WO2025212841A1 - Compositions and methods for treating diseases and disorders associated with muscle weakness - Google Patents

Abstract

The present invention provides compositions and methods of their use in treating dystroglycanopathy, muscular dystrophy and other disorders. In particular, a method of treating a disorder associated with a mutation or loss of function in a fukutin related protein (FKRP) gene and/or a disorder associated with a defect in glycosylation of α-DG in a subject is provided, comprising administering to the subject an effective amount of a ribitol and a selective estrogen receptor modulator (SERM).

Description

COMPOSITIONS AND METHODS FOR TREATING DISEASES AND DISORDERS

ASSOCIATED WITH MUSCLE WEAKNESS

STATEMENT OF PRIORITY

[0001] This application claims the benefit, under 35 U.S.C. § 119(e), of U.S. Provisional Application No. 63/574,452, filed April 4, 2024, and U.S. Provisional Application No. 63/632,671, filed April 11, 2024, the entire contents of each of which are incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The present invention is directed to pharmaceutical formulations and methods of use thereof in treating dystroglycanopathy, muscular dystrophy, and other disorders.

BACKGROUND OF THE INVENTION

[0003] Dystroglycanopathies are a subset of muscular dystrophies characterized by a secondary defect in glycosylation of alpha-dystroglycan (a-DG). The diseases have been linked to autosomal-recessive mutations in at least 18 different genes. They include fukutin-related protein (FKRP fukutin, like-acetylglucosaminyltransferase (LARGE), POMGnTl, P0MT1, P0MT2, Isoprenoid Synthase Domain Containing (ISPD), Transmembrane protein 5 (TMEM5), l,3-N- acetylglucosaminyltransferase 1 (B3GNT1), glycosyltransferase-like domain containing 2 (GTDC2), p3-N-acetylgalactosaminyltransferase 2 (B3GALNT2), DOLK, GMPPB, DMP2 , DMP3 and SGK196. Biochemical studies have established direct evidence for involvement of a number of the genes in glycosylation modifications of a-DG. Fukutin and Fukutin related protein (FKRP) genes have been recently proposed as Ribitol-5-P transferase that transfers the phosphorated ribitol to the core sugar chain of a-DG. LARGE protein acts as a bifunctional glycosyltransferase, xylosyltransferase and glucuronyltransferase, producing repeating units of [- 3-xylose- a 1,3 -glucuronic acid- 1-] that is the functional glycan chain linking cell membrane protein and extracellular matrix proteins. This LARGE glycan chain is linked to the core O- mannosyl glycans by tandem ribitols. This linkage is critical for muscle health and lack of FKRP function as the result of gene mutations therefore prevents the production of functional glycosylation of a-DG, and disrupts normal interaction between membrane and connective tissues, leading to muscle fiber damage and muscular dystrophy. [0004] Mutations in the FKRP gene cause a wide spectrum of disease from a milder form of limbgirdle muscular dystrophy (LGMD2I) to severe Walker-Warburg syndrome (WWS), muscle-eye- brain disease (MEB), and congenital muscular dystrophy type ID (MDC1D). However, little progress has been made for the treatment of the diseases. There is no effective therapy available and only physical therapy and palliative care are being routinely provided as treatment.

[0005] Currently, there is no approved treatment specifically for the FKRP mutation-related diseases. At the time of submitting this document, there are two experimental therapies undergoing clinical trials. One is the Adeno-associated virus (AAV)-mediated gene therapy and the other is ribitol treatment. Specifically, AAV gene therapy has demonstrated significant effect to delay and even to stop disease progression in mouse models bearing mutations detected in patients. However, there are still significant challenges to AAV gene therapy including most critically the dose-related toxicity and variation (heterogeneity) in transgene expression from muscle to muscle and between muscle fibers within a single muscle. Clinical trials have reported fatality closely related to doses at or higher than IxlO¹⁴ AAV virus particles/kg body weight (BW). Yet, such high doses are critical for achieving significant efficacy. Further, even under such conditions, once-for-lifetime efficacy may still not be achievable due to huge variation in transgene expression in muscles. Overall, long-term efficacy of AAV gene therapy to muscular dystrophies remains to be established.

[0006] On the other hand, dietary supplementation with the five-carbon sugar alcohol ribitol can be readily converted into CDP-ribitol, the substrate of FKRP glycosyltransferase, to add the critical ribitol-5-phosphate to form the functional product of glycan on a-DG. Studies have shown that ribitol can rescue glycosylation (matriglycan) defects caused by FKRP mutations, preserve muscle function, and attenuate fibrosis in affected muscles in dystrophic mouse models. Further, preclinical and phase I and II clinical trial have shown that ribitol is well tolerated with no obvious side effect. A phase III clinical trial run by the ML Bio Solutions and Bridge Biopharma, USA, is well in progress for LGMD2I patients. However, the effect of ribitol treatment relies on the residual function of mutant FKRPs. This places limits on capacity of ribitol treatment to achieve sufficient matriglycan expression to stop disease progression at least for a proportion of patient population with greatly diminished function. Fortunately, ribitol restores homogenous expression of glycosylation in both cardiac and skeletal muscles, and this distribution provides better protection from contraction-related muscle damage when compared to effect from the same levels of matriglycan expression by AAV gene therapy. Nevertheless, long-term-efficacy of ribitol treatment remains to be established in clinics.

[0007] Other interesting drugs with potential to treat FKRP mutation-related diseases are tamoxifen and raloxifene which belong to the class of selective estrogen receptor modulator (SERM) which are known to reduce inflammation and improve bone strength. Earlier studies in animal models showed that the combination of these two SERM drugs are able to alleviate disease progression with improved muscle function, however at doses higher than that currently used in clinics for diseases such as osteoporosis and cancer prevention. Such high doses, especially with tamoxifen, are also associated with side effects such as atrophy of male sex organs and female endometrial hyperplasia with increased risk in cancer occurrence. The potential of the SERMs for muscular dystrophies can therefore only be realized if the doses of these drugs can be reduced to that lower than currently used in clinics. Thus, there is a need for the development of effective treatments for FKRP -related diseases.

SUMMARY OF THE INVENTION

[0008] The present invention is based on the finding that a combination therapy of a ribitol and a SERM provides superior therapeutic effects in treating FKRP-mutation related diseases and/or a disorder associated with a defect in glycosylation of a-DG than the individual therapies do alone. Further, the combination permits lower doses of each agent to be administered, reducing potential side effects.

