WO2016014337A1

WO2016014337A1 - Drug delivery nanoemulsion systems

Info

Publication number: WO2016014337A1
Application number: PCT/US2015/040751
Authority: WO
Inventors: Srinivas Ganta; Timothy P. Coleman
Original assignee: Nemucore Medical Innovations Inc
Current assignee: Nemucore Medical Innovations Inc
Priority date: 2014-07-25
Filing date: 2015-07-16
Publication date: 2016-01-28
Anticipated expiration: 2017-01-25

Abstract

Provided are nanoemulsion formulations comprising a drug delivery system and a chemotherapeutic agent for cancer treatment and diagnosis and which do not elicit multi¬ drug resistance. Also provided are methods of preparing the same and of treating or imaging cancer.

Description

DRUG DELIVERY NANOEMULSION SYSTEMS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of U.S. provisional patent application number 62/029, 144, filed on July 25, 2014, the entire content of which is hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Part of the work leading to this invention was carried out with United States Government support provided under a grant from the National Institutes of Health, Grants No. R01CA158881, R43CA144591, U43CA162454 and U54CA151881. Therefore, the U.S. Government has certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present disclosure relates to medicine and pharmacology, and more particularly, to cancer therapy and drug delivery systems for cancer therapeutics.

BACKGROUND

[0004] Chemotherapeutic agents are widely used in cancer therapy. However, in most cases these treatments do not cure the disease. Challenges for effective therapy are the serious side- effects of many cancer drugs, insufficient concentration and short residence time of therapeutic agents at the site of disease, multi-drug resistance (MDR) and the hydrophobicity of pharmaceutical agents. One challenge for effective therapy is that drug efficacy is often undermined by serious side-effects resulting from drug toxicities to normal tissues. Also, some treatment failures may be due to the lack of sufficient concentration and short residence time of therapeutic agents at the site of disease. (Jain (2003) Nat. Med. 9(6):685-693).

Additionally, lack of target specificity contributes to systemic toxicity as the therapeutic agent builds up in non-diseased tissues. In addition, tumors often inherently have or acquire resistance to a chemotherapeutic agent after rounds of treatment and are resistant not only to the original agent, but to other related and unrelated agents, a phenomenon known as multidrug resistance (MDR).

[0005] A number of delivery systems have been developed to address these problems. For example, nanodelivery systems with site-specific binding moieties have been developed with various levels of success to increase concentration of drug in the tumors and decrease side effects. Some delivery vehicles have been devised that improve drug delivery to tumors, for example Doxil (also known as Caelyx), comprising doxorubicin in polyethylene glycol (PEG)-coated liposomes. Another example is poly(epsilon-caprolactone) (PCL)

nanoparticles. (Chawla et al. (2003) AAPS PharmSci. 5(l):28-34). The alkyl structure of the polymer encapsulates hydrophobic compounds. Surface modification of the colloidal carrier with an agent such as a poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO-PEO) triblock copolymer can improve the solubility of the nanoparticle. However, serious side-effects of chemotherapeutic agents to normal tissue remains a challenge for effective cancer treatment.

[0006] MDR is a major obstacle impeding the delivery of chemotherapeutic agents to tumors, and therefore the treatment of cancer.. Many first line chemotherapeutic agents elicit a response and tumors shrink, but often these tumors develop resistance to the

chemotherapeutic agent before affecting a cure. For instance, platinum compounds (including cisplatin, carboplatin, and oxaliplatin), used as therapies for many types of cancers (including head and neck, testicular, ovarian, cervical, lung, colorectal, and relapsed lymphoma) have a good initial response, but later the patients relapse because of the development of resistance. (Siddick et al. (2003) Oncogene 22:7265-7279).

[0007] Effective cancer therapy also suffers from the lack of early data on the delivery of a particular pharmaceutical agent to tumors and thus effectiveness. Patients often proceed with a course of treatment for an extended period of time, while suffering associated side-effects and poor quality of life, only to find out that the particular treatment is not effective.

[0008] Hydrophobicity of pharmaceutical agents limits the range of therapies for cancer treatment. Almost one third of the drugs in the United States Pharmacopeia (http:// www.usp.org/) are hydrophobic and are either insoluble or poorly soluble. (Savic et al.

(2006) J. Drug Target. 14(6):343-55). As a result of poor solubility, many potential new chemical entities are being dropped in the early phases of development.

[0009] Approaches for administering hydrophobic drugs include the use of co-solvents, incorporation of complexing or solubilizing agents, chemical modification of the drug, use of micellar delivery systems such as niosomes, liposomes, and their formulation of the drug in an oily vehicle, for oral, parenteral, nasal, rectal or ophthalmic delivery. However, many of these formulations employ surfactants or co-solvents having associated toxic side-effects, and frequently, stability, sterility, and mass commercial production issues as well.

[0010] Accordingly, there exists a need for delivery systems, which can efficiently deliver therapeutic levels of drug to disease sites with fewer or no side-effects while blocking or avoiding multi-drug resistance pathways. There is also a need to expand the range of therapeutics that can be used for cancer treatment. In addition, a need also exists for imaging capabilities that will allow for quick determination as to whether a patient should proceed with a particular course of treatment.

SUMMARY

[0011] It has been discovered that certain drug delivery systems are able to deliver chemotherapeutic agents to cancer cells without eliciting a multidrug resistance response. The use of these drug delivery systems can increase the efficacy and efficiency of chemotherapeutic treatments of cancer. Resistance is a natural cellular self-defense mechanism developed by evolution to protect cells from toxic natural products and other environmental stressors. The nanoemulsion formulation bypasses or overcomes these defense mechanisms, allowing for the delivery of chemotherapeutic agents directly into the cancer cells of patients with resistant cancer. Therefore, the present disclosure provides a less toxic and more effective system to treat patients with cancer, including multidrug resistant cancers.

[0012] This discovery has been exploited to develop the present disclosure, which, in one aspect, is a nanoemulsion formulation comprising a drug delivery system and a

chemotherapeutic agent. The drug delivery system comprises an oil phase, an interfacial surface membrane, an aqueous phase, and a chemotherapeutic agent comprising a chemotherapeutic agent which is not carboplatin, cisplatin, a di-fatty acid derivative of platinum, a salicylate derivative of platinum, NMI-300, or NMI-500, and wherein the chemotherapeutic agent is dispersed in the oil phase.

[0013] In some embodiments, the oil phase of the drug delivery system comprises flaxseed oil, omega-3 polyunsaturated fish oil, omega-6 polyunsaturated fish oil, safflower oil, olive oil, pine nut oil, cherry kernel oil, soybean oil, pumpkin oil, pomegranate oil, primrose oil, or combinations thereof. [0014] In certain embodiments, the interfacial surface membrane phase of the drug delivery system comprises an emulsifier and/or a stabilizer. In some embodiments, the emulsifier of the interfacial surface membrane comprises egg lecithin, egg phosphatidyl choline, soy lecithin, phosphatidyl ethanolamine, phosphatidyl inositol, dimyristoylphosphatidyl choline, dimyristoylphosphatidyl ethyl-N-dimethyl propyl ammonium hydroxide, hydrogenated soy phosphatidylcholine, l,2-distearoyl-sn-glycero-3-phosphocholine or combinations thereof. In other embodiments, the stabilizer of the interfacial surface membrane comprises a polyethylene glycol derivative, a phosphatide, a polyglycerol mono oleate, or combinations thereof. In certain embodiments the polyethylene glycol derivative of the interfacial surface membrane is PEG₂₀₀₀DSPE, PEG₃₄₀₀DSPE, PEG₅₀₀₀DSPE, or combinations thereof. In some embodiments, the polyethylene glycol derivative has a molecular weight of from 1 kDa to 20 kDa, from 5 kDa to 20 kDa, or from 10 kDa to 20 kDa.

[0015] In some embodiments, the chemotherapeutic agent dispersed in the oil phase of the nanoemulsion formulation is an ionophore, a DNA metabolism inhibitor, a cytoskeletal inhibitor, a kinase inhibitor, an inflammatory signal inhibitor, and/or combinations thereof.

[0016] In some embodiments, the nanoemulsion formulation further comprises a chemopotentiator. In particular embodiments, the chemopotentiator comprises ceramide (CER) or a derivative thereof. In certain embodiments, the chemopotentiator comprises C6- ceramide.

[0017] In some embodiments, the nanoemulsion formulations the chemotherapeutic agent further comprises C6-ceramide.

[0018] In some embodiments, the nanoemulsion formulation further comprises a targeting ligand. In certain embodiments the targeting ligand comprises an EGFR-targeting ligand, a folate receptor-targeting ligand, or a combination thereof. In particular embodiments, the EGFR-targeting ligand comprises peptide 4, an anti-EGFR immunoglobulin or EGFR- binding fragment thereof, EGal-PEG, or combinations thereof. In other embodiments, the folate receptor-targeting ligand comprises DSPE-PEG-cysteine-folic acid, DSPE-PEG(2000) folate, DSPE-PEG(3400) folate, DSPE-PEG(5000) folate, an anti-folate receptor immunoglobulin or folate receptor-binding fragment thereof, or combinations thereof. In some embodiments, the PEG in the targeting ligand EGal-PEG has a molecular weight of from 1 kDa to 20 kDa, from 5 kDa to 20 kDa, or from 10 kDa to 20 kDa.

[0019] In some embodiments, the nanoemulsion formulation further comprises an imaging agent. In particular embodiments, the imaging agent is an MRI contrasting moiety. In certain embodiments the MRI contrasting moiety comprises gadolinium, iron oxide, iron platinum, manganese, or combinations thereof.

[0020] In another aspect, the present disclosure provides a method of inhibiting the growth of, or killing, a cancer cell, comprising contacting the cancer cell with an amount of the nanoemulsion formulation as described above that is toxic to, or which inhibits the growth of, or which kills, the cancer cell. In some embodiments, the cancer cell is in a mammal, and the contacting step comprises administering to the mammal a therapeutically effective amount of the nanoemulsion formulation.

[0021] In yet another aspect, the present disclosure provides a method of imaging a cancer cell, comprising contacting the cancer cell with the nanoemulsion formulation of as described above. In some embodiments, the cancer cell is in a mammalian subject, and the contacting step comprises administering to the subject an amount of the nanoemulsion formulation sufficient to image the cancer cell.

