WO2022241331A2 - Polymer-lipid hybrid nanoparticles of emricasan and use thereof - Google Patents

Abstract

The invention provides novel polymer-lipid nanopartides comprising emricasan, pharmaceutical compositions thereof, as well as methods for their preparation and use in cancer treatment.

Description

POLYMER-LIPID HYBRID NANOPARTICLES OF EMRICASAN AND USE THEREOF

Priority Claims and Related Applications

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63/188,224, filed May 13, 2021, the entire content of which is incorporated herein by reference for all purposes.

Technical Fields of the Invention

[0002] The invention generally relates to pharmaceuticals and therapeutic methods. More particularly, the invention provides novel polymer-lipid nanoparticles comprising emricasan and methods for their preparation and use in cancer treatment.

Background of the Invention

[0003] Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Cancer remains a serious global health threat. Both locoregional and metastatic cancer treatments need improvement. Historically, few cancer drugs were designed for locoregional use (Oladeru OT, et al. JAMA Oncol. 2020;6:1863), Instead, they were extensions of metastatic cancer medicines. The therapeutic window for metastatic cancer drugs tends to be narrow, resulting in cost of life quality for the moderate gain of life span. Immunotherapies, especially immune checkpoint inhibitors (ICI), are poised to drastically change the landscape. The response rate for ICI, however, is low. New therapies that can enhance and expand the efficacy of ICI are urgently needed (Kubli SP, et al. Nat Rev Drug Discov. 2021:1).

[0004] In situ vaccination (ISV, defined as any chemical agent or other therapeutic intervention that is able to stimulate a treated tumor lesion to release systemically active anti-cancer chemicals and/or activate cancer-killing immune cells) represents a rare opportunity in this context (Kepp O, et al. Nat Rev Clin Oncol. 2020;17:49-64.). ISVs may be noncytotoxic themselves and can be given intratumorally, which may create large enough therapeutic windows for early-stage cancer treatment. The immune memory may also prevent relapse and metastasis, which is a desirable attribute for locoregional cancer therapies. In the metastatic settings, ISVs can produce systemic cancer control and enhance the efficacy of ICI. Acute infection-associated spontaneous remission and abscopal effects observed in radiation therapy suggest that ISV can be a practical approach (Siva S, et al. Cancer Lett. 2015;356: 82). The launch of oncolytic virus therapy T-Vec serves as a proof-of- concept to validate this strategy with several other ISVs showing early clinical efficacy (Franke V, et al. Intl J Cancer. 2019;145:974-8.).

[0005] Unlike the current ISV approaches encompassing oncolytic virus, innate immune activators such as STING and TLR agonists, the present invention takes advantage of the immune modulating functions of caspases during tumor cell deaths and tumor antigen release. Apoptosis is an immune silent cell death that is preferred by tumor cells under the assault of chemoradiotherapy to minimize the collateral damage to the tumor tissue. Some tumors hijack the process to promote tumor growth (apoptosis induced proliferation, AiP. Rosenbaum SR, et al. Cancer Discov.

2021;11:266).

[0006] Caspases are the executioners of apoptosis but several nonapoptotic functions are emerging (Arama E, et al. FEES J. 2020). Cells rarely survive post the key event of mitochondrial outer membrane permeabilization (MOMP) regardless of the activity of caspases. It was discovered recently that blockade of caspase activity after MOMP switches apoptosis to caspase-independent cell deaths (CICD). Depending on the nature of CICD, innate and adapt immune systems can be activated to various degrees (Giampazolias E, et al. Nat Cell Biol. 2017;19:1116-29). Caspase inhibitors in combination with BCL inhibitors or radiation can activate NF-kB and STING pathways (Han C, et al. Nat Immunol. 2020;21 : 546), respectively, and resulted in the cure of several cancers in syngeneic and humanized mouse models (Rodriguez-Ruiz ME, et al. Oncoimmunology.

2019;8:el 655964).

[0007] Emricasan (IDN-6556, (S)-3-((S)-2-(2-((2-(tert-butyl)phenyl)amino)-2- oxoacetamido)propanamido)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid) is a pan-caspase inhibitor that has been advanced to clinical trials to treat liver diseases as an oral agent (Harrison SA, et al. J Hepatology. 2020;72:816-27).

[0008] Emricasan was discovered to induce type I interferon and MHC1 surface expression and reduced the PGE2 release in apoptotic cancer cells. In syngeneic mouse tumor models, emricasan alone or in combination with chemotherapies when injected intratumorally controlled tumor growth effectively with most tumors being eradicated. The dramatic tumor control observed was likely a result of switching apoptosis to CICD, which is more immunogenic and damaging to the tumor tissue. This mode of action effectively renders emricasan an ISV.

[0009] Emricasan is barely soluble in water and cannot be formulated in an aqueous solution for injection. Its sodium salt has been given intravenously in a phase 1 trial (Valentino KL, et al. Inti J Clin Pharmacol Therap. 2003;41:441-9). Emricasan, however, is very unstable under even slightly basic conditions and can be easily epimerized. This added complexity renders emricasan salt less attractive. Moreover, small molecule drugs delivered intratumorally tend to escape the tumor tissue before they fully engage the therapeutic targets and are therefore less effective as an ISV.

[0010] Owing to the tumor tissue retention, slow releasing, and other favorable properties, ISV intratumorally delivered in nanoformulations could be used to control tumors in syngeneic models (Ni K, et al. Sci Adv. 2020;6:eabb5223).

[0011] Poly-lactic-co-gly colic acid (PLGA) is an FDA approved copolymer that has been used extensively in drug delivery systems (PanditaD, et al. Drug Discov Today. 2015;20:95-104). PLGA-based nanoparticles are featured by their small size, high structural integrity, stability, tunable properties and versatility in surface functionalization. Their biodegradability, biosafety, biocompatibility, and versatility in formulation and functionalization allow a sustained and targeted delivery as well as enhanced cell penetration of drugs encapsulated (Bala I, et al. Critical Rev Thera Drug Carrier Sys. 2004;21). Certain limitations, however, such as low drug loading for lipophilic compounds, high burst release, phagocytic uptake, short half-life, and immunogenicity, limit their use.

[0012] One approach to improve the performance of the PLGA-based nanoparticles for lipophilic drugs delivery is to introduce different lipid shells. There are a large variety of biocomparable lipids. Significant challenges exist in identifying and controlling the physical and biological properties of the hybrid nanocarriers (Ghitman J, et al. Materials & Design. 2020:108805). Depending on the nature of the drugs encapsulated, the properties relevant to clinical use such as the particle size and homogeneity, drug loading capacity, re-suspension stability and technologies required, and releasing kinetic remain challenging to optimize. Moreover, the lyophilization and storage of lipid-covered nanoparticles may lead to aggregation. Suitable lyoprotectants are required for the integrity of the nanoparticles (Susa F, et al. Cryobiology. 2021;98:46). The selection and optimization of lyoprotectants are empirical processes as well since few rules exist (Amis TM, et al. Pharmaceutics. 2020;12:892).

[0013] The therapeutics and methods currently available for treating cancer are inadequate. There remains an urgent and ongoing need for novel and improved formulations to fully exploit the therapeutic potential of emricasan in injectable form, especially for intratumoral injection and retention. Summary of the Invention

[0014] The invention is based, in part, on the unexpected discovery of nanoformulation of emricasan in injectable form, that is especially suited for intratumoral injection and retention. The disclosed nanoformulation of emricasan is neutral or slightly acidic and may further comprise additional active agents.

[0015] In one aspect, the invention generally relates to a nanoparticle composition, wherein the nanoparticles comprise: a nanoparticle core comprising one or more polymers and at least one active ingredient comprising emricasan or active metabolites thereof; at least one layer of one or more lipids on a surface of the polymer core; and at least one lyoprotectant.

[0016] In the disclosed nanoparticle composition, a neutral polymer and a phospholipid shell was employed to encapsulate emricasan (and further active agents) to control drug release and reduce immunogenicity. Sugar excipients were added as lyoprotectants to further stabilize the nanoformulation during lyophilization and ensure re-suspension quality and reproducibility.

[0017] Emricasan, represented by Formula I below, when encapsuled in a polymer such as PLGA covered by phospholipid shells and a lyoprotectant such as trehalose, remains stable for at least 6 hours when resuspended as nanoparticles sized 50 nm - 400 nm in aqueous solutions, its effects on immune signaling molecule release in tumor cells, and the remarkable in vivo tumor control efficacy in syngeneic tumor models.

Formula I

[0018] Some embodiments described herein are nanoparticle compositions comprising one or more polymers, at least one active ingredient comprising emricasan or its active metabolites, at least one layer of one or more phospholipids on a surface of the polymer core, and at least one lyoprotectant.

[0019] In some embodiments, one or more polymers is poly (lactic-co-gly colic acid) (PLGA). In some embodiments, one or more phospholipids present in the nanoparticle composition is L-α- phosphatidylcholine (L-α-PC). In some embodiments, the lyoprotectant is trehalose. [0020] In some embodiments, the nanoparticle composition further comprises an active agent comprising an anti-cancer drug, an immune stimulating agent, or combinations thereof.

[0021] In some embodiments, the nanoparticles of the nanoparticle composition have a size of about 50 nm to about 400 nm and remain stable after resuspension in aqueous solutions. In some embodiments, a drug load of emricasan in the nanoparticle composition is about 1% to about 50% by weight of the composition.

[0022] In some embodiments, the nanoparticle composition is administered intratumorally or intravenously to a subject in need thereof. In some embodiments, the nanoparticle composition inhibited tumor growth in syngeneic mouse models.

[0023] In some embodiments, no local radiation therapy on the injected or noninjected lesions, either simultaneously or sequentially, is administered.

[0024] In some embodiments, local radiation therapy on the injected or noninjected lesions, simultaneously or sequentially, is administered.

[0025] In some embodiments, anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies, simultaneously or sequentially, are administered.

[0026] In some embodiments, the treated cancer is breast cancer, non small cell lung cancer, prostate cancer, head and neck squamous cell carcinoma, renal cell carcinoma, hepatocellular carcinoma, and melanoma.

