Processes for preparing nanoparticles

Abstract

The present disclosure provides methods for preparing nanoparticles comprising a polymer (e.g., PLGA) and a therapeutics agent (e.g., dibenzazepine), the nanoparticles prepared by such methods, as well as their uses.

Description

PROCESSES FOR PREPARING NANOPARTICLESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 662,551, filed June 21, 2024, the entire contents of which are incorporated by reference herein for all purposes.BACKGROUND

[0002] The obesity epidemic has posed a major concern for public health in modem society due to its association with a spectrum of metabolic diseases including type 2 diabetes (T2D), heart diseases, hyperglycemia and multiple cancers. Obesity is morphologically characterized by the excessive lipid storage in the white adipose tissue (WAT) and featured as a complex disorder of energy imbalance wherein intake outpaces expenditure. Considerable efforts have been devoted to exploring the pathological mechanisms as well as the cellular and molecular therapeutic targets to combat obesity.

[0003] Currently, there are few medications available for obesity treatment. Mostly, those therapies are dedicated to decreasing energy intake by either suppressing appetite through stimulating the central nervous system or reducing nutrient digestion and absorption within the gastrointestinal tract. However, those medications only produce modest effects and are usually accompanied with unpleasant, potentially harmful side effects. Thus, alternative approaches to overcoming obesity are under extensive exploration. One such approach is through boosting energy expenditure. Recently, this strategy through stimulating thermogenesis has been emerging as an appealing alternative.

[0004] Brown adipose tissue (BAT) is the primary site of thermogenesis through uncoupling mitochondrial respiration and ATP synthesis via uncoupling protein 1 (Ucpl). While producing heat, BAT consumes not only free fatty acid but also large amounts of glucose, thus providing benefits to metabolic health. Classical BAT is abundant in neonates but diminishes with age leading to limited quantities in adult humans. This compromises the therapeutic value of BAT in clinical intervention of obesity.

[0005] The recent discovery of inducible BAT (iBAT), also called beige adipose tissue, in WAT depots reignites the promise of obesity treatment through stimulating energy expenditure. Beige adipocytes also possess robust thermogenic capacity.

[0006] Current strategies for enhancement of beige adipocyte biogenesis and function involve genetic and systemic pharmacological manipulations, which are associated with significant translational challenges due to the risks of having unintended adverse consequences on other cells / tissues, and lack of control over the location and temporal extent of beige adipocyte biogenesis. Thus, there is a need to provide an effective polymer-based drug delivery system that induces beige adipocyte biogenesis in a spatio-temporally controlled manner, and methods for making such systems.SUMMARY

[0007] In some aspects, the present disclosure provides methods for preparing a nanoparticle composition, comprising: i) a precipitation step, comprising: i-a) mixing an organic solution comprising a polymer and a therapeutic agent, with an aqueous solution, thereby forming an intermediate mixture; and ii-b) removing at least a portion of a solvent from the intermediate mixture, thereby forming an intermediate nanoparticle suspension comprising a nanoparticle comprising the polymer and the therapeutic agent.

[0008] In some embodiments, the method further comprises: ii) a modifying step, comprising: ii-a) isolating the nanoparticle from the intermediate nanoparticle suspension, thereby obtaining an isolated nanoparticle; and ii-b) mixing the isolated nanoparticle with a cryoprotectant, thereby forming a prelyophilized nanoparticle suspension.

[0009] In some embodiments, the method further comprises: iii) a lyophilization step, comprising: iii-a) lyophilizing the pre-lyophilized nanoparticle suspension, thereby forming a lyophilized nanoparticle formulation.

[0010] In some embodiments, the method further comprises: iv) a reconstitution step, comprising: iv-a) adding an aqueous media to the lyophilized nanoparticle formulation, thereby forming a reconstituted nanoparticle formulation.

[0011] In some aspects, the present disclosure provides nanoparticle compositions (e.g., a nanoparticle suspension, a pre-lyophilized nanoparticle suspension, a lyophilized nanoparticleformulation, or a reconstituted nanoparticle formulation) being prepared by a method disclosed herein.

[0012] In some aspects, the present disclosure provides pharmaceutical compositions comprising a nanoparticle composition (e.g., a nanoparticle suspension, a pre-lyophilized nanoparticle suspension, a lyophilized nanoparticle formulation, or a reconstituted nanoparticle formulation) disclosed herein.

[0013] In some aspects, the present disclosure provides pharmaceutical kits comprising a nanoparticle composition (e.g., a nanoparticle suspension, a pre-lyophilized nanoparticle suspension, a lyophilized nanoparticle formulation, or a reconstituted nanoparticle formulation) disclosed herein.

[0014] In some aspects, the present disclosure provides a method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a nanoparticle composition (e.g., a nanoparticle suspension, a pre-lyophilized nanoparticle suspension, a lyophilized nanoparticle formulation, or a reconstituted nanoparticle formulation) disclosed herein.

[0015] In some aspects, the present disclosure provides nanoparticle compositions (e.g., a nanoparticle suspension, a pre-lyophilized nanoparticle suspension, a lyophilized nanoparticle formulation, or a reconstituted nanoparticle formulation) disclosed herein for use in treating or preventing a disease or disorder in a subject.