DESCRIPTION OF THE FIGURES

[0022] The foregoing and other objects of the present disclosure, the various features thereof, as well as the invention itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

[0023] FIG. 1 is a diagrammatic representation of a generic nanoemulsion formulation of the present disclosure;

[0024] FIG. 2 is a diagrammatic representation of one non-limiting method of preparing a nanoemulsion formulation;

[0025] FIG. 3 is a graphic representation of a DLS plot of a blank nanoemulsion formulation showing the particle size;

[0026] FIG. 4 is a graphic representation of a DLS plot of an SP600125 nanoemulsion formulation showing the particle size;

[0027] FIG. 5 is a graphic representation of a DLS plot of a sulforaphane nanoemulsion formulation showing the particle size;

[0028] FIG. 6 is a graphic representation of a DLS plot of a salinomycin nanoemulsion formulation showing the particle size;

[0029] FIG. 7 is a graphic representation of a DLS plot of a docetaxel nanoemulsion formulation showing the particle size;

[0030] FIG. 8 is a graphic representation of a DLS plot of a docetaxel + ceramide nanoemulsion showing the particle size;

[0031] FIG. 9 is a graphic representation of a DLS plot of a docetaxel + SP600125 nanoemulsion showing the particle size;

[0032] FIG. 10 is a graphic representation of a DLS plot of a docetaxel + salinomycin nanoemulsion showing the particle size;

[0033] FIG. 11 is a graphic representation of a DLS plot of an etoposide nanoemulsion formulation showing the particle size;

[0034] FIG. 12 is a graphic representation of a DLS plot of a cyclopamine nanoemulsion formulation showing the particle size;

[0035] FIG. 13 is a graphic representation of a DLS plot of a noscapine nanoemulsion formulation showing the particle size; [0036] FIG. 14 is a graphic representation of a DLS plot of a docetaxel, ceramide, and salinomycin nanoemulsion formulation showing the particle size;

[0037] FIG. 15 is a graphic representation of a DLS plot of a docetaxel, sulforaphane, and salinomycin nanoemulsion formulation showing the particle size;

[0038] FIG. 16 is a graphic representation of a DLS plot of a abamectin nanoemulsion formulation showing the particle size;

[0039] FIG. 17 is a graphic representation of a DLS plot of a BAY 11-7082 nanoemulsion formulation showing the particle size;

[0040] FIG. 18 is a schematic representation of one non-limiting method of synthesizing EGFR-MAL-PEG-DSPE;

[0041] FIG. 19 is a schematic representation of one non-limiting mode of synthesizing DSPE-PEG-Cys-FA;

[0042] FIG. 20 is a schematic representation of one non-limiting method of preparing Gd ³- DTPA-PE;

[0043] FIG. 21 A is a representation of the NMR spectra of a DSPE-PEG-MAL standard;

[0044] FIG. 2 IB is a representation of the NMR spectra of a EGFR-binding peptide standard;

[0045] FIG. 21C is a representation of the NMR spectra of the DSPE-PEG-MAL-EGFRbp conjugate; and

[0046] FIG. 22 is a series of representations of magnetic resonance images (MRIs) of mice treated with Gd³+-DTPA-PE, Magnevist™ (Mag), Gd³+-DTPA-PE, non-targeted nano-

3+

emulsion formulation (NT), or Gd -DTPA-PE, EGFR-targeted nanoemulsion formulation (T).

DESCRIPTION

[0047] Throughout this application, various patents, patent applications, and publications are referenced. The disclosures of these patents, patent applications, and publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. The instant disclosure will govern in the instance that there is any inconsistency between the patents, patent applications, and publications and this disclosure.

Definitions

[0048] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. 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 to which this invention belongs. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated.

[0049] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0050] The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise.

[0051] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" or "approximately" is used herein to modify a numerical value above and below the stated value by a variance of 20%.

[0052] The expression "at least one" is used herein to mean one or more and thus includes individual components as well as mixtures/combinations.

[0053] "Anticancer agent" or "chemotherapeutic agent" is an agent that prevents or inhibits the development, growth or proliferation of malignant cells.

[0054] "Cancer" is the uncontrolled growth of abnormal cells. [0055] "Stable chemotherapeutic formulation" is a formulation containing a chemotherapeutic agent or ion wherein the agent or ion is stable for transformation for a time sufficient to be therapeutically useful.

[0056] "Stabilizer" is an agent that prevents or slows the transformation or deactivation of a chemotherapeutic agent or ion in a chemotherapeutic agent formulation.

[0057] "Patient" is a human or animal in need of treatment for cancer.

[0058] "CDDP" Cis-dichlorodiammine Pt (II)

[0059] |[SKI]A "combination index" is defined as an isobologrm equation to study combination drug effects on cells and to determine whether the drug combination produced enhanced efficacy in the form of an additive, synergistic, or antagonistic effect on cells.

[0060] "Stabilizer" as used herein means an agent that prevents or slows the transformation or deactivation of a chemotherapeutic agent or other active pharmaceutical ingredient, such as a Pt-containing compound or ion in a Pt-containing nanoemulsion formulation.

[0061] |[SK2]"Nanoemulsion formulation" as used herein means a novel nanoemulsion (NE) comprising an oil phase; an interfacial surface membrane; an aqueous phase; and a a chemotherapeutic agent dispersed in the oil phase.

[0062] "Nanoemulsion" as used herein means a colloidal dispersion comprised of omega-3, -6 or -9 fatty acid rich oils in an aqueous phase and thermo-dynamically stabilized by amphiphilic surfactants, which make up the interfacial surface membrane, produced using a high shear microfluidization process usually with droplet diameter within the range of about 80-220 nm.

[0063] "Oil phase" as used herein means the internal hydrophobic core of the

nanoemulsion in which a chemotherapeutic agent is dispersed and refers either to a single pure oil or a mixture of different oils present in the core. The oil phase is comprised of generally regarded as safe grade, parenterally injectable excipients generally selected from omega-3, omega-6 or omega-9 polyunsaturated unsaturated fatty acid (PUFA) or

monounsaturated fatty acid rich oils.

[0064] "Aqueous phase" is comprised of isotonicity modifiers and pH adjusting agents in sterile water for injection and forms as an external phase of the nanoemulsion formulation in which the oil phase is dispersed.

[0065] "Amphiphilic molecule or amphiphilic compound" as used herein means any molecule of bipolar structure comprising at least one hydrophobic portion and at least one hydrophilic portion. The hydrophobic portion distributes into the oil phase and hydrophilic portion distributes into aqueous phase forming an interfacial surface membrane and has the property of reducing the surface tension of water (γ < 55 mN/m) and of reducing the interface tension between water and an oil phase. The synonyms of amphiphilic molecule are, for example, surfactant, surface-active agent and emulsifier.

[0066] "Amphiphilic or amphiphile" as used herein means a molecule with both a polar, hydrophilic portion and a non-polar, hydrophobic portion.

[0067] "Primary emulsifiers" as used herein means amphiphilic surfactants that constitute a major percentage of amphiphilic surfactants of the nanoemulsion formulation wherein they stabilize the formulation by forming an interfacial surface membrane around oil droplets dispersed in water, and further allow for surface modification with targeting ligands and imaging agents.

[0068] "Co-emulsifiers" as used herein means amphiphilic surfactants used in conjunction with primary emulsifiers where they associate with the interfacial surface membrane, effectively lowering the interfacial tension between oil and water, and help in the formation of stable nanoemulsion formulations.

[0069] "Stabilizers" or "stealth agents" as used herein mean lipidated polyethylene glycols (PEG) where the lipid tail group distributes into the oil phase and hydrophilic PEG chains distribute into the aqueous phase of a nanoemulsion formulation, providing steric hindrance to mononuclear phagocytic system (MPS) cell uptake during the blood circulation, thus providing longer residence time in the blood and allowing for enhanced accumulation at tumor site through leaky tumor vasculature, a phenomenon termed as enhanced permeability and retention effect, largely present in wide variety of solid tumors. Other representative examples are a phosphatide, and a polyglycerol mono oleate,

[0070] "Targeting agents" as used herein are molecules, which direct a nanoemulsion particle towards a tumor/cancer cell. Such targeting agents allow for interaction with tumor cells in vivo, forming a ligand-receptor complex, which is taken up by the tumor cells.

[0071] "Isotonicity modifiers" as used herein means agents that provide an osmolality (285-310 mOsm/kg) to the nanoemulsion formulation, thus maintaining isotonicity for parenteral injection.

[0072] "pH modifiers" as used herein means buffering agents that adjust the pH of nanoemulsion formulation to a value of about pH 6-7.4, thus preventing the hydrolysis of phospholipids upon storage. [0073] "Preservatives" as used herein means antimicrobial agents that when added to the nanoemulsion formulation at about 0.001-0.005% w/v prevent bacterial growth during the storage of nanoemulsion formulation.

[0074] "Antioxidants" as used herein means agents that stop oxidation of oils comprised of fatty acids, thus preventing rancidification of oil phase and destabilization of the

nanoemulsion formulation.

[0075] "Chemopotentiator" as used herein means a drug or chemotherapeutic agent used in combination with other drugs or chemotherapeutic agents to enhance, increase or strengthen the effect, for instance decreases the IC5₀, and thus increasing the efficacy, of the drug or chemotherapeutic agent.

1. Nanoemulsion Formulations

[0076] The present disclosure provides novel nanoemulsion formulations useful for treatment of tumors and cancer cells. These nanoemulsion formulations inhibit or bypass multidrug resistant pathways. This formulation comprises a drug delivery system and a

chemotherapeutic agent. The drug delivery system comprises an oil phase, an interfacial surface membrane, and an aqueous phase. The chemotherapeutic agent is dispersed within the oil phase and is not carboplatin, cisplatin, a di-fatty acid derivative of platinum, or a salicylate derivative of platinum.

[0077] FIG. 1 is a non-limiting schematic representation of a nanoemulsion formulation of the present disclosure. In this figure, 4 represents a chemotherapeutic agent comprising chemotherapeutic agents dispersed in the oil phase 5 of the nanoemulsion formulation. 5 is encapsulated within the interfacial membrane 7 which comprise emulsifiers 8 and stabilizers 3. The polar, hydrophilic portions of the amphiphiles of the interfacial surface membrane project into the aqueous phase 9, and the non-polar, hydrophobic portions of these amphiphiles project into the oil phase 5. 1 represents a targeting ligand linked to stabilizers 3 in the interfacial surface membrane, 2 represents an imaging agent attached to an emulsifier 8 in the interfacial surface membrane 7 and 6 represents a chemopotentiator dispersed in the oil phase 5 of the nanoemulsion formulation.

[0078] The nanoemulsion formulation of the present disclosure may also comprise a co- agent, a co-emulsifier, a preservative, an antioxidant, a pH adjusting agent, an isotonicity modifier, or any combination thereof. [0079] Various non-limiting examples of the drug delivery system components of the nanoemulsion formulations and their corresponding proportions are provided in Table I and discussed below.

Table I

A. Chemotherapeutic Agents

[0080] The novel nanoemulsion formulations according to the disclosure contain certain chemotherapeutic agents, which are ionophores, DNA metabolism inhibitors, cytoskeletal inhibitors, kinase inhibitors, and/or inflammatory signal inhibitors. These are listed in Table II. Table II

NA = Not available

[0081] Non-limiting examples of ionophores are salinomycin, nigericin, and/or abamectin. Without being limited to any mechanism of action, salinomycin inhibits the Wnt signaling pathway by interfering with lipoprotein receptor related protein 6 (LRP6) phosphorylation that is critical to the self-renewal of cancer stem cells (CSC), and also inhibits P-glycoprotein (gp) function. Nigericin inhibits the phosphorylation of the Wnt coreceptor lipoprotein receptor related protein 6, resulting in blockade of CSC self-renewal process. Abamectin inhibits cell proliferation by delaying of the cells cycle start (lag-phase prolongation) and blocking of the mitotic cycle. It is also an inhibitor of multi-drug resistance gpl70 causing down-regulation of Pgp, and as a result cells become sensitized to chemotherapy.

