tracked using a compliance questionnaire, and empty packs and vials were collected at each study visit. Subjects self-administered each study drug.

[0146] Testosterone Enanthate: Subjects received vials of appropriate doses of Testosterone Enanthate and were administered as a single intramuscular injection into the gluteal muscle. When administered appropriately, intramuscular injection of Testosterone Enanthate was well tolerated. Care was taken to slowly and deeply inject the preparation into the gluteal muscle, following the usual precautions for intramuscular administration, such as avoiding intravascular injection.

[0147] GENOTROPIN (registered trademark): Subjects were given the GENOTROPIN PEN (registered trademark) 5 delivery device with a 5 mg cartridge. This device had a dial for selecting the appropriate dose of the study drug and a direct dose display on the pen. Participants were instructed on how to administer the correct drug dose using the dial. Participants received GENOTROPIN (registered trademark) via subcutaneous injection, based on their weight at screening / baseline visit, using these pens. Each subject pinched the skin fold at the injection site, inserted the pen at a 90-degree angle, pressed the black / white injection knob until a clicking sound was heard, waited for 5 seconds, and then withdrew the pen. Participants were given a document detailing the use of the GENOTROPIN PEN (registered trademark) 5 and were taught how to use the pen during their hospital stay.

[0148] Example 3 This example describes the clinical and laboratory evaluations. An overview of the schedule of activities and evaluations is provided in Table 2. TIFF2025524901000008.tif227170

[0149] After informed consent, each participant had a screening visit. Participants who met all eligibility criteria were enrolled and received serial inpatient evaluations at baseline, 8 weeks, 16 weeks, 24 weeks, and 36 weeks. Throughout the study, participants received weekly phone calls from the clinical coordinator to address any study-related difficulties. All subjects were followed for 36 weeks as needed, regardless of protocol violation or withdrawal from the study drug.

[0150] Screening and Baseline Assessment Screening and baseline assessment were conducted at the same visit to the University of Rochester Clinical Research Center, where subjects were required to stay overnight. Screening assessments were performed after the subject signed the informed consent document. Baseline assessments were performed after the inclusion / exclusion criteria were met (e.g., fasting serum glucose, liver function tests, hematocrit, serum prostate specific antigen, and 6-minute walk test).

[0151] During this visit, subjects were thoroughly informed about all aspects of the study, including all scheduled visits and activities, and were required to sign and date the informed consent prior to any study-related procedures.

[0152] Subjects were evaluated for study eligibility by the principal investigator or coordinator. All inclusion criteria must be met, and none of the exclusion criteria may apply. All results from the screening procedures must be available before determining the subject's eligibility for the study.

[0153] The following procedures were performed at the screening visit. ● Obtain written informed consent (if not already obtained) ● Assign a subject identification number ● Review inclusion / exclusion criteria ● Medical history ● Demographics ● Review use of concomitant medications ● Obtain vital sign measurements (height, weight, blood pressure, body mass index, pulse, respiratory rate, waist circumference) at the time of vital sign acquisition ● Obtain blood samples for laboratory tests (total testosterone level, free testosterone level, total IGF-1, PSA, fasting glucose, comprehensive metabolic panel, CK, lipid profile, fasting insulin level, TSH, and free T4 level, luteinizing hormone, FSH, CBC) ● Physical examination ● 6-minute walk test ● International Prostate Symptom Score ● Digital rectal examination ● Electrocardiogram (EKG) was performed.

[0154] After the eligibility criteria were met, the following baseline visit procedures were performed. ● Registration for the target study ● Final review of eligibility was conducted. ● 6-minute walk test ● Quantitative muscle test ● Manual muscle test ● FSHD Clinical Outcome Measure (FSHD-COM) ● Forced vital capacity ● FSHD-Health Index (FSHD-HI) ● PROMIS-57 ● Beck Depression Inventory (BDI) ● Fatigue Severity Scale ● Dual-energy X-ray absorptiometry (DEXA) ● Fundus examination ● Laboratory studies (if not performed within 7 days as part of the screening visit) ● Quantitative insulin sensitivity check index ● Side effect questionnaire ● International Physical Activity Questionnaire ● Administration of the initial dose of each therapy

[0155] After the initial dose of each therapy was administered, the following activities were performed. ● Vital signs were obtained (blood pressure, pulse, respiratory rate). ● Questions were asked about any immediate side effects (e.g., nausea, dizziness, gastrointestinal discomfort). ● A complete dose management log was kept to record the administration of the first dose. ● Instructions were given to the subjects regarding the correct administration of the study drug. ● Instructions were given to the subjects to bring any unused study drug to each study visit and report any adverse events immediately to the investigator or coordinator.