[0016] In some aspects, the present disclosure provides uses of a nanoparticle composition (e.g., a nanoparticle suspension, a pre-lyophilized nanoparticle suspension, a lyophilized nanoparticle formulation, or a reconstituted nanoparticle formulation) disclosed herein in the manufacture of a medicament for treating or preventing a disease or disorder in a subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a graph showing the size distribution of six exemplary batches of nanoparticles prepared by methods disclosed herein.DETAILED DESCRIPTIONMethods of the Present Disclosure

[0018] In some aspects, the present disclosure provides a method for preparing a nanoparticle composition, comprising: i) a precipitation step, comprising:i-a) mixing an organic solution comprising a therapeutic agent and a polymer, with an aqueous solution, thereby forming an intermediate mixture; and ii-b) removing at least a portion of a solvent from the intermediate mixture, thereby forming an intermediate nanoparticle suspension comprising a nanoparticle comprising the polymer and the therapeutic agent.

[0019] In some embodiments, the method further comprises: ii) a modifying step, comprising: ii-a) isolating the nanoparticle from the intermediate nanoparticle suspension, thereby obtaining an isolated nanoparticle; and ii-b) mixing the isolated nanoparticle with a cryoprotectant, thereby forming a prelyophilized nanoparticle suspension.

[0020] In some embodiments, the method further comprises: iii) a lyophilization step, comprising: iii-a) lyophilizing the pre-lyophilized nanoparticle suspension, thereby forming a lyophilized nanoparticle formulation.

[0021] In some embodiments, the method further comprises: iv) a reconstitution step, comprising: iv-a) adding an aqueous media to the lyophilized nanoparticle formulation, thereby forming a reconstituted nanoparticle formulation.Therapeutic Agents and Polymers

[0022] In some embodiments, the therapeutic agent is an inhibitor of the Notch signaling pathway.

[0023] In some embodiments, the therapeutic agent is capable of upregulating uncoupling protein-1 (UCP1) expression.

[0024] In some embodiments, the therapeutic agent is a y-secretase inhibitor.

[0025] In some embodiments, the therapeutic agent is a compound.

[0026] In some embodiments, the therapeutic agent is DBZ, a pharmaceutically acceptable salt thereof, a prodrug thereof, or a derivative thereof.

[0027] In some embodiments, the therapeutic agent is DBZ, a pharmaceutically acceptable salt thereof, or a prodrug thereof.

[0028] In some embodiments, the therapeutic agent is DBZ, or a pharmaceutically acceptable salt thereof.

[0029] In some embodiments, the therapeutic agent is DBZ.

[0030] It is understood that dibenzazepine (DBZ) is known as YO-01027, and has the following structure:

[0031] It is further understood that dibenzazepine (DBZ) may be described by CAS Registry No. 209984-56-5, and / or by IUPAC Name (S)-2-(2-(3,5-difhiorophenyl)acetamido)-N-((S)- 5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)propanamide.

[0032] Those of skill in the art will appreciate that dibenzazepine (DBZ) may be present in any suitable chiral form. In some embodiments, DBZ is present in the S, S form. In some embodiments, DBZ is present in the S, R form. In some embodiments, DBZ is present in the R, R form.

[0033] In some embodiments, the polymer comprises a biodegradable polymer.

[0034] In some embodiments, the polymer comprises poly (aliphatic ester) (e.g. poly(lactide) (PLA)), poly(s-caprolactone) (PCL), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(trimethylene carbonate) (PTMC), polydioxanone (PDS), poly(ortho ester), polyanhydride, poly(anhydride-co-imide), poly(anhydride-esters), polyurethanes (e.g. Degrapol), poly(glycerol sebacate), poly(ethylene imine), poly(acrylic acid) (PAA), polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(N-isopropylacrylamide) (PNIPAm), poly(oxazoline) (e.g. poly(2-methyloxazoline or poly(2-ethyl-2-oxazoline), oligo(ethylene glycol) fumarate (OPF), polypropylene fumarate), poly(alkyl cyanoacrylate), polyacrylic amide, poly(amino acid) (e.g. poly (L-glutamic acid) (L-PGA) or poly (aspartic acid)), polyphosphazene, or poly(phosphoester).

[0035] In some embodiments, the polymer comprises poly(lactic-co-glycolic acid) (PLGA).Organic and Aqueous Solutions

[0036] In some embodiments, the organic solution comprises the therapeutic agent, the polymer, and an organic solvent.

[0037] In some embodiments, the organic solvent is water-miscible.

[0038] In some embodiments, the organic solvent comprises acetone.

[0039] In some embodiments, the therapeutic agent is present in the organic solution at a concentration of about 0.10±0.05% w / v, about 0.10±0.04% w / v, about 0.10±0.03% w / v, about 0.10±0.0.2% w / v, or about 0.10±0.01% w / v (e.g., about 0.10% w / v).

