Poly(d,l-lactide-co-glycolide)-retinoic acid microparticle composition for treating muscle atrophy

Abstract

Pharmaceutical compositions comprising all-trans retinoic acid encapsulated by a microparticle formed from a poly(D,L-lactide-co-glycolide), which can be manufactured from an oil-in-water emulsion method and can be used to treat muscle atrophy.

Description

Attorney Docket No. 37759.0595P1POLY(D,L-LACTIDE-CO-GLYCOLIDE)-RETINOIC ACID MICROPARTICLE COMPOSITION FOR TREATING MUSCLE ATROPHYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 684,264, filed August 16, 2024, the entirety of which is incorporated into this application by reference.STATEMENT REGARDING GOVERNMENT SUPPORT

[0002] This invention is related to federally sponsored research and development under CAREER Award Number 2144087, awarded by the National Science Foundation, contract number R56NS116174, awarded by the National Institutes of Health. SPiRE Award Number I21RX003191, and Merit Award Number I01RX004564, both awarded by the United States Department of Veterans Affairs Rehabilitation Research and Development Service. The government has certain rights in the invention.BACKGROUND

[0003] Muscle atrophy is a phenomenon that occurs from disuse, aging, or illness, leading to a decrease in muscle strength, lower quality of life, and higher mortality. The current standard of treatment involves dietary changes, physical therapy, and exercise, requiring months to years for recovery depending on severity and ability. Patients undergoing recovery after atrophy become more susceptible to falls and additional injury, prolonging recovery time. An effective therapy is needed to aid in the muscle recovery' progress. No pharmaceutical therapies for muscle recovery after atrophy have been approved. This disclosure addresses this need in the art.SUMMARY

[0004] The disclosed method of treating a subject having muscle atrophy comprises administering to the subject by parenteral injection a pharmaceutical composition comprising all-traws retinoic acid encapsulated by a microparticle formed from a poly(D,L-lactide-co- glycolide).

[0005] The microparticle composition comprising aW-t rants retinoic acid encapsulated by a microparticle formed from a poly(D.L-lactide-co-glycolide) can be made by: preparing an oil phase comprising all-trans retinoic acid and the poly(D,L-lactide-co-glycolide) in an organic solvent; combining the oil phase with a water phase comprising water and anAttorney Docket No. 37759.0595P1 emulsifying agent; and removing the organic solvent to provide the microparticle composition.

[0006] The disclosed pharmaceutical composition for parenteral administration comprises (i) a \-trans retinoic acid encapsulated by a microparticle formed from a poly(D,L-lactide-co- glycolide), and (ii) a pharmaceutically carrier suitable for parenteral administration; wherein the microparticle comprises more than 50 pg aW-lmn retinoic acid per mg of the microparticle.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The foregoing summary, as well as the following description of the disclosure, is better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, the drawings illustrate some, but not all, alternative embodiments. This disclosure is not limited to the precise arrangements and instrumentalities shown. The following figures, which are incorporated into and constitute part of the specification, assist in explaining the principles of the disclosure.

[0008] Figure 1 is a plot showing the impact of ATRA and AM580 on IGF-1 secretion by RAW macrophages. RAW macrophages were treated with 0. 1, 1, or 10 pM of ATRA or AM580. A DMSO vehicle control was included. After 24 hours, media was collected and IGF-1 was measured with ELISA. Two-way ANOVA with Tukey's multiple comparison test w as conducted with drug and dosage as sources of variation. ## and #### indicates p < 0.01 and p < 0.0001, respectively, compared to equivalent AM580 dose. *, **, ***, and **** indicates p < 0.05, 0.01, 0.001, and 0.0001, respectively. Data is representative of three independent experiments with three technical replicates per treatment condition. Data are expressed as mean ± SD.

[0009] Figure 2A is a microscopy image of PLG particles with no ATRA. Scale bar 5pm.

[0010] Figure 2B is a microscopy image of ATRA-PLG microparticles. Scale bar 5pm.

[0011] Figure 2C is a histogram plot of particle size for PLG particles with no ATRA (left) and ATRA-PLG microparticles (right).

[0012] Figure 2D is a table showing the characterization of drug loading and particle size for PLG (no ATRA) and ATRA-PLG microparticles.

[0013] Figure 2E is a plot of ATRA drug release profile from particles over 14 days. Linear regression is indicated by the dashed line. Data is representative of two independent experiments with three technical replicates per time point. Data is mean ± standard deviation.Attorney Docket No. 37759.0595P1

[0014] Figure 3A is a plot of IGF-1 concentration versus particle dose (per cell seeded) measured after RAW macrophages were treated for 24 hours with PLG or ATRA-PLG particles at 0.5, 5, or 10 particles per cell seeded, or with vehicle control (media). IFG-1 in the media was measured by ELISA and normalized to the vehicle control, which was 21± 1 pg / mL. **** indicates p < 0.0001 versus PLG particles at the equivalent dose. ** and *** indicate p < 0.01 and 0.001, respectively, between the groups as indicated. Data is representative of 5 independent experiments each conducted with three technical replicates per condition. Data is mean ± SD.

[0015] Figure 3B is a plot of cell density (cell count per field) versus particle dose (per cell seeded) after RAW macrophages were treated for 24 hours with PLG or ATRA-PLG particles at 0.5. 5, or 10 particles per cell seeded, or with vehicle control (media). Two-way ANOVA with Tukey’s multiple comparison was conducted between PLG and ATRA-PLG with particle type and dose as sources of variation. Data is representative of 5 independent experiments each conducted with three technical replicates per condition. Data is mean ± SD.

[0016] Figure 3C is a plot of IGF-1 Concentration (fold change) vs. Particle Dose (per cell seeded) generated after RAW macrophages w ere treated with ATRA-PLG or PLG particles that w ere stored at room temperature under vacuum and in the dark for one w eek.

[0017] Figure 3D is a plot of IGF-1 Concentration (fold change) vs. Particle Dose (per cell seeded) generated three months later relative to the data shown in Figure 3C. For both experiments, particle treatment was for 24 hours at doses of 1, 10, or 25 particles per cell seeded. IGF-1 concentration in cell media was measured by ELISA. Two-way ANOVA with Tukey’s multiple comparison was conducted between PLG and ATRA-PLG with particle type and dose as sources of variation. #, ##, and #### indicate p < 0.05, 0.01. and 0.0001 versus PLG particles at the equivalent dose. *indicates p < 0.05 as indicated. Data is from one experiment with 3 technical replicates per condition. Data are mean ± SD.

[0018] Figure 4A is a timeline of the experiment to study the impact of conditioned media on C2C12 Myoblast formation. The middle four arrows indicated media changes.

[0019] Figure 4B show s representative immunofluorescence images of myotubes after conditioned media treatment, probed with an anti-myosin heavy chain and counterstained with Hoechst nuclear stain. Scale bar 100 pm.

[0020] Figure 4C is a plot showing the number of myotubes per field view . One-w ay ANOVA was conducted between means where #### indicates p < 0,0001 compared to (i) and *, **, ***, and **** indicates p < 0.05, 0.01, 0.001. and 0.0001 respectively, between indicated groups.Attorney Docket No. 37759.0595P1

[0021] Figure 4D is a plot showing the number of nuclei per myotube where horizontal lines denote mean and standard deviation. One-way ANOVA was conducted between means where #### indicates p < 0,0001 compared to (i) and *, **, ***, and **** indicates p < 0.05, 0.01, 0.001, and 0.0001 respectively, between indicated groups.

[0022] Figure 4E is a plot showing the number of myotubes when using an IGF-1- neutralizing antibody or an IgG control. Two-way ANOVA was conducted between means where #### indicates p < 0,0001 compared to (i) and *. **. ***, and **** indicates p < 0.05, 0.01, 0.001, and 0.0001 respectively, between indicated groups.

[0023] Figure 5A shows a flow cytometry graph of side scatter vs. CD45 after cell culture and treatment with BMDMs with fluorescent Coumarin-6 particles.

