+

WO2016014337A1 - Systèmes de nanoémulsions d'administration de médicaments - Google Patents

Systèmes de nanoémulsions d'administration de médicaments Download PDF

Info

Publication number
WO2016014337A1
WO2016014337A1 PCT/US2015/040751 US2015040751W WO2016014337A1 WO 2016014337 A1 WO2016014337 A1 WO 2016014337A1 US 2015040751 W US2015040751 W US 2015040751W WO 2016014337 A1 WO2016014337 A1 WO 2016014337A1
Authority
WO
WIPO (PCT)
Prior art keywords
nanoemulsion
nanoemulsion formulation
oil
formulation
peg
Prior art date
Application number
PCT/US2015/040751
Other languages
English (en)
Inventor
Srinivas Ganta
Timothy P. Coleman
Original Assignee
Nemucore Medical Innovations, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nemucore Medical Innovations, Inc. filed Critical Nemucore Medical Innovations, Inc.
Publication of WO2016014337A1 publication Critical patent/WO2016014337A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1806—Suspensions, emulsions, colloids, dispersions
    • A61K49/1809—Micelles, e.g. phospholipidic or polymeric micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • A61K47/6909—Micelles formed by phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers

Definitions

  • the present disclosure relates to medicine and pharmacology, and more particularly, to cancer therapy and drug delivery systems for cancer therapeutics.
  • 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.
  • MDR multi-drug resistance
  • 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).
  • MDR multidrug resistance
  • a number of delivery systems have been developed to address these problems.
  • 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.
  • Doxil also known as Caelyx
  • PEG polyethylene glycol
  • PCL poly(epsilon-caprolactone)
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the interfacial surface membrane phase of the drug delivery system comprises an emulsifier and/or a stabilizer.
  • 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.
  • the stabilizer of the interfacial surface membrane comprises a polyethylene glycol derivative, a phosphatide, a polyglycerol mono oleate, or combinations thereof.
  • the polyethylene glycol derivative of the interfacial surface membrane is PEG 2000 DSPE, PEG 3400 DSPE, PEG 5000 DSPE, or combinations thereof.
  • 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.
  • 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.
  • the nanoemulsion formulation further comprises a chemopotentiator.
  • the chemopotentiator comprises ceramide (CER) or a derivative thereof.
  • the chemopotentiator comprises C6- ceramide.
  • the nanoemulsion formulations the chemotherapeutic agent further comprises C6-ceramide.
  • the nanoemulsion formulation further comprises a targeting ligand.
  • the targeting ligand comprises an EGFR-targeting ligand, a folate receptor-targeting ligand, or a combination thereof.
  • the EGFR-targeting ligand comprises peptide 4, an anti-EGFR immunoglobulin or EGFR- binding fragment thereof, EGal-PEG, or combinations thereof.
  • 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.
  • 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.
  • the nanoemulsion formulation further comprises an imaging agent.
  • the imaging agent is an MRI contrasting moiety.
  • the MRI contrasting moiety comprises gadolinium, iron oxide, iron platinum, manganese, or combinations thereof.
  • 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.
  • the cancer cell is in a mammal, and the contacting step comprises administering to the mammal a therapeutically effective amount of the nanoemulsion formulation.
  • the present disclosure provides a method of imaging a cancer cell, comprising contacting the cancer cell with the nanoemulsion formulation of as described above.
  • 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.
  • FIG. 1 is a diagrammatic representation of a generic nanoemulsion formulation of the present disclosure
  • FIG. 2 is a diagrammatic representation of one non-limiting method of preparing a nanoemulsion formulation
  • FIG. 3 is a graphic representation of a DLS plot of a blank nanoemulsion formulation showing the particle size
  • FIG. 4 is a graphic representation of a DLS plot of an SP600125 nanoemulsion formulation showing the particle size
  • FIG. 5 is a graphic representation of a DLS plot of a sulforaphane nanoemulsion formulation showing the particle size
  • FIG. 6 is a graphic representation of a DLS plot of a salinomycin nanoemulsion formulation showing the particle size
  • FIG. 7 is a graphic representation of a DLS plot of a docetaxel nanoemulsion formulation showing the particle size
  • FIG. 8 is a graphic representation of a DLS plot of a docetaxel + ceramide nanoemulsion showing the particle size
  • FIG. 9 is a graphic representation of a DLS plot of a docetaxel + SP600125 nanoemulsion showing the particle size
  • FIG. 10 is a graphic representation of a DLS plot of a docetaxel + salinomycin nanoemulsion showing the particle size
  • FIG. 11 is a graphic representation of a DLS plot of an etoposide nanoemulsion formulation showing the particle size
  • FIG. 12 is a graphic representation of a DLS plot of a cyclopamine nanoemulsion formulation showing the particle size
  • FIG. 13 is a graphic representation of a DLS plot of a noscapine nanoemulsion formulation showing the particle size
  • FIG. 14 is a graphic representation of a DLS plot of a docetaxel, ceramide, and salinomycin nanoemulsion formulation showing the particle size
  • FIG. 15 is a graphic representation of a DLS plot of a docetaxel, sulforaphane, and salinomycin nanoemulsion formulation showing the particle size
  • FIG. 16 is a graphic representation of a DLS plot of a abamectin nanoemulsion formulation showing the particle size
  • FIG. 17 is a graphic representation of a DLS plot of a BAY 11-7082 nanoemulsion formulation showing the particle size
  • FIG. 18 is a schematic representation of one non-limiting method of synthesizing EGFR-MAL-PEG-DSPE;
  • FIG. 19 is a schematic representation of one non-limiting mode of synthesizing DSPE-PEG-Cys-FA
  • FIG. 20 is a schematic representation of one non-limiting method of preparing Gd 3 - DTPA-PE
  • FIG. 21 A is a representation of the NMR spectra of a DSPE-PEG-MAL standard
  • FIG. 2 IB is a representation of the NMR spectra of a EGFR-binding peptide standard
  • FIG. 21C is a representation of the NMR spectra of the DSPE-PEG-MAL-EGFRbp conjugate.
  • FIG. 22 is a series of representations of magnetic resonance images (MRIs) of mice treated with Gd 3 +-DTPA-PE, MagnevistTM (Mag), Gd 3 +-DTPA-PE, non-targeted nano-
