KR20110056042A - Nano particles for tumor-targeting and processes for the preparation thereof - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing nanoparticles in an aqueous medium is provided to enable active and passive targeting of an anticancer drug. CONSTITUTION: A pharmaceutical composition of nano-dispersion for targeting contains nanoparticles having 10-1,000 nm of average particle size in an aqueous medium. The nanoparticle contains therapeutically effective amount of anticancer drug; bivalent or trivalent transition metal ion or alkali earth metal ion; and oil. Hyaluronic acid or salt thereof is conjugated on the surface of the nanoparticles. The aqueous medium is distilled water, injection solution, saline solution, dextrose, or amino acid liquid.

Description

Nanoparticles for tumor-targeting and processes for the preparation etc

The present invention relates to a pharmaceutical composition comprising an anticancer agent capable of passive targeting and active targeting. More specifically, a therapeutically effective amount of an anticancer agent; Divalent or trivalent transition metal ions or alkaline earth metal ions; oil; And nanoparticles capable of passive target orientation and active target orientation including hyaluronic acid or salts thereof.

Conventional cancer treatment using an anticancer agent (or antitumor agent) is known to cause various side effects because it affects not only cancer cells but also normal cells in the human body. In order to improve this, various target targeting methods including passive targeting and active targeting have been studied, but are not yet satisfactory.

Passive target orientation means that the microparticles are selectively accumulated only in cancer cells with abnormal blood vessels by continuous blood circulation in the body. For example, cancer cells are enlarged at abnormal growth rates unlike normal cells, so they are larger in size than normal aligned endothelial pores or vary in size from about 10 nm to 1000 nm. It has Therefore, when microparticles containing anticancer agents are circulated continuously in the blood, nanoparticles accumulate only in cancer cells and do not penetrate the vascular endothelium of normal cells, which enhances permeation and retention effect (Enhanced permeation and retention effect). , "EPR" effect. As an example of the passive target-oriented method, a method for delivering an anticancer agent to cancer cells using small particle liposome aerosol (WO 1999/15153) is known.

Active target-oriented methods employ drug delivery systems using receptors that specifically recognize molecules overexpressed on the surface of cancer cells, such as lectins, growth factors, cytokines, hormones, unsaturated fatty acids, low density lipoproteins, and folic acid. And a method for transporting an anticancer agent to cancer cells (SP Vyas., Et. Al., Advanced Drug Delivery Review 43 (2000) 101-164). Various drug delivery systems are known in which an anticancer agent is bound to and / or encapsulated in a biodegradable polymer or liposome, followed by attachment of a receptor that specifically recognizes an overexpressed molecule on a cancer cell surface. For example, US Pat. No. 6,593,308 discloses a drug delivery system comprising an encapsulating delivery vehicle such as liposomes and a hyaluronan ligand. In the drug delivery system, the anticancer agent is encapsulated in the vehicle and uses metal ions as a linking agent or chelating agent for attaching a ligand to the vehicle. In addition, US Pat. No. 6,699,471 discloses injectable preparations in gel form comprising benzyl esters of hyaluronic acid or auto-cross-linked derivatives of hyaluronic acid. In addition, targeting methods using monoclonal antibodies known to have a very high affinity to cancer cells (Stanislv J., et al., Bioorg. Medic. Chem. (2005) 13, 5043-5054) have also been reported.

In addition, US Pat. No. 6,890,901 discloses a formulation in which hyaluronic acid is attached to liposomes on a surface by chemical bonding with histidine, and US Pat. No. 6,593,308 discloses a formulation in which hyaluronic acid is coated on a liposome surface by chemical bonding method. Doing. However, in the case of liposome formulations, double lipid membranes are unstable, resulting in poor long-term stability, being caught by macrophages, degraded by phagocytosis, and complex manufacturing processes that induce chemical bonds with hyaluronic acid. I have a problem.

US Patent Publication No. 2006/0188578 discloses a method of ionically coupling hyaluronic acid to the surface of a conventional chitosan nanoparticle manufacturing method using chitosan and tripolyphosphate, in which case hyaluronic acid is a buffer such as blood. It has the disadvantage of dissolving easily in solution, so that the affinity with cancer cells is easily consumed within a short time, and the drug release proceeds rapidly due to the use of chitosan, which is a natural water-soluble polymer, and thus the drug release does not occur. In addition, US Patent Publication No. 2007/0036728 discloses a transdermal drug transport method using hyaluronic acid nanoemulsion of O / W type by mixing hyaluronic acid and hydrazide.

In addition, US 2007/0031503 discloses a modification of hyaluronic acid to which polylactic acid, polyglycolic acid, or copolymers thereof are bound. However, when the hyaluronic acid modified according to the US patent is prepared, the amount of hyaluronic acid bound to the polymer is significantly less, the ability of targeting to cancer cells, there is a problem that the formulation itself is unstable. In addition, there is a problem that the resulting hyaluronic acid modification is affected by the ionic strength, pH, etc. of the drug used. In addition, International Patent Publication No. WO00 / 041730 discloses to increase anticancer treatment effect by simple mixing of a water-soluble anticancer agent and hyaluronic acid, and US Pat. No. 7,371,738 discloses hyaluronic acid, hydrazide, and a crosslinking agent. After admixing using an emulsification system, a method of obtaining nanoparticle precipitates by adding a certain amount of alcohol has been disclosed.

The conventional target-oriented method is designed for passive target-oriented or active target-oriented only, and achieves a target-oriented method that can simultaneously treat not only solid cancer but also metastatic cancer, namely passive target-oriented and active target-oriented. What is needed is a targeted method. Furthermore, there is a need in the art for a target-oriented method that can solve manufacturing problems (eg, complexity of the manufacturing process) resulting from the design of a separate vehicle such as liposomes for the transport of anticancer agents.

The present inventors have developed a drug delivery system for target orientation using hyaluronic acid or a salt thereof that can simultaneously achieve passive target orientation and active target orientation (Korean Patent Registration No. 774,925, and Korean Patent Publication No. 10-). 2009-0040979). The drug delivery system is prepared by forming a mixture of an anticancer agent (antitumor agent) and hyaluronic acid or a salt thereof by forming nanoparticles using metal ions and / or water-soluble biodegradable polymers, thereby improving permeation / residual enhancement effect (Enhanced permeation). and active targeting of anticancer agents by anti-cancer agents by " EPR " and simultaneous hyaluronic acid.

The present invention provides a drug delivery system for targeting by using hyaluronic acid or a salt thereof, which can simultaneously achieve passive targeting and active targeting. That is, by preparing an anticancer agent (antitumor agent) in the form of nanoparticles with metal ions, oils, and the like, and binding the hyaluronic acid or a salt thereof to the surface of the nanoparticles by interacting with the metal ions, a permeability / residual enhancement effect ( Passive targeting of the anticancer agent by the enhanced permeation and retention effect ("EPR" effect) and active targeting of the anticancer agent by hyaluronic acid can be simultaneously achieved, and the encapsulation efficiency of the anticancer agent ( It provides a drug delivery system that can significantly increase the encapsulation efficiency.

Accordingly, an object of the present invention is to provide a pharmaceutical composition in the form of nano-dispersion liquid, which can simultaneously achieve passive target orientation and active target orientation, and also includes nanoparticles with high encapsulation efficiency of anticancer agents. It is also an object of the present invention to provide a method for preparing a pharmaceutical composition in the form of a nano-dispersion containing the nanoparticles.

According to one aspect of the invention, there is provided a pharmaceutical composition for target orientation in the form of nano-dispersions comprising nanoparticles having an average particle diameter of 10 to 1,000 nm in an aqueous medium, wherein the nanoparticles are in a therapeutically effective amount of an anticancer agent; Divalent or trivalent transition metal ions or alkaline earth metal ions; And an oil, and a hyaluronic acid or a salt thereof is bound to a surface of the nanoparticles.

