WO2011062420A2 - Nanoparticles for tumor-targeting and processes for the preparation thereof - Google Patents

Nanoparticles for tumor-targeting and processes for the preparation thereof Download PDF

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WO2011062420A2
WO2011062420A2 PCT/KR2010/008149 KR2010008149W WO2011062420A2 WO 2011062420 A2 WO2011062420 A2 WO 2011062420A2 KR 2010008149 W KR2010008149 W KR 2010008149W WO 2011062420 A2 WO2011062420 A2 WO 2011062420A2
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pharmaceutical composition
nanoparticles
nano
oil
composition according
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French (fr)
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WO2011062420A3 (en
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Seong-Bae Park
Joo-Eun Kim
Jun-Mo Yang
Mi-Hong Min
Hyun-Ho Choi
Min-Na Woo
Byung-Nak Ahn
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Yuhan Corporation
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    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/02—Inorganic compounds
    • 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
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a pharmaceutical composition containing an anticancer agent, which is capable of both passive and active targeting. More specifically, the present invention relates to nanoparticles capable of both passive and active targeting, which comprises a therapeutically effective amount of an anticancer agent; a divalent or trivalent transition metal ion or alkali earth metal ion; an oil; and hyaluronic acid or its salt.
  • Anticancer agents for cancer therapy affect normal cells as well as cancer cells in the body, thereby resulting in various side effects.
  • various targeting methods including passive targeting or active targeting.
  • targeting methods of satisfactory level still remain to be elucidated.
  • Passive targeting methods are based on the fact that fine-particles may be selectively accumulated in the cancer cells having abnormal blood vessels, during the continuous blood circulation in the body.
  • cancer cells since cancer cells become enlarged due to their abnormal growth rate, they have larger endothelial pores than normal cells; and/or various sizes of endothelial pores (e.g., 10 nm to 1000 nm) while normal cells have aligned endothelial pores. Therefore, when micronized particulate carriers having an anticancer agent are allowed to continuously circulate in the blood, the micronized particles (e.g., nanoparticles) may be accumulated only in cancer cells, while those cannot penetrate into the endothelial pores of normal cells. This mechanism of action is referred to enhanced permeation and retention (EPR) effect.
  • EPR enhanced permeation and retention
  • Examples of the passive targeting method include a method for delivering an anticancer agent using small particle liposome aerosols (see WO1999/15153).
  • Active targeting methods refer to a method for delivering an anticancer agent to cancer cells through a drug delivery system that is prepared by using a receptor specifically recognizing the molecule over-expressed at cancer cell surface, the molecule of which includes e.g., lectin, growth factor, cytokine, hormone, unsaturated fatty acid, low-density lipoprotein, folic acid (S.P Vyas., et. al., Advanced Drug Delivery Review 43 (2000) 101-164).
  • a receptor specifically recognizing the molecule over-expressed at cancer cell surface the molecule of which includes e.g., lectin, growth factor, cytokine, hormone, unsaturated fatty acid, low-density lipoprotein, folic acid.
  • US patent no. 6,593,308 has disclosed a drug delivery system comprising an encapsulating delivery vehicle (e.g., liposome) and hyaluronan ligands.
  • an anticancer agent is encapsulated in the vehicle; and metal ion is used as a linking agent or a chelating agent in order to attach the ligands to the vehicle.
  • metal ion is used as a linking agent or a chelating agent in order to attach the ligands to the vehicle.
  • a targeting method using a monoclonal antibody having high affinity to cancer cells (Stanislv J., et al., Bioorg. Medic. Chem. (2005) 13, 5043-5054).
  • each hyaluronan ligand is chemically attached to the delivery vehicle (e.g., liposome).
  • the delivery vehicle e.g., liposome
  • liposome has very low long-term stability due to instability of the lipid lamella; and may be decomposed through phagocytosis by macrophages.
  • the chemical attachment of hyaluronan ligands to liposome is very complicated and gives unwanted side products during the preparation.
  • US patent no. 6,699,471 has disclosed an injectable composition in the form of a gel comprising a benzyl ester of hyaluronic acid or an auto-cross-linked derivative of hyaluronic acid.
  • US patent publication no. 2006/0188578 has disclosed a method of obtaining nanoparticles using chitosan and tripolyphosphate, in which hyaluronic acid is attached to the surface thereof via ionic bond.
  • US patent publication no. 2007/0036728 has disclosed a method of transdermal drug delivery using an oil-in-water emulsion, which has hyaluronic acid nanoparticles obtained from hyaluronic acid and a hydrazide.
  • 2007/0031503 has disclosed a hyaluronic acid modification product obtained by binding hyaluronic acid with polylactic acid, polyglycolic acid, or lactic acid-glycolic acid copolymer.
  • WO00/041730 has disclosed a method for enhancing the effectiveness of a cytotoxic or anti-neoplastic agent, comprising co-administering said agent with hyaluronan.
  • the present inventors have developed nanoparticles using hyaluronic acid or its salt, which is capable of both passive and active targeting (Korean patent no. 10-744,925 and Korean patent publication no. 10-2009-0040979).
  • the nanoparticles are formed from a mixture of an anticancer agent (anti-tumor agent) and hyaluronic acid or its salt, using a metal ion and/or a water-insoluble biodegradable polymer.
  • the nanoparticles can accomplish both passive targeting through EPR effect and active targeting through hyaluronic acid.
  • the present invention provides an improved drug delivery system in the form of a nano-dispersion using hyaluronic acid or its salt, which is capable of both passive and active targeting.
  • the drug delivery system comprises nanoparticles in an aqueous medium, wherein the nanoparticles include an anticancer agent (anti-tumor agent) along with a metal ion and an oil, and wherein hyaluronic acid or its salt is bound on a surface of the nanoparticles through interaction with the metal ion.
  • the drug delivery system can accomplish both passive targeting through EPR effect and active targeting through hyaluronic acid. And also, the drug delivery system can remarkably increase the encapsulation efficiency of an anticancer agent.
  • a pharmaceutical composition for tumor-targeting in the form of a nano-dispersion comprising nanoparticles having a mean particle size of 10 to 1,000 nm in an aqueous medium, wherein the nanoparticles comprise a therapeutically effective amount of an anticancer agent; a divalent or trivalent transition metal ion or alkali earth metal ion; and an oil, and wherein hyaluronic acid or its salt is bound on a surface of the nanoparticles.
  • nanoparticles for tumor-targeting obtained by drying the pharmaceutical composition in the form of a nano-dispersion, through lyophilizing, rotary evaporation drying, spray drying or fluidized-bed drying.
  • a process for preparing a pharmaceutical composition for tumor-targeting in the form of a nano-dispersion which comprises: (a) providing an oil phase comprising a therapeutically effective amount of an anticancer agent, a divalent or trivalent transition metal ion or alkali earth metal ion and an oil, (b) providing an aqueous phase by dissolving hyaluronic acid or its salt in an aqueous medium, and (c) mixing the oil phase obtained in Step (a) and the aqueous phase obtained in Step (b) and then homogenizing the resulting mixture to form nanoparticles having a mean particle size of 10 to 1,000 nm, the surface of which hyaluronic acid or its salt is bound on.
  • a process for preparing nanoparticles for tumor-targeting which comprises drying the pharmaceutical composition in the form of a nano-dispersion through lyophilizing, rotary evaporation drying, spray drying or fluidized-bed drying.
  • the pharmaceutical composition in the form of a nano-dispersion comprising nanoparticles in an aqueous medium according to the present invention is a drug delivery system in which the nanoparticles are obtained by binding hyaluronic acid or its salt on the surface of nanoparticles comprising an anticancer agent, a metal ion, and an oil.
  • the drug delivery system of the present invention makes it possible to accomplish both passive tumor-targeting through EPR effect and active tumor-targeting through affinity of hyaluronic acid to cancer cells. That is, the nanoparticles of the present invention selectively interact with cancer cells, thereby being able to selectively interact with metastatic tumors as well as solid tumors.
  • the pharmaceutical composition of the present invention can increase the encapsulation efficiency of an anticancer agent, to wanted level of amount (i.e., almost to 100% of wanted amount). And also, the pharmaceutical composition in the form of a nano-dispersion of the present invention has excellent biocompatibility and physicochemical stability.
  • FIG. 1 shows a schematic example of the nanoparticles according to the present invention.
  • FIG. 2 shows a Cyro-TEM image of the nanoparticles according to the present invention.
  • FIG. 3 shows the results of CD44-binding affinity test for the nanoparticles according to the present invention.
  • FIG. 3A and 3C show the results of in vitro affinity test using OVCAR-3 cells.
  • FIG. 3B and 3D show the results of in vitro affinity test using SK-OV-3 cells.
  • FIG. 4A and 4B show the results of evaluation of anticancer efficacy by active and passive tumor-targeting, for the nanoparticles according to the present invention.
  • FIG. 4A shows changes of tumor size in the cancer-transplanted CD44-overexpressed SK-OV-3 models after test materials or controls were respectively administered.
  • FIG. 4B shows changes of tumor size in the cancer-transplanted CD44-not-expressed OVCAR-3 models after test materials or controls were respectively administered.
  • FIG. 5 shows the results of safety evaluation for the nanoparticles according to the present invention.
  • the body weights were monitored for 2 weeks after the test materials were administered.
  • FIG. 6 shows the results of dissolution test for the nanoparticles according to the present invention.
  • nanoparticles refers to particles having a mean particle size of about 1000 nm or less, for example, 10 to 1,000 nm, preferably 50 to 500 nm, more preferably 50 to 300 nm.
  • nano-dispersion refers to a drug delivery system obtained by dispersing the nanoparticles in an aqueous medium.
  • the nano-dispersion may be in the form of a nano-emulsion or in the form of a nano-suspension.
  • nano-emulsion refers to a drug delivery system of an emulsion form, wherein the nanoparticles are dispersed in oil forms (e.g., in droplets) at room temperature (about 25 °C), in an aqueous medium.
  • nano-suspension refers to a drug delivery system of a suspension form, wherein the nanoparticles are dispersed in solid particle forms at room temperature (about 25 °C), in an aqueous medium.
  • the nanoparticles in the nano-dispersion has a mean particle size suitable for providing EPR effect, for example a mean particle size of 10 to 1,000 nm, preferably 50 to 500 nm, more preferably 50 to 300 nm.
  • the present invention provides a pharmaceutical composition for tumor-targeting in the form of a nano-dispersion comprising nanoparticles having a mean particle size of 10 to 1,000 nm in an aqueous medium, wherein the nanoparticles comprise a therapeutically effective amount of an anticancer agent; a divalent or trivalent transition metal ion or alkali earth metal ion; and an oil, and wherein hyaluronic acid or its salt is bound on a surface of the nanoparticles.
  • the pharmaceutical composition of the present invention comprises nanoparticles in an aqueous medium, wherein the nanoparticles include an anticancer agent (anti-tumor agent) along with a metal ion and an oil, and wherein hyaluronic acid or its salt is bound on a surface of the nanoparticles through interaction between hyaluronic acid and the metal ion (e.g., chelate bond).
  • the hyaluronic acid or its salt is present on the exterior of nanoparticles, while the metal ion is present in interior of nanoparticles (see FIGs 1 and 2).
  • Hyaluronic acid one of the hydrophilic polysaccharides in the body, is present in the form of a high molecular weight hetero-polysaccharide in which disaccharide unit of D-glucuronic acid and N-glucosamine (molecular weight: 379) are linked each other to form a long chain.
  • Hyaluronic acid in extracellular matrix provides various functions in proliferation, differentiation and migration of cells. It has been described that the various functions are associated with hyaluronic acid-binding receptors such as CD44 (one of the cell surface glycoproteins); receptors for hyaluronic acid-mediated motility (RHAMM); and other receptors having hyaluronic acid-binding motifs (S.
  • Molecular weight of the hyaluronic acid may range from 379 to 10,000,000 daltons, preferably from 1,000 to 4,000,000 daltons, more preferably from 1,000 to 1,500,000 daltons, but not limited thereto.
  • Hyaluronic acid may be in a salt form, for example an inorganic salt form such as cobalt hyaluronate, magnesium hyaluronate, zinc hyaluronate, calcium hyaluronate, potassium hyaluronate, or sodium hyaluronate; or an organic salt form such as tetrabutylammonium hyaluronate.
  • hyaluronic acid free base and/or sodium hyaluronate may be used.
  • Each of hyaluronic acid or its salt may be bound on the surface of nanoparticles or a mixture of hyaluronic acid and its salt may be bound on the surface of nanoparticles.
  • An amount of the hyaluronic acid or its salt may range from 0.01 to 1.0 w/w%, preferably from 0.1 to 0.5 w/w%, based on the total weight of the composition.
  • the nanoparticles comprise a divalent or trivalent transition metal ion or alkali earth metal ion.
  • the divalent or trivalent transition metal ion or alkali earth metal ion may be at least one selected from the group consisting of Cu 2+ , Cu 3+ , Zn 2+ , Zn 3+ , Ni 2+ , Ni 3+ , Mg 2+ , Mg 3+ , Ca 2+ , Ca 3+ , Co 2+ , Co 3+ , Ba 2+ , Ba 3+ , Al 2+ , Al 3+ , Fe 2+ , and Fe 3+ .
  • the metal ion may be derived from various metal salts.
  • the metal ion may be derived from, but limited to, copper nitrate, copper sulfate, copper chloride, zinc acetate, zinc sulfate, zinc nitrate, zinc chloride, nickel sulfate, nickel nitrate, nickel chloride, magnesium acetate, magnesium sulfate, magnesium nitrate, magnesium chloride, calcium acetate, calcium sulfate, calcium nitrate, calcium chloride, cobalt acetate, cobalt sulfate, cobalt chloride, barium acetate, barium sulfate, barium nitrate, barium chloride, aluminum sulfate, aluminum chloride, ferric (or ferrous) acetate, ferric (or ferrous) sulfate, ferric (or ferrous) nitrate, or ferric (or ferrous) chloride.
