Compositions and methods for localized adipocyte cell death

Abstract

Provided herein are compositions containing, e.g., particles comprising a curcuminoid and cinnamaldehyde or a derivative thereof. For example, provided herein are compositions comprising a liposome comprising curcumin and cinnamaldehyde or a derivative thereof. Such compositions can be useful for, for example, inducing cell death of adipocytes in a subject and / or reducing subcutaneous fat in a subject.

Description

Attorney Docket No.57394-0003WO1 COMPOSITIONS AND METHODS FOR LOCALIZED ADIPOCYTE CELL DEATH CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from U.S. Provisional Application No.63 / 758,920, filed on February 14, 2025, U.S. Provisional Application No. 63 / 725,301, filed on November 26, 2024, U.S. Provisional Application No. 63 / 642,435, filed on May 3, 2024, which are incorporated herein by reference in their entireties. TECHNICAL FIELD

[0002] The present disclosure relates to pharmaceutical compositions comprising a particle comprising a curcuminoid and cinnamaldehyde and / or a derivative thereof. Such compositions can be useful for, for example, inducing cell death of adipocytes in a subject and / or reducing subcutaneous fat in a subject. BACKGROUND

[0003] Surgical and non-surgical procedures for reducing fat and improving appearance are common as populations age and gain weight. For example, weight fluctuations and aging can lead to an altered distribution of subcutaneous fat that may be cosmetically undesirable and resistant to dieting and / or exercise. Liposuction is a common cosmetic surgical procedure involving the surgical removal of fat deposits using suction. However, surgical procedures are invasive, time consuming, can be complicated due to underlying anatomic structures. Furthermore, surgical procedures such as liposuction can have side effects such as bruising, fluid buildup, numbness, infection, internal puncture, fat embolisms, kidney and heart problems, and lidocaine toxicity. There is a need for alternative, non-surgical options for reducing fat and / or improving appearance. SUMMARY

[0004] Provided herein are pharmaceutical compositions comprising a particle comprising curcumin.

[0005] In some embodiments, the pharmaceutical composition further comprises cinnamaldehyde or a derivative thereof. In some embodiments, the cinnamaldehydeAttorney Docket No.57394-0003WO1 derivative comprises cinnamaldehyde, 4-hydroxy-3-methoxy cinnamaldehyde, 2-hydroxy cinnamaldehyde, or a mixture thereof. In some embodiments, the pharmaceutical composition further comprises resveratrol.

[0006] In some embodiments, the particle is a liposome. In some embodiments, the liposome encapsulates the curcumin. In some embodiments, the liposome encapsulates the cinnamaldehyde or derivative thereof. In some embodiments, the liposome encapsulates the resveratrol.

[0007] In some embodiments, the liposome comprises distearoylphosphatidylcholine (DSPC) and cholesterol. In some embodiments, the DSPC is present in an amount of about 45% to about 65% w / w of the total lipid content of the liposome. In some embodiments, the DSPC is present in an amount of about 55% w / w of the total lipid content of the liposome. In some embodiments, the cholesterol is present in an amount of about 30% to about 50% w / w of the total lipid content of the liposome. In some embodiments, the cholesterol is present in an amount of about 40% w / w of the total lipid content of the liposome.

[0008] In some embodiments, the liposome comprises 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC). In some embodiments, the liposome comprises 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and cholesterol. In some embodiments, the DMPC is present in an amount of about 75% to about 95% w / w of the total lipid content of the liposome. In some embodiments, the DMPC is present in an amount of about 80% w / w of the total lipid content of the liposome. In some embodiments, the DMPC is present in an amount of about 85% w / w of the total lipid content of the liposome. In some embodiments, the DMPC is present in an amount of about 90% w / w of the total lipid content of the liposome. In some embodiments, the DMPC is present in an amount of about 95% w / w of the total lipid content of the liposome. In some embodiments, the cholesterol is present in an amount of about 1% to about 20% w / w of the total lipid content of the liposome. In some embodiments, the cholesterol is present in an amount of about 15% w / w of the total lipid content of the liposome. In some embodiments, the cholesterol is present in an amount of about 10% w / w of the total lipid content of the liposome. In some embodiments, the cholesterol is present in an amount of about 5% w / w of the total lipid content of the liposome.Attorney Docket No.57394-0003WO1

[0009] In some embodiments, the liposome further comprises an anionic lipid or a cationic lipid. In some embodiments, the anionic lipid is 1,2-distearoyl-sn-glycero-3- phosphoglycerol sodium salt (DSPG-Na) or 1,2-dimyristoyl-sn-glycero-3- phosphoglycerol sodium salt (DMPG-Na). In some embodiments, the cationic lipid is N- [1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP-Cl). In some embodiments, the liposome further comprises DSPG-Na, DMPG-Na, DOTAP-Cl, polyethylene glycol-2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE- PEG2000), or mixtures thereof. In some embodiments, the DSPG-Na is present in an amount of about 0.1% to about 15% w / w of the total lipid content of the liposome. In some embodiments, the DSPG-Na is present in an amount of about 5% w / w of the total lipid content of the liposome. In some embodiments, the DOTAP-Cl is present in an amount of about 0.1% to about 15% w / w of the total lipid content of the liposome. In some embodiments, the DOTAP-Cl is present in an amount of about 5% w / w of the total lipid content of the liposome. In some embodiments, the DSPE-PEG2000 is present in an amount of about 0.1% to about 15% w / w of the total lipid content of the liposome. In some embodiments, the DSPE-PEG2000 is present in an amount of about 5% w / w of the total lipid content of the liposome. In some embodiments, the DMPG-Na is present in an amount of about 1% to about 20% w / w of the total lipid content of the liposome. In some embodiments, the DMPG-Na is present in an amount of about 1% to about 10% w / w of the total lipid content of the liposome. In some embodiments, the DMPG-Na is present in an amount of about 10% w / w of the total lipid content of the liposome. In some embodiments, the DMPG-Na is present in an amount of about 5% w / w of the total lipid content of the liposome.

[0010] In some embodiments, the molar ratio of the curcumin to lipid is about 1:10 to about 1:14. In some embodiments, the molar ratio of the curcumin to lipid is about 1:12. In some embodiments, the molar ratio of the curcumin and cinnamaldehyde or derivative thereof to lipid is about 1:10 to about 1:14. In some embodiments, the molar ratio of the curcumin and cinnamaldehyde or derivative thereof to lipid is about 1:12.

[0011] In some embodiments, the pharmaceutical composition comprises a liposome comprising curcumin, wherein the liposome encapsulates the curcumin. In some embodiments, the liposome comprises a neutral phospholipid and an anionic lipid. In someAttorney Docket No.57394-0003WO1 embodiments, the neutral phospholipid comprises DMPC and the anionic lipid comprises DMPG-Na. In some embodiments, the DMPC is present in an amount of about 85% to about 95% w / w of the total lipid content of the liposome and the DMPG-Na is present in an amount of about 1% to about 20% w / w of the total lipid content of the liposome. In some embodiments, the DMPC is present in an amount of about 90% w / w of the total lipid content of the liposome. In some embodiments, the DMPG-Na is present in an amount of about 10% w / w of the total lipid content of the liposome. In some embodiments, the DMPC and DMPG-Na are present at a molar ratio of about 90:10 DMPC:DMPG-Na. In some embodiments, the molar ratio of curcumin to lipid is about 1:10 to about 1:14. In some embodiments, the molar ratio of the curcumin to lipid is about 1:12.

[0012] In some embodiments, the liposome further comprises cinnamaldehyde. In some embodiments, the cinnamaldehyde is present at a weight ratio of about 1:6 to about 1:3 cinnamaldehyde:curcumin. In some embodiments, the cinnamaldehyde is present at a weight ratio of about 1:5 cinnamaldehyde:curcumin. In some embodiments, the molar ratio of the curcumin and cinnamaldehyde to lipid is about 1:10 to about 1:14. In some embodiments, the molar ratio of the curcumin and cinnamaldehyde to lipid is about 1:12.

[0013] In some embodiments, the size of the particle is about 20 nm to about 600 nm. In some embodiments, the size of the particle is about 20 nm to about 200 nm. In some embodiments, the size of the particle is about 50 nm to about 100 nm. In some embodiments, the size of the particle is about 50 nm to about 70 nm. In some embodiments, the size of the particle is about 100 nm. In some embodiments, the size of the particle is about 60 nm.

[0014] Also provided herein is a pharmaceutical composition comprising a liposome comprising curcumin present at a molar ratio of about 1:10 to about 1:14 curcumin:lipid.

[0015] In some embodiments, the liposome comprises a neutral phospholipid and an anionic lipid.

[0016] In some embodiments, the neutral phospholipid is present in an amount of about 88% to about 96% w / w of the total lipid content of the liposome. In some embodiments, the neutral phospholipid is present in an amount of about 90% to about 94% w / w of the total lipid content of the liposome.Attorney Docket No.57394-0003WO1

[0017] In some embodiments, the neutral phospholipid comprises distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC), and 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dierucoyl-sn-glycero-3- phosphocholine (DEPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or a combination thereof. In some embodiments, the neutral phospholipid comprises DMPC.

[0018] In some embodiments, the anionic phospholipid comprises 1,2-dipalmitoyl-sn- glycero-3-phosphoglycerol sodium salt (DPPG-Na), 1,2-dimyristoyl-sn-glycero-3- phosphoglycerol sodium salt (DMPG-Na), 1,2-distearoyl-sn-glycero-3-phosphoglycerol sodium salt (DSPG-Na), or a combination thereof.

[0019] In some embodiments, the neutral phospholipid and anionic phospholipid are present at a molar ratio of about 92:8 neutral phospholipid:anionic phospholipid.

[0020] In some embodiments, the neutral phospholipid comprises DMPC and the anionic lipid comprises DMPG-Na. In some embodiments, the DMPC is present in an amount of about 88% to about 96% w / w of the total lipid content of the liposome and the DMPG-Na is present in an amount of about 6% to about 12% w / w of the total lipid content of the liposome. In some embodiments, the DMPC and DMPG-Na are present at a molar ratio of about 92:8 DMPC:DMPG-Na.

[0021] In some embodiments, the molar ratio of the curcumin to lipid is about 1:12.

[0022] In some embodiments, the size of the liposome is about 50 nm to about 100 nm. In some embodiments, the size of the liposome is about 80 nm.

[0023] In some embodiments, the pharmaceutical composition comprises an acetate buffer. In some embodiments, the pharmaceutical composition comprises sodium chloride. In some embodiments, the pharmaceutical composition has a pH of about 5. In some embodiments, the pharmaceutical composition comprises 10 mM acetate buffer and 0.9% w / w NaCl.

[0024] In some embodiments, the pharmaceutical composition has a PDI of about 0.1. In some embodiments, the pharmaceutical composition has a Z-average of about -40 mV.

[0025] In some embodiments, the liposome encapsulates the curcumin.Attorney Docket No.57394-0003WO1

[0026] In some embodiments, the curcumin comprises at least about 99% by weight of the total curcuminoids present in the pharmaceutical composition.

[0027] Also provided herein are methods for inducing cell death of a plurality of adipocytes in a treatment area in a subject, the method comprising administering to the subject an effective amount of any of the pharmaceutical compositions described herein. In some embodiments, the cell death is due to apoptosis and / or necrosis. In some embodiments, the cell death is due to apoptosis.

[0028] Also provided herein are methods for inhibiting adipogenesis in a treatment area in a subject, the method comprising administering to the subject an effective amount of any of the pharmaceutical compositions described herein.

[0029] Also provided herein are methods of reducing subcutaneous fat in a treatment area in a subject, the method comprising administering to the subject an effective amount of any of the pharmaceutical compositions described herein.

[0030] In some embodiments, the method comprises inducing apoptosis of a plurality of adipocytes in the treatment area in the subject.

[0031] Also provided herein are methods of improving the appearance of convexity or fullness associated with fat tissue in a subject, the method comprising administering to the subject an effective amount of any of the pharmaceutical compositions described herein.

[0032] In some embodiments, the treatment area is in the abdomen, a flank, an inner thigh, an outer thigh, jowls, lower face, an arm, buttocks, a calf, breast, chest, or the back of the subject. In some embodiments, the treatment area is in the back of the arm. In some embodiments, the treatment area is in the submental area, jowls, or buccal area of the lower face.

[0033] In some embodiments,the treatment area in the subject has cellulite, a lipoma, or liposarcoma. In some embodiments, the treatment area in the subject has a subcutaneous fat deposit associated with temporal fat pad hyperplasia, pseudo herniation of orbital fat, Graves’ disease, Dercum's disease, multiple hereditary lipomatosis, familial multiple lipomatosis, Gardner syndrome, adiposis dolorosa, PTEN hamartoma tumor syndrome, Cowden syndrome, Madelung disease, or fatty infiltration.

[0034] In some embodiments, the pharmaceutical composition is locally administered to the treatment area in the subject. In some embodiments, the pharmaceutical compositionAttorney Docket No.57394-0003WO1 is locally administered to the treatment area in the subject via injection. In some embodiments, the pharmaceutical composition is administered to the treatment area in the subject via at least two injections. In some embodiments, the pharmaceutical composition is administered to the treatment area in the subject via at least 5 injections. In some embodiments, the pharmaceutical composition is locally administered to the treatment area in the subject via a cannula.

[0035] In some embodiments, the pharmaceutical composition is administered to one or more sites surrounding the treatment area in the subject. In some embodiments, the pharmaceutical composition is administered to two or more sites in the treatment area in the subject. In some embodiments, the pharmaceutical composition is administered to two or more sites surrounding the treatment area.

[0036] Definitions

[0037] In some embodiments, the term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%.

[0038] The term “pharmaceutical composition” refers to a mixture of a compound described herein with other chemical components (referred to collectively herein as “excipients”), such as carriers, stabilizers, diluents, dispersing agents, suspending agents, and / or thickening agents. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to: rectal, oral, intravenous, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

[0039] The term “excipient” or “pharmaceutically acceptable excipient” refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, carrier, or solvent. See, e.g., Remington: The Science and Practice of Pharmacy, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rdAttorney Docket No.57394-0003WO1 ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009.

[0040] The term “effective amount” as used herein, refers to a sufficient amount of a chemical entity (e.g., a compound exhibiting apoptotic activity in an adipocyte) being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result includes reduction and / or alleviation of the signs, symptoms, or causes of a disease or condition, or any other desired alteration of a biological system. For example, an “effective amount” is the amount of the composition comprising a curcuminoid as disclosed herein required to provide a measurable reduction in fat and / or a visible improvement in appearance. An appropriate “effective” amount in any individual case is determined using any suitable technique, such as a dose escalation study or a study that assesses dose relative to subcutaneous fat quantity. For example, the dose of an active pharmaceutical ingredient (API) may be increased for various quantities of subcutaneous fat until the dose that results in the highest fat reduction for each different quantity of subcutaneous fat is found. Such studies can be used to estimate the optimal dose(s) of an API for different quantities of subcutaneous fat. It will be appreciated that the optimal dose may be adjusted based on other factors, e.g., to minimize one or more side effects. For example, the optimal dose can be the dose at which the highest level of fast reduction is observed for a quantity of subcutaneous fat with minimal or no side effects. Quantities of fat can be determined using, e.g., the methods described herein.

[0041] When referring to combinations of compounds (e.g., a curcuminoid as disclosed herein, the combination may be “therapeutically effective” even when one or more of the compounds in the combination is administered at a dose that would be sub-therapeutic when the compound is administered alone. Indeed, the combination of compounds, or pharmaceutically acceptable salts or solvates of the foregoing, can be an additive combination, or can be a synergistic combination. Synergy, as described, for example, by Chou and Talalay, Advances in Enzyme Regulation (1984), 22, 27-55, occurs when the effect of the compounds when administered in combination is greater than the additive effect of the compounds when administered alone as a single agent.

[0042] As used herein, “subject,” “individual,” or “patient,” are used interchangeably and refer to any animal, including mammals such as mice, rats, other rodents, rabbits, dogs,Attorney Docket No.57394-0003WO1 cats, swine, cattle, sheep, goats, horses, primates, and humans. In some embodiments, the patient is a human. In some embodiments, the subject has experienced and / or exhibited at least one symptom of the disease or disorder to be treated and / or prevented.

[0043] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As used herein, the singular form “a”, “an”, and “the” include plural references unless indicated otherwise. For example, “an” excipient includes one or more excipients. It is understood that aspects and variations of the invention described herein include “consisting of” and / or “consisting essentially of” aspects and variations.

[0044] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features and advantages will be apparent from the description and drawings, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. DESCRIPTION OF DRAWINGS

[0045] FIG.1 are representative fluorescent images of DAPI (blue), cleaved caspase- 3 (yellow) and viability dye (magenta) in a subset of compounds including: Controls (Vehicle, 10% DMSO, 0.2% Triton X-100), curcumin (150 ppm), curcumin / CAPE (150 ppm / 60 ppm), curcumin / resveratrol (150 ppm / 40 ppm), curcumin / 2- hydroxycinnamaldehyde (150 ppm / 25 ppm) and curcumin / 4-hydroxy-3- methoxycinnamaldhehyde (150 ppm / 100 ppm) treatment groups. Scale bar: 150 μm.

[0046] FIG.2 are representative fluorescent images of DAPI (blue), cleaved caspase- 3 (yellow) and viability dye (magenta) in a subset of compounds including:Attorney Docket No.57394-0003WO1 cinnamaldehyde (100, 150 and 200 ppm) and curcumin / cinnamaldehyde (150 ppm / 50 and 100 ppm) treatment groups. Scale bar: 150 μm.

[0047] FIG.3 is a graph showing results of cleaved caspase-3 labeling for controls and treatment groups at 48-hours post-compound addition in treatment groups. Data represent mean + / - standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Stars (*) alone specify comparison to the vehicle control. Stars with a bar indicate comparison between those groups.

[0048] FIG. 4 is a graph showing results of viability dye labeling for controls and treatment groups at 48-hours post-compound addition in treatment groups. Data represent mean + / - standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Stars (*) alone specify comparison to the vehicle control. Stars with a bar indicate comparison between those groups.

[0049] FIG.5 is a graph showing results of cleaved caspase-3 labeling for controls and treatment groups. *** p < 0.001.

[0050] FIG. 6 is a graph showing results of viability dye labeling for controls and treatment groups. *** p < 0.001.

[0051] FIG.7 is a graph showing results of cleaved caspase-3 labeling for controls and treatment groups. *** p < 0.001.

[0052] FIG. 8 is a graph showing results of viability dye labeling for controls and treatment groups. *** p < 0.001.

[0053] FIG. 9A is a plot showing the body weight over Days -5 to 15 for ZDF- Leprfa / Crl male rats administered Vehicle or a Test Article (TA) as indicated.

[0054] FIG.9B is a plot showing the percent change of body weight over Days 1 to 15 compared to Day 1 for ZDF-Leprfa / Crl male rats that were administered Vehicle or a Test Article (TA) as indicated.

[0055] FIG.10 is a plot showing food consumption for ZDF-Leprfa / Crl male rats that were administered Vehicle or a Test Article (TA) as indicated.

[0056] FIG.11A is a plot showing fat mass on Days -3 and 14 for ZDF-Leprfa / Crl male rats that were administered Vehicle or a Test Article (TA) as indicated.

