KR20120126356A - Nanoparticles comprising amphiphilic low molecular weight hyaluronic acid complex and a process for the preparation thereof - Google Patents

Abstract

PURPOSE: A nanoparticle containing a hydrophobic drug in a hyaluronic acid-bile acid complex is provided to ensure excellent anticancer effect and to be used as a novel drug delivery system for treating cancer. CONSTITUTION: A low molecular hyaluronic acid-bile acid complex is an amphiphilic complex in which hydrophobic bile acid is conjugated to low molecular hyaluronic acid and forms a self-aggregation in the water. The hydrophobic bile acid is 5-beta-cholanic acid, cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, or 7-oxo-lithocholic acid. The complex is prepared by covalent bond of amino ethyl 5-beta cholanoamide to the low molecular hyaluronic acid.

Description

Nanoparticles comprising amphiphilic low molecular weight hyaluronic acid complex and a process for the preparation

The present invention relates to a drug carrier composition comprising hyaluronic acid nanoparticles bound to bile acids, and more particularly, nanoparticles prepared from high molecular weight hyaluronic acid by encapsulating a drug in a low molecular weight hyaluronic acid-bile acid complex. Compared to the present invention, the present invention relates to nanoparticles having improved solubility, low toxicity, high drug encapsulation, easy sterile filtration, and excellent targeting effect against cancer cells, and methods for preparing the same.

Amphiphilic polymers have both hydrophilic and hydrophobic compartments to form self-aggregates or micelles for stabilization of interfacial energy in aqueous solutions. Self-assembly or micelles formed by amphiphilic polymers are known to exhibit different particle sizes, distributions, rheological properties and thermodynamic stability depending on the degree of hydrophilicity and hydrophobicity. In addition, it has been reported that a variety of hydrophobic drugs can be enclosed in the hydrophobic region inside the auto assembly to induce selective and effective delivery of drugs (Adv. Drug, Deliv. Rev. 2001. 47, 113-131). Nanoparticles prepared by encapsulating hydrophobic drugs in such self-assembly exhibit prolonged circulating time in the body and regulate drug release, as well as specific accumulation in cancer tissue by EPR (Enhanced permeation and retention) effect. . Thus, by significantly reducing the toxicity and increasing the efficacy of the hydrophobic drug, the patient's convenience is increased and side effects are reduced.

In order to encapsulate drugs with high efficiency while forming self-assembly with amphiphilic polymers, it is very important to maintain an appropriate balance of hydrophilic and hydrophobic compartments in aqueous solution. To this end, many polymers have been developed to produce nanoparticles, such as diblock synthetic polymers, natural polymers modified with hydrophobic materials, and amphiphilic lipids. Currently commercialized nanoparticles are a synthetic copolymer of hydrophilic PEG in hydrophobic PLA and Genexol PM (Samyang) encapsulated with paclitaxel, and abraxane (Abraxane, encapsulated with paclitaxel using a hydrophobic pocket of albumin). Abraxis). Lipids are representative of the liposomes, and doxyl (Doxil, Alza), which contains doxorubicin in PEGylated lipids. Many researchers have developed amphiphilic polymers that form self-aggregates, and have been studying nanoparticle formulations to enclose anticancer drugs to significantly reduce side effects and significantly improve drug efficacy. However, the only products exemplified above are substantially the development of new polymers and commercialized into nanoparticle formulations, mainly due to technical difficulties in solubility, viscosity, sterilization, mass production, etc., in the development of injectable formulations.

Hyaluronic acid (HA) is a linear polysaccharide polymer having a molecular weight ranging from 1 x 10 ⁵ to 1 x 10 ⁷ Da, wherein (β, 1-4) -D-glucuronic acid (GlcUA) And a repeating sequence of (β, 1-3) -N-acetyl-D-glucosamine (N-acetyl-D-glucosamine, GlcNAc). It is found in the extracellular matrix and cell surface of most human tissues, especially in synovial fluid and cartilage. Therefore, hyaluronic acid is biocompatible and is biodegradable by hyaluronatease (hyaluronidase), which is a hyaluronic acid enzyme in the blood, and thus is used as a biomaterial such as drug carriers and tissue engineering supports. In particular, hyaluronic acid binds to CD44, RHAMM, which is overexpressed on the surface of cancer cells or metastatic cancer cells, and is internalized through endocytosis, and is degraded in a low pH environment such as lysosomes. By using these properties, a number of hyaluronic acid-anticancer complexes have been developed that have a targeting effect on cancer cells or metastatic cancer cells and can improve the cellular uptake rate of anticancer drugs. For example, mitomycin and epirubicin combined with hyaluronic acid and injected into lung cancer model mice showed excellent anticancer effects. The well-known hyaluronic acid-paclitaxel conjugate showed very high specificity to cancer cells and enhanced the therapeutic effect. In addition, a number of studies have been made to use hyaluronic acid as an amphiphilic drug and use it as a drug carrier.Effect of a hyaluronic acid into an amphiphilic polymer produced by combining a hydrophobic monomolecular or hydrophobic polymer with a carboxyl group of hyaluronic acid as an anticancer agent There is an example that proves that.

The disadvantage of hyaluronic acid as a drug carrier is that it is soluble only in water, so that when combined with hydrophobic substances, the reaction efficiency is low, the viscosity is high, and parenteral administration and mass production are difficult. In particular, a high dose is required to use hyaluronic acid as an inclusion body of the drug, and when the drug delivery solution is prepared according to the effective concentration of the drug, the viscosity of the solution is often high, making it difficult to inject. Korean Patent Publication No. 10-2010-0037494 discloses a method for preparing an amphiphilic complex by binding bile acid to hyaluronic acid. The molecular weights of hyaluronic acid used in this method are 250 KDa and 135 KDa, which are formulated at high concentrations, that is, at concentrations substantially suitable for the clinical dose of the drug, increase viscosity and make them unsuitable for injection. In addition, when the amount of hyaluronic acid is increased to increase the amount of bile acid bound to form nanoparticles, a large amount of bile acid may cause toxicity to the human body. In addition, while the high molecular weight hyaluronic acid-bile acid complex can encapsulate a relatively large amount of the drug, the particle size becomes 400 nm or more, which lowers the EPR effect and the intracellular absorption efficiency, and the range of particle distribution is widened. Reproducibility becomes low. If industrial production is required, it is difficult to manufacture and sterilize due to the problem of viscosity, there was a disadvantage that mass production is impossible.

Accordingly, the present inventors have prepared amphiphilic complexes using low molecular weight hyaluronic acid and hydrophobic bile acids to solve the above-mentioned disadvantages of the existing high molecular weight hyaluronic acid-bile acid complexes. Injectable nanoparticle formulations have been developed, and further, lowering the molecular weight of hyaluronic acid and controlling the binding rate of bile acids to the optimal level results in improved drug encapsulation efficiency and dosage, particle size and distribution, and targeting effects on cancer tissue. The present invention was completed by developing a nanoparticle formulation.

Accordingly, it is an object of the present invention to provide a low molecular weight hyaluronic acid-bile acid complex and a nanoparticle in which a drug is enclosed in the complex.

Another object of the present invention is to provide a low molecular weight hyaluronic acid-bile acid complex and a method for preparing nanoparticles according to the present invention.

Still another object of the present invention is to provide an injection comprising the nanoparticles according to the present invention.

In order to achieve the above object, the present invention provides a low-molecular weight hyaluronic acid-bile acid complex for drug delivery as an amphiphilic complex in which a hydrophobic bile acid is bonded to a low molecular weight hyaluronic acid of 5 to 20 KDa to form an autogenous aggregate in water. .

In another aspect, the present invention provides a nanoparticle drug is enclosed in the low molecular weight hyaluronic acid-bile acid complex. Preferably, the diameter of the nanoparticles is 100 to 500 nm, the hydrophobic bile acid is bound with a degree of substitution (%) of 1 to 20 with respect to the hyaluronic acid monomer, the drug is in the content of 10 to 40% by weight inside the complex It is enclosed.