  • Fe 2+ and/or Fe 3+ ion may be preferably used and the Fe 2+ and/or Fe 3+ ion may be derived from ferric (or ferrous) acetate, ferric (or ferrous) sulfate, ferric (or ferrous) nitrate, or ferric (or ferrous) chloride.
  • the metal ion is reacted mostly with the carboxylic groups of glucuronic acid within hyaluronic acid to form chelate bonds.
  • the metal ion may be also reacted with the hydroxyl groups between hyaluronic acid chains; or reacted with the amine groups of N-acetylglucosamine, so as to form chelate bonds.
  • Molar ratio of the divalent or trivalent transition metal ion or alkali earth metal ion per disaccharide unit of the hyaluronic acid or its salt may range from 0.001 to 2.0, preferably from 0.001 to 0.5.
  • the nanoparticles comprise a biocompatible oil as a lipophilic medium for dissolving an anticancer agent.
  • the oil may be a liquid form or a solid form at room temperature.
  • the oil may be a liquid form at room temperature.
  • the oil having a liquid form at room temperature may be at least one selected from the group consisting of mono-, di-, or tri-glycerides (for example, Myvacet 9-45K); glyceryl mono- or tri-stearates; glyceryl mono-, di-, or tri-acetates (for example, triacetin); alpha-tocopherol (including d-alpha-tocopherol and/or dl-alpha-tocopherol) or its salt; alpha-tocopheryl acetate (including d-alpha-tocopheryl acetate and/or dl-alpha-tocopheryl acetate); alpha-tocopherol succinate (including d-alpha-tocopherol succinate or dl-alpha-tocopherol succinate); C4:0 to C6:0 triglycerides (for example,
  • the oil may be a solid form at room temperature.
  • the oil having a solid form at room temperature may be at least one selected from the group consisting of hydrogenated soybean oil, cacao butter, cetyl alcohol, stearyl alcohol, cetyl palmitate, carnauba wax, white beeswax, tricaprin (for example, Migriol 812), trilaurin, trimyristin, tripalmitin, tristearin, and tribehenin.
  • the oil may be at least one selected from the group consisting of glyceryl mono-, di-, or tri-acetates; alpha-tocopherol (including d-alpha-tocopherol and/or dl-alpha-tocopherol) or its salt; alpha-tocopheryl acetate (including d-alpha-tocopheryl acetate and/or dl-alpha-tocopheryl acetate); alpha-tocopherol succinate (including d-alpha-tocopherol succinate or dl-alpha-tocopherol succinate); tributyrin; and tricaproin.
  • alpha-tocopherol including d-alpha-tocopherol and/or dl-alpha-tocopherol
  • alpha-tocopherol succinate including d-alpha-tocopherol succinate or dl-alpha-tocopherol succinate
  • tributyrin and tricaproin.
  • An amount of the oil may range from 1 to 50 w/w%, preferably from 1 to 30 w/w%, based on the total weight of the composition
  • the nanoparticles also comprise a chemical or biological material having an inhibitory activity against proliferation or metastasis of cancer cells, i.e., anticancer agent (anti-tumor agent), the types of which are not limited.
  • anticancer agent include, but not limited to, paclitaxel or its derivative, uracil, 5-fluorouracil, tegafur, methotrexate, melphalan, mitoxantrone, camptothecin, topotecan, docetaxel, capecitabine, imatinib mesylate, rituximab, doxifluridine, toremifene citrate, doxorubicin, gemcitabine, irinotecan, oxaliplatin, or chlorambucil.
  • the anticancer agent used may be paclitaxel or its derivative, docetaxel, uracil, 5-fluorouracil, tegafur, methotrexate, melphalan, mitoxantrone, camptothecin, or topotecan.
  • Therapeutically effective amount of each anticancer agent may be easily determined from the arts by a skilled person.
  • the pharmaceutical composition of a nano-dispersion form according to the present invention may further comprise a surfactant for obtaining stable dispersion of the nanoparticles (i.e. for increasing stability of the nano-dispersion).
  • the surfactant may be incorporated into the nanoparticles and/or added to the aqueous medium.
  • the surfactant may be at least one selected from the group consisting of alpha-tocopherol polyethylene glycol succinate (TPGS), macrogol 15 hydroxystearate (for example, Solutol HS15), caprylocaproyl macrogolglycerides (for example, Labrasol), sorbitan fatty acid esters (for example, sorbitan monooleate (e.g., Span 20)), polysorbates (for example, Tween 20, Tween 80), polyoxyethylene-polyoxypropylene block copolymer (for example, Poloxamer 188, Poloxamer 407), egg lecithin, and soybean lecithin; but not limited thereto.
  • An amount of the surfactant may be selected according to the type of oil used. For example, the amount of the surfactant may ranges from 1 to 50 w/w%, based on the total weight of the composition, but not limited thereto.
  • the aqueous medium may be at least one selected from the group consisting of distilled water, water for injection, saline, solution for sodium chloride injection, solution for dextrose injection, and solution for amino acid injection; but not limited thereto.
  • the nanoparticles may further comprise a solubilizing agent therein, in order to avoid any precipitation of anticancer agent originated from the nanoparticles.
  • the solubilizing agent may be at least one selected from the group consisting of polyethylene glycol, dimiristoyl phosphatidyl ethanolamine-polyethylene glycols (DMPE-PEGs) (for example, DMPE-PEG 2000 ), cholesterol, propylene glycol, glycofurol, and tricaprylin.
  • An amount of the solubilizing agent may range from 0.05 to 10 w/w%, preferably from 0.05 to 5 w/w%, based on the total weight of the composition.
  • the nanoparticles may further comprise an amino acid having carboxyl group as a low-molecular ligand, in order to stably bind hyaluronic acid or its salt on the surface of nanoparticles.
  • the low-molecular ligand may be at least one selected from the group consisting of glutamic acid, aspartic acid, asparagine, histidine, and alanine.
  • the low-molecular ligand may be glutamic acid and/or aspartic acid.
  • An amount of the low-molecular ligand may range from 0.005 to 0.2 w/w%, based on the total weight of the composition.
  • the pharmaceutical composition of a nano-dispersion form may comprise a pH-controlling agent such as an organic acid or an N-acety amino acid, in order to increase chemical stability.
  • pH of the pharmaceutical composition is controlled in the range of 2.0 to 6.0, using the pH-controlling agent.
  • the pH-controlling agent may be added to the nanoparticles and/or the aqueous medium.
  • the pH-controlling agent may be at least one selected from the group consisting of an organic acid such as citric acid, acetic acid, phosphoric acid, lactic acid, benzoic acid, maleic acid, succinic acid, tartaric acid; and an N-acety amino acid such as N-acetyl cysteine, N-acetyl valine, N-acetylproline, and N-acetylalanine.
  • an organic acid such as citric acid, acetic acid, phosphoric acid, lactic acid, benzoic acid, maleic acid, succinic acid, tartaric acid
  • an N-acety amino acid such as N-acetyl cysteine, N-acetyl valine, N-acetylproline, and N-acetylalanine.
  • the pharmaceutical composition of a nano-dispersion form may further comprise at least one dispersion-stabilizing agent selected from the group consisting of polyvinylpyrrolidone, glycerin, glucose, sucrose, lactose, sorbitol, mannitol, and trehalose.
  • the dispersion-stabilizing agent helps not only maintaining dispersability of the composition of a nano-dispersion form, but also re-dispersing in a dispersing medium the nanoparticles obtained by drying the nano-dispersion.
  • the dispersion-stabilizing agent may be used in an amount of 1 to 20 w/w%, preferably 3 to 7 w/w%, based on the total weight of the composition, but not limited thereto.
  • the present invention also provides nanoparticles for tumor-targeting obtained by drying the pharmaceutical composition in the form of a nano-dispersion, through lyophilizing, rotary evaporation drying, spray drying or fluidized-bed drying.
  • the present invention also provides a process for preparing a pharmaceutical composition for tumor-targeting in the form of a nano-dispersion, which comprises: (a) providing an oil phase comprising a therapeutically effective amount of an anticancer agent, a divalent or trivalent transition metal ion or alkali earth metal ion and an oil, (b) providing an aqueous phase by dissolving hyaluronic acid or its salt in an aqueous medium, and (c) mixing the oil phase obtained in Step (a) and the aqueous phase obtained in Step (b) and then homogenizing the resulting mixture to form nanoparticles having a mean particle size of 10 to 1,000 nm, the surface of which hyaluronic acid or its salt is bound on.
  • a process for preparing pharmaceutical composition according to the present invention may further comprise adding a surfactant to the oil phase and/or the aqueous phase.
  • the anticancer agent the divalent or trivalent transition metal ion or alkali earth metal ion; the oil, the surfactant; and hyaluronic acid or its salt are the same as described in the above.
  • the oil phase may be obtained by (i) dissolving an anticancer agent and optionally a surfactant in an organic solvent, (ii) dissolving a divalent or trivalent transition metal ion or alkali earth metal ion in an oil, and (iii) mixing the solution obtained in Step (i) and the solution obtained in Step (ii) and then drying the resulting mixture to remove the organic solvent.
  • the organic solvent may be methanol, ethanol, or acetonitile, preferably absolute ethanol.
  • the oil phase may further comprise at least one solubilizing agent selected from the group consisting of polyethylene glycol, dimiristoyl phosphatidyl ethanolamine-polyethylene glycols (DMPE-PEGs), cholesterol, propylene glycol, glycofurol, and tricaprylin.
  • DMPE-PEGs dimiristoyl phosphatidyl ethanolamine-polyethylene glycols
  • cholesterol propylene glycol
  • glycofurol glycofurol
  • tricaprylin tricaprylin.
  • the aqueous phase may be obtained by further dissolving an amino acid having carboxyl group as a low-molecular ligand. Types and amounts thereof are the same as described in the above. And also, if necessary, the aqueous phase may be obtained by further dissolving at least one dispersion-stabilizing agent selected from the group consisting of polyvinylpyrrolidone, glycerin, glucose, sucrose, lactose, sorbitol, mannitol, and trehalose. Amount of the dispersion-stabilizing agent is the same as described in the above.
  • the homogenizing may be performed by conventional homogenization methods, for example, using a homogenizer, an ultrasonic homogenizer, a high-pressure homogenizer, to form uniform nanoparticles having a mean particle size of 10 to 1,000 nm, preferably 20 to 200 nm. If necessary, a sterilization step may be further preformed using a sterilizing filter having about 0.22 ⁇ m of pore size.
  • the present invention also provides a process for preparing nanoparticles for tumor-targeting, which comprises drying the pharmaceutical composition in the form of a nano-dispersion obtained by the process in the above.
  • the drying may be performed by lyophilizing, rotary evaporation drying, spray drying or fluidized-bed drying.
  • a nano-emulsion was prepared according to the components and contents listed in Table 1 below.
  • Paclitaxel was added to absolute ethanol in the concentration of 10 %(w/v), and then Polysorbate 80 was added thereto.
  • the resulting mixture was subject to low-shear stirring at a temperature of 40 to 45 °C until obtaining complete dissolution.
  • a solution obtained by completely dissolving ferric chloride (III) in a mixed oil of dl- ⁇ -tocopheryl acetate and refined soybean oil at room temperature was added to the solution.
  • the resulting solution was vacuum dried at 40 to 45 °C under low-shear stirring so as to completely remove ethanol therein, resulting in an oil phase.
  • Sodium hyaluronate (weight average molecular weight: 1,067,000 daltons, Bioiberica, Spain) was dissolved in water for injection at a room temperature to obtain an aqueous phase.
  • the aqueous phase warmed to 40 to 45 °C was mixed with the oil phase to obtain a mixture having 100 g of the total weight, which was then homogenized to obtain a nano-emulsion.
  • the obtained nano-emulsion was subject to sterile-filtration.
  • the temperature was controlled to about 40 °C or more in the whole procedures for preparing the nano-emulsion.
  • the homogenization was carried out by 10 cycles with a high-pressure homogenizer (Microfluidizer M-110S, Microfluidics Inc., USA) under a pressure of about 15 kpsi, while maintaining a temperature of 15 to 25 °C.
  • the sterile-filtration was carried out with a filter having 0.22 ⁇ m of pore size (Acro 50 Vent Devices with Emflon Membrane II, Pall Co. USA).
  • the crystal nucleus of anticancer agent partially formed in preparing the nano-emulsion was removed.
  • the nano-emulsion has a mean particle size of 70 to 80 nm.
  • mannitol was added to the obtained nano-emulsion in the concentration of 5 % (w/v).
  • the resulting mixture was lyophilized to obtain nanoparticles.
  • a nano-suspension was prepared according to the components and contents listed in Table 2 below.
  • Paclitaxel was added to absolute ethanol in the concentration of 10 %(w/v), and then a mixture of TPGS and Poloxamer 407 was added thereto.
  • the resulting mixture was subject to low-shear stirring at a temperature of 55 to 60 °C until obtaining complete dissolution.
  • a solution obtained by completely dissolving ferric chloride (III) in a mixed oil of hydrogenated soybean oil and Migriol 812 at 55 to 60 °C was added to the solution.
  • the resulting solution was vacuum dried at 55 to 60 °C under low-shear stirring so as to completely remove ethanol therein, resulting in an oil phase.
  • Sodium hyaluronate (weight average molecular weight: 1,067,000 daltons, Bioiberica, Spain) was dissolved in water for injection at a room temperature to obtain an aqueous phase.
  • the aqueous phase warmed to 45 to 65 °C was mixed with the oil phase to obtain a mixture having 100 g of the total weight, which was then homogenized to obtain a nano-emulsion.
  • the obtained nano-emulsion was added to an ice-water bath of 1 to 3 °C, to obtain a nano-suspension.
  • the obtained nano-suspension was was subject to sterile-filtration, for injectable use.