[0156] Follow-up visit After the subject completed screening / baseline visit, the subject returned to the clinical research center for in-person visits at 8 weeks, 16 weeks, and 24 weeks of treatment. The subject was also evaluated at week 36, 12 weeks after washout. The subject was contacted weekly to ask about general condition and to resolve any issues with the study medications. The in-person visit took about 6 hours.

[0157] During on-site follow-up visits, the following activities were completed. These visits could be conducted within ±4 days of the target date.

[0158] Week 8 Visit The in-person visit at week 8 was accompanied by the following. ● Blood specimens for clinical safety (total testosterone level, free testosterone level, total IGF-1, PSA, fasting glucose, comprehensive metabolic panel, CK, lipid profile, HGA1c, fasting insulin level, TSH, and free T4 level, LH, FSH, CBC, CRP) ● 6-minute walk test ● Quantitative muscle testing ● Manual muscle testing ● FSHD Clinical Outcome Measure (FSHD-COM) ● Forced vital capacity ● FSHD-Health Index (FSHD-HI) ● PROMIS-57 ● Beck Depression Inventory (BDI) ● Fatigue Severity Scale ● Vital signs (height, weight, blood pressure, body mass index, pulse, respiratory rate, waist circumference) ● Fundus examination ● Physical examination ● Digital rectal examination ● International Prostate Symptom Score ● Quantitative insulin sensitivity check index ● Side effect questionnaire ● International Physical Activity Questionnaire ● EKG ● Review of concomitant medications and adverse events

[0159] Participants without clinically significant adverse events or laboratory abnormalities maintain the daily dosage of the study drug. Participants receive the study drug by mail for the next four weeks.

[0160] Visit at the 16th week The in-person visit at the 16th week was accompanied by the following. ● Blood specimens for clinical safety (total testosterone level, free testosterone level, total IGF-1, PSA, fasting glucose, comprehensive metabolic panel, CK, lipid profile, HGA1c, fasting insulin level, TSH, and free T4 level, LH, FSH, CBC, CRP) ● 6-minute walk test ● Quantitative muscle test ● Manual muscle test ● FSHD Clinical Outcome Measure (FSHD-COM) ● Forced vital capacity ● FSHD-Health Index (FSHD-HI) ● PROMIS-57 ● Beck Depression Inventory (BDI) ● Fatigue Severity Scale ● Dual-energy X-ray absorptiometry (DEXA) ● Vital signs (height, weight, blood pressure, body mass index, pulse, respiratory rate, waist circumference) ● Fundus examination ● Physical examination ● Digital rectal examination ● International Prostate Symptom Score ● Quantitative insulin sensitivity check index ● Side effect questionnaire ● International Physical Activity Questionnaire ● EKG ● Review of concomitant medications and adverse events

[0161] Those participants without clinically significant adverse events or laboratory abnormalities maintain the daily dosage of the study drug.

[0162] Visit at the 24th week The in-person visit at the 24th week was accompanied by the following. ● Blood samples for clinical safety (total testosterone level, free testosterone level, total IGF-1, PSA, fasting glucose, comprehensive metabolic panel, CK, lipid profile, HbA1c, fasting insulin level, TSH, and free T4 level, LH, FSH, CBC, CRP) ● Six-minute walk test ● Quantitative muscle test ● Manual muscle test ● FSHD Clinical Outcome Measure (FSHD-COM) ● Forced vital capacity ● FSHD-Health Index (FSHD-HI) ● PROMIS-57 ● Beck Depression Inventory (BDI) ● Fatigue Severity Scale ● Dual-energy X-ray absorptiometry (DEXA) ● Vital signs (height, weight, blood pressure, body mass index, pulse, respiratory rate, waist circumference) ● Fundus examination ● Physical examination ● Digital rectal examination ● International Prostate Symptom Score ● Quantitative insulin sensitivity check index ● Side effect questionnaire ● International Physical Activity Questionnaire ● EKG ● Review of concomitant medications and adverse events

[0163] After this hospital visit, the participants will not use the study drug.