[0082] Non-limiting examples of DNA metabolism inhibitors are etoposide and/or camptothecin. Etoposide inhibits topoisomerase II, which aids in DNA breakdown, resulting in arrest of cell growth. Camptothecin inhibits DNA enzyme topoisomerase I by forming a hydrogen bond, which results in DNA damage and apoptosis.

[0083] A non-limiting example of cytoskeletal inhibitors is noscapine, which binds to tubulin and alters its conformation, resulting in a disruption of the dynamics of microtubule assembly, thereby arresting cell growth and inducing apoptosis.

[0084] Non-limiting examples of kinase inhibitors are UCN-01, staurosporine, and/or SP600125. UCN-01 is a potent Chkl inhibitor that binds to the ATP -binding pocket of Chkl, which abrogates the G2/M checkpoint, resulting in cell cycle arrest. Staurosporine is a Chkl inhibitor that binds to the ATP -binding pocket of Chkl abrogating the G2/M checkpoint, resulting in cell cycle arrest. SP600125 inhibits Jun N-terminal kinase and mediates downstream effects resulting in inhibition of cell proliferation. It is also a mullerian- inhibiting substance (MIS) agonist and activates the MIS transduction pathway by binding to MIS type II receptors, resulting in cell growth inhibition.

[0085] A non-limiting example of inflammatory signal inhibitors is BAY 1 1-7082, which inhibits transcription factor NFkB that controls cell growth, apoptosis and differentiation, resulting in CSCs cell death.

[0086] These chemotherapeutic agents are lipophilic, forming stable nanoemulsions either alone or in combination with chemopotentiators or co-agents, and thus are available for use as anticancer agents having a high specificity and selectivity to cancer cells. Moreover, their liposolubility makes them useful as slowly and steadily released and sustained

nanomedicines. The encapsulation of chemotherapeutic agents, including those in combination with chemopotentiators and co-agents in the nanoemulsion formulations of the present disclosure, aid in mitigating undesirable side-effects known to sometimes accompany their use. They also inhibit and/or bypass the mechanisms that cause multi-drug resistance in cancer cells and can protect the chemotherapeutic agents from destruction while circulating through the body.

[0087] The nanoemulsion formulations of the present disclosure may comprise a chemopotentiator, such as an apoptosis enhancer. Non-limiting examples for apoptosis enhancers are ceramide (CER), cyclopamine, sulforaphane, curcumin, or ceramide or curcumin derivatives. These chemopotentiators enhance apoptosis and the increase the ability of chemotherapeutic agents to kill cancer cells.

[0088] The structure of C-6 CER is as follows :

[0089] Over-expression of Glucosylceramide synthase is associated with decreased rates of apoptosis in many cancer types. Endogenous addition of CER can significantly enhance the apoptotic potential of chemotherapies, thus improving efficacy. (Shabbits et al. (2003)

Biochim..Biophvs. Acta, 1612:98-106.). Replacement of CER mediates induction of apoptosis via the inhibition of Akt pro-survival pathways, mitochondrial dysfunction, and stimulation of caspase activity, ultimately leading to DNA fragmentation.

[0090] Cyclopamine inhibits the Hedgehog signaling pathway by directly binding to a membrane receptor smoothened, resulting in apoptosis and supression of renewal of CSCs. Sulforaphane inhibits Akt pro-survival pathways and down regulates the Wnt/ -catenin pathway that is critical to the self-renewal of CSCs and differentiation. These events lead to cell growth arrest, overcoming drug resistance, and causing apoptosis.

[0091] Curcumin interferes with the NFkB, Akt/mTOR/p70S6K molecular signaling pathways and drug efflux pumps, resulting in apoptosis and sensitization of cells to chemotherapy. It also nhibits Wnt patway invovled in CSCs growth and self-renewal.

[0092] While chemopotentiators seem to enhance the efficacy of chemotherapeutic agents, there are obstacles to the delivery of these compounds. First, their effectiveness is limited due to their hydrophobicity and possible precipitation when administered in aqueous solutions. In addition, the structures of the chemopotentiators, such as the existence of a second aliphatic chain of CER, can hinder cellular permeability. Also, some of the free chemopotentiators are susceptible to metabolic inactivation by specific enzymes in the systemic circulation. Accordingly, measures, which avoid these obstacles are useful. In this regard, the present nanoemulsion formulations exploit the benefits of the chemopotentiators/apoptosis enhancers by providing increased solubility, intracellular permeability, and protection from systemic enzymatic degradation.

B. Oil Phase

[0093] The oil phase of the nanoemulsion formulation according to the present disclosure comprises individual oil droplets. The average diameter of the oil droplets in the oil phase ranges from about 5 nm to 500 nm. This component is the internal hydrophobic or oil core and may be a single entity or a mixture.

[0094] A wide variety of oils and methods for forming nanoemulsion formulations therefrom are known in the art of drug delivery. The oil phase of the disclosed nanoemulsion formulations may include at least one polyunsaturated fatty acid (PUFA)-rich oil, for example, a first oil that may contain a polyunsaturated oil, for example linolenic acid, and optionally an oil that may be for example a saturated fatty acid, for example icosanaic acid.

[0095] Any oil can be used in accordance with the present invention. Oils can be natural or unnatural (synthetic) oils. Oils can be homogeneous or oils comprising two or more monounsaturated fatty acid or PUFA-rich oils. Contemplated oils may be biocompatible and/or biodegradable.

[0096] Biocompatible oils do not typically induce an adverse response (such as, but not limited to, an immune response with significant inflammation and/or acute rejection) when inserted or injected into a living subject. Accordingly, the therapeutic nanoemulsion formulations contemplated herein can be non-immunogenic.

[0097] One simple test to determine biocompatibility is to expose a nanoemulsion formulation to cells in vitro. Useful biocompatible oils in the nanoemulsion formulation do not result in significant cell death at moderate concentrations, e.g., 50 μg/10⁶ cells. For example, these biocompatible oils can cause less than about 20% cell death when exposed to or taken up by, fibroblasts or epithelial cells. Non-limiting examples of biocompatible oil useful in nanoemulsion formulations of the present disclosure include alpha linolenic acid, pinolenic acid, gamma linolenic, linoleic acid, oleic acid, icosenoic acid, palmitic acid, stearic acid, icosanaic acid, and derivatives thereof. [0098] The biocompatible oils may be biodegradable, i.e., able to degrade, chemically and/or biologically, within a physiological environment, such as within the body. As used herein, "biodegradable" oils are those that, when introduced into cells, are broken down by the cellular machinery (biologically degradable) and/or by a chemical process, such as hydrolysis, (chemically degradable) into components that the cells can either reuse or dispose of without significant toxic effect on the cells. Both the biodegradable oils and their degradation byproducts can be biocompatible.

[0099] Another useful oil is flaxseed oil which is a biocompatible and biodegradable oil of alpha linolenic, linoleic, and oleic. Useful forms of this oil can be characterized by the ratio of alpha linolenic:linoleic:oleic. The degradation rate of flaxseed oil can be adjusted by altering the alpha linolenic:linoleic:oleic ratio, e.g., having a molar ratio of about 65:5:30, about 65:20: 15, about 55: 15:30, or about 55:20:25.

[00100] The nanoemulsion formulations may include an oil phase of saturated fatty acid, monounsaturated fatty acid or PUFA rich oils that are biocompatible and/or biodegradable.

[00101] Oil compositions suitable for use as the oil phase of the nanoemulsion formulations according to the present disclosure can be from any source rich in mono-saturated or PUFAs, such as plant or animal sources. Chemically or enzymatically derivatized, or completely synthetic, monounsaturated or PUFAs are included within the scope of suitable components for the oil phase of the nanoemulsion formulations of the present disclosure. The

concentration of the mono-unsaturated or PUFA in the oil phase can range from about 2% to about 100% (w/w), from about 5% to about 100% (w/w), or greater than 10% from about 20% to about 80% (w/w). The concentration of the oil phase, in the nanoemulsion formulation can vary from about 5% to about 40% (w/w), or from about 5% to about 30% (w/w). The concentration of the chemotherapeutic agent soluble in the oil phase can range from about 0.01% to about 90% (w/w), from about 0.1% to about 45% (w/w), or greater than 0.5%, or from about 1% to about 30% (w/w). For example, the oils may contain high concentrations of mono-saturated or PUFAs such as a concentration of greater than or equal to 10% (w/w) of at least one mono-unsaturated or PUFA of the omega-3, omega-6 or omega- 9 family. A useful oil is one that can solubilize high concentrations of a chemotherapeutic agent, such as those containing high concentrations of linolenic or linoleic acid (e.g., oils of flax seed oil, black currant oil, pine nut oil or borage oil), and fungal oils such as spirulina and the like, alone or in combination. C. Aqueous Phase

[00102] The aqueous phase of the nanoemulsion formulations according to the disclosure is purified and/or ultrapure water. This aqueous phase can also contain isotonicity modifiers such as, but not limited to, glycerine, low molecular weight polyethylene glycol (PEG), sorbitol, xylitol, or dextrose. The aqueous phase can alternatively or also contain pH adjusting agents such as, but not limited to, sodium hydroxide, hydrochloric acid, free fatty acids (oleic acid, linoleic acid, stearic acid, palmitic acid) and their sodium and potassium salts, preservative parabens, such as, but not limited to, methyl paraben or propyl paraben; antioxidants such as, but not limited to, ascorbic acid, a-tocopherol, and/or butylated hydroxy anisole. The concentration of the aqueous phase in the present nanoemulsion formulations can vary from about 30% to about 95% (w/w).

D. Interfacial Surface Membrane

[00103] The term "interfacial surface membrane" as used herein applies to the interface of the oil and aqueous phase and may refer either to a single pure emulsifier or a mixture of different emulsifiers and/or a mixture of emulsifiers and other components, such as stealth agents (stabilizers) present in the interfacial surface membrane of the nanoemulsion formulation. The interfacial surface membrane or corona can comprise degradable lipids or emulsifiers bearing neutral, cationic and/or anionic side chains. The average surface area of the interfacial surface membrane corona on the nanoemulsion formulations described herein from may range from 100 nm² to 750,000 nm².

[00104] The interfacial surface membrane component of the drug delivery system of the present nanoemulsion formulations comprises an emulsifier and may also comprise a stabilizer (stealth agent).

(1) Emulsifiers

[00105] At least one emulsifier forms part of the interface between the hydrophobic or oil phase and the aqueous phase. They comprise individual amphiphilic lipids and/or amphiphilic polymers. The emulsifier can be an amphiphilic molecule such as a nonionic and ionic amphiphilic molecule. For example, the emulsifier can consist of neutral, positively-charged, or negatively-charged, natural or synthetic phospholipids molecules such as, but not limited to, natural phospholipids including soybean lecithin, egg lecithin, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, sphingomyelin, diphosphatidylglycerol, phosphatidyls erine, phosphatidylcholine and cardiolipin; synthetic phospholipids including dimyristoylphosphatidylcholine,

dimyristoylphosphatidyl ethyl-N-dimethyl propyl ammonium hydroxide,

dimyristoylphosphatidylglycerol, distearoylphos-phatidylglycerol and

dipalmitoylphosphatidylcholine; and hydrogenated or partially hydrogenated lecithins and phospholipids, e.g., from a natural source are used. The concentration of amphiphilic lipid in the nanoemulsion formulations can vary from about 0.5% to about 15% (w/v), or from about 1% to about 10% (w/v).