  • the homogenization was carried out by 5 cycles with a high-pressure homogenizer (Microfluidizer M-110S, Microfluidics Inc., USA) under a pressure of about 15 kpsi, while maintaining a temperature of 55 to 65 °C.
  • the sterile-filtration was carried out with a filter having 0.22 ⁇ m of pore size (Acro 50 Vent Devices with Emflon Membrane II, Pall Co. USA).
  • the crystal nucleus of anticancer agent partially formed in preparing the nano-suspension was removed.
  • the nano-suspension has a mean particle size of 100 to 140 nm.
  • mannitol was added to the obtained nano-suspension in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • Nano-emulsions were prepared in the same manner as in Example 1 according to the components and contents listed in Table 3 below. TPGS was added to both of the oil phase and aqueous phase as shown in Table 3. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • Nano-emulsions were prepared in the same manner as in Example 1, further using a solubilizing agent and a dispersion-stabilizing agent according to the components and contents listed in Table 4 below. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • Nano-emulsions were prepared in the same manner as in Example 1, using other surfactant according to the components and contents listed in Table 5 below. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • Nano-emulsions were prepared in the same manner as in Example 1 according to the components and contents listed in Table 6 below. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • Nano-emulsions were prepared in the same manner as in Example 1 according to the components and contents listed in Table 7 below. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • Example 24 Oil phase paclitaxel 0.600 0.600 ferric chloride (III) 0.003 0.003 Myvacet 9-45K 12.000 12.000 TPGS 1.850 1.850 soybean lecithin 1.500 - Aqueous phase water for injection 81.950 81.950 soybean lecithin - 1.500 sodium hyaluronate 0.247 0.247
  • Nano-emulsions were prepared in the same manner as in Example 1, using sodium hyaluronates having various molecular weights as in Table 8 below.
  • the droplet sizes in the nano-emulsions were in the ranges of 50 to 250 nm.
  • mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • Nano-emulsions were prepared in the same manner as in Example 1, using a metal ion derived from ferric (or ferrous) nitrate, ferric (or ferrous) acetate, copper sulfate, nickel chloride and cobalt chloride as in Table 9 below, in an amount of 0.018 mmol, respectively.
  • the droplet sizes in the nano-emulsions were in the ranges of 60 to 140 nm.
  • mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • Nano-emulsions were prepared in the same manner as in Example 1, using sodium hyaluronate in various amounts as in Table 10 below.
  • the droplet sizes in the nano-emulsions were in the ranges of 60 to 80 nm.
  • mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • Example 40 Example 41 sodium hyaluronate (g) 0.080 0.160 0.319
  • Nano-emulsions were prepared in the same manner as in Example 1, using ferric chloride (III) in various amounts as in Table 11 below.
  • the droplet sizes in the nano-emulsions were in the ranges of 60 to 90 nm.
  • mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • a nano-emulsion was prepared in the same manner as in Example 1, using hyaluronic acid free base.
  • the droplet size in the nano-emulsion was in the ranges of 60 to 80 nm.
  • mannitol was added to the obtained nano-emulsion in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • the free base form of hyaluronic acid was obtained by the following: 0.5 %(w/v) sodium hyaluronate was dissolved in a mixed solvent of 0.1 M HCl and absolute ethanol (3:7 (v/v)). The resulting solution was slowly stirred at room temperature for 24 hours and then filtered with a 200 to 400 mesh filter to recover the precipitant on the filter as a free base form of hyaluronic acid. The resulting product was washed with absolute ethanol 3 times and then dried at room temperature to obtain a pure free base form of hyaluronic acid. The free base form of hyaluronic acid was subjected to pH determination (pH 2.65) and IR spectrometry for confirming deletion of the salt.
  • a nano-emulsion was prepared in the same manner as in Example 1, using docetaxel as an anticancer agent, according to the components and contents listed in Table 12 below.
  • the droplet size in the nano-emulsion was in the ranges of 70 to 80 nm.
  • mannitol was added to the obtained nano-emulsion in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • Example 48 Preparation of Nano-emulsion and Nanoparticles comprising PEGylated phospholipids and 5-fluorouracil
  • a nano-emulsion was prepared in the same manner as in Example 1, using 5-fluorouracil as an anticancer agent, according to the components and contents listed in Table 13 below.
  • the droplet size in the nano-emulsion was in the ranges of 90 to 130 nm.
  • mannitol was added to the obtained nano-emulsion in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • Nano-emulsions were prepared in the same manner as in Example 1, using citric acid or N-acetyl cysteine as a pH-controlling agent, according to the components and contents listed in Table 14 below.
  • the droplet sizes in the nano-emulsions were in the ranges of 70 to 80 nm.
  • mannitol was added to the obtained nano-emulsion in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
  • Example 49 Oil phase paclitaxel 0.300 0.300 ferric chloride (III) 0.002 0.002 dl- ⁇ -tocopheryl acetate 7.200 7.200 refined soybean oil 0.800 0.800 Polysorbate 80 8.000 8.000 citric acid 0.002 - N-acetyl cysteine - 0.001 Aqueous phase water for injection 83.445 83.446 sodium hyaluronate 0.251 0.251
  • Nano-emulsions were prepared in the same manner as in Example 1 according to the components and contents listed in Table 15 below.
  • Example 2 The nano-emulsion obtained in Example 1 was ultra-centrifuged at 20 °C for 2 hours. The resulting precipitate was washed with water for injection 3 times and then dried at room temperature to obtain an emulsion aggregate where hyaluronic acid is bound. The amount of hyaluronic acid bound was analyzed in accordance with the method for quantitative determination of hyaluronic acid described in the European pharmacopoeia and the detailed procedures are as follows:
  • Distilled water was added to 0.170 g of the emulsion aggregate to become 100 g of solution in total, which was then diluted to 40-fold to prepare a test solution.
  • Distilled water was added to 0.1 g of D-glucuronic acid to become 100 g of solution in total.
  • 5 standard solutions were prepared so as to contain 6.5 to 65 ug of D-glucuronic acid per g.
  • a solution of 0.95 w/v% borax in sulfuric acid (5 mL) was added to the test solution (1.0 mL) and the standard solutions (1.0 mL), respectively. The mixtures were allowed to stand in a water bath for 15 minutes; and then cooled in ice water.
  • Amounts of sodium hyaluronate (%) (Cg/Cs) x Z x [100/(100-h)] x (401.3/194.1)
  • SK-OV-3 cells ATCC: HTB-77, Rockville, MD, USA
  • OVCAR-3 cells Korean Cell Line Bank, KCLB-00000287
  • CD44 is expressed on the surface of SK-OV-3 cells, while it is not expressed on the surface of OVCAR-3 cells.
  • RPMI 1640 medium HA free medium, Gibco, Carlsbad, USA
  • FBS fetal bovine serum
  • nano-emulsions (obtained in Example 1 and Comparative examples 1 to 3) labeled with FITC were added to each of 1 x 10 6 cells, which was then incubated at 37 °C for 4 hours.
  • the labeled nano-emulsions were obtained by dissolving 0.2 mg of FITC in the nano-droplets.
  • Non-labeled fluorescent material was removed with a dialysis membrane (Pierce, MWCO 2,000).
  • the cells were washed with phosphate buffered saline (PBS) 2 times, treated with 0.1 % trypsin for about 1 minute, and then washed with PBS containing 0.2 % FBS and 0.02 % sodium azide 3 times.
  • PBS phosphate buffered saline
  • the recovered cells were re-dispersed in a solution of 4 % paraformaldehyde (400 uL).
  • the affinities to CD44 were determined using FACS (Fluorescence-activated cell sorter) and epi-fluorescent microscopy.
  • Binding of hyaluronic acid nanoparticles to the ovarian cancer cells was measured by fluorescence scanning at 488 nm using a FACScalibur scanner (Beckton-Dickinson, Mansfield, MA, USA). The results thereof are shown in FIG. 3.
  • FIG. 3A and 3C show the results of in vitro affinity test using OVCAR-3 cells.
  • FIG. 3B and 3D show the results of in vitro affinity test using SK-OV-3 cells.
  • substantial non-specific binding was not observed in all of the samples.
  • substantial specific binding to CD44 on the surface of the cells was not observed.
  • specific binding to CD44 was observed, with difference in binding affinities. Specifically, there was no specific binding to CD44 in the emulsion of Comparative Example 1 and the emulsion containing a metal ion of Comparative Example 2.
  • Example 3 A little affinity to CD44 was observed in the emulsion simply mixed with hyaluronic acid (Comparative Example 3).
  • the nano-emulsion of Example 1 containing nano-droplets coated with hyaluronic acid according to the present invention showed an excellent affinity to CD44. Therefore, it can be seen that the nano-emulsion of the present invention is an excellent drug delivery system for active targeting.
  • Anti-cancer efficacy through active and passive targeting was evaluated using SK-OV-3 cells (ATCC: HTB-77, Rockville, MD, USA) over-expressing CD44 on the surface thereof and OVCAR-3 Cell (Korean Cell Line Bank, KCLB-00000287) not expressing CD44 on the surface thereof as ovarian cancer cells.
  • the SK-OV-3 cells and the OVCAR-3 cells were respectively cultured under 5% CO 2 atmosphere at 37 °C in RPMI 1640 medium supplemented with 10 % FBS, to obtain enough cells for transplantation into test animals, i.e., 7x10 8 or more of SK-OV-3 cells of and 4x10 8 or more of OVCAR-3 cells.
  • the RPMI 1640 medium was prepared by supplementing with 0.1 mM non-essential amino acid, 1 mM sodium pyruvate, 1.5 g/L of sodium bicarbonate and a certain amount of antibiotic/mycotic agent; and then filtering the mixture, which was then stored in a refrigerator. The medium was warmed to 37 °C when used.
  • the SK-OV-3 cells and the OVCAR-3 cells were respectively treated with a trypsin/EDTA solution and then isolated from the culture flask. 1x10 7 of the respective cells were suspended in 0.3 mL of RPMI 1640. 0.3 mL of the respective cell suspensions were subcutaneously injected into the flank of nude mice (BALB/cAnNCrjBgi-nu/nu, female) using a 23G x 1 mL-syringe, for transplantation of the cancer cells.
  • G2 group Taxol ® (positive control) were administered via the caudal vein of the mice.
  • G3, G4, and G5 group the nano-emulsion of Example 1, the nano-suspension of Example 2, and the nano-emulsion of Comparative Example 1 were respectively administered via the caudal vein of the mice of each group.
  • the doses of paclitaxel (active ingredient) were equally 20 mg/kg.
  • G1 group (control group) only hypertonic saline was administered via the caudal vein thereof, for monitoring cancer cell growth.
  • changes of tumor size in the test animals were determined 2-3 times in a week, at intervals of about 3 days.
  • the tumor sizes were calculated from their length and width measured with vernier calipers, according to the following formula.
  • the changes of tumor size on time elapsed were comparatively evaluated for each test group and then the results are represented in FIGs. 4A and 4B.
  • Tumor Size (mm3) length (mm) x [width (mm)] 2 / 2
  • FIG. 4A shows changes of tumor size in the cancer-transplanted CD44-overexpressed SK-OV-3 models after test materials or controls were respectively administered.
  • the groups treated with the nano-emulsion of Example 1 and the nano-suspension of Example 2 represented more excellent inhibitory effects against cancer cell growth than the groups treated with Taxol ® and the nano-emulsion of Comparative Example 1.
  • the nano-dispersions of Example 1 and 2 have nanoparticles which hyaluronic acid has been bound on, these results show that the nano-dispersions of the present invention exhibit an active targeting effect through specific binding of hyaluronic acid to CD44.
  • the emulsion of Comparative Example 1 (i.e., without hyaluronic acid coating) showed very low inhibitory effect against cancer cell growth, which means that the emulsion of Comparative Example 1 represents only a passive targeting effect through EPR effect, not an active targeting effect through specific binding to CD44.
  • FIG. 4B shows changes of tumor size in the cancer-transplanted CD44-not-expressed OVCAR-3 models after test materials or controls were respectively administered.
  • the groups treated with the nano-emulsion of Example 1, the nano-suspension of Example 2, and the emulsion of Comparative Example 1 represented more excellent inhibitory effects against cancer cell growth than the negative control group and the group treated with Taxol ® . Since CD44 was not expressed in the cancer-transplanted OVCAR-3 model, only a passive targeting effect through EPR effect was exhibited.
  • the healthy test animals through breeding for adaptation were selected for repeated-dose toxicity study.
  • the test materials were well shaken just before the administration; and then administered via the tail vein of the mice using a sterilized disposable syringe.
  • the administrations were performed 3 times in total, at intervals of 3 consecutive days.
  • the death and clinical sign of the test animals were monitored.
  • the clinical sign was intermittently monitored according to the status till 4 hours after the administration; and then monitored 1 time per day to the end of monitoring.
  • the body weights of the test animals were measured 1 time per day from the day initiating the administration to the end of monitoring. The results thereof are represented in FIG. 5.
  • the nude mice receiving 3 repeated-administrations with 30 mg/kg of Taxol ® showed rapid body weight loss to an extent of about 20 % and serious toxicity. Among the test animals, 3 mice were died during or after the administration. Also, all the Taxol ® -administered groups showed the symptoms such as dyspnea, lethargy, and ataxia regardless of the doses of paclitaxel. However, the groups receiving 3 repeated-administrations with 20 to 50 mg/kg (as a paclitaxel concentration) of the nano-emulsion of Example 1 showed body weight changes only within 10 % or less and therefore relatively safe toxicity-values in comparison to the positive control (i.e., Taxol ® ). These results mean that the maximum tolerance dose of nano-emulsion of Example 1 is at least 50mg/kg or more in the nude mouse. And also, there was not observed any death or specific clinical sign in all the groups administered with the nano-emulsion of Example 1.