[0027] In another aspect, the invention generally relates to a method for treating cancer, compromising administering to a subject in need thereof a nanoparticle composition disclosed herein. [0028] In yet another aspect, the invention generally relates to a method for in situ vaccination for systemically inhibiting or reducing cancer growth, compromising administering to a subject in need thereof a nanoparticle composition disclosed herein.

[0029] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising the nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier. [0030] In yet another aspect, the invention generally relates to a unit dosage form comprising a pharmaceutical composition disclosed herein.

[0031] In yet another aspect, the invention generally relates to a method of forming a nanoparticle composition disclosed herein. The method comprises: (a), forming an organic phase by combining one or more PLGA polymers, one or more solvents, and at least one of emricasan or its metabolites; (b). mixing the organic phase with water thereby forming a nanosuspension via self- assembly of micelles; (c). forming a liposome by combining a desiccated thin film of one or more phospholipids with an aqueous solution; (d). mixing the nanosuspension with the liposome and sonicating the mixture; (e). adding a lyoprotectant; (f). spray drying or freeze drying the suspension; and (g). resuspending in aqueous solutions.

[0032] In some embodiments, the manufactured nanoparticles are produced in a substantially uniform size with substantially uniform physicochemical properties.

[0033] In yet another aspect, the invention generally relates to use of a nanoparticle composition disclosed herein for the manufacture of a medicament for the treatment of cancer, or a related disease or condition.

[0034] In yet another aspect, the invention generally relates to use of a nanoparticle composition, a pharmaceutical composition, or a unit dosage form disclosed herein for treating cancer.

[0035] In yet another aspect, the invention generally relates to use of a nanoparticle composition, a pharmaceutical composition, or a unit dosage form disclosed herein for in situ vaccination for systemic inhibition or reduction of cancer growth.

Brief Description of the Drawings

[0036] For a more complete understanding of the features and advantages of the present disclosure, reference is now made to the detailed description of the disclosure along with the accompanying figures and in which:

[0037] FIG. 1 shows exemplary data of emricasan blocking apoptosis in MC38 cells.

[0038] FIG. 2 shows exemplary data of emricasan enhancing IFNβ production in MC38 cells.

[0039] FIG. 3 shows exemplary data of exemplary data of emricasan reducing PGE2 production in MC38 cells.

[0040] FIG. 4 shows exemplary data of in vivo efficacy of intratumoral injections of emricasan nanoparticles in the MC38 model.

[0041] FIG. 5 shows exemplary data of in vivo efficacy of intravenous injections of emricasan nanoparticles in the MC38 model.

[0042] FIG. 6 shows exemplary data of in vivo efficacy of the injection site tumor control of intratumoral injections of emricasan nanoparticles in the MC38 model.

[0043] FIG. 7 shows exemplary data of in vivo efficacy of the distal site tumor control of intratumoral injections of emricasan nanoparticles in the MC38 model. [0044] FIG. 8 shows exemplary data of the resuspension stability and particle size of emricasan (4 mg of emricasan/mL) nanoparticles (mass ratio of emricasan: polymer: lyoprotectant: lipid=~4 : 4 : 40 : 1 ) in PBS with 5 minutes of sonication.

[0045] FIG. 9 shows exemplary data of the resuspension stability and particle size of emricasan (4 mg of emricasan/mL) nanoparticles (mass ratio of emricasan: polymer: lyoprotectant: lipid=~4:4:40:1) in PBS without sonication.

[0046] FIG. 10 shows exemplary data of the resuspension stability and particle size of emricasan (4 mg of emricasan/mL) nanoparticles (mass ratio of emricasan: polymer: lyoprotectant: lipid =~3:3:140:1) in PBS.

[0047] FIG. 11 shows exemplary data of the resuspension stability and particle size of emricasan (8 mg of emricasan/mL) nanoparticles (mass ratio of emricasan: polymer: lyoprotectant: lipid =~3:3:140:1) in PBS.

[0048] FIG. 12 shows exemplary data of the chemical stability of emricasan in THF/deionized water (pH6).

[0049] FIG. 13 shows exemplary data of the chemical stability of emricasan in PLGA.

[0050] FIG. 14 shows exemplary data of the chemical stability of emricasan in PEG-PCL.

Detailed Description of the Invention

[0051] While the making and using of various embodiments of the present disclosure are discussed in detail below, it should be appreciated that the present disclosure provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the disclosure and do not limit the scope of the disclosure.

General Terms

[0052] Certain technical and scientific terms are specifically defined below. 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 disclosure relates. General principles of organic chemistry, as well as specific functional moieties and reactivity, are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 2006. In case of conflict, the present specification, including definitions, will control. [0053] To facilitate the understanding of this disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present disclosure.

[0054] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0055] The terms “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.

[0056] Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting.

[0057] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.

[0058] As used herein, “at least” a specific value is understood to be that value and all values greater than that value. The terms “at least one” item or “one or more” item each include a single item selected from the list as well as mixtures of two or more items selected from the list.

[0059] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. [0060] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), and “including” (and any form of including, such as “includes” and “include”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0061] In certain embodiments, the present disclosure may also include methods and compositions in which the transition phrase “consisting essentially of’ or “consisting of’ may also be used. The term “consisting essentially of’, when used to define compositions and methods, shall mean that the compositions and methods include the recited elements and exclude other elements of any essential significance to the compositions and methods. For example, “consisting essentially of’ refers to administration of the pharmacologically active agents expressly recited and excludes pharmacologically active agents not expressly recited. The term consisting essentially of does not exclude pharmacologically inactive or inert agents, e.g., pharmaceutically acceptable excipients, carriers or diluents. The term “consisting of’, when used to define compositions and methods, shall mean excluding trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

[0062] The terms “active ingredient” or “active pharmaceutical ingredient” as used herein refer to a pharmaceutical agent, active ingredient, compound, or substance, or mixtures thereof. The active ingredient may be in the form of pharmaceutically acceptable uncharged or charged molecules, molecular complexes, solvates, or hydrates thereof, and, if relevant, single isomers, enantiomers, racemic mixtures, or mixtures thereof. Furthermore, the active pharmaceutical ingredient may be in any of its crystalline, polymorphous, semi-crystalline, amorphous, or polyamorphous forms, or mixtures thereof.

[0063] The terms “active pharmaceutical ingredient load” or “drug load” as used herein refers to the quantity (mass) or weight percentage (wt%) of the active pharmaceutical ingredient comprised in the nanoparticle compositions described herein.

[0064] As used herein, the terms “administration” and “administering” of a disclosed compound refer to the delivery to a subject of a compound as described herein, or a prodrug or other pharmaceutically acceptable form thereof, using any suitable formulation or route of administration, as discussed herein. The term “administered individually” as used herein in relation to the administration of medicaments refers to the administration of individual medicaments (via the same or an alternative route) at different times. The term “administered simultaneously” as used herein in relation to the administration of medicaments refers to the administration of medicaments such that the individual medicaments are present within a subject at the same time. The term “systemically administered” means a drug is given orally or parenterally. As used herein, the term “intratumorally administered” means that a solution or suspension is injected (e.g., through a needle) directly into the tumor lesion or mass.

[0065] As used herein, the terms a “combination thereof’ and “combinations thereof’ refer to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that have repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0066] The terms "cancer", "cancerous", or "malignant", as used herein, refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. As used herein, the term “tumor” as it applies to a subject diagnosed with, or suspected of having, a cancer refers to a malignant or potentially malignant neoplasm or tissue mass of any size, and includes primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors.

[0067] As used herein, the term “emricasan” or “emri” refers to any pharmacologically active form or prodrug form of emricasan, including any salt, crystalline, polymorphous, semi-crystalline, amorphous, or polyamorphous forms thereof. Further, as used herein, the phrase “active metabolite of emricasan” or the phrase “emricasan or its active metabolites” is intended to include any metabolite of emricasan that is generated by the body of a subject following administration of emricasan via intratumoral, parenteral or non-parenteral means.

[0068] As used herein, the "effective amount" refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of nanoparticle compositions of emricasan may vary depending on such factors as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration, etc. For example, the effective amount of the nanoparticle having emricasan for treating cancer might be the amount that results in a reduction in tumor size by a desired amount over a desired period of time. Additional factors, which may be considered, include the severity of the disease state, age, weight and gender of the patient being treated, diet, time and frequency of administration, drug combinations, reaction sensitivities, and tolerance/response to therapy.

[0069] The phrase “enhanced bioavailability,” “improved bioavailability,” or “better bioavailability” as used herein refers to the increased proportion of an active pharmaceutical ingredient that enters the systemic circulation when introduced into the body as compared to a reference active pharmaceutical’s bioavailability. Bioavailability can be determined by comparing the rate and extent of absorption of a drug with a reference drug when administered at the same molar dose of the active therapeutic ingredient under similar experimental conditions in either a single dose or multiple doses. Typical pharmacokinetic parameters can be used to demonstrate enhanced bioavailability compared to the reference drug.

[0070] As used herein, the term “epimerization” refers to the chemical reaction that leads to the inversion of the configuration of one stereogenic center in a molecule containing at least two stereogenic centers.

[0071] As used herein, the term “in situ vaccination” or “ISV” refers to any chemical agent or therapeutic intervention that is able to stimulate a treated tumor lesion to release systemicaliy active anti-cancer chemicals and/or activate cancer-killing immune cells)

[0072] As used herein, the term "polymer" is given its ordinary meaning as used in the art, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units of polymer suitable for the nanoparticle compositions described herein may all be identical, or in some cases, there may be more than one type of repeat unit present within the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer. Non-limiting examples of biopolymers include peptides or proteins (i.e., biopolymers of various amino acids), or nucleic acids such as DNA or RNA. In some cases, additional moieties may also be present in the polymer, for example biological moieties such as those described below. If more than one type of repeat unit is present within the polymer, then the polymer is said to be a "copolymer." It is to be understood that in any embodiment employing a polymer, the polymer being employed may be a copolymer. The repeat units forming the copolymer may be arranged in any fashion. For example, the repeat units may be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers may have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

[0073] As used herein, the term “radiation therapy”, also called radiation oncology, radiation therapy, or therapeutic radiology refers to the use of ionizing radiation to destroy cancer cells.