[0040] In some embodiments, the polymer is present in the organic solution at a concentration of about 2.0±1.0% w / v, about 2.0±0.5% w / v, about 2.0±0.4% w / v, about 2.0±0.3% w / v, or about 2.0±0.2% w / v (e.g., about 2.0% w / v).

[0041] In some embodiments, the organic solution is prepared by mixing a solution of the polymer in the organic solvent, with a solution of the therapeutic agent in the organic solvent.

[0042] In some embodiments, the organic solution is prepared by adding the therapeutic agent to a solution of the polymer in the organic solvent.

[0043] In some embodiments, the organic solution is prepared by adding the polymer to a solution of the therapeutic agent in the organic solvent.

[0044] In some embodiments, the organic solution is prepared by adding the therapeutic agent and the polymer to the organic solvent.

[0045] In some embodiments, the aqueous solution comprises water (e.g., deionized water).

[0046] In some embodiments, the aqueous solution comprises water (e.g., deionized water) and a surfactant.

[0047] In some embodiments, the surfactant is an alcohol.

[0048] In some embodiments, the alcohol is polyvinyl alcohol (PVA).

[0049] In some embodiments, the alcohol is present in the aqueous solution at a concentration of about 0.5±0.3% w / v, about 0.5±0.2% w / v, about 0.5±0.1% w / v, or about 0.5±0.05% w / v (e.g., about 0.5% w / v).The Precipitation Step

[0050] In some embodiments, the precipitation step comprises i-a) mixing an organic solution comprising a therapeutic agent and a polymer with an aqueous solution, thereby forming an intermediate mixture.

[0051] In some embodiments, the mixing is performed at a temperature of about 25 °C±5 °C, about 25 °C±4 °C, about 25 °C±3 °C, about 25 °C±2 °C, about 25 °C±1 °C (e.g., about 25 °C).

[0052] In some embodiments, the mixing comprises adding the organic solution to the aqueous solution.

[0053] In some embodiments, the mixing comprises adding the organic solution to the aqueous solution at a rate of about 1.0±0.8 mL / min, about 1.0±0.7 mL / min, about 1.0±0.6mL / min, about 1.0±0.5 mL / min, about 1.0±0.4 mL / min, about 1.0±0.3 mL / min, about 1.0±0.2 mL / min, about 1.0±0.1 mL / min (e.g., about 1.0 mL / min).

[0054] In some embodiments, the precipitation step further comprises i-b) removing at least a portion of the solvent from the intermediate mixture, thereby forming an intermediate nanoparticle suspension comprising a nanoparticle comprising the polymer and the therapeutic agent.

[0055] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the solvent is removed.

[0056] In some embodiments, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, or greater than 99% of the solvent is removed.

[0057] In some embodiments, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, less than 95%, or less than 99% of the solvent is removed.

[0058] Various combinations of the above-referenced ranges are also contemplated. For example, in some embodiments, ranging from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 40%, from about 10% to about 50%, from about 10% to about 60%, from about 10% to about 70%, from about 10% to about 80%, from about 10% to about 90%, from about 10% to about 95%, or from about 10% to about 99% of the solvent is removed.Intermediate Nanoparticle Suspensions

[0059] In some embodiments, the intermediate nanoparticle suspension comprises a nanoparticle comprising the polymer and the therapeutic agent.

[0060] In some embodiments, the polymer and the therapeutic agent are present in the intermediate nanoparticle suspension at mass ratio of about 5:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, or about 50:1.

[0061] In some embodiments, the polymer and the therapeutic agent are present in the intermediate nanoparticle suspension at mass ratio of greater than 5:1, greater than 10:1, greater than 15:1, greater than 20:1, greater than 25:1, greater than 30:1, greater than 35:1, greater than 40: 1 , greater than 45 : 1 , or greater than 50:1.

[0062] In some embodiments, the polymer and the therapeutic agent are present in the intermediate nanoparticle suspension at mass ratio of less than 5:1, less than 10:1, less than15:1, less than 20:1, less than 25:1, less than 30:1, less than 35:1, less than 40:1, less than 45:1, or less than 50:1.

[0063] Various combinations of the above-referenced ranges are also contemplated. For example, in some embodiments, the polymer and the therapeutic agent are present in the intermediate nanoparticle suspension at mass ratio ranging from about 5:1 to about 50:1, from about 5:1 to about 45:1, from about 5:1 to about 40:1, from about 5:1 to about 35:1, from about 5:1 to about 30:1, from about 5:1 to about 25:1, from about 5:1 to about 20:1, from about 5:1 to about 15:1, or from about 5:1 to about 10:1.

[0064] In some embodiments, the intermediate nanoparticle suspension further comprises a surfactant.The Modifying Step

[0065] In some embodiments, the modifying step comprises ii-a) isolating the nanoparticle from the intermediate nanoparticle suspension, thereby obtaining an isolated nanoparticle.

[0066] In some embodiments, isolating the nanoparticle from the intermediate nanoparticle suspension comprises centrifuging the intermediate nanoparticle suspension.

[0067] In some embodiments, centrifuging is performed at about 4°C±4°C, about 4°C±3°C, about 4°C±2°C, or about 4°C±1°C (e.g., 4°C).