In addition,

(a) synthesizing hydrophobic bile acid having a cholanic acid structure with aminoethyl colonanoamide;

(b) dissolving 5-20 KDa low molecular weight hyaluronic acid in an aqueous solvent to prepare a hyaluronic acid solution;

(c) dissolving the aminoethyl colonanoamide of step (a) in an organic solvent to prepare a collagenoamide solution;

(d) preparing a reaction solution by adding the collagenoamide solution of step (c) to the hyaluronic acid solution of step (b);

(e) obtaining a hyaluronic acid-bile acid complex from the reaction solution and obtaining it; And

(f) it provides a method for producing nanoparticles comprising the step of encapsulating a drug in the hyaluronic acid-bile acid complex.

The present invention also provides an injectable preparation comprising the nanoparticles.

The nanoparticles encapsulated with a hydrophobic drug in the low molecular weight hyaluronic acid-bile acid complex according to the present invention have excellent solubility in water than conventional drug carriers and can be commercialized as an injection, and can be encapsulated with high efficiency and high dose. have. In addition, the size and distribution of the nanoparticles is significantly improved, and can be developed by a reproducible and mass-producing manufacturing method. The hyaluronic acid-bile acid composite nanoparticles prepared as described above have a specific distribution and accumulation effect in cancer tissues and have been shown to have an excellent anticancer effect compared to existing products, and thus, may be used as a component of a new drug delivery system for cancer treatment. Can be.

1 is a graph showing the hydrogen nuclear magnetic resonance ( ¹ H NMR) analysis spectrum of the hyaluronic acid-bile acid complex prepared according to an embodiment of the present invention. The binding rate of bile acids is measured by the spectral area representing the indicated methyl group.
FIG. 2 is a graph showing the particle size and distribution of the hyaluronic acid-bile acid conjugate encapsulated with paclitaxel in Experimental Example 3 using a dynamic light scattering method. FIG. The size of the measured nanoparticles is 259.8 ± 14.3 nm.
FIG. 3 is a photograph of the cancer tissue targeting effect of the hyaluronic acid-bile acid complex nanoparticles conjugated with the fluorescent material Cy5.5 in Experimental Example 6 after the administration of cancer tissues by near-infrared irradiation.
Figure 4 is a graph showing the measurement of the size change of cancer when the paclitaxel-containing hyaluronic acid-bile acid complex nanoparticles according to the present invention administered to the cancer model in Experimental Example 7.
FIG. 5 is a graph showing the relative cancer growth inhibition rate when the paclitaxel-containing hyaluronic acid-bile acid composite nanoparticles according to the present invention were administered to a cancer model rat in Experimental Example 7. FIG.
6 is a graph showing changes in body weight when the hyaluronic acid-bile acid composite nanoparticles encapsulated with paclitaxel according to the present invention were administered to a female model mouse in Experimental Example 7. FIG.

Hereinafter, the present invention will be described in detail.

The present invention provides a low molecular weight hyaluronic acid-bile acid complex which forms a self-aggregate in water as an amphiphilic complex in which hydrophobic bile acid is bonded to low molecular weight hyaluronic acid of 5 or more and less than 20 KDa.

In the present invention, as a result of the study to overcome the problems of the nanoparticles formed by the self-assembly of the high-molecular weight hyaluronic acid-bile acid complex encapsulated with the conventional drug, low molecular weight hyaluronic acid of 5 to 20 KDa when preparing the hyaluronic acid-bile acid complex When solubility is increased, the solubility is increased and the viscosity is reduced, so it is easy to use and mass production as an injectable preparation, and the particle size is reduced to promote targeting and intracellular absorption into cancer tissue, and even lower the bile acid conjugation rate It has been found that toxicity problems can be solved by showing sex. When preparing a composite using a high molecular weight hyaluronic acid larger than the above range, the viscosity is difficult to formulate into an injection, there is a toxicity problem by increasing the bile acid conjugation rate, and when using a hyaluronic acid having a lower molecular weight than the above range Not only is the drug encapsulation efficiency low, but the nanoparticles are less stable.

Specifically, when the low molecular weight hyaluronic acid according to the present invention is used, the amount of bile acids used and the degree of substitution required to prepare amphiphilic complexes suitable for forming nanoparticles are shown to be lower than those of the composite prepared with high molecular weight hyaluronic acid. Table 3 Reduces the risk of toxicity caused by bile acids. In addition, the low molecular weight (7KDa, 16KDa) hyaluronic acid-bile acid complex of the present invention has a lower viscosity than the high molecular weight (60, 135 and 235KDa) hyaluronic acid-bile acid complex while its water solubility is significantly higher than 50 mg / mL. Increasing (Table 4), the possibility of commercialization as an injectable preparation was confirmed. When the nanoparticles are prepared by encapsulating paclitaxel in the complex, the amount of paclitaxel varies depending on the size of the nanoparticles and the amount of drug used, but in certain embodiments of the present invention, the encapsulation rate shows up to about 30%. And it confirmed that it was enclosed in high dose. As a result of measuring the size of the nanoparticles prepared as described above, the average particle diameter of the low molecular weight hyaluronic acid-bile acid composite nanoparticles according to the present invention is distributed in the range of 250 to 340 nm, nanoparticles made of high molecular weight hyaluronic acid Was found to decrease compared to greater than about 400 nm (Table 5). This reduction in particle size reduces the particle distribution (FIG. 2), increasing the reproducibility in production and drug expression, and causing the effect of improving targeting and intracellular absorption efficiency for cancer tissues, inflammatory tissues, and the like.

Particles such as nanoparticles are larger in size than single drug molecules and thus exhibit specific distribution behavior in vivo, and thus can be used as carriers for targeting drugs to lesion sites. The most important factor influencing the distribution of the fine particles is the capillary wall permeability according to the particle size, which is due to the fact that the capillary properties differ by organ or lesion. In general, particles with a diameter of 0.3 to 3 μm are detected by the reticulum endothelial tissue (RES) of the liver or filtered by the kidney, and particles having a diameter of 0.2 μm or less leak into the blood vessels of a cancer or inflammation site, thereby reducing particle size. As a result, the effect of selective accumulation into cancer or inflammatory tissue can be enhanced. In Patent Publication No. 10-2010-0037494, when the nanoparticles are prepared from the high molecular weight hyaluronic acid-bile acid complex, it is specified that the average size of the particles is 400 nm or more. The present inventors have observed that the particle size was reduced by controlling the hyaluronic acid molecular weight and the degree of bile acid substitution, and in fact, the administration of paclitaxel-embedded nanoparticles according to the present invention to a cancer-causing mouse model showed that it showed a significant accumulation in cancer tissue. Could (Figure 3). Furthermore, the size change of the cancer was measured to evaluate the actual anticancer efficacy, and showed a better cancer growth inhibition rate than Genexol, a commercial formulation encapsulated with paclitaxel in the tested dose range (FIGS. 4 and 5). This demonstrates that the low molecular weight hyaluronic acid-bile acid nanoparticles according to the present invention not only exhibit a cancer tissue specific distribution but also have an excellent uptake efficiency by cancer cells.

Accordingly, the low molecular weight hyaluronic acid-bile acid complex of the present invention has improved solubility and reduced viscosity in aqueous solution, and thus can be used and mass-produced as an injectable preparation. Since particle size, drug encapsulation efficiency and encapsulation can be controlled, hydrophobic drugs can be easily encapsulated, and accumulation and absorption efficiency in cancer tissues can be increased, and thus can be used as a component of a new drug delivery system.

Hyaluronic acid used in the complex of the present invention consists of a monomer represented by the following formula (1).

As mentioned above, CD44, a receptor for hyaluronic acid, is known to be produced in large quantities on the surface of various cancer cells. In addition, cell lines isolated from some cancers (such as prostate cancer, lung cancer, and cancer cell lines isolated from cervical cancer) and many bacteria produce hyaluronidase capable of breaking down hyaluronic acid, which is the enzyme of hyaluronic acid. Hydrolyze glucoside bonds. Therefore, the complex of the present invention containing hyaluronic acid as a main component can be selectively captured in cancer cells by CD44, a specific receptor present in large quantities on the surface of cancer cells, and also by hyaluronidase secreted from cancer cells. The complex is broken down and the drug enclosed inside can exert specific anticancer effects at the cancer site. Therefore, in the present invention, hyaluronic acid simultaneously plays a role as a protective film from blood components and as a ligand for targeting to a target site. The hyaluronic acid is also a low toxicity polymer and is approved by the FDA for injection, so it can be used safely as a material for drug delivery complexes in the present invention.

