WO2022183281A1 - Compounds and treatments for muscular dystrophy - Google Patents

Abstract

Among other things, in general, compositions and methods of treatment for muscular dystrophy and other diseases are disclosed. A method of treatment of a subject is disclosed, in which fatty acid utilization is improved. The method includes administering to the subject in need thereof therapeutically effective amounts of a peroxisome proliferator-activated receptor (Ppar) agonist and of choline, or pharmaceutically acceptable salts or prodrugs thereof. Compositions including a Ppar agonist and choline are also provided.

Description

COMPOUNDS AND TREATMENTS FOR MUSCULAR DYSTROPHY

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/155,855, filed March 3, 2021, the contents of which are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

[01] Muscular dystrophy, congenital, megaconial type (OMIM 602541; www.omim.org) is an autosomal recessive dystrophy caused by loss of function of the CHKB gene and can cause a muscular dystrophy. The most common types of muscular dystrophy result from mutations in genes coding for members of protein complexes which act as linkers between the cytoskeleton of the muscle cell and the extracellular matrix that provides mechanical support to the plasma membrane during myofiber contraction. Muscular dystrophies result in fibrofatty replacement of muscle tissue, progressive muscle weakness, functional disability and often early death.

[02] Phosphatidylcholine (PC) is the major phospholipid present in mammalian cells, comprising approximately 50% of phospholipid mass. Choline kinase catalyzes the phosphorylation of choline to phosphocholine and is the first enzymatic step in the synthesis of PC. There are two genes that encode human choline kinase enzymes, CHKA and CHKB. Monomeric choline kinase proteins combine to form homo- or hetero-dimeric active forms. CHKA and CHKB proteins share similar structures and enzyme activity but display some distinct molecular structural domains and differential tissue expression patterns. Knock-out of the murine Chka gene leads to embryonic lethality. Clikb deficient ( Chkb^-/- ) mice are viable, but noticeably smaller than their wild type counterparts, and show severe bowing of the ulna and radius at birth. By 2-3 months of age Chkb^-/- mice lose hindlimb motor control, while the forelimbs are spared. Inactivation of the Clikb gene in mice would be predicted to decrease PC level, however, reports indicate no, or a very modest, decrease in PC level in Chkb^A mice, and this decrease is similar in both forelimb and hindlimb muscle. The very small decrease in PC mass, and the fact that there is no rostral-to-caudal change in PC, suggest a poor correlation of the anticipated biochemical defects and observed rostral-to-caudal phenotype of this muscular dystrophy.

[03] Historically, it has been unclear how a defect in a gene required for the synthesis of the major phospholipid in mammalian cells causes a muscular dystrophy, especially in light of the fact that global inactivation of the CHKB/Chkb gene (human or mouse) does not affect the level of the product of its biochemical pathway, PC. The etiology is therefore not well understood, and this has hamstrung efforts for finding therapeutic approaches in muscular dystrophy and CHKB associated muscular dystrophy. SUMMARY OF THE INVENTION

[04] In general, in an aspect, a method of treatment of a subject is disclosed, in which fatty acid utilization is improved. The method includes administering to the subject in need thereof therapeutically effective amounts of a peroxisome proliferator-activated receptor (Ppar) agonist and of choline, or pharmaceutically acceptable salts or prodrugs thereof. Implementations may include one or more of the following. The Ppar agonist is a fibrate. The fibrate is ciprofibrate. The fibrate is fenofibrate. The fibrate is bezafibrate. The fibrate is cardarine. The choline is choline bitartrate. The choline is supplied as a prodrug. The prodrug is citicoline. The prodrug is phospho-choline. The subject is in need thereof because the subject suffers from a muscular dystrophy. The subject suffers from a muscular dystrophy caused by loss of function of the CHKB gene. The subject suffers from a myopathy caused by CPT2 deficiency. The subject suffers from a myopathy or cardiomyopathy caused by loss of function of TAG lipase.

[05] In general, in an aspect, a pharmaceutical composition is provided, the composition including a fibrate or pharmaceutically relevant salt or prodrug thereof (with the proviso that the salt is not choline), a choline or pharmaceutically relevant salt of prodrug thereof, and a pharmaceutically acceptable carrier or excipient.

[06] With the foregoing and other advantages and features of the invention that will become hereafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the invention and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[07] Figure 1. Choline kinase deficient mice display hallmark muscular dystrophy phenotypes. Legend is Chkb^+/+ (green circle), Chkb^+-/- (blue square), and Chkb^-/- (purple triangle). (A) Body weight was recorded each week at similar times over the entire duration of phenotyping experiment for Chkb^+/+, Chkb^+-/-, and Chkb^-/- mice. (B) Grip strength measurements were performed at 3 different timepoints and normalized to body weight (BW). (C) Total distance run during an exhaustion test for all experimental groups at 3 different timepoints. (D) Serum creatine kinase (CK) level measurements of 15-week-old Chkb^+/+, Chkb^+-/- and Chkb^-/- mice. (E) Loss in muscle force as a result of repeated contractions of EDL muscles by direct stimulation of the nerve for each genotype. (F) Maximal specific force generated by freshly isolated extensor digitorum longus (EDL) muscle for each genotype All values are expressed as means ± SEM; n=6-13 animals per group. Significance was calculated using one-way ANOVA with Tukey’s multiple comparison test for each specific time point. *P < 0.01 vs. all the other groups and #P<0.05 vs. Chkb^+/+ group at each specific timepoint. Chkb^+/+, Chkb^+-/- and Chkb^-/- respectively labeled with green circles, blue squares, and purple triangles. [08] Figure 2. Chka protein expression is inversely correlated with the rostro-caudal gradient of severity in Chkb-mediated muscular dystrophy. Transmission electron microscopy (TEM) appearance of the (A) forelimb (triceps) and (B) hindlimb (quadriceps) of 115-day old Chkb^-/- mice showing extensive injury in hindlimb not the forelimb. Western blot of (C) forelimb (triceps) and (D) hindlimb (quadriceps) samples from three distinct (lanes 1-3) Chkb^+/+, four distinct (lanes 4-7) Chkb^+/~ and three distinct (lanes 8-10) Chkb^-/- mice probed with anti-Chka, anti-Chkb, and anti-Gapdh antibodies. Bottom: densitometry of the WB data shows the ratio of Chka and Chkb to Gapdh. Chka signal is not significantly different in forelimb and hindlimb samples from Chkb^+/~ mice compared to the wild type. Chka is upregulated in forelimb muscles and downregulated in hindlimb muscles from Chkb^-/- mice. Chkb signal is decreased in hindlimb and forelimb muscle samples of Chkb^+/ mice and is absent in muscle samples of Chkb^-/- mice. Values are means ± SD; n=3-4 per group. *P<0.01 vs Chkb^+/+, **P< 0.01 vs all the other groups (one-way ANOVA with Tukey’s multiple comparison test). #P< 0.05 vs Chkb^+/+ (Student’s t- test). Mitochondrial profile quantification. (E) transmission electron microscopy (TEM) appearance of the mitochondrial profile of hindlimbs from 12-day old and 60 days old wild-type (' Chkb^+/+ ) and Chkb-deficient ( Chkb ^-/-) mice (representative of 3 mice per group). (F) At 12 days of age hindlimbs from wild type and Chkb ^-/- mice had the same number of mitochondria per imaged field however, the volume density of the Chkb ^-/- mitochondria was increased and the cristae density was preserved. At 115 days of age, Chkb ^-/- mitochondria were fewer in number, and had markedly reduced cristae density and were much larger in size. The increased size of the mitochondria at this age accounts for the preserved volume density.

[09] Figure 3. Loss of Chkb activity exerts a major effect on neutral lipid abundance. Comparison of expression levels of major glycerophospholipids and AcCa between the Chkb^+/+ and Chkb ^-/- mice. The analysis was performed on (A-B), 12-day old hindlimb (quadriceps) and (C-D), 30 days old hindlimb (quadriceps) samples; A and C are respectively marked as green for Chkb^+/+ and purple for Chkb ^-/- and presented as green (left side) / purple (right side) for each of the major glycerophospholipids and AcCa. (B and D) Summary of fold change and statistical tests performed on major glycerophospholipids. n=3 mice per group. Pairwise Wilcoxon signed rank test with Bonferroni correction was used to determine the significance of a median pair wise fold-increase in lipid amounts at an overall significance level of 5%. As the Bonferroni correction is fairly conservative, significant differences are reported at both pre-correction (*) and post-correction (***) significance levels. AcCa, acylcarnitine; TG, triacylglycerol; DG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine. (E) Transmission electron microscopy (TEM) appearance of the hindlimb muscle samples (quadriceps) of Chkb^+/+ and Chkb^-/- mice at 12 days and 115 days of age (representative of 3 mice per group). LD=Lipid droplets. M=Mitochondria. *=Disrupted sarcomeres. (F) Quadriceps muscle sections of 30 days old Chkb^+/+ and Chkb^-/- mice were fixed and stained with BODIPY-493/503 to visualize LDs (Green dot-like morphologies), which were seen throughout the Chkb^-/- sections but not in the Chkb^+/+. Concanavalin A dye conjugate (CF™ 633) and DAPI were used to stain membrane (Red) and nucleus (Blue) respectively (representative of 3 mice per group) and appear in both types of sections as web-like connections.

[10] Figure 4. Chkb regulates the gene expression of the members of the Ppar family as well as Ppar target genes. (A) Relative gene expression of the Ppar family members. (B) Western blot of hindlimb (quadriceps) samples from three distinct (lanes 1-3) Chkb^+/+, four distinct (lanes 4-7) Chkb^+-/- and three distinct (lanes 8-10) Chkb^-/- mice probed with anti-Ppara, anti- Pparb, anti-Pparg, anti-Cptlb and anti-Gapdh antibodies. Bottom: densitometry of the western blot data shows the ratio of Ppara, Pparb, Pparg and Cptlb to Gapdh. Values are means ± SD; n=3-4 per group. *P<0.01 vs Chkb^+/+, **P< 0.01 vs all the other groups (one-way ANOVA with Tukey’s multiple comparison test). (C) Fold-Change (2^L (- Delta Delta CT)) is the normalized gene expression (2^L(- Delta CT)) in the Chkb deficient hindlimb sample divided the normalized gene expression (2^L (- Delta CT)) in the control sample. Fold-change values greater than one indicates a positive- or an up-regulation. Fold-change values less than one indicate a negative or down-regulation, and the fold-regulation is the negative inverse of the fold-change. The p values are calculated based on a Student’s t-test of the replicate 2^L (- Delta CT) values for each gene in the Chkb^+/+ group and Chkb^-/- groups. *p < 0.05, **p<0.01. N=3 samples per group. (D) The clustergram of the Ppar family, Rxr family and Ppar coactivators across three genotypes (shown as min/green, avg/black, max/red across a color continuum from green to red, per the legend).