[0068] In some embodiments, the modifying step further comprises ii-b) mixing the isolated nanoparticle with a cryoprotectant, thereby forming a pre-lyophilized nanoparticle suspension.

[0069] In some embodiments, the mixing comprises suspending the isolated nanoparticle in an aqueous solution comprising a cryoprotectant, thereby forming the pre-lyophilized nanoparticle suspension.

[0070] In some embodiments, the cryoprotectant comprises a polyol (e.g., a diol or a triol such as propylene glycol (i.e., 1 ,2-propanediol), 1,3 -propanediol, glycerol, (+ / -)-2-methyl- 2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a nondetergent sulfobetaine (e.g., NDSB-201 (3 -(l-pyridino)-l -propane sulfonate), an osmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K 15), pentaerythritol propoxylate, or polypropylene glycol P 400), an organic solvent (e.g., dimethyl sulfoxide (DMSO) orethanol), a sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, or D-(+)-glucose monohydrate), or a salt (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof) or any combination thereof.

[0071] In some embodiments, the cryoprotectant comprises sucrose.

[0072] In some embodiments, the cryoprotectant is present in the pre-lyophilized nanoparticle suspension at an amount of about 5 w / v% ± 4 w / v%, about 5 w / v% ± 3 w / v%, about 5 w / v% ± 2 w / v%, or about 5 w / v% ± 1 w / v% (e.g., about 5 w / v%).

[0073] In some embodiments, the polymer and the therapeutic agent are present in the prelyophilized nanoparticle suspension at mass ratio of about 5:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, or about 50:1.

[0074] In some embodiments, the polymer and the therapeutic agent are present in the prelyophilized nanoparticle suspension at mass ratio of greater than 5:1, greater than 10:1, greater than 15:1, greater than 20:1, greater than 25:1, greater than 30:1, greater than 35:1, greater than 40: 1 , greater than 45 : 1 , or greater than 50:1.

[0075] In some embodiments, the polymer and the therapeutic agent are present in the prelyophilized nanoparticle suspension at mass ratio of less than 5:1, less than 10:1, less than 15:1, less than 20:1, less than 25:1, less than 30:1, less than 35:1, less than 40:1, less than 45:1, or less than 50:1.

[0076] Various combinations of the above-referenced ranges are also contemplated. For example, in some embodiments, the polymer and the therapeutic agent are present in the prelyophilized nanoparticle suspension at mass ratio ranging from about 5:1 to about 50:1, from about 5:1 to about 45:1, from about 5:1 to about 40:1, from about 5:1 to about 35:1, from about 5:1 to about 30:1, from about 5:1 to about 25:1, from about 5:1 to about 20:1, from about 5:1 to about 15:1, or from about 5:1 to about 10:1.The Lyophilization Step & Lyophilized Nanoparticle Formulations

[0077] In some embodiments, the lyophilization step comprises lyophilizing the prelyophilized nanoparticle suspension, thereby forming a lyophilized nanoparticle formulation.

[0078] In some embodiments, lyophilizing the pre-lyophilized nanoparticle suspension is performed for about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, or about 48 hours.

[0079] In some embodiments, lyophilizing the pre-lyophilized nanoparticle suspension is performed for at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours, at least about 42 hours, or at least about 48 hours.

[0080] In some embodiments, the polymer and the therapeutic agent are present in the lyophilized nanoparticle formulation at mass ratio of about 5:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, or about 50:1.

[0081] In some embodiments, the polymer and the therapeutic agent are present in the lyophilized nanoparticle formulation at mass ratio of greater than 5:1, greater than 10:1, greater than 15:1, greater than 20:1, greater than 25:1, greater than 30:1, greater than 35:1, greater than 40: 1 , greater than 45 : 1 , or greater than 50:1.

[0082] In some embodiments, the polymer and the therapeutic agent are present in the lyophilized nanoparticle formulation at mass ratio of less than 5:1, less than 10:1, less than 15:1, less than 20:1, less than 25:1, less than 30:1, less than 35:1, less than 40:1, less than 45:1, or less than 50:1.

[0083] Various combinations of the above-referenced ranges are also contemplated. For example, in some embodiments, the polymer and the therapeutic agent are present in the lyophilized nanoparticle formulation at mass ratio ranging from about 5:1 to about 50:1, from about 5:1 to about 45:1, from about 5:1 to about 40:1, from about 5:1 to about 35:1, from about 5:1 to about 30:1, from about 5:1 to about 25:1, from about 5:1 to about 20:1, from about 5:1 to about 15:1, or from about 5:1 to about 10:1.The Reconstitution Step & Reconstituted Nanoparticle Formulations

[0084] In some embodiments, the reconstitution step comprises adding an aqueous media to the lyophilized nanoparticle formulation, thereby forming a reconstituted nanoparticle formulation.

[0085] In some embodiments, the aqueous media has a pH of about 3.6, about 3.8, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.2, about 8.4, about 8.6, about 8.8, or about 9.0.