[0074] As used herein, the term “subject” (alternatively “patient”) as used herein refers to a mammal that has been the object of treatment, observation, or experiment. The mammal may be male or female. The mammal may be one or more selected from the group consisting of humans, non- human primates, bovine (e.g., cows), porcine (e.g., pigs), ovine (e.g., sheep), capra (e.g., goats), equine (e.g., horses), canine (e.g., domestic dogs), feline (e.g., house cats), Lagomorpha (rabbits), rodents (e.g., rats or mice), Procyon lotor (e.g., raccoons). In particular embodiments, the subject is human.

[0075] As used herein, the term “substantially” refers to a great or significant extent, but not completely.

[0076] The term “treating” refers to administering a therapy in an amount, manner, or mode effective (e.g., a therapeutic effect) to improve a condition, symptom, disorder, or parameter associated with a disorder, or a likelihood thereof. More particularly, the terms “treatment” of or “treating” a disease or disorder refer to a method of reducing, delaying or ameliorating such a condition before or after it has occurred. Treatment may be directed at one or more effects or symptoms of a disease and/or the underlying pathology. Treatment is aimed to obtain beneficial or desired results including, but not limited to, therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disorder. For prophylactic benefit, the pharmaceutical compounds and/or compositions can be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made. The treatment can be any reduction and can be, but is not limited to, the complete ablation of the disease or the symptoms of the disease. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.

[0077] In particular, “treatment” of or “treating” cancer refers to achieving at least one positive therapeutic effect, such as for example, reduced number of cancer cells, reduced tumor size, reduced rate of cancer cell infdtration into peripheral organs, or reduced rate of tumor metastasis or tumor growth. Such “treatment” may result in a slowing, interrupting, arresting, controlling, or stopping of the progression of a cell-proliferation disorder as described herein but does not necessarily indicate a total elimination of the cell-proliferation disorder or the symptoms of the cell-proliferation disorder. Positive therapeutic effects achieved may be any of PR (partial response), CR (complete response), OR (overall response), PFS (progression free survival), DFS (disease free survival), and OS (overall survival). PFS, also referred to as “Time to Tumor Progression” indicates the length of time during and after treatment that the cancer does not grow, and includes the amount of time patients have experienced a CR or PR, as well as the amount of time patients have experienced SD. DFS refers to the length of time during and after treatment that the patient remains free of disease. OS refers to a prolongation in life expectancy as compared to I or untreated individuals or patients.

[0078] As used herein, the term “in combination” in the context of treatment methods refers to the use of more than one therapies (e.g., a caspase inhibitor and other agents). The use of the term “in combination” does not restrict the order in which therapies (e.g., a caspase inhibitor and other agents) are administered to a subject with a disorder. A first therapy (e.g., an agent that initiates apoptosis and other agents) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, lweek, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, lhour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, lweek, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of other therapy (e.g., a caspase inhibitor and other agents) to a subject with a disorder.

[0079] All compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure.

Nanoparticle Compositions of Emricasan

[0080] Owing to the nature of caspase binding pockets their inhibitors tend to have low aqueous solubility and poor chemical stability. Emricasan is a pan-caspase inhibitor that has progressed to phase 2 trials but has not been approved for any indication by the regulatory agencies. Emricasan was reported to enhance the efficacy of radiation therapy and induce abscopal effects when used in combination with an anti-PD-L1 antibody in preclinical mouse tumor models (Han C, et al. Nat Immunol. 2020;21:546). Caspase inhibition using other tools was reported to have induced abscopal effect when used in combination with radiation therapy and an anti-CTLA-4 antibody (Rodriguez- Ruiz ME, et al. Oncoimmunology, 2019;8:el655964).

[0081] Small molecule drugs such as emricasan administered intratumorally cannot be retained in the tumor effectively and often escape to the systemic circulation before the targets are fully engaged. To overcome these problems, it is important to deliver the drug in the right vehicle in injectable formulations to the site of cancer. The ideal formulation should stay in the tumor tissue for a sufficiently long period of time and protect the therapeutic drugs from the assault of metabolic enzymes, acids, bases, and other catalysis.

[0082] The herein disclosed polymer/lipid hybrid nanoparticles fulfill these requirements. The present disclosure provides compositions and methods of producing stable nanoparticles of emricasan with well-controlled physicochemical properties such as size and surface properties. Stability and size of nanoparticles are influenced by loading level of emricasan, the nature and amount of lyoprotectant used, stoichiometry of the polymer and lipid, and the process of preparation. After thorough optimization, several constructs showed good stability for at least 6 hours at room temperature after resuspension in aqueous solutions without the assistance of somcation were invented.

[0083] In some embodiments, the nanoparticle compositions described herein comprise a nanoparticle core comprising one or more polymers and one or more active agents comprising emricasan or its metabolite(s). In some embodiments, the one or more polymers comprise at least one of poly(lactic-co-gly colic acid) (PLGA) or its PEGylated form PEG-PLGA, polylactic acid (PLA) or its PEGylated form PEG-PLA, polygly colic acid (PGA) or its PEGylated form PEG-PGA, poly-L-lactide-co-ε-caprolactone (PLCL) or its PEGylated form PEG-PLCL, Hyaluronic acid (HA) or its PEGylated form PEG-HA, poly(-L-lysine) (PLL) or its PEGylated form PEG-PLL, polyacrylic acid (PAA) or its PEGylated form PEG-PAA, polyphosphate (polyP), poly(acrylic acid-co-maleic acid), poly(butylene succinate), poly(alkyl cyanoacrylate) (PAC) or its PEGylated form PEG-PAC, or combinations thereof.

[0084] In some embodiments, the nanoparticle composition has a mass ratio of at least one active ingredient (e.g., emricasan) to polymer of about 90:10 to about 10:90. In some embodiments, the mass ratio of at least one active ingredient to polymer is about 90:10, about 80:20, about 70:30, about 50:50, about 60:40, about 40:60, about 30:70, about 20:80, or about 10:90. In some embodiments, the mass ratio of at least one active ingredient to polymer is about 80:20. In some embodiments, the mass ratio of at least one active ingredient to polymer is about 60:40. In some embodiments, the mass ratio of at least one active ingredient to polymer is about 50:50. In some embodiments, the mass ratio of at least one active ingredient to polymer is about 40:60.

[0085] In some embodiments, the lipids comprise at least one of 1,2-Didecanoyl-sn-glycero-3- phosphocholine (DDPC), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), 1,2-dimyristoyl-sn- glycero-3-phosphoethanolamine (DMPE-PEG), 1-palmitoyl-2-myristoyl-sn-glycero-3- phosphocholine (PMPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE-PEG), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPP A), 1-palmitoy 1-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-stearoyl-sn-glycero-3- phosphocholine (PSOC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DEPE- PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[dibenzocyclooctyl(polyethylene glycol)-2000] (DSPE-PEG), L-α-phosphatidylcholine (L-α-PC), 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2- distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-Distearoyl-3-trimethylammonium- propane (DSTAP), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero- 3 -phosphate (DOPA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-stearoyl-2-oleoyl- sn-glycero-3-phosphocholine (SOPC), or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or combinations thereof. In some embodiments, the one or more lipids comprise 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC). In some embodiments, the one or more lipids comprise 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG). In some embodiments, the one or more lipids comprise L-α-phosphatidylcholine (L-α-PC). In some embodiments, the one or more lipids comprise 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

[0086] In some embodiments, the nanoparticle composition has a mass ratio of lipid to PEGylated lipid of about 100:0 to about 20:80. In some embodiments, the mass ratio of the lipid to the PEGylated lipid is about 100:0 to about 50:50. In some embodiments, the mass ratio of lipid to PEGylated lipid is about 100:0, about 95:5, about 90:10, about 80:20, about 70:30, about 50:50, about 60:40, about 40:60, about 30:70, or about 20:80. In some embodiments, the mass ratio of total lipid to PEGylated lipid is about 100:0 (i.e., the nanoparticle composition does not contain any or substantially no pegylated lipid). In some embodiments, the mass ratio of lipid to pegylated lipid is about 60:40. In some embodiments, the mass ratio of lipid to pegylated lipid is about 30:70.

[0087] In some embodiments, the nanoparticle composition has a mass ratio of saturated lipid to unsaturated lipid of about 100:0 to about 10:90. In some embodiments, the mass ratio of saturated lipid to unsaturated lipid is about 100:0 to about 25:75. In some embodiments, the mass ratio of saturated lipid to unsaturated lipid is about 100:0, about 95:5, about 90:10, about 80:20, about 70:30, about 50:50, about 60:40, about 40:60, about 30:70, about 20:80, or about 10:90.

[0088] In some embodiments, the nanoparticle composition has a mass ratio of at least one active ingredient (e.g., emricasan) to lipid of about 100: 0 to about 20:80. In some embodiments, the nanoparticle composition has a mass ratio of at least one active ingredient (e.g., emricasan) to lipid of about 90: 10 to about 30:70. In some embodiments, the mass ratio of at least one active ingredient to lipid is about 90:10, about 80:20, about 70:30, about 50:50, about 60:40, about 40:60, about 30:70, or about 20:80. In some embodiments, the mass ratio of at least one active ingredient to lipid is about 80:20. In some embodiments, the mass ratio of at least one active ingredient to lipid is about 50:50. [0089] In various embodiments, the nanoparticle composition may comprise 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE- PEG), L-α-phosphatidylcholine (L-α-PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or combinations thereof.