[0009] Accordingly, one aspect of the invention relates to a method of treating a disorder associated with a mutation or loss of function in a FKRP gene and/or a disorder associated with a defect in glycosylation of a-DG in a subject, comprising administering to the subject a therapeutically effective amount of a ribitol and a SERM, thereby treating the disorder in the subject.

[0010] Another aspect of the invention relates to a method of treating or inhibiting the development of muscle weakness in a subject that is a carrier of a mutated FKRP gene and/or a gene associated with a defect in glycosylation of a-DG, comprising administering to the subject a therapeutically effective amount of ribitol and a SERM, thereby treating or inhibiting the development of muscle weakness in the subject. [0011] A further aspect of the invention relates to a therapeutically effective amount of ribitol and a SERM for use in treatment of a disorder associated with a mutation or loss of function in a FKRP gene and/or a disorder associated with a defect in glycosylation of a-DG in a subject or for use in treatment or inhibition of development of muscle weakness in a subject that is a carrier of a mutated FKRP gene and/or a gene associated with a defect in glycosylation of a-DG.

[0012] An additional aspect of the invention relates to the use of a therapeutically effective amount of ribitol and a SERM in the manufacture of a medicament for treatment of a disorder associated with a mutation or loss of function in a FKRP gene and/or a disorder associated with a defect in glycosylation of a-DG in a subject or for treatment or inhibition of development of muscle weakness in a subject that is a carrier of a mutated FKRP gene and/or a gene associated with a defect in glycosylation of a-DG.

[0013] These and other aspects of the invention are set forth in more detail in the description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure l is a series of microscopy and Western blot images showing the effect of 12-month treatments of tamoxifen (Tam), ribitol (rib), and a combination of tamoxifen and ribitol (Tam+rib) in FKRP P448L mutant mice (Untreated) compared to control C57 mice on the glycosylation of a-DG (via detection with a IIH6 antibody) in the tibialis anterior (TA), diaphragm (dia), or heart (Heart) muscle.

[0015] Figure 2 is a series of microscopy images showing the effect of 12-month treatments of tamoxifen (Tam), ribitol (rib), and a combination of tamoxifen and ribitol (Tam+rib) in FKRP P448L mutant mice (Untreated) compared to control C57 mice on the histology in the tibialis anterior (TA) or diaphragm muscle.

[0016] Figure 3 is a series of graphs showing the body weight and treadmill exercise tests after 12-month treatments of tamoxifen (Tam), ribitol, and a combination of tamoxifen and ribitol (Tam+ribitol) in FKRP P448L mutant mice (P448L-saline) compared to control C57 mice.

[0017] Figure 4, panel A is a series of microscopy images with H&E staining showing the effect of 12-month treatments of low-dose tamoxifen, ribitol, a combination of low-dose tamoxifen and ribitol, low-dose raloxifene, and a combination of low-dose raloxifene and ribitol in FKRP P448L mutant mice compared to control C57 (Control) mice on the histology in the tibialis anterior (TA), diaphragm (Diaph), or heart (Heart) muscle. Figure 4, panel B is a series of graphs showing the change in centrally nucleated fibers (% CNF) in the in the tibialis anterior (TA) or diaphragm (Diaph) after 12-month treatments of low-dose tamoxifen (Tam), ribitol (Rib), a combination of low-dose tamoxifen and ribitol (Tam/Rib), low-dose raloxifene (Rai), and a combination of low- dose raloxifene and ribitol (Ral/Rib) in FKRP P448L mutant mice compared to control C57 (Cont) mice. * = P<0.05 compared to untreated control.

[0018] Figure 5, panel A is a series of microscopy images with Mason’s Tri chrome staining showing the effect of 12-month treatments of low-dose tamoxifen, ribitol, a combination of low- dose tamoxifen and ribitol, low-dose raloxifene, and a combination of low-dose raloxifene and ribitol in FKRP P448L mutant mice compared to control C57 (Control) mice on the histology in the tibialis anterior (TA), diaphragm (Diaph), or heart (Heart) muscle. Figure 5, panel B is a series of graphs showing the change in fibrosis (% Fibrosis) in the in the tibialis anterior (TA), diaphragm (Diaph), or heart (Heart) muscle after 12-month treatments of low-dose tamoxifen (Tam), ribitol (Rib), a combination of low-dose tamoxifen and ribitol (Tam/Rib), low-dose raloxifene (Rai), and a combination of low-dose raloxifene and ribitol (Ral/Rib) in FKRP P448L mutant mice compared to control C57 (Cont) mice. * = P<0.05 compared to untreated control.

[0019] Figure 6 is a series of graphs showing the forelimb and hindlimb grip force after 6- or 12- month treatments of tamoxifen (Tam), ribitol (Rib), and a combination of tamoxifen and ribitol (Tam/Rib), low-dose raloxifene (Rai), and a combination of low-dose raloxifene and ribitol (Ral/Rib) in FKRP P448L mutant mice compared to control C57 (Cont) mice. * = P<0.05 compared to untreated control.

[0020] Figure 7 is a series of graphs showing the body weight and treadmill exercise tests after 6- or 12-month treatments of tamoxifen (Tam), ribitol (Rib), and a combination of tamoxifen and ribitol (Tam/Rib), low-dose raloxifene (Rai), and a combination of low-dose raloxifene and ribitol (Ral/Rib) in FKRP P448L mutant mice compared to control C57 (Cont) mice. * = P<0.05 compared to untreated control.

[0021] Figure 8 is a series of graphs showing whole body plethysmography tests after 6- or 12- month treatments of tamoxifen (Tam), ribitol (Rib), and a combination of tamoxifen and ribitol (Tam/Rib), low-dose raloxifene (Rai), and a combination of low-dose raloxifene and ribitol (Ral/Rib) in FKRP P448L mutant mice compared to both untreated FKRP P448L mutant mice , with C57 mice as controls (Cont). Whole body plethysmography parameters include inspiratory time (Ti), expiratory time (Te), peak inspiratory flow (PIF), peak expiratory flow (PEF), end inspiratory pause (EIP), end expiratory pause (EEP), frequency (F), minute volume (MV), and enhanced pause (Penh). * = P<0.05 compared to untreated control.

[0022] Figure 9 is a series of graphs showing cardiac function and morphology after treatments of tamoxifen (Tam), ribitol (Rib), and a combination of tamoxifen and ribitol (Tam/Rib), low-dose raloxifene (Rai), and a combination of low-dose raloxifene and ribitol (Ral/Rib) in FKRP P448L mutant mice compared to both untreated FKRP P448L mutant mice, with C57 mice as controls (Cont). Cardiac output function and morphology parameters include cardiac output (CO), ejection fraction (EF), stroke volume (SV), and myocardial thickness (Myo-thick).