[00106] One non-limiting example of a nanoemulsion formulation of the present disclosure comprises oil and amphiphilic compounds of the interfacial surface membrane which surround or are dispersed within the oil and which form a continuous or discontinuous monomolecular layer. The interfacial surface membrane lowers the interfacial tension between the oil and aqueous phases, thereby enhancing the stability of the dispersed oil droplets in the surrounding aqueous phase. Further, the interfacial surface membrane of the nanoemulsion formulation localizes drugs, thereby providing therapeutic advantages by releasing the encapsulated chemotherapeutic drug at predetermined, appropriate times.

[00107] An amphiphilic compound may have a polar head attached to a long hydrophobic tail. The polar portion is soluble in water, while the non-polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. Exemplary amphiphilic compounds include, for example, one or a plurality of the following: naturally derived lipids, surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties.

[00108] Non-limiting examples of amphiphilic compounds making up a representative emulsifier include phospholipids, such as 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), and dilignoceroylphatidylcholine (DLPC), incorporated at a ratio of about 0.5% to about 2.5% (weight lipid/w oil), about between 1.0% to about 1.5% (weight lipid/w oil). Phospholipids, which may be used, include, but are not limited to, phosphatide acids, phosphatidyl cholines with both saturated and unsaturated lipids, phosphatidyl ethanolamines, phosphatidylglycerols, phosphatidylserines,

phosphatidylinositols, lysophosphatidyl derivatives, cardiolipin, and β-acyl-y-alkyl phospholipids. Examples of phospholipids include, but are not limited to,

phosphatidylcholines such as dioleoylphosphati-dylcholine, dimyristoylphosphatidylcholine, dipentadecanoylphosphatidylcholine dilauroylphos-phatidylcholine,

dipalmitoylphosphatidylcholine (DPPC), distearoylphos-phatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcho-line (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC); and phosphatidylethanolamines such as dioleoylphosphatidylethanolamine or l-hexadecyl-2- palmitoylglycerophos-phoethanolamine. Synthetic phospholipids with asymmetric acyl chains (e.g., with one acyl chain of 6 carbons and another acyl chain of 12 carbons) may also be used.

[00109] An amphiphilic compound of the interfacial membrane may include lecithin or phosphatidylcholine.

(2) Stabilizers

[00110] If the interfacial surface membrane comprises a stabilizer or stealth agent, it can be added with the emulsifier when preparing a nanoemulsion formulation of the present disclosure. The stabilizer may be an amphiphilic molecule.

[00111] One representative stabilizer is a PEGylated lipid. Some useful phospholipid molecules are natural phospholipids including polyethylene glycol (PEG) repeat units, which can also be referred to as a "PEGylated" lipid or lipidated PEG. Such PEGylated lipids 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 (RES) and/or the mononuclear phagocyte system (MPS), due to the presence of the poly(ethylene glycol) groups, PEGylated soybean lecithin, PEGylated egg lecithin, PEGylated phosphati-dylglycerol, PEGylated phosphatidylinositol, PEGylated phosphatidylethanolamine, PEGylated phosphatidic acid, PEGylated sphingomyelin, PEGylated diphosphatidylglycerol, PEGylated phosphatidylserine, PEGylated

phosphatidylcholine and PEGylated cardiolipin; synthetic phospholipids including PEGylated dimyristoylphosphatidylcholine, PEGylated dimyristoylphosphatidylglycerol, PEGylated distearoylphosphatidylglycerol and PEGylated dipalmitoylphosphatidylcholine; and hydrogenated or partially hydrogenated PEGylated lecithins and PEGylated phospholipids. Such amphiphilic PEGylated lipids can be used alone or in combination. The concentration of amphiphilic PEGylated lipid in the nanoemulsions can vary from about 0.01% to 15% (w/v), or from about 0.05% to 10% (w/v).

[00112] Exemplary lipids that can be part of the PEGylated lipid include, but are not limited to, fatty acids such as long chain (e.g., C8-C50), substituted, or unsubstituted hydrocarbons. A fatty acid group can be a C10-C20 fatty acid or salt thereof, a C15-C20 fatty acid or salt thereof, or a fatty acid can be unsaturated, monounsaturated, or polyunsaturated. For example, a fatty acid group can be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, lignoceric, palmitoleic, oleic, vaccenic, linoleic, alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic,

eicosapentaenoic, docosahexaenoic, or erucic acid.

[00113] Other exemplary stabilizers are phosphatide, a polyglycerol mono oleate,

PEGioooDSPE, PEG₂₀ooDSPE, PEG₃₄₀₀DSPE, PEG₅₀₀₀DSPE, or any combination thereof. Useful stabilizers are a PEG derivative, a phosphatide, and/or polyglycerol mono oleate and useful non-limiting PEG derivatives are PEGioooDSPE, PEG₂₀₀₀DSPE, PEG₃₄₀₀DSPE, PEGsoooDSPE.

[00114] The PEGylation density may be varied as necessary to facilitate long-circulation in the blood (Perry et al. (2012) Nano. Lett. 12:5304-5310). In some cases, the addition of PEG repeat units may increase plasma half-life of the nanoemulsion formulation, for instance, by decreasing the uptake of the nanoemulsion formulation by the MPS, while decreasing transfection/uptake efficiency by cells. Those of ordinary skill in the art will know of methods and techniques for PEGylating a lipid, for example, by using EDC (l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to react a lipid that will be in the corona of the nanoemulsion formulation to a PEG group terminating in an amine, by ring opening polymerization techniques (ROMP), or the like.

[00115] PEG may include a terminal end group, for example, when PEG is not conjugated to a ligand. For example, PEG may terminate in a hydroxyl, a methoxy or other alkoxyl group, a methyl or other alkyl group, an aryl group, a carboxylic acid, an amine, an amide, an acetyl group, a guanidino group, or an imidazole. Other contemplated end groups include azide, alkyne, maleimide, aldehyde, hydrazide, hydroxylamine, alkoxyamine, or thiol moieties. [00116] The molecular weight of the PEG on interfacial membrane surface of the nanoemulsion formulation can be optimized for effective treatment as disclosed herein. For example, the molecular weight of a PEG may influence particle degradation rate (such as adjusting the molecular weight of a biodegradable PEG), solubility, water uptake, and drug release kinetics. For example, the molecular weight of the PEG can be adjusted such that the particle biodegrades in the subject being treated within a period of time ranging from a few hours, to 1 week to 2 weeks, 3 weeks to 4 weeks, 5 weeks to 6 weeks, 7 weeks to 8 weeks, etc. One useful nanoemulsion formulation comprises a copolymer PEG conjugated to a lipid, the PEG having a molecular weight of about 1 kDa to about 20 kDa, about 5 kDa to about 20 kDa, or about 10 kDa to about 20 kDa, and the lipid can have a molecular weight of about 200 D to about 3 kDa, about 500 D to about 2.5 kDa, or about 700 D to about 1.5 kDa. An exemplary nanoemulsion formulation includes about 5 weight percent (wt %) to about 30 wt % monounsaturated or polyunsaturated fatty acid rich oil, or about 0.5 wt % to about 5 wt % primary emulsifier, or about 0.1 wt % to about 1.0 wt % co-emulsifiers, or about 0.1 wt % to about 0.75 wt %, PEG-derivatives. Exemplary lipid-PEG copolymers can include a number average molecular weight of about 1.5 kDa to about 25 kDa, or about 2 kDa to about 20 kDa.

[00117] The ratio of oil to emulsifier to stabilizer in the nanoemulsion formulation for example, flax seed oil to emulsifier to PEGylated lipid stabilizer, may be selected to optimize certain parameters such as size, chemotherapeutic agent release, and/or nanoemulsion formulation degradation kinetics.

[00118] An alternative stabilizer may contain poly(ester-ether)s. For example, the interfacial membrane surfaces of the nanoemulsion formulation can have repeat units joined by ester bonds (e.g., R— C(O)— O— R' bonds) and ether bonds (e.g., R— O— R' bonds). A biodegradable component of the interfacial membrane surface of the nanoemulsion formulation, such as a hydrolyzable biopolymer containing carboxylic acid groups, may be conjugated with poly(ethylene glycol) repeat units to form a poly(ester-ether) coating on the interfacial membrane surface of the nanoemulsion formulation.

E. Targeting Ligands

[00119] The nanoemulsion formulation of the present disclosure may further comprise a targeting ligand or molecule, which is specific for a receptor or protein or molecule on the cancer cells to be treated or imaged. Using a targeting ligand the nanoemulsion formulation is delivered more accurately to the cells having the target, such as targeting ligand receptors that are found in greater amounts on cancer cells than on normal cells. Representative useful targeting ligands are, i.e., a low-molecular weight ligand, protein, carbohydrate, or nucleic acid.

[00120] One such useful target is epidermal growth factor receptor (EGFR) (see, e.g., Magadala et al. (2008) AAPS. J. 10:565-576). EGFR is a member of the human epidermal growth factor receptor HER/erb family of receptor tyrosine kinases, which plays important roles in both cell growth and differentiation. Overexpression of EGFR is associated negatively with progression-free and overall survival in a wide variety of human cancers, including lung, breast, bladder, and ovarian cancers. Its positive signaling causes increased proliferation, decreased apoptosis, and enhanced tumor cell motility and angiogenesis.

[00121] Useful EGFR-targeting ligands include, but are not limited to, the amino acid peptide Y-H-W-Y-G-Y-T-P-Q-N-V-I (SEQ ID. NO: l, peptide 4) or an anti-EGFR immunoglobulin, e.g., a nanobody such as EGal-PEG.

[00122] Another useful target is the folate receptor. Folate receptor alpha (FR-a) is one of four isoforms: a, β, γ, and δ. FR-α is a 38 kDa glycosyl-phosphatidylinositol-anchored glycoprotein that binds folic acid (and internalizes it) with a Kd of less than 1 nM, and is highly expressed in a number of human tumors including ovarian (> 85%), lung (> 75%), breast (> 60%) renal cell (> 65%), brain, head, and neck. (Fisher et al. (2008) J. Nucl. Med. 49:899-906). In normal tissue its expression is much lower and limited to kidney tubuli, lung epithelium in the apical cell, the choroid plexus, and placenta. FR-a over expression is negatively associated with overall survival in ovarian and other cancers. However, with over 85% of ovarian tumors expressing FR-a it is difficult to correlate expression with mortality. As a predictor of response rate to chemotherapy, complete or partial remission, patients with FR-a greater than median levels had a 15 times higher likelihood of negative response.

[00123] Useful folate-targeting ligands include, but are not limited to, 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000] -cysteine- folic acid (DSPE-PEG(2000)-cysteine-folic acid), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-3400] -folic acid (DSPE-PEG(3400)-cysteine-folic acid), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000]

(ammonium salt) (DSPE-PEG(2000) folate), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[folate(polyethylene glycol)-5000] (ammonium salt) (DSPE- PEG(5000) folate) (Avanti Polar Lipids, Inc. Alabaster, Alabama), and any combinations thereof.