[0090] In some embodiments, the nanoparticle composition has a mass ratio of polymer to lipid of about 100:0 to about 10:90. In some embodiments, the nanoparticle composition has a mass ratio of polymer to lipid of about 90: 10 to about 20:80. In some embodiments, the mass ratio of polymer to lipid is about 100:0, about 95:5, about 90:10, about 80:20, about 70:30, about 50:50, about 60:40, about 40:60, about 30:70, about 20:80, or about 10:90. In some embodiments, the mass ratio of polymer to lipid is about 80:20. In some embodiments, the mass ratio of polymer to lipid is about 70:30. In some embodiments, the mass ratio of lipid to polymer is about 50:50.

[0091] In some embodiment, the nanoparticle composition further comprises at least one lyoprotectant. In some embodiments, the lyoprotectant is selected from sucrose, glucose, trehalose, mannitol, polyethylene glycol, hyaluronic acid, alginic acid, leucine and cyclodextrin. In some embodiments, the lyoprotectant is trehalose.

[0092] In some embodiment, the nanoparticle composition has a mass ratio of at least one active ingredient (e.g., emricasan) to lyoprotectant is about 1:99 to about 50:50. In some embodiments, the mass ratio of at least one active ingredient to lyoprotectant is about 2:98 to about 40:60. In some embodiments, the mass ratio of at least one active ingredient to lyoprotectant is about 5:95 to about 30:70. In some embodiments, the mass ratio of at least one active ingredient to lyoprotectant is about 10:90 to about 20:80. In some embodiments, the mass ratio of at least one active ingredient to lyoprotectant is about 1:99. In some embodiments, the mass ratio of at least one active ingredient to lyoprotectant is about 2:98. In some embodiments, the mass ratio of at least one active ingredient to lyoprotectant is about 5:95. In some embodiments, the mass ratio of at least one active ingredient to lyoprotectant is about 10:90. In some embodiments, the mass ratio of at least one active ingredient to lyoprotectant is about 20:80. In some embodiments, the mass ratio of at least one active ingredient to lyoprotectant is about 30:70. In some embodiments, the mass ratio of at least one active ingredient to lyoprotectant is about 40:60. In some embodiments, the mass ratio of at least one active ingredient to lyoprotectant is about 50:50.

[0093] In some embodiments, the one or more active agents comprise emricasan and/or its metabolite(s).

[0094] In some embodiments, the drug load of active ingredient (e.g., emricasan and/or its metabolite(s)) present in the nanoparticle composition is about 1% to about 50% by weight of the composition. In some embodiments, the drug load is about 2% to about 25%. In some embodiments, the drug load is about 5% to about 15%. In some embodiments, the drug load of active ingredient is about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, or about 50%.

[0095] In some embodiments, the nanoparticle composition has an average nanoparticle size of about 50 nm to about 400 nm. In some embodiments, the nanoparticle composition has an average nanoparticle size of about 100 nm to about 300 nm. In some embodiments, the nanoparticle composition has an average nanoparticle size of about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 300 nm, or about 400 nm. In some embodiments, the nanoparticle composition has an average nanoparticle size of about 150 nm.

[0096] In some embodiments, the pharmaceutical composition is characterized by a concentration of nanoparticle composition of emricasan in the range of about 1 mg/mL to about 40 mg/mL (e.g., about 1 mg/mL to about 25 mg/mL, about 1 mg/mL to about 10 mg/mL, about 5 mg/mL to about 40 mg/mL, about 10 mg/mL to about 40 mg/mL), and a pH in the range of about 5.0 to about 7.0 (e.g., about 5.5 to about 7.0, about 5.0 to about 6.5, about 5.5 to about 6.5).

[0097] In some embodiments, the active agent further comprises at least one anti-cancer drug. In some embodiments, the active agent further comprises at least one immune stimulating drug. In some embodiments, the nanoparticle composition has high bioavailability.

[0098] In some embodiments, the nanoparticles further comprise at least one targeting agent, wherein the targeting agent selectively delivers the nanoparticle to diseased tissue/cells, thereby minimizing whole body dose. In some embodiments, the targeting agent comprises an antibody or functional fragment thereof that is capable of recognizing a target antigen; and/or selected from an antibody, a small molecule, a peptide, a carbohydrate, an siRNA, a protein, a nucleic acid, an aptamer, a second nanoparticle, a cytokine, a chemokine, a lymphokine, a receptor, a lipid, a lectin, a ferrous metal, a magnetic particle, a linker, or an isotope or combinations thereof. The targeting agents may be attached by insertion of hetero/homo bifunctional spacer capable of reacting with amines of lipids and targeting moieties.

[0099] Certain embodiments can be described as intratumoral and/or intravenous and/or subcutaneous administration of a novel formulation of emricasan bound to PLGA and a liposome. Such formulation is designed to offer a sustained release of emricasan as active agent. Reference is made to the reduction of side effects due to the incorporation of a polymer and liposomal components of the formulation.

Polymers

[00100] A wide variety of polymers and methods for forming particles therefrom are known in the art of drug delivery. In some embodiments of the disclosure, the matrix of a particle comprises one or more polymers. Any polymer may be used in accordance with the present disclosure. Polymers may be natural or unnatural (synthetic) polymers. Polymers may be homopolymers or copolymers comprising two or more monomers. In terms of sequence, copolymers may be random, block, or comprise a combination of random and block sequences. Typically, polymers in accordance with the present disclosure are organic polymers.

[00101] Various embodiments of the present disclosure are directed to copolymers, which, in particular embodiments, describes two or more polymers (such as those described herein) that have been associated with each other, usually by covalent bonding of the two or more polymers together. Thus, a copolymer may comprise a first polymer and a second polymer, which have been conjugated together to form a block copolymer where the first polymer is a first block of the block copolymer and the second polymer is a second block of the block copolymer. Those of ordinary skill in the art will understand that a block copolymer may, in some cases, include multiple blocks of polymer, and that a "block copolymer," as used herein, is not limited to only block copolymers having only a single first block and a single second block. For instance, a block copolymer may comprise a first block comprising a first polymer, a second block comprising a second polymer, and a third block comprising a third polymer or the first polymer, etc. In some cases, block copolymers can include any number of first blocks of a first polymer and second blocks of a second polymer (and in certain cases, third blocks, fourth blocks, etc.). In addition, it should be noted that block copolymers can also be formed, in some instances, from other block copolymers. For example, a first block copolymer may be conjugated to another polymer (which may be a homopolymer, a biopolymer, another block copolymer, etc.), to form a new block copolymer including multiple types of blocks, and/or to other moieties (e.g., to non-polymeric moieties). Alternatively, as described below, a copolymer can be formed using a lipid linker (e.g., DSPE).

[00102] In one set of embodiments, a polymer (e.g., copolymer, e.g., block copolymer) of the present disclosure includes a biocompatible polymer, i.e., the polymer that does not typically induce an adverse response when inserted or injected into a living subject, for example, without significant inflammation and/or acute rejection of the polymer by the immune system, for instance, via a T-cell response. Accordingly, the nanoparticles of the present disclosure can be "non-immunogenic." The term "non-immunogenic" as used herein refers to endogenous growth factor in its native state, which normally elicits no, or only minimal levels of, circulating antibodies, or reactive immune cells, and which normally does not elicit in the individual an immune response against itself.

[00103] It will be recognized that "biocompatibility" is a relative term, and some degree of immune response is to be expected even for polymers that are highly compatible with living tissue. However, as used herein, "biocompatibility" refers to the acute rejection of material by at least a portion of the immune system, i.e., a non-biocompatible material implanted into a subject provokes an immune response in the subject that is severe enough such that the rejection of the material by the immune system cannot be adequately controlled, and often is of a degree such that the material must be removed from the subject. One simple test to determine biocompatibility is to expose a polymer to cells in vitro; biocompatible polymers are polymers that typically will not result in significant cell death at moderate concentrations, e.g., at concentrations of 50 micrograms/10 cells. For instance, a biocompatible polymer may cause less than about 20% cell death when exposed to cells such as fibroblasts or epithelial cells, even if phagocytosed or otherwise up-taken by such cells. Non-limiting examples of biocompatible polymers that may be useful in various embodiments of the present disclosure include polydioxanone (PDO), polyhydroxyalkanoate, polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide, polylactide, PLGA, polycaprolactone, or copolymers or derivatives including these and/or other polymers.

[00104] In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body. For instance, the polymer may be one that hydrolyzes spontaneously upon exposure to water (e.g., within a subject), the polymer may degrade upon exposure to heat (e.g., at temperatures of above 37 °C). Degradation of a polymer may occur at varying rates, depending on the polymer or copolymer used. For example, the half-life of the polymer (the time at which 50% of the polymer is degraded into monomers and/or other nonpolymeric moieties) may be on the order of days, weeks, months, or years, depending on the polymer. The polymers may be biologically degraded, e.g., by enzymatic activity or cellular machinery, in some cases, for example, through exposure to a lysozyme (e.g., having relatively low pH). In some cases, the polymers may be broken down into monomers and/or other nonpolymeric moieties that cells can either reuse or dispose of without significant toxic effect on the cells (for example, polylactide may be hydrolyzed to form lactic acid, polyglycolide may be hydrolyzed to form glycolic acid, etc.).

[00105] Further suitable polymers include polyesters, including copolymers comprising lactic acid and glycolic acid units, such as poly(lactic acid-co-gly colic acid) and poly(lactide-co-glycolide), collectively referred to herein as "PLGA"; and homopolymers comprising glycolic acid units, referred to herein as "PGA," and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as "PLA." Exemplary polyesters suitable for the embodiments described herein include, for example, polyhydroxyacids; PEGylated polymers and copolymers of lactide and glycolide (e.g., PEGylated PLA, PEGylated PGA, PEGylated PLGA, and derivatives thereof). Additional polyesters include, for example, polyanhydrides, poly(ortho ester) PEGylated poly(ortho ester), poly(caprolactone), PEGylated poly(caprolactone), polylysine, PEGylated polylysine, poly(ethylene inline), PEGylated poly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine ester), poly(4- hydroxy-L-proline ester), poly [a-(4-aminobutyl)-L-gly colic acid], and derivatives thereof.