[0023] Figure 10 is a series of graphs showing the change in body weight, treadmill distance, and grip force after 6- or 12-month treatments of tamoxifen (Tam), ribitol (Rib), and a combination of tamoxifen and ribitol (Tam/Rib), low-dose raloxifene (Rai), and a combination of low-dose raloxifene and ribitol (Ral/Rib) in FKRP P448L mutant mice compared to control C57 (Cont) mice. * = P<0.05 compared to untreated control.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0025] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

[0027] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0028] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0029] Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0030] The term “about,” as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0031] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.” [0032] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold and/or can be expressed in the enhancement and/or increase of a specified level and/or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. [0033] The term “inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0034] A “therapeutically effective” amount as used herein is an amount that provides some improvement or benefit to the subject. Alternatively stated, a “therapeutically effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject (e.g., in the case of a FKRP -mutation related disease improved walking and/or gait, reduced joint stiffness, improved respiratory function, reduced incidence of a neuronal migration abnormality, enhanced glycosylation of a-DG or other cell membrane proteins, etc.). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0035] By the term “treat,” “treating,” or “treatment of’ (or grammatically equivalent terms) is meant to reduce or to at least partially improve or ameliorate the severity of the subject’s condition and/or to alleviate, mitigate or decrease in at least one clinical symptom and/or to delay the progression of the condition.

[0036] As used herein, the term “prevent,” “prevents,” or “prevention” (and grammatical equivalents thereof) means to delay or inhibit the onset of a disease. The terms are not meant to require complete abolition of disease, and encompass any type of prophylactic treatment to reduce the incidence of the condition or delay the onset of the condition.

[0037] A “prevention effective” amount as used herein is an amount that is sufficient to prevent and/or delay the onset of a disease, disorder and/or clinical symptoms in a subject and/or to reduce and/or delay the severity of the onset of a disease, disorder and/or clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.

[0038] A “subject” may be any vertebrate organism in various embodiments. A subject may be individual to whom an agent is administered, e.g., for experimental, diagnostic, and/or therapeutic purposes or from whom a sample is obtained or on whom a procedure is performed. In some embodiments a subject is a mammal. A mammalian subject may include, but is not limited to, a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, primate, etc.), a farm or commercial animal (e.g., cattle, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, gerbil, hamster, etc.). In some embodiments, a mammalian subject may be a primate, or a non-human primate (e.g., a chimpanzee, baboon, macaque (e.g., rhesus macaque, crab-eating macaque, stumptailed macaque, pig-tailed macaque), monkey (e.g., squirrel monkey, owl monkey, etc.), marmoset, gorilla, etc.). In some embodiments, a mammalian subject may be a human. In some embodiments a human subject is a neonate, child, adult, or geriatric subject. In some embodiments, the subject is a female human. In some embodiments, the subject is a male human.

[0039] A “subject in need” of the methods of the invention can be any subject known or suspected of having increased risk of developing a disease related to a FKRP -mutation and/or a disorder associated with a defect in glycosylation of a-DG described herein to which administering a composition as described herein may provide beneficial health effects.

[0040] “Pharmaceutically acceptable,” as used herein, means a material that is not biologically or otherwise undesirable, z.c., the material can be administered to an individual along with the compositions of this invention, without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The material would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art (see, e g., Remington's Pharmaceutical Science,' 21^st ed. 2005). The carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a tablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight of the active compound. One or more active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well-known techniques of pharmacy comprising admixing the components, optionally including one or more accessory ingredients. Exemplary pharmaceutically acceptable carriers for the compositions of this invention include, but are not limited to, phosphate buffered saline (PBS), sterile pyrogen-free water, and other sterile pyrogen-free physiological saline solutions.

[0041] Furthermore, a "pharmaceutically acceptable" component such as a salt, carrier, excipient or diluent of a composition according to the present invention is a component that (i) is compatible with the other ingredients of the composition in that it can be combined with the compositions of the present invention without rendering the composition unsuitable for its intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are "undue" when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable components include any of the standard pharmaceutical carriers such as saline solutions, water, emulsions such as oil/water emulsion, microemulsions and various types of wetting agents.

[0042] Grammatical variations of “administer,” “administration,” and “administering” to a subject include any route of introducing or delivering to a subject an agent/active compound (e.g., a ribitol and a SERM). Administration can be carried out by any suitable route, including oral, buccal (e.g., sub-lingual), topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intrajoint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intracerebroventricular, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. In some embodiments, the administration is oral administration. [0043] “ Concurrent administration,” “administration in combination,” “simultaneous administration,” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time, overlapping in time, or one following the other. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another. In some embodiments, a composition of the present invention can be administered orally or intravenously or subcutaneously to a subject daily, weekly, biweekly or monthly. [0044] ‘ ‘Isolated”, as used herein, means that a substance (e.g., a ribitol and/or a SERM) is sufficiently free of contaminants or cell components with which said substance may occur. “Isolated” does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the substance in a form in which it can be used therapeutically.

[0045] “Monodispersed” is used herein to describe a mixture of compounds wherein about 100 percent of the compounds in the mixture have the same molecular weight. “Substantially monodispersed” is used herein to describe a mixture of compounds wherein at least about 95 percent of the compounds in the mixture have the same molecular weight. “Purely monodispersed” is used herein to describe a mixture of compounds wherein about 100 percent of the compounds in the mixture have the same molecular weight and have the same molecular structure. Thus, a purely monodispersed mixture is a monodispersed mixture, but a monodispersed mixture is not necessarily a purely monodispersed mixture. “Substantially purely monodispersed” is used herein to describe a mixture of compounds wherein at least about 95 percent of the compounds in the mixture have the same molecular weight and have the same molecular structure. Thus, a substantially purely monodispersed mixture is a substantially monodispersed mixture, but a substantially monodispersed mixture is not necessarily a substantially purely monodispersed mixture.