  • Dissolution tests of the nano-dispersion of the present invention were carried out according to the dissolution test method (Dissolution Test Method No. 2) described in the Korean Pharmacopoeia as follows:
  • Example 1 The nano-emulsion of Example 1, the nano-suspension of Example 2 and Taxol ® injection (Bristol Myers Squibb) were respectively put into dialysis membranes (Spectra/Por) having 12,000 to 14,000 of a molecular weight cut off (MWCO), each of which was added to 1,000 mL of a dissolution medium (0.1 % Polysorbate 80-phospate buffer, pH 7.08).
  • the dialysis membranes were stabilized by immersing them in the dissolution medium for about 30 minutes before use.
  • Each dialysis membrane was fixed to a support and then placed in a sink condition in the dissolution medium.
  • the paddle was rotated at 100 rpm, maintaining a temperature of 37 °C.
  • the nano-dispersion of the present invention is very useful for efficient delivery of a drug.
  • Encapsulation efficiency and re-dispersability were evaluated for the nano-dispersion and the nanoparticles obtained through lyophilization thereof in Example 1.
  • the same evaluations were also carried out for the nano-particles in accordance with the prior arts of the present inventors, i.e., Korean patent no. 10-744,925 (Reference example 1) and Korean patent publication no. 10-2009-0040979 (prepared in Example 93 thereof, Reference example 2).
  • the nano-dispersion and the nanoparticles of Reference example 1 were obtained by the followings: 0.135 g of paclitaxel and 0.045 g of hyaluronic acid was dissolved in a solution of 0.02 M glutamic acid in 70 % ethanol (45 mL).
  • a solution of 0.05 M ferric nitrate (III) (0.84 mL) was added under stirring at 300 rpm to the solution, which was then stirred for 2 hours to obtain the nano-dispersion.
  • the resulting nano-dispersion was lyophilized to obtain the nanoparticles having about 120 nm of a mean particle size.
  • Example 1 and Reference examples 1 and 2 Each of the non-entrapped, precipitated paclitaxel of Example 1 and Reference examples 1 and 2 was isolated by filtration through a 0.22 ⁇ m filter (Acro 50 Vent Devices with Emflon Membrane ® II, Pall Co. USA). Each amount of paclitaxel in the collected filtrates was measured according to the method for quantitative determination of paclitaxel described in the European Pharmacopoeia, to evaluate the encapsulation efficiency thereof. And also, the mean particle sizes of the nanoparticles in the nano-dispersions of Example 1 and Reference examples 1 and 2 were also measured according to the same method as in Example 1.
  • Each of the lyophilized nanoparticles was reconstituted in water for injection. From the reconstituted products (i.e., nano-dispersions), the encapsulation efficiency thereof and mean particle sizes were also evaluated in the same manners in the above.
  • the nano-dispersion and the nanopartices according to the present invention show the highest encapsulation content and efficiency of paclitaxel. And also, the encapsulation content and the mean particle size thereof were maintained after being reconstituted in dispersion medium (water for injection).
  • Example 1 The nanoparticles obtained through lyophilization in Example 1 were subjected to evaluation of physicochemical stability tests for 6 months at room temperature (25 ⁇ 2°C, 60 ⁇ 5 %RH) and at accelerated condition (40 ⁇ 2°C, 75 ⁇ 5 %RH). After reconstitution of lyophilized nanoparticles, the encapsulation contents of paclitaxel and the nanoparticle sizes were measured by the same method as in Test Example 6. Each measuring was repeated three times and then the average value thereof was calculated. The results are represented in Table 18.

Abstract

The present invention provides a pharmaceutical composition for tumor-targeting in the form of a nano-dispersion comprising nanoparticles having a mean particle size of 10 to 1,000 nm in an aqueous medium, wherein the nanoparticles comprise a therapeutically effective amount of an anticancer agent; a divalent or trivalent transition metal ion or alkali earth metal ion; and an oil, and wherein hyaluronic acid or its salt is bound on a surface of the nanoparticles. And also, the present invention provides nanoparticles for tumor-targeting obtained by drying the pharmaceutical composition in the form of a nano-dispersion. And also, the present invention provides a process for preparing the pharmaceutical composition in the form of a nano-dispersion and a process for preparing the nanoparticles.

Description

NANOPARTICLES FOR TUMOR-TARGETING AND PROCESSES FOR THE PREPARATION THEREOF
The present invention relates to a pharmaceutical composition containing an anticancer agent, which is capable of both passive and active targeting. More specifically, the present invention relates to nanoparticles capable of both passive and active targeting, which comprises a therapeutically effective amount of an anticancer agent; a divalent or trivalent transition metal ion or alkali earth metal ion; an oil; and hyaluronic acid or its salt.
Anticancer agents (or anti-tumor agents) for cancer therapy affect normal cells as well as cancer cells in the body, thereby resulting in various side effects. In order to avoid such problem, there have been reported various targeting methods including passive targeting or active targeting. However, targeting methods of satisfactory level still remain to be elucidated.
Passive targeting methods are based on the fact that fine-particles may be selectively accumulated in the cancer cells having abnormal blood vessels, during the continuous blood circulation in the body. In general, since cancer cells become enlarged due to their abnormal growth rate, they have larger endothelial pores than normal cells; and/or various sizes of endothelial pores (e.g., 10 nm to 1000 nm) while normal cells have aligned endothelial pores. Therefore, when micronized particulate carriers having an anticancer agent are allowed to continuously circulate in the blood, the micronized particles (e.g., nanoparticles) may be accumulated only in cancer cells, while those cannot penetrate into the endothelial pores of normal cells. This mechanism of action is referred to enhanced permeation and retention (EPR) effect. Examples of the passive targeting method include a method for delivering an anticancer agent using small particle liposome aerosols (see WO1999/15153).
Active targeting methods refer to a method for delivering an anticancer agent to cancer cells through a drug delivery system that is prepared by using a receptor specifically recognizing the molecule over-expressed at cancer cell surface, the molecule of which includes e.g., lectin, growth factor, cytokine, hormone, unsaturated fatty acid, low-density lipoprotein, folic acid (S.P Vyas., et. al., Advanced Drug Delivery Review 43 (2000) 101-164). There have been reported drug delivery systems wherein an anticancer agent is bound with biodegradable polymer or encapsulated in liposome and wherein a receptor specifically recognizing the molecule over-expressed at cancer cell surface is further attached to the polymer or liposome. For example, US patent no. 6,593,308 has disclosed a drug delivery system comprising an encapsulating delivery vehicle (e.g., liposome) and hyaluronan ligands. In the drug delivery system, an anticancer agent is encapsulated in the vehicle; and metal ion is used as a linking agent or a chelating agent in order to attach the ligands to the vehicle. In addition, there has been reported a targeting method using a monoclonal antibody having high affinity to cancer cells (Stanislv J., et al., Bioorg. Medic. Chem. (2005) 13, 5043-5054).
Various active targeting methods using hyaluronic acid or its derivatives are disclosed in the arts, such as US patent no. 6,593,308 mentioned in the above. In the drug delivery system disclosed in US patent no. 6,593,308, each hyaluronan ligand is chemically attached to the delivery vehicle (e.g., liposome). However, liposome has very low long-term stability due to instability of the lipid lamella; and may be decomposed through phagocytosis by macrophages. And also, the chemical attachment of hyaluronan ligands to liposome is very complicated and gives unwanted side products during the preparation.
In addition, US patent no. 6,699,471 has disclosed an injectable composition in the form of a gel comprising a benzyl ester of hyaluronic acid or an auto-cross-linked derivative of hyaluronic acid. US patent publication no. 2006/0188578 has disclosed a method of obtaining nanoparticles using chitosan and tripolyphosphate, in which hyaluronic acid is attached to the surface thereof via ionic bond. And also, US patent publication no. 2007/0036728 has disclosed a method of transdermal drug delivery using an oil-in-water emulsion, which has hyaluronic acid nanoparticles obtained from hyaluronic acid and a hydrazide. US patent publication no. 2007/0031503 has disclosed a hyaluronic acid modification product obtained by binding hyaluronic acid with polylactic acid, polyglycolic acid, or lactic acid-glycolic acid copolymer. And also, WO00/041730 has disclosed a method for enhancing the effectiveness of a cytotoxic or anti-neoplastic agent, comprising co-administering said agent with hyaluronan.
Most of targeting methods disclosed in the arts were designed for accomplishing only passive targeting or only active targeting. Therefore, there are needs to develop a targeting method capable of both passive targeting and active targeting, thereby being able to treat metastatic tumors as well as solid tumors. And also, there are needs to develop a method capable of avoiding various problems encountered in preparing the delivery systems (for example, complicated procedures for preparation), which are originated from designing a vehicle (e.g., liposome) for delivery of an anticancer agent.
The present inventors have developed nanoparticles using hyaluronic acid or its salt, which is capable of both passive and active targeting (Korean patent no. 10-744,925 and Korean patent publication no. 10-2009-0040979). The nanoparticles are formed from a mixture of an anticancer agent (anti-tumor agent) and hyaluronic acid or its salt, using a metal ion and/or a water-insoluble biodegradable polymer. The nanoparticles can accomplish both passive targeting through EPR effect and active targeting through hyaluronic acid.
The present invention provides an improved drug delivery system in the form of a nano-dispersion using hyaluronic acid or its salt, which is capable of both passive and active targeting. The drug delivery system comprises nanoparticles in an aqueous medium, wherein the nanoparticles include an anticancer agent (anti-tumor agent) along with a metal ion and an oil, and wherein hyaluronic acid or its salt is bound on a surface of the nanoparticles through interaction with the metal ion. The drug delivery system can accomplish both passive targeting through EPR effect and active targeting through hyaluronic acid. And also, the drug delivery system can remarkably increase the encapsulation efficiency of an anticancer agent.
Therefore, it is an object of the present invention to provide a pharmaceutical composition for both passive and active tumor-targeting in the form of a nano-dispersion, which comprises nanoparticles having high encapsulation efficiency of an anticancer agent. And also, it is another object of the present invention to provide a process for preparing the pharmaceutical composition in the form of a nano-dispersion comprising the nanoparticles.
In accordance with an aspect of the present invention, there is provided a pharmaceutical composition for tumor-targeting in the form of a nano-dispersion comprising nanoparticles having a mean particle size of 10 to 1,000 nm in an aqueous medium, wherein the nanoparticles comprise a therapeutically effective amount of an anticancer agent; a divalent or trivalent transition metal ion or alkali earth metal ion; and an oil, and wherein hyaluronic acid or its salt is bound on a surface of the nanoparticles.
In accordance with another aspect of the present invention, there is provided nanoparticles for tumor-targeting obtained by drying the pharmaceutical composition in the form of a nano-dispersion, through lyophilizing, rotary evaporation drying, spray drying or fluidized-bed drying.
In accordance with still another aspect of the present invention, there is provided a process for preparing a pharmaceutical composition for tumor-targeting in the form of a nano-dispersion, which comprises: (a) providing an oil phase comprising a therapeutically effective amount of an anticancer agent, a divalent or trivalent transition metal ion or alkali earth metal ion and an oil, (b) providing an aqueous phase by dissolving hyaluronic acid or its salt in an aqueous medium, and (c) mixing the oil phase obtained in Step (a) and the aqueous phase obtained in Step (b) and then homogenizing the resulting mixture to form nanoparticles having a mean particle size of 10 to 1,000 nm, the surface of which hyaluronic acid or its salt is bound on.
In accordance with still another aspect of the present invention, there is provided a process for preparing nanoparticles for tumor-targeting, which comprises drying the pharmaceutical composition in the form of a nano-dispersion through lyophilizing, rotary evaporation drying, spray drying or fluidized-bed drying.
The pharmaceutical composition in the form of a nano-dispersion comprising nanoparticles in an aqueous medium according to the present invention is a drug delivery system in which the nanoparticles are obtained by binding hyaluronic acid or its salt on the surface of nanoparticles comprising an anticancer agent, a metal ion, and an oil. The drug delivery system of the present invention makes it possible to accomplish both passive tumor-targeting through EPR effect and active tumor-targeting through affinity of hyaluronic acid to cancer cells. That is, the nanoparticles of the present invention selectively interact with cancer cells, thereby being able to selectively interact with metastatic tumors as well as solid tumors. Especially, the pharmaceutical composition of the present invention can increase the encapsulation efficiency of an anticancer agent, to wanted level of amount (i.e., almost to 100% of wanted amount). And also, the pharmaceutical composition in the form of a nano-dispersion of the present invention has excellent biocompatibility and physicochemical stability.
FIG. 1 shows a schematic example of the nanoparticles according to the present invention.
FIG. 2 shows a Cyro-TEM image of the nanoparticles according to the present invention.
FIG. 3 shows the results of CD44-binding affinity test for the nanoparticles according to the present invention. FIG. 3A and 3C show the results of in vitro affinity test using OVCAR-3 cells. FIG. 3B and 3D show the results of in vitro affinity test using SK-OV-3 cells.
FIG. 4A and 4B show the results of evaluation of anticancer efficacy by active and passive tumor-targeting, for the nanoparticles according to the present invention. FIG. 4A shows changes of tumor size in the cancer-transplanted CD44-overexpressed SK-OV-3 models after test materials or controls were respectively administered. FIG. 4B shows changes of tumor size in the cancer-transplanted CD44-not-expressed OVCAR-3 models after test materials or controls were respectively administered.
FIG. 5 shows the results of safety evaluation for the nanoparticles according to the present invention. In the evaluation, the body weights were monitored for 2 weeks after the test materials were administered.
FIG. 6 shows the results of dissolution test for the nanoparticles according to the present invention.
As used herein, the term "nanoparticles" refers to particles having a mean particle size of about 1000 nm or less, for example, 10 to 1,000 nm, preferably 50 to 500 nm, more preferably 50 to 300 nm.