  • MRIs magnetic resonance images
  • NT emulsion formulation
  • T EGFR-targeted nanoemulsion formulation
  • Anticancer agent or “chemotherapeutic agent” is an agent that prevents or inhibits the development, growth or proliferation of malignant cells.
  • “Cancer” is the uncontrolled growth of abnormal cells.
  • “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.
  • Stabilizer is an agent that prevents or slows the transformation or deactivation of a chemotherapeutic agent or ion in a chemotherapeutic agent formulation.
  • Patient is a human or animal in need of treatment for cancer.
  • [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.
  • 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.
  • 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.
  • 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.
  • Oil phase as used herein means the internal hydrophobic core of the
  • nanoemulsion in which a chemotherapeutic agent is dispersed 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
  • 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.
  • 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 ( ⁇ ⁇ 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.
  • Amphiphilic or amphiphile as used herein means a molecule with both a polar, hydrophilic portion and a non-polar, hydrophobic portion.
  • 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.
  • 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.
  • 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,
  • Targeting agents 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.
  • 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.
  • 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.
  • 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.
  • 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
  • “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 IC5 0 , and thus increasing the efficacy, of the drug or chemotherapeutic agent.
  • 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
  • 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.
  • FIG. 1 is a non-limiting schematic representation of a nanoemulsion formulation of the present disclosure.
  • 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
  • 6 represents a chemopotentiator dispersed in the oil phase 5 of the nanoemulsion formulation.
  • 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.
  • a co- agent e.g., a preservative, an antioxidant, a pH adjusting agent, an isotonicity modifier, or any combination thereof.
  • 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
  • Non-limiting examples of ionophores are salinomycin, nigericin, and/or abamectin.
  • 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.
  • LRP6 lipoprotein receptor related protein 6
  • gp P-glycoprotein
  • 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.
  • 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.
  • 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.
  • 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.
  • MIS mullerian- inhibiting substance
  • 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.
  • 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
  • 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.
  • the nanoemulsion formulations of the present disclosure may comprise a chemopotentiator, such as an apoptosis enhancer.
  • 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.
  • 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.
  • 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.
  • chemopotentiators seem to enhance the efficacy of chemotherapeutic agents, there are obstacles to the delivery of these compounds.
  • their effectiveness is limited due to their hydrophobicity and possible precipitation when administered in aqueous solutions.
  • the structures of the chemopotentiators such as the existence of a second aliphatic chain of CER, can hinder cellular permeability.
  • 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.
  • the present nanoemulsion formulations exploit the benefits of the chemopotentiators/apoptosis enhancers by providing increased solubility, intracellular permeability, and protection from systemic enzymatic degradation.
  • 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.
  • 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.
  • PUFA polyunsaturated fatty acid
  • 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.
  • 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.
  • 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 ⁇ g/10 6 cells.
  • these biocompatible oils can cause less than about 20% cell death when exposed to or taken up by, fibroblasts or epithelial cells.
  • 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.
  • the biocompatible oils may be biodegradable, i.e., able to degrade, chemically and/or biologically, within a physiological environment, such as within the body.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • concentration of the aqueous phase in the present nanoemulsion formulations can vary from about 30% to about 95% (w/w).
  • 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 nm 2 to 750,000 nm 2 .
  • 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).
  • 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.
  • 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,
  • dipalmitoylphosphatidylcholine dipalmitoylphosphatidylcholine; and hydrogenated or partially hydrogenated lecithins and phospholipids, e.g., from a natural source are used.
  • 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).
  • 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.
  • the interfacial surface membrane of the nanoemulsion formulation localizes drugs, thereby providing therapeutic advantages by releasing the encapsulated chemotherapeutic drug at predetermined, appropriate times.
  • 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.
  • the polar portion may have either a formal positive charge, or a formal negative charge.
  • 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.
  • 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).
  • DSPE dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • DAPC diarachidoylphosphatidylcholine
  • 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,
  • 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
  • phosphatidylethanolamines such as dioleoylphosphatidylethanolamine or l-hexadecyl-2- palmitoylglycerophos-phoethanolamine.
  • An amphiphilic compound of the interfacial membrane may include lecithin or phosphatidylcholine.
  • 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.