According to another aspect of the invention, the pharmaceutical composition in the form of nano-dispersions is obtained by drying by lyophilization, rotary evaporation drying, spray drying, or fluidized-bed drying Nanoparticles for target orientation are provided.

According to another aspect of the present invention, there is provided a method of preparing an oil phase comprising (a) preparing an oil phase comprising a therapeutically effective amount of an anticancer agent, a divalent or trivalent transition metal ion or an alkaline earth metal ion, and an oil; (b) dissolving hyaluronic acid or a salt thereof in an aqueous medium to obtain an aqueous phase; And (c) mixing and homogenizing the oil phase obtained in step (a) and the water phase obtained in step (b) to form nanoparticles having hyaluronic acid or a salt thereof bound on the surface as nanoparticles having an average particle diameter of 10 to 1,000 nm. Provided is a method of preparing a pharmaceutical composition in nano-dispersion form for target orientation, comprising the step.

According to another aspect of the invention, there is provided a method for producing nanoparticles for target orientation, comprising the step of drying the pharmaceutical composition in the form of nano-dispersion by lyophilization, rotary evaporation, spray drying, or fluidized bed drying Is provided.

Pharmaceutical compositions in the form of nano-dispersions comprising nanoparticles in the aqueous medium of the present invention are drug delivery obtained by combining hyaluronic acid or a salt thereof on the surface of the nanoparticles including an anticancer agent (antitumor agent), a metal ion, and an oil. System. The drug delivery system according to the present invention enables the active target orientation of the anticancer agent through the manual target orientation of the anticancer agent and the affinity between the hyaluronic acid and the cancer cell through the permeability / residual enhancement effect. That is, the nanoparticles according to the present invention is a nano-drug delivery system that can selectively act on cancer cells, and can selectively act on metastatic cancer as well as solid cancer. In particular, the pharmaceutical composition of the nano-dispersion form of the present invention can increase the encapsulation efficiency of the anticancer agent by a desired amount (ie, by a desired amount of almost 100%). In addition, the pharmaceutical compositions in the form of nano-dispersions of the present invention are excellent in biocompatibility and have excellent physicochemical stability.

As used herein, the term "nanoparticles" refers to particles having a 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. In addition, in the present invention, the term "nano-dispersion" refers to a drug carrier obtained by dispersing the nanoparticles in an aqueous medium, and refers to a nano-emulsion or a nano-suspension. Include the form of. The "nano-emulsion" refers to a drug carrier in the form of an emulsion in which the nanoparticles are dispersed in an aqueous medium in oil form at room temperature (about 25 ° C). In addition, the "nano-suspension" refers to a drug carrier in the form of a suspension in which the nanoparticles are dispersed in an aqueous medium in the form of solid particles at room temperature (about 25 ℃). In the nano-dispersion, the average of the nanoparticles The particle diameter has a size that enables the EPR effect, for example, 10 to 1,000 nm, preferably 50 to 500 nm, more preferably 50 to 300 nm.

The present invention provides a pharmaceutical composition for target orientation in the form of nano-dispersions comprising nanoparticles having an average particle diameter of 10 to 1,000 nm in an aqueous medium, wherein the nanoparticles are in a therapeutically effective amount of an anticancer agent; Divalent or trivalent transition metal ions or alkaline earth metal ions; And an oil, and hyaluronic acid or a salt thereof is bound to a surface of the nanoparticles.

The pharmaceutical composition according to the present invention is formed by forming a nanoparticle with an anticancer agent together with a metal ion and oil, and then hyaluronic acid is bound to the nanoparticle surface by the interaction (chelate bond) between the hyaluronic acid and the metal ion. Here hyaluronic acid is present outside the nanoparticles and metal ions are present inside the nanoparticles. (See FIGS. 1 and 2).

The hyaluronic acid is a hydrophilic polysaccharide present in the human body, and has a long basic unit of disaccharide (Dsaccharide, molecular weight 379) of D-glucuronic acid (D-glucuronic acid) and N-glucosamine (N-glucosamine). It exists in the form of high molecular weight hetero-polysaccharides and is responsible for various functions in the extratracelluar matrix such as cell growth, differentiation, and migration. The widespread activity of hyaluronic acid can be attributed to a number of hyaluronic acid-binding receptors such as CD44, the cell surface glycoprotein, receptors for hyaluronic acid-mediated motility (RHAMM), and other hyaluronic acid-binding elements (motifs). (S. Jaracz et al., Recent advances in tumor-targeting anticancer drug conjugates, Bioorg. Med. Chem. 13 (2005) 5043-5054). In particular, various tumors such as epithelial, ovarian, colon, gastrointestinal, and acute leukemia are known to overexpress hyaluronic acid-binding receptors CD44 and RHAMM. Known (Day, AJ; Prestwich, GDJ Biol. Chem. 2002, 277, 4585; and Turley, EA; Belch, AJ; Poppema, S .; Pilarski, LM Blood 1993, 81, 446) and consequently these tumor cells Show enhanced binding and internalization of hyaluronic acid (Hua, Q .; Knudson, CB; Knudson, WJ Cell Sci. 1993, 106, 365). Hyaluronic acid is also involved in the metastasis process of tumor cells [Sleeman, J. et al., Cancer. Res. (1996) 56, 3134. Therefore, the pharmaceutical composition in the form of a nano-dispersion solution for target orientation containing nanoparticles according to the present invention will act as a drug delivery system that uses hyaluronic acid as an energy source or is specific for tumor cells, that is, active target orientation. .

The molecular weight of the hyaluronic acid is not particularly limited, for example, the average molecular weight of the hyaluronic acid may be 379 to 10,000,000 Daltons, preferably 1,000 to 4,000,000 Daltons, more preferably 1,000 to 1,500,000 Daltons. In addition, the salt of the hyaluronic acid may be in various salt forms, for example, inorganic salts such as cobalt hyaluronic acid, magnesium hyaluronic acid, zinc hyaluronic acid, calcium hyaluronic acid, potassium hyaluronate, sodium hyaluronate and tetra hyaluronic acid Organic salts such as butylammonium. Preferably, hyaluronic acid free base or sodium hyaluronate can be used. The surface of the nanoparticles may be combined by combining a salt of hyaluronic acid or a hyaluronic acid, respectively, or by mixing a salt of hyaluronic acid and a hyaluronic acid. The content of the hyaluronic acid or a salt thereof may be in the range of 0.01 to 1.0% by weight, preferably 0.1 to 0.5% by weight based on the total weight of the composition.

The nanoparticles comprise divalent or trivalent transition metal ions or alkaline earth metal ions. The divalent or trivalent transition metal ions or alkaline earth metal ions are Cu ²⁺ , Cu ³⁺ , Zn ²⁺ , Zn ³⁺ , Ni ²⁺ , Ni ³⁺ , Mg ²⁺ , Mg ³⁺ , Ca ²⁺ , At least one selected from the group consisting of 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, for example, 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, iron acetate, iron sulfate, It may be derived from iron nitrate, or iron chloride, but is not limited thereto. In addition, preferably, among the metal ions, Fe ²⁺ and / or Fe ³⁺ ions may be preferably used, and the Fe ²⁺ and / or Fe ³⁺ ions may be formed from iron acetate, iron sulfate, iron nitrate, or iron chloride. Can be derived. The metal ion reacts with a carboxyl group of glucuronic acid in hyaluronic acid to form a chelate bond. Incidentally, it also reacts with the hydroxy group between the hyaluronic acid sugar chains or chelates with the amine group of N-acetylglucosamine. The molar ratio of the divalent or trivalent transition metal ions or alkaline earth metal ions per disaccharide unit of the hyaluronic acid or a salt thereof is in the range of 0.001 to 2.0, more preferably in the range of 0.001 to 0.5.