[0057] FIG. 11B is a plot showing lean mass on Days -3 and 14 for ZDF-Leprfa / Crl male rats that were administered Vehicle or a Test Article (TA) as indicated.Attorney Docket No.57394-0003WO1

[0058] FIG.12 is schematic showing a generalized process flow diagram of the DIANT Lift Skid for the formation of curcumin liposomes.

[0059] FIG.13 is a plot showing the relationship between z-average and PDI for different ethanol purities.

[0060] FIG.14A is a plot showing the impact of cholesterol on z-average.

[0061] FIG.14B is a plot showing the impact of cholesterol on PDI.

[0062] FIG. 15A is a plot showing the body weight over Days -3 to 15 for ZDF- Leprfa / Crl male rats administered Vehicle or a Sample as indicated.

[0063] FIG.15B is a plot showing the percent change of body weight over Days 1 to 15 compared to Day 1 for ZDF-Leprfa / Crl male rats that were administered Vehicle or a Sample as indicated.

[0064] FIG.16 is a plot showing food consumption for ZDF-Leprfa / Crl male rats that were administered Vehicle or a Sample as indicated.

[0065] FIG. 17A is a plot showing inguinal fat pad weight for ZDF-Leprfa / Crl male rats that were administered Vehicle or a Sample as indicated.

[0066] FIG.17B is a plot showing % inguinal fat pad tissue weight for ZDF-Leprfa / Crl male rats that were administered Vehicle or a Sample as indicated. DETAILED DESCRIPTION

[0067] The present disclosure is based, at least in part, on compositions that can, e.g., induce adipocyte cell death. For example, provided herein are pharmaceutical compositions that contain a curcuminoid, a flavonoid, a non-flavonoid polyphenol, or a mixture thereof. Such compositions can contain particles that encapsulate one or more of the curcuminoid, flavonoid, non-flavonoid polyphenol, or a mixture thereof. For example, a pharmaceutical composition provided herein includes (i) a particle containing a curcuminoid; (ii) a particle containing a flavonoid (e.g., any of the flavonoids described herein); (iii) a particle containing a non-flavonoid polyphenol (e.g., resveratrol); (iv) a particle containing a flavonoid and resveratrol; (v) a particle containing a curcuminoid and a flavonoid, a non-flavonoid polyphenol (e.g., resveratrol), or a mixture thereof; or (vi) any mixture of particles thereof. Mixtures of such particles can include, for example, (vii) a particle containing a curcuminoid and a particle containing a flavonoid and / or a non-Attorney Docket No.57394-0003WO1 flavonoid polyphenol (e.g., resveratrol); (viii) a particle containing a flavonoid and a particle containing a non-flavonoid polyphenol (e.g., resveratrol); (ix) a particle containing a curcuminoid, a particle containing a flavonoid, and a particle containing a non-flavonoid polyphenol (e.g., resveratrol); (x) or a mixture thereof.

[0068] In some embodiments, provided herein are pharmaceutical compositions that include a particle (e.g., any of the particles described herein) containing a curcuminoid. In some embodiments, the particle further includes cinnamaldehyde, a cinnamaldehyde derivative, resveratrol or a mixture thereof.

[0069] The pharmaceutical compositions provided herein can include a plurality of particles (e.g., any of the particles or mixtures of particles described herein).

[0070] The particles of the pharmaceutical compositions provided herein can be used to deliver the curcuminoid, flavonoid, non-flavonoid polyphenol, or combination thereof to a local site in a subject. In some embodiments, the particles of the pharmaceutical compositions provided herein can be used to deliver the curcuminoid or the curcuminoid and cinnamaldehyde, cinnamaldehyde derivative, or mixture thereof to a local site in a subject.

[0071] In some embodiments, particles of a pharmaceutical composition provided herein can be used to deliver the curcuminoid, flavonoid, non-flavonoid polyphenol, or combination thereof to adipocytes in a subject. The cell surface charge of human adipocytes can play a role in cell uptake properties. See, e.g., Perry et al. J. Am. Chem. Soc. 138(9):3152-60, 2016. Abdominal subcutaneous adipose tissue cells in physiological conditions have been shown to have a net negative cell surface charge. See, e.g., Perry et al. J. Am. Chem. Soc. 138(9):3152-60, 2016. Additionally, the charge of lipids in lipid nanoparticles can affect the surface charge of the lipid nanoparticles under different pH condition, which can influence, e.g., plasma protein absorption, blood clearance, and tissue distribution. See, e.g., Immordino et al. Int. J. Nanomedicine. 1(3):297–315, 2006; Fröhlich, Int. J. Nanomedicine. 7:5577-91, 2012. Surprisingly, substantial adipocyte cell death was observed in subcutaneous fat, which is highly lipophilic, for pharmaceutical compositions described herein containing a charged liposome (e.g., a liposome containing anionic lipids). The present disclosure advantageously provides methods for reducing fat in a treatment area in a subject and / or improving the appearance of convexity or fullnessAttorney Docket No.57394-0003WO1 associated with fat tissue in a subject. Such methods can include administering to the subject an effective amount of any of the pharmaceutical compositions described herein. Pharmaceutical Compositions

[0072] A pharmaceutical composition provided herein includes a particle containing a curcuminoid. Curcuminoids are natural phenol compounds and can be isolated from turmeric. Non-limiting examples of a curcuminoid include curcumin, demethoxycurcumin, and bisdemethoxycurcumin. In some embodiments of any of the pharmaceutical compositions described herein, the curcuminoid is curcumin.

[0073] In some embodiments, a particle of a pharmaceutical composition provided herein contains a flavonoid. Non-limiting examples of flavonoids include flavanols, flavanones, flavones, and isoflavones.

[0074] Cinnamaldehyde is a naturally occurring flavonoid. Non-limiting examples of a cinnamaldehyde derivative include 4-hydroxy-3-methoxy cinnamaldehyde and 2- hydroxy cinnamaldehyde. In some embodiments, a particle of a pharmaceutical composition provided herein contains cinnamaldehyde, a cinnamaldehyde derivative, or a mixture thereof. In some embodiments, a particle of a pharmaceutical composition provided herein contains a curcuminoid and further contains cinnamaldehyde, a cinnamaldehyde derivative, or a mixture thereof.

[0075] In some embodiments, wherein a pharmaceutical composition provided herein includes a curcuminoid and cinnamaldehyde and / or a derivative thereof, the weight ratio of the cinnamaldehyde and / or derivative thereof to the curcuminoid (e.g., any of the curcuminoids described herein) is about 1:30 to about 9:1. For example, the weight ratio of the cinnamaldehyde and / or derivative thereof to the curcuminoid can be about 1:25 to about 9:1, 1:20 to about 9:1, about 1:15 to about 9:1, about 1:10 to about 9:1, 1:5 to about 9:1, 1:1 to about 9:1, about 1:30 to about 8:1, about 1:30 to about 7:1, about 1:30 to about 6:1, about 1:30 to about 5:1, about 1:30 to about 4:1, about 1:30 to about 3:1, about 1:30 to about 2:1, or about 1:30 to about 1:1. In some embodiments, the weight ratio of the cinnamaldehyde and / or derivative thereof to the curcuminoid is about 1:6 to about 1:3. In some embodiments, the weight ratio of the cinnamaldehyde and / or derivative thereof to the curcuminoid is about 1:5, about 1:6, about 1:7, about 1:8, or about 1:9.Attorney Docket No.57394-0003WO1

[0076] In some embodiments, wherein a pharmaceutical composition provided herein includes cinnamaldehyde and / or a derivative thereof, the weight ratio of the cinnamaldehyde and / or derivative thereof to the curcuminoid (e.g., any of the curcuminoids described herein) is about 99:1 to about 65:45. For example, the weight ratio of the cinnamaldehyde and / or derivative thereof to the curcuminoid can be about 75:1 to about 65:45, about 50:1 to about 65:45, about 99:3 to about 65:45, about 25:1 to about 65:45, about 50:3 to about 65:45, about 75:5 to about 65:45, about 25:3 to about 65:45, about 85:15 to about 65:45, about 25:6 to about 65:45, about 2:1 to about 65:45, about 60:40 to about 65:45, about 99:1 to about 60:40, about 75:1 to about 60:40, about 50:1 to about 60:40, about 99:3 to about 60:40, about 25:1 to about 60:40, about 50:3 to about 60:40, about 75:5 to about 60:40, about 25:3 to about 60:40, about 85:15 to about 60:40, about 25:6 to about 60:40, about 2:1 to about 60:40, about 99:1 to about 2:1, about 75:1 to about 2:1, about 50:1 to about 2:1, about 99:3 to about 2:1, about 25:1 to about 2:1, about 50:3 to about 2:1, about 75:5 to about 2:1, about 25:3 to about 2:1, about 85:15 to about 2:1, about 25:6 to about 2:1, about 99:1 to about 25:6, about 75:1 to about 25:6, about 50:1 to about 25:6, about 99:3 to about 25:6, about 25:1 to about 25:6, about 50:3 to about 25:6, about 75:5 to about 25:6, about 25:3 to about 25:6, about 85:15 to about 25:6, about 99:1 to about 85:15, about 75:1 to about 85:15, about 50:1 to about 85:15, about 99:3 to about 85:15, about 25:1 to about 85:15, about 50:3 to about 85:15, about 75:5 to about 85:15, about 25:3 to about 85:15, about 99:1 to about 25:3, about 75:1 to about 25:3, about 50:1 to about 25:3, about 99:3 to about 25:3, about 25:1 to about 25:3, about 50:3 to about 25:3, about 75:5 to about 25:3, about 99:1 to about 75:5, about 75:1 to about 75:5, about 50:1 to about 75:5, about 99:3 to about 75:5, about 25:1 to about 75:5, about 50:3 to about 75:5, about 99:1 to about 50:3, about 75:1 to about 50:3, about 50:1 to about 50:3, about 99:3 to about 50:3, about 25:1 to about 50:3, about 99:1 to about 25:1, about 75:1 to about 25:1, about 50:1 to about 25:1, about 99:3 to about 25:1, about 99:1 to about 99:3, about 75:1 to about 99:3, about 50:1 to about 99:3, about 99:1 to about 50:1, about 75:1 to about 50:1, or about 99:1 to about 50:1. In some embodiments, the weight ratio of the cinnamaldehyde and / or derivative thereof to the curcuminoid is about 85:15 to about 60:40.Attorney Docket No.57394-0003WO1

[0077] In some embodiments, a particle of a pharmaceutical composition provided herein contains a non-flavonoid polyphenol. Non-limiting examples of a non-flavonoid polyphenol include resveratrol.

[0078] In some embodiments, a particle of a pharmaceutical composition provided herein contains resveratrol. In some embodiments, a particle of a pharmaceutical composition provided herein contains a curcuminoid and further contains cinnamaldehyde, a cinnamaldehyde derivative, resveratrol, or a mixture thereof.

[0079] In some embodiments, wherein a pharmaceutical composition provided herein includes a curcuminoid and resveratrol, the weight ratio of resveratrol to a curcuminoid (e.g., any of the curcuminoids described herein) is about 1:30 to about 9:1. For example, the weight ratio of resveratrol to the curcuminoid can be about 1:25 to about 9:1, 1:20 to about 9:1, about 1:15 to about 9:1, about 1:10 to about 9:1, 1:5 to about 9:1, 1:1 to about 9:1, about 1:30 to about 8:1, about 1:30 to about 7:1, about 1:30 to about 6:1, about 1:30 to about 5:1, about 1:30 to about 4:1, about 1:30 to about 3:1, about 1:30 to about 2:1, or about 1:30 to about 1:1. In some embodiments, the weight ratio of resveratrol to the curcuminoid is about 1:5, about 1:6, about 1:7, about 1:8, or about 1:9.

[0080] The particles of a pharmaceutical composition provided herein can encapsulate the curcuminoid. For example, a particle of a pharmaceutical composition provided herein can partially or fully encapsulate the curcuminoid. In some embodiments, particles of a pharmaceutical composition provided herein encapsulate (e.g., partially or fully) the curcuminoid and cinnamaldehyde, cinnamaldehyde derivative, resveratrol, or mixture thereof. In some embodiments, a particle of a pharmaceutical composition provided herein encapsulates (e.g., partially or fully) the curcuminoid and the cinnamaldehyde, cinnamaldehyde derivative, or mixture thereof. In some embodiments, a particle of a pharmaceutical composition provided herein encapsulates (e.g., partially or fully) the curcuminoid and resveratrol.

[0081] The curcuminoid and cinnamaldehyde, cinnamaldehyde derivative, resveratrol, or mixture thereof can be co-encapsulated in a single particle of a pharmaceutical composition provided herein. Co-encapsulation of two or more active ingredients (e.g., a curcuminoid and cinnamaldehyde, cinnamaldehyde derivative, resveratrol, or mixture thereof) can decrease the difficulty of manufacturing pharmaceutical compositionsAttorney Docket No.57394-0003WO1 comprising the active ingredients and / or improve accuracy in delivering the active ingredients. Co-encapsulation of two or more active ingredients can also enhance therapeutic efficacy. For example, co-encapsulation of two or more active ingredients can increase the additive or synergistic effect exhibited by the combination of the active ingredients compared to the effect of the active ingredients when administered alone as a single agent or when administered in a single composition where the active ingredients are not co-encapsulated in particles. In some embodiments, co-encapsulated active ingredients are delivered to the target (e.g., a cell such as an adipocyte) simultaneously.

[0082] An active pharmaceutical ingredient (API) encapsulated in a particle can be present within the core of the particle and / or within one or more layers of a particle. For example, an API encapsulated in a liposome can be in the aqueous core of the liposome and / or within the lipid bilayer of the liposome. Encapsulation can be determined using any method known in the art. For example, the amount of a non-encapsulated active ingredient (e.g., a curcuminoid and / or cinnamaldehyde and / or a derivative thereof) can be determined and compared to the total amount of the active ingredient. See, e.g., J. Pharm. Biomed. Anal. 236:115751 (2023). In some embodiments, polarized light microscopy (PLM) can be used to visualize crystallization of an API (e.g., one or more of a curcuminoid, cinnamaldehyde, cinnamaldehyde derivative, and resveratrol). PLM can be used for bio, e.g., analyze crystal morphology and / or identify different crystal types. In some embodiments, PLM is used for birefringence imaging in which crystals with anisotropic structures refract light differently depending on the direction light passes through the crystal.

[0083] In some embodiments, a pharmaceutical composition provided herein contains about 41 µM to about 4.1 mM of encapsulated curcuminoid. For example, a pharmaceutical composition provided herein can contain about 41 µM to about 100 µM, about 41 µM to about 250 µM, about 41 µM to about 500 µM, about 41 µM to about 750 µM, about 41 µM to about 1 mM, about 41 µM to about 1.25 mM, about 41 µM to about 1.5 mM, about 41 µM to about 1.75 mM, about 41 µM to about 2 mM, about 41 µM to about 2.25 mM, about 41 µM to about 2.5 mM, about 41 µM to about 2.75 mM, about 41 µM to about 3 mM, about 41 µM to about 3.25 mM, about 41 µM to about 3.5 mM, about 41 µM to about 3.75 mM, about 41 µM to about 4 mM, about 100 µM to about 250 µM, about 100 µM toAttorney Docket No.57394-0003WO1 about 500 µM, about 100 µM to about 750 µM, about 100 µM to about 1 mM, about 100 µM to about 1.25 mM, about 100 µM to about 1.5 mM, about 100 µM to about 1.75 mM, about 100 µM to about 2 mM, about 100 µM to about 2.25 mM, about 100 µM to about 2.5 mM, about 100 µM to about 2.75 mM, about 100 µM to about 3 mM, about 100 µM to about 3.25 mM, about 100 µM to about 3.5 mM, about 100 µM to about 3.75 mM, about 100 µM to about 4 mM, about 250 µM to about 500 µM, about 250 µM to about 750 µM, about 250 µM to about 1 mM, about 250 µM to about 1.25 mM, about 250 µM to about 1.5 mM, about 250 µM to about 1.75 mM, about 250 µM to about 2 mM, about 250 µM to about 2.25 mM, about 250 µM to about 2.5 mM, about 250 µM to about 2.75 mM, about 250 µM to about 3 mM, about 250 µM to about 3.25 mM, about 250 µM to about 3.5 mM, about 250 µM to about 3.75 mM, about 250 µM to about 4 mM, about 500 µM to about 750 µM, about 500 µM to about 1 mM, about 500 µM to about 1.25 mM, about 500 µM to about 1.5 mM, about 500 µM to about 1.75 mM, about 500 µM to about 2 mM, about 500 µM to about 2.25 mM, about 500 µM to about 2.5 mM, about 500 µM to about 2.75 mM, about 500 µM to about 3 mM, about 500 µM to about 3.25 mM, about 500 µM to about 3.5 mM, about 500 µM to about 3.75 mM, about 500 µM to about 4 mM, about 750 µM to about 1 mM, about 750 µM to about 1.25 mM, about 750 µM to about 1.5 mM, about 750 µM to about 1.75 mM, about 750 µM to about 2 mM, about 750 µM to about 2.25 mM, about 750 µM to about 2.5 mM, about 750 µM to about 2.75 mM, about 750 µM to about 3 mM, about 750 µM to about 3.25 mM, about 750 µM to about 3.5 mM, about 750 µM to about 3.75 mM, about 750 µM to about 4 mM, about 1 mM to about 1.25 mM, about 1 mM to about 1.5 mM, about 1 mM to about 1.75 mM, about 1 mM to about 2 mM, about 1 mM to about 2.25 mM, about 1 mM to about 2.5 mM, about 1 mM to about 2.75 mM, about 1 mM to about 3 mM, about 1 mM to about 3.25 mM, about 1 mM to about 3.5 mM, about 1 mM to about 3.75 mM, about 1 mM to about 4 mM, about 1.25 mM to about 1.5 mM, about 1.25 mM to about 1.75 mM, about 1.25 mM to about 2 mM, about 1.25 mM to about 2.25 mM, about 1.25 mM to about 2.5 mM, about 1.25 mM to about 2.75 mM, about 1.25 mM to about 3 mM, about 1.25 mM to about 3.25 mM, about 1.25 mM to about 3.5 mM, about 1.25 mM to about 3.75 mM, about 1.25 mM to about 4 mM, about 1.5 mM to about 1.75 mM, about 1.5 mM to about 2 mM, about 1.5 mM to about 2.25 mM, about 1.5 mM to about 2.5 mM, about 1.5 mM to about 2.75 mM, about 1.5 mM to aboutAttorney Docket No.57394-0003WO1 3 mM, about 1.5 mM to about 3.25 mM, about 1.5 mM to about 3.5 mM, about 1.5 mM to about 3.75 mM, about 1.5 mM to about 4 mM, about 1.75 mM to about 2 mM, about 1.75 mM to about 2.25 mM, about 1.75 mM to about 2.5 mM, about 1.75 mM to about 2.75 mM, about 1.75 mM to about 3 mM, about 1.75 mM to about 3.25 mM, about 1.75 mM to about 3.5 mM, about 1.75 mM to about 3.75 mM, about 1.75 mM to about 4 mM, about 2 mM to about 2.25 mM, about 2 mM to about 2.5 mM, about 2 mM to about 2.75 mM, about 2 mM to about 3 mM, about 2 mM to about 3.25 mM, about 2 mM to about 3.5 mM, about 2 mM to about 3.75 mM, about 2 mM to about 4 mM, about 2.25 mM to about 2.5 mM, about 2.25 mM to about 2.75 mM, about 2.25 mM to about 3 mM, about 2.25 mM to about 3.25 mM, about 2.25 mM to about 3.5 mM, about 2.25 mM to about 3.75 mM, about 2.25 mM to about 4 mM, about 2.5 mM to about 2.75 mM, about 2.5 mM to about 3 mM, about 2.5 mM to about 3.25 mM, about 2.5 mM to about 3.5 mM, about 2.5 mM to about 3.75 mM, about 2.5 mM to about 4 mM, about 2.75 mM to about 3 mM, about 2.75 mM to about 3.25 mM, about 2.75 mM to about 3.5 mM, about 2.75 mM to about 3.75 mM, about 2.75 mM to about 4 mM, about 3 mM to about 3.25 mM, about 3 mM to about 3.5 mM, about 3 mM to about 3.75 mM, about 3 mM to about 4 mM, about 3.25 mM to about 3.5 mM, about 3.25 mM to about 3.75 mM, about 3.25 mM to about 4 mM, about 3.5 mM to about 3.75 mM, about 3.5 mM to about 4 mM, or about 3.75 mM to about 4 mM of encapsulated curcuminoid.