[0024] Figure 5B shows a flow cytometry graph of CD1 lb vs F4 / 80 cells 4 days after harvest and differentiation.

[0025] Figure 5C is a plot showing the proportion of CD1 lb+ and F4 / 80+ cells among total cell population.

[0026] Figure 5D shows live images of fluorescent C6-PLG particles (GFP) in BMDMs over 8 days in culture. Scale bar = 50pm. Images are representative of three independent experiments.

[0027] Figure 6A is a plot of IGF-1 content in media during 12 days of culturing ATRA- PLG with BMDMs. Two-way ANOVA was conducted. Data is representative of two independent experiments with three technical replicates per condition at each time point. Data are expressed as means ± SD.

[0028] Figure 6B is a plot of cell density measurements after 12 days. One-way ANOVA w as conducted. Data is representative of two independent experiments with three technical replicates per condition at each time point. Data are expressed as means ± SD.

[0029] Figure 6C is a plot of IGF-1 concentration over 12 days after treating BMDMs with 5 ng / mL of IL-4 on day 0. Two-way ANOVA w as conducted. Data is representative of one independent experiment with three technical replicates per condition at each time point. Data are expressed as means ± SD.

[0030] Figure 7A is a plot showing IGF-1 content in BMDM media after 24 hours of treating BMDMs with ATRA in the absence of M-CSF. Data is from two independent experiments with three technical replicates per condition. Data is mean ± SD.

[0031] Figure 7B is a plot showing IGF-1 content in media after 24 hours of treating primary murine peritoneal macrophages with ATRA. One-way ANOVA was conducted where *, **, ***, and **** indicates p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 comparedAttorney Docket No. 37759.0595P1 to vehicle, respectively, unless otherwise stated. Data is from one independent experiment with three technical replicates per condition. Data is mean ± SD.

[0032] Figure 7C is a plot showing TNF-a, IL-10 and IGF-1 levels in culture media of RAW macrophages.

[0033] Figure 7D is a plot showing TNF-a, IL-10 and IGF-1 levels in culture media of BMDM. For Figures 7C and 7D, cytokines were measured by ELISA after 24-hours of culture, n.d. means not detected.

[0034] Figure 7E is a plot showing IGF-1 levels in culture media from BMDMs treated with LPS (1 ng / mL) for 6 hours and then treated with ATRA (1 or 10 ng / mL) for 66 hours. IGF-1 measured by ELISA. Data is from 2 independent experiments with 3 technical replicates per experiment. Data are mean ± SD.

[0035] Figure 8 A shows representative western blots of RARa, RAR[3, and RARy in RAW, BMDMs and Peritoneal macrophages.

[0036] Figure 8B is a plot of intensity of RAR(3 signal divided by relative ponceau stain intensity. One-way ANOVA with Tukey’s was conducted where * indicates p < 0.05. n = 4 independent experiments. Data are expressed as means ± SD.DETAILED DESCRIPTION

[0037] The muscle recovery process starts in muscle stem cells called satellite cells. During maintenance or repair, satellite cells proliferate, differentiate into myocytes, then fuse with existing myofibers. This process is regulated, in part, by macrophages. Macrophages are immune cells that play a significant role in establishing a local environment that not only maintains local muscle homeostasis in healthy muscle, but are key regulators of satellite cells and existing muscle fibers to promote muscle regeneration.

[0038] Macrophages are immune cells that are professional phagocytic cells. They reside in all organs and tissues to maintain tissue integrity and homeostasis, particularly after injury or to provide innate immunity after infection. Macrophages from different tissues are transcriptionally and functionally diverse. Of primary macrophages collected from mice for cultured experiments made in vitro, the most commonly used are peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM). PMs are generated by injecting thioglycolate into the peritoneum and collecting the macrophages that are differentiated from the recruited circulating monocytes. BMDMs are generated by harvesting bone marrow and differentiating myeloid progenitor cells with macrophage colony-stimulating factor (M-CSF).Attorney Docket No. 37759.0595P1

[0039] One of the main muscle-supportive factors that macrophages express to the local environment during normal use and for recovery is insulin-like growth factor-1 (IGF-1). IGF- 1 is a potent coordinator of skeletal muscle growth, regulating muscle cell proliferation and differentiation, which contribute to muscle mass. Exogenous, local delivery of IGF-1 promotes regeneration follow ing severe muscle damage. How ever, the delivery' of proteins like IGF-1 or Interleukin-4 (a potent IGF-1 inducer) to muscle is difficult due to their short half-life, off-target effects, and high cost. Researchers have been able to induce endogenous expression of IGF-1 in the skeletal muscle of mice through virus-mediated transfer of IGF-1 several months prior to muscle atrophy. While this preemptive IGF-1 transferal w as able to accelerate recovery' after casting- or hindlimb-induced muscle atrophy, this method would be difficult to translate to clinical application considering the difficulty’, cost, and regulatory barriers associated with gene therapy in humans. This disclosure is based on the discovery of a small molecule inducer of IGF-1 in a suitable delivery' system which should overcome these difficulties.

[0040] Retinoid Acid Receptors (RARs) are nuclear receptors on gene promotor and enhancer regions, that when activated by small molecule ligands, turn on transcription of its associated genes. These nuclear receptors are expressed in macrophages and control gene expression. It w as hypothesized that a selective RAR agonist could increase IGF-1 expression. All-lrans retinoic acid (“ATRA"’) is a known RAR agonist and has been FDA approved for several indications including acne and leukemia but it has not been suggested that ATRA could be useful for treating muscle-related conditions. ATRA is a metabolite of vitamin A and a ligand for all three RAR isoforms. It is currently being used in active NIH- registered clinical trials in the treatment of multiple forms of cancer, allergies and inflammation, skin conditions, and fibrodysplasia ATRA has been established to regulate gene expression involving cell sternness and wound repair, as well as showing promising results when researched as a feed additive for increasing meat yield in farm animals.

[0041] Though commonly administered to humans, ATRA has a short half-life in vivo when administered orally or intravenously, has poor solubility’ in aqueous solution, and is sensitive to light and oxidation. Chronic and high systemic doses of ATRA cause side effects, such as hypertriglyceridemia, mucocutaneous dryness, as well as ATRA resistance. This disclosure is therefore based on the need for a method to deliver and release ATRA to the skeletal muscle that extends its presence in the tissue and limits off-organ side effects.

[0042] Biodegradable biomaterials have been used as drug delivery carriers and present an attractive method for storing and delivering drugs to the intended site. The rationale to useAttorney Docket No. 37759.0595P1 biomaterial microparticles as a drug deliver}' system is to improve stability of the delivered molecule, release the drug over time, and direct localization of the drug to specific tissues within the body. Poly(lactide-co-glycolide) (PLG) is an FDA-approved biomaterial with tunable biodegradability and biocompatibility properties. ATRA has yet to be used or investigated in a biodegradable microparticle formulation for the purpose of regenerating skeletal muscle. This disclosure describes ATRA-PLG microparticles for the encapsulation and release of ATRA over two weeks. ATRA was selected because of its pro-myogenic effects, extensive track record of safety in humans, and its potential to increase IGF-1 production in macrophages. Mouse macrophages were treated with these particles, and the secreted media was used to enhance myotube growth in mouse myoblasts.Pharmaceutical Compositions

[0043] The disclosed pharmaceutical composition for parenteral administration includes (i) all- / ram retinoic acid encapsulated by a microparticle formed from a poly(D,L-lactide-co- glycolide), and (ii) a pharmaceutical carrier suitable for parenteral administration.

[0044] A\\-trans retinoic acid has the structure shown below.ATRA (all-frans retinoic acid)In one embodiment, the microparticle comprises more than 50 pg aW-t / 'ans retinoic acid per mg of the microparticle. In a further embodiment, the microparticle comprises at least 70 pg aA-trans retinoic acid per mg of the microparticle. Exemplar}' ranges of a\\-trans retinoic acid loading include 50-100 pg aA-trans retinoic acid per mg of the microparticle, 60-90 pg alltram retinoic acid per mg of the microparticle, 70-80 pg ai\-trans retinoic acid per mg of the microparticle, or 75-80 pg a\\-trans retinoic acid per mg of the microparticle. In some embodiments, the microparticle comprises a \-trans retinoic acid and no other retinoid, i.e., the retinoid in the microparticle consists of al I - / ram retinoic acid.