(E) Fold change, normalized gene expression for the genes involved in peroxisomal and mitochondrial beta oxidation in the Chkb deficient hindlimb sample divided the normalized gene expression in the control sample. (F) The clustergram of the Ppar family, Rxr family and Ppar coactivators across three genotypes. Average arithmetic means of the expression of 4 housekeeping genes ( Actb , B2m, Gusb and Hsp90abl) were used to normalize the expression of all the studied genes.

[11] Figure 5. Chkb deficiency results in decreased fatty acid usage and increased lipid droplet accumulation in differentiated myocytes in culture. (A) Representative image of isolated skeletal myoblasts from Chkb^+/+ and Chkb^-/- mice, cultured on Matrigel® coated culture flasks. At day 0, when the cells reached 80% confluency, the medium was replaced by differentiation medium and maintained in differentiation media for up to 8 days. (B) Western blot of differentiated Chkb^+/+ and Chkb^-/- myocytes probed with anti-Chka, anti-Chkb, and anti-Gapdh antibodies. (C) RT-qPCR analysis of gene expression in isolated myocytes from Chkb^+/+ and Chkb^'-/- mice at day 5 of differentiation. Values are means ± SD; n=3 independent experiments. *P<0.05, **P< 0.01. Student’s t-test. Formaldehyde fixed and immunostained myotubes were categorized into three groups (1 to 3 nuclei, 4 to 10 nuclei, and >10 nuclei per myotube). (D) The number of multinuclear myotubes in the two groups and the distribution of nuclei were calculated to compare differentiation in primary Chkb^+/+ and CM/Amyocytes. Representative traces of oxygen consumption rates (OCRs) of primary Chkb^+/+ and Chkb^'-/- myocytes at day 4 (E-F) and day 8 (G-H) of differentiation driven with glucose/glutamine/pyruvate or palmitate as stated. Bovine serum albumin (BSA) alone was used as control for palmitate-BSA complex driven OCRs. Oligomycin(O), FCCP(F), Rotenone (R) and Antimycin A (AA) were sequentially injected to assess mitochondrial respiratory states. 4-6 technical replicates per group. Data are mean ± SD. *p < 0.05. Student’s t-test. In (E-H), Chkb^+/+ and Chkb^'-/- are respectively labeled with green circles and purple triangles. More specifically in F and H, green circles represent Palmitate-BSA Chkb^+/+ while blue circles represent BSA Chkb^+/+ (OCR values higher in green series); and purple triangles represent Palmitate-BSA Chkb^'-/- while red triangles represent BSA Chkb^'-/- (OCR values higher in purple series). (I) Isolated primary myocytes from Chkb^+/+ and Chkb^'-/- mice were fixed 5 days after differentiation and stained with BODIPY -493/503 to visualize LDs (Green). DAPI was used to stain nucleus (Blue). (J) The corrected total cell fluorescence intensity of lipid droplets was significantly enhanced in Chkb^'-/- myotubes. For D, I and J, total- 100 cells were quantified per group in 3 independent experiments. Data are mean ± SD. *p < 0.05. Student’s t-test.

[12] Figure 6. Ppar activation rescues defective fatty acid utilization and lipid droplet accumulation in differentiated Chkb^'-/- myocytes in culture. (A) Kinetic graph of the fatty acid oxidation of primary Chkb^'-/- myocytes at day 7 of differentiation. The cells were treated with or without ciprofibrate (50 mM; squares), bezafibrate (500 mM; triangles) and fenofibrate (25 mM; inverted triangles) in the medium 72 hours prior to measurement. (B-C) Quantification of basal respiration and maximal respiration which quantifies maximal electron transport activity induced by the chemical uncoupler FCCP. 6 technical replicates per group. Values are means ± SD; n=4- 5 per group. *P<0.05, **P< 0.01, #P<0.05 vs Chkb^+/+ group, ##P<0.01 vs Chkb^+/+ group (oneway ANOVA with Tukey’s multiple comparison test). Ppara agonist bars are ciprofibrate, bezafibrate, fenofibrate from left to right. (D) Kinetic graph of the fatty acid oxidation of primary Chkb^'-/- myocytes at day 7 of differentiation. The cells were treated with or without specific Pparb/d agonist GW501516 (2.5 mM; inverted triangles) in the medium 72 hours prior to measurement. (E-F) Quantification of basal respiration and maximal respiration (OCR) which quantifies maximal electron transport activity induced by the chemical uncoupler FCCP. Six technical replicates per group. Values are means ± SD; n=4-5 per group. *P<0.05, **P< 0.01, #P<0.05 vs Chkb^+/+ group, ##P<0.01 vs Chkb^+/+ group (one-way ANOVA with Tukey’s multiple comparison test). (G) To study the effect of Ppar agonist treatments on exogenous fatty acid utilization and storage, 4 days after differentiation, myocytes were treated with or without bezafibrate (500 mM) or GW501516 (2.5 mM) in the medium for 48 hours. On day 6 of differentiation the medium with or without drugs was supplemented with Oleate-BSA (400 mM) overnight. On day 7 of differentiation cells were labeled with mitotracker® Red CMXRos (50nM) for 30 min, washed with PBS, fixed and stained with BODIPY 493/503 BODIPY- 493/503 to visualize LDs (dot-like morphologies). DAPI was used to stain nucleus. LDs are seen to be much reduced in treated cells. (H) RT-qPCR analysis of Chka gene expression in differentiated Chkb^-/- myocytes treated with ciprofibrate (50 mM), bezafibrate (500 mM) or GW501516 (2.5 mM) for 48 hours on day 4 of differentiation. For this experiment the media was supplemented with ImM choline. (I) RT-qPCR analysis of Icaml gene expression in differentiated Chkb⁺⁺ myocyte and Chkb^-/- myocytes treated with ciprofibrate (50 mM), bezafibrate (500 mM) or GW501516 (2.5 mM) for 48 hours on day 4 of differentiation. Values are means ± SD; n=3 independent experiments. **P< 0.01 vs Chkb^-/- myocytes, #P<0.01 vs all the other groups (one-way ANOVA with Tukey’s multiple comparison test)

[13] Figure 7. Increased intramyocellular lipid droplet accumulation in skeletal muscles from Chkb^-/- mice. (A) Comparison of expression levels of major glycerophospholipids and AcCa between the Chkb^+/+ and Chkb^-/- mice; 12 days old forelimb (triceps). Summary of fold change and statistical tests performed on major glycerophospholipids (B) as described in Figure 3. (C) Quadriceps muscle sections of 30 days old Chkb^+/+ and Chkb^-/- mice were fixed and stained with Nile Red-500 / 640 nm to visualize LDs (Red) (Upper Panel) or with BODIPY-493/503 to visualize LDs (Green) and Concanavalin A dye conjugate (CF 633) to stain membrane (Red). DAPI was used to stain nucleus (Blue). (D) TEM appearance of the hindlimb muscle samples of 12 days old Chkb^-/- mice showing the high prevalence of peri-droplet mitochondria in hindlimb muscles (representative of 3 mice per group). Scale bar 1 micron. LD=lipid driplets, M=mitochondria, *=disrupted sarcomeres.

[14] Figure 8. Protein expression of the members of the Ppar family in forelimb samples. (A) Western blot of forelimb (triceps) samples from three distinct (lanes 1-3) Chkb^+/+, four distinct (lanes 4-7) Chkb^+-/- and three distinct (lanes 8-10) Chkb^-/- mice probed with anti-Ppara, anti- Pparb, anti-Pparg, anti-Cptlb and anti-Gapdh antibodies. (B) densitometry of the WB data shows the ratio of Ppara, Pparb, Pparg and Cptlb to Gapdh. Values are means +/- standard deviation; n=3-4 per group. No significant difference was observed among groups using one-way ANOVA with Tukey’s multiple comparison test. [15] Figure 9. Chkb regulates the expression of the members of the Ppar family as well as Ppar target genes. Clustergram showing non-supervised hierarchical clustering to display a heat map with dendrograms indicating co-regulated genes across groups or individual samples.

Sample dimension: ID. Join Type: Average. Color Coded: Average Genes. Magnitude of gene expression is shown as min/green, avg/black, max/red across a color continuum from green to red, per the legend.

[16] Figure 10. (A-C) RT-qPCR analysis of Chka gene expression in differentiated Chkb^+/+, Chkb ^-/- and Chkb ^-/- myocytes treated with or without Ciprofibrate 50 (mM) (G), GW501516 (2.5 mM) (H) or bezafibrate (500 mM) (I) in the medium for 48 hours on day 4 of differentiation with or without choline (1 mM) supplementation. For (A-C), n = 3 independent samples per group. One-way ANOVA with Tukey’s multiple comparison test, p < 0.0001. **P< 0.01 (D-F)

Targeted metabolomic profiling of Chkb^+/+, Chkb ^-/- and Chkb ^-/- myocytes treated with bezafibrate (500 mM) for 48 hours on day 4 of differentiation with or without choline (1 mM) supplementation. Ppar activation increases phosphocholine (p-Choline) level (E) and normalizes acylcarnitine (AcCa) level (F) in differentiated Chkb ^-/- myocytes. Values are means ± SEM. For (D and E), n = 6 samples for each group. One-way ANOVA with Tukey’s multiple comparison test p < 0. 0001. Each experiment was repeated independently 3 times with similar results. For (F), n= 15 AcCa species from 3 independent samples for each group. One-way ANOVA with Tukey’s multiple comparison test. P < 0. 0001. Each experiment was repeated independently 3 times with similar results. * p < 0.05 and ** p < 0.01.

DETAILED DESCRIPTION OF THE INVENTION

[17] The compositions and methods herein described relate to the discovery that progression of muscular dystrophy is driven by changes in fatty acid utilization and neutral lipid metabolism.

[18] CHKB encodes one of two mammalian choline kinase enzymes that catalyze the first step in the synthesis of the major membrane phospholipid, phosphatidylcholine (PC). In humans, inactivation of the CHKB gene causes a recessive form of a rostral-to-caudal congenital muscular dystrophy. Using Chkb knockout mice, we reveal that at no stage of the disease is PC level significantly altered. Instead, at early stages of the disease the level of mitochondrial specific lipids acylcarnitine (AcCa) and cardiolipin (CL) increase 15-fold and 10-fold, respectively, in affected muscle. As the disease progresses, AcCa and CL levels normalize and there is a 12-fold increase in the neutral storage lipid triacylgycerol and a 3 -fold increase in its upstream lipid diacylglycerol. Chkb deficiency decreases the expression of peroxisome proliferator-activated receptors (Ppars) and target genes which reinforce the observed changes in lipid levels in Chkb ^-/- affected muscle. Chkb deficient myocytes in culture have reduced capacity to utilize fatty acids for oxygen production, increased lipid droplet accumulation and enhanced markers of myocyte injury. Ppar activation rescues defective fatty acid utilization for mitochondrial respiration and lipid droplet accumulation in differentiated Chkb^-/- myocytes. Our findings indicate that the major change in lipid metabolism upon loss of function of Chkb is not a change in PC level, but instead is an initial inability to utilize fatty acids for energy resulting in shunting of fatty acids into triacyglycerol.