  • PEGylated lipid 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.
  • PEG polyethylene glycol
  • 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
  • RES reticuloendothelial system
  • MPS mononuclear phagocyte system
  • PEGylated soybean lecithin PEGylated egg lecithin
  • PEGylated phosphati-dylglycerol PEGylated phosphatidylinositol
  • PEGylated phosphatidylethanolamine PEGylated phosphatidic acid
  • PEGylated sphingomyelin PEGylated diphosphatidylglycerol
  • PEGylated phosphatidylserine PEGylated
  • amphiphilic PEGylated lipids can be used alone or in combination.
  • 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).
  • 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.
  • 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.
  • exemplary stabilizers are phosphatide, a polyglycerol mono oleate,
  • PEGioooDSPE PEG 20 ooDSPE, PEG 3400 DSPE, PEG 5000 DSPE, 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, PEG 2000 DSPE, PEG 3400 DSPE, PEGsoooDSPE.
  • the PEGylation density may be varied as necessary to facilitate long-circulation in the blood (Perry et al. (2012) Nano. Lett. 12:5304-5310).
  • 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.
  • EDC l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • PEG may include a terminal end group, for example, when PEG is not conjugated to a ligand.
  • 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.
  • the molecular weight of the PEG on interfacial membrane surface of the nanoemulsion formulation can be optimized for effective treatment as disclosed herein.
  • 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.
  • 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.
  • 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.
  • An alternative stabilizer may contain poly(ester-ether)s.
  • 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.
  • 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.
  • 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.
  • EGFR epidermal growth factor receptor
  • HER/erb 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.
  • 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.
  • FR-a Folate receptor alpha
  • FR- ⁇ 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.
  • 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.
  • 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]
  • 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.
  • 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).
  • FIG. 2 shows a representative synthesis scheme for one non-limiting, EGFR- targeted, Gd-labeled nanoemulsion formulation of the present disclosure.
  • 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.
  • 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
  • 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.
  • the nanoemulsion formulations of the present disclosure may have a substantially spherical or non-spherical shape.
  • 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 ⁇ , where the characteristic dimension of a nanoemulsion formulation is the diameter of a perfect sphere having the same volume as the nanoemulsion formulation.
  • 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.
  • Docetaxel to co-Agent ratio in the NE formulation is 1 :5.
  • Docetaxel to co- x ratio in the NE formulation is 1 :5.
  • Ratio of Chemotherapeutic Agent to co-Agent is 1 :5, where the NE formulation is a combination of drugs
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • a compound such as a targeting moiety ligand
  • a compound 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.
  • the interior of the nanoemulsion formulation is more hydrophobic than the surface of the nanoemulsion formulation.
  • 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).
  • 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.
  • An exemplary nanoemulsion formulation may have a PEG derivative corona with a density of about 1.065 g/cm 3 , or about 1.01 g/cm 3 to about 1.10 g/cm 3 .
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the nanoemulsion formulations of the present disclosure can be used to inhibit the growth of, or kill, cancer cells.
  • 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.
  • 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.
  • 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).
  • 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.
  • inventive targeted or non-targeted particle such as a nanoemulsion formulation
  • Nanoemulsion formulations disclosed herein may be combined with pharmaceutical acceptable carriers to form a pharmaceutical composition.
  • 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.
  • 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.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono or di-glycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the nanoemulsion formulations of this disclosure can be administered to a patient by any means known in the art including oral and parenteral routes.
  • patient refers to humans as well as non-humans, including, for example, mammals, birds, reptiles, amphibians, and fish.
  • parenteral routes are chosen since they avoid contact with the digestive enzymes that are found in the alimentary canal.
  • 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).
  • injection e.g., intravenous, subcutaneous or intramuscular, intraperitoneal injection
  • rectally rectally
  • vaginally topically
  • topically as by powders, creams, ointments, or drops
  • inhalation as by sprays.
  • 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.
  • Suitable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono or di-glycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the nanoemulsion formulations may also be administered orally.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • 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
  • solution retarding agents such as paraffin
  • absorption accelerators such as quaternary ammonium compounds
  • wetting agents such as, for example, cetyl alcohol and glycerol monostearate
  • absorbents such as kaolin and bentonite clay
  • lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.
  • the dosage form may also comprise buffering agents.
  • 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.
  • the "effective amount" of a nanoemulsion formulation refers to the amount that elicits the desired biological response.
  • 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.
  • 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.