Compositions

[0046] The present invention relates to administering to a subject a therapeutically effective amount of a ribitol and a SERM. Thus, one aspect of the invention relates to compositions comprising a ribitol and/or a SERM. In some embodiments, the ribitol and the SERM are in the same composition. In some embodiments, the ribitol and the SERM are in separate compositions. [0047] In some embodiments, the ribitol and/or the SERM may comprise a polyalkylene glycol moiety coupled or linked thereto. “Polyalkylene glycol” means straight or branched polyalkylene glycol polymers including, but not limited to, polyethylene glycol (PEG), polypropylene glycol (PPG), and polybutylene glycol (PBG), as well as co-polymers of PEG, PPG and PBG in any combination, and includes the monoalkylether of the polyalkylene glycol. Thus, in various embodiments of this invention, the polyalkylene glycol in the compositions of this invention can be, but is not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and any combination thereof. In some embodiments, the polyalkylene glycol of the composition is polyethylene glycol or “PEG.” The term “PEG subunit” refers to a single polyethylene glycol unit, i.e., — (CH2CH2O)— . Thus, the ribitol and/or the SERM can be “pegylated.” In some embodiments, the PEG can have a molecular weight from about 10,000 g/mol to about 30,000 g/mol. In some embodiments, the polyalkylene glycol (e.g., PEG) can be non-poly dispersed, monodispersed, substantially monodispersed, purely monodispersed, or substantially purely monodispersed.

[0048] The ribitol and SERM of this invention can be in a composition (e.g., in the same or in separate compositions) comprising a pharmaceutically acceptable carrier. In some embodiments, the ribitol and the SERM can be separately mixed or combined with any substance for improved delivery, absorption, etc. In some embodiments, the ribitol and the SERM can be mixed or combined together with any substance for improved delivery, absorption, etc.

[0049] The amount of the ribitol and the SERM administered to a subject will vary from subject to subject, depending on the nature of the disclosed compositions, the species, gender, age, weight and general condition of the subject, the mode of administration, and the like. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the disclosed compositions are those large enough to produce the desired effect (e.g., to reduce pain, treat a respiratory illness, reduce fever, etc.). The dosage should not be so large as to outweigh benefits by causing extensive or severe adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like, although some adverse side effects may be expected. The dosage can be adjusted by the individual clinician in the event of any counterindications.

[0050] In some embodiments, a SERM of this invention (e.g., tamoxifen and/or raloxifene) can be delivered in a dosage range of about 0.01 mg/kg to about 100 mg/kg, e.g., about 0.01 mg/kg to about 10 mg/kg (e.g., 0.01. 0.05. 0.075, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 mg/kg) per treatment. In some embodiments, a SERM of this invention is tamoxifen and is delivered in a dose of about 1 mg/kg. In some embodiments, a SERM of this invention is raloxifene and is delivered in a dose of about 10 mg/kg. Nonlimiting examples of dosage ranges of this invention include from about O.Olmg/kg to about 0. Img/kg, from about 1 mg/kg to about 10 mg/kg, from about 10 mg/kg to about 50 mg/kg a day, etc. In some embodiments, for daily use, the dose can range from about 0.01 mg/kg to about 0.1 mg/kg, or from about 1 mg/kg to about 10 mg/kg a day. In some embodiments, e.g., for weekly or monthly treatment, the dose can range from about 0.1 mg/kg to about 1 mg/kg or from about 10 mg/kg to about 50 mg/kg per treatment. In some embodiments, the SERM is tamoxifen, raloxifene, asofoxifene, bazedoxifene, toremifene, ospemifene, methyl- piperidino-pyrazole (MPP), or any combination thereof.

[0051] In some embodiments, a ribitol of this invention is ribitol (adonitol) pentose alcohol, with or without modifications such as tri-acetylated ribitol (Ribitol(OAc)3, per-acetylated ribitol (Ribitol(OAc)s, a precursor thereof, such as ribose, a polysaccharide thereof, a phosphate form thereof, a non-phosphated form thereof, any precursor of a phosphate form, such as Ribose-5-P, any nucleotide form of ribitol (e.g., a nucleotide-alditol having cytosine or other bases as the nucleobase with 1, 2 or 3 phosphate groups and ribitol as the alditol portion), such as CDP-ribitol, CDP-ribitol-OAc2, or any combination or derivative or modification thereof. In some embodiments, the ribitol can be any other form that can be converted to ribitol, or ribitol phosphate, or nucleotide-ribitol. In some embodiments, the ribitol can be delivered orally in a formulation containing from about 0.1 to about 100% concentration of the drug as many times as desirable, e.g., from about 1 time to about 100 times a day. The ribitol can also be taken as a tablet or a capsule about 1 to about 10 times daily. The total amount of the ribitol for daily use can be from about 0.001g to about 500 g depending on the nature and formulation of the drug, the ribitol or modified ribitol with enhanced effect, etc. In some embodiments, the daily dose of the ribitol is about 1 mg/kg to about 3 mg/kg. In some embodiments, the daily dose of the ribitol is about 2 mg/kg. In some embodiments, ribitol can be administered or delivered to a subject in combination with (e.g., simultaneously, before and/or after) cytidine triphosphate (CTP) and/or any other nucleotide in an amount effective for enhancing the effect of ribitol on glycosylation of a-DG or other proteins.

[0052] Ribitols form in many plants and especially in the plant, Adonis vernalis, also known as spring pheasant's eye, or false hellebore, or yellow pheasant's eye and others. Adonis vernalis belongs to the buttercup family Ranunculaceae . Plants containing ribitols can be administered as the drug for treating FKRP -related diseases and subjects with FKRP mutation and other diseases. Such plants can be directly used as a food supplement, and/or ribitol can be extracted from the plants for administration as described herein. [0053] Compositions suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of one or more active compounds (e.g., a ribitol and/or a SERM); as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such compositions may be prepared by any suitable method known in the art which includes the step of bringing into association one or more active compounds and a suitable carrier. In general, the compositions of the invention are prepared by uniformly and intimately admixing one or more active compounds with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding a powder or granules containing one or more active compounds, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, one or more active compounds in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, one or more powdered, active compounds moistened with an inert liquid binder.

[0054] Compositions suitable for buccal (sub-lingual) administration include lozenges comprising one or more active compounds in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising one or more active compounds in an inert base such as gelatin and glycerin or sucrose and acacia.

[0055] Compositions of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of one or more active compounds, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents. The compositions may be presented in unit\dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-inj ection immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. For example, in one aspect of the present invention, there is provided an injectable, stable, sterile composition comprising one or more active compounds, or a salt thereof, in a unit dosage form in a sealed container. The one or more active compounds or salt is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 10 mg to about 10 g (e.g., about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, or about 10 g) of the compound or salt. When the one or more active compounds or salt is substantially water-insoluble, a sufficient amount of emulsifying agent which is physiologically acceptable may be employed in sufficient quantity to emulsify the one or more active compounds or salt in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

[0056] Compositions suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing one or more active compounds with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture. [0057] Compositions suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

[0058] Compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Compositions suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous solution of one or more active compounds. Suitable compositions comprise citrate or bi s\tri s buffer (pH 6) or ethanol/water and contain from about 0.1 to about 5 M (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or about 5 M) of one or more active compounds.