[0084] In some embodiments, a pharmaceutical composition provided herein contains about 41 µM, 200 µM, 300 µM, 350 µM, 360 µM, 370 µM, 380 µM, 390 µM, 400 µM, 410 µM, 420 µM, 430 µM, 440 µM, 450 µM, 500 µM, 750 µM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, or about 4.1 mM of encapsulated curcuminoid.

[0085] In some embodiments, a particle of a pharmaceutical composition provided herein has a size of about 20 nm to about 1000 µm. For example, a particle of a pharmaceutical composition provided herein can have a size of about 20 nm to about 60 nm, about 20 nm to about 80 nm, about 20 nm to about 100 nm, about 20 nm to about 200 nm, about 20 nm to about 400 nm, about 20 nm to about 600 nm, about 20 nm to about 800 nm, about 20 nm to about 1 µm, about 20 nm to about 200 µm, about 20 nm to about 400 µm, about 20 nm to about 600 µm, about 20 nm to about 800 µm, about 200 nm to about 400 nm, about 200 nm to about 600 nm, about 200 nm to about 800 nm, about 200 nm toAttorney Docket No.57394-0003WO1 about 1 µm, about 200 nm to about 200 µm, about 200 nm to about 400 µm, about 200 nm to about 600 µm, about 200 nm to about 800 µm, about 200 nm to about 1000 µm, about 400 nm to about 600 nm, about 400 nm to about 800 nm, about 400 nm to about 1 µm, about 400 nm to about 200 µm, about 400 nm to about 400 µm, about 400 nm to about 600 µm, about 400 nm to about 800 µm, about 400 nm to about 1000 µm, about 600 nm to about 800 nm, about 600 nm to about 1 µm, about 600 nm to about 200 µm, about 600 nm to about 400 µm, about 600 nm to about 600 µm, about 600 nm to about 800 µm, about 600 nm to about 1000 µm, about 800 nm to about 1 µm, about 800 nm to about 200 µm, about 800 nm to about 400 µm, about 800 nm to about 600 µm, about 800 nm to about 800 µm, about 800 nm to about 1000 µm, about 1 µm to about 200 µm, about 1 µm to about 400 µm, about 1 µm to about 600 µm, about 1 µm to about 800 µm, about 1 µm to about 1000 µm, about 200 µm to about 400 µm, about 200 µm to about 600 µm, about 200 µm to about 800 µm, about 200 µm to about 1000 µm, about 400 µm to about 600 µm, about 400 µm to about 800 µm, about 400 µm to about 1000 µm, about 600 µm to about 800 µm, about 600 µm to about 1000 µm, or about 800 µm to about 1000 µm. In some embodiments, a particle of a pharmaceutical composition provided herein has a size of about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 200 nm, about 400 nm, about 600 nm, about 800 nm, about 1 µm, about 200 µm, about 400 µm, about 600 µm, about 800 µm, or about 1000 µm.

[0086] In some embodiments, a particle of a pharmaceutical composition provided herein is a nanoparticle (e.g., has a size of less than about 1 µm). In some embodiments, the nanoparticle has a size of about 20 nm to about 200 nm. In some embodiments, the nanoparticle has a size of about 45 nm to about 65 nm, about 50 nm to about 60 nm, or about 50 nm to about 100 nm. For example, a particle of a pharmaceutical composition provided herein can have a size of about 45 nm, about 50 nm, about 51 nm, about 52 nm, about 53 nm, about 54 nm, about 55 nm, about 56 nm, about 57 nm, about 58 nm, about 59 nm, about 60 nm, or about 65 nm.

[0087] In some embodiments, a particle of a pharmaceutical composition provided herein is a microparticle (e.g., has a size of about 1 µm to about 1000 µm).

[0088] In some embodiments, a pharmaceutical composition provided herein includes a plurality of particles (e.g., a plurality of any of the particles described herein).Attorney Docket No.57394-0003WO1

[0089] The Polydispersity Index (PDI Index) is a parameter that indicates the particle size distribution. In some embodiments, the plurality of particles in a pharmaceutical composition provided herein has a Polydispersity Index (PDI) of about 0.25 or less. For example, the plurality of particles can have a PDI of about 0.22 or less, about 0.2 or less, about 0.18 or less, about 0.16 or less, about 0.14 or less, or about 0.12 or less. In some embodiments, the plurality of particles in a pharmaceutical composition provided herein has a Polydispersity Index (PDI) of about 0.1 or less. For example, the plurality of particles can have a PDI of about 0.09 or less, about 0.08 or less, about 0.07 or less, about 0.06 or less, about 0.05 or less, about 0.04 or less, about 0.03 or less, or about 0.02 or less.

[0090] In some embodiments, the plurality of particles in a pharmaceutical composition provided herein has a PDI of about 0.001 to about 0.25. For example, the plurality of particles in a pharmaceutical composition provided herein can have a PDI of about 0.001 to about 0.005, about 0.001 to about 0.01, about 0.001 to about 0.05, about 0.001 to about 0.1, about 0.001 to about 0.15, about 0.001 to about 0.2, about 0.005 to about 0.01, about 0.005 to about 0.05, about 0.005 to about 0.1, about 0.005 to about 0.15, about 0.005 to about 0.2, about 0.005 to about 0.25, about 0.01 to about 0.05, about 0.01 to about 0.1, about 0.01 to about 0.15, about 0.01 to about 0.2, about 0.01 to about 0.25, about 0.05 to about 0.1, about 0.05 to about 0.15, about 0.05 to about 0.2, about 0.05 to about 0.25, about 0.1 to about 0.15, about 0.1 to about 0.2, about 0.1 to about 0.25, about 0.15 to about 0.2, about 0.15 to about 0.25, or about 0.2 to about 0.25. In some embodiments, the plurality of particles has a PDI of about 0.01 to about 0.02, about 0.01 to about 0.04, about 0.01 to about 0.06, about 0.01 to about 0.08, about 0.01 to about 0.1, about 0.01 to about 0.14, about 0.01 to about 0.16, about 0.02 to about 0.04, about 0.02 to about 0.06, about 0.02 to about 0.08, about 0.02 to about 0.1, about 0.06 to about 0.14, about 0.06 to about 0.16, about 0.04 to about 0.06, about 0.04 to about 0.08, about 0.04 to about 0.1, about 0.04 to about 0.14, about 0.4 to about 0.16, about 0.06 to about 0.08, about 0.06 to about 0.1, about 0.06 to about 0.14, about 0.06 to about 0.16, about 0.08 to about 0.1, about 0.08 to about 0.14, or about 0.08 to about 0.16. In some embodiments, the plurality of particles has a PDI of about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, or about 0.15.Attorney Docket No.57394-0003WO1

[0091] The zeta potential is a parameter that indicates the electric potential at the shear plane. In some embodiments, the plurality of particles in a pharmaceutical composition provided herein has a zeta potential of about -15 mV to about -45 mV. For example, the plurality of particles in a pharmaceutical composition provided herein can have a zeta potential of about -15 mV to about -20 mV, about -15 mV to about -25 mV, about -15 mV to about -30 mV, about -15 mV to about -35 mV, about -15 mV to about -40 mV, about -20 mV to about -25 mV, about -20 mV to about -30 mV, about -20 mV to about -35 mV, about -20 mV to about -40 mV, about -20 mV to about -45 mV, about -25 mV to about -30 mV, about -25 mV to about -35 mV, about -25 mV to about -40 mV, about -25 mV to about -45 mV, about -30 mV to about -35 mV, about -30 mV to about -40 mV, about -30 mV to about -45 mV, about -35 mV to about -40 mV, about -35 mV to about -45 mV, or about -40 mV to about -45 mV. In some embodiments, the plurality of particles in a pharmaceutical composition provided herein has a zeta potential of about -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, -40 mV, or -45 mV.

[0092] In some embodiments, the particle is a lipid particle. Non-limiting examples of a lipid particle include a liposome, a nanoemulsion, a solid lipid nanoparticle (SLN), a nanostructured lipid carrier, and a lipid polymer hybrid nanoparticle. The particles of a pharmaceutical composition provided herein can include any combination of lipids. Non- limiting examples of lipids that can be used in a lipid particle include sterols, fatty acids, phospholipids, glycerides, sphingolipids, and prenols. Non-limiting examples of glycerides include monoglycerides, diglycerides, and triglycerides (e.g., triolein (glyceryl trioleate) and tricaprylin). Non-limiting examples of sphingolipids include sphingomyelin. In some embodiments, the sphingomyelin is egg sphingomyelin.

[0093] Also provided herein are pharmaceutical compositions including a liposome containing a curcuminoid (e.g., any of the curcuminoids described herein). In some embodiments, provided herein are pharmaceutical compositions including liposomes containing a curcuminoid (e.g., any of the curcuminoids described herein) and cinnamaldehyde, a cinnamaldehyde derivative, resveratrol, or a mixture thereof. The curcuminoid and cinnamaldehyde, cinnamaldehyde derivative, resveratrol, or mixture thereof can be co-encapsulated in a liposome or the curcuminoid can be encapsulated inAttorney Docket No.57394-0003WO1 one liposome and the cinnamaldehyde, cinnamaldehyde derivative, resveratrol, or mixture thereof can be encapsulated in another liposome. In some embodiments, provided herein are pharmaceutical compositions including a liposome containing a curcuminoid (e.g., any of the curcuminoids described herein) and cinnamaldehyde and / or a derivative thereof. In some embodiments, provided herein are pharmaceutical compositions including a liposome containing a curcuminoid (e.g., any of the curcuminoids described herein) and resveratrol.

[0094] In some embodiments, the curcuminoid is encapusulated within the liposome. In some embodiments, the encapsulated curcuminoid is encapsulated within the lipid bilayer of the liposome. In some embodiments, the curcuminoid and cinnamaldehyde, cinnamaldehyde derivative, resveratrol, or mixture thereof are encapusulated within the liposome. In some embodiments, the encapsulated curcuminoid and encapsulated cinnamaldehyde, cinnamaldehyde derivative, resveratrol, or mixture thereof are encapsulated within the lipid bilayer of the liposome.

[0095] In some embodiments, the molar ratio of the API (e.g., the curcuminoid or the curcuminoid and cinnamaldehyde, cinnamaldehyde derivative, resveratrol, or mixture thereof) to the lipid (e.g., the lipid(s) of any of the particles described herein) is about 1:20 to about 10:1. For example, the molar ratio of the API to the lipid can be about 1:20 to about 8:1, about 1:20 to about 6:1, about 1:20 to about 4:1, about 1:20 to about 2:1, about 1:20 to about 1:1, about 1:15 to about 10:1, about 1:15 to about 8:1, about 1:15 to about 6:1, about 1:15 to about 4:1, about 1:15 to about 2:1, about 1:15 to about 1:1, about 1:10 to about 10:1, about 1:10 to about 8:1, about 1:10 to about 6:1, about 1:10 to about 4:1, about 1:10 to about 2:1, about 1:10 to about 1:1, about 1:5 to about 10:1, about 1:5 to about 8:1, about 1:5 to about 6:1, about 1:5 to about 4:1, about 1:5 to about 2:1, about 1:5 to about 1:1, about 1:1 to about 10:1, about 1:1 to about 8:1, about 1:1 to about 6:1, about 1:1 to about 4:1, or about 1:1 to about 2:1. In some embodiments, the molar ratio of the API to the lipid is about 1:10 to about 1:14. In some embodiments, the molar ratio of the API to the lipid is about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, or about 1:15. In some embodiments, the molar ratio of the API to the lipid is about 1:12.Attorney Docket No.57394-0003WO1

[0096] In some embodiments, the molar ratio of the curcuminoid (e.g., any of the curcuminoids described herein) to the lipid (e.g., the lipid(s) of any of the particles described herein) is about 1:20 to about 10:1. For example, the molar ratio of the curcuminoid to the lipid can be about 1:20 to about 8:1, about 1:20 to about 6:1, about 1:20 to about 4:1, about 1:20 to about 2:1, about 1:20 to about 1:1, about 1:15 to about 10:1, about 1:15 to about 8:1, about 1:15 to about 6:1, about 1:15 to about 4:1, about 1:15 to about 2:1, about 1:15 to about 1:1, about 1:10 to about 10:1, about 1:10 to about 8:1, about 1:10 to about 6:1, about 1:10 to about 4:1, about 1:10 to about 2:1, about 1:10 to about 1:1, about 1:5 to about 10:1, about 1:5 to about 8:1, about 1:5 to about 6:1, about 1:5 to about 4:1, about 1:5 to about 2:1, about 1:5 to about 1:1, about 1:1 to about 10:1, about 1:1 to about 8:1, about 1:1 to about 6:1, about 1:1 to about 4:1, or about 1:1 to about 2:1. In some embodiments, the molar ratio of the curcuminoid to the lipid is about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, or about 1:15. In some embodiments, the molar ratio of the curcuminoid to the lipid is about 1:12.

[0097] In some embodiments, the weight ratio of the curcuminoid (e.g., any of the curcuminoids described herein) to the lipid (e.g., the lipid(s) of any of the particles described herein) is about 5:1 to about 15:1. For example, the weight ratio of the curcuminoid to the lipid can be about 6:1 to about 15:1, about 7:1 to about 15:1, about 8:1 to about 15:1, about 9:1 to about 15:1, about 10:1 to about 15:1, about 11:1 to about 15:1, about 12:1 to about 15:1, about 13:1 to about 15:1, about 14:1 to about 15:1, about 5:1 to about 14:1, about 6:1 to about 14:1, about 7:1 to about 14:1, about 8:1 to about 14:1, about 9:1 to about 14:1, about 10:1 to about 14:1, about 11:1 to about 15:1, about 12:1 to about 14:1, about 13:1 to about 14:1, about 5:1 to about 13:1, about 6:1 to about 13:1, about 7:1 to about 13:1, about 8:1 to about 13:1, about 9:1 to about 13:1, about 10:1 to about 13:1, about 11:1 to about 13:1, about 12:1 to about 13:1, about 5:1 to about 12:1, about 6:1 to about 12:1, about 7:1 to about 12:1, about 8:1 to about 12:1, about 9:1 to about 12:1, about 10:1 to about 12:1, about 11:1 to about 12:1, about 5:1 to about 11:1, about 6:1 to about 11:1, about 7:1 to about 11:1, about 8:1 to about 11:1, about 9:1 to about 11:1, about 10:1 to about 11:1, about 5:1 to about 10:1, about 6:1 to about 10:1, about 7:1 to about 10:1, about 8:1 to about 10:1, about 9:1 to about 10:1, about 5:1 to about 9:1, about 6:1 to about 9:1, about 7:1 to about 9:1, about 8:1 to about 9:1, about 5:1 to about 8:1, about 6:1 toAttorney Docket No.57394-0003WO1 about 8:1, about 7:1 to about 8:1, about 5:1 to about 7:1, about 6:1 to about 7:1, or about 5:1 to about 6:1.

[0098] In some embodiments, the curcuminoid comprises curcumin. In some embodiments, the curcumin comprises at least about 80% by weight of the total curcuminoids present in the pharmaceutical composition. For example, the curcumin can comprise about 80% to about 100%, about 82% to about 100%, about 84% to about 100%, about 86% to about 100%, about 88% to about 100%, about 90% to about 100%, about 92% to about 100%, about 94% to about 100%, about 96% to about 100%, about 98% to about 100%, about 99% to about 100%, about 80% to about 98%, about 82% to about 98%, about 84% to about 98%, about 86% to about 98%, about 88% to about 98%, about 90% to about 98%, about 92% to about 98%, about 94% to about 98%, about 96% to about 98%, about 80% to about 96%, about 82% to about 96%, about 84% to about 96%, about 86% to about 96%, about 88% to about 96%, about 90% to about 96%, about 92% to about 96%, about 94% to about 96%, about 80% to about 94%, about 82% to about 94%, about 84% to about 94%, about 86% to about 94%, about 88% to about 94%, about 90% to about 94%, about 92% to about 94%, about 80% to about 92%, about 82% to about 92%, about 84% to about 92%, about 86% to about 92%, about 88% to about 92%, about 90% to about 92%, about 80% to about 90%, about 82% to about 90%, about 84% to about 90%, about 86% to about 90%, about 88% to about 90%, about 80% to about 88%, about 82% to about 88%, about 84% to about 88%, about 86% to about 88%, about 80% to about 86%, about 82% to about 86%, about 84% to about 86%, about 80% to about 84%, about 82% to about 84%, about 80% to about 82%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 97%, at least about 98%, at least about 99% by weight of the total curcuminoids present in the pharmaceutical composition. In some embodiments, the curcumin comprises at least about 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% by weight of the total curcuminoids present in the pharmaceutical composition.

[0099] In some embodiments, a particle of a pharmaceutical composition provided herein can include a phospholipid (e.g., any of the phospholipids described herein) and a sterol (e.g., any of the sterols described herein). For example, a liposome of a pharmaceutical composition provided herein can formed by a phospholipid (e.g., any ofAttorney Docket No.57394-0003WO1 the phospholipids described herein) and a sterol (e.g., any of the sterols described herein). Non-limiting examples of phospholipids include phosphatidylcholines (e.g., distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC), and 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)), phosphatidylglycerols (e.g., a 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) salt, a 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) salt, and a 1,2- distearoyl-sn-glycero-3-phosphoglycerol (DSPG) salt), phosphatidylethanolamines (e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)), and phosphatidylinositols. Non-limiting examples of sterols include cholesterol, β‑sitosterol, ergosterol, and stigmasterol.

[0100] In some embodiments, a phospholipid in a particle of a pharmaceutical composition provided herein is present in an amount of about 35% to about 99% w / w of the total lipid content of the particle (e.g., about 45% to about 90%, about 50% to about 80% w / w, or about 60% to about 70% of the total lipid content of the particle). In some embodiments, a phospholipid in a particle of a pharmaceutical composition provided herein is present in an amount of about 70% to about 100% w / w of the total lipid content of the particle. For example, a phospholipid in a particle of a pharmaceutical composition provided herein can be present in an amount of about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, 70% to about 99%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 99%, about 75% to about 100%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 99%, about 80% to about 100%, about 85% to about 90%, about 85% to about 95%, about 85% to about 99%, about 85% to about 100%, about 90% to about 95%, about 90% to about 99%, about 90% to about 100%, about 95% to about 99%, about 95% to about 100%, or about 99% to about 100% w / w of the total lipid content of the particle. In some embodiments, a phospholipid in a particle of a pharmaceutical composition provided hereinAttorney Docket No.57394-0003WO1 is present in an amount of about 92% to about 98% w / w of the total lipid content of the particle.