[0086] In some embodiments, the aqueous media has a pH of great than 3.6, great than 3.8, great than 4.0, great than 4.2, great than 4.4, great than 4.6, great than 4.8, great than 5.0, great than 5.2, great than 5.4, great than 5.6, great than 5.8, great than 6.0, great than 6.2, great than 6.4, great than 6.6, great than 6.8, great than 7.0, great than 7.2, great than 7.4,great than 7.6, great than 7.8, great than 8.0, great than 8.2, great than 8.4, great than 8.6, great than 8.8, or great than 9.0.

[0087] In some embodiments, the aqueous media has a pH of less than 3.6, less than 3.8, less than 4.0, less than 4.2, less than 4.4, less than 4.6, less than 4.8, less than 5.0, less than 5.2, less than 5.4, less than 5.6, less than 5.8, less than 6.0, less than 6.2, less than 6.4, less than 6.6, less than 6.8, less than 7.0, less than 7.2, less than 7.4, less than 7.6, less than 7.8, less than 8.0, less than 8.2, less than 8.4, less than 8.6, less than 8.8, or less than 9.0.

[0088] Various combinations of the above-referenced ranges are also contemplated. For example, in some embodiments, the aqueous media has a pH ranging from about 3.6 to about 9.0, about 3.6 to about 8.8, about 3.6 to about 8.6, about 3.6 to about 8.4, about 3.6 to about8.2, about 3.6 to about 8.0, about 3.6 to about 7.8, about 3.6 to about 7.6, about 3.6 to about7.4, about 3.6 to about 7.2, about 3.6 to about 7.0, about 3.6 to about 6.8, about 3.6 to about6.6, about 3.6 to about 6.4, about 3.6 to about 6.2, about 3.6 to about 6.0, about 3.6 to about5.8, about 3.6 to about 5.6, about 3.6 to about 5.4, about 3.6 to about 5.2, about 3.6 to about5.0, about 3.6 to about 4.8, about 3.6 to about 4.6, about 3.6 to about 4.4, about 3.6 to about4.2, about 3.6 to about 4.0, or about 3.6 to about 3.8.

[0089] In some embodiments, the aqueous media is a buffer (e.g., a tris buffer, a phosphate buffer, an acetate buffer, a citrate buffer, water, Ringer's solution, or sodium chloride solution).

[0090] In some embodiments, the polymer and the therapeutic agent are present in the reconstituted nanoparticle formulation at mass ratio of about 5:1, about 10:1, about 15:1, about 20: 1 , about 25: 1 , about 30:1, about 35:1, about 40: 1 , about 45: 1 , or about 50:1.

[0091] In some embodiments, the polymer and the therapeutic agent are present in the reconstituted nanoparticle formulation at mass ratio of greater than 5:1, greater than 10:1, greater than 15:1, greater than 20:1, greater than 25:1, greater than 30:1, greater than 35:1, greater than 40: 1 , greater than 45 : 1 , or greater than 50:1.

[0092] In some embodiments, the polymer and the therapeutic agent are present in the reconstituted nanoparticle formulation at mass ratio of less than 5:1, less than 10:1, less than 15:1, less than 20:1, less than 25:1, less than 30:1, less than 35:1, less than 40:1, less than 45:1, or less than 50:1.

[0093] Various combinations of the above-referenced ranges are also contemplated. For example, in some embodiments, the polymer and the therapeutic agent are present in the reconstituted nanoparticle formulation at mass ratio ranging from about 5:1 to about 50:1, from about 5:1 to about 45:1, from about 5:1 to about 40:1, from about 5:1 to about 35:1,from about 5:1 to about 30:1, from about 5:1 to about 25:1, from about 5:1 to about 20:1, from about 5:1 to about 15:1, or from about 5:1 to about 10:1.Pharmaceutical Compositions and Kits

[0094] In some embodiments, the nanoparticle described herein is combined with a pharmaceutically suitable or acceptable carrier (also referred to herein as a pharmaceutically suitable (or acceptable) excipient, physiologically suitable (or acceptable) excipient, or physiologically suitable (or acceptable) carrier) selected on the basis of a chosen route of administration and standard pharmaceutical practice as described, for example, in Remington: The Science and Practice of Pharmacy (Gennaro, 21stEd. Mack Pub. Co., Easton, PA (2005)).

[0095] Accordingly, the present disclosure provides pharmaceutical compositions comprising a nanoparticle described herein, or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof, and a pharmaceutically acceptable excipient.

[0096] In some aspects, the present disclosure provides a nanoparticle composition (e.g., a nanoparticle suspension, a pre-lyophilized nanoparticle suspension, a lyophilized nanoparticle formulation, or a reconstituted nanoparticle formulation) being prepared by a method disclosed herein.

[0097] In some aspects, the present disclosure provides a pharmaceutical composition comprising a nanoparticle composition (e.g., a nanoparticle suspension, a pre-lyophilized nanoparticle suspension, a lyophilized nanoparticle formulation, or a reconstituted nanoparticle formulation) disclosed herein.

[0098] In some aspects, the present disclosure provides a pharmaceutical kit comprising a nanoparticle composition e.g., a nanoparticle suspension, a pre-lyophilized nanoparticle suspension, a lyophilized nanoparticle formulation, or a reconstituted nanoparticle formulation) disclosed herein.