In addition, the nanoparticles contain a biocompatible oil as a lipophilic medium capable of dissolving the anticancer agent. In addition, the oil may include both liquid and solid at room temperature.

In addition, when the pharmaceutical composition is in the form of a nano-emulsion, the oil is mono-, di-, or tri-glycerides (e.g., Mivaset, which are liquid at room temperature). 9-45K (Myvacet 9-45K, etc.); Glyceryl mono- or tri-stearate; Glyceryl mono-, di-, or tri-acetate (eg, triacetin, etc.); Alpha-tocopherol (including d-alpha-tocopherol or dl-alpha-tocopherol forms) or salts thereof; Alpha-tocopherol acetic acid esters (including d-alpha-tocopherol acetic acid ester or dl-alpha-tocopherol acetic acid ester forms); Alpha-tocopherol succinate (including d-alpha-tocopherol succinate or dl-alpha-tocopherol succinate forms); C4: 0 to C6: 0 triglycerides (for example, tributyrin etc.); C8: 0 to C14: 0 triglycerides (for example, tricaproin or caprylic / capric triglyceride, etc.); It may be selected from the group consisting of castor oil, corn oil, olive oil, cottonseed oil, peppermint oil, sesame oil and soybean oil (including refined soybean oil).

In addition, when the pharmaceutical composition is in the form of a nano-suspension, the oil is a solid soybean oil (hydrogenated soybean oil), cacao butter, cetyl alcohol, stearyl alcohol, cetyl palmitate, carnauba wax, pewter (white) at room temperature bee wax), tricaprine (e.g., Migriol 812, etc.), trilaurin, trimyristin, tripalmitin, tristearin, and tribehenin.

More preferably, the oil is glyceryl mono-, di-, or tri-acetate (glyceryl mono-, di-, or tri-acetate); Alpha-tocopherol or salts thereof; Alpha-tocopherol acetic acid esters (including d-alpha-tocopherol acetic acid ester or dl-alpha-tocopherol acetic acid ester forms); Alpha-tocopherol succinate (including d-alpha-tocopherol succinate or dl-alpha-tocopherol succinate forms); Tributyrin; And tricaproin may be selected one or more.

The content of the oil may be in the range of 1 to 50% by weight, preferably 1 to 30% by weight, based on the total weight of the composition.

The nanoparticles also include chemical and / or biological substances having an activity of inhibiting growth, metastasis, etc. of cancer cells, that is, anticancer agents (antitumor agents), and the anticancer agents are not limited in kind. Examples of anticancer agents contained in the nanoparticles of the present invention include paclitaxel, paclitaxel derivatives, uracil, 5-fluorouracil, tegafur, methotrexate, and melparan. (Melphalan), Mitoxantrone, Camptothecin, Topotecan, Docetaxel, Capecitabine, Imatinib mesylate, Rituximab, Doxifluridine, toremifene citrate, doxorubicin, gemcitabine, irinotecan, oxaliplatin, or chlorambucil . Preferably, the anticancer agent includes paclitaxel, paclitaxel derivatives, docetaxel, uracil, 5-fluorouracil, tegaper, methotrexate, melfaran, mitoxantrone, camptothecin, or topotecan. The therapeutically effective amount of the anticancer agent can be easily determined by those skilled in the art from known literature.

In addition, the pharmaceutical composition in the form of nano-dispersion may further include a surfactant for stable dispersion of the nanoparticles, and may selectively add a surfactant to the nanoparticles and / or the aqueous medium. The type of surfactant may be appropriately selected for effective dispersion stability of the nanoparticles. For example, the surfactant may be alpha-tocopherol polyethylene glycol succinate (TPGS), macrogol 15 hydroxystearate (eg, solutol HS15, etc.), caprylocaproyl macrogolglycerides caprylocaproyl macrogolglycerides (e.g., labrazol, etc.), sorbitan fatty acid esters (e.g., sorbitan monooleate (e.g., Span 20), etc.), polysorbates [For example, tween 20, tween 80, etc.], polyoxyethylene-polyoxypropylene block copolymer (for example, poloxamer 188, poloxamer 407, etc.), egg yolk lecithin , And soybean lecithin may be selected from one or more species, but is not limited thereto. The amount of the surfactant may also be appropriately selected depending on the type of oil used, for example, but may be in the range of 1 to 50% by weight based on the total weight of the composition, but is not limited thereto.

The aqueous medium contained in the pharmaceutical composition of the present invention may be selected from one or more selected from the group consisting of distilled water, water for injection, saline solution, glucose solution, and amino acid solution, but is not particularly limited.

If necessary, the nanoparticles may be formed of polyethylene glycol, dimyristoyl phosphatidyl ethanolamine-polyethylene glycol (DMPE-PEGs) (eg , DMPE-PEG ₂₀₀₀ ), polypropylene glycol, glycofurol, and tricapryline may further comprise at least one dissolution aid selected from the group consisting of: The content of the dissolution aid may be in the range of 0.05 to 10% by weight, preferably 0.05 to 5% by weight, based on the total weight of the composition.

In addition, the nanoparticles may further include an amino acid having a carboxyl group as a low molecular ligand in order to stably bind hyaluronic acid or a salt of hyaluronic acid to the nanoparticle surface. The low molecular ligand may be at least one selected from the group consisting of glutamic acid, aspartic acid, aspartic acid, histidine, and alanine, and more preferably glutamic acid and / or aspartic acid. The content of the low molecular ligand may be in the range of 0.005 to 0.2% by weight based on the total weight of the composition.

In addition, the pharmaceutical composition of the nano-dispersion form may be adjusted to the pH of the composition to 2.0 to 6.0 by adding an organic acid or N-acetyl amino acid as a pH adjusting agent to improve the chemical stability of the anticancer agent. The pH adjusting agent may optionally be added to the nanoparticles and / or the aqueous medium. The pH adjusting agent may be at least one selected from the group consisting of citric acid, acetic acid, phosphoric acid, lactic acid, benzoic acid, maleic acid, succinic acid and tartaric acid as organic acids, and N-acetyl cysteine, N-acetyl valine, N as N-acetyl amino acid. It can be used by selecting one or more from the group consisting of -acetyl proline and N-acetyl alanine.

If necessary, the pharmaceutical composition in the form of a nano-dispersion according to the present invention is one from the group consisting of polyvinylpyrrolidone, glycerin, glucose, sucrose, lactose, sorbitol, mannitol, and trehalose. It may further comprise a dispersion stabilizer selected above. The dispersion stabilizer not only helps maintain the dispersibility of the pharmaceutical composition in the form of nano-dispersions, but also serves to facilitate the redispersion of the nanoparticles obtained by drying the nano-dispersions in the dispersion medium. The amount of the dispersion stabilizer is not particularly limited, but may be used in an amount of 1 to 20% by weight, preferably 3 to 7% by weight, based on the total weight of the composition.

The invention also provides nanoparticles for target orientation, obtained by drying the pharmaceutical composition in the form of nano-dispersions by lyophilization, rotary evaporation, spray drying, or fluid bed drying.

The present invention also includes the steps of (a) preparing an oil phase comprising a therapeutically effective amount of an anticancer agent, a divalent or trivalent transition metal ion or an alkaline earth metal ion, and an oil; (b) dissolving hyaluronic acid or a salt thereof in an aqueous medium to obtain an aqueous phase; And (c) mixing and homogenizing the oil phase obtained in step (a) and the water phase obtained in step (b) to form nanoparticles having hyaluronic acid or a salt thereof bound on the surface as nanoparticles having an average particle diameter of 10 to 1,000 nm. It provides a method of preparing a pharmaceutical composition in the form of nano-dispersion for the target orientation, comprising the step.