[0101] In some embodiments, a phospholipid in a particle of a pharmaceutical composition provided herein is present in an amount of about 35% to about 65% w / w of the total lipid content of the particle. For example, the phospholipid can be present in a particle of a pharmaceutical composition provided herein in an amount of about 35% to about 40%, about 35% to about 45%, about 35% to about 50%, about 35% to about 55%, about 35% to about 60%, about 40% to about 45%, about 40% to about 50%, about 40% to about 55%, about 40% to about 60%, about 40% to about 65%, about 45% to about 50%, about 45% to about 55%, about 45% to about 60%, about 45% to about 65%, about 50% to about 55%, about 50% to about 60%, about 50% to about 65%, about 55% to about 60%, about 55% to about 65%, or about 60% to about 65% w / w of the total lipid content of the particle. In some embodiments, the phospholipid is present in a particle of a pharmaceutical composition provided herein in an amount of about 45% to about 65% w / w of the total lipid content of the particle. In some embodiments, the phospholipid is present in a particle of a pharmaceutical composition provided herein in an amount of about 45% to about 55% w / w of the total lipid content of the particle.

[0102] In some embodiments, the phospholipid (e.g., any of the phospholipids described herein) is present in a particle of a pharmaceutical composition provided herein in an amount of about 35%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% w / w of the total lipid content of the particle. In some embodiments, the phospholipid is present in a particle of a pharmaceutical composition provided herein in an amount of about 55% w / w of the total lipid content of the particle. In some embodiments, the phospholipid is present in a particle of a pharmaceutical composition provided herein in an amount of about 95% w / w of the total lipid content of the particle.

[0103] In some embodiments, the phospholipid includes DMPC. In some embodiments, the phospholipid includes DMPC and a DMPG salt.Attorney Docket No.57394-0003WO1

[0104] In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DMPC.

[0105] The phospholipid can include a neutral phospholipid. In some embodiments, the phospholipid is a neutral phospholipid. In some embodiments, a neutral lipid is a lipid that is either uncharged or has a neutral zwitterionic form at a selected pH (e.g., at physiological pH). Non-limiting examples of neutral lipids include distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC), and 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dierucoyl-sn-glycero-3- phosphocholine (DEPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0106] In some embodiments, the neutral phospholipid includes DSPC, DOPC, DMPC, DPPC, POPC, DOPE, HSPC, DEPC, or a mixture thereof. In some embodiments, the phospholipid is a neutral phosphatidylcholine.

[0107] A neutral phospholipid can be present in a particle of a pharmaceutical composition provided herein in an amount of about 70% to about 99% w / w of the total lipid content of the particle. For example, a neutral phospholipid can be present in a particle of a pharmaceutical composition provided herein in an amount of about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 99%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 99%, about 85% to about 90%, about 85% to about 95%, about 85% to about 99%, about 90% to about 95%, about 90% to about 99%, or about 95% to about 99% w / w of the total lipid content of the particle. In some embodiments, the neutral phospholipid in a particle of a pharmaceutical composition provided herein is present in an amount of about 82% to about 88% w / w of the total lipid content of the particle. In some embodiments, the neutral phospholipid in a particle of a pharmaceutical composition provided herein is present in an amount of about 88% to about 95% w / w of the total lipid content of the particle. In some embodiments, the neutral phospholipid in a particle of a pharmaceutical composition provided herein is present in an amount of about 88% to about 96% w / w of the total lipid content of the particle. In someAttorney Docket No.57394-0003WO1 embodiments, the neutral phospholipid in a particle of a pharmaceutical composition provided herein is present in an amount of about 90% to about 94% w / w of the total lipid content of the particle.

[0108] In some embodiments, a neutral phospholipid in a particle of a pharmaceutical composition provided herein is present in an amount of about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 95%, or about 99% w / w of the total lipid content of the particle. In some embodiments, the neutral phospholipid in a particle of a pharmaceutical composition provided herein is present in an amount of about 92% w / w of the total lipid content of the particle.

[0109] In some embodiments, a sterol in a particle of a pharmaceutical composition provided herein is present in an amount of about 0.1% to about 60% w / w of the total lipid content of the particle (e.g., about 0.1% to about 25%, about 1% to about 40%, or about 10% to about 50% w / w of the total lipid content of the particle).

[0110] In some embodiments, a sterol in a particle of a pharmaceutical composition provided herein is present in an amount of about 0.1% to about 20% w / w of the total lipid content of the particle. For example, the sterol can be present in a particle of a pharmaceutical composition provided herein in an amount of about 0.1% to about 1%, about 0.1% to about 2%, about 0.1% to about 4%, about 0.1% to about 6%, about 0.1% to about 8%, about 0.1% to about 10%, about 0.1% to about 12%, about 0.1% to about 14%, about 0.1% to about 16%, about 0.1% to about 18%, about 0.1% to about 20%, about 1% to about 2%, about 1% to about 4%, about 1% to about 6%, about 1% to about 8%, about 1% to about 10%, about 1% to about 12%, about 1% to about 14%, about 1% to about 16%, about 1% to about 18%, about 1% to about 20%, about 2% to about 4%, about 2% to about 6%, about 2% to about 8%, about 2% to about 10%, about 2% to about 12%, about 2% to about 14%, about 2% to about 16%, about 2% to about 18%, about 2% to about 20%, about 4% to about 6%, about 4% to about 8%, about 4% to about 10%, about 4% to about 12%, about 4% to about 14%, about 4% to about 16%, about 4% to about 18%, about 4% to about 20%, about 6% to about 8%, about 6% to about 10%, about 6% to about 12%, about 6% to about 14%, about 6% to about 16%, about 6% to about 18%, about 6% to about 20%, about 8% to about 10%, about 8% to about 12%, about 8% to about 14%, aboutAttorney Docket No.57394-0003WO1 8% to about 16%, about 8% to about 18%, about 8% to about 20%, about 10% to about 12%, about 10% to about 14%, about 10% to about 16%, about 10% to about 18%, about 10% to about 20%, about 12% to about 14%, about 12% to about 16%, about 12% to about 18%, about 12% to about 20%, about 14% to about 16%, about 14% to about 18%, about 14% to about 20%, about 16% to about 18%, about 16% to about 20%, or about 18% to about 20% w / w of the total lipid content of the particle.

[0111] In some embodiments, the sterol is present in a particle of a pharmaceutical composition provided herein in an amount of about 1% to about 15% w / w of the total lipid content of the particle. In some embodiments, the sterol is present in a liposome of a pharmaceutical composition provided herein in an amount of about 12% to about 18% w / w of the total lipid content of the liposome. In some embodiments, the sterol is present in a particle of a pharmaceutical composition provided herein in an amount of about 1% to about 10% w / w of the total lipid content of the particle. In some embodiments, the sterol is present in a particle of a pharmaceutical composition provided herein in an amount of about 2% to about 8% w / w of the total lipid content of the particle.

[0112] In some embodiments, a sterol in a particle of a pharmaceutical composition provided herein is present in an amount of about 20% to about 60% w / w of the total lipid content of the particle. For example, the sterol can be present in a particle of a pharmaceutical composition provided herein in an amount of about 20% to about 25%, about 20% to about 30%, about 20% to about 35%, about 20% to about 40%, about 20% to about 45%, about 20% to about 50%, about 20% to about 55%, about 25% to about 30%, about 25% to about 35%, about 25% to about 40%, about 25% to about 45%, about 25% to about 50%, about 25% to about 55%, about 25% to about 60%, about 30% to about 35%, about 30% to about 40%, about 30% to about 45%, about 30% to about 50%, about 30% to about 55%, about 30% to about 60%, about 35% to about 40%, about 35% to about 45%, about 35% to about 50%, about 35% to about 55%, about 35% to about 60%, about 40% to about 45%, about 40% to about 50%, about 40% to about 55%, about 40% to about 60%, about 45% to about 50%, about 45% to about 55%, about 45% to about 60%, about 50% to about 55%, about 50% to about 60%, or about 55% to about 60% w / w of the total lipid content of the particle.Attorney Docket No.57394-0003WO1

[0113] In some embodiments, the sterol is present in a particle of a pharmaceutical composition provided herein in an amount of about 30% to about 50% w / w of the total lipid content of the particle. In some embodiments, the sterol is present in a particle of a pharmaceutical composition provided herein in an amount of about 35% to about 45% w / w of the total lipid content of the particle.

[0114] In some embodiments, the sterol (e.g., any of the sterols described herein) is present in a particle of a pharmaceutical composition provided herein in an amount of about 0.1%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% w / w of the total lipid content of the particle. In some embodiments, the sterol is present in a particle of a pharmaceutical composition provided herein in an amount of about 5% w / w of the total lipid content of the particle. In some embodiments, the sterol is present in a particle of a pharmaceutical composition provided herein in an amount of about 40% w / w of the total lipid content of the particle.

[0115] In some embodiments, the sterol is cholesterol.

[0116] In some embodiments, a particle of a pharmaceutical composition provided herein can include a cationic lipid and / or an anionic lipid. A cationic lipid can have a positive or partial positive charge at physiological pH. Non-limiting examples of cationic lipids include a N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP) salt (e.g., DOTAP-Cl) and a N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) salt (e.g., DOTMA-Cl). In some embodiments, an anionic lipid has a negative or partial negative charge at physiological pH. Non-limiting examples of anionic lipids include 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol sodium salt (DPPG-Na), 1,2- dimyristoyl-sn-glycero-3-phosphoglycerol sodium salt (DMPG-Na), and 1,2-distearoyl- sn-glycero-3-phosphoglycerol sodium salt (DSPG-Na). The ionization of a cationic lipid or an anionic lipid can affect the surface charge of lipid nanoparticles under different pH conditions. See, e.g., Immordino et al. Int. J. Nanomedicine.1(3):297–315, 2006; Fröhlich, Int. J. Nanomedicine.7:5577-91, 2012.Attorney Docket No.57394-0003WO1

[0117] In some embodiments, a particle of a pharmaceutical composition provided herein can include a PEGylated lipid. A PEGylated lipid refers to a lipid modified with polyethylene glycol (PEG).

[0118] In some embodiments, a particle of a pharmaceutical composition provided herein can include a neutral phospholipid (e.g., any of the neutral phospholipids described herein) and one or more of an anionic lipid (e.g., any of the anionic lipids described herein), a cationic lipid (e.g., any of the cationic lipids described herein), and a PEGylated lipid (e.g., any of the PEGylated lipids described herein). For example, a liposome of a pharmaceutical composition provided herein can formed by a neutral phospholipid (e.g., any of the neutral phospholipids described herein) and one or more of an anionic lipid (e.g., any of the anionic lipids described herein), a cationic lipid (e.g., any of the cationic lipids described herein), and a PEGylated lipid (e.g., any of the PEGylated lipids described herein).

[0119] In some embodiments, the molar ratio of the neutral phospholipid (e.g., any of the neutral phospholipids described herein) to the anionic lipid (e.g., any of the anionic lipids described herein), cationic lipid (e.g., any of the cationic lipids described herein), or PEGylated lipid (e.g., any of the PEGylated lipids described herein) is about 90:30 to about 90:1. For example, the weight ratio of the neutral phospholipid to the the anionic lipid, cationic lipid, or PEGylated lipid can be about 90:30 to about 90:25, about 90:30 to about 90:20, about 90:30 to about 90:15, about 90:30 to about 90:10, about 90:30 to about 90:5, about 90:30 to about 90:1, about 90:25 to about 90:20, about 90:25 to about 90:15, about 90:25 to about 90:10, about 90:25 to about 90:5, about 90:25 to about 90:1, about 90:20 to about 90:15, about 90:20 to about 90:10, about 90:20 to about 90:5, about 90:20 to about 90:1, about 90:15 to about 90:10, about 90:15 to about 90:5, about 90:15 to about 90:1, about 90:10 to about 90:5, about 90:10 to about 90:1, about 90:5 to about 90:1. In some embodiments, the weight ratio of the weight ratio of the neutral phospholipid to the the anionic lipid, cationic lipid, or PEGylated lipid is about 90:30, about 90:25, about 90:20, about 90:15, about 90:10, about 90:5, or about 90:1. In some embodiments, the weight ratio of the weight ratio of the neutral phospholipid to the the anionic lipid, cationic lipid, or PEGylated lipid is about 92:8.Attorney Docket No.57394-0003WO1

[0120] In some embodiments, a particle of a pharmaceutical composition provided herein can further include a sterol. In some embodiments, a particle of a pharmaceutical composition provided herein can include a neutral phospholipid (e.g., any of the neutral phospholipids described herein), a sterol (e.g., any of the sterols described herein), and one or more of an anionic lipid (e.g., any of the anionic lipids described herein), a cationic lipid (e.g., any of the cationic lipids described herein), and a PEGylated lipid (e.g., any of the PEGylated lipids described herein). For example, a liposome of a pharmaceutical composition provided herein can formed by a neutral phospholipid (e.g., any of the neutral phospholipids described herein), a sterol (e.g., any of the sterols described herein), and one or more of an anionic lipid (e.g., any of the anionic lipids described herein), a cationic lipid (e.g., any of the cationic lipids described herein), and a PEGylated lipid (e.g., any of the PEGylated lipids described herein).

[0121] In some embodiments, a particle of a pharmaceutical composition provided herein includes a neutral phospholipid (e.g., any of the neutral phospholipids described herein), a sterol (e.g., any of the sterols described herein), and an anionic lipid (e.g., any of the anionic lipids described herein). In some embodiments, a particle of a pharmaceutical composition provided herein includes a neutral phospholipid (e.g., any of the neutral phospholipids described herein), a sterol (e.g., any of the sterols described herein), and a cationic lipid (e.g., any of the cationic lipids described herein). In some embodiments, a particle of a pharmaceutical composition provided herein includes a neutral phospholipid (e.g., any of the neutral phospholipids described herein), a sterol (e.g., any of the sterols described herein), and a PEGylated lipid (e.g., any of the PEGylated lipids described herein).

[0122] In some embodiments, an anionic lipid, a cationic lipid, or a mixture thereof in a particle of a pharmaceutical composition provided herein is present in an amount of about 0.1% to about 15% w / w of the total lipid content of the particle. For example, the anionic lipid, cationic lipid, or mixture thereof can be present in a particle of a pharmaceutical composition provided herein in an amount of about 0.1% to about 1%, about 0.1% to about 2%, about 0.1% to about 4%, about 0.1% to about 6%, about 0.1% to about 8%, about 0.1% to about 10%, about 0.1% to about 12%, about 0.1% to about 14%, about 1% to about 2%, about 1% to about 4%, about 1% to about 6%, about 1% to about 8%, about 1% to aboutAttorney Docket No.57394-0003WO1 10%, about 1% to about 12%, about 1% to about 14%, about 1% to about 15%, about 2% to about 4%, about 2% to about 6%, about 2% to about 8%, about 2% to about 10%, about 2% to about 12%, about 2% to about 14%, about 2% to about 15%, about 4% to about 6%, about 4% to about 8%, about 4% to about 10%, about 4% to about 12%, about 4% to about 14%, about 4% to about 15%, about 6% to about 8%, about 6% to about 10%, about 6% to about 12%, about 6% to about 14%, about 6% to about 15%, about 8% to about 10%, about 8% to about 12%, about 8% to about 14%, about 8% to about 15%, about 10% to about 12%, about 10% to about 14%, about 10% to about 15%, about 12% to about 14%, about 12% to about 15%, or about 14% to about 15% w / w of the total lipid content of the particle. In some embodiments, an anionic lipid, a cationic lipid, or a mixture thereof is present in a particle of a pharmaceutical composition provided herein in an amount of about 4% to about 6% w / w of the total lipid content of the particle. In some embodiments, an anionic lipid, a cationic lipid, or a mixture thereof is present in a particle of a pharmaceutical composition provided herein in an amount of about 5% to about 12% w / w of the total lipid content of the particle.

[0123] In some embodiments, an anionic lipid (e.g., any of the anionic lipids described herein), a cationic lipid (e.g., any of the cationic lipids described herein), or a mixture thereof is present in a particle of a pharmaceutical composition provided herein in an amount of about 0.1%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% w / w of the total lipid content of the particle. In some embodiments, an anionic lipid, a cationic lipid, or a mixture thereof is present in a particle of a pharmaceutical composition provided herein in an amount of about 10% w / w of the total lipid content of the particle. In some embodiments, an anionic lipid, a cationic lipid, or a mixture thereof is present in a particle of a pharmaceutical composition provided herein in an amount of about 8% w / w of the total lipid content of the particle. In some embodiments, an anionic lipid, a cationic lipid, or a mixture thereof is present in a particle of a pharmaceutical composition provided herein in an amount of about 5% w / w of the total lipid content of the particle.

[0124] In some embodiments, a particle of a pharmaceutical composition provided herein includes an anionic lipid (e.g., any of the anionic lipids described herein). In someAttorney Docket No.57394-0003WO1 embodiments, the anionic lipid is DSPG-Na. In some embodiments, the anionic lipid is DMPG-Na.

[0125] In some embodiments, a particle of a pharmaceutical composition provided herein includes a cationic lipid (e.g., any of the cationic lipids described herein). In some embodiments, the anionic lipid is DOTAP-Cl.

[0126] In some embodiments, a PEGylated lipid in a particle of a pharmaceutical composition provided herein is present in an amount of about 0.1% to about 15% w / w of the total lipid content of the particle. For example, the PEGylated lipid can be present in a particle of a pharmaceutical composition provided herein in an amount of about 0.1% to about 1%, about 0.1% to about 2%, about 0.1% to about 4%, about 0.1% to about 6%, about 0.1% to about 8%, about 0.1% to about 10%, about 0.1% to about 12%, about 0.1% to about 14%, about 1% to about 2%, about 1% to about 4%, about 1% to about 6%, about 1% to about 8%, about 1% to about 10%, about 1% to about 12%, about 1% to about 14%, about 1% to about 15%, about 2% to about 4%, about 2% to about 6%, about 2% to about 8%, about 2% to about 10%, about 2% to about 12%, about 2% to about 14%, about 2% to about 15%, about 4% to about 6%, about 4% to about 8%, about 4% to about 10%, about 4% to about 12%, about 4% to about 14%, about 4% to about 15%, about 6% to about 8%, about 6% to about 10%, about 6% to about 12%, about 6% to about 14%, about 6% to about 15%, about 8% to about 10%, about 8% to about 12%, about 8% to about 14%, about 8% to about 15%, about 10% to about 12%, about 10% to about 14%, about 10% to about 15%, about 12% to about 14%, about 12% to about 15%, or about 14% to about 15% w / w of the total lipid content of the particle.

[0127] In some embodiments, a PEGylated lipid is present in a particle of a pharmaceutical composition provided herein in an amount of about 4% to about 6% w / w of the total lipid content of the particle. In some embodiments, a PEGylated lipid is present in a particle of a pharmaceutical composition provided herein in an amount of about 0.1%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% w / w of the total lipid content of the particle. In some embodiments, a PEGylated lipid is present in a particle of a pharmaceutical composition provided herein in an amount of about 5% w / w of the total lipid content of the particle.Attorney Docket No.57394-0003WO1

[0128] In some embodiments, the PEGylated lipid is a PEGylated phospholipid. For example, the PEGylated lipid can be a PEGylated phosphatidylethanolamine (e.g., any of the phosphatidylethanolamines described herein). In some embodiments, the PEGylated lipid is DSPE-PEG2000.