[0059] In addition to one or more active compound, the pharmaceutical compositions described herein may contain other additives, such as pH-adjusting additives. In particular, useful pH- adjusting agents include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate. Further, the compositions may contain microbial preservatives. Useful microbial preservatives include methylparaben, propylparaben, and benzyl alcohol. The microbial preservative is typically employed when the formulation is placed in a vial designed for multidose use. Of course, as indicated, the pharmaceutical compositions of the present invention may be lyophilized using techniques well known in the art.

[0060] In some embodiments of this invention, the compound of this invention is present in an aqueous solution for subcutaneous administration. In some embodiments, the compound is provided as a lyophilized powder that is reconstituted and administered subcutaneously.

[0061] The active compounds of this invention can be present in one or more pharmaceutical formulations (e.g., compositions) that comprise substances and/or agents that are not natural products. As a nonlimiting example, the active compounds of this invention can be present in a pharmaceutical composition with polyethylene glycol (PEG), which in some embodiments can have a molecular weight (MW) in a range of about 200 to about 500. In some embodiments, a pharmaceutical composition of this invention can comprise glucose.

Methods of Use

[0062] One aspect of the invention relates to a method of treating a disorder associated with a mutation or loss of function in a fukutin related protein (FKRP) gene and/or a disorder associated with a defect in glycosylation of a-DG in a subject, comprising administering to the subject a therapeutically effective amount of a ribitol and a selective estrogen receptor modulator (SERM), thereby treating the disorder in the subject. In some embodiments, the disorder associated with a mutation or loss of function in the FKRP gene is a limb-girdle muscular dystrophy- dystroglycanopathy (e.g., limb-girdle muscular dystrophy type 21 (LGMD2I) or limb-girdle muscular dystrophy type R9 (LGMDR9)), Walker-Warburg syndrome (WWS), muscle-eye-brain disease (MEB), congenital muscular dystrophy type 1C (MDC1C), congenital muscular dystrophy type ID (MDC1D), or any combination thereof.

[0063] Another aspect of the invention relates to a method of treating or inhibiting the development of muscle weakness in a subj ect that is a carrier of a mutated FKRP gene, comprising administering to the subject a therapeutically effective amount of ribitol and a SERM, thereby treating or inhibiting the development of muscle weakness in the subject. In some embodiments, the muscle weakness can be in skeletal muscle, cardiac muscle and/or respiratory muscle, singly or in any combination. [0064] In some embodiments, the ribitol and the SERM are administered concurrently, e.g., in the same composition or in separate compositions. In some embodiments, the ribitol and the SERM are administered separately. In some embodiments, the SERM is administered at a dose that is about 0.4 to about 0.8 times (e.g., about 0.4, 0.5, 0.6, 0.7, or about 0.8 times) the dose used for treating cancer (e.g., breast cancer). In some embodiments, the SERM (e.g., tamoxifen) is administered at a dose of about 0.5 mg/kg to about 20 mg/kg (e.g., about 0.5, 1, 1.5, 2, 2.5, 3, 3.5,

4. 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 mg/kg). In some embodiments, the ribitol is administered at a dose of about 1 g/kg to about 5 g/kg (e.g., 1,

1.5, 2, 2.5, 3, 3.5, 4, 4.5, or about 5 g/kg). In some embodiments, the ribitol is administered at a dose of about 2 g/kg.

[0065] In some embodiments, the ribitol and/or the SERM can be administered from about every 1 hour to about every 12 hours (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or about every 12 hours). In some embodiments, the ribitol and/or the SERM can be administered from about every 1 week to about every 7 days (e.g., about every 1, 2, 3, 4, 5, 6, or about every 7 days). In some embodiments, the ribitol and/or the SERM can be administered from about every 1 week to about every 4 weeks (e g., about every 1, 2, 3, or about every 4 weeks). In some embodiments, the ribitol and/or the SERM can be administered from about every 1 month to about every 6 months (e.g., about every 1, 2, 3, 4, 5, or about every 6 months).

[0066] The method of this invention can include a step of additionally administering to the subject an additional therapeutic agent, in combination with the ribitol and the SERM (concurrently, before and/or after the ribitol and the SERM administration). Nonlimiting examples of a therapeutic agent of this invention include a phosphodiesterase type 5 (PDE 5) inhibitor, a nonsteroidal anti-inflammatory agent, a metabolite supplement, or any combination thereof. In some embodiments, the ribitol and the SERM can be co-administered with (prior to, simultaneously and/or after) a bisphosphonate (e.g., Alendronate), an angiotensin converting enzyme inhibitor (ACE inhibitor), an angiotensin receptor blocker (e.g., rosartan), singly or in any combination. In some embodiments, the ribitol and the SERM can be administered with any other therapy and/or therapeutic agent (simultaneously, before and/or after), such as steroid therapy and/or FKRP gene therapy to enhance or increase the therapeutic effect.

[0067] In some embodiments, the subject of this invention is a female subject and in some embodiments, the subject of this invention is a male subject. In some embodiments, the SERM is tamoxifen and the subject is a female subject. In some embodiments, the SERM is raloxifene and the subject is a male or female subject that has or is at increased risk of having muscular dystrophy. [0068] The methods of this invention can also be used to treat non-muscular dystrophy diseases for which restoration of and/or enhanced glycosylation of a-DG would be beneficial and/or therapeutic.

[0069] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.