[00124] In some nanoemulsion formulations, the targeting moieties are attached, e.g., covalently bonded, to a lipid component of the nanoemulsion formulation. One exemplary nanoemulsion formulation comprises a chemotherapeutic agent, an oil core comprising functionalized and non-functionalized oils, an interfacial surface membrane or corona, and a low-molecular weight targeting ligand, wherein the targeting ligand is covalently bonded, to the lipid component of the nanoemulsion formulation's interfacial surface membrane.

2. Preparation of Nanoemulsion Formulations

[00125] The nanoemulsion formulations of the present disclosure can be prepared from various intermediates and component constituents, for example, as described in Examples 1-4 below, and can be made using a microfluidizer (Microfluidics Corp., Newton, MA).

[00126] FIG. 2 shows a representative synthesis scheme for one non-limiting, EGFR- targeted, Gd-labeled nanoemulsion formulation of the present disclosure. In this figure, 1 is a chemotherapeutic agent. 2 is C6-ceramide, a proapoptotic agent. 3 represents the compounds 1 and 2 being dissolved in chloroform and added to flax seed oil. Chloroform is removed using nitrogen, and mixture is then heated at 60°C for 2 minutes resulting in oil phase formation. 4 is the imaging moiety Gd-DTPA-PE. 5 is the targeting ligand EGFRBP-PEG- DSPE. 6 represents the compounds of 4 and 5 being added to egg lecithin and PEG2000DSPE in glycerol water solution, and mixture is then heated at 60°C for 2 minutes resulting in aqueous phase formation 6. 7 represents the oil phase of 3 and aqueous phase of 6 being combined, and mixed for 5 minutes to form the coarse emulsion. 8 represents the coarse emulsion of 7 being emulsified using a high pressure homogenizer (LV1 Microfluidizer) at 25,000 psi for 10 cycles to obtain nanoemulsion formulation droplets of a size below 150 nm. 9 is the resulting nanoemulsion formulation of one embodiment of the present disclosure with a size below 150 nm. 10 is a representative drawing of an individual resulting nanoemulsion formulation.

[00127] An initial screening step for chemotherapeutic agents that may be suitable candidates for the present nanoemulsion formulations is to test their solubility in various oils that can be used to form the oil phase of the nanoemulsion formulation. The solubility of representative chemotherapeutic agents is shown in Table III. Table III

Drug Solubility Screening in Oils for Nanoemulsion Formulation

N (No) - Insoluble

[00128] The solubility of these drugs was estimated by visual observation. The

nanoemulsion formulations of the present disclosure were prepared with a concentration of chemotherapeutic agent from about 0.5 mg/ml to about 20 mg/ml.

3. Characterization of Nanoemulsion Formulations

[00129] The nanoemulsion formulations of the present disclosure may have a substantially spherical or non-spherical shape. For instance, the nanoemulsion formulations initially may appear to be spherical, but upon shrinkage, may adopt a non-spherical configuration. These nanoemulsion formulations may have a characteristic dimension of less than about 1 μιη, where the characteristic dimension of a nanoemulsion formulation is the diameter of a perfect sphere having the same volume as the nanoemulsion formulation. For example, the characteristic dimensions of the nanoemulsion formulation can be less than about 300 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, or less than about 50 nm. Some disclosed nanoemulsion formulations may have a diameter of about 50 nm to 200 nm, about 50 nm to about 180 nm, about 80 nm to about 160 nm, or about 80 nm to about 150 nm. The size of the nanoemulsion formulation particles can be determined by dynamic light scattering (DLS) (Zetasizer ZS, Malvern Instruments Ltd., Worcestershire, United Kingdom). The figures show the size of particles making up a blank (control; FIG. 3) or representative (FIGS. 4-17) nanoemulsion according to the disclosure. Size distribution and zeta potential values are shown below of a control blank formulation (Table IV) or specific nanoemulsion formulations (Tables V-IX) according to the disclosure.

Table IV

Size and Charge of SP600125, Sulforaphane, and Salinomycin Nanoemulsion Formulations

• All the samples have droplet size below 100 nm with narrow distribution (PDI <0.1).

Table V

Size and Charge of Docetaxel and Docetaxel Combination Nanoemulsion Formulations

• Docetaxel to co-Agent ratio in the NE formulation is 1 :5.

Table VI

• Docetaxel to co- x ratio in the NE formulation is 1 :5.

• All the samples have droplet size below below 100 nm with narrow distribution (PDI <0.1).

Table VII

Size and Charge of Etoposide, Cyclopamine, and Noscapine Nanoemulsion Formulations

Table VIII

Size and Charge of Abamectin and BAY 11 -7082 Nanoemulsion Formulations

Table IX

Size and Charge of Nanoemulsion Formulations

• Ratio of Chemotherapeutic Agent to co-Agent is 1 :5, where the NE formulation is a combination of drugs

• All the samples have droplet size below 150 nm with narrow distribution (PDI <0.1).

[00130] These nanoemulsion formulations were prepared using Microfluidics LVI (10 cycles of 25,000 PSI). Nanoemulsion samples were diluted 1 : 1000 in distilled water for and charge analysis. Size and charge were measured using Malvern Zetasizer ZS. The average particle size of the control nanoemulsion formulation containing no

chemotherapeutic agent was below 200 nm in diameter. The incorporation of

chemotherapeutic agents alone or with chemopotentiators and/or co-agents in the nanoemulsion formulations did not significantly change the hydrodynamic particle size and size remained below 200 nm. The average surface charges of the nanoemulsion formulations were in the range of about -38 mV to -56 mV.

[00131] Nanoemulsion formulation size distribution of control blank nanoemulsion formulation, and nanoemulsion formulations with chemotherapeutic agents alone or with chemopotentiators and/or co-agents were determined using Zetasizer ZS (Malvern

Instruments, Worcestershire, United Kingdom) at 4°C for up to one month. The results are shown in below Tables X - XIII.

Table X

Physical Stability of SP600125, Sulforaphane, and Salinomycin NE Formulations

Table XI

Physical Stability of Docetaxel, Salinomycin, and Sulforaphane Nanoemulsion Formulations

• Docetaxel to co- x ratio is 1 :5 mM

Table XII

Physical Stability of Docetaxel, SP600125, and Salinomycin Combination Nanoemulsion

Formulations

• Docetaxel to co-Rx ratio is 1 :5 mM

Table XIII

Physical Stability of Etoposide, Cyclopame, and Noscapine Nanoemulsion Formulations

• Particle size values are shown as Avg (nm) ± SD, n=3.

• Polydispersity index values are shown in the brackets.

[00132] In these tables, the particle size values are shown as Avg (nm) ± SD, n=3. Polydispersity index values are shown in the brackets. The average particle size of the blank nanoemulsion formulation containing chemotherapeutic agents remained below 200 nm in diameter for up to 1 month. The incorporation of chemotherapeutic agents alone or with chemopotentiators and/or co-agent in the nanoemulsion formulations did not significantly change the hydrodynamic particle size and size remained below 200 nm for up to 1 month indicating that the nanoemulsion formulations were stable at 4°C for up to 1 month.

[00133] The nanoemulsion formulations of the present disclosure may have an interior and a surface, where the surface has a composition different from the interior, i.e., there may be at least one compound present in the interior but not present on the surface (or vice versa), and/or at least one compound is present in the interior and on the surface at differing concentrations. For example, a compound, such as a targeting moiety ligand, may be present in both the interior and the surface of the nanoemulsion formulation, but at a higher concentration on the surface than in the interior of the nanoemulsion formulation, although in some cases, the concentration in the interior of the nanoemulsion formulation may be essentially nonzero, i.e., there is a detectable amount of the compound present in the interior of the nanoemulsion.

[00134] In some cases, the interior of the nanoemulsion formulation is more hydrophobic than the surface of the nanoemulsion formulation. For instance, the interior of the nanoemulsion formulation may be relatively hydrophobic with respect to the surface of the nanoemulsion formulation, and a drug or other payload may be hydrophobic, and readily associates with the relatively hydrophobic center of the nanoemulsion formulation. The drug or other payload can thus be contained within the interior of the nanoemulsion formulation, which can shelter it from the external environment surrounding the nanoemulsion formulation (or vice versa). For example, a chemotherapeutic drug or other payload contained within the delivery system of the nanoemulsion formulation administered to a subject will be protected from a subject's body, and the body may also be substantially isolated from the drug for at least a period of time.

[00135] An exemplary nanoemulsion formulation may have a PEG derivative corona with a density of about 1.065 g/cm³, or about 1.01 g/cm³ to about 1.10 g/cm³.

[00136] The nanoemulsion formulations of the present disclosure may have controlled release properties, e.g., may be capable of delivering an amount of active agent to a patient, for example to a specific site in a patient, over an extended period of time, for example over 1 day, 1 week, or more. Some disclosed nanoemulsion formulations substantially immediately release (for example over about 1 minute to about 30 minutes), less than about 2% in 6 hours, less than about 4% in 24 hours, less than about 7% in 48 hours, or less than about 10% of a chemotherapeutic agent in 72 hours, for example when placed in a phosphate buffer saline solution at room temperature and/or at 37° C.

4. Method of Treatment

[00137] The nanoemulsion formulation in accordance with the present disclosure may be used to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a cancer or tumor.

[00138] The term "cancer" includes pre-malignant as well as malignant cancers. Cancers include, but are not limited to, ovarian, breast, prostate, gastric cancer, colorectal cancer, skin cancer, e.g., melanomas or basal cell carcinomas, lung cancer, cancers of the head and neck, bronchus cancer, pancreatic cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and the like. Cancer cells can be in the form of a tumor or exist alone within a subject (e.g., leukemia cells).

[00139] In certain cases, targeted nanoemulsion formulation may be used to treat any cancer where EGFR or folate receptor is expressed on the surface of cancer cells or in the tumor neovasculature, including the neovasculature of ovarian or non-ovarian solid tumors.

Examples of the EGFR- or folate receptor-related indications include, but are not limited to, breast, ovarian, esophageal, and oropharyngeal cancers.

[00140] When treating cancer, a therapeutically-effective amount of the nanoemulsion formulation of the present disclosure is administered and is that amount effective for treating, alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of the cancer.

[00141] As will be appreciated by those of ordinary skill in this art, the effective amount of the nanoemulsion formulation 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 nanoemulsion formulation is 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 taken into account include the severity of the disease state; age, weight and gender of the patient being treated; diet, time and frequency of administration; reaction sensitivities; and tolerance/response to therapy.

[00142] The nanoemulsion formulations of the present disclosure can be used to inhibit the growth of, or kill, cancer cells. As used herein, the term "inhibits growth of cancer cells" or "inhibiting growth of cancer cells" refers to any slowing of the rate of cancer cell proliferation and/or migration, arrest of cancer cell proliferation and/or migration, or killing of cancer cells, such that the rate of cancer cell growth is reduced in comparison with the observed or predicted rate of growth of an untreated control cancer cell. The term "inhibits growth" can also refer to a reduction in size or disappearance of a cancer cell or tumor, as well as to a reduction in its metastatic potential. Such an inhibition at the cellular level may reduce the size, deter the growth, reduce the aggressiveness, or prevent or inhibit metastasis of a cancer in a patient. Those skilled in the art can readily determine, by any of a variety of suitable indicia, whether cancer cell growth is inhibited.