[00106] In some embodiments, the polymer is PLGA. PLGA is a biocompatible and biodegradable co-polymer of lactic acid and glycolic acid, and various forms of PLGA are characterized by the ratio of lactic acid:gly colic acid. Lactic acid can be in the form of L-lactic acid, D-lactic acid, or D, L-lactic acid. The degradation rate of PLGA can be adjusted by altering the lactic acid-glycolic acid ratio. In some embodiments, PLGA is characterized by a lactic acid: glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.

[00107] In some embodiments, the polymer is PEG-PCL. Emricasan is unstable in solutions to undergo a fast epimerization reaction leading to the formation of an undesired epimer. In some embodiments, a PEG-PCL encapsulated emricasan nanoparticle is provided with enhanced chemical stability upon reconstitution in an aqueous-based solvent system. The isomerization of aspartic acid and its derivatives is a longstanding problem in drug research and polymer-encapsulated nanoparticles provide a potential solution to this problem.

[00108] In particular embodiments, by optimizing the ratio of lactic acid to glycolic acid monomers in the polymer of the nanoparticle (e.g., the PLGA block copolymer or PLGA-PEG block copolymer), nanoparticle parameters such as water uptake, therapeutic agent release (e.g., "controlled release") and polymer degradation kinetics can be optimized. In some embodiments, the polymer is acrylic polymers. Suitable and non-limiting acrylic polymers include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acid polyacrylamide, aminoalkyl methacrylate copolymer, glycidyl methacrylate copolymers, poly cyanoacrylates, and combinations comprising one or more of the foregoing phospholipids. The acrylic polymer may comprise fully-polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.

[00109] In some embodiments, the polymer is a cationic polymer. In general, cationic polymers are able to condense and/or protect negatively charged strands of nucleic acids (e.g. DNA, RNA, or derivatives thereof). Amine-containing polymers such as poly(lysine) (Zauner et al. 1998, Adv Drug Dev Rev, 30:97; and Kabanov et al. 1995, Bioconjugate Chem, 6:7), polyethylene imine (PEI; Boussif et al. 1995, Proc Natl Acad Sci, USA, 1995, 92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo et al. 1996, Proc Natl Acad Sci, USA, 93:4897; Tang et al. 1996, Bioconjugate Chem, 7:703; and Haensler et al. 1993, Bioconjugate Chem, 4:372) are positively charged at physiological pH, form ion pairs with nucleic acids, and mediate transfection in a variety of cell lines.

[00110] In some embodiments, the polymer is a degradable polyester bearing cationic side chains (Putnam et al. 1999, Macromolecules, 32:3658; Barrera et al. 1993, J Am Chem Soc, 115:11010; Kwonef et al. 19%9, Macromolecules, 22325, Um et al. 1999, J A Chem. Soc, 121:5633; and Zhou et al, 1990, Macromolecules, 23:3399). Examples of these polyesters include poly(L-lactide-co-L- lysine) (Barrera et al, 1993, J Am Chem Soc, 115:11010), poly(serine ester) (Zhou et al, 1990, Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam et ah, 1999, Macromolecules, 32:3658; and Lim et al, 1999, J Am Chem Soc, 121:5633). Poly(4-hydroxy-L-proline ester) was demonstrated to condense plasmid DNA through electrostatic interactions, and to mediate gene transfer (Putnam et al, 1999, Macromolecules, 32:3658; and Lim et al, 1999, J Am Chem Soc, 121:5633). These new polymers are less toxic than poly(lysine) and PEI, and they degrade into non toxic metabolites. A polymer (e.g., copolymer, e.g., block copolymer) having poly(ethylene glycol) repeat units is also referred to as a "PEGylated" polymer. Such polymers can control inflammation and/or immunogenicity (i.e., the ability to provoke an immune response) and/or lower the rate of clearance from the circulatory system via the reticuloendothelial system, due to the presence of the poly(ethylene glycol) groups. PEGylation may also be used, in some cases, to decrease charge interaction between a polymer and a biological moiety, e.g., by creating a hydrophilic layer on the surface of the polymer, which may shield the polymer from interacting with the biological moiety. In some cases, the addition of poly(ethylene glycol) repeat units may increase plasma half-life of the polymer (e.g., copolymer, e.g., block copolymer), for instance, by decreasing the uptake of the polymer by the phagocytic system while decreasing transfection/uptake efficiency by cells. Those of ordinary skill in the art will know of methods and techniques for PEGylating a polymer, for example, by using EDC (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N- hydroxysuccinimide) to react a polymer to a PEG group terminating in an amine, by ring opening polymerization techniques (ROMP), or the like.

[00111] In addition, certain embodiments of the disclosure are directed towards copolymers having poly(ester-ether)s, e.g., polymers having repeat units joined by ester bonds (e.g., R-C(0)-0-R' bonds) and ether bonds (e.g., R-O-R' bonds). In some embodiments of the disclosure, a biodegradable polymer, such as a hydrolyzable polymer, having carboxylic acid groups, is conjugated with poly(ethylene glycol) repeat units to form a poly(ester-ether).

[00112] In a particular embodiment, the molecular weight of the polymers of the nanoparticles of the disclosure is optimized for effective treatment of diseases, e.g., cancer. For example, the molecular weight of the polymer influences nanoparticle degradation rate (particularly when the molecular weight of a biodegradable polymer is adjusted), solubility, water uptake, and drug release kinetics (e.g. "controlled release"). As a further example, the molecular weight of the polymer can be adjusted such that the nanoparticle biodegrades in the subject being treated within a reasonable period of time (ranging from a few hours to 1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks, etc.). [00113] In particular embodiments is a nanoparticle composition comprising a copolymer of PEG and PLGA or PCL. In certain embodiments, the PEG has a molecular weight of about 1,000-20,000 Da, about 5,000-20,000 Da, or about 10,000-20,000 Da, the PLGA has a molecular weight of about 5,000-100,000 Da, about 20,000-70,000 Da or about 20,000-50,000 Da, the PCL has a molecular weight of about 5,000-100,000 Da, about 20,000-70,000 Da or about 20,000-50,000 Da. Lipids

[00114] In some embodiments, the lipid used in the nanoparticle composition is an oil. In general, any oil known in the art can be conjugated to the polymers used in the disclosure. In some embodiments, an oil may comprise one or more fatty acid groups or salts thereof. In some embodiments, a fatty acid group may comprise digestible, long chain (e.g., Cg-Cso), substituted or unsubstituted hydrocarbons. In some embodiments, a fatty acid group may be a C10-C20 fatty acid or salt thereof. In some embodiments, a fatty acid group may be a C15-C20 fatty acid or salt thereof. In some embodiments, the fatty acid is saturated. In some embodiments, the fatty acid is unsaturated. In some embodiments, a fatty acid group is monounsaturated. In some embodiments, a fatty acid group is polyunsaturated. In some embodiments, a double bond of an unsaturated fatty acid group is in the cis conformation. In some embodiments, a double bond of an unsaturated fatty acid is in the trans conformation.

[00115] In some embodiments, a fatty acid group can be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid. In some embodiments, a fatty acid group may be one or more of palmitoleic, oleic, vaccenic, linoleic, alpha- linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic, or erucic acid. Exemplary and non-limiting lipids are provided in Table 1.

Table 1. List of lipids and their properties at pH 7

Lyoprotectants

[00116] Formation of aggregates can occur during lyophilization due to the dehydration of the surface of the particles. This dehydration can be avoided using lyoprotectants, such as disaccharides, in the suspension before lyophilization. There are no general rules in rationally selecting lyoprotectants or cryoprotectants and a suitable formulation with acceptable size and stability after resuspension for clinical use has to be found using empirical methods. Suitable disaccharides include sucrose, lactulose, lactose, maltose, trehalose, or cellobiose, and/or mixtures thereof. Other contemplated disaccharides include kojibiose, nigerose, isomaltose, b,b-trehalose, a,b-trehalose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiase, melibiose, melibiulose, rutinose, rutinulose, and xylobiose..

[00117] In some embodiments, one or more ionic halide salts may be used as an additional lyoprotectant to a sugar, such as sucrose, trehalose or mixtures thereof. Sugars may include disaccharides, monosaccharides, trisaccharides, and/or polysaccharides, and may include other excipients, e.g. glycerol and/or surfactants. Optionally, a cyclodextrin may be included as an additional lyoprotectant. The cyclodextrin may be added in place of the ionic halide salt. Alternatively, the cyclodextrin may be added in addition to the ionic halide salt. Optionally, a leucine may be included as an additional lyoprotectant.

Methods of Manufacture

[00118] Some embodiments described herein are methods for forming a nanoparticle composition comprising forming an organic phase by combining one or more PLGA, one or more solvents and at least one of emricasan or its metabolites in a multi-inlet vortex mixer, whereby self-assembly of micelles occurs; forming a liposome in aqueous phase by combining a desiccated thin film of one or more phospholipids with an aqueous solution; mixing the nanosuspension with the aqueous liposome; adding a lyoprotectant to improve lyophilization stability; spray drying or freeze drying the suspension and recycling the organic solvents; and resuspending the nanoparticles in aqueous solutions and wherein the emricasan or its metabolites nanoparticles provide sustained release of active components when provided to a subject. In some embodiments, the solvent is selected from ethanol, methanol, tetrahydrofuran, acetonitrile, acetone, tert-butyl alcohol, dimethyl formamide, and hexafluroisopropanol. In some embodiments, the one or more active agents comprise emricasan or its metabolites.

[00119] In certain embodiments, mixing the organic phase with an aqueous phase comprises vigorous micromixing and/or vertexing and/or sonicating. In certain embodiments, mixing nanosuspension with liposome comprises vigorous micromixing and/or vertexing and/or sonicating. In certain embodiments, the method further comprises adding a lyoprotectant. certain embodiments, the method further comprises organic solvent removal; and/or dialysis. In certain embodiments, the method further comprises freezing the nanoparticles; and/or lyophilizing the nanoparticles; and/or spray-drying the particles. In certain embodiments, the method further comprises attaching a targeting agent to the nanoparticles; and/or attaching at least one targeting agent, wherein the targeting agent selectively delivers the nanoparticle to diseased tissue/cells, thereby minimizing whole body dose; and/or attaching at least one targeting agent to the nanoparticles, wherein the targeting agent comprises an antibody or functional fragment thereof that is capable of recognizing a target antigen.

