KR20090058420A - Dextran acetate microparticles preparation for drug delivery and drug delivery method using the same - Google Patents

Abstract

The present invention provides a method for producing dextran acetate microparticles having excellent biodegradability and bioavailability, and excellent in poorly soluble drug and radiolabelled substance delivery properties. In addition, by providing a method for encapsulating poorly soluble drugs in the dextran acetate microparticles prepared according to this method to provide a new method for producing a dextran acetate drug carrier.

Description

Preparation method of Dextran Acetate Nanoparticles for Drug Delivery and of Drug Delivery Media using Dextran Acetate Nanoparticles}

The present invention relates to a method for preparing an effective drug carrier and a radiolabeled carrier, wherein the manufacturing process belongs to the field of biochemistry and the fields of application belong to the field of diagnostic and therapeutic medicine.

Cyclosporin A is an immunosuppressive agent and is a very important drug applied to organ transplant patients. Cyclosporine A is Tolypocladium inflatum It is a substance extracted from Gams mold, and 7 amino acids out of 11 amino acid cyclic polypeptides are methylated and have three hydrogen bonds in the structure. Cyclosporin A is highly administered to patients with tissue transplantation because of its high immunosuppressive effect. Cyclosporin A, however, is poorly soluble and bioavailable, which limits its application to treatment. Oral administration of cyclosporin A can cause side effects, such as hemochromatogenic anemia and lymphocytosis. Therefore, in order to increase the therapeutic effect and eliminate side effects of cyclosporin A, a drug carrier that can increase bioavailability and drug delivery efficiency is required.

Dextran is water-soluble and highly biocompatible, and is used as a carrier for small molecule size drugs, proteins and peptides. However, when the hydrophobic agent is introduced into the dextran, the hydrophilicity of the dextran causes the drug to be delivered without condensation, thereby limiting its use. Because of the hydrophilic nature of the dextran, various dextran derivatives, such as dextran polymer materials, have been proposed as a method for application as a drug carrier.

There are also attempts to use dextran as a carrier to deliver radiolabels. However, dextran has a problem in that it cannot be applied as a label carrier because of colloidal contamination and low labeling efficiency. In order to improve this, there is a great interest in developing various dextran derivatives, such as a method of making dextran derivatives by combining cysteamine and the like with dextran.

Dextran derivatives, which improve the shortcomings of dextran, can be used as carriers for poorly soluble drugs and can be applied as carriers for radiolabelled substances, and thus have high utility value. Therefore, it is very important to develop a dextran derivative having excellent function.

Cyclosporin A has a very low absorption rate oral bioavailability and oral variation between individuals upon oral administration. In addition, the effective blood concentration range is very narrow, and high concentrations have serious limitations such as nephrotoxicity, hypertension, hepatotoxicity and neurotoxicity.

In order to improve these problems, drug delivery vehicles using liposomes or polysaccharides have been proposed. However, liposomes are not widely applied clinically because of low drug encapsulation rate and stability, and the polysaccharide microparticles are absorbed through the small intestine upon oral administration.

Recently, interest in developing nanoparticles using natural polymers for the development of effective drug carriers has been increasing. Biodegradable nanoparticles using natural polymers do not need to be surgically removed after most administration, and can target the drug or control the release to specific sites, which can reduce side effects due to toxicity or allergy and improve bioavailability. It can have many advantages.

Biodegradable and biocompatible natural polymers include dextran, chitosan and gelatin. Among these, dextran is a linear polysaccharide composed of α-1,6-glucopyranose bonds with α-1,3 binding molecules, and is toxic, immune, and biocompatible. It is also widely used as a parenteral carrier. However, since dextran is water soluble and cannot be used as a carrier for poorly soluble drugs, it is necessary to convert it into poorly soluble by chemical simulation.

The present invention solves the problem of dextran to provide a hydrophobic drug such as cyclosporin A to the body efficiently, and to provide a label transporter with high label delivery efficiency and label stability for radiolabeling for imaging To solve this problem by providing.

In the present invention, to solve the above problems by providing a method for producing poorly soluble dextran acetate from water-soluble dextran by chemical conversion reaction. In addition, the present invention provides a method for producing a drug carrier that can be delivered by encapsulating poorly soluble drugs in dextran acetate microparticles to provide a drug carrier with excellent biodegradability and biocompatibility. In addition, to solve the above problems by adopting the dextran acetate microparticles according to the present invention to deliver the drug by lymphatic vessel administration. By using the dextran acetate microparticles according to the present invention, a radiolabeled material for imaging can be loaded and delivered through lymphatic vessels, and also poorly soluble drugs such as cyclosporin A can be applied to diagnosis and treatment. Do it.