In addition, in the above production method of the present invention, the oil phase and / or water phase may further include a surfactant.

In the preparation method of the present invention, the anticancer agent, divalent or trivalent transition metal ion or alkaline earth metal ion, oil, surfactant, hyaluronic acid or salt thereof is as mentioned above.

The oil phase is (i ') dissolving the anticancer agent and optionally a surfactant in an organic solvent, (ii') dissolving the divalent or trivalent transition metal ions or alkaline earth metal ions in the oil, and (iii ') Can be obtained by drying the mixture of the solution obtained in step (i') and the solution obtained in step (ii ') to remove the organic solvent. The organic solvent may be methanol, ethanol, acetonitrile, acetone, and the like, preferably anhydrous ethanol.

In addition, the oil phase is one or more from the group consisting of polyethylene glycol, dimyristoyl phosphatidyl ethanolamine-polyethylene glycol (DMPE-PEGs), polypropylene glycol, glycofurol, and tricapryline It may further comprise a selected dissolution aid, the amount of the dissolution aid is as described above.

The aqueous phase may be obtained by further dissolving an amino acid having a carboxyl group as a low molecular ligand, and the type and amount of the low molecular ligand are as described above. In addition, the aqueous phase further dissolves a dispersion stabilizer selected from the group consisting of polyvinylpyrrolidone, glycerin, glucose, sucrose, lactose, sorbitol, mannitol, and trehalose as necessary. The dispersion stabilizer may be used as described above.

The homogenization can be carried out according to a conventional homogenization method, for example, homogenizer, by homogenizing using an ultrasonic homogenizer, high pressure homogenizer, etc., an average particle diameter of 10 to 1,000 nm, preferably 20 to 200 nm. It is possible to form a uniform fine droplet having a. As described above, the homogenized fine droplets may be subjected to a sterile process by passing through a sterile filter having a pore of 0.22 μm as necessary.

The invention also comprises the step of drying (eg, lyophilization, rotary evaporation drying, spray drying, or fluidized bed drying) of the pharmaceutical composition in the form of nano-dispersions obtained by the above method, nano for target orientation. Provided are methods for preparing the particles.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example 1 Preparation of Nano-Emulsions and Nanoparticles

According to the components and contents of Table 1 below, nano-emulsions were prepared. That is, paclitaxel was added to anhydrous ethanol at a concentration of 10% (w / v), followed by mixing with polysorbate 80 and low shear stirring at a temperature of 40 to 45 ° C. until completely dissolved. To the mixed oil of dl-alpha-tocopheryl acetate and refined soybean oil, a solution obtained by completely dissolving iron (III) chloride at room temperature was added thereto, followed by vacuum drying at 40 to 45 ° C. under low shear stirring to completely remove ethanol. An oil phase was prepared. An aqueous phase was prepared by dissolving sodium hyaluronate (average molecular weight: 1,067,000 Daltons, Bioiberica, Spain) in water for injection at room temperature. The aqueous phase warmed to 40-45 ° C. was added to the oil phase to adjust the total mass to 100 g and then homogenized to prepare a nano-emulsion, and the resulting nano-emulsion was sterile filtered. The temperature of the entire process of preparing the nano-emulsion was maintained at about 40 ° C. or more to prevent gelation of the oil phase, and the homogenization was performed using a high pressure homogenizer (Microfluidizer M-110S, Microfluidics Inc, USA) at a pressure of about 15 kpsi. A total of 10 cycles were carried out while maintaining a temperature of 15-18 ° C. under the conditions. In addition, the sterile filtration is a filter having an air gap of 0.22 ㎛ (Acro 50 Vent Devices with Emflon Membrane  II, Pall Co. USA), wherein some of the anticancer drug nuclei formed during the emulsion preparation were removed. The particle size of the droplets in the obtained nano-emulsion was measured using a Zetasizer NanoZS (Malvern Co. USA) analyzer, the average particle diameter of about 70 ~ 80 nm.

division ingredient Content (g) Paid Paclitaxel 0.300 Ferric chloride (Ⅲ) 0.002 dl-alpha-tocopheryl acetate 7.200 Refined Soybean Oil 0.800 Polysorbate 80 8.000 Awards Water for injection 83.447 Sodium hyaluronate 0.251

In addition, mannitol was added to the nano-emulsion obtained above at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

Example 2. Preparation of Nano-Suspensions and Nanoparticles

According to the components and contents of Table 2 below, nano-suspensions were prepared. Paclitaxel was added to anhydrous ethanol at a concentration of 10% (w / v), mixed with TPGS and poloxamer 407, and dissolved by low shear stirring at a temperature of 55 to 60 ° C. Iron (III) chloride was added to a mixed oil of soybean cured oil and Migriol 812, and then a solution completely dissolved at 55 to 60 ° C. was added to the solution containing paclitaxel, followed by vacuum at 55 to 60 ° C. under low shear agitation. Drying completely removed the ethanol to prepare an oil phase. An aqueous phase was prepared by dissolving sodium hyaluronate (average molecular weight: 1,067,000 Daltons, Bioiberica, Spain) in water for injection at room temperature. The water phase warmed to 55-65 ° C. was then added to the oil phase to bring the total mass to 100 g and then homogenized to produce nano-emulsions. The resulting nano-emulsion was added to a cold water bath at 1 to 3 ° C. to prepare a solidified lipid nano-suspension. The prepared nano-suspension was made available for injection through sterile filtration. The homogenization was carried out over a total of five cycles using a high pressure homogenizer (Microfluidizer M-110S, Microfluidics Inc, USA) maintaining a temperature of 55-65 ° C. at about 15 kpsi pressure. In addition, the sterile filtration is a filter having an air gap of 0.22 ㎛ (Acro 50 Vent Devices with Emflon Membrane  II, Pall Co. USA), wherein some of the anticancer drug nuclei formed during the suspension preparation process were removed. The particle diameter of the obtained nano-suspension was measured using a Zetasizer NanoZS (Malvern Co. USA) analyzer, the average particle diameter of about 100 to 140 nm.

division ingredient Content (g) Paid Paclitaxel 0.300 Ferric chloride (Ⅲ) 0.008 Soybean Cured Oil (Akosol) 19.000 Migriol 812 0.950 TPGS 4.750 Poloxamer 407 4.750 Awards Water for injection 70.031 Sodium hyaluronate 0.211

In addition, mannitol was added to the nano-suspension obtained above at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

Examples 3-7. Preparation of Nano-Emulsions and Nanoparticles

According to the components and contents of Table 3 below, nano-emulsions were prepared in the same manner as in Example 1. However, TPGS was divided | segmented and applied to the oil phase and the water phase as shown in Table 3. In addition, mannitol was added to the obtained nano-emulsion at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

division ingredient Content (g) Example 3 Example 4 Example 5 Example 6 Example 7 Paid 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 Awards 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-Emulsions and Nano Particles

According to the components and contents of Table 4 below, a dissolution aid and a dispersion stabilizer were further used to prepare a nano-emulsion in the same manner as in Example 1. In addition, mannitol was added to the obtained nano-emulsion at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

division ingredient Content (g) Example 8 Example 9 Example 10 Example 11 Example 12 Example 13 Paid 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-alpha-tocopherol - - 6.900 9.600 - - Glycofurol - - 5.100 2.400 - - Tricapryline - - - - 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 - - Awards 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 14-18. Preparation of Nano-Emulsions and Nanoparticles

According to the component and the content of Table 5 below, the surfactant was changed to prepare a nano-emulsion in the same manner as in Example 1. In addition, mannitol was added to the obtained nano-emulsion at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

division ingredient Content (g) Example 14 Example 15 Example 16 Example 17 Example 18 Paid 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-alpha-tocopherol 5.000 2.500 - - - Egg yolk lecithin 4.000 4.800 - - - TPGS - - 2.250 2.250 3.500 Solutol HS-15 - - 1.500 - - Awards 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-Emulsions and Nanoparticles