[0164] Hospital visit at week 36 (12 weeks after washout) The in-person hospital visit at week 36 was accompanied by the following. ● Blood samples for clinical safety (total testosterone level, free testosterone level, total IGF-1, PSA, fasting glucose, comprehensive metabolic panel, CK, lipid profile, HbA1c, fasting insulin level, TSH, and free T4 level, LH, FSH, CBC, CRP) ● Six-minute walk test ● Quantitative muscle test ● Manual muscle test ●FSHD Clinical Outcome Measurement (FSHD-COM) ●Forced vital capacity ●FSHD-Health Index (FSHD-HI) ●PROMIS-57 ●Beck Depression Inventory (BDI) ●Fatigue Severity Scale ●Dual-energy X-ray absorptiometry (DEXA) ●Vital signs (height, weight, blood pressure, body mass index, pulse, respiratory rate, waist circumference) ●Fundus examination ●Physical examination ●Rectal examination ●International Prostate Symptom Score ●Quantitative insulin sensitivity check index ●Side effect questionnaire ●International Physical Activity Questionnaire ●EKG ●Review of concomitant medications and adverse events

[0165] Participants received weekly phone calls to assess any side effects or changes in medical health over time.

[0166] Example 4 This example describes the assessment of subjects and other evaluations. The efficacy evaluation was performed using the 6-minute walk test (6MWT) measurement, quantitative muscle testing (QMT), manual muscle testing (MMT), and FSHD Clinical Outcome Measurement (FSHD-COM). The functional evaluation was performed using forced vital capacity, Epworth Sleepiness Scale, Fatigue Severity Scale, and dual-energy X-ray absorptiometry (DEXA). The self-reported evaluations included the FSHD-Health Index (FSHD-HI), PROMIS-57, individualized neuromuscular quality of life questionnaire (INQoL), and Beck Depression Inventory (BDI).

[0167] For safety evaluation and adverse event evaluation, various clinical safety laboratory tests were performed. For treatment-specific evaluation, subjects were monitored for the following treatment-related events. ●Peripheral edema ●Carpal tunnel syndrome ●Gynecomastia ● Insulin resistance ● Joint pain ● Increased erythropoiesis ● Reduction of HDL cholesterol ● Elevation of prostate-specific antigen ● Increase in blood pressure

[0168] Physical examinations were performed to evaluate peripheral edema, carpal tunnel symptoms, and joint pain, including funduscopy using a pan-optic ophthalmoscope. Participants were also monitored using the International Prostate Symptom Score (Barry MJ, et al. The American Urological Association symptom index for benign prostatic hyperplasia. The Measurement Committee of the American Urological Association. J Urol 1992;148:1549,57; discussion 1564) and the Quantitative Insulin Sensitivity Check Index (Quicki) to assess signs of prostate dysfunction or diabetes. See Blackman MR, et al. JAMA 2002;288:2282-92, and Sattler FR, et al. J Clin Endocrinol Metab 2009;94:1991-2001. Patients completed a treatment-specific questionnaire designed for early detection of adverse events related to the study drug continuously at each study visit and were contacted weekly by study personnel to monitor for any adverse events (Giannoulis MG, et al. J Clin Endocrinol Metab 2006;91:477-84).