[0045] The poly(D,L-lactide-co-glycolide) (“PLG”) can form microparticles capable of encapsulating or adsorbing the all-tram retinoic acid. The use of a non-toxic PLG allows for a subject to safely receive the particles, which can be metabolized after delivery. The PLG polymers are also sterilizable, allowing for the preparation of pharmaceutical grade formulations. Useful PLG polymers include those having a lactide / glycolide molar ratio ranging from 20:80 to 80:20, for example. 25:75, 40:60, 45:55, 50:50, 55:45, 60:40, or 75:25.Attorney Docket No. 37759.0595P1In one embodiment, the poly(D,L-lactide-co-glycolide) has a lactide / glycolide molar ratio ranging from 45:55 to 55:45. Useful PLG polymers include those having a weight-average molecular weight (Mw) in the range of 5,000-200,000 Daltons, for example, 5,000-100,000 Daltons, 5,000-70,000 Daltons, 5,000-40,000 Daltons, 5,000-30,000 Daltons, 5,000-20,000 Daltons, or 7,000-17,000 Daltons. The PLG polymer can have any suitable end group at the terminal ends of the polymer chain, such as an ester.

[0046] Microparticles formed from the PLG polymer can have a particle size which allows for passively targeting macrophages for internalization, to enable the release and immunomodulatory effects of encapsulated all-traw.? retinoic acid to be enacted intracellularly. For example, the microparticle can have an average particle size of 0.05-8 microns as determined from light microscopy, for example. 0.05-6 microns, 0.05-5 microns. 0.05-4 microns, 1-6 microns, 1-5 microns, 1-4 microns, 1-3 microns, 2-6 microns, 2-5 microns, 2-4 microns, or 2-3 microns.

[0047] The pharmaceutical composition will generally be administered directly to a subject by parenteral administration. Delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue). Injection may be via a needle (e.g., a hypodermic needle) but needle-free injections may also be used. A typical intramuscular dose is 0.5 ml. Intramuscular injections can be administered in different body muscles, including the deltoid, dorsogluteal, ventrogluteal, rectus femoris, or vastus lateralis muscles. Pharmaceutical carriers suitable for parenteral administration are known in the art.

[0048] Additional embodiments of the pharmaceutical composition includes those in which the composition is present in a kit or commercial package. In one embodiment, the kit or commercial package includes the pharmaceutical composition as a drug product, e.g., a lyophilized drug product. The kit or commercial package can also include one or more of: instructions for treating muscle atrophy in a subj ect, one or more syringes or other devices suitable for administering the pharmaceutical composition, or pharmaceutical carriers suitable for parenteral administration, or any combination of these additional items.

[0049] Also described are uses of the pharmaceutical composition described above for the manufacture of a medicament for the treatment of muscle atrophy. Similarly described is the pharmaceutical composition described above for use in the treatment of muscle atrophy.Attorney Docket No.37759.0595P1Methods for Making the Microparticle Compositions

[0050] The microparticle compositions described above can be made using a single oil- in-water emulsification process followed by solvent removal to provide the microparticle composition. In one embodiment, the method comprises (i) preparing an oil phase comprising al 1 -trans retinoic acid and the PLG polymer in an organic solvent, (ii) combining the oil phase with a water phase comprising water and an emulsifying agent, and (iii) and removing the organic solvent to provide the microparticle composition. It was found that drug loading into PLG microparticles using single emulsions may be dependent on several factors, including PLG composition and molecular weight, the weight ratio of a -trans retinoic acid and PLG in the oil phase, and the solvent used in the oil phase.

[0051] In one embodiment, microparticles particularly suited for intracellular delivery can be prepared by selecting a suitable ratio of aW-trans retinoic acid and the PLG polymer in the oil phase used in the manufacturing method. For example, the oil phase can comprise all- trans retinoic acid and the PLG polymer in a weight ratio of 1:2 to 1:20, e.g., 1:2-1:15, 1:3- 1:15, 1:4-1:15, 1:5-1:15, 1:8-1:15, 1:8-1:12, or about 1:10.

[0052] The concentration of the a\\-trans retinoic acid in the oil phase can also affect microparticle size, drug loading, and other characteristics. In one embodiment, the method utilizes a relatively high concentration of aX-trans retinoic acid in the oil phase. In some embodiments, the oil phase comprises 2-10 mg / mL of al-lrans retinoic acid, with the all- trans retinoic acid and the PLG polymer being present in the oil phase in a weight ratio of 1 :2 to 1:20, , e.g., 1:2-1:15, 1:3-1:15, 1:4-1:15, 1:5-1:15, 1:8-1:15, 1:8-1:12, or about 1:10. In a further embodiment, the oil phase comprises 3-8 mg / mL of a\\-irans retinoic acid, with the a\\-trans retinoic acid and the PLG polymer being present in the oil phase in a weight ratio of 1:2 to 1:20, , e.g., 1:2-1:15, 1:3-1:15, 1:4-1:15, 1:5-1:15, 1:8-1:15, 1:8-1:12, or about 1:10. In a further embodiment, the oil phase comprises 4-8 mg / mL of a\\-trans retinoic acid, with the a\\-trans retinoic acid and the PLG polymer being present in the oil phase in a weight ratio of 1:2 to 1:20, , e.g., 1:2-1:15, 1:3-1:15, 1:4-1:15, 1:5-1:15, 1:8-1:15, 1:8-1:12, or about 1:10. . In a further embodiment, the oil phase comprises 4-6 mg / mL of a\\-irans retinoic acid, with the all-iraw.’ retinoic acid and the PLG polymer being present in the oil phase in a weight ratio of 1:2 to 1:20, , e.g., 1:2-1:15, 1:3-1:15, 1:4-1:15, 1:5-1:15, 1:8-1:15, 1:8-1:12, or about 1:10. In a further embodiment, the oil phase comprises about 5 mg / mL of a\\-irans retinoic acid, with the a\\-lrans retinoic acid and the PLG polymer being present in the oil phase in a weight ratio of 1:2 to 1:20. , e.g., 1:2-1:15, 1:3-1:15, 1:4-1:15, 1:5-1:15, 1:8-1:15, 1:8-1:12, or about 1:10.Attorney Docket No. 37759.0595P1

[0053] As with the pharmaceutical compositions described above, useful PLG polymers for the oil-in-water emulsification method include those having a lactide / glycolide molar ratio ranging from 20:80 to 80:20, for example, 25:75, 40:60, 45:55, 50:50, 55:45, 60:40, or 75:25. In one embodiment, the poly(D,L-lactide-co-glycolide) used in the manufacturing method has a lactide / glycolide molar ratio ranging from 45:55 to 55:45. Useful PLG polymers for the manufacturing method include those having a weight-average molecular weight (Mw) in the range of 5.000-200,000 Daltons, for example. 5,000-100.000 Daltons, 5,000-70,000 Daltons, 5,000-40,000 Daltons, 5,000-30,000 Daltons, 5,000-20,000 Daltons, or 7,000-17,000 Daltons. The PLG polymer used in the manufacturing method can have any suitable end group at the terminal ends of the polymer chain, such as an ester. Microparticles formed from the manufacturing method can have an average particle size of 0.05-8 microns as determined from light microscopy, for example, 0.05-6 microns, 0.05-5 microns, 0.05-4 microns, 1-6 microns, 1-5 microns, 1-4 microns, 1-3 microns, 2-6 microns, 2-5 microns, 2-4 microns, or 2-3 microns.