  • the nanoemulsion formulation of the present disclosure may be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form 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
  • 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., ED 50 (the dose is therapeutically effective in 50% of the population) and LD 50 (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, LD5 0 /ED5 0 .
  • 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.
  • 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.
  • 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.
  • a proapoptotic agent such as ceramide, sulforaphane, curcumin or cyclopanine
  • 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).
  • 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.
  • Nanoemulsion formulations of the present disclosure can further include imaging or contrast agents.
  • 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.
  • Gd-DTPA-PE l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- 1,4,7, 10-tetraazacyclod
  • 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.
  • 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.
  • imaging moieties are soluble in the oil phase.
  • 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.
  • 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 (Gd 3+ ), 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.
  • paramagnetic elements such as gadolinium ions (Gd 3+ ), iron oxide, iron, platinum, or manganese
  • the nanoemulsion formulations combined with an appropriate imaging agent can act as MRI contrast agents to enhance tissue image resolution.
  • Contrast agents such as Gd 3+ 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.
  • the nanoemulsion formulations according to the disclosure can serve as a new Gd 3+ 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.
  • 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.
  • 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.
  • the nanoemulsion formulation of the disclosure includes a paramagnetic metal ion (e.g., Gd 3+ ).
  • 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 PEG2 000 -DSPE construct.
  • the EGFR B p conjugate was then purified by dialysis against deionized distilled water at RT using a 3500 molecular weight cut-off membrane (Spectrapore, Spectrum Laboratories, Collinso 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.
  • EGFR BP -PEG2 000 DSPE conjugate formation was confirmed by nuclear magnetic resonance spectroscopy (NMR) analysis.
  • NMR nuclear magnetic resonance spectroscopy
  • the DSPE-PEG-Mal complex was prepared according to the scheme in FIG. 19.
  • DSPE-PEG-Mal 100 mg, 1.3596 mM
  • cysteine 8.24 mg, 2.72 mM
  • HEPES buffer 25 ml
  • 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.
  • 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 ⁇ , ⁇ '-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.
  • 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 ⁇ 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 ⁇ 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.
  • DMF dry Dimethylformamide
  • EDCI l-ethyl-3-(3-dimethylamino- propyl)carbodiimide
  • NHS N
  • Gd +3 -DTPA-PE chelate was prepared according to the scheme shown in FIG. 20.
  • 30 ⁇ 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.
  • DTPA anhydride diethylene triaminepentacetic dianhydride
  • 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
  • ninhydrin as a visualizing reagent.
  • reactants DTPA, PE
  • complex DTPA-PE
  • Ninhydrin solution was then sprayed and the spots and their retention times were compared for the formation of the complex.
  • 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.
  • 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.
  • 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
  • PEG 2000 DSPE (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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Pgp P-glycoprotein
  • 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 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.
  • 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 ⁇ g/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 ⁇ g/well) for 2 hr. The resulting formazan crystals were dissolved in dimethyl sulfoxide (150 ⁇ g/well) and measured at 570 nm in the Plate reader (Synergy HT, Biotek Instruments, Winooski, VT).
  • the concentration of drug that inhibits fifty percent of growth is known as the 50% growth inhibitory concentration (IC5 0 ).
  • IC5 0 concentration of drug that inhibits fifty percent of growth
  • the IC5 0 values were calculated. All IC5 0 values were obtained by analyzing the MTT assays results using Graphpad Prism 5 scientific data analysis software. The results are shown in Table XVIII.
  • the IC5 0 was decreased when non-targeted nanoemulsion formulations of the present disclosure were used. Most significant is the decrease in IC5 0 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).
  • 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)
  • CI (a /A) + (b/B) where, "a” is the primary therapeutic IC5 0 in combination with secondary therapeutic at concentration "b.” "A” is the primary therapeutic IC5 0 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.
  • the ratio of Pt : CER was determined in order to identify ratios that could reduce the IC5 0 of Pt.
  • 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.
  • MagnevistTM 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 MagnevistTM control rapidly accumulated in the tumor over the first hour, but then cleared and resolved to near baseline by the 6th hr.
  • 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.
  • 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.
  • nanoemulsion formulations allow for more efficient treatment of multidrug resistant tumors.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Dispersion Chemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Nanotechnology (AREA)
  • Radiology & Medical Imaging (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dermatology (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention concerne des formulations de nanoémulsions comprenant un système d'administration de médicaments et un agent chimiothérapeutique pour le traitement et le diagnostic du cancer et qui ne provoquent pas de résistance pléiotrope. L'invention concerne également des méthodes de préparation de celles-ci ainsi que de traitement ou d'imagerie du cancer.
PCT/US2015/040751 2014-07-25 2015-07-16 Systèmes de nanoémulsions d'administration de médicaments WO2016014337A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462029144P 2014-07-25 2014-07-25
US62/029,144 2014-07-25