[0129] In some embodiments, provided herein are pharmaceutical compositions including a liposome containing: any of the active ingredients described herein (e.g., a curcuminoid, a cinnamaldehyde, a cinnamaldehyde derivative, resveratrol, or any mixture thereof); a neutral phospholipid (e.g., any of the neutral phospholipids described herein) present in an amount of about 75% to about 95% w / w of the total lipid content of the liposome and an anionic lipid (e.g., any of the anionic lipids described herein) or a cationic lipid (e.g., any of the cationic lipids described herein) present in an amount of about 1% to about 20% w / w of the total lipid content of the liposome.

[0130] In some embodiments, the neutral phospholipid is present in an amount of about 78% to about 82%, about 82% to about 88%, about 88% to about 92%, or about 90% to about 94% w / w of the total lipid content of the liposome.

[0131] In some embodiments, the neutral phospholipid includes DMPC.

[0132] In some embodiments, the anionic lipid or cationic lipid is present in an amount of about 2% to about 8%, about 6% to about 10%, or about 12% to about 18% w / w of the total lipid content of the liposome.

[0133] In some embodiments, wherein the anionic lipid or cationic lipid is a cationic lipid, the cationic lipid is DOTAP-Cl. In some embodiments, wherein the anionic lipid or cationic lipid is an anionic lipid, the anionic lipid is DMPG-Na.

[0134] In some embodiments, the liposome further includes a sterol present in an amount of about 1% to about 20%, about 12% to about 18%, or about 2% to about 8% w / w of the total lipid content of the liposome. In some embodiments, the sterol is cholesterol.

[0135] In some embodiments, a liposome provided herein includes: any of the active pharmaceutical ingredients described herein (e.g., a curcuminoid, a cinnamaldehyde, a cinnamaldehyde derivative, resveratrol, or any mixture thereof);Attorney Docket No.57394-0003WO1 DMPC present in an amount of about 88% to about 92% w / w of the total lipid content of the liposome; and DMPG-Na present in an amount of about 8% to about 12% w / w of the total lipid content of the liposome. In some embodiments, the active pharmaceutical ingredient includes curcumin. In some embodiments, the active pharmaceutical ingredient includes curcumin and cinnamaldehyde. In some embodiments, wherein the active pharmaceutical ingredient includes curcumin and cinnamaldehyde, the curcumin and cinnamaldehyde are present at a weight ratio of about 5:1 curcumin:cinnamaldehyde.

[0136] In some embodiments, a liposome provided herein includes: curcumin; a neutral phospholipid (e.g., any of the neutral phospholipids described herein); and an anionic lipid (e.g., any of the anionic lipids described herein), wherein the neutral phospholipid and anionic lipid are present at a molar ratio of about 90:5 to about 90:15 neutral phospholipid:anionic lipid; and wherein the curcumin is present at a molar ratio of about 1:10 to about 1:14 curcumin:lipid. In some embodiments, the curcumin is present at a molar ratio of about 1:12 curcumin:lipid. In some embodiments, the neutral phospholipid and anionic lipid are present at a molar ratio of about 90:10 neutral phospholipid:anionic lipid. In some embodiments, the neutral phospholipid is DMPC. In some embodiments, the anionic lipid is DMPG-Na.

[0137] In some embodiments, a liposome provided herein includes: curcumin; a neutral phospholipid (e.g., any of the neutral phospholipids described herein); and an anionic lipid (e.g., any of the anionic lipids described herein), wherein the neutral phospholipid and anionic lipid are present at a molar ratio of about 92:8 neutral phospholipid:anionic lipid; and wherein the curcumin is present at a molar ratio of about 1:10 to about 1:14 curcumin:lipid. In some embodiments, the curcumin is present at a molar ratio of aboutAttorney Docket No.57394-0003WO1 1:12 curcumin:lipid. In some embodiments, the neutral phospholipid is DMPC. In some embodiments, the anionic lipid is DMPG-Na.

[0138] In some embodiments, a liposome provided herein includes: curcumin; cinnamaldehyde; a neutral phospholipid (e.g., any of the neutral phospholipids described herein); and an anionic lipid (e.g., any of the anionic lipids described herein), wherein the neutral phospholipid and anionic lipid are present at a molar ratio of about 90:5 to about 90:15 neutral phospholipid:anionic lipid; and wherein the curcumin and cinnamaldyhde are present at a molar ratio of about 1:10 to about 1:14 curcumin and cinnamaldehyde:lipid. In some embodiments, the curcumin is present at a molar ratio of about 1:12 curcumin and cinnamaldehyde:lipid. In some embodiments, the neutral phospholipid and anionic lipid are present at a molar ratio of about 90:10 neutral phospholipid:anionic lipid. In some embodiments, the neutral phospholipid is DMPC. In some embodiments, the anionic lipid is DMPG-Na.

[0139] In some embodiments, a liposome provided herein includes: any of the active pharmaceutical ingredients described herein (e.g., a curcuminoid, a cinnamaldehyde, a cinnamaldehyde derivative, resveratrol, or any mixture thereof); DMPC present in an amount of about 82% to about 88% w / w of the total lipid content of the liposome; and DMPG-Na present in an amount of about 8% to about 12% w / w of the total lipid content of the liposome; and cholesterol present in an amount of about 2% to about 8% of the total lipid content of the liposome. In some embodiments, the active pharmaceutical ingredient includes curcumin. In some embodiments, the active pharmaceutical ingredient includes curcumin and cinnamaldehyde. In some embodiments, wherein the active pharmaceutical ingredient includes curcumin and cinnamaldehyde, the curcumin and cinnamaldehyde are present at a weight ratio of about 5:1 curcumin:cinnamaldehyde.

[0140] In some embodiments, a liposome provided herein includes:Attorney Docket No.57394-0003WO1 any of the active pharmaceutical ingredients described herein (e.g., a curcuminoid, a cinnamaldehyde, a cinnamaldehyde derivative, resveratrol, or any mixture thereof); DMPC present in an amount of about 78% to about 82% w / w of the total lipid content of the liposome; DOTAP-Cl present in an amount of about 2% to about 8% w / w of the total lipid content of the liposome; and cholesterol present in an amount of about 12% to about 18% of the total lipid content of the liposome. In some embodiments, the active pharmaceutical ingredient includes curcumin. In some embodiments, the active pharmaceutical ingredient includes curcumin and cinnamaldehyde. In some embodiments, wherein the active pharmaceutical ingredient includes curcumin and cinnamaldehyde, the curcumin and cinnamaldehyde are present at a weight ratio of about 5:1 curcumin:cinnamaldehyde.

[0141] In some embodiments, provided herein are pharmaceutical compositions including a liposome containing: any of the active pharmaceutical ingredients described herein (e.g., a curcuminoid, a cinnamaldehyde, a cinnamaldehyde derivative, resveratrol, or any mixture thereof); a neutral phospholipid (e.g., any of the neutral phospholipids described herein) present in an amount of about 75% to about 95% w / w of the total lipid content of the liposome; and a PEGylated lipid (e.g., any of the PEGylated lipids described herein) present in an amount of about 1% to about 20% w / w of the total lipid content of the liposome.

[0142] In some embodiments, the neutral phospholipid is present in an amount of about 78% to about 82%, about 82% to about 88%, or about 88% to about 92% w / w of the total lipid content of the liposome.

[0143] In some embodiments, the neutral phospholipid includes DMPC.

[0144] In some embodiments, the PEGylated lipid is present in an amount of about 2% to about 8% or about 12% to about 18% w / w of the total lipid content of the liposome.

[0145] In some embodiments, the PEGylated lipid includes a PEGylated neutral phospholipid, a PEGylated cationic lipid, or a PEGylated anionic lipid. In some embodiments, the PEGylated lipid is DSPE-PEG2000.Attorney Docket No.57394-0003WO1

[0146] In some embodiments, the liposome further includes a sterol present in an amount of about 1% to about 20%, about 12% to about 18%, or about 2% to about 8% w / w of the total lipid content of the liposome. In some embodiments, the sterol is cholesterol.

[0147] In some embodiments provided herein are pharmaceutical compositions including a liposome containing: a curcuminoid (e.g., any of the curcuminoids described herein); cinnamaldehyde and / or a derivative thereof (e.g., any of the cinnamaldehyde derivatives described herein); a neutral phospholipid present in an amount of about 45% to about 65% w / w of the total lipid content of the liposome; a sterol present in an amount of about 30% to about 50% w / w of the total lipid content of the liposome; and an anionic lipid (e.g., any of the anionic lipids described herein), a cationic lipid (e.g., any of the cationic lipids described herein), a PEGylated lipid (e.g., any of the PEGylated lipids described herein) or a mixture thereof present in an amount of about 0.1% to about 15% w / w of the total lipid content of the liposome. In some embodiments, the neutral phospholipid is DSPC. In some embodiments, the sterol is cholesterol. In some embodiments, the cationic lipid is DOTAP-Cl. In some embodiments, the anionic lipid is DSPG-Na. In some embodiments, the PEGylated lipid is DSPE- PEG2000.

[0148] In some embodiments provided herein are pharmaceutical compositions including a liposome containing: a curcuminoid (e.g., any of the curcuminoids described herein); cinnamaldehyde and / or a derivative thereof (e.g., any of the cinnamaldehyde derivatives described herein); DSPC present in an amount of about 45% to about 65% w / w of the total lipid content of the liposome; cholesterol present in an amount of about 30% to about 50% w / w of the total lipid content of the liposome; and an anionic lipid (e.g., any of the anionic lipids described herein), a cationic lipid (e.g., any of the cationic lipids described herein), a PEGylated lipid (e.g., any of the PEGylated lipids described herein) or a mixture thereof present in an amount of about 0.1% to about 15% w / w of the total lipid content of the liposome. In some embodiments, the cationic lipid is DOTAP-Cl. In some embodiments, the anionic lipid is DSPG-Na. In some embodiments, the PEGylated lipid is DSPE- PEG2000.

[0149] The pharmaceutical compositions provided herein can be formulated for parenteral administration, e.g., formulated for injection via intramuscular or subcutaneousAttorney Docket No.57394-0003WO1 routes. Such compositions can be prepared as injectables, either as liquid solutions or suspensions. The form must be fluid to the extent that it may be easily injected. The proper fluidity of an injectable solution can be maintained, for example, by the maintenance of the required particle size in the case of dispersion. In some embodiments, such compositions are prepared as solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection. The preparation of such formulations will be known to those of skill in the art in light of the present disclosure.

[0150] The pharmaceutical compositions provided herein can further include one or more pharmaceutically acceptable excipients. Such excipients can include, e.g., isotonic agents (e.g., sugars or sodium chloride) and a solvent or dispersion medium containing, for example, water, a buffer, ethanol, or suitable mixtures thereof. Non-limiting examples of buffers that can be used in a pharmaceutical composition provided herein include acetate buffers, histidine buffers, phosphate buffers, and citrate buffers. Some buffers, buffer concentrations, pHs, and / or sodium chloride concentrations can contribute to injection site pain. See, e.g., Shi et al. Pharm. Res. 38:779-793, 2021. Accordingly, in some embodiments, a pharmaceutical composition formulated for parenteral administration can be formulated to minimize injection site pain by selecting suitable buffers, buffer concentrations, pHs, and / or sodium chloride concentrations. In some embodiments, a buffer in a pharmaceutical composition provided herein includes histidine or acetate. In some embodiments, the buffer does not include citrate.

[0151] In some embodiments, a pharmaceutical composition provided herein is sterile. Sterile injectable solutions can be prepared by incorporating the active ingredients (e.g., a curcuminoid or a curcuminoid and cinnamaldehyde, a cinnamaldehyde derivative, resveratrol, or a mixture thereof) in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. In some embodiments, a pharmaceutical composition provided herein (e.g., a pharmaceutical composition provided herein containing liposomes) having a particle size of about 50 nm to about 150 nm and a PDI of about 0.1 to about 0.2 can be filtered through a 0.22 micron sterile filter.

[0152] An injectable solution should also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms,Attorney Docket No.57394-0003WO1 such as bacteria and fungi. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0153] In some embodiments, a pharmaceutical composition provided herein is prepared by dissolving a curcuminoid (e.g., any of the curcuminoids described herein) or a curcuminoid and cinnamaldehyde, a cinnamaldehyde derivative, resveratrol, or a mixture thereof in a solvent. Non-limiting examples of such solvents include water, an alcohol (e.g., ethanol), and a combination thereof. An aqueous buffer can then be added to the solution containing the curcuminoid or the curcuminoid and cinnamaldehyde, a cinnamaldehyde derivative, resveratrol, or a mixture thereof, to form liposomes. Non-limiting examples of such aqueous buffers include an acetate or histidine buffer. In some embodiments, mixture of the aqueous buffer and solution containing the curcuminoid or the curcuminoid and cinnamaldehyde, a cinnamaldehyde derivative, resveratrol, or a mixture thereof, is mixed. In some embodiments, the mixture is concentrated until the desired concentration of the curcuminoid or the curcuminoid and cinnamaldehyde, a cinnamaldehyde derivative, resveratrol, or a mixture thereof is reached. For example, the mixture can be concentrated to a concentration of about 1 mg / mL to about 10 mg / mL of the curcuminoid. In some embodiments, the mixture is concentrated to a concentration of about 5 mg / mL to about 10 mg / mL of the curcuminoid. The mixture can be filtered as described herein.

[0154] Other pharmaceutical compositions that can be used in the methods described herein include described in U.S. Patent Nos.10,226,503; 10,716,824; and 10,537,548; U.S. Publication Nos.2023 / 0372378; 2016 / 0129068; and 2016 / 0374911. Methods

[0155] Provided herein are methods for methods for inducing cell death of a plurality of adipocytes in a treatment area in a subject and / or inhibiting adipogenesis of a plurality of adipocytes in a treatment area in a subject, the method comprising administering to the subject an effective amount of any of the pharmaceutical compositions described herein.

[0156] In some embodiments, the cell death is due to apoptosis and / or necrosis. In some embodiments, the cell death is due to apoptosis. Cell death through apoptosis ratherAttorney Docket No.57394-0003WO1 than necrosis can help minimize side effects. Compositions that reduce fat locally through necrosis can lead to necrosis of surrounding cells, inflammation, and pain.

[0157] Also provided herein are methods of reducing subcutaneous fat in a treatment area in a subject, the method comprising administering to the subject an effective amount of any of the pharmaceutical compositions described herein. In some embodiments, the method comprises inducing apoptosis of a plurality of adipocytes in the treatment area in the subject.

[0158] Also provided herein are methods of improving the appearance of convexity or fullness associated with fat tissue in a treatment area in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition of any of the pharmaceutical compositions described herein.

[0159] A treatment area can refer to an area of subcutaneous fat in a subject. For example, a treatment area can refer to a fat deposit in a subject’s body such as a fat deposit in the subject’s abdomen, flank, inner thigh, outer thigh, periprostatic adipose tissue, arm, back, buttocks, calf, ankle, jowls, submental area, preplatysmal submental area, breast, chest, lower face, or under an eye. In some embodiments, a fat deposit in the subject’s body includes a facial fat deposit. Non-limiting examples of facial fat deposits include middle forehead fat, central forehead fat, lateral temporal fat, middle cheek fat, medial cheek fat, nasolabial fat, inferior orbital fat, superior orbital fat, lateral orbital fat, retro septal orbital fat, jowl fat, premental fat, subplatysmal fat, retroorbicularis fat, medial suborbicularis fat, lateral suborbicularis fat, buccal fat, and deep medial cheek fat. In some embodiments, a fat deposit in the subject’s abdomen includes intra-abdominal fat. In some embodiments, the treatment area is in the abdomen, a flank, an inner thigh, an outer thigh, an arm, back, buttocks, a breast, chest, a calf, an ankle, jowls, preplatysmal submental area, lower face, or under an eye of a subject. In some embodiments, the treatment area is in the back of an arm of the subject. In some embodiments, wherein the treatment area is in the submental area, jowls, or buccal area of the lower face.

[0160] In some embodiments, the treatment area is in preplatysmal submental area of the subject. In some embodiments, the subject can have moderate to severe submental convexity.Attorney Docket No.57394-0003WO1

[0161] Treatment areas can include subcutantous fat deposits associated with, e.g., cellulite, lipidema, or a subcutaneous tumor of adipose cells. Accordingly, some embodiments of any of the methods provided herein include reducing subcutaneous fat in a treatment area in a subject having one or more of cellulite, lipidema, or a subcutaneous tumor of adipose cells. In some embodiments of any of the methods provided herein, the method includes improving the appearance of one or more of cellulite, a lipoma, lipidema, and liposarcoma in a subject.

[0162] In some embodiments, the subject has cellulite. Cellulite includes subcutaneous fat within fibrous connective tissue that manifests as an uneven surface to the skin, e.g., as skin dimpling and nodularity.

[0163] In some embodiments, the subject has a subcutaneous tumor of adipose cells. Subcutaneous tumors of adipose cells can include lipomas (e.g., benign subcutaneous tumors of adipose cells) and malignant subcutaneous tumors of adipose cells. In some embodiments, a lipoma is a nodular lipoma or a diffuse lipoma. In some embodiments, a lipoma is an intermuscular lipoma, an intramuscular lipoma, a parosteal lipoma, a periosteal lipoma, a lipoma arborescens (synovial lipomatosis), or an intracranial lipoma. Further non-limiting examples of lipomas include angiolipoma, chondroid lipoma, lipoblastoma, myolipoma, pleomorphic lipoma / spindle cell lipoma, intramuscular lipoma, intermuscular lipoma, lipomatosis of nerve, lipoma of the tendon sheath and joint, lipoma arborescens, multiple symmetric lipomatosis, diffuse lipomatosis, adiposis dolorosa, and hibernoma.

[0164] In some embodiments, a fat deposit in a subject is associated with temporal fat pad hyperplasia, pseudo herniation of orbital fat, Graves’ disease, Dercum's disease, multiple hereditary lipomatosis, familial multiple lipomatosis, Gardner syndrome, adiposis dolorosa, PTEN hamartoma tumor syndrome, Cowden syndrome, or Madelung disease. In some embodiments, a fat deposit in a subject is associated with fatty infiltration. For example, a fat deposit in a subject can be associated with accumulation of adipocytes in non-adipose tissue. Non-limiting examples of fatty infiltration include parotid gland fatty infiltration, hepatic fatty infiltration, myocardial fatty infiltration, pancreatic fatty infiltration, fatty kidney disease, and fatty gallbladder disease. Non-limiting examples of aAttorney Docket No.57394-0003WO1 fatty gallbladder disease include lipotoxic cholecystopathy, steatocholecystitis, nonalcoholic fatty gallbladder disease (NAFGBD), and cholecystosteatosis.

[0165] In some embodiments, the subcutaneous tumor of adipose cells is malignant. Non-limiting examples of malignant subcutaneous tumors of adipose cells include liposarcoma, Proteus syndrome, and soft tissue sarcoma.