[19] As such, Ppar activation is a treatment option for muscular dystrophy and particularly in CHKB associated muscular dystrophy. The addition of choline was found to further enhance the therapeutic effect.

[20] As described in the Examples below, mouse and cell models were used to investigate the temporal changes in lipid metabolism in the absence of the Chkb gene. Results demonstrate that PC level remains essentially unchanged. Instead, this genetic defect in PC synthesis drives large fluctuations in mitochondrial lipid metabolism with an inability to use fatty acids for mitochondrial b-oxidation resulting in a temporal shunting of fatty acids into triacylglycerol (TG) and their storage as lipid droplets. This provides insight into the surprising biochemical phenotype whereby a genetic block in a lipid metabolic pathway does not directly affect the product of its pathway, and instead alters tangential pathways in a manner that explains the rostral-to-caudal gradient of a genetic disease. The altered lipid metabolic profile observed resulted in reduced Ppar expression. We go on to demonstrate the therapeutic effect of Ppar agonists, in that their addition increased fatty acid use for energy production resulting in a corresponding decrease in lipid droplet accumulation.

[21] Without wishing to be held to any particular theory, we make the following observations in support of a potential mechanism for therapeutic effect of Ppar agonists and choline. In muscles from Chkb^+,+ mice, there is a balance between storage of fatty acid as TG and usage of fatty acids either as an energy source by mitochondria b-oxidation or membrane phospholipid synthesis. In hindlimb muscles from Chkb^~-/- mice, we have established that an inability to consume diacylglycerol (DG) for PC synthesis results in an imbalance between storage and usage of fatty acids. Although the cells are able to increase PC uptake from plasma to compensate for defective PC synthesis, this genetic defect in PC synthesis drives large fluctuations in lipid metabolism. At an early stage of Chkb mediated muscular dystrophy (Phase

1), there is an approximately 12- to 15-fold increase in the levels of the mitochondrial specific lipids CL and AcCa; the large increase in CL reflects the increase in mitochondrial size at this stage of the disease. The increase in AcCa level is due to the inability to consume DG for PC synthesis, resulting in an accumulation of its precursor - fatty acid which the cell attempts to consume via mitochondria b-oxidation, however there is a concomitant decrease in Ppar mediated expression of genes required for fatty acid conversion to AcCa for its import into mitochondria and consumption by b-oxidation. AcCa level increase suggests there is either a decreased ability to transport of AcCa into mitochondria for subsequent fatty acid b-oxidation, and/or incomplete b-oxidation resulting in a backup of substrate within this pathway. In support of this idea the expression of many of the enzymes required for fatty acid transport into mitochondria and subsequent fatty acid b-oxidation were decreased many fold in affected muscle of Chkb^-/- mice. Based on our analysis of mitochondrial respiration in Chkb^-/- myocytes, at this initial stage the reduced fatty acid oxidation capacity is potentially being compensated by an increase in usage of other sources of fuel (Figure 5E-F). The increase in AcCa level at the early stage of Chkb mediated muscular dystrophy, and the decreased expression of genes required for its synthesis and use, is consistent with an inability to import AcCa into mitochondria for fatty acid b-oxidation.

[22] As the disease progresses (Phase 2), CL level returns to wild type (probably as a result of damaged mitochondrial inner membrane), and an approximately 12-fold increase in the storage lipid TG occurs due to an inability to consume AcCa and the shunting of fatty acids into storage lipid droplets (from energy source to energy storage). These changes at later stage of the disease are consistent with our analysis of mitochondrial respiration where we observed a significant decrease in fatty acid oxidation in differentiated in Chkb^-/- myocytes (Figure 5G-H). This observation is also consistent with other reports showing that inhibition of PC biosynthesis in mouse liver, and cell culture, significantly increased TG level. ~ 80% of the photographed lipid droplets from Chkb^-/- hindlimb muscles photographed in the Examples below were closely associated with mitochondria (Figure 5A and Figure 1C). Peri-droplet mitochondria have enhanced bioenergetic capacity and reduced fatty acid oxidation capacity, and peri-droplet mitochondria promote lipid droplet expansion by providing ATP for triglyceride synthesis. As such, our observation of an increase in peri-droplet mitochondria in Chkb^-/- affected skeletal is consistent with our proposal that there is a reprogramming of muscle lipid metabolism from impaired fatty acid usage for energy to fatty acid storage in TG.

[23] Interestingly, we herein disclose that inactivation of a gene for PC synthesis does not alter PC level. Indeed, the changes in the level of PC do not appear to contribute to the disease phenotype. Our work shows, surprisingly, that a change in PC level is not the major metabolic driver behind this disease despite the fact that the genetic defect lies within the major metabolic pathway for the synthesis of PC. Moreover, there is a rostral to caudal gradient in diseased mice of this type. We propose that the rostral-to-caudal gradient is due to compensatory effects modulated by a second choline kinase isoform, Chka. In hindlimb muscle of Chkb^-/- mice we observed a marked reduction in Chka protein level, while conversely in the forelimb of Chkb^-/- mice there was a compensatory upregulation of Chka. Ppara and Pparb/d protein expression is significantly decreased in Chkb deficient hindlimb muscle compared to the forelimb muscle and since Ppar activation enhances Chka expression (Figure 6H) we propose that the inability of hindlimb muscle to enhance Chka expression can be in part due to decreased Ppar expression/activity in the hindlimb muscles. We further disclose that PC level does not change as PC can be replenished via exogenous PC supply. PC is imported into cells from serum via low density lipoproteins (LDL), and enhanced expression of scavenger receptor-Bl (SR-B1) and low-density lipoprotein receptor (LDLR) was previously observed in muscle of Chkb ^-/- mice, both of which would be expected to enhance the uptake of plasma PC ¹. The expected decrease in the level of PC in hindlimb muscle of Chkb ^-/- mice, due to inactivation of the Ckkb gene and downregulation of Chka gene expression, is not observed in our Examples as this can be compensated for by increased PC uptake from serum. These predictions are consistent with the changes in muscle function along the rostral-to-caudal gradient in Chkb ^-/- mice.

[24] An interesting mechanistic feature of disease progression in the Examples is the transition from an inability to synthesize PC in affected muscle to an increase in AcCa. The synthesis of PC requires the consumption of diacylglycerol (DAG) at the final step in the CDP- choline pathway, and this would not occur in affected muscle as the choline kinase step is either inactivated ( Ckhb ) or downregulated (Chka). DAG requires fatty acids for its synthesis, and an inability to synthesize DAG could result in an inability to utilize fatty acids for subsequent DAG synthesis. Indeed, over time we see an increase in DAG mass in affected muscle. The inability to synthesize PC results in a metabolic defect downstream within this pathway that results in major changes in tangential yet connected lipid metabolic pathways over time, an inability to use excess fatty acid for energy followed by its storage as neutral lipid.

[25] We herein disclose that muscle lipid metabolism in muscular dystrophy (as demonstrated in Chkb ^-/- mice) reflects an imbalance between storage and usage of fatty acids (Figure 7). Accordingly, we further herein disclose that Ppar activation is useful for therapy in muscular dystrophy, particularly CHKB mediated muscular dystrophy, and especially when supplemented with choline (Figure 6H).

[26] In some embodiments, a method of treatment of a subject is disclosed, in which fatty acid utilization is improved, the method comprising administering to the subject in need thereof therapeutically effective amounts of a peroxisome proliferator-activated receptor (Ppar) agonist and of choline, or pharmaceutically acceptable salts or prodrugs thereof. In some embodiments, the Ppar agonist is a fibrate. In some embodiments, the fibrate is ciprofibrate. In some embodiments, the fibrate is fenofibrate. In some embodiments, the fibrate is bezafibrate. In some embodiments, the fibrate is cardarine. [27] In some embodiments, the choline is choline bitartrate. In some embodiments, the choline is supplied as a prodrug, in which the prodrug is citicoline. In some embodiments, the choline is supplied as a prodrug, in which the prodrug is phospho-choline.

[28] In some embodiments, a pharmaceutical composition is provided, the composition comprising a fibrate or pharmaceutically relevant salt or prodrug thereof (with the proviso that the salt is not choline), a choline or pharmaceutically relevant salt of prodrug thereof, and a pharmaceutically acceptable carrier or excipient.

[29] In some embodiments, these compositions and methods are relevant to treatment of muscular dystrophies; particularly muscular dystrophy, congenital, megaconial type; and other diseases characterized by a CHKB deficiency.

[30] In some embodiments, these compositions and methods are relevant to treatment of lipid storage myopathies, a group of genetic disorders characterized by excessive and pathological lipid accumulation in muscles and defined by progressive myopathy with muscle weakness, myalgia, and fatigue which are cause by dysfunction in intracellular neutral lipid metabolism and include the transport of carnitine, AcCa or long chain fatty acids, and defects in mitochondrial fatty acid b-oxidation.

[31] In some embodiments, these compositions and methods are relevant to treatment of myopathy resulting from a decreased ability to metabolize AcCa due to CPT2 deficiency ( CPT2 , OMIM: #600650).

[32] In some embodiments, these compositions and methods are relevant to treatment of myopathy or cardiomyopathy resulting from mutations in PNPLA2 (NLSD, OMIM: #275630), which encodes TAG lipase. Many of the clinical presentations of NLSD including myopathy, cardiomyopathy, global developmental delay, mitochondrial defects, and premature death are also reported in patients with CHKB mutations.

[33] Definitions

[34] Unless otherwise defined, terms as used in the specification refer to the following definitions, as detailed below.

[35] The term “acyl” as used herein means an alkyl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein. Representative examples of acyl include, but are not limited to, acetyl, 1-oxopropyl, 2, 2-dimethyl- 1-oxopropyl, 1- oxobutyl, and 1-oxopentyl.

[36] The terms “administration” or “administering” compound should be understood to mean providing a compound of the present invention to an individual in a form that can be introduced into that individual’s body in an amount effective for prophylaxis, treatment, or diagnosis, as applicable. Such forms may include e.g., oral dosage forms, injectable dosage forms, transdermal dosage forms, inhalation dosage forms, and rectal dosage forms.

[37] The term “hydroxy” as used herein means an — OH group.