[0169] As shown in FIGS. 1A-1I, the results of the above evaluation showed various statistically and clinically relevant improvements after 24 weeks of treatment. The mean 6-minute walk distance increased by 37.6 meters (p = 0.001), the 2-minute walk distance increased by 11.6 meters (p = 0.03), the total fat-free mass (measured by DEXA) increased by 2.2 kg (p < 0.0001), the total body fat (measured by DEXA) decreased by 1.3 kg (p = 0.044), the overall strength from six bilateral muscle groups (standardized QMT, predicted normal mean %) increased by 5.5% (p = 0.032), the upper body strength from four bilateral muscle groups (standardized QMT, predicted normal mean %) increased by 4.9% (p = 0.031), the mean lower limb strength from eight bilateral muscle groups (manual muscle testing) increased by 3.0% (0.13 points, p = 0.004), the physical function measured by FSHD-COM improved by 12.4% (2.4 points, p = 0.006), and the Beck Depression Inventory score improved by 31.6% (1.7 points, p = 0.031). Regarding the PROMIS-57 score, the fatigue score improved by 10.6% from baseline (5.7 points, p = 0.002), and the pain interference score improved by 11.7% (6.5 points, p = 0.005). Regarding the FSHD-HI score, the total score improved by 19.5% from baseline (6.08 points, p = 0.043), the shoulder / arm function score improved by 23.7% (11.4 points, p = 0.024), the core strength / function score improved by 30.3% (10.4 points, p = 0.018), the fatigue score improved by 29.9% (11.5 points, p = 0.017), and the pain score improved by 31.0% (9.5 points, p = 0.011). Importantly, these changes correlated with the change in IGF-1 levels from baseline, and the IGF-1 level increased by 46.5% from baseline (89.79 ng / mL, p < 0.0001). TIFF2025524901000009.tif103170

[0170] These unexpected and excellent results demonstrate in clinical trials that combination therapy of testosterone and growth hormone has significant benefits in functional capacity, disease burden, muscle mass, fat reduction, walking, fatigue, pain, and strength for patients with FSHD. The scope of this benefit has not been demonstrated using any other therapeutic mechanism in FSHD.

[0171] The foregoing description of the embodiments and preferred embodiments is not intended to limit the disclosure as defined by the claims, but should be construed as illustrative. As will be readily understood, numerous variations and combinations of the features described above can be utilized without departing from the disclosure described in the claims. Such variations are not considered to depart from the scope of the disclosure, and it is intended that all such variations be included within the scope of the following claims. All references cited herein are hereby incorporated by reference in their entirety.

Claims

**Claim 1** A method for use in a subject in need of (i) improving muscle mass, muscle strength, or muscle function, or (ii) treating a disorder associated with muscle wasting or muscle weakness, or (iii) reducing fatigue, pain, or obesity, the method comprising: administering to the subject an effective amount of testosterone or a derivative thereof; administering to the subject an effective amount of growth hormone or a derivative thereof. **Claim 2** Use in the manufacture of a medicament for (i) improving muscle mass, muscle strength, or muscle function, (ii) treating a disorder associated with muscle wasting or muscle weakness, or (iii) reducing fatigue, pain, or obesity, of (A) an effective amount of testosterone or a derivative thereof, or (B) an effective amount of growth hormone or a derivative thereof, or (C) both, wherein said improving, treating, or reducing comprises administering to a subject in need thereof an effective amount of testosterone or a derivative thereof and administering to the subject an effective amount of growth hormone or a derivative thereof. **Claim 3** The method or use according to claim 1 or 2, wherein the subject is healthy, has a disorder or condition associated with muscle wasting or muscle weakness, or is at risk of developing said disorder or condition. **Claim 4** The method or use according to claim 2 or 3, wherein the disorder or condition is selected from facioscapulohumeral muscular dystrophy (FSHD), sarcopenia, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, Becker muscular dystrophy, Pompe disease, myotonic dystrophy type 1, myotonic dystrophy type 2, inclusion body myositis, polymyositis, dermatomyositis, ALS, spinal muscular atrophy, Charcot-Marie-Tooth disease, HIV myopathy, wasting in the elderly, deconditioning, nutritional deficiency, injury, cancer-related wasting, muscle dysfunction, nerve dysfunction, neuromuscular junction dysfunction, and motor neuron disease. **Claim 5** The method or use according to claim 4, wherein the disorder is FSHD. **Claim 6** The method or use according to any one of the preceding claims, wherein the derivative is a testosterone ester. **Claim 7** The derivative has the structure of formula (II), The method or use according to claim 6, wherein R is alkyl, alkanediyl, alkenyl, alkenediyl, alknyl, aralkyl, aryl, heteroaryl, or acyl.