[0166] Fat reduction in a treatment area in a subject can be evaluated using methods known in the art. For example, reduction in submental fat can be assessed using the Clinician‐Rated SMF Rating Scale (CR‐SMFRS) or the Subject Self‐Rating Scale (SSRS). See, e.g., Jalian et al. J Cosmet Dermatol. 21(6): 2437–2444 (2022). Fat reduction in a treatment area can also be evaluated using an imaging method such as magnetic resonance imaging (MRI), ultrasound, and computed tomography (CT).

[0167] In some embodiments, one or more of the diameter, surface area, and volume of a subcutantous fat deposit can be reduced. For example, one or more of the diameter, surface area, and volume of a fat deposit can be reduced. In some embodiments, one or more of the diameter, surface area, and volume of a subcutaneous fat deposit is reduced by about 20% to about 50% per treatment (e.g., per dosage unit of a pharmaceutical composition provided herein). For example, one or more of the diameter, surface area, and volume of a subcutaneous fat deposit can be reduced by about 20% to about 25%, about 20% to about 30%, about 20% to about 35%, about 20% to about 40%, about 20% to about 45%, about 25% to about 30%, about 25% to about 35%, about 25% to about 40%, about 25% to about 45%, about 25% to about 50%, about 30% to about 35%, about 30% to about 40%, about 30% to about 45%, about 30% to about 50%, about 35% to about 40%, about 35% to about 45%, about 35% to about 50%, about 40% to about 45%, about 40% to about 50%, or about 45% to about 50% per treatment. Administration

[0168] The pharmaceutical compositions provided herein can be administered to a subject in need thereof by any route which makes the pharmaceutical composition bioavailable (e.g., locally bioavailable). Non-limiting examples of routes of administration include injection, implantable infusion, and topical. Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions,Attorney Docket No.57394-0003WO1 creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0169] In some embodiments, a pharmaceutical composition provided herein is administered topically. In some embodiments, a pharmaceutical composition provided herein is administered topically for transdermal delivery. For example, transdermal delivery of a pharmaceutical composition provided herein can be mediated by peptides, ultrasound, ultrasound (US)-stimulated microbubble (MB) cavitation, ultrasonication, and other energy sources such as thermal heat, microwave, lasers, and radio frequency,

[0170] In some embodiments, a pharmaceutical composition provided herein is administered subcutaneously. For example, the pharmaceutical composition can be administered by injection to subcutaneous fat tissue in the subject. In some embodiments, the pharmaceutical composition contacts subcutaneous fat (e.g., a fat deposit) in the treatment area.

[0171] In some embodiments, a pharmaceutical composition provided herein is administered by one or more injections. For example, a pharmaceutical composition provided herein can be administered using a needle or a cannula. In some embodiments, a pharmaceutical composition provided herein is administered using microneedling and / or a jet injector. In some embodiments, the one or more injections are guided by imaging. For example, imaging can be used to help localize the placement of the one or more injections. In some embodiments, the one or more injections are guided by ultrasound imaging, computerized tomography (CT) scans, or magnetic resonance imaging (MRI).

[0172] A dosage unit (e.g., an injection) of a pharmaceutical composition provided herein can contain an amount of the active ingredient necessary to deliver an effective dose as described herein. In some embodiments, a pharmaceutical composition provided herein is administered as a single dose or as 2 or more doses (e.g., as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more doses).

[0173] In some embodiments, a pharmaceutical composition provided herein is administered as a divided dose (e.g., a dose divided into 2, 3, 4, 5, 10, or 15 or more parts). For example, a dosage unit divided into two or more parts (e.g., two or more injections) ofAttorney Docket No.57394-0003WO1 a pharmaceutical composition provided herein can contain an amount of the active ingredient necessary to deliver an effective dose as described herein.

[0174] In some embodiments, a pharmaceutical composition provided herein is administered to the treatment area in the subject via at least 2 injections (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or 40 or more injections to the treatment area in the subject).

[0175] In some embodiments, a dosage unit (e.g., an injection) of a pharmaceutical composition provided herein is administered daily. In some embodiments, a dosage unit of a pharmaceutical composition provided herein is administered on a non-daily basis. For example, a dosage unit of a pharmaceutical composition provided herein can be administered every other day, every two days, every three days, once per week, twice per week, once every two weeks, once a month, twice a month, or once every two months.

[0176] In some embodiments a pharmaceutical composition provided herein is administered for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 months, about 6 months, about 9 months, about 1 year, or beyond. For example, a dosage unit can be administered weekly for about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, or about 3 months or more.

[0177] In some embodiments, the pharmaceutical compositions provided herein are suitable for local administration. For example, a pharmaceutical composition provided herein can be administered to a particular treatment area so as to provide local administration of the active ingredient(s) to the area in need of treatment. In some embodiments, relatively low systemic exposure of the pharmaceutical compositions provided herein occurs during said local administration. In some embodiments, the pharmaceutical composition is locally administered to the treatment area in the subject via injection.

[0178] In some embodiments, upon administration, the local concentration of a pharmaceutical composition provided herein in the treatment area is higher (e.g., about 2 to about 1,000, about 2 to about 900, about 2 to about 800, about 2 to about 700, about 2 to about 500, about 2 to about 400, about 2 to about 300, about 2 to about 200, about 2 to about 100, about 2 to about 50, about 5 to about 1,000, about 5 to about 900, about 5 to about 800, about 2 to about 700, about 5 to about 500, about 5 to about 400, about 5 toAttorney Docket No.57394-0003WO1 about 300, about 5 to about 200, about 5 to about 100, about 5 to about 50, about 5 to about 25, or about 5 to about 15 times higher) than the concentration of the pharmaceutical composition in an area outside the treatment area.

[0179] In some embodiments, a pharmaceutical composition provided herein is administered to one site in the treatment area. In some embodiments, a pharmaceutical composition provided herein is administered to two or more sites in the treatment area. For example, a pharmaceutical composition provided herein can be administered to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or 40 or more sites in the treatment area.

[0180] The pharmaceutical compositions provided herein can be administered at the same sites, adjacent sites, or nearby sites and at various frequencies and dosages as described herein.

[0181] In some embodiments each site receives a dosage unit of a pharmaceutical composition provided herein. In some embodiments, each dosage unit is administered as an injection. In some embodiments each site receives a divided dosage unit of a pharmaceutical composition provided herein. In some embodiments, each divided dosage unit is administered as an injection.

[0182] In some embodiments, a pharmaceutical composition provided herein is administered to one or more sites surrounding the treatment area in the subject. For example, a pharmaceutical composition provided herein can be administered to 2, 3, 4, 5, 10, or 15 or more sites surrounding the treatment area in the subject. In some embodiments, a pharmaceutical composition provided herein is administered by one or more injections to one or more sites surrounding a treatment area.

[0183] In some embodiments, a pharmaceutical composition provided herein can be administered to sites spaced throughout the treatment area. For example, a pharmaceutical composition provided herein can be administered such that injections of the pharmaceutical composition are spaced about 0.1 cm to about 5 cm apart (e.g., about 0.1 cm to about 1 cm, about 0.1 cm to about 2 cm, about 0.5 cm to about 1.5 cm, about 0.5 cm to about 2.5 cm, or about 1 cm to about 2 cm apart). In some embodiments, injections of the pharmaceutical composition are spaced about 0.1 cm to about 5 cm apart in a grid-like pattern. In some embodiments, a pharmaceutical composition provided herein is administered such thatAttorney Docket No.57394-0003WO1 injections of the pharmaceutical composition are spaced about 0.1 cm, about 0.5 cm, about 1 cm, about 1.5 cm, about 2 cm, about 3 cm, about 4 cm, or about 5 cm apart. Exemplary Embodiments

[0184] Embodiment 1 is a pharmaceutical composition comprising a particle comprising curcumin.

[0185] Embodiment 2 is the pharmaceutical composition of embodiment 1, wherein the pharmaceutical composition further comprises cinnamaldehyde or a derivative thereof.

[0186] Embodiment 3 is the pharmaceutical composition of embodiment 2, wherein the cinnamaldehyde derivative comprises cinnamaldehyde, 4-hydroxy-3-methoxy cinnamaldehyde, 2-hydroxy cinnamaldehyde, or a mixture thereof.

[0187] Embodiment 4 is the pharmaceutical composition of any one of embodiments 1-3, wherein the pharmaceutical composition further comprises resveratrol

[0188] Embodiment 5 is the pharmaceutical composition of any one of embodiments 1-4, wherein the particle is a liposome.

[0189] Embodiment 6 is the pharmaceutical composition of any one of embodiments 1-5, wherein the liposome encapsulates the curcumin.

[0190] Embodiment 7 is the pharmaceutical composition of any one of embodiments 2-6, wherein the liposome encapsulates the cinnamaldehyde or derivative thereof.

[0191] Embodiment 8 is the pharmaceutical composition of any one of embodiments 4-7, wherein the liposome encapsulates the resveratrol.

[0192] Embodiment 9 is the pharmaceutical composition of any one of embodiments 1-8, wherein the liposome comprises distearoylphosphatidylcholine (DSPC) and cholesterol.

[0193] Embodiment 10 is the pharmaceutical composition of embodiment 9, wherein the DSPC is present in an amount of about 45% to about 65% w / w of the total lipid content of the liposome.

[0194] Embodiment 11 is the pharmaceutical composition of embodiments 9 or 10, wherein the DSPC is present in an amount of about 55% w / w of the total lipid content of the liposome.Attorney Docket No.57394-0003WO1

[0195] Embodiment 12 is the pharmaceutical composition of any one of embodiments 9-11, wherein the cholesterol is present in an amount of about 30% to about 50% w / w of the total lipid content of the liposome.

[0196] Embodiment 13 is the pharmaceutical composition of any one of embodiments 9-12, wherein the cholesterol is present in an amount of about 40% w / w of the total lipid content of the liposome.

[0197] Embodiment 14 is the pharmaceutical composition of any one of embodiments 1-8, wherein the liposome comprises 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).

[0198] Embodiment 15 is the pharmaceutical composition of any one of embodiments 1-8, wherein the liposome comprises 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol.

[0199] Embodiment 16 is the pharmaceutical composition of embodiments 14 or 15, wherein the DMPC is present in an amount of about 75% to about 95% w / w of the total lipid content of the liposome.

[0200] Embodiment 17 is the pharmaceutical composition of any one of embodiments 14-16, wherein the DMPC is present in an amount of about 80% w / w of the total lipid content of the liposome.

[0201] Embodiment 18 is the pharmaceutical composition of any one of embodiments 14-16, wherein the DMPC is present in an amount of about 85% w / w of the total lipid content of the liposome.

[0202] Embodiment 19 is the pharmaceutical composition of any one of embodiments 14-16, wherein the DMPC is present in an amount of about 90% w / w of the total lipid content of the liposome.

[0203] Embodiment 20 is the pharmaceutical composition of any one of embodiments 14-16, wherein the DMPC is present in an amount of about 95% w / w of the total lipid content of the liposome.

[0204] Embodiment 21 is the pharmaceutical composition of any one of embodiments 15-20, wherein the cholesterol is present in an amount of about 1% to about 20% w / w of the total lipid content of the liposome.Attorney Docket No.57394-0003WO1

[0205] Embodiment 22 is the pharmaceutical composition of any one of embodiments 15-18, wherein the cholesterol is present in an amount of about 15% w / w of the total lipid content of the liposome.

[0206] Embodiment 23 is the pharmaceutical composition of any one of embodiments 15-21, wherein the cholesterol is present in an amount of about 5% w / w of the total lipid content of the liposome.

[0207] Embodiment 24 is the pharmaceutical composition of any one of embodiments 7-23, wherein the liposome further comprises an anionic lipid or a cationic lipid.

[0208] Embodiment 25 is the pharmaceutical composition of embodiment 24, wherein the anionic lipid is 1,2-distearoyl-sn-glycero-3-phosphoglycerol sodium salt (DSPG-Na) or 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol sodium salt (DMPG-Na).

[0209] Embodiment 26 is the pharmaceutical composition of embodiments 24 or 25, wherein the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP-Cl).

[0210] Embodiment 27 is the pharmaceutical composition of any one of embodiments 5-26, wherein the liposome further comprises DSPG-Na, DMPG-Na, DOTAP-Cl, polyethylene glycol-2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE- PEG2000), or mixtures thereof.

[0211] Embodiment 28 is the pharmaceutical composition of any one of embodiments 25-27, wherein the DSPG-Na is present in an amount of about 0.1% to about 15% w / w of the total lipid content of the liposome.

[0212] Embodiment 29 is the pharmaceutical composition of any one of embodiments 25-28, wherein the DSPG-Na is present in an amount of about 5% w / w of the total lipid content of the liposome.

[0213] Embodiment 30 is the pharmaceutical composition of any one of embodiments 26-29, wherein the DOTAP-Cl is present in an amount of about 0.1% to about 15% w / w of the total lipid content of the liposome.

[0214] Embodiment 31 is the pharmaceutical composition of any one of embodiments 26-30, wherein the DOTAP-Cl is present in an amount of about 5% w / w of the total lipid content of the liposome.Attorney Docket No.57394-0003WO1

[0215] Embodiment 32 is the pharmaceutical composition of any one of embodiments 27-31, wherein the DSPE- PEG2000 is present in an amount of about 0.1% to about 15% w / w of the total lipid content of the liposome.

[0216] Embodiment 33 is the pharmaceutical composition of any one of embodiments 27-32, wherein the DSPE- PEG2000 is present in an amount of about 5% w / w of the total lipid content of the liposome.

[0217] Embodiment 34 is the pharmaceutical composition of any one of embodiments 25-33, wherein the DMPG-Na is present in an amount of about 1% to about 20% w / w of the total lipid content of the liposome.

[0218] Embodiment 35 is the pharmaceutical composition of any one of embodiments 25-34, wherein the DMPG-Na is present in an amount of about 1% to about 10% w / w of the total lipid content of the liposome.

[0219] Embodiment 36 is the pharmaceutical composition of any one of embodiments 25-35, wherein the DMPG-Na is present in an amount of about 10% w / w of the total lipid content of the liposome.

[0220] Embodiment 37 is the pharmaceutical composition of any one of embodiments 25-35, wherein the DMPG-Na is present in an amount of about 5% w / w of the total lipid content of the liposome.

[0221] Embodiment 38 is the pharmaceutical composition of any one of embodiments 1-37, wherein the molar ratio of the curcumin to lipid is about 1:10 to about 1:14.

[0222] Embodiment 39 is the pharmaceutical composition of any one of embodiments 1-38, wherein the molar ratio of the curcumin to lipid is about 1:12.

[0223] Embodiment 40 is the pharmaceutical composition of any one of embodiments 2-39, wherein the molar ratio of the curcumin and cinnamaldehyde or derivative thereof to lipid is about 1:10 to about 1:14.

[0224] Embodiment 41 is the pharmaceutical composition of any one of embodiments 2-40, wherein the molar ratio of the curcumin and cinnamaldehyde or derivative thereof to lipid is about 1:12.

[0225] Embodiment 42 is the pharmaceutical composition of any one of embodiments 1-41, wherein the pharmaceutical composition comprises a liposome comprising curcumin, wherein the liposome encapsulates the curcumin.Attorney Docket No.57394-0003WO1

[0226] Embodiment 43 is the pharmaceutical composition of embodiment 42, wherein the liposome comprises a neutral phospholipid and an anionic lipid.

[0227] Embodiment 44 is the pharmaceutical composition of embodiment 43, wherein the neutral phospholipid comprises DMPC and the anionic lipid comprises DMPG-Na.

[0228] Embodiment 45 is the pharmaceutical composition of embodiment 44, wherein the DMPC is present in an amount of about 85% to about 95% w / w of the total lipid content of the liposome and the DMPG-Na is present in an amount of about 1% to about 20% w / w of the total lipid content of the liposome.

[0229] Embodiment 46 is the pharmaceutical composition of embodiment 45, wherein the DMPC is present in an amount of about 90% w / w of the total lipid content of the liposome.

[0230] Embodiment 47 is the pharmaceutical composition of embodiments 45 or 46, wherein the DMPG-Na is present in an amount of about 10% w / w of the total lipid content of the liposome.

[0231] Embodiment 48 is the pharmaceutical composition of any one of embodiments 44-47, wherein the DMPC and DMPG-Na are present at a molar ratio of about 90:10 DMPC:DMPG-Na.

[0232] Embodiment 49 is the pharmaceutical composition of any one of embodiments 42-48, wherein the molar ratio of curcumin to lipid is about 1:10 to about 1:14.

[0233] Embodiment 50 is the pharmaceutical composition of any one of embodiments 42-49, wherein the molar ratio of the curcumin to lipid is about 1:12.

[0234] Embodiment 51 is the pharmaceutical composition of any one of embodiments 42-50, embodiment 1, wherein the liposome further comprises cinnamaldehyde.

[0235] Embodiment 52 is the pharmaceutical composition of embodiment 51, wherein the cinnamaldehyde is present at a weight ratio of about 1:6 to about 1:3 cinnamaldehyde:curcumin.

[0236] Embodiment 53 is the pharmaceutical composition of embodiment 52, wherein the cinnamaldehyde is present at a weight ratio of about 1:5 cinnamaldehyde:curcumin.

[0237] Embodiment 54 is the pharmaceutical composition of any one of embodiments 51-53, wherein the molar ratio of the curcumin and cinnamaldehyde to lipid is about 1:10 to about 1:14.Attorney Docket No.57394-0003WO1

[0238] Embodiment 55 is the pharmaceutical composition of embodiment 54, wherein the molar ratio of the curcumin and cinnamaldehyde to lipid is about 1:12.

[0239] Embodiment 56 is the pharmaceutical composition of any one of embodiments 1-55, wherein the size of the particle is about 20 nm to about 500 nm.

[0240] Embodiment 57 is the pharmaceutical composition of any one of embodiments 1-56, wherein the size of the particle is about 20 nm to about 200 nm.

[0241] Embodiment 58 is the pharmaceutical composition of any one of embodiments 1-57, wherein the size of the particle is about 50 nm to about 100 nm.

[0242] Embodiment 59 is the pharmaceutical composition of any one of embodiments 1-57, wherein the size of the particle is about 50 nm to about 70 nm.

[0243] Embodiment 60 is the pharmaceutical composition of any one of embodiments 1-58, wherein the size of the particle is about 100 nm.

[0244] Embodiment 61 is the pharmaceutical composition of any one of embodiments 1-59, wherein the size of the particle is about 60 nm.

[0245] Embodiment 62 is a method for inducing cell death of a plurality of adipocytes in a treatment area in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition of any one of embodiments 1-61.

[0246] Embodiment 63 is the method of embodiment 62, wherein the cell death is due to apoptosis and / or necrosis.

[0247] Embodiment 64 is the method of embodiments 62 or 63, wherein the cell death is due to apoptosis.

[0248] Embodiment 65 is a method for inhibiting adipogenesis in a treatment area in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition of any one of embodiments 1-61.

[0249] Embodiment 66 is a method of reducing subcutaneous fat in a treatment area in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition of any one of embodiments 1-61.

[0250] Embodiment 67 is the method of embodiment 66, wherein the method comprises inducing apoptosis of a plurality of adipocytes in the treatment area in the subject.

[0251] Embodiment 68 is a method of improving the appearance of convexity orAttorney Docket No.57394-0003WO1 fullness associated with fat tissue in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition of any one of embodiments 1-61.