[38] Unless otherwise indicated, the term “prodrug” encompasses pharmaceutically acceptable esters, carbonates, thiocarbonates, N-acyl derivatives, N-acyloxyalkyl derivatives, quaternary derivatives of tertiary amines, N-Mannich bases, Schiff bases, aminoacid conjugates, phosphate esters, metal salts and sulfonate esters of compounds disclosed herein. Examples of prodrugs include compounds that comprise a biohydrolyzable moiety (e.g., a biohydrolyzable amide, biohydrolyzable carbamate, biohydrolyzable carbonate, biohydrolyzable ester, biohydrolyzable phosphate, or biohydrolyzable ureide analog). Prodrugs of compounds disclosed herein are readily envisioned and prepared by those of ordinary skill in the art. See, e.g., Design of Prodrugs, Bundgaard, A. Ed., Elsevier, 1985; Bundgaard, “Design and Application of Prodrugs,” A Textbook of Drug Design and Development, Krosgaard-Larsen; Bundgaard, Ed., 1991,

Chapter 5, p. 113-191; and Bundgaard, Advanced Drug Delivery Review, 1992, 8, 1-38 (these references being hereby incorporated by reference in their entireties).

[39] The compounds of the invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids. Pharmaceutically acceptable salt(s) are well- known in the art. For clarity, the term “pharmaceutically acceptable salts” as used herein generally refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases. Suitable pharmaceutically acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N,N'- dibenzylethylenediamine, chloroprocaine, choline (excepting when an active ingredient is choline), diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Suitable non-toxic acids include inorganic and organic acids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonic acid. Specific non-toxic acids include hydrochloric, hydrobromic, phosphoric, sulfuric, and methanesulfonic acids. Examples of specific salts thus include hydrochloride and mesylate salts. Others are well-known in the art. See, e.g., Remington's Pharmaceutical Sciences, 18 th ed. (Mack Publishing, Easton Pa.: 1990) and Remington: The Science and Practice of Pharmacy, 19 th ed. (Mack Publishing, Easton Pa.: 1995); these references are hereby incorporated by reference in their entireties. The preparation and use of acid addition salts, carboxylate salts, amino acid addition salts, and zwitterion salts of compounds of the present invention may also be considered pharmaceutically acceptable if they are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use. Such salts may also include various solvates and hydrates of the compound of the present invention.

[40] The term “pharmaceutically acceptable excipient”, as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of one skilled in the art of formulations.

[41] Unless otherwise indicated, the terms “prevent,” “preventing” and “prevention” contemplate an action that occurs before a patient begins to suffer from the specified disease or disorder, which inhibits or reduces the severity of the disease or disorder or of one or more of its symptoms. The terms encompass prophylaxis.

[42] Unless otherwise indicated, a “prophylactically effective amount” of a compound is an amount sufficient to prevent a disease or condition, or one or more symptoms associated with the disease or condition, or prevent its recurrence. A prophylactically effective amount of a compound is an amount of therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the disease. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

[43] Unless otherwise indicated, a “therapeutically effective amount” of a compound is an amount sufficient to treat a disease or condition, or one or more symptoms associated with the disease or condition. “Therapeutically effective amounts” of two or more compounds may be optimized by varying the administered amount of one compound in the presence of a constant amount of the other. In some embodiments, a therapeutically effective amount of a Ppar agonist by itself is established, and the amount of choline administered is titrated based on patient response to arrive at the therapeutically effective amount of choline. In some embodiments, both the Ppar agonist and choline amounts are varied to arrive at the preferred therapeutically effective amounts.

[44] The term “subject” is intended to include living organisms in which disease may occur. Examples of subjects include humans, monkeys, cows, sheep, goats, dogs, cats, mice, rats, and transgenic species thereof.

[45] The term “substantially pure” means that the isolated material is at least 90% pure, preferably 95% pure, even more preferably 99% pure as assayed by analytical techniques known in the art.

[46] The pharmaceutical compositions can be formulated for oral administration in solid or liquid form, for parenteral intravenous, subcutaneous, intramuscular, intraperitoneal, intra arterial, or intradermal injection, for or for vaginal, nasal, topical, or rectal administration. Pharmaceutical compositions of the present invention suitable for oral administration can be presented as discrete dosage forms, e.g., tablets, chewable tablets, caplets, capsules, liquids, and flavored syrups. Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990); hereby incorporated by reference in its entirety.

[47] Parenteral dosage forms can be administered to patients by various routes including subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses patients' natural defenses against contaminants, parenteral dosage forms are specifically sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. Pharmaceutical compositions for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like, and suitable mixtures thereof), vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate, or suitable mixtures thereof. Suitable fluidity of the composition may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservative agents, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[48] In some cases, in order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[49] Suspensions, in addition to the active compounds, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof. If desired, and for more effective distribution, the compounds of the invention can be incorporated into slow-release or targeted-delivery systems such as polymer matrices, liposomes, and microspheres. They may be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporation of sterilizing agents in the form of sterile solid compositions, which may be dissolved in sterile water or some other sterile injectable medium immediately before use.

[50] Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations also are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

[51] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[52] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, one or more compounds of the invention is mixed with at least one inert pharmaceutically acceptable carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and salicylic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[53] Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using lactose or milk sugar as well as high molecular weight polyethylene glycols. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract in a delayed manner. Examples of materials which can be useful for delaying release of the active agent can include polymeric substances and waxes.

[54] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[55] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. A desired compound of the invention is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention. The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[56] Powders and sprays can contain, in addition to the compounds of this invention, lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.

[57] Compounds of the invention may also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes may be used. The present compositions in liposome form may contain, in addition to the compounds of the invention, stabilizers, preservatives, and the like. The preferred lipid components for liposomal delivery are the natural and synthetic phospholipids and phosphatidylcholines (lecithins) used separately or together. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y., (1976), p 33 et seq; hereby incorporated by reference in its entirety.

[58] Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention can be varied so as to obtain an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient, compositions and mode of administration. The selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[59] An effective amount of one of the compounds of the invention can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form. Alternatively, the compound can be administered as a pharmaceutical composition containing the compound of interest in combination with one or more pharmaceutically acceptable carriers. It will be understood, however, that the total daily usage of the compounds and compositions of the invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; the risk/benefit ratio; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[60] The total daily dose of Ppar agonist as administered to a human or lower animal may range from about 0.0003 to about 30 mg/kg of body weight. For purposes of oral administration, more preferable doses can be in the range of from about 0.0003 to about 1 mg/kg body weight. If desired, the effective daily dose can be divided into multiple doses for purposes of administration; consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. For oral administration, the compositions of the invention are preferably provided in the form of tablets or capsules containing about 1.0, about 5.0, about 10.0, about 15.0, about 25.0, about 50.0, about 100, about 250, or about 500 milligrams of the Ppar agonist.

[61] Established tolerable upper intake levels for choline from food and supplementation in healthy individuals ranges from 1,000 mg/day in individuals aged 1-3 to 3,500 mg/day in individuals aged 19 or older. In some embodiments, the total daily dose of choline as administered to a human or lower animal may range from about 25 mg/day to about 7,000 mg/day. For purposes of oral administration, more preferable doses can be in the range of from about 250 mg/day to about 3,500 mg/day; however, for diseased subjects under a physician’s supervision, doses can exceed 3,500 mg/day. If desired, the effective daily dose can be divided into multiple doses for purposes of administration; consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. For oral administration, the compositions of the invention are preferably provided in the form of tablets or capsules containing about 1.0, about 5.0, about 10.0, about 15.0, about 25.0, about 50.0, about 100, about 250, or about 500 milligrams of choline. In some embodiments, administration of choline comprises administration by use of pharmaceutically relevant salts such as choline bitartrate. In some embodiments, administration of choline comprises administration by use of a prodrug such as citicoline. In some embodiments, administration of choline supplementation is adjusted for expected or measured levels of dietary choline. In some embodiments, the Ppar agonist and choline are both present in the same tablet or capsule for ease of administration.

EXAMPLES

[62] Example 1 - Choline kinase deficient mice display hallmark muscular dystrophy phenotypes

[63] To address the extent that mice lacking Chkb function display gross muscular dystrophy phenotypes, we tested muscle function in Chkb^+/+, Chkb^+/- and Chkb^-/- mice from 6 weeks to 20 weeks of age using a grip strength assay and a total distance run to exhaustion test. Body weight was also recorded each week at similar times over the duration of the phenotyping experiments. Body weight of the Chkb^+/+and Chkb^+/- mice showed no difference between groups (Figure 1A). The Chkb ^-/- mice weighed significantly less than their wild type counterparts at all time points. The average body weight of Chkb ^-/- mice was 33% to 42% less than that of Chkb^+/+ mice at week

6 and week 20, respectively.

[64] Forelimb grip strength measurements were performed at three different timepoints and normalized to body weight. The Chkb-/- mice had significantly lower (less than half) the normalized forelimb strength than wild type mice at all three timepoints (week 6, 12 and 18) (Figure IB). Another measure of neuromuscular function is the resistance to treadmill running, evaluated as the total distance that each mouse is able to run until exhaustion. The test was performed in all groups at three timepoints (Week 7, 13 and 19). The total distance covered by the wild type mice before exhaustion was similar at all 3 time points (Figure 1C). There was no significant difference between Chkb^+/+ and Chkb^+/~ groups, these mice maintained the ability to cover the same total distance before exhaustion (week 7 vs. week 19; non-significant). At week 7, the Chkb ^-/- mice showed a basal level of total distance run that was 50% that of the wild type or Chkb^+/- mice. Moreover, the Chkb^-/- mice showed a decline in running performance from week

7 to week 19, with an almost complete inability to run observed by week 19. Gross measurements of neuromuscular strength in whole mice demonstrate that mice heterozygous for Chkb gene display similar phenotypes to wild type mice. Notably, mice lacking both copies of the Chkb gene display a significant decrease in overt neuromuscular phenotypes.

[65] The level of circulating creatinine kinase (CK), a biomarker of sarcolemmal injury, was determined in Chkb^+/+, Chkb^+/-, and Chkb ^-/- mice. No significant change in the serum level of CK was observed in Chkb^+/- heterozygous mice when compared to the wild type. At week 15, CK activity was 2.5-fold higher in Chkb ^-/- null mice than that of wild type mice (Figure ID).

[66] To determine if the decreased neuromuscular phenotypes observed in the Chkb ^-/- mice were due to a direct effect on muscle itself, maximal specific force generated by freshly isolated extensor digitorum longus (EDL) muscle from the hindlimb of Chkb^+/+, Chkb^+-/-, and Chkb^-/- mice at week 20 was determined. EDL muscle fatigue was measured with 60 isometric contractions for 300 ms each, once every 5 sec, at 250 Hz. There was no significant difference between wild type and heterozygous Chkb mice in regard to specific force decrease during fatigue and specific force generation, (Figure IE and F). Chkb^-/- mice displayed a specific EDL force that was 10% that of Chkb^+/+ or Chkb^+-/- mice. In addition, Chkb^-/- mice were at maximally fatigued levels, that is those observed in Chkb^+/+ or Chkb^+-/- mice after 60 muscle stimulations, at the first stimulation. Hindlimb muscle from Chkb^-/- mice produce less force, and are much more easily fatigued, than that of wild type or Chkb heterozygous mice.