[0099] Pharmaceutical compositions are administered in a manner appropriate to the disease to be treated (or prevented). An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provides the composition(s) in an amount sufficient to provide therapeutic and / or prophylactic benefit (e.g., an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and / or overall survival, or a lessening of symptom severity. Optimal dosesare generally determined using experimental models and / or clinical trials. The optimal dose depends upon the body mass, weight, or blood volume of the patient.

[0100] In some embodiments, the pharmaceutical composition is formulated for oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, intrapulmonary, intradermal, intrathecal and epidural and intranasal administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intravenous injection, oral administration, inhalation, nasal administration, topical administration, or ophthalmic administration. In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition is formulated for intravenous injection. In some embodiments, the pharmaceutical composition is formulated as a liquid, an inhalant, a nasal spray solution, a suppository, a suspension, a gel, a colloid, a dispersion, a suspension, a solution, an emulsion, an ointment, a lotion, an eye drop, or an ear drop.Methods of Use

[0101] In some aspects, the present disclosure provides a method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a nanoparticle composition (e.g., a nanoparticle suspension, a pre-lyophilized nanoparticle suspension, a lyophilized nanoparticle formulation, or a reconstituted nanoparticle formulation) disclosed herein.

[0102] In some aspects, the present disclosure provides a nanoparticle composition (e.g., a nanoparticle suspension, a pre-lyophilized nanoparticle suspension, a lyophilized nanoparticle formulation, or a reconstituted nanoparticle formulation) disclosed herein for treating or preventing a disease or disorder in a subject.

[0103] In some aspects, the present disclosure provides a use of a nanoparticle composition (e.g., a nanoparticle suspension, a pre-lyophilized nanoparticle suspension, a lyophilized nanoparticle formulation, or a reconstituted nanoparticle formulation) disclosed herein in the manufacture of a medicament for treating or preventing a disease or disorder in a subject.

[0104] In some embodiments, the disease or disorder is obesity, type II diabetes, insulin resistance, prediabetes, hyperlipidemia, fatty liver disease optionally nonalcoholic steatohepatitis (NASH), cardiovascular disease, atherosclerosis, hypertension, hyperinsulinemia, hypoinsulinemia, hyperphagia, endocrine abnormalities, triglyceridestorage disease, Bardet-Biedl syndrome, Lawrence-Moon syndrome, or Prader-Labhart-Willi syndrome.

[0105] In some embodiments, the disease or disorder is obesity.

[0106] In some embodiments, the disease or disorder is central obesity (i.e., abdominal obesity).

[0107] In certain embodiments, the subject is a mammal.

[0108] In certain embodiments, the subject is a human.Definitions

[0109] “Pharmaceutically acceptable” means approved or approvable by a regulatory agency of the Federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans.

[0110] “Pharmaceutically acceptable salt” refers to a salt of a compound of the disclosure that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic may be inorganic or organic acid addition salts and base addition salts. Specifically, such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethane-disulfonic acid, 2- hydroxyethanesulfonic acid, benzenesulfonic acid, chlorobenzenesulfonic acid, 2- naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2]-oct-2-ene-l-carboxylic acid, glucoheptonic acid , 3 -phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid , gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion , an alkaline earth ion , or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N- methylglucamine and the like. Salts further include, by way of example only, sodium potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the compound contains a basic functionality, salts of nontoxic organic or inorganic acids,such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

[0111] The term “pharmaceutically acceptable cation” refers to an acceptable cationic counterion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like (see, e.g., Berge, et al., J. Pharm. Sci. 66 (1): 1-79 (January 77).

[0112] “Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient or carrier with which a compound of the disclosure is administered.

[0113] “Pharmaceutically acceptable metabolically cleavable group” refers to a group which is cleaved in vivo to yield the parent molecule of the structural formula indicated herein. Examples of metabolically cleavable groups include -COR, -COOR, -CONR2 and -CH2OR radicals, where R is selected independently at each occurrence from alkyl, trialkylsilyl, carbocyclic aryl or carbocyclic aryl substituted with one or more of alkyl, halogen, hydroxy or alkoxy. Specific examples of representative metabolically cleavable groups include acetyl, methoxycarbonyl, benzoyl, methoxymethyl and trimethylsilyl groups.

[0114] The term "prodrug," as used in this disclosure, means a compound which is convertible in vivo by metabolic means (e.g., by hydrolysis) to a disclosed compound.

[0115] Since prodrugs may enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.), the compounds of the present disclosure (e.g., a compound of any of the formulae or any individual compounds disclosed herein), or pharmaceutically acceptable salts, solvates, stereoisomers, or tautomers thereof can be delivered in prodrug form. Thus, the present disclosure is intended to cover prodrugs of a compound of the present disclosure (e.g., a compound of any of the formulae or any individual compounds disclosed herein), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, methods of delivering the same and compositions containing the same. "Prodrugs" are intended to include any covalently bonded carriers that release an active parent drug of the present disclosure in vivo when such prodrug is administered to a mammalian subject. Prodrugs are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Prodrugs include compounds of the disclosure wherein a hydroxyl or amino, group is bonded to any group that, when the prodrug of the present disclosure is administered to a mammalian subject, it cleaves to form a free hydroxyl or free amino group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups in thecompounds of each of the formulae described herein or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof.