[0252] Embodiment 69 is the method of any one of embodiments 62-68, wherein the treatment area in the subject is in the abdomen, a flank, an inner thigh, an outer thigh, jowls, lower face, an arm, buttocks, chest, breast, back, or any other area of deposited subcutaneous fat of the subject.

[0253] Embodiment 70 is the method of embodiment 68, wherein the treatment area is in the back of the arm of the subject.

[0254] Embodiment 71 is the method of any one of embodiments 62-70, wherein the treatment area in the subject has cellulite, a lipoma, or liposarcoma.

[0255] Embodiment 72 is the method of any one of embodiments 62-71, wherein the treatment area in the subject has a subcutaneous fat deposit associated with temporal fat pad hyperplasia, pseudo herniation of orbital fat, Graves’ disease, Dercum's disease, multiple hereditary lipomatosis, familial multiple lipomatosis, Gardner syndrome, adiposis dolorosa, PTEN hamartoma tumor syndrome, Cowden syndrome, Madelung disease, or fatty infiltration.

[0256] Embodiment 73 is the method of any one of embodiments 62-72, wherein the pharmaceutical composition is locally administered to the treatment area in the subject.

[0257] Embodiment 74 is the method of any one of embodiments 62-73, wherein the pharmaceutical composition is locally administered to the treatment area in the subject via injection.

[0258] Embodiment 75 is the method of any one of embodiments 62-74, wherein the pharmaceutical composition is administered to the treatment area in the subject via at least two injections.

[0259] Embodiment 76 is the method of embodiment 75, wherein the pharmaceutical composition is administered to the treatment area in the subject via at least 5 injections.

[0260] Embodiment 77 is the method of any one of embodiments 62-73, wherein the pharmaceutical composition is locally administered to the treatment area in the subject via a cannula.

[0261] Embodiment 78 is the method of any one of embodiments 62-77, wherein theAttorney Docket No.57394-0003WO1 pharmaceutical composition is administered to one or more sites surrounding the treatment area in the subject.

[0262] Embodiment 79 is the method of any one of embodiments 61-78, wherein the pharmaceutical composition is administered to two or more sites in the treatment area in the subject.

[0263] Embodiment 80 is the method of any one of embodiments 61-79, wherein the pharmaceutical composition is administered to two or more sites surrounding the treatment area. EXAMPLES EXAMPLE 1. Assessment of Cytotoxicity and Apoptosis in Adipocyte-Like Cells. Methods

[0264] Cell Culture. The 3T3-L1 (ATCC, CL-173) mouse embryonic fibroblast cell line was thawed and cultured in maintenance medium consisting of DMEM (Gibco, 11995065) supplemented with 10% bovine calf serum and 1% Pen / Strep (Gibco, 15140122), until the appropriate confluence was reached. The cells were then passaged and seeded into 96-well plates at a seeding density of 1,250 cells / well. Once the cells reached 70-80% confluence, adipocyte differentiation was started. The medium was exchanged for supplemented DMEM medium containing 1% Pen / Strep, 0.5 mM IBMX (Sigma, 15879), 1 μM dexamethasone and 10 μg / mL insulin, then after three days the medium was exchanged for supplemented DMEM containing 1% Pen / Strep and 10 μg / mL insulin. The cells were cultured in the insulin containing medium for three days after which the medium was switched to maintenance medium. Forty-eight hours post-transition to maintenance medium, the cells were observed for adipocyte-like features and dosed. All cell culture occurred in a 37°C / 5% CO2 incubator.

[0265] Treatment. Upon observing adipocyte-like features in the culture, medium was exchanged for medium containing the test articles or controls and cultured for 48 hours. Table 1 shows all test articles and both stock and final concentrations. The vehicle control consisted of 1.25% DMSO; all wells except for the 10% DMSO control and 0.2% Triton X-100 control were normalized to 1.25% DMSO. Table 1. Test article combinations, manufacturer information and final concentrations.Attorney Docket No.57394-0003WO1 Test Article Final Stock Solvent / Concentration(s) Concentration[ ] a y a e ng. e conc us on o ours o es ar c e or con rol treatment, samples were washed three times in 1X PBS and then incubated with a 1:1000 dilution of eBIOSCIENCETMFIXABLE VIABILITY DYE eFLUORTM780 (Thermo Fisher, 65-0865-14) for 30 minutes at room temperature and then washed again three times with 1X PBS. The samples were fixed in 10% phosphate buffered formalin for 15 minutes at room temperature. After fixation, an additional three washes in 1X PBS were performed. Cells positive for the viability dye indicate non-viable cells.

[0267] Labeling. After incubation with the viability dye, the cells were prepared for labeling for cleaved caspase-3 (Cell Signaling Technologies, 9661). Labeling for cleaved caspase-3 allows for the quantification of cells that are actively undergoing apoptosis as caspase-3 is cleaved upon initiation of apoptosis. The labeling for cleaved caspase-3 was performed according to the manufacturer’s instructions. First the samples were blocked with 5% goat serum for 1 hour at room temperature; the primary antibody for cleaved caspase-3 was added at a dilution of 1:400 and allowed to incubate overnight at 4°C. The samples were then washed in PBS three times, and a secondary antibody (Thermo Fisher, A11011) and DAPI (Thermo Fisher, D3571), a nuclear label, were added for two hours atAttorney Docket No.57394-0003WO1 room temperature. The secondary was removed by washing three times with PBS before imaging.

[0268] Imaging. Images were captured in nine fields of view using a 20X objective in widefield mode of a Molecular Devices ImageXpress Micro Confocal High-Content imaging platform. During the scan, laser / dichroic settings were tuned for 405, 568 and 730 nm excitation wavelengths. All images were saved as 16-bit 2048x2048 TIF files for processing.

[0269] Image Analysis. The images were fed into a custom analysis pipeline, and using the nuclear channel, the location and number of nuclei were determined. The nuclear objects were then expanded to mimic the cell body. The fluorescent images (viability dye and cleaved caspase-3) were masked using these objects to determine the mean intensity of the fluorophore in each cell. Due to variable background in the cleaved caspase-3 labeled images, a background subtraction was performed prior to masking the images. The percentage of viability dye positive cells or cleaved caspase-3 positive cells was calculated by setting a threshold cutoff based on perceived positive labeling. Cells above the cutoff were designated as being positive for the respective label. The percentage of positive cells for each label was calculated by normalizing to the total cell number in each well.

[0270] Statistical Analysis. Statistical analysis was performed using GraphPad Prism statistical software. A one-way ANOVA with post hoc analysis via Dunnett’s multiple comparisons test was conducted to compare experimental groups to the vehicle control. Additional comparisons were performed between relevant groups. Values graphed are the mean percent of positive cells for the respective markers. All error bars correspond to standard deviation. Results

[0271] Viability and Apoptosis Labeling. The effect on viability of several treatments was assessed in adipocyte-like cells using a fluorescent viability dye, which allows for quantification of cells that have lost membrane integrity via uptake of the fluorescent dye. Fluorescent labeling was also performed to observe and quantify active apoptosis in treated adipocyte-like cells using cleaved caspase-3. Representative images can be seen in Fig.1 and Fig.2. Data were graphed as the percentage of cleaved caspase-3 positive cells (Fig.Attorney Docket No.57394-0003WO1 3) or percent viability (Fig.4). The vehicle control showed minimal cell death, with more than 90% viable cells and less than 20% apoptotic cells. In comparison, the positive control (0.2% Triton X-100) showed substantial cell death, as expected, and very few apoptotic cells. As seen previously, the 10% DMSO treated group showed a fairly high percentage of apoptotic cells in combination with substantial cell death.

[0272] All treatment groups showed statistically significant decreases in cell viability compared to the vehicle control (Fig. 4). Likewise, groups treated with 150 ppm of curcumin showed statistically significant increases in the percentage of apoptotic cells. Of the three cinnamaldehyde concentrations, only the lowest cinnamaldehyde treatment group (100 ppm) showed a statistically significant increase in apoptotic cells compared to the vehicle control. Co-treatment with curcumin and cinnamaldehyde significantly increased the percentage of apoptotic cells when comparing the 100 ppm cinnamaldehyde groups. Additionally, co-treatment with curcumin and resveratrol, 4-hydroxy-3- methoxycinnamalehyde or cinnamaldehyde resulted in a statistically significant increase in the percentage of apoptotic cells compared to curcumin alone.

[0273] Summary. The effect of select test conditions on cell viability and apoptosis was evaluated in adipocyte-like cells. All controls behaved as expected. All test conditions showed low levels of cell viability with all samples treated with curcumin also showing high levels of apoptosis. Co-treatment of samples with curcumin and three other test compounds (resveratrol, 4-hydroxy-3-methoxycinnamalehyde or cinnamaldehyde) resulted in a statistically significant increase in the percentage of apoptotic cells compared to curcumin alone. EXAMPLE 2. Preparation of Liposome Formulations.

[0274] The active pharmaceutical ingredient (API) and lipid were dissolved in 95% ethanol. See Table 2 for the concentrations of the lipids and API. To form the liposome, a solution of 10 mM acetate buffered solution with a pH of 5 was mixed with the API / lipid 95% ethanol solution. Once the liposome was formed, the solution was concentrated until the API concentration was between 1-10 mg / mL or between 5-10 mg / mL.Attorney Docket No.57394-0003WO1 Table 2. Composition of Liposome Formulations. Composition # Lipids (Molar Ratio) API E. y o ox c y apop os s assay n a pocy e- e ce s.

[0275] 3T3-L1 fibroblasts were differentiated into adipocyte-like cells using the protocol described in Example 1. These cells were then treated with test articles (see Tables 3 and 4) for 48 hrs before being assayed for viability and apoptosis using fluorescence microscopy (see Example 1 for protocol). Image analysis was used to score the proportion of apoptotic and viable cells in the population. See Figs. 5-6 for the test articles in Table 3 and Figs. 7-8 for the test articles in Table 4. The curcuminoid extract was an extract from Curcumin longa and contained about 80% w / w curcumin, about 15% w / w deemethoxycurcumin, and about 5% w / w bisdemethoxycurcumin. Table 3. Test article combinations and final concentrations. Test Article Final Stock Solvent / i i nAttorney Docket No.57394-0003WO1 Curcuminoid extract (Sigma) 150 ppm 1.5% DMSO Curcumin 200 ppm 1.5% DMSOTable 4. Test article combinations and final concentrations. Test Article Final Stock Solvent / Concentration(s) ConcentrationAttorney Docket No.57394-0003WO1 Curcumin / Resveratrol 150 ppm / 50 ppm 1.5% DMSO Curcumin / Resveratrol 150 ppm / 37.5 ppm 1.5% DMSO EXAMwing Test Article Administration via Inguinal Fat Pads Injections.

[0276] This study measured changes in adipose tissue following test article administration into fat pads in ZDF rats. Methods

[0277] Test Materials. The control article (also referred to as the Vehicle) was 10 mM acetate buffered saline. The Test Articles (TAs) were as shown in Table 5. Test Article TA1 was a liposomal curcumin formulation composed of DMPC, DMPG, and cholesterol lipids and 5.4 mg / mL concentration of curcumin. TA2 was a liposomal curcumin formulation composed of DMPC, DMPG, and cholesterol lipids and 7.1 mg / mL of curcumin. TA3 was a liposomal curcumin and resveratrol formulation composed of DMPC, DMPG, and cholesterol and 5.6 mg / mL curcumin and 0.8 mg / mL resveratrol. Table 5. Test Articles. se Level Dos Total Dose Group Do e Dose Volume per b

[0278] The Vehicle and TAs were stored in a refrigerator set to maintain 4 °C until use. Each day of dosing, the Vehicle and TAs were removed from the refrigerator and allowed to warm to ambient temperature for at least 30 minutes before dosing.Attorney Docket No.57394-0003WO1

[0279] Animals.21 (18 plus 3 spares) ZDF-Leprfa / Crl male rats were used in this study. The target age at the initiation of dosing was at least 9-10 weeks. The target weight at the initiation of dosing was 330-400g. LabDiet 5008 was provided ad libitum throughout the study, except during designated procedures. The same diet in meal form may be provided to individual animals as warranted by clinical signs (e.g., broken / damaged incisors or other health changes).

[0280] Experimental Design. Rats were assigned to Groups on Days -2 or -3 (prior to Day 1), so that there would be no significant differences between groups with respect to body weight and fat mass from echoMRI.

[0281] The first day of dosing was designated as Day 1. The test article was administered to animals in bilateral injections in inguinal fat pads. Prior to loading the syringes for each dosing group, test material was mixed by gently swirling and gentle end- to-end inversion at least 5 times (~10 seconds). The area may be shaved prior to dosing. The Test Material (Vehicle or Test Articles (TA) 1, 2, or 3) was administered by direct injection with appropriate size insulin syringe into the fat pads. Groups 1-4 were administered the Test Material on Day 1, Day 3 and Day 5. The Test Material (Vehicle or TA1, TA 2, or TA 3) and dosages for each Group are provided in Table 5.

[0282] Body composition was measured on Day -3, Day 14 for Groups 2-5. The animals were taken from their home cage and placed into a plastic restrainer compatible with the body composition analysis machine (EchoMRI). The restrainer was placed into the EchoMRI, body composition as assessed, and the animal was returned to their home cage. Animals were in the restrainer for no longer than 5 minutes.

[0283] Terminal Procedures. Terminal procedures are summarized in Table 6. Table 6. Terminal Procedures. Scheduled Necropsy Procedures t tAttorney Docket No.57394-0003WO1

[0284] Animals in Groups 1-4 were euthanized via CO2 asphyxiation on Day 15. The terminal body weight was measured. Following CO2asphyxiation, tissue was collected from the animals as indicated below.

[0285] The tissue identified for weighing was the inguinal fat pads (left and right). Tissue weights were not recorded for animals found dead. Inguinal fat tissue (from left and right pads) was collected and weighed (separately) for all animals. Following weight recording, the fat tissue was discarded. Results

[0286] The excised inguinal fat pads were collected and weighed for all animals. The results from the rats are shown below in Tables 7-10. The mean inguinal fat pad data was normalized by the mean weight of the rat and then compared to the normalized Control inguinal fat pad data. The results showed a 24.2% reduction of inguinal fat in test article TA1, a 29.1% reduction of inguinal fat in test article TA2, and a 28.1% reduction of inguinal fat in test article TA3. Table 7. Inguinal Fat Pad Weights for Control. Rat Weight R Fat Pad L Fat Pad ) )Attorney Docket No.57394-0003WO1 SD 33.1 3.95 2.96 Tabe . ngu na a a e g s or . TA2 Rat Weight R Fat Pad L Fat Pad (g) Weight (g) Weight(g)Table 10. Inguinal Fat Pad Weights for TA3. TA3 Weight (g) R Fat Pad L Fat Pad Weight (g) Weight (g)

[0287] Theanimals are shown in FIGS.9A and 9B. Food consumption is shown in FIG.10. Fat and lean mass are shown in FIGS.11A and 11B, respectively.

[0288] These results show that a negatively charged liposome containing curcumin that is injected locally into subcutaneous fat, which is highly lipophilic, can cause cell death of the fat tissue adipocytes. EXAMPLE 5. Liposomal Curcumin Preparation and Characterization - Liposome Charge and Curcumin Degradation. Methods

[0289] All liposome preparation was completed using the DIANT Pharma Inc., Lift System. The Lift system is a continuous liposome formation device that allows for bothAttorney Docket No.57394-0003WO1 particle formation and concentration. Particle formation used the DIANT Jet, a turbulent co-flow ethanol injection.

[0290] Liposome concentration was achieved through a single pass tangential flow filtration (SPTFF) system. All TFF runs utilized Repligen mPES 100 / 300 kDa cassettes. A generalized process flow diagram is shown in FIG.12.

[0291] A Malvern Zetasizer Pro Red was used to determine the particle size, polydispersity index (PDI), and zeta potential of the liposomes. Particle size and PDI measurements used 12 mm o.d. square polystyrene cuvettes with stoppers (Malvern Panalytical Part # DTS0012). Zeta potential measurements used Zetasizer disposable folded capillary cells (Malvern Panalytical Part # DTS1070).

[0292] The Thermo Scientific Vanquish HPLC was used for curcumin (Table 11) and lipid (Table 12) concentration. Table 11. HPLC column information for curcumin concentration. Column Agilent Poroshell 120 EC-C18, 4.6 x 100mm, 2.7μm. . Reversed-phase HPLC BEH (Bridged Ethylene C l H b id l C18 13 i 35Attorney Docket No.57394-0003WO1 Total Runtime 19 min followed by equilibration for 2 min CAD Evaporation Temperature 35°C [ation reported. Results

[0294] Initial trials focused on two factors, liposome charge and curcumin degradation. Three different charged systems were investigated: anionic, cationic, and neutral (Table 13). See Table 14 for the liposome components and ratios. Both 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP) and N-(carbonyl-methoxypolyethylene glycol)- 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-mPEG2000) systems had rapid formulation of curcumin crystals (< 48 hr), large particle size (>> 150 nm), high polydispersity index (PDI) (> 0.2), and a general inability to stably encapsulate curcumin. Table 13. Liposome systems. System Charge Ionized / PEGylated Lipid Anionic DSPG Table 14. Ip p . Lipid Composition (M %) D P

[0295] Curcumigradation in basic and neutral aqueous conditions. Ionic conditions can further impact the rate of degradation. Phosphate buffered saline (PBS) and an acidic 0.9 % NaCl solution were tested for their ability to mitigate hydrolytic degradation (Table 15). 1x PBS at pH 4 showed minimalAttorney Docket No.57394-0003WO1 degradation compared to all other tested solutions. A combination of buffer capacity and acidic pH were required for proper curcumin stability.

[0296] Further studies were conducted to investigate both histidine and acetate buffers. There was no apparent difference between histidine and acetate buffer for curcumin stability.10 mM acetate buffer at pH 4.5 was selected as it was shown to have the lowest injection site pain while still providing buffering capacity and the requisite acidic pH. Table 15. Impact of aqueous phase composition and pH on curcumin stability over time. Purity (%) Formulation pH Day 0 Day 6

[0297] mpact o Curcum n ur ty. rev ous test ng was comp eted us ng off-the-shelf curcumin provided by Millipore Sigma (Product #C7727), which was a mixture of curcuminoids with curcumin content being ≥ 80%. A switch was made to a custom synthesized curcumin from Patheon API Services (Product #AFB). This material had a purity of ≥ 97%. The Patheon curcumin was unable to be encapsulated in the DSPC / DSPG / cholesterol liposome system. It was hypothesized that pure curcumin could compete with cholesterol for space in the bilayer. To test this, liposomes were formulated with various cholesterol levels ranging from 35–50 M %. Crystal growth rate and amount were higher in the liposomes with increased cholesterol.

[0298] Next, liposomes formulated with no cholesterol were compared using DSPC and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) as the main lipids with DSPG as the anionic lipid for both (90:10 M ratio). The shorter acyl chain length of DMPC could have a stabilizing effect as it would allow for increased hydrogen bond interactions between curcumin and the headgroup. Of the tested formulations, crystals only formed in those containing DSPC. Interestingly, a decrease in particle size was seen in all DMPC formulations compared to DSPC (25 nm and 50 nm respectively).Attorney Docket No.57394-0003WO1

[0299] Optimization of DMPC Liposomes. Further testing was run on process conditions for optimization of the DMPC / DSPG liposomes. Factors tested were ethanol purity (95 % vs.100 %), M of acetate buffer (10 mM vs.100 mM), acetate buffer pH (4.5 vs.5) ethanol to buffer flow rate at mix (1:1 vs.5:1), M % DSPG (5% vs 10%).