[00120] In some embodiment, the disclosure provides lipid layer-protected nanoparticles, and methods of making the nanoparticles, wherein one polymer of the polymeric matrix (e.g., PEG), is conjugated to a lipid that will self-assemble with another polymer (e.g., PLGA), such that the polymer of the polymeric matrix are not covalently bound, but are bound through self-assembly. "Self-assembly" refers to a process of spontaneous assembly of a higher order structure that relies on the natural attraction of the components of the higher order structure (e.g., molecules) for each other. Self-assembly typically occurs through random movements of the molecules and formation of bonds based on size, shape, composition, or chemical properties.

Administration

[00121] The current disclosure is based on the discovery that activated caspases play a key role in stimulating tumor growth and evading local and systemic immune surveillance during induced and spontaneous apoptosis. The invention provides a novel approach to cancer treatment using nanoparticle composition of emricasan.

[00122] In some embodiment, the nanoparticle composition of emricasan is administered at a dose in a range of about 0.5 mg to about 250 mg, about 1 mg to about 100 mg, about 5 mg to about 50 mg (e.g., about 0.5 mg, about 1 mg, about 5 mg, about 10 mg, about 20 mg, about 25 mg, about 40 mg, about 80 mg, about 150 mg, about 200 mg, about 250 mg). Such doses may be provided, e.g., intratumorally, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation. Such doses may be for once daily administration, twice daily administration, or every two days administration.

[00123] In some embodiment, the nanoparticle composition of emricasan is administered at a dose of about 0.5 mg delivered intratumorally, about 5 mg delivered intratumorally, about 10 mg delivered intratumorally, about 20 mg delivered intratumorally, about 40 mg delivered intratumorally, about 80 mg delivered intratumorally, about 150 mg delivered intratumorally, about 200 mg delivered intratumorally, about 250 mg delivered intratumorally, e.g. about 250 mg/day. Such doses may be particularly adapted for patients of weight between 50 and 120 kg, e.g. 70 and 100 kg.

[00124] In some embodiments, the nanoparticle composition of emricasan is administered at a dose in a range of about 0.5 mg/day to about 100 mg/day (e.g., 0.5 mg/day, 1 mg/day, about 5 mg/day, about 10 mg/day, about 15 mg/day, about 20 mg/day, about 25 mg/day, about 30 mg/day, about 40 mg/day, about 50 mg/day, about 75 mg/day, about 100 mg/day) delivered intratumor ally. Such doses may be also provided, e.g., intratumorally, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation. Such regimens may be particularly adapted for patients of weight between 50 and 120 kg, e.g. 70 and 100 kg. [00125] In some embodiments of the invention, the nanoparticle composition of emricasan is administered intratumorally at a dose of about 0.5 mg twice daily to about 100 mg twice daily (e.g., about 0.5 mg twice daily, about 5 mg twice daily, about 10 mg twice daily, about 25 mg twice daily, about 50 mg twice daily, about 75 mg twice daily, about 100 mg twice daily). Such doses may be also provided, e.g., intratumorally, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation.

[00126] In some embodiments, local radiation therapy on the injected or noninjected lesions, simultaneously or sequentially, is administered to a subject in need thereof. In some embodiment, the subject in need thereof is administered a biologically equivalent dose (BED) of radiation therapy. In some embodiment, the subject in need thereof is administered time-adjusted BED. In some embodiment, the subject in need thereof is administered a hyperfractionation (reduced fraction size, two times or more per day) dose of radiation therapy. In some embodiment, the subject in need thereof is administered a hypofractionation (fewer fractions, larger dose-per-fraction) dose of radiation therapy.

[00127] In some embodiment, the radiation therapy is administered in a total dose of about 10 Gy to about 200 Gy. In some embodiment, the radiation therapy is administered in a total dose of about 20 Gy to about 150 Gy. In some embodiment, the radiation therapy is administered at a total dose of about 10 Gy, about 20 Gy, about 30 Gy, about 40 Gy, about 50 Gy, about 60 Gy, about 70 Gy, about 80 Gy, about 90 Gy, about 100 Gy, about 110 Gy, about 120 Gy, about 130 Gy, about 140 Gy, about 150 Gy, about 160 Gy, about 170 Gy, about 180 Gy, about 190 Gy, about 200 Gy. Such doses may be administered three times daily, twice daily, once daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly. [00128] In some embodiments, anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies, simultaneously or sequentially, are administered. Anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies may be provided by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly, semiannually, annually etc. Doses may be provided, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation. A total dose for a treatment interval is generally at least 0.05 μg/kg body weight, more generally at least 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg or more. Doses may also be provided to achieve a pre-determined target concentration of anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies in the subject's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/mL or more. In some embodiment, anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies are administered as a 200 mg dose once every 21 days. In some embodiments, anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies are administered subcutaneously or intravenously, on a weekly, biweekly, "every 4 weeks," monthly, bimonthly, or quarterly basis at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg/subject.

Pharmaceutical Compositions

[00129] Certain embodiments described herein are pharmaceutical compositions comprising the nanoparticle composition described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for the intratumoral and/or intravenous and/or subcutaneous administration of a novel formulation of synthesized emricasan in a nanoparticle composition described herein.

[00130] As used herein, the term "pharmaceutically acceptable carrier" means a nontoxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Remington's Pharmaceutical Sciences. Ed. By Gennaro, Mack Publishing, Easton, Pa., 1995 discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Some exemplary and non-limiting materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as TWEEN® 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. If fdtration or other terminal sterilization methods are not feasible, the formulations can be manufactured under aseptic conditions.

[00131] The pharmaceutical compositions of this disclosure can be administered to a patient by any means known in the art including oral and parenteral routes. In some embodiments, intratumoral injections are desirable since systemic side effects can be minimized. In some embodiments, parenteral routes are desirable since they avoid contact with the digestive enzymes that are found in the alimentary canal. According to such embodiments, inventive compositions may be administered by injection (e.g., intratumoral, intravenous, subcutaneous or intramuscular, intraperitoneal injection), orally, rectally, vaginally, topically (as by powders, creams, ointments, or drops), or by inhalation (as by sprays). In a particular embodiment, the nanoparticles of the present disclosure are administered to a subject in need thereof intratumorally, e.g., by intratumoral infusion or injection. [00132] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In one embodiment, the inventive nanoparticle composition is suspended in a carrier fluid comprising 1 % (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) TWEEN® 80. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[00133] In some embodiment the nanoformulation of emricasan or its metabolites can be delivered topically or transdermally to treat certain skin cancer and warts. Dosage forms for topical or transdermal administration of an inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The inventive nanoparticle composition is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulations, eardrops, and eye drops are also contemplated as being within the scope of this disclosure. The ointments, pastes, creams, and gels may include, in addition to the inventive nanoparticle compositions of this disclosure, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof. Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the inventive nanoparticle compositions in a proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the inventive nanoparticle compositions in a polymer matrix or gel.

[00134] Powders and sprays can include, in addition to the inventive nanoparticle compositions of this disclosure, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures thereof. Sprays can additionally include customary propellants such as chlorofluorohydrocarbons. When administered orally, the inventive nanoparticles can be, but are not necessarily, encapsulated. A variety of suitable encapsulation systems are known in the art ("Microcapsules and Nanoparticles in Medicine and Pharmacy," Edited by Doubrow, M., CRC Press, Boca Raton, 1992; Mathiowitz and Langer J. Control. Release 5:13, 1987; Mathiowitz et al. Reactive Phospholipids 1987; 6:275; Mathiowitz et al. J Appl Polymer Sci. 1988;35:755; Langer, Acc. Chem. Res. 2000; 33:94; Langer, J Control Release 1999; 62:7; Uhrich et al. Chem Rev. 1999; 99:3181; Zhou et al. J Control Release 2001;75:27; and Hanes et al. Pharm Biotechnol. 1995;6:389). The inventive nanoparticle compositions may be encapsulated within biodegradable polymeric microspheres or liposomes. Examples of natural and synthetic polymers useful in the preparation of biodegradable microspheres include carbohydrates such as alginate, cellulose, polyhydroxyalkanoates, polyamides, polyphosphazenes, polypropylfumarates, polyethers, polyacetals, polycyanoacrylates, biodegradable polyurethanes, polycarbonates, polyanhydrides, polyhydroxyacids, poly(ortho esters), and other biodegradable polyesters. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Pharmaceutical compositions for oral administration can be liquid or solid. Liquid dosage forms suitable for oral administration of inventive compositions include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to an encapsulated or unencapsulated nanoparticle composition, the liquid dosage forms may include inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[00135] Besides inert diluents, the oral compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. As used herein, the term "adjuvant" refers to any compound, which is a nonspecific modulator of the immune response. In certain embodiments, the adjuvant stimulates the immune response. Any adjuvant may be used in accordance with the present disclosure. A large number of adjuvant compounds is known in the art (Allison, Dev Biol Stand. 1998; 92:3-11; Unkeless et al. Ann Rev Immunol. 1998; 6:251-281; and Phillips et al. Vaccine. 1992;10:151-158).

[00136] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the encapsulated or unencapsulated nanoparticle composition is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, ethyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents.

[00137] Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art.

[00138] It will be appreciated that the exact dosage of the pharmaceutical composition is chosen by the individual physician in view of the patient to be treated, in general, dosage and administration are adjusted to provide an effective amount of the pharmaceutical composition to the patient being treated.