Hereinafter, the present invention will be described in more detail.

At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art. In addition, repeated description of the same technical configuration and operation as in the prior art will be omitted.

The present invention provides a method for preparing a dextran acetate microparticles, a method for producing a drug carrier for encapsulating at least one selected from a hydrophobic drug or a radiolabeled substance in the dextran acetate microparticles prepared therefrom, the dextran acetate microparticles. do. The present invention also more specifically provides a dextran acetate-cyclosporin conjugate in which cyclosporin is bonded as a hydrophobic drug to dextran acetate microparticles as a drug carrier.

The method for preparing dextran acetate microparticles according to the present invention includes the following steps.

a) dissolving dextran in a polar organic solvent to prepare a dextran solution;

b) reacting the dextran solution by adding an organic base and acetic anhydride; And

c) separating the reaction product.

The dextran is a polymer of α-1,6 bonds of D-glucose and has a structure in which α-1,3 or α-1,4 bonds are branched and has a molecular weight in the range of 60,000 to 90,000. .

The polar organic solvent may be formamide, dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), dimethylacetamide (DMAc), dimethylformamide (DMF), dimethylimidazolidone (DMI) or these Mixtures of these may be used, more preferably using formamide.

The organic base may be selected from pyridine or amine compound, and examples of the amine compound include primary amine, secondary amine or tertiary amine compound.

The reaction temperature of step b) is preferably adjusted to the range of 40 ~ 100 ℃, which may be too low when the reaction temperature is less than 40 ℃, reaction by-products when the reaction temperature exceeds 100 ℃ This is because the yield of the reaction can be reduced by production.

Separation of the reaction product of step c) consists of a process of centrifuging the precipitate produced by adding distilled water, followed by washing and dialysis.

The hydroxyl group of dextran is converted to an acetate group through the above preparation step to produce hydrophobic dextran acetate fine particles. Dextran acetate microparticles have an average particle diameter in the range of 0.05 to 1 μm, more specifically 0.1 to 0.5 μm. Dextran acetate microparticles according to a preferred embodiment of the present invention is spherical as shown in Figure 1 has an average particle diameter of 200 ~ 300 nm distribution.

In another aspect, the present invention provides a method for producing a drug carrier using the dextran acetate fine particles prepared by the above method. The method for producing a drug carrier of the present invention specifically includes the following steps.

1) preparing a mixed solution by mixing the dextran acetate microparticles prepared by the above method and at least one selected from a hydrophobic drug or a radiolabel with an organic solvent;

2) dialysis of the mixed solution in the dialysis tube; And

3) lyophilizing the obtained drug carrier.

The hydrophobic drug may be paclitaxel, amphotericin B, camptothecin, biphenyl dimethyl dicarboxylate (DDB), idebenone, or pipefosnel. (piposulfan), danazole, cyclosporin A, taxotere, adriamycin, teniposide, etoposide, itraconazole, azathioprine ), Nystatin, hemoglobin and derivatives thereof, and one or more selected from topotekan, etodolac and omeprazole, and more preferably cyclosporin A ( cyclosporin A).

The organic solvent is dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dimethylacetamide (DMAc), dioxane, chloroform, n-hexane, toluene, dichloromethane, ethyl acetate, chloroform, acetonitrile, acetone or these It can be used from a mixture of.

The radiolabelled material is ^99m Tc pertechnetate, gallium-67 (Ga-67, Gallium-67), rhenium-188 (Re-188, Rhenum-188), or thallium-201 (Tl- 201, Thallium-201).

In another aspect, the present invention provides a dextran acetate-cyclosporine conjugate prepared by combining the dextran acetate microparticles prepared by the above method and the hydrophobic drug cyclosporin, wherein the dextran acetate-cyclosporine conjugate further comprises a radiolabel It may further comprise a substance. The amount of the cyclosporin bound to the dextran acetate particles in the dextran acetate-cyclosporine conjugate is up to 50% by weight based on the weight of dextran acetate, specifically, in the range of 10 to 50% by weight.

As an example of the present invention, the drug release characteristics of the dextran acetate-cyclosporine conjugate showed excellent drug release characteristics in artificial intestinal and artificial gastric juices, as shown in FIG. 3. In addition, as a result of confirming the labeling effect after loading the dextran acetate-cyclosporin conjugate according to the present invention, the technetium-99m pertechnetate as a radiolabel, as shown in FIG. Indicates.

The present invention has the effect of utilizing the properties of natural polymer dextran excellent in biodegradability and biocompatibility and efficiently deliver poorly soluble drugs by preparing dextran acetate microparticles and using it as a drug carrier.