According to the components and contents of Table 6 below, nano-emulsions were prepared in the same manner as in Example 1. In addition, mannitol was added to the obtained nano-emulsion at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

division ingredient Content (g) Example 19 Example 20 Example 21 Example 22 Example 23 Paid 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-alpha-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 - - Awards 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-Emulsions and Nanoparticles

According to the components and contents of the following Table 7, nano-emulsion was prepared in the same manner as in Example 1. In addition, mannitol was added to the obtained nano-emulsion at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

division ingredient Content (g) Example 24 Example 25 Paid 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 - Awards Water for injection 81.950 81.950 Soybean Lecithin - 1.500 Sodium hyaluronate 0.247 0.247

Examples 26 to 31. Preparation of Nano-Emulsions and Nanoparticles

As shown in Table 8, using the sodium hyaluronate having a variety of molecular weight, the nano-emulsion was prepared by the same composition and preparation method as in Example 1. The particle diameter of the droplets in the obtained nano-emulsion was 50 to 250 nm. In addition, mannitol was added to the obtained nano-emulsion at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

Example 26 Example 27 Example 28 Example 29 Example 30 Example 31 Hyaluronic Acid Average Molecular Weight (Dalton) 3,642,000 2,000,000 1,490,000 800,000 15,000 4,280

Examples 32 to 38. Preparation of Nano-Emulsions and Nanoparticles

According to the components and contents of Table 9 below, about 0.018 mmol of metal ions derived from iron nitrate, iron acetate, copper sulfate, nickel chloride, and cobalt chloride was used to prepare a nano-emulsion in the same manner as in Example 1. The particle diameter of the droplets in the obtained nano-emulsion was 60 to 140 nm. In addition, mannitol was added to the obtained nano-emulsion at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

Metal ion Metal salt Example 32 Fe ³⁺ Iron nitrate (Ⅲ) Example 33 Fe ²⁺ Iron nitrate (Ⅱ) Example 34 Fe ₂₊ Iron acetate (II) Example 35 Cu ²⁺ Copper sulfate (Ⅱ) Example 36 Ni ²⁺ Nickel Chloride (II) Example 37 Co ³⁺ Cobalt Chloride (III) Example 38 Co ²⁺ Cobalt Chloride (II)

Examples 39-41. Preparation of Nano-Emulsions and Nanoparticles

According to the components and contents of Table 10 below, nano-emulsions were prepared in the same manner as in Example 1 using various amounts of sodium hyaluronate. The particle diameter of the droplets in the obtained nano-emulsion was 60 to 80 nm. In addition, mannitol was added to the obtained nano-emulsion at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

Example 39 Example 40 Example 41 Sodium hyaluronate (g) 0.080 0.160 0.319

Examples 42-45. Preparation of Nano-Emulsions and Nanoparticles

According to the ingredients and contents of Table 11 below, using the various amounts of iron (III) chloride, nano-emulsion was prepared in the same manner as in Example 1. The particle diameter of the droplets in the obtained nano-emulsion was 60-90 nm. In addition, mannitol was added to the obtained nano-emulsion at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

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-Emulsions and Nanoparticles Using Hyaluronic Acid Free Base

In the same composition and preparation method as in Example 1, the nano-emulsion was prepared using the salt-free hyaluronic acid free base, and the particle diameter of the droplets in the obtained nano-emulsion was 60 to 80 nm. In addition, mannitol was added to the obtained nano-emulsion at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

The hyaluronic acid from which the salt was separated was obtained as follows: 0.5% (w / v) sodium hyaluronate was dissolved in a solution in which 0.1 M hydrochloric acid and ethanol anhydride were mixed at a 3: 7 (v / v) ratio. The solution was slowly stirred at room temperature for 24 hours and then filtered through a 200 to 400 mesh filter to recover the precipitate remaining in the filter to obtain a hyaluronic acid free base. This was washed three times with anhydrous ethanol and dried in vacuo at room temperature to obtain pure hyaluronic acid free base. The hyaluronic acid free base was checked for salt removal by pH measurement (pH 2.65) and infrared spectroscopy.

Example 47. Preparation of Nano-Emulsions and Nanoparticles Using Docetaxel

According to the ingredients and contents of Table 12 below, using docetaxel as an anticancer agent, a nano-emulsion was prepared in the same manner as in Example 1. The particle diameter of the droplets in the obtained nano-emulsion was 70 to 80 nm. In addition, mannitol was added to the obtained nano-emulsion at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

division ingredient Content (g) Paid Docetaxel 1,000 Ferric chloride (Ⅲ) 0.003 dl-alpha-tocopheryl acetate 11.880 Tricapryline 0.120 Polysorbate 80 5.400 Awards Water for injection 75.558 Polyvinylpyrrolidone 1.632 glycerin 4.080 Glutamic acid 0.082 Sodium hyaluronate 0.245

Example 48. Preparation of Nano-Emulsions and Nanoparticles Comprising PEGylated Phospholipids and 5-Fluorouracil

According to the ingredients and contents of Table 13 below, using the 5-fluorouracil (50 mg / mL) as an anticancer agent, a nano-emulsion was prepared in the same manner as in Example 1. The particle diameter of the droplets in the obtained nano-emulsion was 90 to 130 nm. In addition, mannitol was added to the obtained nano-emulsion at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

division ingredient Content (g) Paid 5-fluorouracil 5.000 Ferric chloride (Ⅲ) 0.003 Myvacet 9-45K 11.880 Tricapryline 0.120 TPGS 2.500 DMPE-PEG ₂₀₀₀ 1,000 Awards 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-Emulsions and Nanoparticles

According to the components and contents shown in Table 14 below, nano-emulsions were prepared in the same manner as in Example 1, using citric acid and N-acetyl cysteine as pH adjusting agents. The particle diameter of the nanoparticles in the obtained nano-emulsion was 70 to 80 nm. In addition, mannitol was added to the obtained nano-emulsion at a concentration of 5% (w / v), and then lyophilized to obtain nanoparticles.

division ingredient Content (g) Example 49 Example 50 Paid Paclitaxel 0.300 0.300 Ferric chloride (Ⅲ) 0.002 0.002 dl-alpha-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 Awards Water for injection 83.445 83.446 Sodium hyaluronate 0.251 0.251

Comparative Examples 1 to 3 Emulsions Containing Paclitaxel

According to the component and the content of Table 15 below, a nano-emulsion was prepared in the same manner as in Example 1.

division ingredient Content (g) Comparative Example 1 Comparative Example 2 Comparative Example 3 Paid Paclitaxel 0.300 0.300 0.300 Ferric chloride (Ⅲ) - 0.002 - dl-alpha-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 Awards Water for injection 83.700 83.698 83.700 Sodium hyaluronate - - 0.251

Test Example 1. Measurement of coating amount of hyaluronic acid

After ultracentrifugation of the nanoparticles, namely nano-emulsions, obtained in Example 1 at 20 ° C. for 2 hours, the precipitates were washed three times with water for injection and dried at room temperature to obtain an aggregate of hyaluronic acid-bound emulsions. . The analysis of hyaluronic acid binding amount was performed according to the hyaluronic acid quantitative method published in the European Pharmacopoeia (EP), the specific quantitative method is as follows.