[67] Similar to as reported in humans, mice with one functional copy of the CHKB gene do not possess any obvious overt muscle dysfunction, whereas mice that are homozygous null for functional copies of the Chkb gene display hallmark muscular dystrophy phenotypes.

[68] Example 2 - Chka protein expression is inversely correlated with the rostro-caudal gradient of severity in Chkb-mediated muscular dystrophy

[69] Consistent with the rostral-to-caudal nature of Chkb associated muscular dystrophy, transmission electron micrographs of 115-day old Chkb^~-/- mice show extensive injury in hindlimb (quadriceps and gastrocnemius) but not the forelimb (triceps) (Figure 2A and B). Chkb encodes choline kinase b, the first enzymatic step in the synthesis of PC, the most abundant phospholipid present in eukaryotic membranes. A second choline kinase, Chka is present in mouse (and human) tissues. We investigated whether the lack of dystrophic phenotypes in Chkb^+-/- mice, and the rostro-caudal gradient of muscular dystrophy in Chkb^-/- muscle, can be explained by compensatory changes in Chkb or Chka protein levels using western blot. In Chkb^+-/- mice, there was a -50% decrease in Chkb protein detected in both the forelimb and hindlimb muscles of Chkb^+/- mice compared to wild type (Figure 2C-D). There was no change in Chka protein level in hindlimb muscle of Chkb^+/- mice compared to wild type, and a small but statistically insignificant increase in Chka level in forelimb muscle.

[70] In Chkb^-/- mouse forelimb or hindlimb muscle, Chkb protein expression was undetectable consistent with the allele not producing Chkb protein. In forelimb muscle from Chkb^-/- mice there was a compensatory upregulation of Chka protein expression to almost 3-fold that observed in wild type mice. In contrast, in hindlimb muscle from Chkb^-/- mice Chka protein expression was decreased to less than 10% that observed in wild type mice. A compensatory level of Chka protein expression inversely correlates with the rostro-caudal gradient of severity in Chkb^-/- associated muscular dystrophy.

[71] Congenital muscular dystrophies associated with CHKB loss-of-function mutations display distinct enlarged (megaconial) mitochondria in the peripheries of muscle fiber and absence of mitochondria in the centers, but the dynamic changes in mitochondrial morphology have been previously unstudied. To understand the temporal development of morphological changes in mitochondria in hindlimb muscle of Chkb^-/- mice, we used standard transmission electron microscopy (TEM) stereological methods. The results show that at 12 days of age, the size of mitochondria increased (6.2%±0.5 vs 11.4%±1.6; P<0.01; wild type vs Chkb^-/-) while the number of mitochondria (17.3+2.6 vs 16.3+2.1) and cristae density (21.6+2 vs. 23.3+1.9) remained the same. At 60 days of age, the number of mitochondria (18.1+7.6 vs 1.8+0.5;

P<0.01) and the cristae density (27.7+1.9 vs. 5.8+1.2; P<0.01) decreased significantly while the size did not change (7.3%+0.2 vs 8.2% +2.5). At the early stages of Chkb muscular dystrophy, there is an increase in mitochondrial size but not number or morphology. As the disease progresses, the increase in mitochondrial size remains, however the number of mitochondria, and the cristae within mitochondria, decrease (Figure 2E and F).

[72] Example 3 - Loss of Chkb activity exerts a major effect on neutral lipid abundance

[73] PC synthesis is integrated with the synthesis of other major phospholipid classes, as well as AcCa, fatty acids and the neutral lipids diacylglycerol and triacylglycerol. Lipidomics was used to determine if complete loss of Chkb function, and the associated upregulation of Chka in the forelimb but not hindlimb muscle of Chkb^-/- mice, differentially altered lipid metabolism. The levels of the major glycerophospholipids, neutral lipids and acylcamitine in hindlimb and forelimb muscle isolated from 12-day old and 30-day old Chkb^+/+ and Chkb^-/- mice were quantified.

[74] In the forelimb and hindlimb muscle of both 12-day old and 30-day old Chkb^-/- mice, the level of PC was the same as wild type mice (Figure 3A-B and Figure 7A-B). In 12-day old Chkb ^-/- mice the largest change observed was a 15-fold increase in AcCa level in hindlimb muscle, and to a lesser degree (~2-fold increase) in forelimb, compared to their wild type littermates. The second largest change in 12-day old mice was a 10-fold increase in the level of cardiolipin (CL) in hindlimb muscle that was not present in forelimb muscle of Chkb^-/- mice. Phosphatidylethanolamine (PE) and phosphatidylinositol (PI) levels were also slightly increased (-1.5 fold) in both forelimb and hindlimb muscles of 12-day old Chkb^-/- mice. The large changes in lipid levels in hindlimb muscle, versus forelimb, of Chkb^-/- mice are consistent with the rostral-to-caudal nature of the muscular dystrophy observed in these mice.

[75] Considering the progressive nature of the disease, we tracked the changes in the lipid profile in the hindlimb of 30-day old Chkb^-/- mice, when muscle injury is more pronounced. In sharp contrast to 12-day old mice, AcCa and CL levels were no longer increased and were at the same level as wild type mice. Instead, there was a - 12-fold increase in the neutral storage lipid TG and a 3-fold increase in its precursor DG in the hindlimb samples of Chkb^-/- mice (Figure 3C and D). PE and PS levels were 2-3-fold higher in the hindlimb samples from 30-day old Chkb^-/- mice compared to wild type littermates. There is a temporal shift from a ~12 to 15-fold increase in CL and AcCa, to a similar increase in TG, only in affected muscle in Chkb^-/- mice.

[76] As AcCa levels are many fold higher than wild type mice in the early stage of Chkb^-/- muscular dystrophy, the affected muscles appear to be defective in using fatty acids for the production of cellular energy by mitochondrial b-oxidation. As Chkb^-/- muscular dystrophy progresses, the affected muscles appear to adapt to this inability to consume fatty acids by transitioning toward energy storage indicated by the large increase in TG.

[77] To understand early ultrastructural pathological changes, and to further explore the nature of the accumulation of TG in affected muscle as Chkb^-/- muscular dystrophy progresses, we performed TEM on hindlimb muscles from 12- and 115-day old mice. A closer examination of hindlimb muscle TEM images from 12-day old mice revealed early signs of disrupted sarcomeres, as well as a small increase in the abundance of cytoplasmic lipid droplets, consistent with the small (2-fold) but statistically not significant increase in TG in hindlimb muscle we observed using lipidomics. These lipid droplets were located mainly adjacent to enlarged mitochondria (Figure 3E). Detailed quantification of randomly imaged lipid droplets in hindlimb muscle from 12-day old Chkb^-/- mice determined that 81% were associated with mitochondria (Figure 3E and Figure 7D). In 115-day old Chkb^-/- mice, cytoplasmic lipid droplets increased substantially in size (Figure 3E).

[78] We also evaluated TG accumulation in muscle using confocal microscopy by staining hindlimb muscle sections of 30-day old Chkb^-/- mice with BODIPY 493/503 (Figure 3F). Concanavalin A dye conjugate (CF™ 633) and Dapi were used to stain membrane (Red) and nucleus (Blue) respectively. Consistent with our TEM and lipidomics results, BODIPY-stained lipid droplets were noticeably more frequent and larger in Chkb^-/- hindlimb muscles compared to the wild type littermates. The same pattern of lipid droplet staining was observed using Nile red staining (Figure 1C).

[79] Example 4 - Changes in expression of peroxisome proliferator- activated receptors (Ppars) and target genes reinforce the observed changes in lipid levels in Chkb^-/- hindlimb muscle

[80] Our lipidomic data illustrate a temporal shift from the use of fatty acids for energy to lipid storage in affected muscle of Chkb^-/- mice. To further investigate this metabolic shift, we determined the expression of peroxisome proliferator-activated receptors (Ppars) in affected muscle of 30 day old Chkb^+/+, Chkb^+-/- and Chkb^-/- mice. Peroxisome proliferator-activated receptors (PPARs) are master regulators of lipid metabolism. The endogenous ligands for Ppars are fatty acids and their derivatives. There are three Ppar members, each encoded by distinct genes, designated Ppara, Pparb/d and Pparg. Ppara and Pparb/d primarily regulate the expression of genes required for fatty acid oxidation, with Pparb/d also regulating genes required for mitochondria biogenesis. Pparg is primarily expressed in adipose tissue and regulates insulin sensitivity and glucose metabolism. Using reverse transcription (RT) quantitative polymerase chain reaction (qPCR), we determined that the expression of Ppara and Pparb/d were 4-fold and 6-fold lower, while Pparg was 2-fold higher, in the hindlimb muscle of 30-day old mice Chkb^-/- mice compared to wild type (Figure 4A). Consistent with RT qPCR results, assessment of Ppar protein levels by western blot show decreased Ppara and Pparb/d protein expression, and an increase in Pparg protein expression, in Chkb deficient hindlimb muscle compared to wild type (Figure 4B). Ppar protein levels did not change in Chkb deficient forelimb muscle compared to Chkb^+/+ (Figure 8) indicating that the Ppar changes are isolated to affected muscle. See also Figure 10 for further PCR data.

[81] To further evaluate and validate how the Ppar pathway contributes to the lipid metabolic changes observed in Chkb ^-/- mice, we utilized a microarray of 82 Ppar regulated genes, along with 4 housekeeping genes, to assess transcriptional changes in the hindlimb muscle of 30-day old mice. As expected, there was no change in the expression of the 4 housekeeping genes. For Ppar receptors to bind to Ppar response elements in gene promoters, Ppars form obligate heterodimers with Retinoid X receptors (Rxr). There are three members of the Rxr family, Rxra, Rxrb, and Rxrg, and their expression was reduced 8-, 5-, and 16-fold in hindlimb muscle of Chkb ^-/- mice compared to wild type (Figure 4C and D). Ppar and Rxr heterodimers are bound to DNA with coactivator molecules ²⁷ and the expression of each co-activator was also decreased from 2.8- to 14.4-fold compared to wild type (Figure 4C and D). The several fold decrease in expression of the Ppars, as well as their obligate co-receptors, aligns well with the observed changes in lipid profiles we observed in affected muscle of Chkb^-/- mice that predict a decreased capacity to import and use fatty acids by mitochondria for b-oxidation.