In the present invention, the hydrophobic bile acid is used to prepare an amphiphilic complex capable of imparting hydrophobicity to hydrophilic hyaluronic acid and forming an autoaggregate in water. Hydrophobic bile acids may comprise primary, secondary and tertiary bile acids or derivatives thereof having cholanic acid as a backbone. More specifically, the hydrophobic bile acid is 5-β cholanic acid (5-β cholanic acid), cholic acid (cholic acid), chenodeoxycholic acid (chenodeoxycholic acid), glycocholic acid (glycocholic acid), taurocholic acid (taurocholic acid) ), One or more selected from the group consisting of deoxycholic acid, lithocholic acid, and 7-oxo-lithocholic acid, but is not limited thereto. . In the present invention, 5-β cholanic acid preferably represented by the following Chemical Formula 2 may be used.

In order to bind hyaluronic acid and bile acid, a method of introducing an amine group to bile acid to form an amide bond with a carboxyl group of hyaluronic acid may be used. For this purpose, an aminoethylamide derivative of hydrophobic bile acid may be used. Preferred is aminoethyl 5-β collagenoamide, in which an amine group is introduced into -β cholanic acid.

Another embodiment of the present invention is a nanoparticle in which a drug is enclosed in the low molecular weight hyaluronic acid-bile acid complex. The diameter of the nanoparticles is preferably 100 to 500 nm, more preferably 100 to 400 nm, and most preferably 100 to 300 nm, but is not limited thereto. In addition, the hydrophobic bile acid with respect to the hyaluronic acid monomer may be combined with a substitution of less than 1 to 20, the drug may be encapsulated in an amount of 5 to 40% by weight inside the complex, but is not limited thereto.

In order to form stable nanoparticles in the aqueous solution, the degree of substitution of the hydrophobic bile acid should vary according to the molecular weight of hyaluronic acid. When the hyaluronic acid molecular weight is 100 KDa or more, the substitution degree of bile acid with respect to the hyaluronic acid monomer should be 20% or more in order to form particles having sufficient affinity in the aqueous solution. Lowering the molecular weight of hyaluronic acid should also lower the substitution of hydrophobic bile acids. For low molecular weight hyaluronic acid, more specifically hyaluronic acid monomers having a molecular weight of 5 KDa to 20 KDa, the hydrophobic bile acid can be bound, for example, at a degree of substitution (%) of 1 to 20. When the degree of substitution of bile acid to the hyaluronic acid monomer exceeds 20%, the water solubility of the hyaluronic acid-bile acid complex becomes very low, which is unsuitable for injection, and when the degree of substitution is less than 1%, the cohesive force of the hydrophobic part is low and the nano The formation of particles can be difficult.

The interior of the hyaluronic acid-bile acid complex of the present invention is composed of hydrophobic bile acids, and since some hydrophilic portions of the bile acids are combined with hyaluronic acid, the interior of the complex is very hydrophobic. Therefore, the hydrophobic drug is easily encapsulated inside the hyaluronic acid-bile acid complex of the present invention, which is limited to chemical bonds because the physical inclusion due to the hydrophobicity of both the internal substance and the drug of the complex. Can be increased.

Hyaluronic acid-bile acid composite nanoparticles of the present invention may be prepared to contain hydrophobic drugs in the range of about 5 to 40% by weight, but is not limited thereto. Considering the stability and size of the particles, etc., the encapsulation amount of the drug is most preferably 10 to 30% by weight.

Hydrophobic drugs encapsulated inside the hyaluronic acid-bile acid complex include, but are not limited to, water insoluble drugs. As used herein, the "hydrophobic" drug need not be limited to non-polar or low-polar materials that are not miscible with water, and may include substances that are physically contained within the complex according to the present invention and may be stably present in a hydrophobic internal environment consisting of bile acids. It includes everything. The "drug" may include not only a pharmacologically active therapeutic agent but also all reagents and other medical agents that can be administered to the human body for diagnosis and the like. The hydrophobic drug may be, but is not limited to, antineoplastic, anesthetic, anti-inflammatory, immunosuppressant or hormone.

In certain embodiments of the invention, the hydrophobic drug may be an anti-neoplastic agent. The term "antineoplastic" is used herein in the broadest sense to refer to all pharmaceutically active agents used for the treatment, amelioration or slowing of the progression of a neoplastic disease. Therefore, it includes the concept of a conventional "antitumor agent" or "anticancer agent", can be used in combination with them. Such anti-neoplastic agents include, but are not limited to, alkylating agents, antibiotics, anti-metabolic agents, hormones, and fission inhibitors commonly used in the treatment of neoplastic diseases. For example, anti-neoplastic agents that can be encapsulated in the nanoparticles of the present invention include adriamycin, cyclophosphamide, actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, fluoro Uracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, cisplatin etoposide, interferon, campotesine and derivatives thereof, phenesterin, taxanes, paclitaxel and derivatives thereof, taxotere and derivatives thereof, vinbla Stin, vincristine, tamoxifen, etoposide, piposulfan, docetaxel, anasterazole, gleevec, phloxuridine, leuprolide, flutamide, zoleronate, gemcitabine, streptozosin, carboplatin, topo Tecan, velotecan, irinotecan, vinorelbine, hydroxyurea, valerubicin, retinoic acid series, mechloretamine, chlorambucil, sulfan, doxyfluidine, predney , Testosterone, mitosantron, aspirin, salicylate, ibuprofen, naproxen, phenopropene, indomethacin, phenylbutazone, cyclophosphamide, mecloethamine, dexamethasone, prednisolone, celecoxib, valdecoxib , But not limited to nimesulide, cortisone or corticosteroids.

In another aspect of the present invention, the present invention provides a low molecular weight hyaluronic acid-bile acid complex and a method for preparing nanoparticles comprising the following steps:

(a) synthesizing hydrophobic bile acid having a cholanic acid structure with aminoethyl colonanoamide;

(b) dissolving low molecular weight hyaluronic acid of at least 5 and less than 20 KDa in an aqueous solvent to prepare a hyaluronic acid solution;

(c) dissolving the aminoethyl colonanoamide of step (a) in an organic solvent to prepare a collagenoamide solution;

(d) preparing a reaction solution by adding the collagenoamide solution of step (c) to the hyaluronic acid solution of step (b);

(e) obtaining a hyaluronic acid-bile acid complex from the reaction solution and obtaining it; And

(f) encapsulating the drug in the hyaluronic acid-bile acid complex.

Specifically, in step (a), hydrophobic bile acid is synthesized as a derivative capable of chemically bonding with hyaluronic acid. In an embodiment of the present invention, the coupling of hyaluronic acid and bile acid is performed by reacting ethylenediamine with the carboxyl group of bile acid to introduce an amine group at the end of bile acid, and the amine group and the carboxyl group of hyaluronic acid form an amide bond by a covalent bond. Can be. For example, if the hydrophobic bile acid is 5-β cholanic acid, the step of synthesizing aminoethyl 5-β collagenoamide therefrom can be summarized by the following formula (3).

Formula 4 below briefly shows the step of binding the bile acid derivative prepared in step (a) to hyaluronic acid. This linkage is achieved by forming an amide bond with a carboxyl group of hyaluronic acid and an amine group introduced into the bile acid, which will be described in detail in the following steps (b) to (d).