In addition, dextran acetate microparticles have high delivery efficiency for radiolabelled substances and can be applied to radiolabelled substances delivered through lymphatic vessels. In addition, since the radiolabelled substance and the therapeutic drug can be loaded at the same time, the diagnostic effect by the delivery of the label and the therapeutic effect by the drug delivery can be simultaneously obtained.

Dextran acetate microparticles provided in the present invention can be effectively used for lymphatic vessel delivery, reduce the toxicity of encapsulated drugs and target specific tissue sites, and thus can be used as radiopharmaceuticals as useful materials for imaging. It seems to be.

Hereinafter, a method for preparing a dextran acetate microparticle according to the present invention, a method for encapsulating a drug in the dextran acetate microparticle, a drug delivery effect of a dextran acetate drug carrier, and a radiolabeled dextran acetate drug carrier It will be described in more detail with reference to the phenomenon that the labeling material is delivered through the lymph vessel by loading on. However, the following examples are merely examples of the present invention, and the claims of the present invention are not limited thereto.

Example 1 Preparation of Dextran Acetate Microparticles

A reaction was performed to convert dextran, an aqueous polysaccharide, to dextran acetate, which is water insoluble. 6 g of dextran (weight average molecular weight 70,000, Hayashibara Co., Japan) was dispersed in 60 ml of formamide and dissolved by vigorous stirring at 55 ° C. 18 ml of pyridine and 40 ml of acetic anhydride were added to the reaction solution and reacted at 50 ° C. for 48 hours. After completion of the reaction, 200 ml of distilled water was added to precipitate the precipitate. The precipitate was centrifuged and washed several times. The precipitate was dissolved in dimethylsulfoxide, then dialyzed for 4 days, and then lyophilized for 4 days. Acetate microparticles were prepared.

The synthesized dextran acetate microparticles were synthesized by FT-IR analysis and the shape and size of the particles were measured by scanning electron microscopy (SEM). Referring to the scanning electron micrograph of Figure 1, the dextran acetate microparticles are spherical, the average particle diameter was about 250 nm. Referring to FIG. 2, which is an FT-IR spectrum measured to confirm the formation of dextran acetate, the prepared dextran acetate exhibits a C═O bond band at 1747 cm ⁻¹ unlike the FT-IR spectrum of dextran. Dextran acetate-specific binding bands such as a CH ₃ binding band at 1431 cm ⁻¹ and an OC═O binding band at 604 cm ⁻¹ were observed. From the above results, it was confirmed that dextran was converted to dextran acetate.

Example 2 Preparation of Dextran Acetate Drug Carrier Enclosed with Drug

In order to encapsulate the drug in the dextran acetate microparticles prepared in Example 1, a dextran acetate drug carrier in which the drug was encapsulated was prepared as follows. After dissolving 40 mg of dextran acetate microparticles and 20 mg of cyclosporine A in 10 ml of dimethylsulfoxide (DMSO) for 30 minutes, the drug was placed in a dialysis tube having a molecular weight cutoff (MWCO) of 12,000 g / mol. After the dialysis was repeated while distilled water was changed at intervals of 3 hours for 24 hours, the dialysis drug was filtered through a 0.45 μm membrane filter, and then lyophilized to prepare a dextran acetate drug carrier. The content of cyclosporin encapsulated in the prepared drug carrier was about 50% by weight of dextran acetate.

Drug release characteristics of the dextran acetate drug carrier prepared by the method of the present invention were investigated through the following drug delivery experiments. An artificial intestinal fluid having a pH of 7.4 was prepared using phosphate buffer saline. In addition, a hydrochloric acid solution having a pH of 1.2 was prepared to prepare artificial gastric juice. Each 50 ml of the solution was added to a tube, followed by a dextran acetate drug carrier enclosed with cyclosporin A, followed by a release experiment in an incubator adjusted to 37 ° C. The solution was stirred at a constant speed to remove the concentration boundary layer of the drug, and samples were taken at a predetermined time interval by 1 ml, and the released drug was analyzed by liquid chromatography (HPLC).

3 shows the release of the drug from the dextran acetate microparticles containing cyclosporin A in artificial intestinal fluid (pH 7.4) and artificial gastric fluid (pH 1.2). Cyclosporin A was released about 50% in the intestinal fluid after 2 days, and more than 35% in the gastric juice showed relatively good drug release properties.

Example 3

The labeling effect of the dextran acetate microparticles provided on the present invention was investigated. The cyclosporin A-embedded dextran acetate microparticles prepared in Example 2 were placed in a vacuum vial containing phosphate buffered saline solution. Tartrate was added to a new vial and injected with ^99m Tc-pertechnetate label. This mixed solution was injected into the stomach vacuum vial and mixed at room temperature to load the radiolabel.