[0054] A variety of emulsifying agents can be used, including ionic or non-ionic surfactants, and a number of organic solvents are useful. In one embodiment, the emulsifying agent is polyvinyl alcohol (PVA), polyethylene glycol sorbitan monolaurate (Tween), sorbitan monooleate (Span), or sodium dodecyl sulfate (SDS). In a further embodiment, the organic solvent is dichloromethane, chloroform, hexafluoro-isopropanol, ethyl acetate, isopropanol, methyl ethyl ketone, acetone, or benzyl alcohol. After the emulsification process, solvent may be removed by any suitable method such as by stirring for a suitable amount of time to allow the solvent to evaporate, or by drying under vacuum. After removal of the organic solvent, the particles may be filtered, collected, washed, and optionally lyophilized for storage.

[0055] Also described is a pharmaceutical composition for parenteral administration that includes a \-trans retinoic acid encapsulated by the microparticle formed from the PLG polymer, in the manufacture of a medicament for the therapeutic treatment of muscle atrophy. Similarly, described is a process for the manufacture of a medicament for use in the treatment of muscle atrophy, characterized by the use of the pharmaceutical composition for parenteral administration that includes all-trara retinoic acid encapsulated by the microparticle formed from the PLG polymer.Attorney Docket No. 37759.0595P1Treatment Methods and Uses

[0056] The pharmaceutical compositions are useful for treating muscle atrophy. The method of treatment generally comprises administering to a subject by parenteral injection a pharmaceutical composition comprising all-trans retinoic acid encapsulated by the microparticle formed from the PLG polymer. Muscle atrophy can be a result or symptom of a variety of underlying issues, such as a muscle disease or disorder, or muscle disuse (e.g.. after an injury requiring a cast or other immobilization). Examples of muscle diseases or disorders that may result in muscle atrophy include muscle-wasting, a muscle degenerative disease, a myopathy, an age-related decline in muscle function, frailty or pre-frailty, a neuromuscular disease, Duchenne muscular dystrophy, sarcopenia, cachexia, muscle loss, a muscle function disorder, physical fatigue, muscle fatigue, inclusion body myositis, or sporadic inclusion body myositis.

[0057] As described above, the pharmaceutical composition will generally be administered directly to a subject by parenteral administration. Delivery' may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue). Injection may be via a needle (e.g.. a hypodermic needle) but needle-free injections may also be used. A typical intramuscular dose is 0.5 ml. Intramuscular injections can be administered in different body muscles, including the deltoid, dorsogluteal. ventrogluteal, rectus femoris, or vastus lateralis muscles. Pharmaceutical carriers suitable for parenteral administration are known in the art.

[0058] Because the described microparticle compositions provided for extended release of the encapsulated aU-trans retinoic acid, the dosing regimen can be spaced apart to provide more convenience to the patient, which may also result in adequate patient compliance and less discomfort. In one embodiment, the pharmaceutical composition is administered to the subject once per every two weeks, i.e., at day 1 and again at day 14, and so on. In a further embodiment, the pharmaceutical composition is administered to the subject once per every three weeks, one per every four weeks, once per every five weeks, or once per every six weeks.

[0059] As with the pharmaceutical compositions and manufacturing methods described above, useful PLG polymers for the treatment methods and uses include those having a lactide / glycolide molar ratio ranging from 20:80 to 80:20, for example, 25:75, 40:60, 45:55, 50:50, 55:45. 60:40, or 75:25. In one embodiment, the poly(D,L-lactide-co-glycolide) used in the treatment method or use has a lactide / glycolide molar ratio ranging from 45:55 to 55:45. Useful PLG polymers for treatment method or use include those having a weight-averageAttorney Docket No. 37759.0595P1 molecular weight (Mw) in the range of 5,000-200,000 Daltons, for example, 5,000-100,000 Daltons, 5,000-70,000 Daltons, 5,000-40,000 Daltons, 5,000-30,000 Daltons, 5,000-20,000 Daltons, or 7,000-17,000 Daltons. The PLG polymer used in the treatment method or use can have any suitable end group at the terminal ends of the polymer chain, such as an ester. Microparticles used in the treatment method or use can have an average particle size of 0.05- 8 microns as determined from light microscopy, for example, 0.05-6 microns, 0.05-5 microns, 0.05-4 microns, 1-6 microns. 1-5 microns. 1-4 microns. 1-3 microns, 2-6 microns, 2-5 microns, 2-4 microns, or 2-3 microns.

[0060] In one embodiment, the microparticle used in the treatment method or use comprises more than 50 pg all-Zranv retinoic acid per mg of the microparticle. In a further embodiment, the microparticle used in the treatment method or use comprises at least 70 pg all-traws retinoic acid per mg of the microparticle. Exemplary ranges of all -trans retinoic acid loading for the treatment method or use include 50-100 pg a\\-trans retinoic acid per mg of the microparticle, 60-90 pg a\\-trans retinoic acid per mg of the microparticle, 70-80 pg all- trans retinoic acid per mg of the microparticle, or 75-80 pg a \-trans retinoic acid per mg of the microparticle. In some embodiments, the microparticle used in the treatment method or use comprises ail-trans retinoic acid and no other retinoid, i.e., the retinoid in the microparticle consists of all-trans retinoic acid.

[0061] Also described is the use of a pharmaceutical composition for parenteral administration that includes al\-trans retinoic acid encapsulated by the microparticle formed from the PLG polymer in the treatment of muscle atrophy. Similarly described is the pharmaceutical composition for parenteral administration that includes a -trans retinoic acid encapsulated by the microparticle formed from the PLG polymer for use in the treatment of muscle atrophy.EXAMPLES

[0062] The following examples further illustrate this disclosure. The scope of the disclosure and claims is not limited by the scope of the following examples.Induction of IGF-1 Secretion in RAW Macrophages

[0063] IGF-1 is a key factor in muscle regeneration and growth. Macrophages are sources of IGF-1 in the muscle, and it was determined that activation of retinoic acid receptors in macrophages induces IGF-1 gene expression. The ability of retinoids AM580 and ATRA toAtorney Docket No. 37759.0595P1 induce IGF-1 secretion into the culture media using RAW macrophages was tested (Figure 1). The structures of AM580 and ATRA are shown below.

[0064] With reference to Figure 1 , IGF-1 concentration for the vehicle control (DMSO) was 20 ± 1 pg / mL, and all other measurements were normalized to this value. All tested ATRA concentrations increased IGF-1 expression with the largest increase (3 -fold) achieved at 1 pM. In the same experiments (i.e., on the same well plates), the effect of AM580 was determined. It was found that only 1 and 10 pM AM580 increased IGF-1 over the vehicle control. In addition, at every concentration tested, ATRA induced significantly more IGF-1 than AM580.ATRA-PLG Particle Characterization

[0065] Two particle formulations were produced: PLG particles with no drug loading (PLG) and PLG particles loaded with ATRA (ATRA-PLG). All particles were spherical (Figures 2A-B) with a narrow size range (Figure 2C). The ATRA-PLG particles were yellow in color and appeared darker via microscopy compared to the PLG particles, which were white (Figures 2A-B). Particle characteristics are shown in Figure 2D. The average particle size was 2.7 gm in diameter and mass yield ranged between 57 and 58%. Addition of ATRA to the emulsion did not impact average particle size or mass yield. Drug loading and encapsulation efficiency for ATRA-PLG was 78 pg / mg and 52%, respectively. Having determined that ATRA was encapsulated into PLG particles, the release kinetics of ATRA from the particles were investigated in vitro (Figure 2E). ATRA-PLG particles exhibited a linear drug release of 2.9% per day (r2 = 0.943) between days 1 and 14.

[0066] Having developed ATRA-PLG particles, it was next determined whether the ATRA remained able to induce IGF-1 expression after encapsulation. To test this, RAW macrophages were exposed to a dose range of particles for 24 hours, and the media was collected. The doses investigated were 0.5, 5 and 10 particles per cell seeded. ATRA-PLG particles significantly increased IGF-1 expression over blank particles (Figure 3 A), indicating the ATRA released was bioactive. A dose response was detected for the ATRA-PLG particles, with no difference detected between the two highest doses. Microscopic analysis ofAttorney Docket No. 37759.0595P1 cell morphology suggested that the amount of ATRA released was well tolerated by the cells, and cell density’ measurements made from those images was not impacted (Figure 3B).