In step (b), the hyaluronic acid may be dissolved in an aqueous solvent, and then a catalyst for activating the carboxyl group of hyaluronic acid may be added. 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide; EDC] and N-hydroxysuccinimide (NHS) are preferred, but are not limited thereto. At this time, EDC and NHS serves to induce the amino group and amide bond of bile acid by converting the carboxyl group of hyaluronic acid to NHS ester. The molar ratio of EDC to NHS is suitably 1 to 3 times that of the carboxyl group of hyaluronic acid, respectively. If it is less than that, the NHS ester is hydrolyzed at a high rate, so the binding rate after the reaction is lowered, and if the molar ratio is higher than three times, the purification process is inefficient. On the other hand, the water-soluble solvent may be used any water-soluble solvent that can dissolve hyaluronic acid, preferably distilled water can be used.

The organic solvent of step (c) may be any organic solvent capable of dissolving the amide derivative of hydrophobic bile acid, preferably an organic solvent which can be mixed with water, such as dimethyl formamide, dimethyl sulfoxide, methanol, and the like. . The hydrophobic bile acid is preferably added in an amount of 6 to 60 mg based on 100 mL of the organic solvent, but is not limited thereto.

The reaction of step (d) may be carried out by slowly adding the bile acid solution to the hyaluronic acid solution and then stirring it. Specifically, the mixing ratio of the hyaluronic acid solution and the bile acid solution is 1: 2 (v / v). This is because when the ratio of the hyaluronic acid solution is higher than that of the bile acid solution, bile acid may precipitate on the reaction solution, and when the ratio of the bile acid solution exceeds 1: 2, hyaluronic acid may precipitate. The reaction time after mixing is suitable for 24 hours to 3 days, preferably, the reaction solution can be reacted by stirring for 3 days. The reaction temperature may range from room temperature to 60 ° C., but the present invention is not limited thereto. Preferably, the reaction temperature is 60 ° C. to increase the bonding rate.

In the step (e), the reaction solution is completed to form a hyaluronic acid-bile acid complex is precipitated in an organic solvent to obtain a hyaluronic acid-bile acid complex. The organic solvent used for the precipitation may be any organic solvent capable of precipitating the hyaluronic acid-bile acid complex, for example a single solvent selected from acetone, isopropyl alcohol, isopropyl ether, and dimethyl ether or these A mixed solvent of may be used. The composite obtained after precipitation can be washed several times with the same precipitation solvent and dried in powder form by vacuum drying. This precipitation method can save time and cost compared to commonly used dialysis and lyophilization methods.

After said step (e), an additional step of separating and purifying the obtained complex may be involved. This is to remove hyaluronic acid-bile acid complexes suitable for pharmaceutical standards and tests by removing addition products, residual solvents and unreacted bile acids occurring in the reaction solution. The purification can be performed by methods commonly known to those skilled in the art, including extraction. In a preferred embodiment of the present invention, the hyaluronic acid-bile acid complex obtained in step (e) is isolated and purified by dissolving it in distilled water, followed by extraction by adding dimethyl chloroform or ethyl acetate and other organic solvents that can be separated from water. do.

Step (f) is a step of encapsulating the hydrophobic drug in the hyaluronic acid-bile acid complex, the encapsulation method may be appropriately selected by those skilled in the art according to the type and amount of the drug. Representative methods include film hydration method and solvent evaporation method. In addition, air suspension coating method (eg Wurster method), interfacial polymerization method, spray drying method, phase separation method, etc. The method may be used but is not limited thereto.

In one embodiment of the present invention, when the encapsulated drug is paclitaxel, film hydration can be used. In this case, the encapsulation efficiency is 80% or more and nanoparticles having a size of about 300 nm are formed. Specifically, the anticancer agent is dissolved in an organic solvent, and hyaluronic acid is dissolved in water and mixed with each other. At this time, the type of organic solvent and the mixing ratio with water are important factors controlling the size and encapsulation efficiency of the nanoparticles. The organic solvent capable of dissolving the anticancer agent may be selected from ethanol, methanol, chloroform, dimethylchloromethane, dimethyl formamide, dimethyl sulfoxide, and mixtures thereof, but is not limited thereto. After mixing the anticancer agent solution and hyaluronic acid solution to produce a film while evaporating the solvent using a rotary evaporator (rotary evaporator). After the solvent is evaporated, the resulting film is hydrated using distilled water, water for injection, physiological saline, etc. to form nanoparticles. At this time, during the hydration process, energy can be imparted using an ultrasonic grinder to reduce the distribution of nanoparticles and to reduce the particle size. The film hydration method has an advantage that the encapsulation efficiency is generally higher than the solvent evaporation method, but has a disadvantage in that mass production is difficult.

In another embodiment of the invention, the hydrophobic drug can be enclosed in the hyaluronic acid-bile acid complex by solvent evaporation. The solvent evaporation method has the advantage of being able to mass-produce, but the gap between the drug encapsulation efficiency is greater than the film hydration method. Specifically, the anticancer agent is dissolved in an organic solvent and hyaluronic acid is dissolved in an aqueous solution, and the organic solvent is selected to be able to form an emulsion without mixing with water. For example, the organic solvent may be dimethylchloromethane or chloroform. The mixing ratio of the organic solvent and the aqueous solution may be in the range of 1:50 to 1: 1, and more preferably in the case of 1:10, the encapsulation efficiency is excellent.

In the most preferred embodiment, the method for producing nanoparticles according to the present invention comprises the following steps:

(a) reacting 5-β cholanic acid with ethylenediamine to synthesize aminoethyl 5-β collagenoamide;

(b) 5-20 KDa low molecular weight hyaluronic acid is dissolved in distilled water and 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added Preparing a hyaluronic acid solution;

(c) dissolving the aminoethyl 5-β collagenoamide of step (a) in dimethylformamide to prepare a collagenoamide solution;

(d) adding the collagenoamide solution to the hyaluronic acid solution in a ratio of 1: 2 (v / v) dropwise and stirring to prepare a reaction solution;

(e) depositing the reaction solution in a precipitation solvent to obtain a hyaluronic acid-bile acid complex, which is dried in vacuo;

(f) dissolving the hyaluronic acid-bile acid complex in an aqueous solution and then separating and purifying the complex by extracting with dimethyl chloroform; And

(g) mixing the hydrophobic drug dissolved in the organic solvent with the aqueous solution of the obtained hyaluronic acid-bile acid complex to enclose the hydrophobic drug in the hyaluronic acid-bile acid complex.

In a further aspect of the invention, the invention provides an injection comprising said low molecular weight hyaluronic acid-bile acid complex nanoparticles.

Injections comprising the complex in which the drug is enclosed can be prepared according to conventional methods for preparing injections known in the art. Injectables according to the present invention may be in a form dispersed in a sterile medium so that it can be used as it is when administered to a patient, or may be administered in a form of dispersing it in an appropriate concentration by adding distilled water for injection.

Injectables of the present invention as described above, unlike injections containing nanoparticles prepared using high molecular weight hyaluronic acid, the water solubility of the nanoparticles can be improved to be prepared at a concentration suitable for various dosages of the drug. In addition, the use of low molecular weight hyaluronic acid significantly lowers the viscosity, improving the efficiency of the sterilization process, such as the preparation and filtration of the injection, it is easier to administer to the patient even in the clinical administration stage.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these embodiments are provided to aid understanding of the present invention, and the scope of the present invention is not limited thereto in any sense.

Reference Example : Aminoethyl  5-β Collagenoamide ( cholanoamide ) Produce

10 g of colic acid (Sigma, USA) was dissolved in 600 ml of methanol, and 1.6 ml of hydrochloric acid was added thereto, followed by stirring at 60 ° C. for 6 hours. The solution was then concentrated to 100 ml using a rotary evaporator, precipitated in 2 L of distilled water, filtered, and washed several times with distilled water and lyophilized. 60 ml of ethylenediamine was added to 10 g of methyl-5-β collagen obtained after drying, followed by stirring at 130 ° C. for 8 hours. Dilute with 100 mL methanol at room temperature and precipitate in 2 L distilled water. It was filtered, washed several times with distilled water and lyophilized.