[0116] A “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g. infant, child, adolescent) or an adult subject (e.g., young adult, middle aged adult or senior adult) and / or a non-human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal.

[0117] An “effective amount” means the amount of a compound that, when administered to a subject for treating or preventing a disease, is sufficient to affect such treatment or prevention. The “effective amount” can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated. A “therapeutically effective amount” refers to the effective amount for therapeutic treatment. A “prophylatically effective amount” refers to the effective amount for prophylactic treatment.

[0118] “Preventing”, “prevention” or “prophylactic treatment” refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject not yet exposed to a disease-causing agent, or in a subject who is predisposed to the disease in advance of disease onset).

[0119] The term “prophylaxis” is related to “prevention,” and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Non limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization, and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.

[0120] “Treating” or “treatment” or “therapeutic treatment” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting the disease or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment, “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physicalparameter), or both. In a further embodiment, “treating” or “treatment” relates to slowing the progression of the disease.

[0121] The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability or within statistical experimental error, and thus the number or numerical range, in some instances, will vary between 1% and 15% of the stated number or numerical range. In certain embodiments, the number or numerical range vary by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of the stated number or numerical range. In certain embodiments, the number or numerical range vary by 1%, 2%, 3%, 4%, or 5% of the stated number or numerical range. In certain embodiments, the number or numerical range vary by 1%, 2%, or 3% of the stated number or numerical range.

[0122] The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, “consist of’ or “consist essentially of’ the described features.

[0123] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” may refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0124] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but notboth”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0125] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) may refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0126] While the present teachings have been described in conjunction with various embodiments and examples, it is not intended that the present teachings be limited to such embodiments or examples. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

[0127] While various inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventiveembodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0128] The claims should not be read as limited to the described order or elements unless stated to that effect. It should be understood that various changes in form and detail may be made by one of ordinary skill in the art without departing from the spirit and scope of the appended claims. All embodiments that come within the spirit and scope of the following claims and equivalents thereto are claimed.EXAMPLESExample 1: Nanoparticle Generation via Nanoprecipitation

[0129] Organic phase and Water phase: Organic phase that includes 100 mg PLGA R.G504 (from Sigma), 5 mg dibenzazepine, and 5 mL acetone; and 100 mL of 0.5% PVA in DI water (water phase) were prepared.

[0130] Process: Injection of organic phase was injected into the water phase with a flow rate of 1.25 mL / min, which leads to nanoparticle formation. Stirring has been maintained at 600 RPM for 6 hours.Example 2: Nanoparticle Freeze Drying and Storage

[0131] Centrifugation and Resuspension: Centrifugation was performed at 13,500 RPM at 4 °C for 45 min using Superspeed centrifuges (Beckman Avanti J-E). The nanoparticles were resuspended with 5% sucrose (cryoprotectant).

[0132] Freeze Drying and Storage: The resuspended NPs were transferred to a 4 °C fridge for 30 min; then -20 °C for 30 min, and finally, to -80 °C for overnight. These steps were taken to gradually reduce the temperature of the resuspended NPs to avoid inducing thermal shock to the NPs. The next day, the process of freeze drying the resuspended NPs started and was continued for 48 hours. The freeze-drying conditions: 0.01 mBar and room temperature (21 °C). The temperature of the condenser was at -56 °C. The freeze-dried NPs were stored at -20 °C for future use.Example 3: Nanoparticle Characterization

[0133] Nanoparticle Size was characterized with a random selection among 40 exemplary batches. Results are summarized in Table 1 below.Table 1

[0134] Drug Loading (DL%) and Encapsulation Efficiency (EE%) was determined usingHPLC mass spec, Riley Research building, Indianapolis. Results are summarized in Table 2 below.Table 2EQUIVALENTS

[0135] The foregoing description has been presented only for the purposes of illustration and is not intended to limit the disclosure to the precise form disclosed, but by the claims appended hereto.

[0136] Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above. The implementations should not be limited to the particular limitations described. Other implementations may be possible.

[0137] While the disclosure been illustrated and described in detail in the foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only certain embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. It is intended that the scope of the present methods and apparatuses be defined by the following claims. However, it must be understood that this disclosure may be practiced otherwise than is specifically explained and illustrated without departing from its spirit or scope. It should be understood by those skilled in the art that various alternatives to the embodiments described herein may be employed in practicing the claims without departing from the spirit and scope as defined in the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method for preparing a nanoparticle composition, comprising: i) a precipitation step, comprising: i-a) mixing an organic solution comprising a polymer and a therapeutic agent, with an aqueous solution, thereby forming an intermediate mixture; and ii-b) removing at least a portion of a solvent from the intermediate mixture, thereby forming an intermediate nanoparticle suspension comprising a nanoparticle comprising the polymer and the therapeutic agent.