[82] Among Ppar associated genes, the expression of 44 genes was decreased statistically significantly (P<0.05) by at least 2-fold in Chkb ^-/- mice, while 8 genes were upregulated at least 2-fold (Figure 4C-F, Figure 9, and in the following tables):

[83] Table 1 below: Ppar associated genes under-expressed in Chkb ' hindlimb vs. Chkb^+/+ hindlimb. Fold Regulation cut off =2. p-Value cut off= 0.05. Fold-Change (2^L (- Delta Delta CT)) is the normalized gene expression (2^L(- Delta CT)) in the Test Sample divided the normalized gene expression (2^L (- Delta CT)) in the Control Sample. Fold-change values less than one indicate a negative or down-regulation, and the fold-regulation is the negative inverse of the fold-change p values are calculated based on a Student’s t-test of the replicate 2^L (- Delta CT) values for each gene (control and Chkb deficient groups).

[84] Table 2 below: Ppar associated genes over-expressed in Chkb^'-/- hindlimb vs. Chkb^+/+ hindlimb. Fold Regulation cut off =2. p-Value cut off= 0.05. Fold-Change (2^Λ (- Delta Delta CT)) is the normalized gene expression (2^Λ(- Delta CT)) in the Test Sample divided the normalized gene expression (2^Λ (- Delta CT)) in the Control Sample. Fold-change values more than one indicate a positive or up-regulation. The p values are calculated based on a Student’s t- test of the replicate 2^Λ (- Delta CT) values for each gene (control and Chkb deficient groups).

[85] Carnitine palmitoyltransferase lb (Cptlb), the major muscle isoform of Cpt, is involved in the carnitine shuttle as it catalyzes the conversion of cytoplasmic long-chain fatty acyl-CoA and carnitine into AcCa that are translocated across the inner mitochondrial membrane for subsequent mitochondrial fatty acid b-oxidation. The expression of Cptlb was decreased 7.9-fold in affected muscle of Chkb ^-/- mice. In addition, the expression of enzymes required for mitochondrial fatty acid b-oxidation were also decreased several folds in affected muscle of Chkb ^-/- mice including several fatty acylCoA synthases/ligases, fatty acid binding proteins, and fatty acid b-oxidation enzymes.

[86] Ppara and Ppar b/d are the major transcriptional reporters that regulate expression of fatty acid metabolizing genes. The many-fold decrease in the expression of these Ppars that was specific to affected muscle, along with their coreceptors and downstream target genes corroborate the lipdomics data that show a major change in lipid metabolism in muscular dystrophy (particularly Chkb mediated) is an inability to metabolize fatty acids via mitochondrial b-oxidation resulting in shunting of excess fatty acid into TG rich lipid droplets.

[87] Example 5 - Chkb deficiency results in decreased fatty acid oxidation and increased lipid droplet accumulation in differentiated myocytes in culture

[88] To address if the observed increase in TG in Chkb^-/- mice was due to muscle specific events or was due to larger physiological changes that then impact muscle physiology, we assessed fatty acid utilization and TG level in primary cultured muscle cells subsequent to myoblast differentiation. We first determined if Chkb deficiency alters differentiation in primary myoblasts. Primary muscle cell cultures were examined for their transition from a single cell proliferative condition to differentiated multinucleated myotubes. During the process of differentiation, mononuclear myoblasts fuse to form myocytes (myotubes), which are large multinucleated cells. We isolated skeletal myoblasts from Chkb ^+/+ and Chkb ^-/- mice and induced differentiation by switching to low growth factor serum. Representative light micrographs of cultures of dissociated myogenic cells from skeletal muscle of Chkb^+/+ and Chkb mice at 0, 4 -/- 8 days after switching to differentiation media show a similar degree of myotube formation (Figure 5A). Chkb deficiency resulted in a compensatory upregulation of Chka gene expression as well as a significant increase in the markers of myocyte injury, namely Icaml and Tgfbl ²⁸ (Figure 5C). We calculated the fusion index, which is nuclei distribution, to determine the extent of myotube differentiation, by immunofluorescence staining. There was no difference between the Chkb^+/+ and Chkb^-/- cells in terms of the percentage of nuclei within the myotubes, the average number of nuclei in each myotube, or the distribution of nuclei in myotubes (Figure 5D). Loss of Chkb function does not appear to affect gross myoblast differentiation.

[89] To test whether Chkb deficiency and reduced expression of peroxisome proliferator- activated receptors (Ppars) and target genes in skeletal muscle affected the fatty acid oxidation capacity of these cells, we measured the oxygen consumption rate of primary Chkb ^+/+ and Chkb- ^{/ -}myocytes at day 4 and 8 of differentiation using a Seahorse XF24 extracellular flux analyzer. The same number of primary myoblasts were seeded into different wells of the same Seahorse plate and oxygen consumption was determined using glucose/glutamine/pyruvate as energy source, or fatty acid as energy source, at days 4 and 8 of differentiation. At day 4 of differentiation maximal respiration driven by glucose/glutamine/pyruvate was significantly increased in Chkb^-/- myocytes compared to the wild type (Figure 5E). Contrary to this, there was a slight but significant decrease in maximal respiration driven by long chain fatty acid as fuel (Figure 5F). At day 8 of differentiation there was a larger reduction in maximal respiration driven by fatty acid in Chkb^-/- myocytes (Figure 5H) while the maximal respiration driven by other fuels (glucose/glutamine/pyruvate) was no longer different in Chkb ^{-/ -}myocytes compared to the wild type (Figure 5G). We concluded that Chkb ^-/ -yocytes have reduced fatty acid oxidation capacity by initially normalizing fat oxidation capacity to other sources of fuel, and that mitochondrial respiration in primary Chkb^-/- ym ocytes progressively declines.

[90] To assess whether Chkb deficiency and decreased fatty acid utilization modulates TG storage in isolated myotubes, we stained differentiated myotubes with BODIPY 493/503 to visualize neutral lipid droplets. Lipid droplets (LDs) were noticeably more abundant and larger in Chkb deficient myotubes compared to wild type (Figure 51). Quantification of the corrected total cell fluorescence intensity in Chkb ^-/-myotubes confirmed a 2-fold increase in lipid droplet formation (Figure 5J). The increase in TG level in differentiated muscle cells isolated from Chkb - ^{/ -}mice is in line with the increased TG and lipid droplet levels observed in isolated hindlimb muscle from older Chkb ^-/- mice and shows that the increase in TG in hindlimb muscle due to the loss of Chkb function is a direct effect on lipid metabolism within the muscle cells themselves.

[91] Example 6 - Ppar activation via fibrate administration rescues defective fatty acid utilization and lipid droplet accumulation in differentiated Chkb^-/- myocytes in culture [92] To determine whether the decreased fatty acid utilization and increased lipid droplet accumulation in Chkb^-/- myocytes is mediated by a Ppar signaling pathway(s) we treated Chkb^-/- myocytes with the Ppara agonists ciprofibrate and fenofibrate, the pan Ppar agonist bezafibrate, and the Pparb/d specific agonist cardarine (also known as GW 501516) and assessed the capacity of the myocytes to oxidize fatty acid. Quantification of maximal respiration showed that all of the Ppar agonists enhanced maximal respiration of Chkb^-/- myocytes (Figure 6 A, C, D and F). Cardarine was also able to increase the basal respiration in Chkb^-/- myocytes to the same level as Chkb^+/+ myocytes (Figure 6D-E). In a separate experiment, 4 days after differentiation, Chkb^-/- myocytes were treated with bezafibrate or cardarine in the medium for 48 hours. On day 6 of differentiation the medium was supplemented with Oleate-BSA (400 mM) overnight before labeling cells with mitotracker® Red CMXRos and staining with BODIPY 493/503 to visualize LDs. LDs were noticeably more abundant and larger in Chkb^-/- myocytes compared to the wild type, however, Ppar activation by bezafibrate or cardarine significantly decreased lipid droplets in Chkb deficient myotubes to a level comparable to wild type. One interesting observation was the increased clusters of colocalized LDs and mitochondria in Chkb^-/- myocytes treated with cardarine (Figure 6G). We found that ciprofibrate, bezafibrate or cardarine treatment of Chkb^-/- myocytes for 48 hours resulted in a 2-to-4-fold increase in Chka gene expression and a significant reduction in the marker of myocyte injury ( Icaml ) (Figure 6H and I). The addition of Ppara or Pparb/d agonists correct the defects observed in Chkb^-/- affected muscle cells by increasing mitochondrial fatty acid oxidation, preventing the accumulation of TG in LD, and increasing the expression of the alternate choline kinase isoform Chka. Thus, we came to the surprising conclusion that treatment of an inherited muscular dystrophy that is due to a defect in the synthesis of the major membrane phospholipid could be ameliorated by a Ppara or Pparb/d agonist.

[93] Example 7 - Methods for the previous examples

[94] Mouse strains - All animal procedures were approved by the Dalhousie University’s Committee on Laboratory Animals in accordance with guidelines of the Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals. Chkb mutant mice in C57BL/6J background were originally generated at the Jackson Laboratory (Bar Harbor, Maine, USA). Male Chkb^+-/- mice on the C57BL/6J background were crossed with female Chkb^+-/- on the same background to generate Chkb^+/+, Chkb^-/- and Chkb^+-/- littermates. The mutation identified in Chkb^-/- mice is a 1.6 kb genomic deletion between exon 3 and intron 9 that results in expression of a truncated mRNA and the absence of Chkb protein expression.

[95] Mouse genotyping - The mutation identified in Chkb^-/- mice is a 1.6 kb genomic deletion between exon 3 and intron 9. AccuStart II Mouse Genotyping Kit (Beverly, MA, USA) was used 11 extract DNA from ear punches and to perform PCR analysis. A single genotyping program was used to amplify both the wild type Chkb allele between exons 5 and 9 and the truncated Chkb allele between exons 2 and 10. The primers used for genotyping were purchased from Integrated DNA Technologies (Coralville, IA, USA). The primer sequences to genotype wild type are Forward Primer: 5'-GTG GGT GGC ACT GGC ATT TAT -3'; Reverse Primer: 5'- GTT TCT TCT GTT CCT CTT CGG AGA-3' (amplicon size 753 bp). The primer sequences to genotype the mutants are: Forward Primer: 5'-TAC CCA CGT ACC TCT GGC TTT T -3' Reverse Primer: 5'-GCT TTC CTG GAG GAC GTG AC 3'(amplicon size 486 bp). For each mouse, one PCR reactions was performed using both the primer sets. If two bands were observed, the mouse was characterized as a heterozygous.