EXAMPLES

[0070] Currently, each of the potential treatments for FKRP mutation-related diseases have their own inherent weaknesses. AAV gene therapy poses dose-related toxicity, highly variable transgene expression, and uncertain long-term genome toxicity. Ribitol treatment alone can only restore limited levels of glycosylation despite its desirable distribution of the restored glycosylation. The use of tamoxifen and raloxifene at the reported dosage risks severe, known side effects. Without wishing to be bound by any particular theory, the combination treatment of ribitol and SERM can be beneficial in treating FKRP mutation-related diseases. Specifically, without wishing to be bound by any particular theory, ribitol restores glycosylation, reduction of which is the cause of the diseases, while the SERMs prevent any muscle damage-related secondary inflammatory response in diseased muscles. The use of SERM also improves osteoporosis due to disuse of muscles, thus preventing bone fractures which often causes dire consequences to patients. Without wishing to be bound by any particular theory, the combined treatment of ribitol and tamoxifen or raloxifene may provide preferable treatment to the FKRP mutation-related diseases. [0071] Here, tamoxifen and raloxifene were tested at doses similar to and lower than the doses currently used in clinics for treating human diseases in long-term (e.g., 12 month) animal model studies. Without wishing to be bound by any particular theory, lower doses of tamoxifen and raloxifene could also provide therapeutic effect to diseased muscles of patients with FKRP mutations. With doses lower than that currently used in clinics for human diseases, these two drugs can be safely applied for long-term treatment of FKRP mutation-related diseases. Without wishing to be bound by any particular theory, combined treatment of ribitol with clinically applicable doses of either tamoxifen or raloxifene may be able to achieve better efficacy than any drug alone.

[0072] The results demonstrated that 1 year tamoxifen treatment alone at the daily dose of 2mg/kg, equivalent to a human dose of about lOmg for 60kg body weight, had limited but detectable therapeutic effect in the P448L mutant mice (FIGS. 1-3). However, combination of 2mg/kg tamoxifen with 5g/kg ribitol significantly improved muscle pathology and function. FIG. 1 shows the glycosylation that was detected with the IIH6 antibody on samples of FKRP P448L mutant mice treated for 12 months. A loss of glycosylation was seen in control tissues with high background staining most notable in the diaphragm. Ribitol treatment, but not tamoxifen alone, clearly enhanced glycosylation. The combined treatment showed the most preferable preservation of histology and glycosylation with IIH6 staining homogeneously, with the pattern similar to that in normal control C57 mice. A positive signal for matriglycan was barely detectable in the saline treated control in the Western blot, and no difference seen in the sample of tamoxifen and raloxifene treatments alone. A marked increase in IIH6 staining was seen in the heart and TA in combination treatment groups.

[0073] In FIG. 2, the areas of fibrosis (white grey patches) were evident in the muscles of untreated mutant mice. Ribitol treatment reduced the fibrosis, but the clearest reduction was observed in the muscles from the combined treatment. Analysis of the muscle function by treadmill exercise in FKRP P448L mutant mice demonstrated that ribitol (5 g/kg) and tamoxifen (2 mg/kg) treatment alone improved muscle function (FIG. 3). However, the biggest improvement was seen from the mice treated with the combination of the two drugs. There was no clear body weight change with single treatment, but the bodyweight was reduced in the combined treatment group. [0074] While a dose of 2 mg/kg tamoxifen is close to the dose currently used in clinics for treating cancer, we further explored an even lower dose of tamoxifen for these combined treatment experiments. Importantly, raloxifene was also included to analyze if other, similar compounds, could have similar therapeutic effects at clinically applicable dose and in combination with ribitol. One year treatment with these therapies demonstrated their efficacy.

[0075] H&E staining in P448L mutant mice showed that the TA of untreated control mice had clusters of centrally nucleated fibers with mononuclear cell infiltration (FIG. 4, panel A). Reduced numbers of mononuclear cells were observed in all treated TA samples. Large pockets of mononuclear cell and fat infiltration were noted in the diaphragm of untreated controls with the remaining muscle fibers sporadically isolated. An increase in the number of muscle fibers was observed in ribitol treated diaphragm due to a reduction in fibrosis and fat replacement. Mice treated with tamoxifen with and without ribitol showed improved numbers of muscle fibers but retained clear fibrosis and streaks of mononuclear cells. Raloxifene with and without ribitol showed the greatest improvement in diaphragm histology with the least streaks of fibrosis and mononuclear cells. FIG. 4, panel B demonstrated a reduction in centrally nucleated fibers in the TA and diaphragm in all treatment groups with combination treatments showing fewer centrally nucleated fibers than single treatment.

[0076] Mason’s Trichrome staining in P448L mutant mice showed that fibrotic streaks (stained in blue) were seen in all treated samples, but were reduced in the treated samples with ribitol, raloxifene and tamoxifen with ribitol (FIG. 5, panel A). The reduction was especially clear in diaphragm with raloxifene and raloxifene in combination with ribitol. Although only a small percentage of the heart was fibrotic in untreated mice, a reduction was noted in all samples after treatments. Quantification of fibrosis showed minimal reduction in fibrosis in the TA with all ribitol, raloxifene and tamoxifen and combined treatments (FIG. 5, panel B). Reduction in fibrosis was clearly detected in the heart, most noticeably in the raloxifene treated samples. All treated samples showed a reduction in fibrosis in the diaphragm with the most notable reduction in the raloxifene and ribitol combination treatments.

[0077] Grip force measurements at 6-months post treatment showed improved forelimb grip strength in all treated samples except ribitol and improvements in hindlimb strength in both raloxifene treatments and ribitol with minor improvement in tamoxifen treatments (FIG. 6). 12 months post treatment showed improvements in all treatment groups, except ribitol alone, with statistically significant improvements in both combination therapies and raloxifene alone. Statistically significant improvements were seen in all treatment group for hindlimb grip strength. [0078] Treadmill and body weight measurements at 6- and 12-months post treatment in P448L mutant mice showed significantly lower weight in both raloxifene treated groups (FIG. 7). An increase in overall running distance and time were noted in all treatment groups, with the farthest distance and time noted in the raloxifene treated mice whereas tamoxifen and ribitol alone had only a slight improvement. Similar to 6-months posttreatment, 12-month treatments showed improved treadmill distance and time in all groups with statistical significance in groups of raloxifene treatments, combined tamoxifen and ribitol group over untreated controls. The greatest improvements in distance and time were noted in the combination raloxifene and ribitol treatment. [0079] Whole body plethysmography was used to measure pulmonary function after 12-months of treatments (FIG. 8). C57 controls in addition to the P448L diseased model control were used for references to evaluate the nature of changes. Almost all parameters changed towards levels seen in the C57 mice after treatments, indicating improvement. Inspiratory time (Ti) was significantly reduced in tamoxifen with ribitol as well as raloxifene with and without ribitol, though not to the level of C57. Expiratory time (Te) was significantly increased in the same three treatments with the combination treatment of raloxifene with ribitol reaching similar level to C57. Peak inspiratory flow (PIF) was only slightly improved in the combination treatments with no change in peak expiratory flow (PEF), though raloxifene alone showed further reduction. End inspiratory pause (EIP) was significantly reduced in all treatment groups with only a slight reduction in end expiratory pause (EEP) in the tamoxifen and raloxifene alone treatments. EIP reduction was the most consistent feature for improvement after gene therapy, ribitol and SERM treatments. Reduced frequency (F) was seen in the combination therapies with overall minute volume (MV) being reduced in tamoxifen alone as well as raloxifene with and without ribitol. Enhanced pause (Penh) was significantly reduced in all treatment groups except ribitol, though not to the level of C57.