Ⅰ. Hyaluronic Acid Bile acid  Manufacture of Composites

To prepare a hyaluronic acid-bile acid complex combining hyaluronic acid and bile acid according to the composition of Table 1 below. The molecular weight of hyaluronic acid was determined based on a commercially available hyaluronic acid product (Lifecore, USA), 7 KDa and 16 KDa hyaluronic acid as a low molecular weight hyaluronic acid used in the present invention, 60 KDa, 135 as a comparative example High molecular weight hyaluronic acid of KDa and 250 KDa was used. The amount of bile acid used was measured by weight.

Hyaluronic Acid Bile acid  Composite manufacturing Hyaluronic acid molecular weight ( Da ) For hyaluronic acid monomer
Bile acid  usage (%) Example  One 7K 10 Example  2 16K 10 Example  3 7K 20 Example  4 16K 20 Example  5 7K 30 Example  6 16K 30 Example  7 16K 40 Comparative example  One 60K 10 Comparative example  2 135 K 10 Comparative example  3 250 K 10 Comparative example  4 60K 30 Comparative example  5 135 K 30 Comparative example  6 250 K 30 Comparative example  7 60K 40 Comparative example  8 135 K 40 Comparative example  9 250 K 40

Example  1-2: Low molecular weight  Hyaluronic acid-10% Bile acid  Composite manufacturing

1 g of 7K (Example 1) and 16K (Example 2) hyaluronic acid was added to 160 ml of distilled water and stirred to dissolve. 1.57 g of 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC) is added to the solution and 0.941 g of N-hydroxysuccinimide (NHS) is 1 ml dimethylformimide. After dissolving in (DMF), the solution was added to the solution to activate the carboxyl group of hyaluronic acid with stirring. 125 mg of aminoethyl 5-β collagenoamide prepared in Reference Example was dissolved in 320 ml DMF, and the hyaluronic acid solution was added dropwise while stirring in the reactor. At this time, the reaction temperature was 60 ℃, the reaction time was 3 days.

The hyaluronic acid-bile acid complex was obtained in the form of a powder while the reaction mixture was dipped in the precipitation solvent. Isopropyl alcohol and isopropyl ether were mixed 1: 1 and used as a precipitation solvent, and the ratio of the precipitation solvent and the reaction liquid was 4: 1. The reaction solution was added dropwise to the precipitation solvent and stirred, then washed three times with the same solvent as the precipitation solvent and dried in vacuo. The hyaluronic acid-bile acid complex obtained after vacuum drying was purified. In order to remove impurities and residual solvent, the complex was dissolved in 100 ml of distilled water, placed in a separator, and 100 ml of dichloromethane was added thereto to separate and purify. At this time, the pH of the distilled water was to be 8 or more, and the process was repeated several times, and only the aqueous layer was taken and lyophilized.

Example  3-4: Low molecular weight  Hyaluronic Acid-20% Bile acid  Composite manufacturing

1 g of 7K (Example 3) and 16K (Example 4) hyaluronic acid was added to 160 mL distilled water and stirred to dissolve. 1.57 g of 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC) was added to the solution, and 0.941 g of N-hydroxysuccinimide (NHS) was added to 1 ml dimethylformimide ( DMF) and added to the solution to activate the carboxyl group of hyaluronic acid with stirring. 250 mg of aminoethyl 5-β collagenoamide prepared in Reference Example was dissolved in 320 ml DMF, and the hyaluronic acid solution was added dropwise while stirring in the reactor. Since the preparation step was carried out in the same manner as in Example 1-2.

Example  5-6: Low molecular weight  Hyaluronic acid-30% Bile acid  Composite manufacturing

1 g of 7K (Example 5) and 16K (Example 6) hyaluronic acid was added to 160 mL distilled water, stirred and dissolved. 1.57 g of 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC) was added to the solution and 0.941 g of N-hydroxysuccinimide (NHS) was added to 1 ml dimethylformimide (DMF). ) And added to the solution while stirring to activate the carboxyl group of hyaluronic acid. Hyaluronic acid solution was added dropwise while dissolving 375 mg of aminoethyl 5-β collagenoamide prepared in Reference Example in 320 ml DMF. Since the preparation step was carried out in the same manner as in Example 1-2.

Example  7: Low molecular weight  Hyaluronic Acid-40% Bile acid  Composite manufacturing

Since 7K hyaluronic acid was found to be insoluble in the preparation of the 30% bile acid complex (Example 5), a 40% bile acid complex was prepared using only 16K hyaluronic acid.

1 g of 16K hyaluronic acid was added in 160 ml distilled water, stirred to dissolve. 1.57 g of 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC) was added to the solution and 0.941 g of N-hydroxysuccinimide (NHS) was added to 1 ml dimethylformimide (DMF). After dissolving in, it was added to the solution and stirred to activate the carboxyl group of hyaluronic acid. 625 mg of aminoethyl 5-β collagenoamide prepared in Reference Example was dissolved in 320 ml DMF, and the hyaluronic acid solution was added dropwise while stirring in the reactor. Since the preparation step was carried out in the same manner as in Example 1-2.

Comparative example  1-3: High molecular weight  Hyaluronic acid-10% Bile acid  Composite manufacturing

1 g of 60K (Comparative Example 1), 135K (Comparative Example 2) and 250K (Comparative Example 3) hyaluronic acid was added to 160 ml of distilled water and dissolved with stirring. 125 mg of aminoethyl 5-β collagenoamide prepared in Reference Example was dissolved in 320 ml DMF, and the hyaluronic acid solution was added dropwise while stirring in the reactor. Since the preparation step was carried out in the same manner as in Example 1-2.

Comparative example  4-6: High molecular weight  Hyaluronic acid-30% Bile acid  Composite manufacturing

1 g of 60K (Comparative Example 4), 135K (Comparative Example 5) and 250K (Comparative Example 6) hyaluronic acid was added to 160 ml of distilled water and dissolved with stirring. 375 mg of aminoethyl 5-β collagenoamide prepared in Reference Example was dissolved in 320 ml of DMF, and then the hyaluronic acid solution was added dropwise while stirring in the reactor. Since the preparation step was carried out in the same manner as in Example 1-2.

Comparative example  7-9: High molecular weight  Hyaluronic Acid-40% Bile acid  Composite manufacturing

1 g of 60K (Comparative Example 7), 135K (Comparative Example 8) and 250K (Comparative Example 9) hyaluronic acid was added to 160 ml of distilled water, stirred and dissolved. 625 mg of aminoethyl 5-β collagenoamide prepared in Reference Example was dissolved in 320 ml DMF, and then hyaluronic acid solution was added dropwise while stirring in the reactor. Since the preparation step was carried out in the same manner as in Example 1-2.

Ⅱ. The drug Enclosed  Hyaluronic Acid Bile acid  Preparation of Composite Nanoparticles

According to the composition of Table 2 was prepared nanoparticles in which paclitaxel is encapsulated inside the low molecular weight hyaluronic acid-bile acid complex, the nanoparticles made of high molecular weight hyaluronic acid-bile acid complex in the experimental example described below Comparison was made with the particles. The film hydration method was used to encapsulate paclitaxel inside the complex.

 Hyaluronic Acid Bile acid  In the complex Paclitaxel Enclosed  Preparation of Nanoparticles Hyaluronic acid
Molecular Weight( Da ) Bile acid
usage(%) Complex
Manufacturing example Paclitaxel
Encapsulation (weight%) Example  8 7K 10 Example  One 10 Example  9 16K 30 Example  6 10 Example  10 7K 10 Example  One 20 Example  11 16K 30 Example  6 20 Example  12 7K 10 Example  One 30 Example  13 16K 30 Example  6 30 Example  14 7K 10 Example  One 40 Example  15 16K 30 Example  6 40 Comparative example  10 135 K 30 Comparative example  5 30 Comparative example  11 250 K 40 Comparative example  9 30

Example  8-9: Low molecular weight  Hyaluronic Acid Bile acid  10 wt% in the composite Paclitaxel  Enclosed

90 mg of the complexes of Examples 1 and 6, which were bound to 7K and 16K hyaluronic acid using 10% and 30% of bile acids, respectively, were weighed out and dissolved in 10 ml of distilled water. 10 mg paclitaxel was dissolved in 30 ml ethanol and then mixed with the complex aqueous solution and stirred in a round flask. The solvent was evaporated using a rotary evaporator and a film was formed around the round flask. After confirming that the film was completely formed, hydrated by adding 50 ml of distilled water. After confirming that the film was fully hydrated, the nanoparticles were dispersed using an ultrasonic mill.