[00143] Inhibition of cancer cell growth may be evidenced, for example, by arrest of cancer cells in a particular phase of the cell cycle, e.g., arrest at the G2/M phase of the cell cycle. Inhibition of cancer cell growth can also be evidenced by direct or indirect measurement of cancer cell or tumor size. In human cancer patients, such measurements generally are made using well known imaging methods such as magnetic resonance imaging, computerized axial tomography and X-rays. Cancer cell growth can also be determined indirectly, such as by determining the levels of circulating carcinoembryonic antigen, prostate specific antigen or other cancer-specific antigens that are correlated with cancer cell growth. Inhibition of cancer growth is also generally correlated with prolonged survival and/or increased health and well- being of the subject.

[00144] Also provided herein are therapeutic protocols that include administering a therapeutically effective amount of a disclosed therapeutic nanoemulsion formulation to a healthy individual (i.e., a subject who does not display any symptoms of cancer and/or who has not been diagnosed with cancer). For example, healthy individuals may be "immunized" with an inventive targeted or non-targeted particle, such as a nanoemulsion formulation, prior to development of cancer and/or onset of symptoms of cancer; at risk individuals (for example, patients who have a family history of cancer; patients carrying one or more genetic mutations associated with development of cancer; patients having a genetic polymorphism associated with development of cancer; patients infected by a virus associated with development of cancer; patients with habits and/or lifestyles associated with development of cancer; etc.) can be treated substantially contemporaneously with (for example, within 48 hours, within 24 hours, or within 12 hours of) the onset of symptoms of cancer. Individuals known to have cancer may receive inventive treatment at any time.

[00145] Nanoemulsion formulations disclosed herein may be combined with pharmaceutical acceptable carriers to form a pharmaceutical composition. As would be appreciated by one of skill in this art, the carriers may be chosen based on the route of administration as described below, the location of the target issue, the drug being delivered, the time course of delivery of the drug, etc.

[00146] 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 parentally 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 conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

5. Methods of Administration

[00147] The nanoemulsion formulations of this disclosure can be administered to a patient by any means known in the art including oral and parenteral routes. The term "patient," as used herein, refers to humans as well as non-humans, including, for example, mammals, birds, reptiles, amphibians, and fish. Sometimes parenteral routes are chosen since they avoid contact with the digestive enzymes that are found in the alimentary canal. These

compositions may be administered by injection (e.g., intravenous, subcutaneous or intramuscular, intraperitoneal injection), rectally, vaginally, topically (as by powders, creams, ointments, or drops), or by inhalation (as by sprays).

[00148] For example, the nanoemulsion formulations of the present disclosure may be administered to a subject in need thereof systemically, e.g., by intravenous infusion or injection. 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 parentally 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 conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[00149] The nanoemulsion formulations may also be administered orally. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the encapsulated or unencapsulated conjugate 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, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents.

[00150] It will be appreciated that the exact dosage of the nanoemulsion formulation of the present disclosure 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 nanoemulsion formulation to the patient being treated. As used herein, the "effective amount" of a nanoemulsion formulation refers to the amount that elicits the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of the nanoemulsion formulations 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 nanoemulsion formulation is 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 taken into account include the severity of the disease state; age, weight and gender of the patient being treated; diet, time and frequency of administration; reaction sensitivities; and tolerance/response to therapy.

[00151] The nanoemulsion formulation of the present 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 the nanoemulsion formulation appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the nanoemulsion formulations of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. For any

nanoemulsion formulation, 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 the nanoemulsion formulations 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. Nanoemulsion

formulations, 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 dosage for human use.

[00152] For example, the nanoemulsion formulation may contain chemotherapeutic agents at a concentration of about 0.001% to about 2% (about 0.01 mg/ml to about 20 mg/ml). The dosage administered by injection may contain chemotherapeutic agents in the range of about 5 mg to about 1000 mg in the first day of every 1 week to 4 weeks depending upon the patient. One might administer a dosage of about O.Olmg/kg of patient body weight to about 500 mg/kg patient body weight in the first day of every 1 week to 4 weeks. Such dosages may prove useful for patients having a body weight outside this range. The nanoemulsion formulation may also contain a proapoptotic agent, such as ceramide, sulforaphane, curcumin or cyclopanine, that act to enhance the cytotoxicity of other chemotherapeutic agents in the cancer cells. The concentration of proapoptotic agent in the composition is about 0.001% to about 2% (about 0.01 mg/ml to about 20 mg/ml).

[00153] The nanoemulsion for oral administration are of about the same volume as those used for injection. However, when administering the drug orally, higher doses may be used when administering by injection. For example, a dosage containing about 10 mg to about 1500 mg chemotherapeutic agent in the first day of every 1 week to 4 weeks may be used. In preparing such liquid dosage form, standard making techniques may be employed.

6. Imaging Moieties

[00154] Nanoemulsion formulations of the present disclosure can further include imaging or contrast agents. The use of imaging agents on the nanoemulsion formulation of the present disclosure allows physicians to track in real time the amount of chemotherapeutic agent actually reaching the site of disease. Physicians can then quickly decide whether a particular patient should continue with treatment. Useful imaging agents include paramagnetic agents such as gadolinium (Gd), iron oxide, iron platinum, and manganese. Useful gadolinium derivatives include 1 ,2-dimyristoyl-sn-glycero-3 -phosphoethanolamine-N-diethylene- triaminepentaacetic acid (Gd-DTPA-PE), l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N- 1,4,7, 10-tetraazacyclododecane-l, 4,7, 10-tetraacetic acid (Gd- DOTA-PE), and 1 ,2-dimyristoyl-sn-glycero-3 -paraazoxyphenetole-N- 1,4,7,10- tetraazacyclododecane-l,4,7, 10-tetraacetic acid (Gd-PAP-DOTA) (Avanti Polar Lipids, Inc. Alabaster, Alabama). These gadolinium-based MRI contrast moieties can be prepared or obtained and incorporated into a nanoemulsion formulation as described herein. Useful imaging agents are gadolinium, iron oxide, iron, platinum, and manganese. Examples of suitable gadolinium imaging agents are Gd-DTPA-PE, Gd-DOTA-PE, Gd-PAP-DOTA.

[00155] Accordingly, a representative nanoemulsion formulation comprises an imaging moiety attached, e.g., covalently bonded, to a lipid component of the nanoemulsion formulation. One exemplary nanoemulsion formulation comprises a chemotherapeutic agent, an oil phase comprising functionalized and non-functionalized oils, an interfacial surface membrane or corona, an EGFR targeting ligand, and an imaging agent, wherein the imaging agent is covalently bonded to the lipid component of the nanoemulsion formulation's interfacial surface membrane.

[00156] In another exemplary nanoemulsion formulation, imaging moieties are soluble in the oil phase. For example, a nanoemulsion formulation comprises a chemotherapeutic agent, an oil phase comprising functionalized and non-functionalized oils, an interfacial surface membrane, an EGFR targeting ligand, and an imaging agent, wherein the imaging agent is soluble in the oil phase.

7. Imaging Methods

[00157] The nanoemulsion formulations in accordance with the present disclosure may be used to image tumors or cancer cells. These nanoemulsion formulations are small enough to travel into minute body regions and, when coupled with paramagnetic elements, such as gadolinium ions (Gd³⁺), iron oxide, iron, platinum, or manganese, can enhance tissue contrast in an MRI. Once the nanoemulsion formulation has reached the cancer site, its efficacy is determined, which can be done using an in vivo imaging modality such as MRI. Image- guided therapy using nanoemulsion formulations couples drug delivery with tissue imaging to allow clinicians to efficiently deliver chemotherapeutic agents, while simultaneously localizing the drugs and visualizing their physiological effects.

[00158] The nanoemulsion formulations combined with an appropriate imaging agent can act as MRI contrast agents to enhance tissue image resolution. Contrast agents such as Gd³⁺ have unpaired electrons that interact with surrounding water molecules to decrease their proton spin time, also referred to as TV Relaxation time is defined as the period it takes for a proton to return to its equilibrium position following a magnetization pulse. MRI can measure Ti by creating a magnetic field that reverses the sample's magnetization, and then recording the time required for the spin directions to realign in their equilibrium positions again. The decreased Ti relaxation time of the target tissue allows an MRI machine to better distinguish between it and its surrounding aqueous environment.

[00159] The nanoemulsion formulations according to the disclosure can serve as a new Gd³⁺ chelated, EGFR- or folate receptor-targeted nanoemulsion formulation that not only exhibits MRI contrast but also carries an encapsulated chemotherapeutic agent and a

chemopotentiator to the target tissue for successful image-guided therapy. To examine the MRI contrast potential of these nanoemulsions, in vivo studies were conducted using MRI, while cell uptake and trafficking as well as efficacy studies were conducted to examine the drug delivery potential of the nanoemulsion formulation.

[00160] The method of imaging includes administering to a patient or subject to be imaged a diagnostically effective amount of a nanoemulsion formulation according to the disclosure. The nanoemulsion formulation can be administered by a variety of techniques including subcutaneously and intravenously. The method is effective for imaging cancers, such as breast, ovarian, esophageal, and oropharyngeal cancers and other cancers accessible by the lymphatic or vascular (blood) systems. For magnetic resonance imaging methods, the nanoemulsion formulation of the disclosure includes a paramagnetic metal ion (e.g., Gd³⁺).

[00161] The following examples provide specific exemplary methods of the invention, and are not to be construed as limiting the invention to their content.

EXAMPLES

EXAMPLE 1

Synthesis of EGFRRP-PEG-DSPE

[00162] EGFRBP-PEG-DSPE was prepared according to the scheme shown in FIG. 18. Briefly, the synthetic EGFR-targeting peptide Y-H-W-Y-G-Y-T-P-Q-N-V-I (SEQ ID NO: l) with a linker sequence G-G-G-G-C (SEQ ID NO:2) was synthesized by standard peptide organic synthesis methods. The carboxyl group of terminal cysteine of the peptide was reacted with the maleimide of the PEG2₀₀₀-DSPE construct. To accomplish this reaction, 9.4 mg of EGFR-binding peptide Y-H-W-Y-G-Y-T-P-Q-N-V-I G-G-G-G-C (SEQ ID NO:3) was added to 14.7 mg MAL-PEG2₀₀₀-DSPE (2.942 kDa mol. wt.) dissolved in HEPES (4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid ) buffer (pH 7.4) solution at 1 : 1 molar ratio while mixing at 400 rpm) under nitrogen at 4°C for 24 hr.

[00163] The EGFR_Bp conjugate was then purified by dialysis against deionized distilled water at RT using a 3500 molecular weight cut-off membrane (Spectrapore, Spectrum Laboratories, Rancho Dominguez, CA). The purified sample was then transferred into tubes and freeze-dried for 24 hr. The sample was stored at -20°C until use.

[00164] EGFR_BP-PEG2₀₀₀DSPE conjugate formation was confirmed by nuclear magnetic resonance spectroscopy (NMR) analysis. A 2 mg sample was dissolved in 1 ml DMSO (dimethyl sulfoxide) and the NMR spectra was recorded using a Varian 400AS Spectrometer (400 MHz, Varian Inc., Palo Alto, CA).