[00139] The nanoparticles of the disclosure may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to a physically discrete unit of nanoparticle appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. For any nanoparticle, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity of nanoparticles can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED₅₀ (the dose is therapeutically effective in 50% of the population) and LD₅₀ (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀/ ED₅₀. Pharmaceutical compositions, which exhibit large therapeutic indices, may be useful in some embodiments. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for human use. [00140] The present disclosure also provides any of the above-mentioned compositions in kits, optionally with instructions for administering any of the compositions described herein by any suitable technique as previously described, for example, orally, intravenously, pump or implantable delivery device, or via another known route of drug delivery. "Instructions" can define a component of promotion, and typically involve written instructions on or associated with packaging of compositions of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner. The "kit" typically defines a package including any one or a combination of the compositions of the disclosure and the instructions, but can also include the composition of the disclosure and instructions of any form that are provided in connection with the composition in a manner such that a clinical professional will clearly recognize that the instructions are to be associated with the specific composition. The kits described herein may also include one or more containers, which may include the inventive composition and other ingredients as previously described. The kits also may include instructions for mixing, diluting, and/or administrating the compositions of the disclosure in some cases. The kits also can include other containers with one or more solvents, surfactants, preservative and/or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose) as well as containers for mixing, diluting or administering the components in a sample or to a subject in need of such treatment.

[00141] The compositions of the kit may be provided as any suitable form, for example, as liquid solutions or as dried powders. When the composition provided is a dry powder, the composition may be reconstituted by the addition of a suitable solvent, which may also be provided. In embodiments where liquid forms of the composition are used, the liquid form may be concentrated or ready to use. The solvent will depend on the nanoparticle and the mode of use or administration. Suitable solvents for drug compositions are well known, for example as previously described, and are available in the literature. The solvent will depend on the nanoparticle and the mode of use or administration.

Methods

[00142] The invention also provides, in another aspect, a method for treating cancer, compromising administering to a subject in need thereof a nanoparticle composition disclosed herein. Some embodiments described herein are methods for treating a subject having a disease (e.g., cancer) including identifying a subject suspected of having a disease, and administering an effective amount of the nanoparticle composition or pharmaceutical composition comprising it in an effective amount to treat the disease. In certain embodiments, the administration of the composition reduces one or more of the side effects comprising nausea, vomiting, dermatitis, bone-marrow depression, cardiotoxicity or diarrhea or a combination thereof when provided to a subject compared to administration of emricasan that is not formulated in the nanoparticle composition described herein. [00143] In further embodiments described herein, the compositions described herein may be used for the treatment of neoplastic diseases (cancer), and neurologic-auto-immunological degenerative diseases (Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS, sequel, behavioral and cognitive disorders, autism spectrum, and depression). In certain embodiments, the compositions of the present disclosure are administered intratumorally, intramuscularly, subcutaneously, orally, or intravascularly.

[00144] The nanoparticle composition or pharmaceutical compositions described herein can be administered, for example, to a subject, or a subject in need thereof. In certain embodiments, the subject is a mammal, or a mammal in need thereof. In certain embodiments, the subject is a human, or human in need thereof. In certain embodiments, the subject is a human. In certain embodiments, the subject is a child (~0-9 years old) or an adolescent (~10-17 years old). In certain embodiments, the subject is from about 0 to about 9 years of age. In certain embodiments, the subject is from about 10 years to about 17 years of age. In certain embodiments, the subject is over 17 years of age. In certain embodiments, the subject is an adult (≥18 years of age).

[00145] Some additional embodiments are a method of manufacturing a medicament comprising the nanoparticle composition of emricasan for the treatment of one or more diseases described herein. The medicament can subsequently be administered to a patient in need thereof.

[00146] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

Examples

[00147] The disclosure is further illustrated by the following examples, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting.

In vitro activity of emricasan and in vivo efficacy of emricasan nanoparticles

[00148] Cell lines and drugs : MC38 cell line was derived from C57BL6 murine colon adenocarcinoma. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and lOOU/mL penicillin/streptomycin.

[00149] Apoptosis flow cytometry assay: FITC Annexin V Apoptosis Detection Kit with PI (BioFegend) was used for apoptosis detection. The MC38 cells were seeded in 24-well plate with 1X10^{^}5 cells/well and cultured overnight. The DMSO drug stocks were diluted in DMEM to make the working solution (10 mM Emricasan). After 24 hours treatment, cells were collected and dissociated as single cell suspensions for Annexin V / PI staining. In briefing, cells were washed twice with cold BioFegend’ s Cell Staining Buffer, then resuspended in 100 mL Annexin V Binding Buffer. 5 mL FITC Annex V and 10 mL Propidium Iodide (PI) Solution were added in each sample. The samples were gently vortexed and incubated for 15 minutes at room temperature in the dark. Wash the samples with Cell Staining Buffer one time and resuspend cells in 500 mL Cell Staining Buffer for flow cytometry analysis. The data were recorded on BD Accuri™ C6 plus flow Cytometer and analyzed with FlowJo software.

[00150] Interferon-beta ( IFNβ ) ELISA assay: The MC38 cell culture supernatants were collected

24 hours post-drug treatment and stored at -80°C for IFNβ ELISA measurement. The concentration of IFNβ was measured with VeriKine -HS Interferon Beta Serum ELISA Kit (PBL Assay Science) in accordance with the manufacturer’s instruction. The data of absorbance was recorded at 450 nm by using BioTek synergy 4 plate reader and analyzed with GraphPad software.

[00151] In vivo studies: Female C57BL6 mice of 9 weeks of age were obtained from the Jackson lab, ME. Passages were made twice weekly with a 1:2 split and cultured in DMEM:F12 supplemented with 10% FBS (HyClone, Ft. Collins, CO). For inoculation, approximately 5x10⁵ cells were suspended in 100 μL of PBS (Becton Dickinson Labware, Bedford, MA) and injected subcutaneously into the flanks of mice. Most mice developed palpable tumors within 5 days of inoculation. Tumor bearing mice are grouped in cohorts of 5-8 mice for dosing studies. Mice with tumor sizes of 50-100 mm³ are randomized between treatment groups. Baseline tumor volumes were established and dosing initiation began on day 0. Emricasan nanoparticle in PBS was administered intratumorally or intravenously (q.d.) for 3-4 days. Tumor volumes were measured twice a week using standard calipers and calculated as (length x width²)/2, with the length and width defined as the long and short axes, respectively. Measurement of body weight was initiated on day 0 and repeated once a week.

[00152] Our results demonstrated that caspase inhibitor emricasan partially blocked the spontaneous apoptosis and enhanced the IFNβ production and reduced PGE2 release in MC38 cells as shown in FIG. 1, FIG. 2, and FIG. 3, respectively. Intratumorally administered emricasan nanoparticles were able to completely control MC38 tumors at the site of injection as shown in FIG. 4 and intravenously administered emricasan nanoparticles (3 injections, q.d.) partially controlled MC38 tumors as shown in FIG. 5. When an anti-PD-L1 antibody was used, emricasan nanoparticles were able to control larger tumors as shown in FIG. 6 and induce abscopal effects in controlling untreated contralateral tumors as shown in FIG. 7.

[00153] IFNβ is a key early innate immune response signal that is responsible for several critical steps in anti-tumor immunity such as cross priming of CD8 T cells and is an integral component in STING and Toll-like receptor agonism as cancer therapeutics. PGE2 is an immune suppressant and was reported to promote tumor repopulation. These data suggest emricasan may function as an ISV in tumor control and adding checkpoint inhibitors may induce abscopal effect for metastatic cancer treatment.

[00154] Most cancers are diagnosed at the localized stage, but rationally designed anti-cancer drugs specifically targeting this large population are lacking. Most drugs were designed for metastatic diseases with the safety profiles incompatible with the treatment of early-stage cancer. Emricasan is a very safe compound and the efficacious dose for tumor control is well below its maximum tolerant dose. As a covalent inhibitor Emricasan is suitable for local administration as a short period of drug coverage would be sufficient to fully disable apoptotic caspases in the tumor tissue. Its immune stimulating activity may prevent the relapse and metastasis after the locoregional cancer treatment. These features rend emricasan nanoformulation an ideal therapy for locoregional cancer treatments.

Nanoparticle preparation and characterization

[00155] Liposomes (10 mM) having L-α-PC was prepared using thin film dehydration method. The details are as follows: 310 μL of L-α-PC (25 mg/mL in chloroform) was pipetted in a scintillation vial and dried under a gentle stream of Argon gas. The dried film was then placed under vacuum for an additional 2 hours to remove any residual traces of organic solvent. The desiccated film was subsequently rehydrated with 1 mL of phosphate buffer saline (PBS) and bath-sonicated for approximately 3 minutes. The solution was then extruded 21 times with 200 nm polycarbonate membrane and stored at 4°C for later use.

[00156] Nanoparticles of PLGA (7k-17k) encapsulating emricasan were generated by flash nanoprecipitation method. One of the four inlet streams of MIVM (stream 1) containing 0.1 wt% PLGA and 0.1 wt% emricasan dissolved in THF. The other three inlet streams contained deionized water as anti-solvent to precipitate the polymer and the drug. The flowrate of stream 1 and stream 2 was set to be 13.5 mL/minute while flowrate of stream 3 and 4 was 54 mL/minute. The nanoparticle suspension was collected into a beaker having 267 μL solution of liposome (10 mM) solution under stirring. The mixture was then bath-sonicated for 1 minute for the liposomes to wrap around polymeric nanoparticles. The solution was immediately cooled in an ice bath while trehalose was added into the nanoparticle suspension. Lor each vial having 7 mL volume of nanoparticle suspension, 140 μL, 280 μL and 560 μL of 50 mg/mL trehalose was added reach the ratios of 5 times, 10 times and 20 times trehalose to solid nanoparticles, respectively

[00157] The nanoparticles solution was frozen in a -80°C freezer for two days and freeze-dried for 72 hours to produce stable solid powder. Nanoparticle resuspension

[00158] Deionized water was added to the dried nanoparticle powder. Each sample was vortexed for 30 seconds. Some samples required immediately sonication for 5 minutes. The nanoparticle solution has a white, opaque appearance.