Distilled water was added to 0.170 g of the emulsion aggregate obtained after ultracentrifugation to adjust to 100 g, and then diluted 40 times to prepare a sample solution. Distilled water was added to 0.1 g of D-glucuronic acid and adjusted to 100 g. Five standard solutions containing 6.5 to 65 µg of D-glucuronic acid were prepared per 1 g. 5 mL of a sulfuric acid solution of 0.95 w / v% borax was added to 1.0 mL of the prepared sample solution and the standard solution, and the mixture was allowed to stand in a water bath for 15 minutes and then cooled in ice water. 0.2 mL of 0.125 w / v% carbazole-containing ethanol solution was added to the cooled solution, followed by mixing in a water bath for 15 minutes and cooling to room temperature. The solution was measured for absorbance at 530 nm according to the ultraviolet visible absorbance method. This was performed three times, and the average concentration of D-glucuronic acid in the sample solution was calculated using the calibration curve prepared from the standard solution. The results are shown in Table 16.

Amount of sodium hyaluronate (%) = (Cg / Cs) × Z × [100 / (100-h)] × (401.3 / 194.1)

Cg: average concentration of D-glucuronic acid in the samples (mg / g)

-Cs: the average concentration of Fernoxib in mg (mg / g)

-Z: content of D-glucuronic acid (%)

h: loss on drying (%)

401.3: Relative molecular weight of disaccharides

194.1: Relative molecular weight of glucuronic acid

Bound hyaluronic acid content Average particle size of nano-emulsion 0.8 ± 0.1 wt% 74 ± 3 nm

Test Example 2 In vitro Affinity Test for CD44

To measure CD44 affinity, SK-OV-3 cells (ATCC: HTB-77, Rockville, MD) and OVCAR-3 cells (Korea Cell Line Bank, KCLB-00000287) were used. SK-OV-3 expresses CD44 on the cell surface and OVCAR-3 does not express CD44 on the cell surface. The cells were treated with 5% CO ₂ , in RPMI 1640 medium (HA free medium, Gibco, Carlsbad, USA) containing 100 U / mL penicillin, 0.1 mg / mL streptomycin and 10% fetal bovine serum (FBS). Incubated at 37 ° C. The cells incubated in 1 × 10 ⁶ cells were charged with FITC-labeled nano-emulsions (Example 1, Comparative Examples 1 to 3), and then reacted at 37 ° C. for 4 hours. The labeled nano-emulsions were obtained by dissolving 0.2 mg of FITC in nanodroplets, and some unlabeled fluorescent material was removed using a dialysis membrane (Pierce, MWCO 2,000).

After the reaction, the cells were washed twice with phosphate buffered saline (PBS), treated with 0.1% trypsin for about 1 minute, 0.2% fetal bovine serum (FBS) and 0.02% sodium azide (Sodium) Azide) was washed three times with PBS solution. The recovered cell-sample was redispersed in 400 uL of 4% paraformaldehyde and the CD44 affinity was measured using a Fluorescence-activated cell sorter (FACS) and epi-fluorescent microscopy. Whether hyaluronic acid nanocapsules were bound to breast cancer cells was measured by fluorescence scanning at 488 nm with a FACScalibur (Beckton-Dickinson, Mansfield, MA) scanner. The result is shown in FIG. 3.

A and C of Figure 3 is an OVCAR-3 cell line, B and D of Figure 3 is the result of measuring the binding and affinity to SK-OV-3 cell line. As a result of the measurement, all of the above samples showed little non-specific binding, and the samples containing no hyaluronic acid showed little specific binding with CD44 on the surface of the cell line, and the composition of the hyaluronic acid-containing composition There was a difference of, but specific binding with CD44 appeared. Specifically, the emulsion (Comparative Example 1) and the emulsion containing the metal ion (Comparative Example 2) did not show a specific binding phenomenon with CD44, and in the case of an emulsion in which only hyaluronic acid was simply mixed (Comparative Example 3), to some extent, The affinity with CD44 was shown, and the affinity with CD44 was most excellent for the nano-emulsion of the present invention (Example 1) containing nanodroplets having a hyaluronic acid coating layer. Thus, it can be seen that the nano-emulsions of the present invention are excellent drug carriers for active target orientation.

Test Example 3 In vivo Efficacy Test

In order to evaluate the anticancer efficacy by active and passive target-oriented, SK-OV-3 cells (ATCC: HTB-77, Rockville, MD), which are overexpressed CD44 on the cell surface, and CD44 are not expressed on the cell surface. OVCAR-3 cells (Korea Cell Line Bank, KCLB-00000287) were used.

SK-OV-3 cells and OVCAR-3 cells, each using a RPMI 1640 medium containing 10% FBS, were transplanted into test animals in an incubator at 37 ° C. and 5% CO ₂ , ie, SK. -OV-3 cells were cultured until 7 × 10 ⁸ or more and OVCAR-3 cells reached 4 × 10 ⁸ or more. The RPMI 1640 medium contains 0.1 mM non-essential amino acid, 1 mM sodium pyruvate, 1.5 g / L sodium bicarbonate, and an amount of antibiotic (antibiotic / mycotic agent). It was prepared to contain, stored in the refrigerator after filtration, and warmed to 37 ℃ each time used.

After reaching a certain number of SK-OV-3 cells and OVCAR-3 cells were treated with trypsin / EDTA solution, separated from the culture flasks, and then 1 × 10 ⁷ cells were suspended in 0.3 mL of RPMI 1640. In addition, the cancer cells were transplanted by injecting the suspended cancer cell solution 0.3 mL at the subcutaneous side of nude mice (BALB / cAnNCrjBgi-nu / nu, Female) using a 1 mL syringe of 23G. After cancer cell transplantation, when the tumor size reached about 100 mm 3 or more, test substances were administered (n = 7) by dividing into five groups (G1-G5) so that the mean value of the tumor size was similar. In the G2 test group, Taxol, a positive control, was administered intravenously, the G3 test group was administered the nano-emulsion of Example 1, the G4 test group was administered the nano-suspension of Example 2, and the G5 test group. The nano-emulsion of Comparative Example 1 was administered. The dose of paclitaxel, the main component of the G2-G5 test group, was constant at 20 mg / kg, and the G1 control group did not administer the main component and the excipient to observe the growth type of cancer cells. The test was performed with only (Hypertonic saline). In order to evaluate anticancer activity, tumor size changes of test animals were measured 2-3 times a week at intervals of about 3 days from when cancer growth was observed to some extent. Tumor size was measured using the Vernier calipers and the tumor size and length were calculated by the following equation. The change of was evaluated. The results are the same as in Figs. 4a and 4b.

Tumor (Tumor Volume) (㎣) = length (㎜) X [width (㎜)] ^2/2

Figure 4a is a result of tumor size change over time after administration of the test drug and the control drug in SK-OV-3 cancer transplantation model overexpressed CD44. Examples of the nano-emulsion and nano-suspension administration groups of Examples 1 and 2 were Taxol  Compared with the administration group and the nano-emulsion administration group of Comparative Example 1 showed an excellent cancer growth inhibitory effect. Examples 1 and 2 are nanoparticles to which hyaluronic acid is bound, and it is shown that the specific target of hyaluronic acid and CD44 has an active target-directing effect of the drug. On the other hand, the composition of Comparative Example 1 can be seen that the anti-hyaluronic acid-coated emulsion formulation is significantly reduced cancer growth inhibitory effect, which indicates that the composition of Comparative Example 1 shows only the passive target-oriented effect of the drug by the EPR effect CD44 It is considered to be a phenomenon that does not exhibit an active target orientation effect by specific binding of.

Figure 4b shows the change in tumor size over time after administration of the test drug and the control drug in OVCAR-3 cancer transplantation model that does not express CD44. The nano-emulsion administration group of Example 1, the nano-suspension administration group of Example 2, and the emulsion administration group of Comparative Example 1 showed higher cancer growth inhibitory effect than the negative control group and the Taxol administration group. In the OVCAR-3 cancer transplantation model, since CD44 is not expressed, the active-targeting effect of the nano-emulsion of Example 1 does not appear, but only the passive-targeting effect by the EPR effect.