The labeling effect was measured using thin layer chromatography. Thin layer chromatography analysis was performed using saline and acetate mobile phases. To compare the labeling effect on textan acetate particles, the labeling effect was compared by introducing the same labeling material into human serum albumin. Immediately after labeling, labeling effects were examined every 30 minutes, 1 hour, 2 hours, 4 hours and 12 hours. The labeling efficiency of the dextran acetate microparticles irradiated in this manner is shown in FIG. 4. The labeling efficiency was over 80%, and the labeling efficiency was over 80% for 12 hours, indicating stable labeling efficiency.

0.1 ml (approximately 0.25 mCi) of cyclosporin A and radiolabelled dextran acetate microparticles were injected into the left ankle of each mouse (NTacSam: SD, female, 16 weeks, 250 g) through the lymphatic vessel, respectively. To be delivered. Human serum albumin was introduced with the same label and injected into mice in the same manner. An image of the delivered radiolabeled image was taken using a gamma camera. An image photographed in this manner is shown in FIG. 5. As a result of confirming the degree of delivery of the labeling material over time, the radioactive labeling material (FIG. 5 (a)) introduced into dextran acetate microparticles and the labeling material loaded on human serum albumin which are widely used (FIG. 5 (b)) was able to confirm that the time passed to the inguinal part after 40 minutes through the image. Seventy minutes after injection, both carriers delivered labeling to the kidneys. The dextran acetate microparticles prepared by the method provided by the present invention were encapsulated with a radiolabeling agent and a poorly soluble drug, and delivered through the lymphatic vessel. As a result, the labeling substance was delivered to a degree similar to that of human serum albumin.

1 is a scanning electron micrograph of dextran acetate microparticles,

2 is an FT-IR spectrum of dextran (a) and dextran acetate microparticles (b),

Figure 3 shows the results of the release in artificial intestinal fluid (a) and gastric juice (b) of cyclosporin A encapsulated in dextran acetate microparticles,

4 is a result of sustaining label efficiency (label stability) of radioactive waste material encapsulated in dextran acetate microparticles,

FIG. 5 is an image photograph taken by a gamma camera of a label material transfer phenomenon, (a) is a delivery image of a label material loaded on dextran acetate, and (b) is a delivery image of a label material loaded on human serum albumin.

Claims (12)

a) dissolving dextran in a polar organic solvent to prepare a dextran solution; b) reacting the dextran solution by adding an organic base and acetic anhydride; And c) separating the reaction product; Dextran acetate microparticles manufacturing method comprising a. The method of claim 1, The polar organic solvent is any one of formamide, dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), dimethylacetamide (DMAc), dimethylformamide (DMF), and dimethylimidazolidone (DMI). Or dextran acetate fine particles. The method of claim 1, The organic base is a method for producing dextran acetate microparticles, characterized in that selected from pyridine or amine compound. The method of claim 1, The b) step reaction temperature is a dextran acetate microparticles manufacturing method, characterized in that 40 ~ 100 ℃. The method of claim 1, Separation of step c) is a method for producing dextran acetate microparticles, characterized in that the precipitate produced by the addition of distilled water, followed by washing and dialysis. 1) preparing a liquid mixture by mixing dextran acetate microparticles prepared by the method of any one of claims 1 to 5 and at least one selected from hydrophobic drugs or radiolabels with an organic solvent. ; 2) dialysis of the mixed solution in the dialysis tube; And 3) freeze drying the obtained drug carrier; Dextran acetate drug delivery method comprising a. The method of claim 6, The hydrophobic drug is a method for producing a dextran acetate drug carrier, characterized in that the cyclosporin. The method of claim 6, The radiolabelled material is ^99m Tc pertechnetate, gallium-67 (Ga-67, Gallium-67), rhenium-188 (Re-188, Rhenum-188), or thallium-201 (Tl- 201, Thallium-201). The method of claim 6, The organic solvent is any one of dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMAc), dioxane, chloroform, n-hexane, toluene, dichloromethane, ethyl acetate, chloroform, acetonitrile and acetone. A method for preparing a dextran acetate drug carrier, characterized in that it is selected from one or a mixture thereof. Textan acetate-cyclosporine conjugate in which cyclosporine is bound to dextran acetate particles. The method of claim 10, And said conjugate further comprises a radiolabelled substance. The method of claim 11, The radiolabelled material is ^99m Tc pertechnetate, gallium-67 (Ga-67, Gallium-67), rhenium-188 (Re-188, Rhenum-188), or thallium-201 (Tl- 201, Thallium-201). Textan acetate / cyclosporine conjugate.

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