[0080] Thus, the results shown here demonstrated that a low-dose, below the currently used in clinics, of tamoxifen had efficacy in treating the FKRP mutation related disease. Similarly, raloxifene doses lower than currently used in clinics achieved significant therapeutic effect whereas earlier studies reported limited therapeutic effect at doses 5 to 10 times higher than as described herein. Without wishing to be bound by any particular theory, this suggests that drug dosage is critical, especially to be used long-term for chronic diseases. The superior effect seen herein of raloxifene in combination with ribitol was also unexpected from the results of earlier, higher dose testing.

[0081] Cardiac function and morphology were examined using high-frequency ultrasound. (FIG. 9). C57 controls in addition to the P448L diseased model control were used for references to evaluate the nature of changes. The P448L mutant mouse showed a reduced ejection fraction and stroke volume as well as an increase in myocardial thickness compared to C57 controls, which lead to reduced cardiac output. EF was increased with combination treatment tamoxifen and raloxifene compared to untreated controls with significant increase for the Raloxifene alone group. However, myocardial thickness is reduced across all treatment groups with the biggest reduction noted in the raloxifene alone group. These changes result in an improved CO in all treatment groups with combined treatments showing CO levels above those of drug alone.

[0082] Changes in body weight, treadmill distance and grip force were observed from 6 to 12 months post treatment. (FIG. 10). C57 controls in addition to the P448L diseased model control were used for references to evaluate the nature of changes. While there is considerable variation between animals, a significant reduction in treadmill performance at 12-month posttreatment was seen when compared to 6-month post treatment in the control mice. A smaller reduction in treadmill performance was seen between two post treatments in the tamoxifen treated groups with and without ribitol. A minimal reduction was observed in the group with ribitol alone, especially in the raloxifene-treated groups, without and with ribitol. The control mice showed an increase in body weight from 6 to 12 months post treatment, while ribitol treated mice showed a smaller increase. Tamoxifen treated mice also increased body weight with and without ribitol. The mice treated with Raloxifene alone and in combination also increased in body weight between 6 to 12 months post treatment. However, body weight increased much slower during the first 6 months of raloxifene treatment. As a result, the raloxifene groups maintained an overall lower body weight compared to all the other groups. It’s also noted that the raloxifene groups did not appear to be underweight as the average weight at 6 months post treatment was about 30 g with raloxifene alone and about 33 g with combined treatment with ribitol after 12 months of treatment (FIG. 7), which is supported by the better maintenance of muscle function of the raloxifene treated mice than all other groups.

Materials

[0083] The FKRP-P448Lneo- (P448L) mutant mouse was used with C57B1/6 mice as controls. The P448L mutant mouse contains a c. 1343OT point mutation in the fukutin related protein (FKRP) gene resulting in an amino acid change from proline to leucine at the 448 position. In each treatment group, 10 P448L or C57BL/6 mice (five female and five male mice), aged 8 weeks, were used. The mice were randomly assigned to control and different treatment groups, with littermates split between control and treatment groups.

[0084] Treated P448L mice were given either Img/kg tamoxifen (Letco Medical, Decatur, AL) or 10 mg/kg Raloxifene (Teva Pharmaceuticals, North Wales, PA) daily, 6 days per week, for 12 months via oral gavage with or without 2 g/kg Ribitol (Biosynth, Gardner, MA). Control P448L mice were gavaged with the same amount of saline. Mice were sacrificed at scheduled time points, and muscles and organs snap frozen in cold isopentane (-80°C) and stored at -80°C. All attempts were made to analyze experiments blind to the treatment groups.

[0085] Tissue sections (6 pm thick) were stained with monoclonal antibody IIH6C4 (Millipore, Temecula, CA) and Alexa Fluor 594-labelled goat-anti -mouse IgM (Invitrogen, Eugene, OR) for detection of functionally glycosylated a-DG. Sections were stained with hematoxylin and eosin or Masson’s trichrome for histologic assessment of fiber size and centranucleation using the MetaMorph Basic Offline software version 7.7.0.0 (Molecular Devices LLC, Sunnyvale, CA). The percentages of fibrosis in diaphragm, heart, and biceps muscles were measured from Masson's trichrome staining (ImageJ software version 1.42; NIH, Bethesda, MD). The level of serum components was determined by Charles Riverside Laboratories International (Wilmington, MA). [0086] Total proteins were extracted from each muscle shaving using TX-100 buffer [1% Triton X-100, 50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, and 0.1% SDS] supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany). Samples were homogenized in TX-100 buffer, and the supernatants were collected by centrifugation at 16,000 x g for 10 minutes. Protein concentration was determined by Bradford assay (Bio-Rad DC protein assay). The lysates were then loaded onto 4% to 20% Tris-glycine gel (Invitrogen, Carlsbad, CA). The proteins were transferred to polyvinylidene difluoride membranes with constant ampere at 200 mA for 2 hours in cold room (4°C). Polyvinylidene difluoride membranes were incubated with protein-free T20 blocking buffer (Pierce, Rockford, IL). The antibodies against a-DG (IIH6C4) and a-actin (Sigma, St. Louis, MO) were incubated in 20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, and 0.1% Tween 20 at 1 :2000 dilutions. a-DG and a-actin antibodies were detected by horseradish peroxidase-goat anti-mouse IgM (Invitrogen, Carlsbad, CA) and goat anti-rabbit IgG-horseradish peroxidase conjugate (Bio-Rad, Hercules, CA), respectively. Blots were developed with electrochemiluminescence (PerkinElmer, Waltham, MA), and the images were exposed and processed by an LAS-4000 imaging system (Fujifdm, Valhalla, NY).