EXAMPLE 2

Synthesis of Folate-Targeting Ligand DSPE-PEG-FA

[00165] The DSPE-PEG-Mal complex was prepared according to the scheme in FIG. 19. DSPE-PEG-Mal (100 mg, 1.3596 mM) was added to cysteine (8.24 mg, 2.72 mM) in a 1 :2 molar ratio in HEPES buffer (25 ml) and the coupling reaction was carried out overnight at 4 °C under a nitrogen environment. The next day, excess cysteine was dialyzed out for 24 hr using 2000 Da cut-off dialysis bags. The outside water was changed every 2 hr to facilitate dialysis.

[00166] A purified sample was freeze-dried and characterized by NMR. 51 mg of DSPE- PEG-Cys was dissolved in 6 ml dry DMSO containing 13 mg folic acid. 3 ml pyridine was added to the solution followed by 16 mg of Ν,Ν'-dicyclohexylcarbodiimide. The coupling was carried out for 4 hr at RT with continuous mixing. The sample was dialyzed in water using 2 kDa cut-off dialysis bags. Outside water was changed every 2 hr for 24 hr to facilitate dialysis. Purified sample was freeze-dried and characterized by NMR.

[00167] Alternatively the DSPE-PEG-FA was prepared as follows. Folic acid (10.3 mg; 0.023 mol) was weighed in a 20 ml glass vial followed by the addition of 1600 μΐ of dry Dimethylformamide (DMF). 10.6 mg (0.051 mol) of l-ethyl-3-(3-dimethylamino- propyl)carbodiimide (EDCI) and 6.4 mg (0.056 mol) of N-hydroxysuccinimide (NHS) were weighed in two different 2ml plastic tubes. EDCI was dissolved in 400 μΐ of DMF; the solution was transferred to NHS and mixed. The combined clear solution was added to the folic acid solution. The resulting mixture was sonicated 1-2 min on bath sonicator until completely clear. The reaction mixture was stirred 4.5 hours at 25°C.

[00168] 92.9 mg (0.022 mol) of DSPE-PEG₃₄oo-NH₂ was dissolved in 1500 μΐ of DMF in a 7 ml glass vial. 20 μΐ of triethylamine (Et₃N) was added to this solution. Combined solution was added to pre-activated folic acid solution. Reaction mixture was stirred 48 hours at 25 °C.

[00169] 50 ml of phosphate buffer saline (PBS) was added to the reaction mixture. The mixture was dialyzed 2 hours against a 5% DMF solution in PBS, then 2 hours against PBS, then 48 hours against deionized (DI) water with frequent water change. The solution was transferred from dialysis bag to 50ml tubes, frozen and freeze-dried for 48 hours. Dry product was dissolved in 7 ml of dry chloroform. Chloroform solution was filtered through Wathman Resist (chemically inert) syringe filters using glass syringe (filters were changed 3-4 times during filtration as they became clogged). The chloroform was evaporated first on rotary evaporator and then under the stream of nitrogen gas. The residue was freeze-dried for 1 hour to remove trace amounts of chloroform. The product was dissolved in 6 ml of Dl-water, filtered, frozen and freeze-dried overnight to obtain 84.8 mg (78.8%) of yellow powder.

EXAMPLE 3

Synthesis of Gd⁺³ -DTPA-PE

[00170] Gd⁺³ -DTPA-PE chelate was prepared according to the scheme shown in FIG. 20. 30 μΐ of triethylamine (Sigma) was added to 100 mg of L-a-phosphatidylethanolamine, transphosphatidylated (egg chicken) (841 118C, Avanti Polar Lipids, Birmingham, AL) dissolved in 4 ml of chloroform (extra dried). This solution was then added drop-wise to 400 mg (1 mM) of diethylene triaminepentacetic dianhydride (DTPA anhydride) (Sigma) in 20 ml of dimethylsulfoxide and the mixture was stirred for 3 hr under nitrogen atmosphere at RT. Nitrogen was then blown on to a sample to remove the chloroform.

[00171] The DTPA-PE conjugate was then purified by dialysis against deionized distilled water at RT using a 3 kDa molecular weight cut-off membrane (Spectrapore, Spectrum Laboratories). The purified sample was then transferred into tubes and freeze-dried for 48 hr. The DTPA-PE complex formation and purity of the complex were monitored by thin layer chromatography (TLC) using a mobile phase of chloroform: methanol: water at a

3.25: 1.25:0.5 (v/v) ratio and using ninhydrin as a visualizing reagent. For this, reactants (DTPA, PE) and complex (DTPA-PE) were dissolved in chloroform and placed on a TLC plate and developed in the mobile phase. Ninhydrin solution was then sprayed and the spots and their retention times were compared for the formation of the complex.

[00172] 18.5 mg (10.0 mM) of gadolinium (III) chloride hexahydrate (Sigma) in 0.1 ml of water was then added drop-wise to the 100 mg of DTPA-PE complex dissolved in 20 ml of DMSO and the reaction mixture was stirred (400 rpm) for 1 hr.

[00173] An Arsenazo III assay was used to monitor the reaction and formation of the Gd- DTPA-PE complex. 10 μΐ of reaction mixture was added to 0.2 mM of Arsenazo III (Pointe Scientific) in water and observed for the color change (pink to blue). No change in solution color indicated that Gd conjugated to DTPA-PE (free Gd turns Arsenazo III solution to a blue color).

[00174] The resulting Gd⁺³-DTPA-PE conjugate was purified by dialysis against deionized distilled water at RT using a 3000 molecular weight cut-off membrane (Spectrapore, Spectrum Laboratories). The purified sample was then transferred into tubes and freeze-dried for 48 hr. The conjugate was stored at -20°C until use.

EXAMPLE 4

Preparation of a Nanoemulsion Formulation

[00175] The oil phase of this oil-in-water nanoemulsion was prepared as follows. 10 mg of SP600125 was dissolved in chloroform (extra dry) in a glass scintillation vial. Flax seed oil (1 g) was placed in a scintillation glass vial. The SP600125 solution was added and nitrogen gas was blown on the sample to evaporate chloroform and to form the oil phase.

[00176] The aqueous phase of this oil-in-water nanoemulsion was prepared as follows. 120 mg egg lecithin (Lipoid E 80, Lipoid GMBH, Ludwigshafen, Germany), 15 mg

PEG₂₀₀₀DSPE (Genzyme, Cambridge, MA) was added to 4 ml of 2.21% w/v glycerol (Sigma) solution in a glass scintillation vial made in water for injection. The mixture was stirred (400 rpm) for 1 hr to achieve complete dissolution of these excipients.

[00177] The aqueous and oil phases from above steps were heated to 60°C for 2 min in a water bath, and the aqueous phase was added to the oil phase, and vortex mixed for 1 min. The resulting mixture was passed through a LV1 Microfluidizer (Microfluidics Corp., Newton, MA) at 25,000 psi for 10 cycles. Product entered the microfluidizer system via the inlet reservoir and was powered by a high-pressure pump into the interaction chamber at speeds up to 400 m/s. It was then effectively cooled and collected in the output reservoir.

[00178] These steps resulted in the production of a stable SP600124 nanoemulsion formulation.

[00179] The quantity of ingredients for other nanoemulsion formulations (NE) of the present disclosure and made according to the procedure above are listed in Tables XIV - XVII.

Table XIV

- SP600125, Sulforaphane, Salinomycin

Table XV

Drugs: DTX - Docetaxel, CER - C6-Ceramide, SFN - Sulforaphane, SNM - Salinomycin.

Ratio of DTX to co-RX is 1 :5 mmol, where the NE formulation consisting of combination of drugs. Table XVI

Table XVII

EXAMPLE 5

Cellular Uptake Studies

[00180] The following assay demonstrates the effect that targeted nanoemulsion formulations have on cellular uptake. Uptake is measured in cancer cells using fluorescence. The cells growing on cover slips in 6-well plate at 3000 cells/well are incubated with the fluorescently labeled nanoemulsion formulations for 5 min, 15 min, or 30 min. At the end of incubation period, cells are washed thrice with phosphate buffered saline (PBS) and incubated with Lyso Tracker and DAPI for 10 min, which stains lysosomes and nucleus of the cells, respectively. Cells are further washed with PBS, inverted and mounted on glass slides using Flouromount G mounting media. [00181] DIC/Fluorescent images of fluorescently labeled cells treated with nanoemulsion formulation according to the present disclosure are acquired using a Confocal Zeiss LSM 700 microscope with an object 63 x oil immersion over a 30 min period.

[00182] A fluorescently labeled agent, such as 0.01% NBD-Ceramide at 0.01% w/v is incorporated in all formulations. Lyso Tracker and DAPI were used to monitor the co- localization of the nanoemulsion formulations in the SKOV3 cells.

[00183] These images show fluorescently labeled agent, such as NBD-CER co-localized with Lyso Tracker, indicating the entry of fluorescently labeled agent into lysosomes. The study demonstrates that the nanoemulsion formulations of the present disclosure are able to evade the drug degradative lysosomal pathway, thereby enhancing drug concentrations in cells in vitro.

EXAMPLE 6

Efficacy of Nanoemulsion Formulations

[00184] In order to determine if the nanoemulsion formulations according to the disclosure produce a cytotoxic effect on cancer cells, including multidrug resistant cancer cells, the following experiments were done on SKOV3, SKOV3_TR, A2780, A2780 CP, PEO-1, PEO-4, ES-2 and PANC-1 cells. SKOV3 and SKOV3TR cells are human ovarian cancer cells. The SKOV3TR cells express P-glycoprotein (Pgp), a multi-drug resistant transporter, which produces chemotherapeutic agent efflux out of the cell and is associated with multidrug resistant cancer cells. A2780 and A2780 CP cells are human ovarian cancer cell lines. The A2780 CP cells are resistant to cis-Platin. The PEO-1 and PEO-4 cells were developed from the ascites of patients with ovarian adenocarcinoma - the PEO- 1 cells from patients that were untreated and the PEO-4 cells from patients that developed resistance to the

chemotherapeutic agents CDDP, 5-FU and chlorambucil. ES-2 cells are human ovarian cancer cells expressing low levels of P glycoprotein and are reported to be moderately resistance to a number of chemotherapeutic agents including doxorubicin, cisplatin, carmustine, etoposide and cyanomorpholinodoxorubicin (MRA-CN), and PANC- 1 is a human pancreatic cancer cell line.

[00185] A tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) assay was performed, which measures the activity of cellular enzymes that reduces the MTT dye to insoluble formazan. Polyethylenimine at 50 μg/ml was used as a positive control for cytotoxicity. The effect of chemotherapeutic agents in solution, and

nanoemulsion formulations of the disclosure on the viability of cancer cells were studied and measured after 72 hr treatment. After the completion of treatment, cells were incubated with MTT reagent (50 μg/well) for 2 hr. The resulting formazan crystals were dissolved in dimethyl sulfoxide (150 μg/well) and measured at 570 nm in the Plate reader (Synergy HT, Biotek Instruments, Winooski, VT).

[00186] The concentration of drug that inhibits fifty percent of growth is known as the 50% growth inhibitory concentration (IC5₀). Using the dose response curves (not shown), the IC5₀ values were calculated. All IC5₀ values were obtained by analyzing the MTT assays results using Graphpad Prism 5 scientific data analysis software. The results are shown in Table XVIII.