[96] In vivo grip strength and fatigability measurements - Forelimb grip strength was measured using a grip strength meter (Columbus Instruments, Columbus, OH, USA) at 3 time points (6, 12, 18 weeks old). All mice were acclimated for a period of five consecutive days before testing. For each time point, Force measurements were collected in the morning hours over a 5-day period, with maximum values for each day over this period averaged to obtain absolute GSM values (Kgf) or normalized to BW (recorded on the first day of testing) for normalized GSM values (Kgf/kg). For the treadmill exhaustion assay, mice are subjected to an enforced running paradigm that tests the resistance level of fatigue in mice. The exhaustion test was performed at 3 time points (7, 13, 19 weeks old) in each group. Groups of mice were made to run on a horizontal treadmill for 5 min at 5 m/min, followed by an increase in the speed of lm/min each minute. The total distance run by each mouse until exhaustion was measured. Exhaustion was defined as the inability of the mouse to continue running on the treadmill for 30 seconds, despite repeated gentle stimulation.

[97] Ex vivo force measurement - At the end of the in vivo phase (Week 19), mice were deeply anesthetized with ketamine and xylazine (80 and 10 mg/kg). The extensor digitorum longus (EDU) muscle of the right hindlimb was removed for comparison of Ex vivo force contractions between groups as previously described. Briefly, the EDU muscle was securely tied with braided surgical silk at both tendon insertions to the lever arm of a servomotor/force transducer (model 305B) (Aurora Scientific, Aurora, Ontario, Canada) and the proximal tendon was fixed to a stationary post in a bath containing buffered Ringer solution (composition in mM: 137 NaCl, 24 NaHCO₃, 11 glucose, 5 KC1, 2 CaC1₂, 1MgO₄, 1 NaH₂P0₄ and 0.025 turbocurarine chloride) maintained at 25°C and bubbled with 95% 0₂ - 5% CO2 to stabilize pH at 7.4. At optimal muscle length, the maximal force developed was measured during trains of stimulation (300 milliseconds, ms) with increasing frequencies up to 250 Hz or until the highest plateau was achieved. The force generated to obtain the highest plateau was used to determine specific force (maximal force normalized to cross-sectional area of the muscle). Finally, the muscle was subjected to a fatigue protocol consisting of 60 isometric contractions for 300 ms each, once every 5 seconds. The frequency at which the EDL muscles were stimulated is 250 Hz. The force was recorded every 10th contraction during the repetitive contractions and again at 5 and 10 min afterward to measure recovery.

[98] Creatine kinase (CK) serum levels - CK was determined from serum taken from blood samples withdrawn by cheek bleed at 3 time points (5, 10 and 15 weeks old). Blood was centrifuged for 3000 g for 10 min at 4°C to obtain the serum. CK determination was performed by standard spectrophotometric analysis, using a CK diagnostic kit (Cat. no. C7522-450,

PONITS CIENTIFIC, Canton, MI, USA).

[99] Total RNA isolation, cDNA generation, and RT qPCR - Isolated tissue samples were incubated overnight in pre-chilled RNAlater® (Cat. no. R0901, Sigma- Aldrich, Ontario, Canada) at 4°C. Tissues were then homogenized in TRIzol reagent (Cat. no. 15596026, Invitrogen, MA, USA) and total RNA was isolated according to the manufacturer’s protocol. Nine hundred nanograms of total RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Cat. no. 4368814, Applied Biosystems, MA, USA). Quantitative real-time RT-PCR assays were performed on the Bio-Rad CFX96 Touch Real-Time PCR Detection (Bio- Rad, California, USA) System using TaqMan Fast Advanced Master Mix (Cat. no. 4444557) and TaqMan Gene Expression Assays (Cat. no. 4331182, ThermoFisher Scientific) for Chka (RRID: Mm00442759_m 1 ) , Chkb Cptlb (Exon boundary7-8) (RRID: Mm01308102_gl), Cnsk2a2 (RRID: Mm01243455_ml), Cptlb (RRID: Mm00487191_gl), Gapdh (RRID: Mm99999915_gl), Icaml (RRID: Mm00516023_ml), Ppara (RRID: Mm00440939_ml), Ppard (RRID: Mm00440940_m 1 ), Pparg (RRID: Mm00440940_ml) and Tgfbl (RRID:

MmOl 1778820_ml). Reactions were run in triplicate.

[100] Microarray analysis of Ppar targets - Mature RNA was isolated using a Qiagen RNeasy Plus Mini Kit (Cat. no 74134). RNA quality was determined using a spectrophotometer and was reverse transcribed using a cDNA conversion kit. The cDNA was used on the real-time RT2 Profiler PCR Array (QIAGEN, Cat. no. PAMM-149Z) in combination with RT2 SYBR® Green qPCR Mastermix (Cat. no. 330529). CT values were exported to an Excel file to create a table of CT values. This table was then uploaded on to the data analysis web portal at http://www.qiagen.com/geneglobe. Samples were assigned to controls and test groups. CT values were normalized based on a manual selection of reference genes. The data analysis web portal calculates fold change/regulation using delta-delta CT method, in which delta CT is calculated between gene of interest (GOI) and an average of reference genes (HKG), followed by delta-delta CT calculations (delta CT (Test Group)-delta CT (Control Group)). Fold Change is then calculated using 2^L (-delta delta CT) formula. The data analysis web portal was used to plot scatter clustergram and heat map.

[101] Lipid extraction - We performed lipid extractions using the modified Bligh and Dyer extraction for LC-MS analysis of lipids protocol (Bligh & Dyer, Can J Biochem Physiol Aug 1959;37(8):911-7, hereby incorporated by reference in its entirety). All reagents were of LC- MS grade. Briefly, the muscle tissue (~10mg) was homogenized with a steel bead in 1 ml of cold 0.1 N HChmethanol (1:1, v/v) using a TissueLyser II instrument (Qiagen) set at 30 strokes/s for 2-4 min. Based on protein quantification results, all samples were adjusted to the final concentration of 700 pg/ml and spiked with 10 mΐ of internal standard (Avanti Polar Lipids Inc; Catalog Number-330707). 500 mΐ of chloroform was added to each sample, vortexed for 30 minutes and centrifuged to separate phases (5 minutes at 6000 rpm). The bottom organic phase was transferred into a new Eppendorf and dried under a nitrogen stream. Samples were stored at -80°C until ready for analysis.

[102] UHPLC method for lipid analysis - The Accucore C30 column (250 x 2.1 mm I.D., particle size: 2.8 pm) was obtained from ThermoFisher Scientific (ON, Canada). The mobile phase system consisted of solvent A (acetonitrile: H20 60:40 v/v) and solvent B (isopropanol: acetonitrile: water 90:10:1 v/v) both containing 10 mM ammonium formate and 0.1% formic acid. C30-RPLC separation was carried out at 30°C (column oven temperature) with a flow rate of 0.2 mL/min, and 10 pL of the lipid extraction suspended in the mobile phase solvents mixtures (A:B, 70:30%) was injected onto the column. The following system gradient was used for separating the lipid classes and molecular species: 30% solvent B for 3 min; then solvent B increased to 50% over 6 min, then to 70% B in 6 min, then kept at 99% B for 20 min, and finally the column was re-equilibrated to starting conditions (30% solvent A) for 5 min prior to each new injection.

[103] High resolution tandem mass spectrometry and lipidomics - Lipid analyses were carried out using a Q-Exactive Orbitrap mass spectrometer controlled by X-Calibur software 4.0 (ThermoScientific, MO, USA) with an acquisition HPLC system. The following parameters were used for the Q-Exactive mass spectrometer - sheath gas: 40, auxiliary gas: 5, ion spray voltage: 3.5 kV, capillary temperature: 250 °C; mass range: 200-2000 m/z; full scan mode at a resolution of 70,000 m/z; top-1 m/z and collision energy of 35 (arbitrary unit); isolation window: 1 m/z; automatic gain control target: le5. The instrument was externally calibrated to 1 ppm using ESI negative and positive calibration solutions (ThermoScientific, MO, USA). Tune parameters were optimized using a mixture of lipid standards (Avanti Polar Lipids, Alabama, USA) in both negative and positive ion mode Thermo Scientific™ LipidSearch™ software version 4.2 was used for lipid identification and quantitation. First, the individual data files were searched for product ion MS/MS spectra of lipid precursor ions. MS/MS fragment ions were predicted for all precursor adduct ions measured within ±5 ppm. The product ions that matched the predicted fragment ions within a ±5 ppm mass tolerance was used to calculate a match-score, and those candidates providing the highest quality match were determined. Next, the search results from the individual positive or negative ion files from each sample group were aligned within a retention time window (±0.2 min) and the data were merged for each annotated lipid.

[104] Data cleanup and statistical analysis of lipids - Lipid concentrations extracted from the LipidSearch software were further analyzed with an in-house script using the R programming language. The data was filtered to exclude any peak concentration estimates with a signal to noise ratio (SNR parameter) of less than 2.0 or a peak quality score (PQ parameter) of less than 0.8. If this exclusion resulted in the removal of two observation within a biological triplicate, the remaining observation was also excluded. The individual concentrations were then gathered together by lipid identity (summing together the concentration of multiple mass spectrometry adducts where these adducts originated from the same molecular source and averaging together biological replicates) and grouped within the broader categories of AcCa, TG, DG, PC, PE, PG, CL, PI, PS. The result was nine groups containing multiple lipid concentrations corresponding to specific lipid identities, which were then compared between wild type and KO samples using a (paired, non-parametric) Wilcoxon signed-rank test at an overall significance level of 5%

(using the Bonferroni correction to account for the large number of tests performed). As the Bonferroni correction is fairly conservative, significant differences are reported at both pre correction (*) and post-correction (***) significance levels.