Publications (1)

Publication Number Publication Date
WO2016014337A1 true WO2016014337A1 (fr) 2016-01-28

Family

ID=55163571

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/040751 WO2016014337A1 (fr) 2014-07-25 2015-07-16 Systèmes de nanoémulsions d'administration de médicaments

Country Status (1)

Country Link
WO (1) WO2016014337A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106750252A (zh) * 2016-12-05 2017-05-31 山东大学齐鲁医院 二硬脂酰基磷脂酰乙醇胺‑聚乙二醇2000‑双叶酸及其制备方法和应用
CN109289057A (zh) * 2018-11-27 2019-02-01 中国人民解放军陆军军医大学 一种靶向治疗类风湿性关节炎的地塞米松纳米制剂及其制备方法
CN109395101A (zh) * 2018-09-27 2019-03-01 复旦大学附属华山医院 靶向血脑屏障和脑胶质瘤的磁共振对比剂的制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8216607B2 (en) * 2008-03-17 2012-07-10 Roger Williams Hospital Combination of ceramide and gemcitabine for inducing cell death and uses thereof in treating cancer
US20140105829A1 (en) * 2012-10-15 2014-04-17 Nemucore Medical Innovations, Inc. Therapeutic nanoemulsion formulation for the targeted delivery of docetaxel and methods of making and using the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8216607B2 (en) * 2008-03-17 2012-07-10 Roger Williams Hospital Combination of ceramide and gemcitabine for inducing cell death and uses thereof in treating cancer
US20140105829A1 (en) * 2012-10-15 2014-04-17 Nemucore Medical Innovations, Inc. Therapeutic nanoemulsion formulation for the targeted delivery of docetaxel and methods of making and using the same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ALTINTAS, I ET AL.: "Nanobody-Albumin Nanoparticles (NANAPs) For The Delivery of a Multikinase Inhibitor 17864 to EGFR Overexpressing Tumor Cells", JOURNAL OF CONTROLLED RELEASE., vol. 165, 2013, pages 110 - 118 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106750252A (zh) * 2016-12-05 2017-05-31 山东大学齐鲁医院 二硬脂酰基磷脂酰乙醇胺‑聚乙二醇2000‑双叶酸及其制备方法和应用
CN106750252B (zh) * 2016-12-05 2018-11-30 山东大学齐鲁医院 二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000-双叶酸及其制备方法和应用
CN109395101A (zh) * 2018-09-27 2019-03-01 复旦大学附属华山医院 靶向血脑屏障和脑胶质瘤的磁共振对比剂的制备方法
CN109289057A (zh) * 2018-11-27 2019-02-01 中国人民解放军陆军军医大学 一种靶向治疗类风湿性关节炎的地塞米松纳米制剂及其制备方法
CN109289057B (zh) * 2018-11-27 2022-02-15 中国人民解放军陆军军医大学 一种靶向治疗类风湿性关节炎的地塞米松纳米制剂及其制备方法