[0087] Grip strength was measured using a horizontal forelimb mesh and hindlimb angled mesh grip strength meter (Columbus Instruments, Columbus, OH). Five successful forelimb and hind limb strength measurements were recorded within 2 minutes were recorded with the highest and lowest measurements removed. Data was averaged and normalized to body weight (BW). [0088] Treadmill test was performed on LE8700 treadmill (Panlab/Harvard Apparatus, Barcelona, Spain). Mice were acclimated with a 0-degree plane for 5 minutes at a speed of 7 cm/second. After the acclimation the speed was increased by 1 cm/second every 30 seconds until a maximum speed of 25 cm/second was reached. A 0.2-mA shock grid was used throughout the procedure for motivation. Mice were considered exhausted if they remained on the shock grid for 10 consecutive seconds without getting off or 50% on/off within 1 -minute period. Mice were also monitored for any signs of distress or injury and removed promptly if any signs appeared.

[0089] Whole body plethysmography was performed using the Emka Technologies Whole Body Plethysmograph with IOX Respiratory function analysis software (Emka Technologies, Falls Church, VA). Mice were acclimated to individual chambers for 5 minutes prior to recording respiratory data. Immediately following the acclimation period the data collection was begun and continued for 15 minutes with continuous exchange of air.

[0090] Echocardiography was performed as described by Blaeser et al (Blaeser 2016) using the BioScan SonixTablet High Frequency Ultrasound (Analogic Ultrasound, Peabody, MA).

[0091] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

What is claimed is:

1. A method of treating a disorder associated with a mutation or loss of function in a fukutin related protein (FKRP) gene and/or a disorder associated with a defect in glycosylation of a-DG in a subject, comprising administering to the subject an effective amount of a ribitol and a selective estrogen receptor modulator (SERM), thereby treating the disorder in the subject.

2. The method of claim 1, wherein the ribitol and the SERM are administered concurrently.

3. The method of claim 2, wherein the ribitol and the SERM are administered in the same composition.

4. The method of claim 1, wherein the ribitol and the SERM are administered separately.

5. The method of any preceding claim, wherein the disorder associated with a mutation or loss of function in the FKRP gene is limb-girdle muscular dystrophy type 21 (LGMD2I), Walker- Warburg syndrome (WWS), muscle-eye-brain disease (MEB), congenital muscular dystrophy type 1C (MDC1C), congenital muscular dystrophy type ID (MDC1D), or any combination thereof.

6. The method of any preceding claim, wherein the SERM is tamoxifen, raloxifene, asofoxifene, bazedoxifene, toremifene, ospemifene, methyl-piperidino-pyrazole (MPP), or any combination thereof.

7. The method of any preceding claim, wherein the SERM is administered at a dose that is about 0.4 to about 0.8 times the dose used for treating cancer (e.g., breast cancer).

8. The method of claim 6, wherein the SERM is tamoxifen and is administered at a dose of about 1 mg/kg.

9. The method of claim 6, wherein the SERM is raloxifene and is administered at a dose of about 10 mg/kg.

10. The method of any preceding claim, wherein the ribitol is ribitol (adonitol) pentose alcohol, a precursor thereof, a polysaccharide thereof, a phosphate form thereof, a non-phosphated form thereof, a nucleotide form thereof, a precursor of a phosphate form thereof, Ribose-5-P, ribose, or any combination thereof.

11. The method of any preceding claim, wherein the ribitol is administered at a dose of about 2 g/kg.

12. A method of treating or inhibiting the development of muscle weakness in a subject that is a carrier of a mutated FKRP gene and/or a gene associated with a defect in glycosylation of a-DG, comprising administering to the subject an effective amount of ribitol and a SERM, thereby treating or inhibiting the development of muscle weakness in the subject.

13. The method of claim 12, wherein the muscle weakness is in skeletal muscle, cardiac muscle and/or respiratory muscle, singly or in any combination.

14. The method of claim 12 or 13, wherein the SERM is tamoxifen, raloxifene, asofoxifene, bazedoxifene, toremifene, ospemifene, methyl-piperidino-pyrazole (MPP), or any combination thereof.

15. The method of any one of claims 12-14, wherein the SERM is administered at a dose that is about 0.4 to about 0.8 times the dose used for treating breast cancer.

16. The method of claim 14, wherein the SERM is tamoxifen and is administered at a dose of about 1 mg/kg.

17. The method of claim 14, wherein the SERM is raloxifene and is administered at a dose of about 10 mg/kg.

18. The method of any one of claims 12-17, wherein the ribitol is ribitol (adonitol) pentose alcohol, a precursor thereof, a polysaccharide thereof, a phosphate form thereof, a non-phosphated form thereof, a nucleotide form thereof, a precursor of a phosphate form thereof, Ribose-5-P, ribose, or any combination thereof.

19. The method of any one of claims 12-18, wherein the ribitol is administered at a dose of about 2 g/kg.

20. The method of any one of claims 12-19, wherein the ribitol and the SERM are administered concurrently.

21. The method of claim 20, wherein the ribitol and the SERM are administered in the same composition.

22. The method of any one of claims 12-19, wherein the ribitol and the SERM are administered in separate compositions.

23. The method of any one of claims 12-19, wherein the ribitol and the SERM are administered separately.

24. A composition comprising a ribitol and a SERM.

25. The composition of claim 24, wherein the SERM is tamoxifen, raloxifene, asofoxifene, bazedoxifene, toremifene, ospemifene, methyl-piperidino-pyrazole (MPP), or any combination thereof.

26. The composition of claim 24 or 25, wherein the ribitol is ribitol (adonitol) pentose alcohol, a precursor thereof, a polysaccharide thereof, a phosphate form thereof, a non-phosphated form thereof, a nucleotide form thereof, a precursor of a phosphate form thereof, Ribose-5-P, ribose, or any combination thereof.

27. The composition of any one of claims 24-26, wherein the ribitol and/or the SERM comprises a polyalkylene glycol moiety coupled or linked thereto.

28. A pharmaceutical composition comprising a therapeutically effective amount of ribitol, a therapeutically effective amount of a SERM, and a pharmaceutically acceptable carrier.

29. The pharmaceutical composition of claim 28, wherein the SERM is tamoxifen, raloxifene, asofoxifene, bazedoxifene, toremifene, ospemifene, methyl-piperidino-pyrazole (MPP), or any combination thereof.

30. The pharmaceutical composition of claim 28 or 29, wherein the ribitol is ribitol (adonitol) pentose alcohol, a precursor thereof, a polysaccharide thereof, a phosphate form thereof, a non- phosphated form thereof, a nucleotide form thereof, a precursor of a phosphate form thereof, Ribose-5-P, ribose, or any combination thereof.

31. The pharmaceutical composition of any one of claims 28-30, wherein the ribitol and/or the SERM comprises a polyalkylene glycol moiety coupled or linked thereto.