The term "nano-dispersion" refers to a drug delivery system obtained by dispersing the nanoparticles in an aqueous medium. The nano-dispersion may be in the form of a nano-emulsion or in the form of a nano-suspension. The term "nano-emulsion" refers to a drug delivery system of an emulsion form, wherein the nanoparticles are dispersed in oil forms (e.g., in droplets) at room temperature (about 25 ℃), in an aqueous medium. The term "nano-suspension" refers to a drug delivery system of a suspension form, wherein the nanoparticles are dispersed in solid particle forms at room temperature (about 25 ℃), in an aqueous medium. The nanoparticles in the nano-dispersion has a mean particle size suitable for providing EPR effect, for example a mean particle size of 10 to 1,000 nm, preferably 50 to 500 nm, more preferably 50 to 300 nm.
The present invention provides a pharmaceutical composition for tumor-targeting in the form of a nano-dispersion comprising nanoparticles having a mean particle size of 10 to 1,000 nm in an aqueous medium, wherein the nanoparticles comprise a therapeutically effective amount of an anticancer agent; a divalent or trivalent transition metal ion or alkali earth metal ion; and an oil, and wherein hyaluronic acid or its salt is bound on a surface of the nanoparticles.
The pharmaceutical composition of the present invention comprises nanoparticles in an aqueous medium, wherein the nanoparticles include an anticancer agent (anti-tumor agent) along with a metal ion and an oil, and wherein hyaluronic acid or its salt is bound on a surface of the nanoparticles through interaction between hyaluronic acid and the metal ion (e.g., chelate bond). The hyaluronic acid or its salt is present on the exterior of nanoparticles, while the metal ion is present in interior of nanoparticles (see FIGs 1 and 2).
Hyaluronic acid, one of the hydrophilic polysaccharides in the body, is present in the form of a high molecular weight hetero-polysaccharide in which disaccharide unit of D-glucuronic acid and N-glucosamine (molecular weight: 379) are linked each other to form a long chain. Hyaluronic acid in extracellular matrix provides various functions in proliferation, differentiation and migration of cells. It has been described that the various functions are associated with hyaluronic acid-binding receptors such as CD44 (one of the cell surface glycoproteins); receptors for hyaluronic acid-mediated motility (RHAMM); and other receptors having hyaluronic acid-binding motifs (S. Jaracz et al., Recent advances in tumor-targeting anticancer drug conjugates, Bioorg. Med. Chem. 13 (2005) 5043-5054). Especially, it has been reported that various tumors, including epithelial tumor, ovarian tumor, colon tumor, stomach tumor, and acute leukemia, over-express CD44 and RHAMM which are hyaluronic acid-binding receptors (Day, A. J.; Prestwich, G. D. J. Biol. Chem. 2002, 277, 4585; and Turley, E. A.; Belch, A. J.; Poppema, S.; Pilarski, L. M. Blood 1993, 81, 446), and that those tumor cells show increase in hyaluronic acid-binding and internalization of hyaluronic acid (Hua, Q.; Knudson, C. B.; Knudson, W. J. Cell Sci. 1993, 106, 365). And also, it has been reported that hyaluronic acid also participate in metastasis of tumor cells (Sleeman, J. et al., Cancer. Res.(1996) 56, 3134). Therefore, the pharmaceutical composition according to the present invention specifically interacts with tumor cells, thereby being able to function as a drug delivery system for active tumor-targeting.
Molecular weight of the hyaluronic acid may range from 379 to 10,000,000 daltons, preferably from 1,000 to 4,000,000 daltons, more preferably from 1,000 to 1,500,000 daltons, but not limited thereto. Hyaluronic acid may be in a salt form, for example an inorganic salt form such as cobalt hyaluronate, magnesium hyaluronate, zinc hyaluronate, calcium hyaluronate, potassium hyaluronate, or sodium hyaluronate; or an organic salt form such as tetrabutylammonium hyaluronate. Preferably, hyaluronic acid free base and/or sodium hyaluronate may be used. Each of hyaluronic acid or its salt may be bound on the surface of nanoparticles or a mixture of hyaluronic acid and its salt may be bound on the surface of nanoparticles. An amount of the hyaluronic acid or its salt may range from 0.01 to 1.0 w/w%, preferably from 0.1 to 0.5 w/w%, based on the total weight of the composition.
The nanoparticles comprise a divalent or trivalent transition metal ion or alkali earth metal ion. The divalent or trivalent transition metal ion or alkali earth metal ion may be at least one selected from the group consisting of Cu2+, Cu3+, Zn2+, Zn3+, Ni2+, Ni3+, Mg2+, Mg3+, Ca2+, Ca3+, Co2+, Co3+, Ba2+, Ba3+, Al2+, Al3+, Fe2+, and Fe3+. The metal ion may be derived from various metal salts. For example, the metal ion may be derived from, but limited to, copper nitrate, copper sulfate, copper chloride, zinc acetate, zinc sulfate, zinc nitrate, zinc chloride, nickel sulfate, nickel nitrate, nickel chloride, magnesium acetate, magnesium sulfate, magnesium nitrate, magnesium chloride, calcium acetate, calcium sulfate, calcium nitrate, calcium chloride, cobalt acetate, cobalt sulfate, cobalt chloride, barium acetate, barium sulfate, barium nitrate, barium chloride, aluminum sulfate, aluminum chloride, ferric (or ferrous) acetate, ferric (or ferrous) sulfate, ferric (or ferrous) nitrate, or ferric (or ferrous) chloride. Among them, Fe2+ and/or Fe3+ ion may be preferably used and the Fe2+ and/or Fe3+ ion may be derived from ferric (or ferrous) acetate, ferric (or ferrous) sulfate, ferric (or ferrous) nitrate, or ferric (or ferrous) chloride. The metal ion is reacted mostly with the carboxylic groups of glucuronic acid within hyaluronic acid to form chelate bonds. In addition, the metal ion may be also reacted with the hydroxyl groups between hyaluronic acid chains; or reacted with the amine groups of N-acetylglucosamine, so as to form chelate bonds. Molar ratio of the divalent or trivalent transition metal ion or alkali earth metal ion per disaccharide unit of the hyaluronic acid or its salt may range from 0.001 to 2.0, preferably from 0.001 to 0.5.
The nanoparticles comprise a biocompatible oil as a lipophilic medium for dissolving an anticancer agent. The oil may be a liquid form or a solid form at room temperature.
When the pharmaceutical composition is in the form of a nano-emulsion, the oil may be a liquid form at room temperature. The oil having a liquid form at room temperature may be at least one selected from the group consisting of mono-, di-, or tri-glycerides (for example, Myvacet 9-45K); glyceryl mono- or tri-stearates; glyceryl mono-, di-, or tri-acetates (for example, triacetin); alpha-tocopherol (including d-alpha-tocopherol and/or dl-alpha-tocopherol) or its salt; alpha-tocopheryl acetate (including d-alpha-tocopheryl acetate and/or dl-alpha-tocopheryl acetate); alpha-tocopherol succinate (including d-alpha-tocopherol succinate or dl-alpha-tocopherol succinate); C4:0 to C6:0 triglycerides (for example, tributyrin); C8:0 to C14:0 triglycerides (for example, tricaproin or caprylic/capric triglyceride); castor oil, corn oil, olive oil, cottonseed oil, peppermint oil, sesame oil, and soybean oil (including refined soybean oil).
When the pharmaceutical composition is in the form of a nano-suspension, the oil may be a solid form at room temperature. The oil having a solid form at room temperature may be at least one selected from the group consisting of hydrogenated soybean oil, cacao butter, cetyl alcohol, stearyl alcohol, cetyl palmitate, carnauba wax, white beeswax, tricaprin (for example, Migriol 812), trilaurin, trimyristin, tripalmitin, tristearin, and tribehenin.
Preferably, the oil may be at least one selected from the group consisting of glyceryl mono-, di-, or tri-acetates; alpha-tocopherol (including d-alpha-tocopherol and/or dl-alpha-tocopherol) or its salt; alpha-tocopheryl acetate (including d-alpha-tocopheryl acetate and/or dl-alpha-tocopheryl acetate); alpha-tocopherol succinate (including d-alpha-tocopherol succinate or dl-alpha-tocopherol succinate); tributyrin; and tricaproin.
An amount of the oil may range from 1 to 50 w/w%, preferably from 1 to 30 w/w%, based on the total weight of the composition
The nanoparticles also comprise a chemical or biological material having an inhibitory activity against proliferation or metastasis of cancer cells, i.e., anticancer agent (anti-tumor agent), the types of which are not limited. Examples of the anticancer agent include, but not limited to, paclitaxel or its derivative, uracil, 5-fluorouracil, tegafur, methotrexate, melphalan, mitoxantrone, camptothecin, topotecan, docetaxel, capecitabine, imatinib mesylate, rituximab, doxifluridine, toremifene citrate, doxorubicin, gemcitabine, irinotecan, oxaliplatin, or chlorambucil. Preferably, the anticancer agent used may be paclitaxel or its derivative, docetaxel, uracil, 5-fluorouracil, tegafur, methotrexate, melphalan, mitoxantrone, camptothecin, or topotecan. Therapeutically effective amount of each anticancer agent may be easily determined from the arts by a skilled person.
The pharmaceutical composition of a nano-dispersion form according to the present invention may further comprise a surfactant for obtaining stable dispersion of the nanoparticles (i.e. for increasing stability of the nano-dispersion). The surfactant may be incorporated into the nanoparticles and/or added to the aqueous medium. The surfactant may be at least one selected from the group consisting of alpha-tocopherol polyethylene glycol succinate (TPGS), macrogol 15 hydroxystearate (for example, Solutol HS15), caprylocaproyl macrogolglycerides (for example, Labrasol), sorbitan fatty acid esters (for example, sorbitan monooleate (e.g., Span 20)), polysorbates (for example, Tween 20, Tween 80), polyoxyethylene-polyoxypropylene block copolymer (for example, Poloxamer 188, Poloxamer 407), egg lecithin, and soybean lecithin; but not limited thereto. An amount of the surfactant may be selected according to the type of oil used. For example, the amount of the surfactant may ranges from 1 to 50 w/w%, based on the total weight of the composition, but not limited thereto.
In the pharmaceutical composition of the present invention, the aqueous medium may be at least one selected from the group consisting of distilled water, water for injection, saline, solution for sodium chloride injection, solution for dextrose injection, and solution for amino acid injection; but not limited thereto.
If necessary, in the pharmaceutical composition of the present invention, the nanoparticles may further comprise a solubilizing agent therein, in order to avoid any precipitation of anticancer agent originated from the nanoparticles. The solubilizing agent may be at least one selected from the group consisting of polyethylene glycol, dimiristoyl phosphatidyl ethanolamine-polyethylene glycols (DMPE-PEGs) (for example, DMPE-PEG2000), cholesterol, propylene glycol, glycofurol, and tricaprylin. An amount of the solubilizing agent may range from 0.05 to 10 w/w%, preferably from 0.05 to 5 w/w%, based on the total weight of the composition.
And also, the nanoparticles may further comprise an amino acid having carboxyl group as a low-molecular ligand, in order to stably bind hyaluronic acid or its salt on the surface of nanoparticles. The low-molecular ligand may be at least one selected from the group consisting of glutamic acid, aspartic acid, asparagine, histidine, and alanine. Preferably, the low-molecular ligand may be glutamic acid and/or aspartic acid. An amount of the low-molecular ligand may range from 0.005 to 0.2 w/w%, based on the total weight of the composition.
The pharmaceutical composition of a nano-dispersion form may comprise a pH-controlling agent such as an organic acid or an N-acety amino acid, in order to increase chemical stability. Typically, pH of the pharmaceutical composition is controlled in the range of 2.0 to 6.0, using the pH-controlling agent. The pH-controlling agent may be added to the nanoparticles and/or the aqueous medium. The pH-controlling agent may be at least one selected from the group consisting of an organic acid such as citric acid, acetic acid, phosphoric acid, lactic acid, benzoic acid, maleic acid, succinic acid, tartaric acid; and an N-acety amino acid such as N-acetyl cysteine, N-acetyl valine, N-acetylproline, and N-acetylalanine.
If necessary, the pharmaceutical composition of a nano-dispersion form may further comprise at least one dispersion-stabilizing agent selected from the group consisting of polyvinylpyrrolidone, glycerin, glucose, sucrose, lactose, sorbitol, mannitol, and trehalose. The dispersion-stabilizing agent helps not only maintaining dispersability of the composition of a nano-dispersion form, but also re-dispersing in a dispersing medium the nanoparticles obtained by drying the nano-dispersion. The dispersion-stabilizing agent may be used in an amount of 1 to 20 w/w%, preferably 3 to 7 w/w%, based on the total weight of the composition, but not limited thereto.
The present invention also provides nanoparticles for tumor-targeting obtained by drying the pharmaceutical composition in the form of a nano-dispersion, through lyophilizing, rotary evaporation drying, spray drying or fluidized-bed drying.
The present invention also provides a process for preparing a pharmaceutical composition for tumor-targeting in the form of a nano-dispersion, which comprises: (a) providing an oil phase comprising a therapeutically effective amount of an anticancer agent, a divalent or trivalent transition metal ion or alkali earth metal ion and an oil, (b) providing an aqueous phase by dissolving hyaluronic acid or its salt in an aqueous medium, and (c) mixing the oil phase obtained in Step (a) and the aqueous phase obtained in Step (b) and then homogenizing the resulting mixture to form nanoparticles having a mean particle size of 10 to 1,000 nm, the surface of which hyaluronic acid or its salt is bound on.
A process for preparing pharmaceutical composition according to the present invention may further comprise adding a surfactant to the oil phase and/or the aqueous phase.
In the process of the present invention, the anticancer agent; the divalent or trivalent transition metal ion or alkali earth metal ion; the oil, the surfactant; and hyaluronic acid or its salt are the same as described in the above.