[0067] The long-term stability of ATRA-PLG was also assessed. It was found that particles stored at room temperature under vacuum and in the dark for 3 months induce an IGF-1 response comparable to particles stored for only 1 week (Figures 3C-3D). The data indicate an ATRA-PLG shelf life of at least 3 months when stored under dry conditions protected from light.C2C12 Myotube Formation from Conditioned Media Collected from ATRA-PLG- Treated RAW Macrophages

[0068] ATRA-PLG treatment of RAW macrophages induced IGF-1 secretion (Figure 3A), which is a protein important for satellite cell proliferation and differentiation into mature muscle. The impact of media from RAW macrophages treated with ATRA-PLG on myotube formation using C2C12 myoblasts was further studied. RAW macrophages were treated with ATRA-PLG particles for 24 hours and then the conditioned media was collected, depleted of particles via filtration, and stored for later treatment of C2C12 cells (this media is referred to as media “iv”). Additionally, unconditioned media (i), RAW macrophage conditioned media without particles (ii) and ATRA-PLG conditioned media (iii) were generated as controls.

[0069] As shown in the timeline in Figure 4A, after one day of differentiation, the unconditioned (i) or conditioned medias (ii-iv) were used to differentiate myoblasts for 3 days. Immunofluorescence microscopy was performed for myosin heavy chain and nuclei (Figure 4B). Differentiation was terminated after 4 days instead of later time points (e.g., 7 days). The conditioned media caused the C2C12 cells to form myotubes rapidly, and by 5 days the mature myotubes detached from the cell culture dish making them difficult to image. Media collected from macrophages treated with ATRA-PLG particles (media iv) induced C2C12 cells to fuse more readily into myotubes compared to all other treatments, as quantified by the number of myotubes per image field (Figure 4C) and the number of nuclei per myotube (Figure 4D). The data indicate that all conditioned medias enhanced C2C12 myotube formation. Media from RAW macrophages treated with ATRA-PLG was superior.

[0070] Superior myotube growth with media from RAW cells treated with ATRA-PLG (media iv) was hypothesized to be due to IGF-1. To test this, the same myotube formation assay was conducted with an IGF-1 -neutralizing antibody or antibody without IGF-1 specificity. IGF-1 neutralization significantly decreased myotube formation when C2C12 cells were treated with conditioned media from RAW macrophages treated ith ATRA-PLGAttorney Docket No. 37759.0595P1(Figure 4E. media iv). In contrast, the other groups exhibited a non-significant decrease in myotube formation when IGF-1 was neutralized. The data indicate that increased IGF-1 content is responsible for the increased myotube formation observed using conditioned media from RAW macrophages treated with ATRA-PLG (media iv). It is important to consider that IGF-1 antibody blockade tends to decrease myotube formation when the C2C12 cells are treated with control medias (particularly medias i and ii, Figure 4E); although, the differences in means were not statistically significant. These findings suggest that C2C 12 cells may also be a source of IGF-1 that impacts myotube formation. Thus, the significant reduction in myotube formation for media iv may be due to antibody blockade antagonizing IGF-1 growth signals from both C2C12 cells and macrophages.Long-Term Culture of Bone Marrow Derived Macrophages with Florescent Particles

[0071] One application of ATRA-PLG particles is delivery to muscle tissue where they may be taken up by macrophages, reside in the cytosol, and release ATRA for an extended period of time. The fate of particles cultured with terminally-differentiated bone marrow derived macrophages was determined (BMDM), which do not divide and can be cultured for days without passage (in contrast to RAW macrophages). To aid in visualization, the particles encapsulated coumarin 6, a fluorescent dye with comparable molecular weight and hydrophobicity to ATRA. Bone marrow cell differentiation consistently yielded cultures in which greater than 95% of the cells expressed F4 / 80 and CD1 lb (Figures 5 A-C), which are accepted markers for BMDM. In addition, BMDMs could be cultured without passage for up to 21 days with 1 / 2 media changes ever}' two days.

[0072] BMDMs were treated with C6-PLG particles (size = 2.7 pm) at a dose of 50 particles per cells seeded. Two days later, the unbound / non-intemalized particles were removed during a 1 / 2 media change, and the cells were imaged using fluorescence microscopy (Figure 5D). The 1 / 2 media changes and fluorescence imaging continued every' two days until day 8. After 8 days in culture, fluorescent C6-PLG particles were still intact. Particle fluorescence waned over time and required higher excitation light intensity' for detection (Figure 5D). This was likely' due to release of the coumarin 6.Attorney Docket No. 37759.0595P1Impact of ATRA-PLG on IGF-1 Secretion from Bone Marrow Derived Macrophages

[0073] The impact of ATRA-PLG particles on secretion of IGF-1 from BMDM cultures over time was investigated. BMDM was treated with a dose range of ATRA-PLG particles in the same manner as Figure 5. Cells were treated with particles for 48 hours (2 days) and then unbound particles were removed during a 1 / 2 media change. Then, every 2 days for 12 days. 1 / 2 media changes were carried out while 1 / 2 of the conditioned media was frozen. At day 12, it was confirmed that particles were visible in the ATRA-PLG treated BMDM and the experiment was terminated.

[0074] An IGF-1 ELISA was performed on the media from all time points. Changes in IGF-1 levels in comparison to Vehicle-treated cells were not detected at any concentration between days 2 and 10. At day 12, there was a trend of increased IGF-1 for the ATRA-PLG treated cells (Figure 6A). Analysis of images taken on day 12 indicated that cell density was not impacted over the course of the experiment (Figure 6B), indicating that a decreased cell count did not play a role in the IGF-1 response. The BMDMs could increase expression of IGF-1 in response to IL-4 treatment (Figure 6C), ruling out the possibility that BMDM could not increase IGF-1 production. IL-4 treated BMDMs were able to maintain high levels of IGF-1 for up to 12 days with a single, two-day treatment of IL-4, which was followed by 1 / 2 media changes every 2 days with fresh media that did not contain IL-4 (Figure 6C).Conditions in Which ATRA Induces IGF-1 Production by Primary Macrophages

[0075] Data indicated that either ATRA or ATRA-PLG induces IGF-1 secretion by RAW macrophages (Figure 1 and 3B), when a similar IGF-1 response was not observed by BMDMs (Figure 6A). M-CSF, a growth factor needed for the differentiation of BMDMs, induces IGF-1 expression in these cells. It was determined that once the BMDMs were fully differentiated, M-CSF could be removed from the culture for up to 72 hours before viability was affected. BMDMs were treated, which had not been exposed to MCSF for 24 hours, with ATRA. BMDMs did not exhibit a significant increase in IGF-1 content in media (Figure 7A). To determine if ATRA can induce IGF-1 production in other primary macrophages, peritoneal macrophages (which do not require M-CSF for culture) were cultured in the presence of ATRA for 24 hours. ATRA did induce IGF-1 in peritoneal macrophages (Figure 7B).