[105] Nile red 550 / 640 nm, BODIPY 493/503 nm and nuclei staining of muscle tissue - Quadriceps and gastrocnemius muscles were embedded in Optimal Cutting Temperature (Sakura Finetek, Torrence, CA), and were frozen in cooled isopentane in liquid nitrogen and stored at - 80°C. Frozen sections (7 pm thick) were thaw-mounted on SuperFrost Microscope slides (Microm International, Kalamazoo, MI) and air dried. Tissue sections were then fixed in 4% (w/v) paraformaldehyde for 15 minutes and incubated with Concanavalin A CF Dye Conjugates CF633 (50-200 pg/mL) for 20 minutes followed by incubation with either Nile red solution in PBS (0.5 pg/mL) or BODIPY 493/503 for 15 minutes. The sections were then washed for 5 times with PBS, each time for 15 minutes and mounted using ProLong Gold Antifade Mountant with DAPI (Thermo Scientific™, Cat. no. P36931) and cured overnight in the dark. Slides were observed under a confocal microscope (Leica TCS SP8 with LIGHTNING) using excitation wavelength 633 for Concanavalin A, 550nm for Nile red, 448 nm for BODIPY and 405 for DAPI. [106] Primary myoblast isolation, culture and differentiation - We followed a protocol outlined in Shahini et al. (Shahini et al, Stem Cell Res Jul 2018;30:122-9; hereby incorporated by reference in its entirety) for isolation of myoblast by enabling the outgrowth of these cells from muscle tissue fragments of Chkb^+/+ and Chkb^-/- mice. Briefly, the mice were euthanized via CO2, were sprayed with 70% ethanol and transferred to a sterile hood. The forelimb and hindlimb muscles were removed, finely minced into small pieces and transferred to a 50 ml conical tube. 1 ml enzymatic solution of PBS containing collagenase type II (500 U/mL), collagenase D (1.5 U/mL), dispase II (2.5 U/mL), and CaCh (2.5 mM) was added to the tube. The muscle mixture was placed in a water bath at 37°C for 60 minutes with agitation every 5 minutes. The suspension was centrifuged for 10 minutes at 300 g. Following centrifugation, the supernatant was removed and discarded, and the pellet was resuspended in proliferation medium. Proliferation medium included high glucose Dulbecco's Modified Eagle Medium (DMEM,

Gibco, Grand Island, NY), 20% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA), 10% horse serum (HS, Gibco), 0.5% chicken embryo extract (CEE, Accurate Chemical and Scientific, Westbury, NY), 2.5 ng/mL bFGF (ORF Genetics, Iceland), 10 pg/mL gentamycin (Gibco), and 1% Antibiotic-Antimitotic (AA, Gibco), and 2.5 pg/mL plasmocin prophylactic (Invitrogen, San Diego, CA). The re-suspended pellet containing small pieces of muscle tissue was plated on Matrigel coated flasks at 10-20% surface coverage and incubated at 37°C and 5% CO2 to allow attachment of the tissues to the surface and subsequent outgrowth and migration of cells. The myogenic cell population was further purified with one round of pre plating on collagen coated dishes to isolate fibroblasts from myoblasts. To induce differentiation into multinucleated myotubes, the cells were seeded at 10000 cells/cm² on plastic coverslip chambers coated with Matrigel and the medium was replaced by differentiation medium containing DMEM with high glucose and 5% HS.

[107] BODIPY 493/503 and nuclei staining of primary myocytes

[108] Isolated skeletal myoblasts were cultured on Matrigel coated glass chamber slides (Thermo Scientific, Cat. no. 154534) and differentiated into myocyte. 3 days after differentiation, the cells were washed two times with PBS and fixed in 4% (w/v) paraformaldehyde for 15 minutes. The cells were washed with PBS for 10 minutes and incubated with BODIPY solution in PBS for 15 minutes, at room temperature on a shaker. The cells were then washed for 3 times with PBS, each time for 15 minutes and mounted using ProLong Gold Antifade Mountant with DAPI (Thermo Scientific, Cat. no. P36931) and cured overnight in the dark. Slides were observed under a confocal microscope (Leica TCS SP8 with LIGHTNING) using excitation wavelength 448 nm for BODIPY and 405 for DAPI. Images were converted to 8-bit and the total corrected cellular fluorescence for the green channel was measured in random 100 cells per group using FIJI (NIH) software. The total corrected cellular fluorescence (TCCF)

= integrated density - (area of selected cell x mean fluorescence of background readings), was calculated and compared between groups. To study the effect of Ppar agonist treatments on exogenous fatty acid utilization and storage, 4 days after differentiation, myocytes were treated with or without ciprofibrate (50 mM), bezafibrate (500 mM) and fenofibrate (25 mM) in the medium for 48 hours. Fibrates were commercially available, though they may also be prepared from commercially available reagents using chemical reactions known in the art. On day 6 of differentiation the medium with or without drugs was supplemented with Oleate-BSA (400 mM) overnight. On day 7 of differentiation cells were labeled with mitotracker Red CMXRos (50nM) for 30 min, washed with PBS, fixed and stained with BODIPY 493/503.

[109] Seahorse analysis of mitochondrial function - Oxygen consumption rate (OCR) was measured using a Seahorse XF24 extracellular flux analyzer (Seahorse Biosciences, North Billeric, MA, USA). Isolated skeletal myoblasts were cultured on Matrigel coated 24-well Seahorse XF24 plates at a density of 40,000 cells/well and differentiated into myocyte as described earlier. Experiments were performed on days 4 and 8 of differentiation in vitro. For fatty acid oxidation analysis, the day prior to the assay growth medium was exchanged for substrate-limited medium (DMEM without glucose, glutamine, sodium pyruvate or bicarbonate (Agilent, Cat. no. 103575-100) supplemented with 0.5 mM glucose, 1 mM glutamate, 0.5 mM carnitine and 1% FBS). On the day of assay sensor calibration was performed and substrate limited medium was exchanged for fatty acid oxidation buffer (111 mM NaCl, 4.7 mM KCL, 1.25 mM CaCh, 2 mM MgS0₄, 1.2 mM NaH₂P0₄, 5 mM HEPES, 2.5 mM glucose and 0.5 mM carnitine) and the plate was equilibrated at 37°C for 1 h before the measurements. Cell mitochondrial stress test was performed after adding BSA control or BSA-palmitate (200 mM final concentration). To study the effect of Ppar agonists on fatty acid oxidation, the cells were treated with or without ciprofibrate (50 mM), bezafibrate (500 mM) and fenofibrate (25 mM) in the medium 72 h prior to measurement. For pyruvate/glucose/glutamine oxidation analysis, calibration sensors were prepared according to the manufacturer’s instructions. On the day of assay, differentiation medium was exchanged for DMEM base medium (Agilent, Cat. no. 103575-100) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose and the plates were equilibrated at 37°C for 1 h before the measurements. For all assays, mitochondrial function was probed by the sequential addition of oligomycin (1 mM), FCCP (carbonyl cyanide- p-trifluoromethoxyphenylhydrazone; 4 mM), rotenone (1 mM) and antimycin A (5 mM); all final concentrations. Three measurements were performed for each condition. All experiments were normalized to total protein as determined by a bicinchoninic acid (BCA) protein quantitation assay. [110] Transmission electronic microscopy - For TEM analysis, ~5x5 mm cubes of quadriceps, gastrocnemius and triceps were with 2.5% glutaraldehyde diluted with 0.1M sodium cacodylate buffer and postfixed with 1% osmium tetroxide in Millonig’s buffer solution for 2 hr, dehydrated, and embedded in epon araldite resin. Ultrathin sections were stained with 2% uranyl acetate for 30 min and lead citrate for 4 min and viewed with a JEOL JEM 1230 transmission electron microscope at 80kV. Images were captured using a Hamamatsu ORCA-HR digital camera. Three mice per genotype for each timepoint were evaluated. The mitochondrial content was determined from the images at 10,000x magnification using Image J software and calculated as mitochondria count/field by blinded investigators. Point counting was used to estimate mitochondrial volume density and mitochondrial cristae density based on standard stereological methods. Only mitochondria profiles of acceptable quality defined as clear visibility and no or few missing spots of the inner membrane were included. Using ImageJ software, a point grid was digitally layered over the micrographic images at 20,000x or 40,000x magnification for mitochondrial volume density and cristae density calculations respectively. Grid sizes of 85 nm x 85 nm and 165 nm x 165 nm were used to estimate mitochondria volume and cristae surface area, respectively. Mitochondria volume density was calculated by dividing the points assigned to mitochondria to the total number of points counted inside the muscle. The mitochondrial cristae surface area per mitochondrial volume (mitochondrial cristae density) was estimated by the formula: mitochondrial cristae density = (4/p) BA, where BA is the boundary length density estimated by counting intersections on test lines multiplied by p/2. In brief, we counted the intersections I(imi) between the inner mitochondrial membrane trace and the test lines and measured the total length of the test line within the mitochondria profile to calculate mitochondrial cristae density =2. 1(imi)/L(mi).

[111] Western blot (WB) analysis and quantification - The muscle tissue (~ lOOmg) was homogenized with a steel bead in 1 ml of cold RIPA buffer containing IX Proteinase Inhibitor Mix (complete Protease Inhibitor Cocktail, Roche, Cat. no.11 697 498001), IX PhosStop (Roche, Mannheim Germany, Cat. no.04906845001) using a TissueLyser II instrument (Qiagen) set at 30 strokes/s for 2-4 min. Based on protein quantification results, all samples were adjusted to the final concentration of 2ug/ul and heat-denatured for 5 min at 99°C in 2X Laemmli buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated in Odyssey blocking solution for 1 h. Total proteins were detected by probing the membranes with appropriate primary antibodies overnight at 4°C. The following antibodies were used: Chka (1:1000, Abeam Cat#ab88053), Ppara (1:1000, Abeam, Cat#Ab24509), Pparb (1:1000, Biorad, Cat#AHP1272), Cptlb (1:1000,

Proteintech®, Cat#22170-l-AP), Chkb (1:250, Santa Cruz, Cat#398957), GAPDH (1:1000, Cell signaling, Cat#398957), Pparg (1:500, Santa Cruz, Cat# sc-7273); Ppara = Ppar alpha; Pparb = Ppar beta; Pparg = Ppar gamma. Proteins were visualized with goat anti-rabbit IRDye-800- or - 680-secondary antibodies (LI-COR Biosciences) or anti-mouse m-IgGK BP-CFL 790 (Santa Cruz, Cat. no. sc-516181) using an Odyssey imaging system and band density were evaluated using FIJI (NIH).

[112] Quantification and statistical analysis - All experiments were repeated 3 or more times. Data are presented as mean ± SEM or mean ± SD, as appropriate. For comparison of two groups the two-tailed Student’s t-test was used unless otherwise specified. Comparison of more than two groups was done by one-way ANOVA followed by the Tukey’s Multiple Comparison test. P values <0.05 were considered significant.

Claims

1. A method of treatment in a subject, wherein fatty acid utilization is improved, the method comprising administering to the subject in need thereof therapeutically effective amounts of a peroxisome proliferator-activated receptor (Ppar) agonist and of choline, or pharmaceutically acceptable salts or prodmgs thereof.

2. The method of claim 1, in which the Ppar agonist is a fibrate.

3. The method of claim 2, in which the fibrate is selected from ciprofibrate, fenofibrate, bezafibrate, and cardarine.

4. The method of claim 3, in which the choline is choline bitartrate.

5. The method of claim 3, in which the choline is supplied as a prodrug, in which the prodrug is selected from citicoline and phospho-choline.

6. The method of any of the preceding claims, in which the subject suffers from muscular dystrophy.

7. The method of claim 6, in which the subject suffers from a muscular dystrophy caused by loss of function of the CHKB gene.

8. The method of claim 6, in which the subject suffers from a myopathy caused by CPT2 deficiency.

9. The method of claim 6, in which the subject suffers from a myopathy or cardiomyopathy caused by loss of function of TAG lipase.

10. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, a fibrate and a choline or pharmaceutically relevant salts or prodrugs thereof.