The oil phase may be obtained by (i) dissolving an anticancer agent and optionally a surfactant in an organic solvent, (ii) dissolving a divalent or trivalent transition metal ion or alkali earth metal ion in an oil, and (iii) mixing the solution obtained in Step (i) and the solution obtained in Step (ii) and then drying the resulting mixture to remove the organic solvent. The organic solvent may be methanol, ethanol, or acetonitile, preferably absolute ethanol.
And also, the oil phase may further comprise at least one solubilizing agent selected from the group consisting of polyethylene glycol, dimiristoyl phosphatidyl ethanolamine-polyethylene glycols (DMPE-PEGs), cholesterol, propylene glycol, glycofurol, and tricaprylin. An amount of the solubilizing agent is the same as described in the above.
The aqueous phase may be obtained by further dissolving an amino acid having carboxyl group as a low-molecular ligand. Types and amounts thereof are the same as described in the above. And also, if necessary, the aqueous phase may be obtained by further dissolving at least one dispersion-stabilizing agent selected from the group consisting of polyvinylpyrrolidone, glycerin, glucose, sucrose, lactose, sorbitol, mannitol, and trehalose. Amount of the dispersion-stabilizing agent is the same as described in the above.
The homogenizing may be performed by conventional homogenization methods, for example, using a homogenizer, an ultrasonic homogenizer, a high-pressure homogenizer, to form uniform nanoparticles having a mean particle size of 10 to 1,000 nm, preferably 20 to 200 nm. If necessary, a sterilization step may be further preformed using a sterilizing filter having about 0.22 ㎛ of pore size.
The present invention also provides a process for preparing nanoparticles for tumor-targeting, which comprises drying the pharmaceutical composition in the form of a nano-dispersion obtained by the process in the above. The drying may be performed by lyophilizing, rotary evaporation drying, spray drying or fluidized-bed drying.
Hereinafter, the present invention will be described in further detail with reference to the following examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1. Preparation of Nano-emulsion and Nanoparticles
A nano-emulsion was prepared according to the components and contents listed in Table 1 below. Paclitaxel was added to absolute ethanol in the concentration of 10 %(w/v), and then Polysorbate 80 was added thereto. The resulting mixture was subject to low-shear stirring at a temperature of 40 to 45 ℃ until obtaining complete dissolution. A solution obtained by completely dissolving ferric chloride (III) in a mixed oil of dl-α-tocopheryl acetate and refined soybean oil at room temperature was added to the solution. The resulting solution was vacuum dried at 40 to 45 ℃ under low-shear stirring so as to completely remove ethanol therein, resulting in an oil phase. Sodium hyaluronate (weight average molecular weight: 1,067,000 daltons, Bioiberica, Spain) was dissolved in water for injection at a room temperature to obtain an aqueous phase. The aqueous phase warmed to 40 to 45 ℃ was mixed with the oil phase to obtain a mixture having 100 g of the total weight, which was then homogenized to obtain a nano-emulsion. The obtained nano-emulsion was subject to sterile-filtration. In order to prevent gelation of the oil phase, the temperature was controlled to about 40 ℃ or more in the whole procedures for preparing the nano-emulsion. The homogenization was carried out by 10 cycles with a high-pressure homogenizer (Microfluidizer M-110S, Microfluidics Inc., USA) under a pressure of about 15 kpsi, while maintaining a temperature of 15 to 25 ℃. The sterile-filtration was carried out with a filter having 0.22 ㎛ of pore size (Acro 50 Vent Devices with Emflon Membrane II, Pall Co. USA). At the sterile-filtration, the crystal nucleus of anticancer agent partially formed in preparing the nano-emulsion was removed. As a result of measuring the particle size (i.e., droplet size) of the obtained nano-emulsion with Zetasizer NanoZS Analyzer (Malvern Co. USA), the nano-emulsion has a mean particle size of 70 to 80 nm.
Table 1
Components Contents (g)
Oil phase paclitaxel 0.300
ferric chloride (III) 0.002
dl-α-tocopheryl acetate 7.200
refined soybean oil 0.800
Polysorbate 80 8.000
Aqueous phase water for injection 83.447
sodium hyaluronate 0.251
And also, mannitol was added to the obtained nano-emulsion in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Example 2. Preparation of Nano-suspension and Nanoparticles
A nano-suspension was prepared according to the components and contents listed in Table 2 below. Paclitaxel was added to absolute ethanol in the concentration of 10 %(w/v), and then a mixture of TPGS and Poloxamer 407 was added thereto. The resulting mixture was subject to low-shear stirring at a temperature of 55 to 60 ℃ until obtaining complete dissolution. A solution obtained by completely dissolving ferric chloride (III) in a mixed oil of hydrogenated soybean oil and Migriol 812 at 55 to 60 ℃ was added to the solution. The resulting solution was vacuum dried at 55 to 60 ℃ under low-shear stirring so as to completely remove ethanol therein, resulting in an oil phase. Sodium hyaluronate (weight average molecular weight: 1,067,000 daltons, Bioiberica, Spain) was dissolved in water for injection at a room temperature to obtain an aqueous phase. The aqueous phase warmed to 45 to 65 ℃ was mixed with the oil phase to obtain a mixture having 100 g of the total weight, which was then homogenized to obtain a nano-emulsion. The obtained nano-emulsion was added to an ice-water bath of 1 to 3 ℃, to obtain a nano-suspension. The obtained nano-suspension was was subject to sterile-filtration, for injectable use. The homogenization was carried out by 5 cycles with a high-pressure homogenizer (Microfluidizer M-110S, Microfluidics Inc., USA) under a pressure of about 15 kpsi, while maintaining a temperature of 55 to 65 ℃. The sterile-filtration was carried out with a filter having 0.22 ㎛ of pore size (Acro 50 Vent Devices with Emflon Membrane II, Pall Co. USA). At the sterile-filtration, the crystal nucleus of anticancer agent partially formed in preparing the nano-suspension was removed. As a result of measuring the particle size of the obtained nano-suspension with Zetasizer NanoZS Analyzer (Malvern Co. USA), the nano-suspension has a mean particle size of 100 to 140 nm.
Table 2
Components Contents (g)
Oil phase paclitaxel 0.300
ferric chloride (III) 0.008
hydrogenated soybean oil (Akosol) 19.000
Migriol 812 0.950
TPGS 4.750
Poloxamer 407 4.750
Aqueous phase water for injection 70.031
sodium hyaluronate 0.211
And also, mannitol was added to the obtained nano-suspension in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Examples 3 to 7. Preparation of Nano-emulsion and Nanoparticles
Nano-emulsions were prepared in the same manner as in Example 1 according to the components and contents listed in Table 3 below. TPGS was added to both of the oil phase and aqueous phase as shown in Table 3. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Table 3
Components Contents (g)
Ex. 3 Ex. 4 Ex. 5 Ex. 6 Ex. 7
Oil phase paclitaxel 0.600 0.600 0.600 0.600 0.600
ferric chloride (III) 0.003 0.003 0.003 0.004 0.005
Myvacet 9-45K 12.000 12.000 12.000 16.000 20.000
TPGS 3.000 1.500 2.000 4.000 4.750
Aqueous phase water for injection 81.153 81.153 83.147 75.170 69.685
TPGS 3.000 4.500 2.000 4.000 4.750
sodium hyaluronate 0.244 0.244 0.250 0.226 0.210
Examples 8 to 13. Preparation of Nano-emulsion and Nanoparticles
Nano-emulsions were prepared in the same manner as in Example 1, further using a solubilizing agent and a dispersion-stabilizing agent according to the components and contents listed in Table 4 below. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Table 4
Components Contents (g)
Ex. 8 Ex. 9 Ex. 10 Ex. 11 Ex. 12 Ex. 13
Oil phase paclitaxel 0.600 0.600 0.600 0.600 0.600 0.600
ferric chloride (III) 0.003 0.003 0.003 0.003 0.003 0.003
Myvacet 9-45K 6.900 9.600 - - 11.880 11.400
dl-α-tocopherol - - 6.900 9.600 - -
glycofurol - - 5.100 2.400 - -
tricaprylin - - - - 0.120 0.600
PEG 400 5.100 2.400 - - - -
TPGS 3.000 3.000 - - 3.000 3.000
Polysorbate 80 - - 8.000 8.000 - -
Aqueous phase water for injection 79.530 79.530 75.201 75.201 79.444 79.444
polyvinylpyrrolidone 1.628 1.628 - - 1.628 1.628
glycerin - - 3.970 3.970 - -
TPGS 3.000 3.000 - - 3.000 3.000
sodium hyaluronate 0.239 0.239 0.226 0.226 0.244 0.244
Examples 14 to 18. Preparation of Nano-emulsion and Nanoparticles
Nano-emulsions were prepared in the same manner as in Example 1, using other surfactant according to the components and contents listed in Table 5 below. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Table 5
Components Contents (g)
Ex. 14 Ex. 15 Ex. 16 Ex. 17 Ex. 18
Oil phase paclitaxel 0.600 0.600 0.600 0.600 0.600
ferric chloride (III) 0.002 0.003 0.003 0.003 0.002
Myvacet 9-45K 4.000 9.500 12.000 12.000 8.000
dl-α-tocopherol 5.000 2.500 - - -
egg lecithin 4.000 4.800 - - -
TPGS - - 2.250 2.250 3.500
Solutol HS15 - - 1.500 - -
Aqueous phase water for injection 86.053 82.266 81.153 81.153 86.139
Aspartic acid 0.086 0.083 - - -
TPGS - - 2.250 2.250 -
Solutol HS-15 - - - 1.500 1.500
sodium hyaluronate 0.259 0.248 0.244 0.244 0.259
Examples 19 to 23. Preparation of Nano-emulsion and Nanoparticles
Nano-emulsions were prepared in the same manner as in Example 1 according to the components and contents listed in Table 6 below. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Table 6
Components Contents (g)
Ex. 19 Ex. 20 Ex. 21 Ex. 22 Ex. 23
Oil phase paclitaxel 0.300 0.300 0.600 0.600 0.600
ferric chloride (III) 0.002 0.002 0.003 0.003 0.003
Myvacet 9-45K - - 12.000 12.000 8.000
dl-α-tocopheryl acetate 7.200 7.200 - - -
Refined soybean oil 0.800 0.800 - - -
Polysorbate 80 8.000 8.000 - - -
TPGS - 2.250 2.250 3.500
Poloxamer 407 - - 1.500 - -
Aqueous phase water for injection - 81.951 82.649 81.153 81.153
Poloxamer 188 1.500 0.800 - - -
Poloxamer 407 - - - 1.500 1.500
TPGS - - 2.250 2.250 -
sodium hyaluronate 0.247 0.249 0.244 0.244 0.259
Examples 24 and 25. Preparation of Nano-emulsion and Nanoparticles
Nano-emulsions were prepared in the same manner as in Example 1 according to the components and contents listed in Table 7 below. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Table 7
Components Contents (g)
Example 24 Example 25
Oil phase paclitaxel 0.600 0.600
ferric chloride (III) 0.003 0.003
Myvacet 9-45K 12.000 12.000
TPGS 1.850 1.850
soybean lecithin 1.500 -
Aqueous phase water for injection 81.950 81.950
soybean lecithin - 1.500
sodium hyaluronate 0.247 0.247
Examples 26 to 31. Preparation of Nano-emulsion and Nanoparticles
Nano-emulsions were prepared in the same manner as in Example 1, using sodium hyaluronates having various molecular weights as in Table 8 below. The droplet sizes in the nano-emulsions were in the ranges of 50 to 250 nm. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Table 8
Ex. 26 Ex. 27 Ex. 28 Ex. 29 Ex. 30 Ex. 31
weight average molecular weight of sodium hyaluronate (Dalton) 3,642,000 2,000,000 1,490,000 800,000 15,000 4,280
Examples 32 to 38. Preparation of Nano-emulsion and Nanoparticles
Nano-emulsions were prepared in the same manner as in Example 1, using a metal ion derived from ferric (or ferrous) nitrate, ferric (or ferrous) acetate, copper sulfate, nickel chloride and cobalt chloride as in Table 9 below, in an amount of 0.018 mmol, respectively. The droplet sizes in the nano-emulsions were in the ranges of 60 to 140 nm. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Table 9
Metal ion Metal salt
Example 32 Fe3+ ferric nitrate (III)
Example 33 Fe2+ ferrous nitrate (II)
Example 34 Fe2+ ferrous acetate (II)
Example 35 Cu2+ copper sulfate (II)
Example 36 Ni2+ nickel chloride (II)
Example 37 Co3+ cobalt chloride (III)
Example 38 Co2+ cobalt chloride (II)
Examples 39 to 41. Preparation of Nano-emulsion and Nanoparticles
Nano-emulsions were prepared in the same manner as in Example 1, using sodium hyaluronate in various amounts as in Table 10 below. The droplet sizes in the nano-emulsions were in the ranges of 60 to 80 nm. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Table 10
Example 39 Example 40 Example 41
sodium hyaluronate (g) 0.080 0.160 0.319
Examples 42 to 45. Preparation of Nano-emulsion and Nanoparticles
Nano-emulsions were prepared in the same manner as in Example 1, using ferric chloride (III) in various amounts as in Table 11 below. The droplet sizes in the nano-emulsions were in the ranges of 60 to 90 nm. And also, mannitol was added to the obtained nano-emulsions in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Table 11
Example 42 Example 43 Example 44 Example 45
Ferric chloride (III) (g) 0.001 0.006 0.010 0.016
Example 46. Preparation of Nano-emulsion and Nanoparticles using Hyaluronic Acid Free Base
A nano-emulsion was prepared in the same manner as in Example 1, using hyaluronic acid free base. The droplet size in the nano-emulsion was in the ranges of 60 to 80 nm. And also, mannitol was added to the obtained nano-emulsion in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
The free base form of hyaluronic acid was obtained by the following: 0.5 %(w/v) sodium hyaluronate was dissolved in a mixed solvent of 0.1 M HCl and absolute ethanol (3:7 (v/v)). The resulting solution was slowly stirred at room temperature for 24 hours and then filtered with a 200 to 400 mesh filter to recover the precipitant on the filter as a free base form of hyaluronic acid. The resulting product was washed with absolute ethanol 3 times and then dried at room temperature to obtain a pure free base form of hyaluronic acid. The free base form of hyaluronic acid was subjected to pH determination (pH 2.65) and IR spectrometry for confirming deletion of the salt.