[0076] Differences in activation states between RAW macrophages and BMDMs were investigated to explain the variations in IGF-1 production observed. To determine theAttorney Docket No. 37759.0595P1 activation state of resting (i.e., unstimulated) RAW macrophages and BMDM, we conducted ELISA for TNF-a, IL-10, and IGF-1 in culture media. RAW macrophages express high levels of TNF-a and low levels of IL-10 and IGF-1 compared to BMDM (Figure 7C and 7D). The data indicate that RAW macrophages exhibit a pro-in fl ammatory activation state compared to BMDM. This finding led us to investigate if inflammatory activation of BMDM with LPS could enhance IGF-1 release after ATRA treatment. However, this was not the case. While LPS treatment tended to increase IGF-1 release by BMDM, ATRA treatment following LPS treatment did not further increase IGF-1 (Figure 7E). Taken together, the data indicate that RAW macrophages exhibit a more pro-inflammatory activation state compared to BMDM in terms of the cytokines measured. However, inflammatory activation of BMDM with LPS is not sufficient to increase IGF-1 release after ATRA treatment.Expression of Retinoic Acid Receptor Proteins in Macrophages

[0077] The differing ATRA-induced IGF-1 response in RAW, BMDMs, and peritoneal macrophages suggested that there may be a difference in expression of retinoic acid receptors (RAR) in these cell types. Western blots for RARa, RARp. and RARy were conducted on whole cell lysate from RAW cells, BMDMs, and Peritoneal macrophages. RARp was detected in all three types of macrophages. By contrast, RARa and RARy w ere only detected in RAW macrophages (Figure 8A). Equivalent protein loading was confirmed with Ponceau- S stain. RAR expression was quantified, and the level in peritoneal macrophages was significantly higher than in RAW macrophages (Figure 8B).Materials and Methods

[0078] The following were purchased from Sigma (St. Louis. MO): 50:50 poly 50:50 poly (D,L-lactide-co-glycolide) (PLG) (RESOMER® 502, Mw 7000-17,000), dichloromethane (DCM), Coumarin-6, Poly (vinyl alcohol) (PVA) (Mw 13,000-23,000, 87- 89% hydrolyzed), Phosphatase Inhibitor Cocktail, insulin from bovine pancreas, goat serum, horse serum, and phalloidin. Trizma-Base, Trizma-HCl, Glycine, sodium dodecyl sulfate, ammonium persulfate, sodium chloride and brewer’s thiogly collate medium.

[0079] The following were purchased from Thermo Fisher (Hampton, NH): Dimethyl sulfoxide (DMSO), ACK lysing buffer, Hoechst, Protease Inhibitor Cocktail and phylmethylsulfonyl fluoride. Pierce BCA Protein Assay Kit, SuperSignal West Pico Chemiluminscent, Pierce R1PA Buffer, Hoescht 33342. Ultrapure water was obtained from a Thermo Scientific Bamstead Nanopure system.Attorney Docket No. 37759.0595P1

[0080] All -trans -Retinoic Acid (ATRA) and AM580 were purchased from CaymanChemical (Ann Arbor, MI).

[0081] The following were purchased from Coming (Coming, NY): DMEM high glucose medium, DMEM / F12 medium, sodium pyruvate, sodium bicarbonate, penicillinstreptomycin and fetal bovine serum, Dulbecco's phosphate buffered saline, and Trypsin with 0.25% EDTA.

[0082] Red Blood Cell Lysis Solution was purchased from Miltenyi Biotec (Bergisch Gladbach, North Rhine-Westphalia, Germany).

[0083] The following were purchased from Biorad (Hercules, CA): Acrylamide / Bis Solution, Laemmli Sample Buffer, Goat anti-mouse IgG (H+L)-HRP Conjugate, mini- PROTEAN Tetra Handcast Systems and Vertical Electrophoresis Cell, Critereon TransBlot, Precision Protein StrepTactin-HRP Conjugate, Precision Plus Protein Dual Color Standards, Goat anti-mouse-HRP Conjugate (STAR207P).

[0084] Recombinant macrophage stimulating factor (GFM8-10) and recombinant mouse IL-4 (GFM18-20) was purchased from Cell Guidance Systems (Cambridge, UK).

[0085] IGF-1 DuoSet ELISA kits. Myosin Heavy Chain Monoclonal Mouse IgG2B Antibody (MAB4470), Mouse IGF-1 Antibody (#AF791) and Normal Goat IgG Control (AB-108-C) were purchased from R&D Systems (Minneapolis, MN).

[0086] The following antibodies were purchased from Invitrogen (Waltham, MA): F(ab’)2-Goat anti-Mouse IgG (H+L) cross-adsorbed secondary antibody conjugated to Alexa Fluor 555 (A-21425).

[0087] The following antibodies w ere purchased from Biolegend (San Diego, CA): TruStain FcX (101319), anti-CD45 clone 30-F11 FITC (103108). anti-F4 / 80 clone BM8 APC (123116), anti CDl lb clone MI / 70 PE-Cy7 (101216), Isotype Control Rat IgG2b Clone RTK4530 (400605), Isotype Control Rat IgG2b Clone RTK4530 PE / Cy7 (400618), Isotype Control Rat IgG2a Clone RTK2758 APC (400512).

[0088] The following antibodies w ere purchased from Abeam (Cambridge, UK): Goat Anti-Rabbit IgG H&L (ab97051). The following antibodies w ere purchased from Cell Signaling Technologies (Danvers. MA): RARa Rabbit mAb clone E6Z6K (62294).

[0089] The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): mouse anti-RAR(32 clone B-12 (sc-514585), mouse anti-RARy clone G-l (sc- 7387).

[0090] The PT3100D homogenizer was purchased from Kinematica (Malters, Switzerland). An Eppendorf Thermomixer R was purchased from Eppendorf (Hamburg,Attorney Docket No. 37759.0595P1Germany). A Spectramax 190 UV-Vis spectrophotometer was purchased from Molecular Devices (San Jose. CA). A Labconco freeze drier was purchased from Labconco (Kansas City, MO). EVOS FL light microscope with light cubes GFP (470nm excitation / 510nm emission wavelength), DAPI (357nm excitation / 447nm emission wavelength) and RFP (530 nm excitation / 590 nm emission wavelength) were purchased from Fisher (Hampton, NH). An iBright CL1500 Imaging System was purchased from Thermo Fisher (Hampton, NH). A FACs Area flow cytometer was purchased from BD Biosciences (San Jose, CA).

[0091] Software used includes ImageJ as developed and provided by the National institutes of Health and Laboratory for Optical and Computational Instrumentation (Bethesda, MD and LOCI, University of Wisconsin, WN), FlowJo from Becton, Dickinson & Company (Franklin Lakes, NJ), and GraphPad Prism software (San Diego, CA). iBright Analysis Software was provided by Thermo Fisher Scientific (Hampton, NH).

[0092] Polymer Particle Formation'. PLG particles were made using a single oil-in-water emulsification / solvent evaporation protocol as follows. For the organic phase, PLG and ATRA were dissolved in DCM at concentrations of 52.8 mg / mL and 5 mg / mL, respectively. When fluorescent particles were made, coumarin 6 was also added to the DCM at a concentration of 0.5 mg / mL. For the emulsion, 0.6 mL of the organic phase was added dropwise to 4 mL of a PVA solution (10 mg / mL) and homogenized at 11,000 rpm for 5 minutes. The emulsion was then added to 80 mL of water and stirred at 80 rpm for 1 hour, allowing the DCM to evaporate and polymer particles to form. The particles were then passed through a 40 pm filter, collected via centrifugation at 250 xg for 10 minutes, and washed 3 times in ultrapure water. Washed particles were frozen at -20°C and subsequently lyophilized overnight. Recovered particles were stored under vacuum in a dark, dry environment at room temperature.

[0093] Particle size was determined by analyzing light microscopy images using ImageJ software. Particles were suspended in complete media at a concentration of 0.25 mg / mL. 400pL of these suspensions was added to a well of a 48-well plate and allowed to settle prior to image acquisition. Images for particle size analysis were taken on an EVOS FL light microscope at 20X. Three representative images were taken of each particle condition and converted to binary (B / W). The Particle Analysis plugin in ImageJ was used to measure particle diameter. This method was validated with monodispered polystyrene beads purchased from Duke Standards.Attorney Docket No. 37759.0595P1

[0094] Mass yield was calculated by Equation 1, where Mtot is the total mass of particles recovered from the emulsion, MPLG is the mass of polymer added to the emulsion, and MRA is the mass of ATRA added to the emulsion.(Equation 1) 100

[0095] ATRA loading in the particles was determined by dissolving 1 mg of particles in 1 rnL DMSO and measuring absorbance at 355 nm using a UV-Vis spectrophotometer. ATRA concentration was then interpolated from a 10-point standard curve generated with standard solutions of ATRA dissolved in DMSO. ATRA loading was calculated by equation 2, where mrais the mass of ATRA measured in a sample of particles weighing mP.(Equation !)