Example 8 is a nanoparticle encapsulated with 10% paclitaxel in 7KHA-10% CA, Example 9 is a nanoparticle encapsulated with 10% paclitaxel in 16KHA-30% CA.

Example  10-11: Low molecular weight  Hyaluronic Acid Bile acid  20 wt% in the composite Paclitaxel  Enclosed

80 mg of 7KHA-10% CA complex (Example 1) and 16KHA-30% CA complex (Example 6) were each weighed and dissolved in 10 ml distilled water. 20 mg paclitaxel was dissolved in 30 ml ethanol and then mixed with the complex aqueous solution and stirred in a round flask. Since the preparation step was carried out in the same manner as in Example 8-9.

Example 10 is a nanoparticle encapsulated in 20% paclitaxel in 7KHA-10% CA and Example 11 is a nanoparticle encapsulated in 20% paclitaxel in 16KHA-30% CA.

Example  12-13: Low molecular weight  Hyaluronic Acid Bile acid  30 wt% in the composite Paclitaxel  Enclosed

70 mg each of the 7KHA-10% CA complex (Example 2) and the 16KHA-30% CA complex (Example 7) were weighed and dissolved in 10 ml of distilled water. 30 mg paclitaxel was dissolved in 30 ml ethanol and then mixed with the complex aqueous solution and stirred in a round flask. Since the preparation step was carried out in the same manner as in Example 8-9.

Example 12 is a nanoparticle encapsulated with 30% paclitaxel in 7KHA-10% CA and Example 13 is a nanoparticle encapsulated with 30% paclitaxel in 16KHA-30% CA.

Example  14-15: Low molecular weight  Hyaluronic Acid Bile acid  40 wt% in the composite Paclitaxel  Enclosed

60 mg of 7KHA-10% CA complex (Example 2) and 16KHA-30% CA complex (Example 7) were each weighed and dissolved in 10 ml distilled water. 40 mg paclitaxel was dissolved in 30 ml ethanol and then mixed with the complex aqueous solution and stirred in a round flask. The preparation step was then carried out in the same manner as in Example 8-9.

Example 14 is a nanoparticle encapsulated 40% paclitaxel in 7KHA-10% CA and Example 15 is a nanoparticle encapsulated 40% paclitaxel in 16KHA-30% CA.

Comparative example  10-11: High molecular weight  Hyaluronic Acid Bile acid  20 wt% in the composite Paclitaxel  Enclosed

As the high molecular weight hyaluronic acid-bile acid complex, 135KHA-30% CA complex (Comparative Example 5) and 250KHA-40% CA complex (Comparative Example 9) were used as shown in Table 2. 80 mg of the complexes of Comparative Example 5 and Comparative Example 9 were weighed in 80 mg each, and dissolved in 10 ml of distilled water. 20 mg paclitaxel was dissolved in 30 ml ethanol and then mixed with the complex aqueous solution and stirred in a round flask. Since the preparation step was carried out in the same manner as in Example 8-9.

Comparative Example 10 is a nanoparticle encapsulated 20% paclitaxel in 135KHA-30% CA and Comparative Example 11 is a nanoparticle encapsulated 20% paclitaxel in 250KHA-40% CA.

Experimental Example  1: according to manufacturing conditions Low molecular weight  Hyaluronic Acid Bile acid  Within the complex gall Acid binding efficiency measurement

In the present experimental example, the substitution degree and the substitution efficiency of the bile acid according to the molecular weight of hyaluronic acid were measured to find the conditions for preparing the hyaluronic acid-bile acid complex suitable for use as an injection. For this purpose, the bile acid content in the hyaluronic acid-bile acid complex was analyzed using hydrogen nuclear magnetic resonance ( ¹ H NMR).

Way

Examples 1-7 and Comparative Examples 1-9, the hyaluronic acid produced according to the mixture of the bile acid _{conjugate, D 2 O / DMSO (1} : 1, v / v) for ¹ H-NMR spectrometer and a NMR solvent ( spectrometer; Bruker, 400 MHz). As shown in FIG. 1, the amount of bile acid bound was determined using the methyl group area of hyaluronic acid to which bile acid was bound on the NMR spectrum. Degree of substitution (DS) and substitution efficiency of bile acids with respect to the monomers of hyaluronic acid were determined according to the following formulas, and the results are shown in Table 3.

Bile acid substitution degree (%)

= (Number of bile acid bonds / number of reactors per molecule of hyaluronic acid) × 100

Bile Acid Substitution Efficiency (%)

= (Bile Acid Substitution / Bile Acid Consumption) × 100

result

According to the ¹ H NMR analysis spectrum represented by Figure 1 it can be confirmed that the hyaluronic acid-bile acid complex was prepared.

Hyaluronic acid molecular weight and Bile acid  According to usage Bile acid  Degree of substitution Hyaluronic acid
Molecular Weight( Da ) Hyaluronic acid
For monomers
Bile acid  usage(%) Hyaluronic acid
For monomers
Bile acid  Degree of substitution (%)
Substitution efficiency (%) Example  One 7K 10 8 80 Example  2 16K 10 8 80 Example  3 7K 20 Insoluble - Example  4 16K 20 10-12 80 Example  5 7K 30 Insoluble - Example  6 16K 30 14 47 Example  7 16K 40 Insoluble - Comparative example  One 60K 10 8 80 Comparative example  2 135 K 10 9 90 Comparative example  3 250 K 10 9 90 Comparative example  4 60K 30 14 46 Comparative example  5 135 K 30 25 83 Comparative example  6 250 K 30 27 90 Comparative example  7 60K 40 Insoluble - Comparative example  8 135 K 40 Insoluble - Comparative example  9 250 K 40 30 75

As shown in Table 3, the degree of bile acid substitution was different depending on the molecular weight of hyaluronic acid and the amount of bile acid used. According to Examples 1-2 and Comparative Examples 1-3, when the bile acid was added 10%, even if the molecular weight of hyaluronic acid increased, there was no significant difference in the degree of substitution. On the other hand, in Example 6 and Comparative Examples 4-6, the use of 30% bile acid showed that the substitution efficiency of bile acid increased as the molecular weight of hyaluronic acid increased.

Considering the solubility of the complex according to the substitution efficiency and degree of substitution, it was intended to find the conditions for preparing the hyaluronic acid-bile acid complex suitable for nanoparticle formation. Therefore, the amount of bile acid used was lower than that of insoluble substitution and the maximum substitution degree of nanoparticles could be formed. From the above results, it can be seen that the amount of bile acid having a maximum degree of substitution varies according to the molecular weight of hyaluronic acid. Specifically, when the hyaluronic acid molecular weight is 7KDa, 10% bile acid is used (Example 1), and 30% when 16KDa is used. The amount of bile acids used (Example 6) was found to be suitable. On the other hand, in the comparative example, 30% bile acid consumption (Comparative Example 4) for 60KDa, 30% bile acid usage (Comparative Example 5) for 135KDa, and 40% bile acid consumption (Comparative Example 9) for 250KDa were found to be suitable. .

Experimental Example  2: Low molecular weight  Hyaluronic Acid Bile acid  Water solubility measurement of the complex

The solubility of the nanoparticles encapsulated in the drug is important for achieving a concentration suitable for the clinical dose of the drug as an injectable preparation. It should be high enough. In this experimental example, the solubility of the hyaluronic acid-bile acid complex according to the molecular weight of hyaluronic acid was measured using distilled water as a solvent.