Test Example 4 Evaluation of In Vivo Toxicity

Only healthy test animals that are recognized as suitable for repeated dose toxicity evaluation through adaptive breeding are selected, and then 5 dogs are placed per breeding box using the stepless randomization method, shake the test substance just before administration, and then a disposable sterile syringe is used. It was administered to the vein of the test animal. According to the body weight measured on the day of administration, each test substance was repeatedly administered three times at intervals of 3 days, and mortality and symptoms according to test substance administration were observed. Symptoms were observed intermittently depending on the situation up to 4 hours after administration, and observed once a day from thereafter until the end of observation, body weight was measured once a day from the start of administration to the end of observation. After the observation period, autopsies and examinations of surviving subjects were performed to evaluate the safety of the test substance, if necessary. The result is shown in FIG.

When the reference drug Taxol was repeatedly administered to nude mice three times at a concentration of 30 mpk, a sudden weight loss of about 20% was observed, and three nude mice died of serious toxicity after the administration or completion of the administration. Also all Taxol  The administration group showed symptoms such as dyspnea, lethargy, and ataxia regardless of paclitaxel concentration. On the other hand, when the composition of Example 1 was repeatedly administered at a concentration of 20 to 50 mpk three times, all the administration groups showed a change in body weight within 10%, indicating a relatively safe toxicity level compared to the reference drug, which is nano- Emulsion means that the maximum tolerated dose of drug in nude mice is 50 mpk or more. In addition, none of the nano-emulsion groups in Example 1 died or exhibited any particular clinical symptoms.

Test Example 5 Drug Release Test

For the nanoparticles prepared according to the present invention, the dissolution test was carried out as follows according to the method of the Korean Pharmacopoeia.

The nano-emulsion obtained in Example 1, the nano-suspension obtained in Example 2 and the Taxol injection of Bristol Myers Squib (BMS) were dialyzed with a molecular weight cutoff (MWCO) of 12,000 to 14,000 (Spectra / It was added to 1,000 mL eluate (0.1% polysorbate 80-phosphate buffer, pH 7.08) in a Por dialysis membrane. The dialysis membrane was immersed in the eluent for about 30 minutes before use to stabilize it. The dialysis membrane was fixed to the support to form a sink condition with sufficient volume of the eluent and the rotation of the paddle (100 rpm), and the temperature was maintained at 37 ° C. After taking 1 mL of the eluate filtered out of the dialysis membrane with time, the elution rate of paclitaxel was calculated using high performance liquid chromatography. The eluate was constantly replenished with the eluent to maintain 1,000 mL. The result is shown in FIG. 6, the dissolution rate was measured for 6 days, and compared with the Taxol injection formulation, it can be seen that the nanoparticles of the present invention continuously release paclitaxel over a long time. It can also be seen that the nano-suspension shows a slightly more sustained release pattern than the nano-emulsion. Since there is an elapsed time for the nanoparticles injected into the human body to reach a target cancer cell, it is advantageous for the therapeutic effect that the drug arrives at the target point of the cancer cell and is released slowly, rather than being released rapidly. Therefore, the nanoparticles according to the present invention are very useful for effective delivery of drugs.

Test Example 6. Evaluation of Encapsulation Efficiency and Redispersibility of Nanoparticles

The encapsulation efficiency and redispersibility of the nanoparticles obtained by lyophilization in Example 1 were evaluated. In addition, as a control formulation, nanoparticles according to the inventors' previous results, that is, nanoparticles according to the Republic of Korea Patent Registration No. 774,925 (Control Example 1) and Patent Publication No. 10-2009-0040979 (Prepared according to Example 93) And Comparative Example 2) were also evaluated in the same manner. The nanoparticles of Comparative Example 1 were prepared as follows: 0.135 g of paclitaxel and 0.045 g of hyaluronic acid were added to 45 mL of a 70% ethanol solution containing 0.02 M glutamic acid to obtain an aqueous solution. While stirring the obtained solution at 300 rpm, 0.84 mL of 0.05 M iron (III) nitrate aqueous solution was added thereto, stirred for 2 hours, and lyophilized to obtain nanoparticles having a particle size of about 120 nm.

The encapsulation efficiency was measured by filtering a dispersion (Example 1, Comparative Examples 1 and 2) dispersed in water at a constant concentration by a filter having a porosity of 0.22 μm (Acro 50 Vent Devices with Emflon Membrane® II, Pall Co. USA). The supernatant was taken by ultracentrifugation, and compared and evaluated according to the paclitaxel content analysis described in the European Pharmacopoeia (EP). Subsequently, the redispersibility of the formulation was evaluated by comparing the encapsulated content and particle size of the paclitaxel obtained by re-dispersing the lyophilized formulation into water for injection and comparing with the paclitaxel content and particle size of the preparation before lyophilization. The results are shown in Table 17 below.

Before lyophilization Redistribution Inclusion content
(mg / mL) Encapsulation efficiency
(%) Particle size
(nm) Inclusion content
(mg / mL) Encapsulation efficiency
(%) Particle size
(nm) Example 1 3.00 ± 0.04 100.3 ± 0.1 70 ± 1 3.01 ± 0.03 100.3 ± 0.1 71 ± 2 Comparative Example 1 0.41 ± 0.11 13.7 ± 3.7 127 ± 9 0.18 ± 0.08 6.0 ± 2.7 862 ± 98 Comparative Example 2 0.22 ± 0.05 36.7 ± 8.3 104 ± 13 0.13 ± 0.03 21.7 ± 5.0 128 ± 34 *

* Determine particle size after removing some clumped particles

As can be seen from Table 17, the nanoparticles according to the present invention had the highest encapsulation content of the drug, paclitaxel, and the encapsulation efficiency was also excellent. In addition, even when redispersed in the dispersion medium (injection water), the content and particle size of the drug were maintained as it is.

Test Example 7 Evaluation of Physical and Chemical Stability

The lyophilized nanoparticles obtained in Example 1 were evaluated for physicochemical stability at room temperature (25 ± 2 ° C., 60 ± 5% RH) and accelerated (40 ± 2 ° C., 75 ± 5% RH) for 6 months. The change in the inclusion content of paclitaxel and the change in the size of the nanoparticles were measured three times in the same manner as in Test Example 6, and the average value was calculated. The results are shown in Table 18.

storage duration
(month)
Room temperature conditions
(25 ± 2 ℃, 60 ± 5% RH) Acceleration condition
(40 ± 2 ℃, 75 ± 5% RH) Encapsulation Content (mg / ml) Particle size (nm) Encapsulation Content (mg / ml) Particle size (nm) Early 3.01 ± 0.03 71 ± 2 3.01 ± 0.03 71 ± 2 One 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 can be seen from Table 18, the particle size and content of the lyophilized formulation redispersed with water for injection was the same as the nano-emulsion before lyophilization, it can be seen that the physicochemical stability is maintained for 6 months.

1 shows a schematic diagram of nanoparticles according to the present invention.

Figure 2 shows a Cyro-TEM image of the nanoparticles according to the present invention.

3 is a result of measuring the CD44 affinity of the nanoparticles according to the present invention. In Figure 3, A and C are OVCAR-3 cell line, B and D is the result of measuring the affinity and affinity for SK-OV-3 cell line.

4a and 4b shows the results of evaluating the anticancer efficacy of the active and passive target orientation of the nanoparticles according to the present invention. 4A and 4B show changes in tumor size over time after administration of the test and control agents, respectively, in the SK-OV-3 cancer transplantation model overexpressing CD44 and the OVCAR-3 cancer transplantation model without CD44 expression, respectively. The results shown.

5 is a result of the safety evaluation of the nanoparticles according to the present invention, which is the result of measuring the weight change for 2 weeks after administration of the test substance.

Figure 6 shows the dissolution test results of the nanoparticles according to the present invention.