Table XVIII follows on page 48.

Table XVIII

• NIR - Not in Range

• NT - Not Targeted

• NPeg - Not Pegylated; Peg - Pegylated

[00187] As shown above, the IC5₀ was decreased when non-targeted nanoemulsion formulations of the present disclosure were used. Most significant is the decrease in IC5₀ values observed when multidrug resistant cells were treated with the nanoemulsion formulations of the present disclosure, indicating that the nanoemulsion formulations are capable of by -passing the multidrug resistant mechanisms that these cells express. The nanoemulsions containing no chemotherapeutic agent did not affect cell viability (data not shown).

[00188] The optimum concentration of multiple chemotherapeutic agents combined in the nanoemulsion formulations of the present disclosure can be determined by calculating the combination index from the dose response curves of the single agents (Chou (2006)

Pharmacol. Rev. 58(3): 621-681). This method uses the isobologram equation below to determine combination index (CI):

CI = (a /A) + (b/B) where, "a" is the primary therapeutic IC5₀ in combination with secondary therapeutic at concentration "b." "A" is the primary therapeutic IC5₀ without secondary therapeutic; and "B" is the secondary therapeutic IC50 in the absence of primary therapeutic. The CI represents the degree of interaction between two drugs regardless of mechanism. A CI value lower than 1.0 indicates synergy, while a CI value greater than 1.0 indicates that the drugs are antagonists. If drugs are synergistic the relative dose needed to get the same effect is reduced and is known as the "dose reduction index" (DRI). DRI is a measure of decrease in drug concentration for a synergistic combination as compared with the concentration of each drug alone.

[00189] For the nanoemulsion formulations of the present disclosure, the ratio of Pt : CER was determined in order to identify ratios that could reduce the IC5₀ of Pt.

EXAMPLE 7

In vivo MRI Studies Using Gd-Labeled Nanoemulsion Formulations

[00190] That a Gd-based MRI contrasting agent is useful in the nanoemulsion formulation according to the disclosure was demonstrated as follows: [00191] Three female Nu/Nu mice (Charles River Laboratories, Cambridge, MA) each weighing approximately 20 g and bearing subcutaneous SKOV3 tumors (human ovary cancer cells, American Type Culture Collection (ATCC, Manassas, VA) approximately 200 mm to 300 mm in size, were used as test subjects. The first mouse was intravenously injected with gadopentetic acid containing a 0.072 mmol/Kg dose of the gadolinium-based MRI contrasting agent Gd-DTPA-PE (Magnevist™). The second mouse was intravenously injected with a non-targeted Gd-labeled nanoemulsion formulation of the present disclosure containing a 0.072 mmol/Kg dose of the gadolinium-based MRI contrasting agent Gd-DTPA- PE. The third mouse was intravenously injected with an EGFR-targeted nanoemulsion formulation of the present disclosure containing a 0.072 mmol/Kg dose of the gadolinium- based MRI contrasting agent Gd-DTPA-PE. All three mice were full body scanned and imaged using a Bruker Biospec 20/70 MRI machine over a period of 24 hr.

[00192] Representations of the resulting MRIs of a mouse injected with Magnevist™ (Magnevist™ (M)), a mouse injected with the non-targeted nanoemulsion formulation (Non- Targeted (NT)), and a mouse injected with the EGFR-targeted nanoemulsion formulation (Targeted (T)) can be seen in FIG. 22. These images show preferential accumulation of the Gd-containing nanoemulsion formulations of the present disclosure in the subcutaneous flank SKOV3 tumors compared to that of the contrasting agent control Magnevist™. The

Magnevist™ control was observed to show contrast enhancement of tumors between 2 hr to 4 hr; whereas the Gd-labeled targeted and non-targeted nanoemulsion formulations of the present disclosure shows contrast enhancement of tumors between 6 hr to 24 hr. The Magnevist™ control rapidly accumulated in the tumor over the first hour, but then cleared and resolved to near baseline by the 6th hr. In contrast, the nanoemulsion formulations of the present disclosure enhanced tumor imaging accumulated and remained in the tumor over a longer period of time, thereby enhancing tumor imaging.

[00193] These studies show that the Gd-containing nanoemulsion formulations of the present disclosure are useful MRI agents, and are effective at imaging tumors over a longer period of time than the pure imaging agent Magnevist™. Additionally, these tumor images can be used to measure the accumulation of chemotherapeutic agent nanoemulsion formulation in the tumor. EXAMPLE 8

Biodistribution Studies of Nanoemulsion Formulations

[00194] In order to determine if the nanoemulsion formulations according to the disclosure produce therapeutic efficacy against cancer in vivo the following experiment is performed. Orthotropic tumors were developed in 30 Nu/Nu female mice, each weighing approximately 20 g (Charles River Laboratories, Cambridge, MA). The mice were injected intraperitoneally (ip) with a cancer cell line, such as 4 x 10⁶ SKOV3 human ovary cancer cells (ATCC, Manassas, VA) suspended in phosphate buffered salinelOO μΐ PBS. The mice are divided into test groups of 10 individual mice. Each mouse is then dosed every 7 d for 5 wk with either nothing (control group) or 1 the test compounds. The test compounds are administered in about 3 cycles to about 7 cycles. The survival time of each group of mice is determined and the median survival time (days) calculated using Graphpad Kaplan-Meyers survival analysis software on the basis of the observed survival time of each mouse.

[00195] The fractional survival for groups treated with the nanoemulsion formulation of the present disclosure are significantly improved compared to that of the control and free chemotherapeutic agent group. Encapsulation of chemotherapeutic agents in the

nanoemulsion formulation sequesters them from normal tissue to reduce therapy related systemic toxicity, while still allowing the chemotherapeutic agent of the nanoemulsion formulation to inhibit the division of cancer cells in tumors. The nanoemulsion formulations of the present disclosure, in which targeting ligands are present, are also useful as anticancer delivery systems. These nanoemulsion formulations allow for a more efficient

chemotherapeutic delivery system, which had reduced systemic toxicity while functioning to inhibit the division of cancer cells. These nanoemulsion formulations allow for more efficient treatment of multidrug resistant tumors.

[00196] Encapsulation of chemotherapeutic agents as part of the oil core of the

nanoemulsion formulation according to the disclosure and inclusion of targeting modified lipids allow for targeting moieties to be attached to an amphiphile of the interfacial surface membrane of the nanoemulsion formulation. EQUIVALENTS

[00197] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

What is claimed is:

1. A nanoemulsion formulation comprising:

a drug delivery system comprising:

an oil phase; and

an interfacial surface membrane; and

an aqueous phase; and

a chemotherapeutic agent comprising a chemotherapeutic agent, which is not carboplatin, cisplatin, a di-fatty acid derivative of platinum, a salicylate derivative of platinum, NMI-300, or NMI-500, and wherein the chemotherapeutic agent is dispersed in the oil phase.

2. The nanoemulsion formulation of claim 1, wherein the oil phase comprises flaxseed oil, omega-3 polyunsaturated fish oil, omega-6 polyunsaturated fish oil, safflower oil, olive oil, pine nut oil, cherry kernel oil, soybean oil, pumpkin oil, pomegranate oil, primrose oil, or combinations thereof.

3. The nanoemulsion formulation of claim 1, wherein the interfacial surface membrane phase comprises an emulsifier and/or a stabilizer.

4. The nanoemulsion formulation of claim 3, wherein the emulsifier comprises egg lecithin, egg phosphatidyl choline, soy lecithin, phosphatidyl ethanolamine, phosphatidyl inositol, dimyristoylphosphatidyl choline, dimyristoylphosphatidyl ethyl-N-dimethyl propyl ammonium hydroxide, hydrogenated soy phosphatidylcholine, l,2-distearoyl-sn-glycero-3- phosphocholine or combinations thereof.

5. The nanoemulsion formulation of claim 3, wherein the stabilizer comprises a polyethylene glycol derivative, a phosphatide, a polyglycerol mono oleate, or combinations thereof.

6. The nanoemulsion formulation of claim 5, wherein the polyethylene glycol derivative is PEG₂₀₀₀DSPE, PEG₃₄₀₀DSPE, PEG₅₀₀₀DSPE, or combinations thereof.

7. The nanoemulsion of claim 5, wherein the polyethylene glycol in the polyethylene glycol derivative has a molecular weight of from 1 kDa to 20 kDa, from 5 kDa to 20 kDa, or from 10 kDa to 20 kDa.

8. The nanoemulsion formulation of claim 1, wherein the chemotherapeutic agent is an ionophore, a DNA metabolism inhibitor, a cytoskeletal inhibitor, a kinase inhibitor, an inflammatory signal inhibitor, and/or combinations thereof.

9. The nanoemulsion formulation of claim 1, further comprising a chemopotentiator.

10. The nanoemulsion formulation of claim 9, wherein the chemopotentiator comprises ceramide or a derivative thereof.

11. The nanoemulsion formulation of claim 10, wherein the chemopotentiator comprises C6-ceramide.

12. The nanoemulsion formulation of claim 8, further comprising C6-ceramide.

13. The nanoemulsion formulation of claim 1, further comprising a targeting ligand.

14. The nanoemulsion formulation of claim 13, wherein the targeting ligand comprises an EGFR-targeting ligand, a folate receptor-targeting ligand, or a combination thereof.

15. The nanoemulsion formulation of claim 14, wherein the EGFR-targeting ligand comprising peptide 4, an anti-EGFR immunoglobulin or EGFR-binding fragment thereof, EGal-PEG, or combinations thereof.

16. The nanoemulsion formulation of claim 14, wherein the folate receptor-targeting ligand comprising DSPE-PEG-cysteine-folic acid, DSPE-PEG(2000) folate, DSPE- PEG(3400) folate, DSPE-PEG(5000) folate, an anti-folate receptor immunoglobulin or folate receptor-binding fragment thereof, or combinations thereof.

17. The nanoemulsion formulation of claim 16, wherein the PEG in the targeting ligand EGal-PEG has a molecular weight of from 1 kDa to 20 kDa, from 5 kDa to 20 kDa, or from 10 kDa to 20 kDa.

18. The nanoemulsion formulation of claim 1, further comprising an imaging agent.

19. The nanoemulsion formulation of claim 18, wherein the imaging agent is an MRI contrasting moiety.

20. The nanoemulsion formulation of claim 19, wherein the MRI contrasting moiety comprises gadolinium, iron oxide, iron platinum, manganese, or combinations thereof.

21. A method of inhibiting the growth of, or killing, a cancer cell, comprising contacting the cancer cell with an amount of the nanoemulsion formulation of claim 1 that is toxic to, or which inhibits the growth of, or which kills, the cancer cell.

22. The method of claim 21, wherein the cancer cell is in a mammal, and the contacting step comprises administering to the mammal a therapeutically effective amount of the nanoemulsion formulation.

23. A method of imaging a cancer cell, comprising contacting the cancer cell with the nanoemulsion formulation of claim 18.

24. The method of claim 23, wherein the cancer cell is in a mammalian subject, and the contacting step comprises administering to the subject an amount of the nanoemulsion formulation sufficient to image the cancer cell.