[00159] The final mass composition for each sample of emricasan is presented in Table 2:

Table 2. Final mass composition of the resuspended nanoparticles

DLS measurement protocol

[00160] Nanoparticle size measurements were measured using a Malvern® Zetasizer instrument. Samples were mixed with a pipette before measured. Each measurement duration was set for 30 seconds. The laser attenuator was set at 7 unless otherwise specified to record the count rate throughout 6 hours. Each data point was the average of 3 to 6 measurements.

Size distribution after mixing and resuspension

[00161] Immediately following mixing, the nanoparticles size was around 150 nm. Right after resuspension, the nanoparticle size increased slightly for 5x and lOx trehalose samples but remained the same for 20 times trehalose samples. Several constructs showed variable stability, but the 5x trehalose construct with 5 minutes of sonication for resuspension and 20x trehalose construct without the assistance of sonication gave desirable and stable particle size for at least 6 hours at room temperature as shown in FIG. 6 and FIG. 7, respectively. To demonstrate the unpredictability of the behaviors of these nanoparticles, FIG. 8 and FIG. 9 show the 5x trehalose construct without sonication and 20x trehalose construct with twice the loading of emricasan, respectively. Both constructs were less stable than the reference.

Chemical stability of emricasan in different media

[00162] Emricasan or polymer-encapsulated emricasan was dissolved in THF and deionized water and allowed to stay at room temperature for indicated amount of time. The mixture was extracted with ethyl acetate and emricasan and its epimer were analyzed by HPLC using a chiral column (Reflect C-Amylose A; Mobile Phase: Hexanes:Isopropanol:Acetic Acid=85:15:0.1; Flowrate: 1.5 mL/minutes; Detection method: 254 nm UV).

[00163] Applicant’s disclosure is described herein in preferred embodiments with reference to the Figures, in which like numbers represent the same or similar elements. Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment. It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and/or “consisting essentially of are also provided. Unless expressly stated to the contrary, all ranges cited herein are inclusive; i.e., the range includes the values for the upper and lower limits of the range as well as all values in between. As an example, dose ranges, percentages, and the like described herein include the upper and lower limits of the range and any value in the continuum there between.

[00164] The described features, structures, or characteristics of Applicant’s disclosure may be combined in any suitable manner in one or more embodiments. In the description, herein, numerous specific details are recited to provide a thorough understanding of embodiments of the invention.

One skilled in the relevant art will recognize, however, that Applicant’s composition and/or method may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the disclosure.

[00165] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Methods recited herein may be carried out in any order that is logically possible, in addition to a particular order disclosed.

Incorporation by Reference [00166] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure.

Equivalents

[00167] The representative examples are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples and the references to the scientific and patent literature included herein. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

What is claimed is: CLAIMS

1. A nanoparticle composition, wherein the nanoparticles comprise: a nanoparticle core comprising one or more polymers and at least one active ingredient comprising emricasan or active metabolites thereof; at least one layer of one or more lipids on a surface of the polymer core; and at least one lyoprotectant.

2. The nanoparticle composition of claim 1, wherein the one or more polymers comprise poly(lactic-co-glycolic acid) (PLGA) or its PEGylated form PEG-PLGA, polylactic acid (PLA) or its PEGylated form PEG-PLA, polyglycolic acid (PGA) or its PEGylated form PEG-PGA, polycaprolactone (PCL) or its PEGylated form PEG-PCL, poly-L-lactide-co- e-caprolactone (PLCL) or its PEGylated form PEG-PLCL, Hyaluronic acid (HA), polyacrylic acid (PAA) or PEG-PAA, polyphosphate (polyP), poly(acrylic acid-co-maleic acid), poly(butylene succinate), poly(alkyl cyanoacrylate) (PAC) or its PEGylated form PEG-PAC, or a combination thereof.

3. The nanoparticle composition of claim 2, wherein the one or more polymers is poly(lactic-co-glycolic acid) (PLGA).

4. The nanoparticle composition of claim 2, wherein the one or more polymers is PEGylated polycaprolactone (PEG-PCL).

5. The nanoparticle composition of any one of claims 1-4, wherein the one or more lipids comprise 1,2-Didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dilauroyl-sn- glycero-3-phosphoethanolamine (DLPE), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE-PEG), 1-palmitoyl-2-myristoyl-sn-glycero-3- phosphocholine (PMPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-( 1 '-rac-glycerol)

(DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine (DPPE-PEG), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1- palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSOC), 1-stearoyl-2-palmitoyl-sn- glycero-3-phosphocholine (SPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (DEPE-PEG), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[dibenzocyclooctyl(polyethylene glycol)-2000] (DSPE-PEG), L-α-phosphatidylcholine (L-α-PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-distearoyl-sn- glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-Distearoyl-3-trimethylammonium- propane (DSTAP), 1,2-dioleoyl-3-trimethylammonium -propane (DOTAP), 1,2-dioleoyl- sn-glycero-3-phosphate (DOPA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), or 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC), or a combination thereof. The nanoparticle composition of claim 5, wherein the one or more lipids is L-α- phosphatidylcholine (L-α-PC). The nanoparticle composition of any one of claims 1-6, wherein the at least one lyoprotectant is selected from the group consisting of sucrose, glucose, trehalose, mannitol, polyethylene glycol, hyaluronic acid, alginic acid, leucine and cyclodextrin. The nanoparticle composition of claim 7, wherein at least one lyoprotectant is trehalose. The nanoparticle composition of any one of claims 1-8, further comprising an active agent selected from an anti-cancer drug, an immune stimulating agent, and a combination thereof. The nanoparticle composition of claims 1-9, wherein a mass ratio of emricasan to the one or more polymers is about 90: 10 to about 10:90. The nanoparticle composition of claims 1-10, wherein a mass ratio of emricasan to the one or more lipids is about 100: 0 to about 20:80. The nanoparticle composition of claims 1-11, wherein a mass ratio of emricasan to the at least one lyoprotectant is about 1:99 to about 50:50. The nanoparticle composition of claims 1-12, wherein the nanoparticle is characterized by a zeta potential from about -80 mV to about 80 mV. The nanoparticle composition of claims 1-13, further comprising at least one targeting agent capable of selectively delivering the nanoparticle to diseased tissue/cells. The nanoparticle composition of claim 14, wherein the at least one targeting agent comprises an antibody or functional fragment thereof, a small molecule, a peptide, a carbohydrate, an siRNA, a protein, a nucleic acid, an aptamer, a second nanoparticle, a cytokine, a chemokine, a lymphokine, a receptor, a lipid, a lectin, a ferrous metal, a magnetic particle, a linker, an isotope, or a combination thereof. The nanoparticle composition of claims 1-15, wherein the nanoparticles have a size of about 50 nm to about 400 nm. The nanoparticle composition of claims 1-16, wherein the nanoparticles remain stable at room temperature for at least 6 hours after resuspension in an aqueous solution. The nanoparticle composition of claims 1-17, wherein a drug load of emricasan is about 1% to about 50% by weight of the composition. The nanoparticle composition of claim 18, wherein the drug load of emricasan is about 10% to about 40% by weight of the composition. The nanoparticle composition of any one of claims 1-19, being in a lyophilized form. The nanoparticle composition of any one of claims 1-19, being an aqueous solution. The nanoparticle composition of claim 21, characterized by improved stability with respect to emricasan epimerization. A pharmaceutical composition comprising a nanoparticle composition according to any one of claims 1-22 and a pharmaceutically acceptable carrier. A unit dosage form comprising the pharmaceutical composition of claim 23. A method of forming a nanoparticle composition of any one of claims 1-21, comprising: a), forming an organic phase by combining one or more PLGA polymers, one or more solvents, and at least one of emricasan or its metabolites; b). mixing the organic phase with water thereby forming a nanosuspension via self-assembly of micelles; c). forming a liposome by combining a desiccated thin film of one or more phospholipids with an aqueous solution; d). mixing the nanosuspension with the liposome and sonicating the mixture; e). adding a lyoprotectant; f). spray drying or freeze drying the suspension; and g). resuspending in aqueous solutions. The method of claim 25, wherein the nanoparticles are characterized by having a substantially uniform size with substantially uniform physicochemical properties. A method for treating cancer, compromising administering to a subject in need thereof a nanoparticle composition, a pharmaceutical composition, or a unit dosage form according to any one of claims 1-24. A method for in situ vaccination for systemically inhibiting or reducing cancer growth, compromising administering to a subject in need thereof a nanoparticle composition, a pharmaceutical composition, or a unit dosage form according to any one of claims 1-24. The method of claim 27 or 28, wherein administration is intratumoral, intramuscular, subcutaneous, intravascular, or intravenous. The method of claim 29, wherein the nanoparticle composition is administered intratum orally to a subject in need thereof. The method of any one of claims 27-30, wherein the subject is not administered, simultaneously or sequentially, local radiation therapy on the injected or noninjected lesions. The method of any one of claims 27-30, wherein the subject is further administered, simultaneously or sequentially, local radiation therapy on the injected or noninjected lesions. The method of any one of claims 27-30, wherein the subject is further administered, simultaneously or sequentially, anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies. The method of any one of claims 27-33, wherein the cancer is selected from breast cancer, small cell lung cancer, non-small cell lung cancer, prostate cancer, gastric cancer, renal cell carcinoma, ovarian cancer, cervical cancer, colorectal cancer, hepatocellular carcinoma, glioblastoma, melanoma, basal cell carcinoma, cutaneous squamous cell carcinoma, Bowen's disease, pancreatic ductal carcinoma, head and neck squamous cell carcinoma, lip squamous cell carcinoma, buccal mucosa squamous cell carcinoma, oral tongue squamous cell carcinoma, oral squamous cell carcinoma, salivary mucoepidermoid carcinoma, and endometrial carcinoma. The method of claims 27-34, wherein the active agent is released in a sustained manner. Use of a nanoparticle composition according to any one of claims 1-22 for the manufacture of a medicament for the treatment of cancer, or a related disease or condition. Use of a nanoparticle composition according to any one of claims 1-22 for treating cancer. Use of a nanoparticle composition according to any one of claims 1-22 for systemically inhibiting or reducing cancer growth.