Similar Documents

Publication Publication Date Title
JP6427097B2 (ja) 癌を処置するための組成物および該組成物を製造するための方法
Martins et al. Brain targeting effect of camptothecin-loaded solid lipid nanoparticles in rat after intravenous administration
US20160166507A1 (en) Nanoemulsions of hydrophobic platinum derivatives
Liang et al. Nanocrystal-loaded liposome for targeted delivery of poorly water-soluble antitumor drugs with high drug loading and stability towards efficient cancer therapy
CA2976526A1 (fr) Compositions et procedes de traitement de tumeur utilisant des nanoparticules
Xing et al. Novel lipophilic SN38 prodrug forming stable liposomes for colorectal carcinoma therapy
US20140105829A1 (en) Therapeutic nanoemulsion formulation for the targeted delivery of docetaxel and methods of making and using the same
An et al. Design, preparation, and characterization of novel calix [4] arene bioactive carrier for antitumor drug delivery
WO2011092708A2 (fr) Liposomes comprenant des médicaments amphipathiques et procédé pour leur préparation
WO2016090092A1 (fr) Nano-émulsions thérapeutiques pour l'administration à et l'imagerie du cerveau et des procédés de leur préparation et utilisation
WO2021226368A1 (fr) Formulations liposomales pour le traitement de cancers et de la résistance aux médicaments de cancers
US20210213051A1 (en) Combined pharmaceutical formulation comprising drug-containing liposome composition and platinum preparation
Abeesh et al. Preparation and characterization of withaferin A loaded pegylated nanoliposomal formulation with high loading efficacy: In vitro and in vivo anti-tumour study
Gill et al. EGFR targeted delivery of paclitaxel and parthenolide co-loaded in PEG-Phospholipid micelles enhance cytotoxicity and cellular uptake in non-small cell lung cancer cells
US20210275489A1 (en) Platinum-based amphiphile prodrugs
Nagdiya et al. Drug delivery systems of gefitinib for improved cancer therapy: A review
WO2008134528A1 (fr) Compositions de conjugués d'agent anticancéreux-acide hyaluronique et procédés
WO2016014337A1 (fr) Systèmes de nanoémulsions d'administration de médicaments
US20250064784A1 (en) Liposome composition comprising liposomal prodrug of mitomycin c and methods of treatment
CN108853056B (zh) 一种叶酸靶向修饰共载盐酸阿霉素和藤黄酸纳米结构脂质载体制剂及其制备方法
Zhang et al. Reversing multi-drug resistance by polymeric metformin to enhance antitumor efficacy of chemotherapy
KR100847626B1 (ko) 수난용성 캄프토테신 함유 리포솜 제제
Fang et al. Liposome-like nanocapsules of dual drug-tailed betaine for cancer therapy
WO2022265880A1 (fr) Méthodes et compositions améliorées pour l'administration de médicament se rapportant à des liquides ioniques
AU2021101145A4 (en) Codelivery of verapamil with cisplatin and paclitaxel nanopalrticulate drug delivery system for ovarian cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15824969

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15824969

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载