[0300] Observations were as follows: ^ 95% ethanol increased particle size by 30%. ^ Buffer M did not have an impact. ^ A flow rate of ~ 2:1 increased particle size by 50%. ^ Neither DSPG amount, nor acetate buffer pH had an impact.

[0301] To further improve the particles, DSPG was replaced with 1,2-dimyristoyl-sn- glycero-3-phospho-(1'-rac-glycerol) (DMPG), which was hypothesized to have a similar stabilizing effect seen when switching from DSPC to DMPC. Processing conditions were further modified to target a liposome in the 60 – 90 nm size range which has previously been shown to be optimal for uptake via endocytosis. Specifically, ethanol to buffer flow rate ratio (40:90 mL / min vs.40:60 mL / min) and the inclusion 0.9 % NaCl in the acetate buffer solution. The combination of acetate buffer saline and lower flow rates, 40:60 mL / min achieved particles ~ 70 nm with PDI ~ 0.1. Solvent optimization was done to improve liposome PDI while maintaining particle size (Error! Reference source not found.13).95% ethanol allowed for greater control over PDI while allowing for a range of particle sizes.

[0302] Inclusion of Cholesterol. With ethanol purity, flow rate, ionized lipid, acetate buffer M and acetate buffer pH conditions selected, the inclusion of cholesterol into the liposome composition was revisited. M % cholesterol had an inverse relationship with size and PDI (Figures 14A and 14B). To further lower PDI, injection temperature was raised from 35 °C to 45 °C. The resultant cholesterol formulations were then stored at 4 °C to investigate stability. After 11 days, curcumin crystal formation occurred in all cholesterol containing liposomes. Discussion

[0303] Processing conditions were as shown in Table 16 with resulting liposome properties shown in Table 17.Attorney Docket No.57394-0003WO1 Table 16. Liposome formation parameters after initial development work. A1 Phase 10 mM Acetate Buffer, pH=5.0 A1 Flow Rate 60 mL / minmation parameters. Z-Average Zeta Potential Curcumin Loading (nm) PDI (mV) (mg / mL) EXAMPL. p p .

[0304] The impact of formation temperature, total lipid:API M ratio, and DMPG M % on the z-average size, PDI, and zeta potential of curcumin liposomes was studied. Methods

[0305] A full factorial, 3x2 design with one center point was utilized to investigate several parameters (Table 18). Levels were chosen based on prior experimentation in which unstable particle formation was observed at temperatures under 35 °C, with ease of formation increasing up to 45 °C. The upper bound of 65 °C was chosen to investigate a previously seen linear relationship between temperature and PDI. Stable particles could not form at a lipid:API M ratio below 9. Particles were unstable above 12.5 M % DMPG and lacked sufficient charge to prevent storage aggregation below 5 M %. A center point was included to give insight to possible non-linear responses. Table 18.3x2 design pattern with associated factor levels. Pattern Formation Lipid:API DMPGAttorney Docket No.57394-0003WO1 +−+ 65 9 10 ++− 65 12 5 Results

[0306] Testing was completed on all samples following sterile filtration of the final product (Table 19), except for Sample 7, which could not be filtered due to large size and high PDI. Table 19. Testing Results. Pattern Sample No. Z-Average Zeta Potential (nm) PDI (mV)

[0307] The results were fit to a full factorial design using JMP. Model simplification followed a hierarchical approach, with non-significant (p > 0.05) higher order terms being removed in a sequential manner. Residuals plots were assessed to ensure removal did not impact normality or homoscedasticity. The resulting final model factors and associated p- values are shown in Table 20. Table 20. Summary of final model factors and associated p-values. Terms p-valueAttorney Docket No.57394-0003WO1

[0308] Model residuals indicated a non-normal response (i.e. tail curvature) in the normal quantile plots for both z-average and zeta potential. This curvature is driven almost exclusively by the midpoint, Sample 9, which points to either a non-linear response or an outlier result. To investigate the possible non-linearity a confirmation run was completed. An intermediate DMPG value was chosen for simplicity’s sake as it showed a statistically significant parameter effect for both variables. Assuming a linear response our model prediction was as follows: z-average – 77.6 nm, PDI – 0.104, zeta potential – -28.3 mV.

[0309] The resulting sample, Sample 10, had properties as follows: z-average – 78.9 nm, PDI – 0.109, zeta potential – -24.8 mV (Table 21). As such, the midpoint, Sample 9, was an outlier as the confirmation run matched expected results from our linear approximation model. To ensure that the midpoint did not further impact normality, it was removed and the model reassessed. Table 21. Model prediction versus experimental result. Z-Average Zeta Potential (nm) PDI (mV)

[0310] Removal of the midpoint, increased effect significance (Table 22), improved residual normality, and tightened 95% confidence intervals (CI) for both z-average and PDI. Table 22. Model effect summary after removal of outlier. Terms p-value *

[0311] Further confirmation runs that varied DMPG M % (temperature – 45 °C; lipid:API – 12:1), were completed (Table 23) and were in-line with model predictions.Attorney Docket No.57394-0003WO1 Table 23. Model predictions versus experimental results. DMPG (M %) Sample No. Z-Average PD Zeta Potential (nm) I (mV) [0031. , were also screened for stability. In brief, after sterile filtration samples were stored at 4 °C, protected from light. Periodically, aliquots of samples were removed and imaged using polarized light microscopy (PLM) to discern if any crystal growth had occurred. Due to its low solubility, 0.6 µg / mL, any free curcumin readily crystallized. During sampling, liposomes were also inspected visually for color change and agglomeration. Free curcumin in solution rapidly breaks down and undergoes a color shift from bright yellow to orange. Additionally, agglomeration shows as settling at the bottom of storage container. A distinct pattern emerged during sample storage, any samples with lower lipid:API ratio (i.e. 9:1) saw major crystal growth within 2-3 weeks of the initiation of storage. Samples at the higher ratio, 12:1, have yet to show major crystal growth. Discussion

[0313] This model has shown high fidelity and reproducibility in the prediction of liposomal formulation properties. DMPG is the main driver behind z-average, PDI, and zeta potential. Temperature influences zeta potential but with a known trade-off with curcumin stability. For this reason, a formation temperature of 45 °C is used. Additionally, appropriate storage stability can only be reached at lipid:API ratios greater than or equal to 12:1 and will thus act as our the lower bound in future formulations. Specific size targets can be met by variation of DMPG M %, with the focus on particles in the size range of 60- 80 nm. This will help to maximize cellular uptake, while also maximizing loading potential of the liposome. Zeta potential is targeted to be below -25 mV to provide repulsion duringAttorney Docket No.57394-0003WO1 storage and avoid agglomeration. Finally, PDI is targeted at < 0.20 to ensure proper particle uniformity.

[0314] Process parameters for the encapsulation of curcumin in stably formed liposomal nanoparticles were successfully determined. The resulting liposomes are highly reproducible with advantageous properties for shelf-life, curcumin loading, and cellular uptake. The resulting parameters and associated liposome properties are listed below (Tables 24 and 25). Table 241. Final process conditions. A1 Phase 10 mM Acetate Buffer, pH=5.0 A1 Flow Rate 60 mL / min HAssociated Liposome Properties EXAMPLE 7. Efficacy Study to Monitor Changes in Adipose Tissue Following Sample Administration via Inguinal Fat Pads Injections.

[0315] The objective of this study was to measure changes in adipose tissue following test article administration into fat pads in ZDF rats. Methods

[0316] Test Materials. The control article (also referred to as the Vehicle) was 10 mM acetate buffered saline. The Samples were as shown in Table 26.Attorney Docket No.57394-0003WO1 Table 26. Samples. Sample 1 Sample 2 Sample 3

[00317] Experimental Design. The study methods were similar to that of Example 4 with the following experimental design: Table 27. Experimental Design. Dose Tota Dose Volume erial Dose Leve l Dose Group Test Mat l Concentration Volume per Site Animal 02 04 04 04

[0318] Dose formulations were administered to animals via bilateral injection into inguinal fat pads as detailed in Table 27. Animals in Groups 1 to 3 were dosed once on Days 1, 3, and 5. Animals in Group 4 were dosed on Day 1 and Day 5.

[0319] Nineteen male ZDF-Leprfa / Crl rats were received from Charles Rivers Laboratories in Raleigh, North Carolina. At the initiation of dosing animals weighted 383- 569 g and were 10-11 weeks of age. Animals were assigned to Groups 1 to 4 to ensure there was no significant differences in body wight. Animals were provided with LabDiet 5008 and municipal tap water ad libitum throughout the study.Attorney Docket No.57394-0003WO1

[0320] Animals were euthanized by carbon dioxide asphyxiation on Day 15 (Groups 1 to 4).

[0321] The following parameters and endpoints were evaluated in this study: mortality, clinical observations, body weights, food consumption, tissue weights (inguinal fat), and macroscopic examinations. Results

[0322] The excised inguinal fat pads were collected and weighed for all animals. The results from the rats are shown below in Tables 28-31. Table 28. Inguinal Fat Pad Weights for Control. Group 1 Terminal Body Adipose Adipose 0 mg / kg per dose Wt (g) Inguinal L (g) Inguinal R (g)Table 29. Inguinal Fat Pad Weights for Sample 1. Group 2 Terminal Adipose Adipose 40 m / k er dose Bod Wt () Inuinal L () Inuinal R (g)abe 30. nguna at ad egts or Sampe . Group 3 Terminal Adipose Adipose InguinalAttorney Docket No.57394-0003WO1 3004 576 16.057 14.234 Mean 547.3 15.9828 15.5723Group 4 Terminal Body Adipose Adipose 40 mg / kg per dose Wt (g) Inguinal L (g) Inguinal R (g)

[0323] Administration of Sample 1, Sample 2, and Sample 3 by subcutaneous injection was well tolerated in ZDF rats at levels of 40 mg / kg / day. EXAMPLE 8. Efficacy Study to Monitor Changes in Adipose Tissue Following Sample Administration via Inguinal Fat Pads Injections.

[0324] The objective of this study was to measure changes in adipose tissue following test article administration into fat pads in ZDF rats. Methods

[0325] Test Materials. The control article (also referred to as the Vehicle) was 10 mM acetate buffered saline. The Samples were as shown in Table 32. Table 32. Samples. Sample 1 Sample 2 Sample 3 Sample 4 nAttorney Docket No.57394-0003WO1 Purity 99.17 99.17 99.17 99.17 5 mg / ml curcumin 5 mg / ml curcumin 4 mg / ml curcumin inwith the following experimental design: Table 33. Experimental Design. Group Dose Total Dose Dose Volume No.SampleDose Level(mg / kg / dose) Concentration Volume per Site Animal No. 4 4 4 4 4 b

[0327] Dose formulations were administered to animals via bilateral injection into inguinal fat pads as detailed in the experimental design table. Animals in Groups 1 to 5 were dosed once on Days 1, 3, and 5.

[0328] Twenty male ZDF-Leprfa / Crl rats were received from Charles Rivers Laboratories in Raleigh, North Carolina. At the initiation of dosing animals weighted 350- 600g and were 9-10 weeks of age. Animals were assigned to Groups 1 to 5 to ensure there was no significant differences in body wight. Animals were provided with LabDiet 5008 and municipal tap water ad libitum throughout the study. Animals were euthanized by carbon dioxide asphyxiation on Day 15 (Groups 1 to 5).

[0329] The following parameters and endpoints were evaluated in this study: mortality, clinical observations, body weights, food consumption, and macroscopic examinations. Results

[0330] The excised inguinal fat pads were collected and weighed for all animals. The results from the rats are shown below in Tables 34-38. The mean inguinal fat pad data was normalized by the mean weight of the rat and then compared to the normalized Control inguinal fat pad data.Attorney Docket No.57394-0003WO1 Table 34. Inguinal Fat Pad Weights for Control. Group 1 (0 mg / kg Terminal Body Adipose Adipose Wt (g) Inguinal L (g) Inguinal R (g)Table 35. Inguinal Fat Pad Weights for Sample 1. Group 2 (50.3 mg / kg Terminal Adipose Adipose B d Wt I i l L I i l R )Table 36. Inguinal Fat Pad Weights for Sample 2. Group 3 (488 m / k Terminal Body Adipose Adipose )Table 37. Inguinal Fat Pad Weights for Sample 3.Attorney Docket No.57394-0003WO1 Group 4 (40.1 mg / kg Terminal Body Adipose Adipose Wt (g) Inguinal L (g) Inguinal R (g)Group 5 (40 mg / kg Terminal Body Adipose Adipose Wt ( ) I i l L ( ) I i l R ( )

[0331] als are shown in FIGS. 15A and 15B. Food consumption is shown in FIG. 16. Fat and lean mass are shown in FIGS.17A and 17B, respectively.

[0332] In conclusion, administration of test articles by bilateral injections in the inguinal fat pads was well tolerated in ZDF rats at levels of 40-50.3mg / kg / dose.

[0333] A summary of the formulations and results from Examples 4, 7, and 8 is provided in Table 39. Table 39. Summary Sample Normalized Particle Fat Size PDI Zeta Lipid:API DMPC DMPG CholesterolAttorney Docket No.57394-0003WO1 OTHER EMBODIMENTS

[0334] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No.57394-0003WO1 WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising a liposome comprising curcumin present at a molar ratio of about 1:10 to about 1:14 curcumin:lipid.

2. The pharmaceutical composition of claim 1, wherein the liposome comprises a neutral phospholipid and an anionic lipid.

3. The pharmaceutical composition of claim 2, wherein the neutral phospholipid is present in an amount of about 88% to about 96% w / w of the total lipid content of the liposome.

4. The pharmaceutical composition of any one of claims 2-3, wherein the neutral phospholipid is present in an amount of about 90% to about 94% w / w of the total lipid content of the liposome.

5. The pharmaceutical composition of any one of claims 2-4, wherein the neutral phospholipid comprises distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero- 3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1-palmitoyl-2-oleoyl-glycero-3- phosphocholine (POPC), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dierucoyl- sn-glycero-3-phosphocholine (DEPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or a combination thereof.

6. The pharmaceutical composition of any one of claims 2-5, wherein the neutral phospholipid comprises DMPC.

7. The pharmaceutical composition of any one of claims 2-6, wherein the anionic phospholipid comprises 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol sodium salt (DPPG-Na), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol sodium salt (DMPG-Na), 1,2- distearoyl-sn-glycero-3-phosphoglycerol sodium salt (DSPG-Na), or a combination thereof.Attorney Docket No.57394-0003WO1 8. The pharmaceutical composition of any one of claims 2-7, wherein the neutral phospholipid and anionic phospholipid are present at a molar ratio of about 92:8 neutral phospholipid:anionic phospholipid.

9. The pharmaceutical composition of any one of claims 2-8, wherein the neutral phospholipid comprises DMPC and the anionic lipid comprises DMPG-Na.

10. The pharmaceutical composition of claim 9, wherein the DMPC is present in an amount of about 88% to about 96% w / w of the total lipid content of the liposome and the DMPG-Na is present in an amount of about 6% to about 12% w / w of the total lipid content of the liposome.

11. The pharmaceutical composition of any one of claims 9-10, wherein DMPC and DMPG-Na are present at a molar ratio of about 92:8 DMPC:DMPG-Na.

12. The pharmaceutical composition of any one of claims 1-11, wherein the molar ratio of the curcumin to lipid is about 1:

12.

13. The pharmaceutical composition of any one of claims 1-12, wherein the size of the liposome is about 50 nm to about 100 nm.

14. The pharmaceutical composition of any one of claims 1-13, wherein the size of the liposome is about 80 nm.

15. The pharmaceutical composition of any one of claims 1-14, wherein the pharmaceutical composition comprises an acetate buffer.

16. The pharmaceutical composition of any one of claims 1-15, wherein the pharmaceutical composition comprises sodium chloride.

17. The pharmaceutical composition of any one of claims 1-16, wherein the pharmaceutical composition has a pH of about 5Attorney Docket No.57394-0003WO1 18. The pharmaceutical composition of any one of claims 1-17, wherein the pharmaceutical composition comprises 10 mM acetate buffer and 0.9% w / w NaCl.

19. The pharmaceutical composition of any one of claims 1-18, wherein the pharmaceutical composition has a PDI of about 0.

1.

20. The pharmaceutical composition of any one of claims 1-19, wherein the pharmaceutical composition has a Z-average of about -40 mV.

21. The pharmaceutical composition of any one of claims 1-20, wherein the liposome encapsulates the curcumin.

22. The pharmaceutical composition of any one of claims 1-21, wherein the curcumin comprises at least about 99% by weight of the total curcuminoids present in the pharmacetucial composition.

23. A method for inducing cell death of a plurality of adipocytes in a treatment area in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition of any one of claims 1-21.

24. The method of claim 22, wherein the cell death is due to apoptosis and / or necrosis.

25. The method of claims 22 or 23, wherein the cell death is due to apoptosis.

26. A method for inhibiting adipogenesis in a treatment area in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition of any one of claims 1-21.

27. A method of reducing subcutaneous fat in a treatment area in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition of any one of claims 1-21.Attorney Docket No.57394-0003WO1 28. The method of claim 26, wherein the method comprises inducing apoptosis of a plurality of adipocytes in the treatment area in the subject.

29. A method of improving the appearance of convexity or fullness associated with fat tissue in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition of any one of claims 1-21.

30. The method of any one of claims 22-28, wherein the treatment area in the subject is in the abdomen, a flank, an inner thigh, an outer thigh, jowls, lower face, an arm, buttocks, chest, breast, back, or any other area of deposited subcutaneous fat of the subject.

31. The method of claim 29, wherein the treatment area is in the submental area, jowls, or buccal area of the lower face.

32. The method of any one of claims 22-30, wherein the treatment area in the subject has cellulite, a lipoma, or liposarcoma.

33. The method of any one of claims 22-31, wherein the treatment area in the subject has a subcutaneous fat deposit associated with temporal fat pad hyperplasia, pseudo herniation of orbital fat, Graves’ disease, Dercum's disease, multiple hereditary lipomatosis, familial multiple lipomatosis, Gardner syndrome, adiposis dolorosa, PTEN hamartoma tumor syndrome, Cowden syndrome, Madelung disease, or fatty infiltration.

34. The method of any one of claims 22-32, wherein the pharmaceutical composition is locally administered to the treatment area in the subject.

35. The method of any one of claims 22-33, wherein the pharmaceutical composition is locally administered to the treatment area in the subject via injection.

36. The method of any one of claims 22-34, wherein the pharmaceutical composition is administered to the treatment area in the subject via at least two injections.Attorney Docket No.57394-0003WO1 37. The method of claim 35, wherein the pharmaceutical composition is administered to the treatment area in the subject via at least 5 injections.

38. The method of any one of claims 22-36, wherein the pharmaceutical composition is locally administered to the treatment area in the subject via a cannula.

39. The method of any one of claims 22-37, wherein the pharmaceutical composition is administered to one or more sites surrounding the treatment area in the subject.

40. The method of any one of claims 22-38, wherein the pharmaceutical composition is administered to two or more sites in the treatment area in the subject.

41. The method of any one of claims 22-39, wherein the pharmaceutical composition is administered to two or more sites surrounding the treatment area.

Images