Example 47. Preparation of Nano-emulsion and Nanoparticles using Docetaxel
A nano-emulsion was prepared in the same manner as in Example 1, using docetaxel as an anticancer agent, according to the components and contents listed in Table 12 below. The droplet size in the nano-emulsion was in the ranges of 70 to 80 nm. And also, mannitol was added to the obtained nano-emulsion in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Table 12
Components Contents (g)
Oil phase docetaxel 1.000
ferric chloride (III) 0.003
dl-α-tocopheryl acetate 11.880
tricaprylin 0.120
Polysorbate 80 5.400
Aqueous phase water for injection 75.558
polyvinylpyrrolidone 1.632
glycerin 4.080
glutamic acid 0.052
sodium hyaluronate 0.245
Example 48. Preparation of Nano-emulsion and Nanoparticles comprising PEGylated phospholipids and 5-fluorouracil
A nano-emulsion was prepared in the same manner as in Example 1, using 5-fluorouracil as an anticancer agent, according to the components and contents listed in Table 13 below. The droplet size in the nano-emulsion was in the ranges of 90 to 130 nm. And also, mannitol was added to the obtained nano-emulsion in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Table 13
Components Contents (g)
Oil phase 5-fluorouracil 5.000
ferric chloride (III) 0.003
Myvacet 9-45K 11.880
tricaprylin 0.120
TPGS 2.500
DMPE-PEG2000 1.000
Aqueous phase water for injection 75.149
polyvinylpyrrolidone 1.540
TPGS 2.500
glutamic acid 0.077
sodium hyaluronate 0.231
Examples 49 and 50. Preparation of Nano-emulsion and Nanoparticles
Nano-emulsions were prepared in the same manner as in Example 1, using citric acid or N-acetyl cysteine as a pH-controlling agent, according to the components and contents listed in Table 14 below. The droplet sizes in the nano-emulsions were in the ranges of 70 to 80 nm. And also, mannitol was added to the obtained nano-emulsion in the concentration of 5 % (w/v). The resulting mixture was lyophilized to obtain nanoparticles.
Table 14
Components Contents (g)
Example 49 Example 50
Oil phase paclitaxel 0.300 0.300
ferric chloride (III) 0.002 0.002
dl-α-tocopheryl acetate 7.200 7.200
refined soybean oil 0.800 0.800
Polysorbate 80 8.000 8.000
citric acid 0.002 -
N-acetyl cysteine - 0.001
Aqueous phase water for injection 83.445 83.446
sodium hyaluronate 0.251 0.251
Comparative Examples 1 to 3. Preparation of Nano-emulsion and Nanoparticles
Nano-emulsions were prepared in the same manner as in Example 1 according to the components and contents listed in Table 15 below.
Table 15
Components Contents (g)
Comparative example 1 Comparative example 2 Comparative example 3
Oil phase paclitaxel 0.300 0.300 0.300
ferric chloride (III) - 0.002 -
dl-α-tocopheryl acetate 7.200 7.200 7.200
refined soybean oil 0.800 0.800 0.800
Polysorbate 80 8.000 8.000 8.000
Aqueous phase water for injection 83.740 83.698 83.700
sodium hyaluronate - - 0.251
Test Example 1. Determination for Binding amounts of Hyaluronic acid
The nano-emulsion obtained in Example 1 was ultra-centrifuged at 20 ℃ for 2 hours. The resulting precipitate was washed with water for injection 3 times and then dried at room temperature to obtain an emulsion aggregate where hyaluronic acid is bound. The amount of hyaluronic acid bound was analyzed in accordance with the method for quantitative determination of hyaluronic acid described in the European pharmacopoeia and the detailed procedures are as follows:
Distilled water was added to 0.170 g of the emulsion aggregate to become 100 g of solution in total, which was then diluted to 40-fold to prepare a test solution. Distilled water was added to 0.1 g of D-glucuronic acid to become 100 g of solution in total. Using the D-glucuronic acid solution, 5 standard solutions were prepared so as to contain 6.5 to 65 ug of D-glucuronic acid per g. A solution of 0.95 w/v% borax in sulfuric acid (5 mL) was added to the test solution (1.0 mL) and the standard solutions (1.0 mL), respectively. The mixtures were allowed to stand in a water bath for 15 minutes; and then cooled in ice water. A solution of 0.125 w/v% carbazole in ethanol (0.2 mL) was added to the cooled solution. The cooled solution was allowed to stand in a water bath for 15 minutes; and then cooled to room temperature. Each absorbance of the solutions was measured at 530 nm by UV/Vis spectrophotometry. The measurement was repeated 3 times and then an average concentration of D-glucuronic acid in the test solution was calculated, using a calibration curve obtained from the standard solutions. The amount of sodium hyaluronate was calculated from the following formula and the result thereof is represented in Table 16.
Amounts of sodium hyaluronate (%) = (Cg/Cs) x Z x [100/(100-h)] x (401.3/194.1)
- Cg: Average concentration of D-glucuronic acid in the test solutions (mg/g)
- Cs: Average concentration of sodium hyaluronate in the test solutions (mg/g)
- Z: Amount of D-glucuronic acid (%)
- h: Loss on drying (%)
- 401.3: Relative molecular weight of disaccharide
- 194.1: Relative molecular weight of D-glucuronic acid
Table 16
Amounts of hyaluronic acid bound Mean particle size of nano-emulsion
0.8±0.1 wt% 74±3 nm
Test Example 2. In vitro Affinity to CD44
SK-OV-3 cells (ATCC: HTB-77, Rockville, MD, USA) and OVCAR-3 cells (Korean Cell Line Bank, KCLB-00000287) were used as ovarian cancer cells for determining affinity to CD44. CD44 is expressed on the surface of SK-OV-3 cells, while it is not expressed on the surface of OVCAR-3 cells. These cells were cultured under 5% CO2 atmosphere at 37 ℃, in RPMI 1640 medium (HA free medium, Gibco, Carlsbad, USA) supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin and 10 % fetal bovine serum (FBS). The nano-emulsions (obtained in Example 1 and Comparative examples 1 to 3) labeled with FITC were added to each of 1 x 106 cells, which was then incubated at 37 ℃ for 4 hours. The labeled nano-emulsions were obtained by dissolving 0.2 mg of FITC in the nano-droplets. Non-labeled fluorescent material was removed with a dialysis membrane (Pierce, MWCO 2,000).
After the incubation, the cells were washed with phosphate buffered saline (PBS) 2 times, treated with 0.1 % trypsin for about 1 minute, and then washed with PBS containing 0.2 % FBS and 0.02 % sodium azide 3 times. The recovered cells were re-dispersed in a solution of 4 % paraformaldehyde (400 uL). The affinities to CD44 were determined using FACS (Fluorescence-activated cell sorter) and epi-fluorescent microscopy. Binding of hyaluronic acid nanoparticles to the ovarian cancer cells was measured by fluorescence scanning at 488 nm using a FACScalibur scanner (Beckton-Dickinson, Mansfield, MA, USA). The results thereof are shown in FIG. 3.
FIG. 3A and 3C show the results of in vitro affinity test using OVCAR-3 cells. FIG. 3B and 3D show the results of in vitro affinity test using SK-OV-3 cells. As shown in FIG. 3, substantial non-specific binding was not observed in all of the samples. In case of the samples not containing hyaluronic acid, substantial specific binding to CD44 on the surface of the cells was not observed. However, in case of the samples containing hyaluronic acid, specific binding to CD44 was observed, with difference in binding affinities. Specifically, there was no specific binding to CD44 in the emulsion of Comparative Example 1 and the emulsion containing a metal ion of Comparative Example 2. A little affinity to CD44 was observed in the emulsion simply mixed with hyaluronic acid (Comparative Example 3). The nano-emulsion of Example 1 containing nano-droplets coated with hyaluronic acid according to the present invention showed an excellent affinity to CD44. Therefore, it can be seen that the nano-emulsion of the present invention is an excellent drug delivery system for active targeting.
Test Example 3. In vivo efficacy test
Anti-cancer efficacy through active and passive targeting was evaluated using SK-OV-3 cells (ATCC: HTB-77, Rockville, MD, USA) over-expressing CD44 on the surface thereof and OVCAR-3 Cell (Korean Cell Line Bank, KCLB-00000287) not expressing CD44 on the surface thereof as ovarian cancer cells.
The SK-OV-3 cells and the OVCAR-3 cells were respectively cultured under 5% CO2 atmosphere at 37 ℃ in RPMI 1640 medium supplemented with 10 % FBS, to obtain enough cells for transplantation into test animals, i.e., 7x108 or more of SK-OV-3 cells of and 4x108 or more of OVCAR-3 cells. The RPMI 1640 medium was prepared by supplementing with 0.1 mM non-essential amino acid, 1 mM sodium pyruvate, 1.5 g/L of sodium bicarbonate and a certain amount of antibiotic/mycotic agent; and then filtering the mixture, which was then stored in a refrigerator. The medium was warmed to 37 ℃ when used.
The SK-OV-3 cells and the OVCAR-3 cells were respectively treated with a trypsin/EDTA solution and then isolated from the culture flask. 1x107 of the respective cells were suspended in 0.3 mL of RPMI 1640. 0.3 mL of the respective cell suspensions were subcutaneously injected into the flank of nude mice (BALB/cAnNCrjBgi-nu/nu, female) using a 23G x 1 mL-syringe, for transplantation of the cancer cells. When tumor sizes in the mice reached about 100 ㎣ or more after the transplantation of the cancer cells, the mice were divided into 5 groups (G1 - G5) (n=7 for each group) each of which had similar average values in tumor size; and then test materials were administered thereto. In G2 group, Taxol® (positive control) were administered via the caudal vein of the mice. In G3, G4, and G5 group, the nano-emulsion of Example 1, the nano-suspension of Example 2, and the nano-emulsion of Comparative Example 1 were respectively administered via the caudal vein of the mice of each group. In G2 to G5 groups, the doses of paclitaxel (active ingredient) were equally 20 mg/kg. In G1 group (control group), only hypertonic saline was administered via the caudal vein thereof, for monitoring cancer cell growth. For evaluation of anticancer activity, changes of tumor size in the test animals were determined 2-3 times in a week, at intervals of about 3 days. The tumor sizes were calculated from their length and width measured with vernier calipers, according to the following formula. The changes of tumor size on time elapsed were comparatively evaluated for each test group and then the results are represented in FIGs. 4A and 4B.
Tumor Size (㎣) = length (mm) x [width (mm)]2 / 2
FIG. 4A shows changes of tumor size in the cancer-transplanted CD44-overexpressed SK-OV-3 models after test materials or controls were respectively administered. The groups treated with the nano-emulsion of Example 1 and the nano-suspension of Example 2 represented more excellent inhibitory effects against cancer cell growth than the groups treated with Taxol® and the nano-emulsion of Comparative Example 1. Considering that the nano-dispersions of Example 1 and 2 have nanoparticles which hyaluronic acid has been bound on, these results show that the nano-dispersions of the present invention exhibit an active targeting effect through specific binding of hyaluronic acid to CD44. However, the emulsion of Comparative Example 1 (i.e., without hyaluronic acid coating) showed very low inhibitory effect against cancer cell growth, which means that the emulsion of Comparative Example 1 represents only a passive targeting effect through EPR effect, not an active targeting effect through specific binding to CD44.
FIG. 4B shows changes of tumor size in the cancer-transplanted CD44-not-expressed OVCAR-3 models after test materials or controls were respectively administered. The groups treated with the nano-emulsion of Example 1, the nano-suspension of Example 2, and the emulsion of Comparative Example 1 represented more excellent inhibitory effects against cancer cell growth than the negative control group and the group treated with Taxol®. Since CD44 was not expressed in the cancer-transplanted OVCAR-3 model, only a passive targeting effect through EPR effect was exhibited.
Test Example 4. In vivo toxicity study
The healthy test animals (nude mice) through breeding for adaptation were selected for repeated-dose toxicity study. The selected nude mice were arranged into each cage (n=5 for each cage) by random order. The test materials were well shaken just before the administration; and then administered via the tail vein of the mice using a sterilized disposable syringe. The administrations were performed 3 times in total, at intervals of 3 consecutive days. The death and clinical sign of the test animals were monitored. The clinical sign was intermittently monitored according to the status till 4 hours after the administration; and then monitored 1 time per day to the end of monitoring. The body weights of the test animals were measured 1 time per day from the day initiating the administration to the end of monitoring. The results thereof are represented in FIG. 5.
The nude mice receiving 3 repeated-administrations with 30 mg/kg of Taxol® (positive control) showed rapid body weight loss to an extent of about 20 % and serious toxicity. Among the test animals, 3 mice were died during or after the administration. Also, all the Taxol®-administered groups showed the symptoms such as dyspnea, lethargy, and ataxia regardless of the doses of paclitaxel. However, the groups receiving 3 repeated-administrations with 20 to 50 mg/kg (as a paclitaxel concentration) of the nano-emulsion of Example 1 showed body weight changes only within 10 % or less and therefore relatively safe toxicity-values in comparison to the positive control (i.e., Taxol®). These results mean that the maximum tolerance dose of nano-emulsion of Example 1 is at least 50mg/kg or more in the nude mouse. And also, there was not observed any death or specific clinical sign in all the groups administered with the nano-emulsion of Example 1.