2. The method of claim 1, wherein the therapeutic agent is a y-secretase inhibitor.

3. The method of claim 2, wherein the therapeutic agent is dibenzazepine (DBZ).

4. The method of any one of claims 1-3, wherein the polymer comprises poly(lactide) (PLA)), poly(s-caprolactone) (PCL), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), or poly(trimethylene carbonate) (PTMC).

5. The method of claim 4, wherein the polymer comprises PLGA.

6. The method of any one of claims 1-5, wherein the aqueous solution comprises water (e.g., deionized water) and an alcohol.

7. The method of claim 6, wherein the alcohol is polyvinyl alcohol.

8. The method of any one of claims 1-7, wherein the mixing comprises adding the organic solution to the aqueous solution at a rate of about 1.0±0.8 mL / min, about 1.0±0.7 mL / min, about 1.0±0.6 mL / min, about 1.0±0.5 mL / min, about 1.0±0.4 mL / min, about 1.0±0.3 mL / min, about 1.0±0.2 mL / min, about 1.0±0.1 mL / min e.g., about 1.0 mL / min).

9. The method of any one of claims 1-8, wherein the polymer and the therapeutic agent are present in the intermediate nanoparticle suspension at mass ratio of about 5:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, or about10. The method of any one of claims 1-9, wherein the method further comprises: ii) a modifying step, comprising: ii-a) isolating the nanoparticle from the intermediate nanoparticle suspension, thereby obtaining an isolated nanoparticle; and ii-b) mixing the isolated nanoparticle with a cryoprotectant, thereby forming a pre-lyophilized nanoparticle suspension.

11. The method of claim 10, wherein isolating the nanoparticle from the intermediate nanoparticle suspension comprises centrifuging the intermediate nanoparticle suspension.

12. The method of claim 10 or 11, wherein the cryoprotectant comprises a polyol (e.g., a diol or a triol such as propylene glycol (i.e., 1,2-propanediol), 1,3-propanediol, glycerol, (+ / -)- 2-methyl-2,4-pentanediol, 1 ,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a nondetergent sulfobetaine e.g., NDSB-201 (3-(l-pyridino)-l -propane sulfonate), an osmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K 15), pentaerythritol propoxylate, or polypropylene glycol P 400), an organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), a sugar (e.g., D-(+)-sucrose, D- sorbitol, mannitol, trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol, myoinositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, or D-(+)-glucose monohydrate), or a salt (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof) or any combination thereof.

13. The method of claim 12, wherein the cryoprotectant comprises sucrose.

14. The method of any one of claims 10-13, wherein the polymer and the therapeutic agent are present in the pre-lyophilized nanoparticle suspension at mass ratio of about 5:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, or about 50:1.

15. The method of any one of claims 1-14, wherein the method further comprises: iii) a lyophilization step, comprising: iii-a) lyophilizing the pre-lyophilized nanoparticle suspension, thereby forming a lyophilized nanoparticle formulation.

16. The method of claim 15, wherein the polymer and the therapeutic agent are present in the lyophilized nanoparticle formulation at mass ratio of about 5:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, or about 50:

117. The method of any one of claims 1-16, wherein the method further comprises: iv) a reconstitution step, comprising: iv-a) adding an aqueous media to the lyophilized nanoparticle formulation, thereby forming a reconstituted nanoparticle formulation.

18. The method of claim 17, wherein the aqueous media has a pH of about 3.6, about 3.8, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.2, about 8.4, about 8.6, about 8.8, or about 9.0.

19. The method of claim 17 or 18, wherein the aqueous media is a buffer (e.g. , a tris buffer, a phosphate buffer, an acetate buffer, a citrate buffer, water, Ringer's solution, or sodium chloride solution).

20. The nanoparticle, isolated nanoparticle, lyophilized nanoparticle formulation, or reconstituted nanoparticle formulation prepared from the method of any one of claims 1-19.

21. A method for treating a disease or disorder, comprising administering to a subject the nanoparticle, isolated nanoparticle, lyophilized nanoparticle formulation, or reconstituted nanoparticle formulation of claim 20.

22. The nanoparticle, isolated nanoparticle, lyophilized nanoparticle formulation, or reconstituted nanoparticle formulation of claim 20 for use in treating a disease or disorder.

23. Use of the nanoparticle, isolated nanoparticle, lyophilized nanoparticle formulation, or reconstituted nanoparticle formulation of claim 20 in the manufacture of a medicament for treating a disease or disorder.

24. The method, use, or nanoparticle, isolated nanoparticle, lyophilized nanoparticle formulation, or reconstituted nanoparticle formulation for use of any one of claims 21-23, wherein the disease or disorder is selected from obesity, type II diabetes, insulin resistance, prediabetes, hyperlipidemia, fatty liver disease optionally nonalcoholic steatohepatitis (NASH), cardiovascular disease, atherosclerosis, hypertension, hyperinsulinemia, hypoinsulinemia, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet- Biedl syndrome, Lawrence-Moon syndrome, and Prader-Labhart- Willi syndrome.

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