Way

Hyaluronic acid-bile acid complexes prepared according to Examples 1, 6, Comparative Examples 4, 5, and 9 (hyaluronic acid molecular weights 7K, 16K, 60K, 135K, and 235K used) were dissolved in distilled water until a saturated solution was reached. The amount of solute required was determined and expressed as concentration. Specifically, 10 mg of the hyaluronic acid-bile acid complex dried in powder state was added to 10 ml of distilled water, and the mixture was dissolved until saturated and filtered. The weight of the complex obtained by lyophilization of the filtrate was measured to define a saturated solution when the addition amount and the yield after filtering and drying were the same. Solubility (mg / ml) was obtained using the amount of the obtained complex, and the results are shown in Table 4.

result

Hyaluronic Acid Bile acid  Water solubility of the complex Molecular Weight( Da ) - Bile acid  usage Water solubility ( mg Of ml ) Example  One 7K-10% 80 Example  6 16K-30% 50 Comparative example  4 60K-30% 30 Comparative example  5 135K-30% 25 Comparative example  9 235K-40% 10

As shown in Table 4, in Examples 1 and 6, which are low molecular weight hyaluronic acid-bile acid complexes, water solubility was 80 and 50 mg / ml, respectively, and high molecular weight hyaluronic acid-bile acid complexes (Comparative Examples 4, 5, and 9). ) Was significantly improved compared to that of 10 to 30 mg / ml.

The clinical dose should be at least 25 mg / ml of the total concentration of the complex formulation, based on 20 wt% of inclusions for paclitaxel. In the case of high molecular weight hyaluronic acid-bile acid complex, the maximum solubility is 30 mg / ml, which is only about 1 times the clinical dose, thus limiting the encapsulation of the drug, especially in the case of a high therapeutically effective drug. On the other hand, the low molecular weight hyaluronic acid-bile acid complex according to the present invention has a maximum solubility of 50 mg / ml or more, which is suitable for clinical use.

Experimental Example  3: Paclitaxel Enclosed Low molecular weight  Hyaluronic Acid Bile acid  Measurement of particle size and distribution of the composite

In drug targeting strategies, the particle size of the drug is important in that the tissue distributed and accumulated depends on the particle diameter after absorption. When the particle size is 400 nm or more, it tends to go to the spleen and kidney rather than accumulate in cancer tissue, and the uptake efficiency of the nanoparticles into cells is also very low. In addition, large particle sizes make it difficult to produce on an industrial scale due to filtering difficulties. In the present experimental example, the size of the nanoparticles prepared using the low molecular weight hyaluronic acid according to the present invention was measured, and the change of the particle size according to the molecular weight and drug usage of the hyaluronic acid was analyzed.

Way

The nanoparticles obtained in Examples 8-15 were homogeneously dispersed in distilled water at a concentration of 2 mg / ml. The particle size was measured by the dynamic light scattering method using the NICOMP 380ZLS, and the results are shown in Table 5.

result

Paclitaxel  Enclosed hyaluronic acid Bile acid  Composite Nanoparticle Size Measurement HA - CA
(Hyaluronic acid molecular weight-
Bile acid  usage) Paclitaxel  usage
(weight%) size( nm ) Example  8 7KHA-10% CA
Example 1 10 791.2 ± 629 Example  9 16KHA-30% CA
(Example 6) 10 318.4 ± 35 Example  10 7KHA-10% CA
(Example 1) 20 337.7 ± 28 Example  11 16KHA-30% CA
(Example 6) 20 316.4 ± 76.8 Example  12 7KHA-10% CA
(Example 1) 30 260.6 ± 113.3 Example  13 16KHA-30% CA
(Example 6) 30 292.4 ± 84.5 Example  14 7KHA-10% CA
(Example 1) 40 Not enclosed Example  15 16KHA-30% CA
(Example 6) 40 250.5 ± 72 Comparative example  10 135KHA-30% CA
(Comparative Example 5) 20 430.7 ± 302.4 Comparative example  11 250KHA-40% CA
(Comparative Example 9) 20 383.4 ± 192.6

The overall size of the hyaluronic acid-bile acid composite nanoparticles when paclitaxel was encapsulated was found to be in the range of 250 to 400 nm. Examples 8 to 15 using low molecular weight hyaluronic acid had a particle size of about 250 as represented by FIG. 2, whereas the particle sizes of Comparative Examples 10 and 11 using high molecular weight hyaluronic acid were about 430.7 and 383.4 nm, respectively. It was distributed in the range from 340 nm to a small particle size and a narrow particle distribution.

Analyzing the particle size according to the drug loading amount, it can be seen from the results of Example 8-15 that the larger the amount of drug (paclitaxel) used, the smaller the particle size is. Specifically, Examples 12 and 13 (about 260.6 and 292.4 nm) at 30% by weight compared to Examples 8 and 9 (about 791.2 and 318.4 nm) at 10% by weight of drug use had a small average particle size and a particle distribution diagram. Narrowly appeared. When comparing low molecular weight hyaluronic acid 7K and 16K, 16K can form nanoparticles and encapsulate the drug when the amount of the drug is used in the amount of 10% to 40% by weight. It is difficult to see the nanoparticles having a substantial EPR effect at a particle size of about 700 nm, and at 40 wt%, no drug encapsulation.

Experimental Example  4: Low molecular weight  Hyaluronic Acid Bile acid  Drugs of the complex Encapsulation  Measure

Based on the results of Experimental Example 3, by selecting Examples 9, 11, 13 and 15 shown to have the appropriate nanoparticle size in the entire range of drug doses tested, the encapsulation efficiency and the amount of encapsulation according to the amount of paclitaxel used Measured.

Way

When paclitaxel was added to the 16K hyaluronic acid-bile acid complexes according to Examples 9, 11, 13 and 15 to 10 wt%, 20 wt%, 30 wt% and 40 wt%, respectively, The amount was measured using HPLC (High performance liquid chromatography, Waters, USA). A C18 column (150 mm L.ⅹ 4.6 mm I.D.) was used for HPLC analysis and the particle size was 5 μm. The solvent and mobile phase of the sample used 80% methanol, the analysis was carried out at 25 ℃ at a flow rate of 1 ml / min. Detection of the drug was measured at 240 nm wavelength using a UV detector.

For quantification, a calibration curve was prepared using the concentration of paclitaxel and the area change of the characteristic peak accordingly, and then the concentration of paclitaxel in the sample was calculated. The encapsulation efficiency and final encapsulation amount of the drug were determined by the following equation, and the results are shown in Table 6.

Encapsulation Efficiency (%)

= (Quantity of drug in nanoparticles) / (initial drug usage) × 100

Final loading (%)

= (Drug amount in nanoparticles) / (total nanoparticle weight) × 100

result

Paclitaxel Encapsulation efficiency  And Encapsulation HA - CA Paclitaxel
Usage (% by weight) Encapsulation efficiency (%) final Encapsulation (%) Example  9 16KHA-30% CA
(Example 6) 10 88.2 8.8 Example  11 16KHA-30% CA
(Example 6) 20 79.6 16 Example  13 16KHA-30% CA
(Example 6) 30 85.2 25.6 Example  15 16KHA-30% CA
(Example 6) 40 77.5 31

Experimental Example  5: in the mouse cancer model Paclitaxel Enclosed Low molecular weight  Hyaluronic Acid-Bile Acid Complex Nanoparticles Cancer tissue  Accumulation effect

The advantage of nanoparticle formulations is that they effectively mask and deliver encapsulated drugs, as well as allow specific distribution of drugs at target sites by controlling particle size. In this experimental example, paclitaxel-embedded low molecular weight hyaluronic acid-bile acid nanoparticles prepared according to Example 12 or 13 were labeled with a fluorescent substance and administered to cancer-inducing mice to evaluate the targeting effect on cancer tissues.

Way

Example 1 (7KHA-10% CA) and Example 6 (16 KHA-30% CA) was conjugated to the fluorescent material Cy5.5 (Sigma aldrich, USA) to prepare a complex labeled with a fluorescent substance on hyaluronic acid-bile acid It was.