Claims (29)

A pharmaceutical composition for target orientation in the form of nano-dispersions comprising nanoparticles having an average particle diameter of 10 to 1,000 nm in an aqueous medium, the pharmaceutical composition comprising a therapeutically effective amount of an anticancer agent; Divalent or trivalent transition metal ions or alkaline earth metal ions; And an oil, wherein a hyaluronic acid or a salt thereof is bound to a surface of the nanoparticle. The pharmaceutical composition according to claim 1, wherein the average molecular weight of hyaluronic acid is 379 to 10,000,000 Daltons. The pharmaceutical composition according to claim 1, wherein the hyaluronic acid salt is cobalt hyaluronic acid, magnesium hyaluronate, zinc hyaluronate, calcium hyaluronate, potassium hyaluronate, sodium hyaluronate, or tetrabutylammonium hyaluronate. The pharmaceutical composition according to claim 1, wherein the content of hyaluronic acid or a salt thereof is 0.01 to 1.0% by weight based on the total weight of the composition. The method of claim 1, wherein the divalent or trivalent transition metal ions or alkaline earth metal ions are Cu ²⁺ , Cu ³⁺ , Zn ²⁺ , Zn ³⁺ , Ni ²⁺ , Ni ³⁺ , Mg ²⁺ , Mg One or more from the group consisting of ³⁺ , Ca ²⁺ , Ca ³⁺ , Co ²⁺ , Co ³⁺ , Ba ²⁺ , Ba ³⁺ , Al ²⁺ , Al ³⁺ , Fe ²⁺ , and Fe ³⁺ Pharmaceutical composition, characterized in that selected. The method of claim 1, wherein the divalent or trivalent transition metal ions or alkaline earth metal ions are selected from the group consisting of copper nitrate, copper sulfate, copper chloride, zinc acetate, zinc sulfate, zinc nitrate, zinc chloride, nickel sulfate, nickel nitrate, nickel chloride and acetic acid. Magnesium, 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, acetic acid A pharmaceutical composition, characterized in that it is derived from iron, iron sulfate, iron nitrate, or iron chloride. The pharmaceutical composition according to claim 1, wherein a molar ratio of the divalent or trivalent transition metal ions or alkaline earth metal ions per disaccharide unit of hyaluronic acid or a salt thereof is 0.001 to 2.0. The method of claim 1, wherein the pharmaceutical composition is in the form of a nano-emulsion, the oil is mono-, di-, or tri-glycerides (mono-, di-, or tri-glycerides); Glyceryl mono- or tri-stearate; Glyceryl mono-, di-, or tri-acetate; Alpha-tocopherol or salts thereof; Alpha-tocopherol acetic acid ester; Alpha-tocopherol succinate; C4: 0 to C6: 0 triglycerides; C8: 0 to C14: 0 triglycerides; Pharmaceutical composition, characterized in that at least one selected from the group consisting of castor oil, corn oil, olive oil, cottonseed oil, peppermint oil, sesame oil, and soybean oil. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is in the form of a nano-suspension and the oil is hydrogenated soybean oil, cacao butter, cetyl alcohol, stearyl alcohol, cetyl palmitate, carnauba wax, white bee wax), tricaprine, trilaurin, trimyristin, tripalmitin, tristearin, and tribehenin. The pharmaceutical composition according to claim 1, wherein the oil content is in the range of 1 to 50% by weight, based on the total weight of the composition. According to claim 1, wherein the anticancer agent Paclitaxel (Paclitaxel), Paclitaxel derivatives, Uracil (Uracil), 5-Fluorouracil (Tegafur), Methotrexate (Methotrexate), Melphalan (Melphalan) , Mitoxantrone, camptothecin, topotecan, topotecan, docetaxel, capecitabine, imatinib mesylate, rituximab, doxifluriri Pharmaceutical composition characterized in that it is doxifluridine, toremifene citrate, doxorubicin, gemcitabine, irinotecan, oxaliplatin, or chlorambucil . The method of claim 1, wherein the pharmaceutical composition is alpha-tocopherol polyethylene glycol succinate, macrogol 15 hydroxystearate, caprylocaproyl macrogolglycerides, sorbitan monooleate ( Surfactant selected from the group consisting of sorbitan monooleate, polysorbates, polyoxyethylene-polyoxypropylene block copolymer, egg lecithin, and soybean lecithin Pharmaceutical composition, characterized in that it further comprises. 13. The pharmaceutical composition of claim 12, wherein the amount of the surfactant is 1-50% by weight based on the total weight of the composition. The pharmaceutical composition according to claim 1, wherein the aqueous medium is selected from the group consisting of distilled water, water for injection, saline solution, glucose solution, and amino acid solution. The dissolution aid according to claim 1, wherein the nanoparticles are at least one selected from the group consisting of polyethylene glycol, dimyristoyl phosphatidyl ethanolamine-polyethylene glycol, cholesterol, polypropylene glycol, glycofurol, and tricapryline. Pharmaceutical composition, characterized in that it further comprises. The pharmaceutical composition according to claim 15, wherein the content of the dissolution aid is 0.05 to 10% by weight based on the total weight of the composition. The pharmaceutical composition according to claim 1, wherein the surface of the nanoparticle further comprises an amino acid having at least one carboxyl group selected from the group consisting of glutamic acid, aspartic acid, aspartic acid, histidine, and alanine as a low molecular ligand. 18. The pharmaceutical composition of claim 17, wherein the content of the low molecular ligand is 0.005 to 0.2 wt% based on the total weight of the composition. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is 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-acetyl proline, N-acetyl alanine Pharmaceutical composition, characterized in that it further comprises one or more selected pH adjusting agent. The pharmaceutical composition of claim 19, wherein the pH of the pharmaceutical composition is 2.0 to 6.0. The pharmaceutical composition of claim 1 further comprising at least one dispersion stabilizer selected from the group consisting of polyvinylpyrrolidone, glycerin, glucose, sucrose, lactose, sorbitol, mannitol, and trehalose. Composition. A pharmaceutical composition in the form of a nano-dispersion according to any one of claims 1 to 21 is subjected to lyophilization, rotary evaporation drying, spray drying, or fluidized-bed drying. Nanoparticles for target orientation, obtained by drying. (a) preparing an oil phase comprising a therapeutically effective amount of an anticancer agent, a divalent or trivalent transition metal ion or an alkaline earth metal ion, and an oil; (b) dissolving hyaluronic acid or a salt thereof in an aqueous medium to obtain an aqueous phase; And (c) mixing and homogenizing the oil phase obtained in step (a) and the water phase obtained in step (b) to form nanoparticles having hyaluronic acid or a salt thereof bound to the surface as nanoparticles having an average particle diameter of 10 to 1,000 nm. step Including a method for producing a pharmaceutical composition in the form of nano-dispersion for the target orientation. The method of claim 23, wherein the pharmaceutical composition further comprises a surfactant. The method of claim 23, wherein the oil phase further comprises a dissolution aid selected from the group consisting of polyethylene glycol, dimyristoyl phosphatidyl ethanolamine-polyethylene glycol, polypropylene glycol, glycofurol, and tricapryline. Manufacturing method characterized in that. The production method according to claim 23, wherein the aqueous phase is obtained by further dissolving an amino acid having a carboxyl group as a low molecular ligand. 27. The composition of any one of claims 23 to 26, wherein the aqueous phase is selected from the group consisting of polyvinylpyrrolidone, glycerin, glucose, sucrose, lactose, sorbitol, mannitol, and trehalose. A method of producing the above-mentioned dispersion stabilizer by further dissolving. 28. A method for preparing nanoparticles for target orientation, comprising the step of drying the pharmaceutical composition in nano-dispersion form obtained by the process according to any one of claims 23 to 27. 29. The method of claim 28, wherein said drying is performed by lyophilization, rotary evaporation drying, spray drying, or fluid bed drying.

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