[0096] Encapsulation Efficiency is calculated by multiplying Drug Loading (Equation 2) by the total mass of particles recovered (Mtot) and dividing by the mass of ATRA MRA) added to the emulsion as indicated by equation 3.(Equation 3)

[0097] In Vitro Release Assay: A pre-weighed mass of ATRA-PLG were dispersed in 1 mL ultrapure water in 1.5 mL tubes and maintained at 37°C and agitated at 600 rpm on an Eppendorf Thermo mixer R while protected from light. At the designated time points, tubes were removed from the thermomixer and particles were collected via centrifugation. The supernatant was removed, and the particle pellet was frozen and lyophilized. Particles were analyzed for ATRA loading as described in Section 2.4. ATRA released (%) was calculated by equation 4, where Lo is the loading of the particles at time point t = 0. and Lt is the loading at time t during the release assay.(Equation 100

[0098] Culture of RAW 264. 7 macrophages: RAW264.7 (RAW) macrophages were obtained from ATCC and cultured as instructed by the company. RAW macrophages were cultured in DMEM high glucose medium containing 10% fetal bovine serum, 1% penicillinstreptomycin, and supplemented with sodium bicarbonate (1.5% w / v) and sodium pyruvate (1 mM). Cells were seeded at a density of 25,000 cells / cm2. Cells were passaged before reaching 60-70% confluency.

[0099] Retinoid treatment of RAW macrophages: Twenty-four hours after seeding, RAW cells were treated with 0. 1-10 LIM of ATRA or AM580 using DMSO as the vehicle (<0.1% v / v). After 24 hours of treatment, media was collected for ELISA analysis.Attorney Docket No. 37759.0595P1

[0100] ELISA analysis of media. Cell culture media was analyzed with Duoset ELISA kit following the manufacturer instructions.

[0101] Particle treatment of RAW macrophages'. ATRA-PLG, PLG, or C6-PLG particles were suspended in RAW culture media (see Section 2.6) and added to wells for final concentrations of 0.5-10 particles per cell seeded. Particle number was calculated assuming spherical geometry with a 2.7 micron diameter and taking the density of PLG to be 1.6 g / mL. After 24 hours of treatment, media was collected, and particles were removed by centrifugation.

[0102] Cell density measurements'. Cell density measurements were made by counting all cells in 500x500 pixel brightfield image taken at 10X. Five images per condition were analyzed and averaged.

[0103] Production of conditioned medias'. RAW macrophage conditioned media (designated as “ii”): RAW macrophages were seeded in T75 flasks and cultured for 24 hours. To generate conditioned media, cells were washed with PBS and 10 mL of fresh media was added to the flasks and placed in the incubator. After 24 hours, the media was collected, sterile-filtered through a 0.2 pm filter, and frozen in aliquots at -20°C.

[0104] ATRA-PLG conditioned media (designated as “iii”): 10 mL of fresh media containing 100 pg of ATRA-PLG particles were added to T75 flasks and placed in the incubator. After 24 hours, the media was collected, sterile-filtered through a 0.2 pm filter, and frozen in aliquots at -20°C.

[0105] RAW macrophage treated with ATRA-PLG conditioned media (designated as “iv”):_RAW macrophages were plated in T75 flasks and cultured for 24 hours. To generate conditioned media, cells were washed with PBS and 10 mL of fresh media containing 100 pg of ATRA-PLG particles were added to flasks and placed in the incubator (100 pg correlates to 10 particles per cell seeded). After 24 hours, the media was collected, sterile-filtered through a 0.2 pm filter, and frozen in aliquots at -20°C.

[0106] C2C12 myoblast culture and myotube formation assay. C2C12 myoblasts were obtained from ATCC and cultured as instructed by the company. C2C12 cells were cultured in DMEM high glucose medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded at a density of 25,000 cells / cm2Media was changed every other day and cells were passaged before reaching 70% confluency. To induce myotube formation, C2C12 cells were cultured to confluence, at which time the media was changed to “Differentiation Media" consisting of DMEM high glucose medium supplemented with 1 pM bovine insulin, 2% horse serum, and 1% penicillin-streptomycin forAttorney Docket No. 37759.0595P1 twenty-four hours. For the following four days, the media was exchanged daily with fresh media consisting of a 1 : 1 ratio of DMEM high glucose medium supplemented 2% horse serum and 1% penicillin-streptomycin and one of the following: (i) “unconditioned media’’, which was fresh RAW macrophage culture media (section 2.6); (ii) media conditioned with RAW macrophages for twenty -four hours (section 2. 11); (iii) media conditioned with ATRA- PLG for twenty -four hours (section 2. 11); or (iv) media conditioned with RAW macrophages treated with ATRA-PLG for twenty-four hours (section 2. 11). After three consecutive days of exposure to treatment media, myotube cultures were fixed in 4% formaldehyde. In certain experiments, an IGF- 1 -neutralizing antibody or Goat IgG was added to the media at 1 pg / mL.

[0107] Immunofluorescence microscopy of myotubes'. Immunofluorescence was conducted on 4% paraformaldehyde-fixed myotubes using a mouse anti-myosin heavy chain primary antibody (1 ug / mL) and a goat anti-mouse antibody conjugated to Alexa Fluor 555 (10 pg / mL). Nuclei were visualized with Hoescht. Light and fluorescent images of cells were taken on an EVOS FL light microscope at 10X and 20X. Fluorescence images were acquired with the DAPI and RFP light cubes.

[0108] Myotube Image Analysis: C2C 12 myotubes were quantified using ImageJ software. The number of nuclei in each myotube was counted and a myotube was defined as a Myosin Heavy Chain-positive tube containing three or more nuclei. For each well, 3-5 random fields were imaged and quantified.

[0109] Harvest and culture of bone marrow -derived macrophages (BMDM): Primary cultures of bone marrow-derived macrophages (BMDM) were generated from the hindlimbs of female ICR mice between 8 and 12 w eeks old. After euthanasia, hindlimbs w ere dissected and marrow' was collected through centrifugation. Red blood cells were lysed, and remaining cells were seeded into non-tissue culture-treated petri dishes at 500,000 cells / cm2. Cells were cultured in DMEM / F12 media containing 10% FBS, 1% Penicillin-Streptomycin (BMDM Complete Media), and supplemented with 10 ng / mL macrophage-colony stimulating factor (M-CSF). After 5 days of differentiation, nonadherent cells were removed and the adherent population was passaged into tissue culture-treated multi-well plates at a seeding density of 50.000 cells / cm2. BMDMs seeded into multi-well plates received A media changes every 48 hours in which 1 / 2 of the media was removed and replaced with an equal volume of fresh BMDM Complete Media.

[0110] Flow cytometry of BMDMs to assess purity: After 5 days of differentiation, BMDMs were trypsinized and washed with MACS buffer (PBS, 0.5 mM EDTA. 30% BSA), and incubated with TruStain FcX followed by antibodies to CD45, CD1 lb, and F4 / 80. AfterAttorney Docket No. 37759.0595P1 antibody incubation, cells were washed, fixed, and analyzed using a FACS Aria flow cytometer. FlowJo software was utilized to compensate and analyze data. FMOs with isotype controls were used to determine specific antibody signal. The gating scheme used in the flow cytometry analysis is depicted in Figure 5A.

[0111] Particle and IL-4 treatment ofBMDMs'. Forty-eight hours after being seeded in multi-well plates, BMDMs were treated with PLG, C6-PLG, or ATRA-PLG at a concentration of 1. 5, 10, or 50 particles per cell seeded. After 48 hours of particle treatment, the media was collected and depleted of particles by centrifugation and particles remaining in the well were removed by gentle washing with PBS. The particle-depleted media w as added back to w ells with fresh BMDM Complete Media at a 1: 1 ratio for a ! media change. The BMDMs were then cultured with ! media changes every 48 hours for up to 12 days. For one experiment, BMDMs were treated with IL-4. After 48 hours of IL-4 treatment, a ! media change w as carried out but no additional IL-4 w as added to the well. These ! media changes continued every 48 hours for 12 days when the experiment was terminated.