20 mg of the hyaluronic acid-bile acid complex of Example 1 was dissolved in phosphate buffer (pH 8) and then EDC was added by the equivalent of the carboxyl group in hyaluronic acid. NHS was then dissolved in 500 μl DMSO by EDC equivalent and added to activate the carboxyl group of hyaluronic acid. 1 mg Cy5.5 was then dissolved in 500 μl phosphate buffer (pH 8) and added to the reaction solution. After reacting for 24 hours at room temperature, the resulting complex was purified by dialysis and lyophilized. 30 wt% paclitaxel was encapsulated in the same manner as in Example 12, and a nanoparticle solution was prepared at a concentration of 2 mg / ml. The hyaluronic acid-bile acid complex of Example 6 was also labeled with a fluorescent material in the same manner as described above, and then, as in Example 13, 30 wt% paclitaxel was encapsulated to prepare a nanoparticle solution.

Nude mice (BALB / c) were used as experimental animals and cancer was induced by injection of squamous cell carcinoma 7 cell line. 100 μl of the nanoparticle solution prepared in the mouse was injected intravenously, and a UV spectrophotometer was used as a control to obtain an amount of Cy5.5 indicating the same amount of fluorescence as the nanoparticles. After a certain period of time, near-infrared irradiation was performed to obtain cancer tissue images (FIG. 3).

result

As a result, as shown in FIG. 3, when the hyaluronic acid-bile acid composite nanoparticles of the present invention were administered, the nanoparticles accumulated significantly in cancer tissues compared to the Cy5.5-administered group, indicating that the target had an excellent targeting effect against cancer. there was.

Experimental Example  6: in the mouse cancer model Paclitaxel Enclosed Low molecular weight  Anticancer Effect of Hyaluronic Acid-Bile Acid Complex Nanoparticles

Pharmaceutical agents acting in cells, such as anticancer agents, must not only be selectively distributed to target tissues but also effectively uptake by cells in tissues in order to express drug efficacy. In this experimental example, the change in tumor size when paclitaxel-embedded hyaluronic acid-bile acid complex prepared in Example 13 was measured was confirmed whether the actual anti-cancer effect expression.

Way

Cancer was induced by transplanting human breast cancer cell line MDA-MB231 into nude mice (BALB / c). Mice were administered 20 mg / kg or 40 mg / kg of paclitaxel-encapsulated hyaluronic acid-bile acid complex prepared in Example 13, three times, once every four days. Controls include commercially available water for injection (negative control) and 20 mg / kg Genexol (Genexol INJ, Manufacture: Samyangsa) (Yangseong Control) was used. Tumor size and weight change were measured while observing mice for 32 days, and the results are shown in FIGS. 4 to 6.

Tumor size and body weight were compared with the control group using t-test for statistical treatment, and p <0.05 was determined to be statistically significant.

result

As shown in Figure 4, compared to the negative control group (injection water administration group), both the hyaluronic acid-bile acid complex and genexol administration group of the present invention significantly inhibited cancer growth. Example 13 showed an effect of inhibiting cancer growth equivalent to that of the index drug Xenxol at both 20 mg / kg and 40 mg / kg doses.

Figure 5 is a measure of cancer size in terms of relative cancer growth inhibition rate, the change in the size of the cancer after administration of the test substance and the control material was expressed as a percentage of the size of the initial cancer. In the case of the Genexol-administered group, the cancer grows again over time (after day 27) .However, when the complex of Example 13 was administered, cancer growth was continuously suppressed regardless of the dose. I didn't see it.

As shown in Figure 6, both the water for injection, Genexol and the complex administration group of Example 13 showed no characteristic body weight change. However, the group of 40 mg / kg of the complex of the present invention had a significant weight loss (p <0.05) compared to Genexol due to a weight loss of about 10%, but began to recover after the 12th day of administration and the control group from the 17th day. There was no significant difference.

Claims (18)

A low molecular weight hyaluronic acid-bile acid complex that forms a self-aggregate in water as an amphiphilic complex in which hydrophobic bile acids are bonded to low molecular weight hyaluronic acid of 5 or more and less than 20 KDa. The low molecular weight hyaluronic acid-bile acid complex according to claim 1, wherein a hydrophobic bile acid is bonded with a degree of substitution of 1 to 20% with respect to the monomer of the low molecular weight hyaluronic acid. The method of claim 1, wherein the hydrophobic bile acid is 5-β cholanic acid (5-β-cholanic acid), cholic acid (cholic acid), chenodeoxycholic acid (chenodeoxycholic acid), glycocholic acid (glycocholic acid), taurocholic acid ( Low molecular weight hyaluronic acid-bile acid complexes selected from the group consisting of taurocholic acid, deoxycholic acid, lithocholic acid and 7-oxo-lithocholic acid. 4. The low molecular weight hyaluronic acid-bile acid complex of claim 3, wherein the hydrophobic bile acid is 5-β cholanic acid. The low molecular weight hyaluronic acid-bile acid complex according to claim 4, wherein the low molecular weight hyaluronic acid is produced by covalent bonding of aminoethyl 5-β colonanoamide. A nanoparticle in which a hydrophobic drug is enclosed in the low molecular weight hyaluronic acid-bile acid complex according to any one of claims 1 to 5. The nanoparticle of claim 6, wherein the particle has a diameter of 100 to 500 nm, and the hydrophobic drug is encapsulated in a content of 5 to 40 wt% in the complex. The nanoparticle of claim 6, wherein the hydrophobic drug is selected from the group consisting of anti-neoplastic, anesthetic, anti-inflammatory, immunosuppressive and hormone. The nanoparticle of claim 8, wherein the hydrophobic drug is an anti-neoplastic agent. The method according to claim 9, wherein the hydrophobic anti-neoplastic agent is adriamycin, cyclophosphamide, actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, fluorouracil, carboplatin, Carmustine (BCNU), methyl-CCNU, cisplatin, cisplatin etoposide, interferon, campotesine and derivatives thereof, phenesterin, taxanes, paclitaxel and derivatives thereof, taxotere and derivatives thereof, vinblastine, vincristine, tamoxifen , Etoposide, piposulfan, docetaxel, anasterazole, gleevec, phloxuridine, leuprolide, flutamide, zoleronate, gemcitabine, streptozosin, carboplatin, topotecan, velotecan, irinotecan , Vinorelbine, hydroxyurea, varubicin, retinoic acid series, mechloretamine, chlorambucil, busulfan, doxyfluidine, prednisone, testosterone, US Tostron, aspirin, salicylate, ibuprofen, naproxen, phenopropene, indomethacin, phenylbutazone, cyclophosphamide, mecloethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nimesulide , Nanoparticles selected from the group consisting of cortisone and corticosteroids. An injection comprising the nanoparticles of claim 6. (a) synthesizing hydrophobic bile acid having a cholanic acid structure with aminoethyl colonanoamide;
(b) dissolving low molecular weight hyaluronic acid of at least 5 and less than 20 KDa in an aqueous solvent to prepare a hyaluronic acid solution;
(c) dissolving the aminoethyl colonanoamide of step (a) in an organic solvent to prepare a collagenoamide solution;
(d) preparing a reaction solution by adding the collagenoamide solution of step (c) to the hyaluronic acid solution of step (b);
(e) obtaining a hyaluronic acid-bile acid complex from the reaction solution and obtaining it; And
(f) encapsulating a drug in the hyaluronic acid-bile acid complex, comprising the nanoparticles of claim 6. The method of claim 12, wherein the hydrophobic bile acid is 5-β cholanic acid. The method according to claim 12, wherein 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added to the hyaluronic acid solution of step (b). Manufacturing method. The process according to claim 12, wherein the organic solvent of step (c) is selected from the group consisting of dimethylacetamide, dimethyl sulfoxide and methanol. The process according to claim 12, wherein the precipitation solvent of step (e) is selected from the group consisting of acetone, isopropyl alcohol, isopropyl ether, dimethyl ether, hexane and combinations thereof. The method according to claim 12, further comprising purifying the hyaluronic acid-bile acid complex by removing residual solvent and impurities after the step (e). 13. The hyaluronic acid-bile acid complex according to claim 12, wherein step (f) comprises dissolving the drug in an organic solvent selected from the group consisting of ethanol, methanol, chloroform, dichloromethane, dimethylsulfoxide and dimethylacetamide. The method comprising the step of mixing an aqueous solution of.

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