8. The method or use according to claim 7, wherein the testosterone derivative is selected from the group consisting of testosterone enanthate, testosterone propionate, testosterone cypionate, testosterone undecanoate, testosterone oleate, and testosterone palmitate.

9. The method or use according to claim 8, wherein the derivative is testosterone enanthate.

10. The method or use according to claim 9, wherein the testosterone or its derivative is administered at about 0.1 mg to 30,000 mg.

11. The method or use according to claim 10, wherein the testosterone or its derivative is administered once every two weeks at about 70 - 170 mg, or once every two weeks at about 110 - 150 mg.

12. The method or use according to claim 10, wherein the testosterone or its derivative is administered once every two weeks at about 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, or 150 mg.

13. The method or use according to claim 12, wherein the testosterone or its derivative is administered once every two weeks at about 140 mg.

14. The method or use according to any one of the preceding claims, wherein the testosterone or its derivative is administered to the subject via intramuscular injection.

15. The method or use according to any one of the preceding claims, wherein the growth hormone or its derivative is administered at about 0.01 μg / kg / day to 250 μg / kg / day / kg / day.

16. The method or use according to claim 14, wherein the growth hormone or its derivative is administered at about 2.5 - 6.0 μg / kg / day, or about 4.0 - 5.5 μg / kg / day.

17. The method or use according to claim 16, wherein the growth hormone or a derivative thereof is administered at about 4.0 μg / kg / day, 4.1 μg / kg / day, 4.2 μg / kg / day, 4.3 μg / kg / day, 4.4 μg / kg / day, 4.5 μg / kg / day, 4.6 μg / kg / day, 4.7 μg / kg / day, 4.8 μg / kg / day, 4.9 μg / kg / day, 5.0 μg / kg / day, 5.1 μg / kg / day, 5.2 μg / kg / day, 5.3 μg / kg / day, 5.4 μg / kg / day, or 5.5 μg / kg / day.

18. The method or use according to claim 17, wherein the growth hormone or a derivative thereof is administered at about 5.0 μg / kg / day.

19. The method or use according to any one of the preceding claims, wherein the growth hormone or a derivative thereof is administered to the subject via subcutaneous injection.

20. The method or use according to any one of the preceding claims, wherein the testosterone or a derivative thereof, or the growth hormone or a derivative thereof is administered for a duration of at least one week.

21. The method or use according to claim 19, wherein the duration is 8 to 36 weeks, 12 to 30 weeks, or about 24 weeks.

22. The method or use according to any one of the preceding claims, further comprising identifying or evaluating the subject.

23. The identifying or evaluating comprises one or more selected from the group consisting of quantitative muscle testing (QMT), manual muscle testing (MMT), FSHD clinical outcome measurement (FSHD - COM), FSHD - health index (FSHD - HI), forced vital capacity (FVC), Epworth sleepiness scale, fatigue severity scale, dual energy X-ray absorptiometry (DXA) fat-free mass (total and local), DXA body fat mass, PROMIS - 57, individualized neuromuscular quality of life questionnaire (INQoL), Beck depression inventory (BDI), and 6-minute walk distance. The method or use according to claim 23.

24. A method for treating FSHD in a subject, comprising: administering to the subject an effective amount of testosterone or a derivative thereof according to any one of the preceding claims; and administering to the subject an effective amount of growth hormone or a derivative thereof according to any one of the preceding claims.

25. A kit for use in a subject in need of (i) improvement of muscle mass, muscle strength, or muscle function, (ii) treatment of disorders associated with muscle wasting or muscle weakness, or (iii) reduction of fatigue, pain, or obesity, the kit comprising: (i) an effective amount of testosterone or a derivative thereof; and (ii) an effective amount of growth hormone or a derivative thereof.

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