[0112] Treatment ofBMDMs with ATRA in the absence ofM-CSF'. Forty-eight hours after BMDMs were seeded into tissue culture-treated multi-well plates (see section 2.15), media was exchanged with fresh BMDM Complete Media without M-CSF. Twenty -four hours later, ATRA (final concentrations: 0.1, 1, or 10 pM) or DMSO (<0.1% v / v) was added to existing media. Media was then collected after 24 hours.

[0113] Harvest, culture and treatment of primary peritoneal macrophages '. Primary cultures of peritoneal macrophages were generated from C57BL / 6J mice. Mice were injected with 1 mL 3.8% brewer’s thioglycolate medium into the peritoneal cavity. Three days later, mice were euthanized and a peritoneal lavage with DPBS w as used to collect peritoneal macrophages. The aspirated cell suspension was centrifuged at 400 g for 10 min at 4°C, and the supernatant was discarded. Red blood cell contamination was removed using Red Blood Cell Lysis Solution. The pellet was resuspended, counted and plated in DMEM + 10% FBS + 1% Penicillin / Streptomycin. Twenty -four hours later, ATRA (final concentrations: 0.1, 1, or 10 pM) or DMSO (<0. 1% v / v) was added to existing media. Media was then collected after 24 hours.

[0114] Western Blot'. Cell pellets were generated from untreated RAW, BMDMs, or Peritoneal macrophages. The cell pellet was lysed in RIPA buffer containing phosphatase inhibitors, protease inhibitors, and ImM phenylmethylsulfonyl fluoride, and centrifuged to generate whole cell lysate. The protein content of the lysate was measured using BCA protein assay kits. For Western blotting, 30 pg of protein was electrophoretically separated on 10%Attorney Docket No. 37759.0595P1SDS-PAGE. The bands were transferred to nitrocellulose membranes, and then Ponceau S staining and imaging was performed. The membrane was then blocked with 5% nonfat milk and incubated overnight at 4°C with one of the following antibodies: anti-RARa (1 :250), anti-RARp (1: 1000), or anti-RARy (1:250). Afterwards, membranes were rinsed and incubated with goat anti-mouse or goat anti-rabbit secondary antibody (1: 1000) diluted in 5% nonfat milk for an hour. Bands were visualized using Chemiluminescence substrate. Images of the membranes were acquired, scanned, and analyzed using iBright Analysis Software.

[0115] Statistical Analysis'. GraphPad Prism software was employed for all statistical analysis. Where appropriate, a t-test, one-way or multiple-way ANOVA test with Tukey’s multiple comparison were carried out to compare differences between means. Data represented as mean ± standard deviation unless otherwise stated. Statistical symbols indicate p-values of * < 0.05, ** < 0.01, *** < 0.001, and **** <0.0001 to show significant difference between the groups unless noted otherwise. Additional details regarding statistical analyses carried out for each data set are described in the figure legend.

[0116] Features and advantages of this disclosure are apparent from the detailed specification, and the claims cover all such features and advantages. Numerous variations will occur to those skilled in the art, and any variations equivalent to those described in this disclosure fall within the scope of this disclosure. Those skilled in the art will appreciate that the conception upon which this disclosure is based may be used as a basis for designing other compositions and methods for carrying out the several purposes of this disclosure. As a result, the claims should not be considered as limited by the description or examples.

Claims

Attorney Docket No. 37759.0595P1CLAIMSWhat is claimed is:

1. A method of treating a subject having muscle atrophy comprising administering to the subject by parenteral injection a pharmaceutical composition comprising all - / ra retinoic acid encapsulated by a microparticle formed from a poly(D,L-lactide-co-glycolide).

2. The method of claim 1, wherein the subject has muscle atrophy as a symptom of a muscle disease or disorder, or muscle disuse.

3. The method of claim 2, wherein the muscle disease or disorder is muscle-wasting, a muscle degenerative disease, a myopathy, an age-related decline in muscle function, frailty or pre-frailty, a neuromuscular disease. Duchenne muscular dystrophy, sarcopenia, cachexia, muscle loss, a muscle function disorder, physical fatigue, muscle fatigue, inclusion body myositis, or sporadic inclusion body myositis.

4. The method of claim 1, wherein the pharmaceutical composition is administered to the subject by subcutaneous or intramuscular injection.

5. The method of claim 1, wherein the pharmaceutical composition is administered to the subject once per every two weeks.

6. The method of claim 1, wherein the microparticle has an average particle size of 0.05- 8 microns as determined from light microscopy.

7. The method of claim 1, wherein the poly(D,L-lactide-co-glycolide) has a lactide / glycolide molar ratio of 45:55 to 55:45.

8. The method of claim 1, wherein the poly(D,L-lactide-co-glycolide) has a weightaverage molecular weight of 5,000 to 200.000 Daltons.

9. The method of claim 1, wherein the poly(D,L-lactide-co-glycolide) is ester- terminated.

10. The method of claim 1, wherein the microparticle comprises at least 70 pg all-Zra s’ retinoic acid per mg of the microparticle.Attorney Docket No. 37759.0595P111. A method for making a microparticle composition comprising a\\-trans retinoic acid encapsulated by a microparticle formed from a poly(D,L-lactide-co-glycolide). the method comprising: preparing an oil phase comprising all-frt retinoic acid and the poly(D,L- lactide-co-glycolide) in an organic solvent; combining the oil phase with a water phase comprising water and an emulsifying agent; and removing the organic solvent to provide the microparticle composition.

12. The method of claim 1 1, wherein the oil phase comprises all-trans retinoic acid and the poly(D,L-lactide-co-glycolide) in a weight ratio of 1 :2 to 1:20.

13. The method of claim 11. wherein the oil phase comprises 2-10 mg / mL of aA-trans retinoic acid, and wherein the a\\-t rants retinoic acid and the poly(D,L-lactide-co-glycolide) are present in the oil phase in a weight ratio of 1 :2 to 1 :20.

14. The method of claim 11. wherein the poly(D,L-lactide-co-glycolide) has a lactide / glycolide molar ratio of 45:55 to 55:45.

15. The method of claim 11, wherein the poly(D,L-lactide-co-glycolide) has a weightaverage molecular weight of 5,000 to 200.000 Daltons.

16. The method of claim 11, wherein the poly(D,L-lactide-co-glycolide) is ester- terminated.

17. The method of claim 1 1, wherein the emulsify ing agent is polyvinyl alcohol (PVA), polyethylene glycol sorbitan monolaurate (Tween), sorbitan monooleate (Span), or sodium dodecyl sulfate (SDS).

18. The method of claim 11, wherein the organic solvent is dichloromethane, chloroform, hexafluoro-isopropanol, ethyl acetate, isopropanol, methyl ethyl ketone, acetone, or benzyl alcohol.

19. A pharmaceutical composition for parenteral administration comprising (i) all-traw. retinoic acid encapsulated by a microparticle formed from a poly(D,L-lactide-co-glycolide), and (ii) a pharmaceutical carrier suitable for parenteral administration; wherein the microparticle comprises more than 50 pg ail-trans retinoic acid per mg of the microparticle.Attorney Docket No. 37759.0595P120. The pharmaceutical composition of claim 19, wherein the microparticle has an average particle size of 0.05-8 microns as determined from light microscopy.

21. The pharmaceutical composition of claim 19, wherein the microparticle comprises at least 70 pg aA-trans retinoic acid per mg of the microparticle.

22. The pharmaceutical composition of claim 19, wherein the poly(D,L-lactide-co- glycolide) has a lactide / glycolide molar ratio of 45:55 to 55:45.

23. The pharmaceutical composition of claim 19, wherein the poly(D,L-lactide-co- glycolide) has a weight-average molecular weight of 5,000 to 200,000 Daltons.

24. The pharmaceutical composition of claim 19, wherein the poly(D,L-lactide-co- glycolide) is ester-terminated.

25. Use of the pharmaceutical composition of any one of claims 19-24 for the manufacture of a medicament for the treatment of muscle atrophy .

26. The pharmaceutical composition of any one of claims 19-24 for use in the treatment of muscle atrophy.

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