Test Example 5. Dissolution test
Dissolution tests of the nano-dispersion of the present invention were carried out according to the dissolution test method (Dissolution Test Method No. 2) described in the Korean Pharmacopoeia as follows:
The nano-emulsion of Example 1, the nano-suspension of Example 2 and Taxol® injection (Bristol Myers Squibb) were respectively put into dialysis membranes (Spectra/Por) having 12,000 to 14,000 of a molecular weight cut off (MWCO), each of which was added to 1,000 mL of a dissolution medium (0.1 % Polysorbate 80-phospate buffer, pH 7.08). The dialysis membranes were stabilized by immersing them in the dissolution medium for about 30 minutes before use. Each dialysis membrane was fixed to a support and then placed in a sink condition in the dissolution medium. The paddle was rotated at 100 rpm, maintaining a temperature of 37 ℃. 1 mL of the dissolution medium was taken at predetermined times and then analyzed with HPLC to obtain the dissolution rates of paclitaxel released from the dialysis membrane. The dissolution medium was continuously supplemented to keep its volume to 1000 mL. The results of dissolution test for 3 days are represented in FIG. 6. From the results of FIG. 6, it can be seen that paclitaxel is sustained-released from the nano-dispersions of the present invention for a long time, which is different from Taxol® injection. And also, it can be seen that the nano-suspension shows more sustained-releasing pattern than the nano-emulsion. Considering the time required for the nanoparticles introduced into the body to reach targeted cancer cells, sustained-releasing pattern is more advantageous rather than rapid-releasing pattern, in order to exhibit more therapeutic effects after arrival at targeted cancer cells. Therefore, the nano-dispersion of the present invention is very useful for efficient delivery of a drug.
Test Example 6. Evaluation for Encapsulation Efficiency and Reconstitutability of Nanoparticles
Encapsulation efficiency and re-dispersability were evaluated for the nano-dispersion and the nanoparticles obtained through lyophilization thereof in Example 1. The same evaluations were also carried out for the nano-particles in accordance with the prior arts of the present inventors, i.e., Korean patent no. 10-744,925 (Reference example 1) and Korean patent publication no. 10-2009-0040979 (prepared in Example 93 thereof, Reference example 2). The nano-dispersion and the nanoparticles of Reference example 1 were obtained by the followings: 0.135 g of paclitaxel and 0.045 g of hyaluronic acid was dissolved in a solution of 0.02 M glutamic acid in 70 % ethanol (45 mL). A solution of 0.05 M ferric nitrate (III) (0.84 mL) was added under stirring at 300 rpm to the solution, which was then stirred for 2 hours to obtain the nano-dispersion. The resulting nano-dispersion was lyophilized to obtain the nanoparticles having about 120 nm of a mean particle size.
Each of the non-entrapped, precipitated paclitaxel of Example 1 and Reference examples 1 and 2 was isolated by filtration through a 0.22 ㎛ filter (Acro 50 Vent Devices with Emflon Membrane®II, Pall Co. USA). Each amount of paclitaxel in the collected filtrates was measured according to the method for quantitative determination of paclitaxel described in the European Pharmacopoeia, to evaluate the encapsulation efficiency thereof. And also, the mean particle sizes of the nanoparticles in the nano-dispersions of Example 1 and Reference examples 1 and 2 were also measured according to the same method as in Example 1.
Each of the lyophilized nanoparticles was reconstituted in water for injection. From the reconstituted products (i.e., nano-dispersions), the encapsulation efficiency thereof and mean particle sizes were also evaluated in the same manners in the above.
The results are represented in table 17.
Table 17
Before freeze-drying Re-dispersion
Encapsulation content (mg/mL) Encapsulation efficiency (%) Mean Particle size (nm) Encapsulation content (mg/mL) Encapsulation efficiency (%) Mean Particle size (nm)
Ex 1 3.00±0.04 100.3±0.1 70±1 3.01±0.03 100.3±0.1 71±2
Ref. ex. 1 0.41±0.11 13.7±3.7 127±9 0.18±0.08 6.0±2.7 862±98
Ref. ex. 2 0.22±0.05 36.7±8.3 104±13 0.13±0.03 21.7±5.0 128±34*
* : Particle size was measured after removing some of aggregated particles
As shown in Table 17, the nano-dispersion and the nanopartices according to the present invention show the highest encapsulation content and efficiency of paclitaxel. And also, the encapsulation content and the mean particle size thereof were maintained after being reconstituted in dispersion medium (water for injection).
Test Example 7. Physicochemical Stability test
The nanoparticles obtained through lyophilization in Example 1 were subjected to evaluation of physicochemical stability tests for 6 months at room temperature (25±2℃, 60±5 %RH) and at accelerated condition (40±2℃, 75±5 %RH). After reconstitution of lyophilized nanoparticles, the encapsulation contents of paclitaxel and the nanoparticle sizes were measured by the same method as in Test Example 6. Each measuring was repeated three times and then the average value thereof was calculated. The results are represented in Table 18.
Table 18
Storage Period (month) at room temperature(25±2℃, 60±5 %RH) accelerated condition(40±2℃, 75±5 %RH)
Encapsulation contents (mg/mL) Mean particle size (nm) Encapsulation contents (mg/mL) Mean particle size (nm)
beginning 3.01±0.03 71±2 3.01±0.03 71±2
1 2.94±0.02 69±3 2.97±0.03 73±2
2 3.01±0.04 68±1 2.98±0.02 71±3
3 2.93±0.06 73±2 2.96±0.01 75±2
4 2.92±0.07 70±2 2.99±0.04 72±1
5 2.91±0.05 68±3 2.93±0.03 74±1
6 2.96±0.03 71±1 2.94±0.04 75±3
As shown in Table 18, it can be seen that there is no significant difference in the particle size and the contents thereof for 6 months, which means that physicochemical stability of the nanoparticles can be maintained for at least 6 months.

Claims (29)

  1. A pharmaceutical composition for tumor-targeting in the form of a nano-dispersion comprising nanoparticles having a mean particle size of 10 to 1,000 nm in an aqueous medium, wherein the nanoparticles comprise a therapeutically effective amount of an anticancer agent; a divalent or trivalent transition metal ion or alkali earth metal ion; and an oil, and wherein hyaluronic acid or its salt is bound on a surface of the nanoparticles.
  2. The pharmaceutical composition according to claim 1, wherein molecular weight of the hyaluronic acid ranges from 379 to 10,000,000 daltons.
  3. The pharmaceutical composition according to claim 1, wherein the salt of hyaluronic acid is cobalt hyaluronate, magnesium hyaluronate, zinc hyaluronate, calcium hyaluronate, potassium hyaluronate, sodium hyaluronate, or tetrabutylammonium hyaluronate.
  4. The pharmaceutical composition according to claim 1, wherein an amount of the hyaluronic acid or its salt ranges from 0.01 to 1.0 w/w%, based on the total weight of the composition.
  5. The pharmaceutical composition according to claim 1, wherein the divalent or trivalent transition metal ion or alkali earth metal ion is at least one selected from the group consisting of Cu2+, Cu3+, Zn2+, Zn3+, Ni2+, Ni3+, Mg2+, Mg3+, Ca2+, Ca3+, Co2+, Co3+, Ba2+, Ba3+, Al2+, Al3+, Fe2+, and Fe3+.
  6. The pharmaceutical composition according to claim 1, wherein the divalent or trivalent transition metal ion or alkali earth metal ion is derived from copper nitrate, copper sulfate, copper chloride, zinc acetate, zinc sulfate, zinc nitrate, zinc chloride, nickel sulfate, nickel nitrate, nickel chloride, magnesium acetate, magnesium sulfate, magnesium nitrate, magnesium chloride, calcium acetate, calcium sulfate, calcium nitrate, calcium chloride, cobalt acetate, cobalt sulfate, cobalt chloride, barium acetate, barium sulfate, barium nitrate, barium chloride, aluminum sulfate, aluminum chloride, ferric (or ferrous) acetate, ferric (or ferrous) sulfate, ferric (or ferrous) nitrate, or ferric (or ferrous) chloride.
  7. The pharmaceutical composition according to claim 1, wherein a molar ratio of the divalent or trivalent transition metal ion or alkali earth metal ion per disaccharide unit of the hyaluronic acid or its salt ranges from 0.001 to 2.0.
  8. The pharmaceutical composition according to claim 1, wherein the composition is in the form of a nano-emulsion, and wherein the oil is at least one selected from the group consisting of mono-, di-, or tri-glycerides; glyceryl mono- or tri-stearates; glyceryl mono-, di-, or tri-acetates; alpha-tocopherol or its salt; alpha-tocopheryl acetate; alpha-tocopherol succinate; C4:0 to C6:0 triglycerides; C8:0 to C14:0 triglycerides; castor oil, corn oil, olive oil, cottonseed oil, peppermint oil, sesame oil, and soybean oil.
  9. The pharmaceutical composition according to claim 1, wherein the composition is in the form of a nano-suspension, and wherein the oil is at least one selected from the group consisting of hydrogenated soybean oil, cacao butter, cetyl alcohol, stearyl alcohol, cetyl palmitate, carnauba wax, white beeswax, tricaprin, trilaurin, trimyristin, tripalmitin, tristearin, and tribehenin.
  10. The pharmaceutical composition according to claim 1, wherein an amount of the oil ranges from 1 to 50 w/w%, based on the total weight of the composition.
  11. The pharmaceutical composition according to claim 1, wherein the anticancer agent is paclitaxel or its derivative, uracil, 5-fluorouracil, tegafur, methotrexate, melphalan, mitoxantrone, camptothecin, topotecan, docetaxel, capecitabine, imatinib mesylate, rituximab, doxifluridine, toremifene citrate, doxorubicin, gemcitabine, irinotecan, oxaliplatin, or chlorambucil.
  12. The pharmaceutical composition according to claim 1, further comprising at least one surfactant selected from the group consisting of alpha-tocopherol polyethylene glycol succinate, macrogol 15 hydroxystearate, caprylocaproyl macrogolglycerides, sorbitan monooleate, polysorbates, polyoxyethylene-polyoxypropylene block copolymer, egg lecithin, and soybean lecithin.
  13. The pharmaceutical composition according to claim 12, wherein an amount of the surfactant ranges from 1 to 50 w/w%, based on the total weight of the composition.
  14. The pharmaceutical composition according to claim 1, wherein the aqueous medium is at least one selected from the group consisting of distilled water, water for injection, saline, solution for sodium chloride injection, solution for dextrose injection, and solution for amino acid injection.
  15. The pharmaceutical composition according to claim 1, further comprising at least one solubilizing agent selected from the group consisting of polyethylene glycol, dimiristoyl phosphatidyl ethanolamine-polyethylene glycol, cholesterol, propylene glycol, glycofurol, and tricaprylin, within the nanoparticles.
  16. The pharmaceutical composition according to claim 15, wherein an amount of the solubilizing agent ranges from 0.05 to 10 w/w%, based on the total weight of the composition.
  17. The pharmaceutical composition according to claim 1, wherein the surface of the nanoparticles further comprises at least one amino acid having carboxyl group selected from the group consisting of glutamic acid, aspartic acid, asparagine, histidine, and alanine as a low-molecular ligand.
  18. The pharmaceutical composition according to claim 17, wherein an amount of the low-molecular ligand ranges from 0.005 to 0.2 w/w%, based on the total weight of the composition.
  19. The pharmaceutical composition according to claim 1, further comprising at least one pH-controlling agent selected from the group consisting of citric acid, acetic acid, phosphoric acid, lactic acid, benzoic acid, maleic acid, succinic acid, tartaric acid, N-acetyl cysteine, N-acetyl valine, N-acetylproline, and N-acetylalanine.
  20. The pharmaceutical composition according to claim 19, wherein the pH of the composition ranges from 2.0 to 6.0.
  21. The pharmaceutical composition according to claim 1, further comprising at least one dispersion-stabilizing agent selected from the group consisting of polyvinylpyrrolidone, glycerin, glucose, sucrose, lactose, sorbitol, mannitol, and trehalose.
  22. Nanoparticles for tumor-targeting obtained by drying the pharmaceutical composition in the form of a nano-dispersion according to any one of claims 1 to 21, through lyophilizing, rotary evaporation drying, spray drying or fluidized-bed drying.
  23. A process for preparing a pharmaceutical composition for tumor-targeting in the form of a nano-dispersion, which comprises:
    (a) providing an oil phase comprising a therapeutically effective amount of an anticancer agent, a divalent or trivalent transition metal ion or alkali earth metal ion and an oil,
    (b) providing an aqueous phase by dissolving hyaluronic acid or its salt in an aqueous medium, and
    (c) mixing the oil phase obtained in Step (a) and the aqueous phase obtained in Step (b) and then homogenizing the resulting mixture to form nanoparticles having a mean particle size of 10 to 1,000 nm, the surface of which hyaluronic acid or its salt is bound on.
  24. The process according to claim 23, further comprising adding a surfactant to the pharmaceutical composition.
  25. The process according to claim 23, wherein the oil phase further comprises at least one solubilizing agent selected from the group consisting of polyethylene glycol, dimiristoyl phosphatidyl ethanolamine-polyethylene glycol, cholesterol, propylene glycol, glycofurol, and tricaprylin.
  26. The process according to claim 23, wherein the aqueous phase is obtained by further dissolving an amino acid having carboxyl group as a low-molecular ligand.
  27. The process according to any one of claims 23 to 26, wherein the aqueous phase is obtained by further dissolving at least one dispersion-stabilizing agent selected from the group consisting of polyvinylpyrrolidone, glycerin, glucose, sucrose, lactose, sorbitol, mannitol, and trehalose.
  28. A process for preparing nanoparticles for tumor-targeting, which comprises drying the pharmaceutical composition in the form of a nano-dispersion obtained by the process according to any one of claims 23 to 26.
  29. The process according to claims 28, wherein the drying is performed by lyophilizing, rotary evaporation drying, spray drying or fluidized-bed drying.
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