KR20220123260A - Chitosan hydrogel-chelator manufacturing method for cancer treatment - Google Patents

Abstract

The present invention relates to a radioactive chitosan hydrogel-chelator, a method for preparing the same, and a composition for cancer treatment and embolic treatment containing the same as an active ingredient. The prepared chitosan hydrogel-chelator labeled with a radionuclide with a size of 60 to 100 μm according to the present invention blocks the flow of blood toward the tumor tissue and blocks the supply of oxygen and nutrients to the tumor cells, thereby embolizing cancer tissue. In particular, by directly labeling radionuclides, free radionuclides can be minimized due to excellent labeling efficiency and labeling stability of radionuclides. Therefore, the therapeutic effect of the lesion can be increased.

Description

Chitosan hydrogel-chelator manufacturing method for cancer treatment

The present invention relates to a method for preparing a chitosan hydrogel-chelator for the treatment of cancer.

In modern society, as the incidence of cancer is steadily increasing along with the death from cancer, there is an urgent need for early detection of cancer and the suggestion of new treatment methods. have.

The method of treating intractable diseases including cancer using radionuclides is very simple and economical compared to surgery, and above all, it not only causes less pain to the patient, but also has a high therapeutic effect. Therefore, a treatment method using a radionuclide is widely used. However, the treatment method using radionuclides has the disadvantage of destroying normal cells by affecting not only diseased sites but also normal tissues.

In order to solve the above problems, it is important to use a biocompatible polymer to selectively destroy tissue only at the disease site to which the radionuclide is administered, and to design so that the radionuclide does not move to another normal site. In addition, it is preferable that the biocompatible polymer containing the administered radionuclide is biodegraded, absorbed and discharged after a sufficient time passes after emitting radiation from the diseased site.

As a topical treatment using radionuclides currently used, there is a treatment for liver cancer using Sirtex's ⁹⁰ Y SIR microspheres, which are usefully used in the treatment of patients with liver cancer that is not easy to remove surgically. In addition, Dong Wha Pharm Co., Ltd.'s liver cancer treatment (milican) using a complex of ¹⁶⁶ holmium and chitosan ( ¹⁶⁶ Ho-chitosan) is known to be useful in treating small liver cancer because it can kill cancer cells in a short time with just one injection. . In addition to this, radionuclides include ¹⁸⁶ Re-tin colloid, ¹⁸⁶ Re-sulfur colloid, ¹⁸⁸ Re-hydroxyapatite, ⁹⁰ Y-colloid, and the like. In addition, Korean Patent Publication No. 10-530276 describes a particulate radionuclide-conjugated polymer, a method for preparing the same, and a kit for the preparation thereof, and Korean Patent Publication No. 10-530276 describes a prostate containing a radioactive material-chitosan complex. A composition for treating cancer and a kit for preparing the composition are described, and Korean Patent Application Laid-Open No. 10-2006-60970 discloses a radioactive material-chitosan complex solution composition with improved gelation stability when injected into the body and a method for preparing the same. However, when a therapeutic agent containing a radionuclide as described above is administered to a lesion in an aqueous solution state, a part of it comes out of the tissue and destroys the normal tissue, which may cause side effects, and does not stay in the administered site but flows to the other side or after administration The form of radioactive particles may become unsuitable for treatment, such as only water being absorbed into the tissue and radionuclides precipitated and gathered to one side, resulting in non-uniform irradiation.

Among the radionuclides, the ¹⁶⁶ Ho-chitosan complex changes into a gel in the body after being injected in an aqueous solution and stays there while maintaining its shape at the lesion site. has a In addition, there is an advantage that not only the radioactivity disappears after a sufficient time passes, but also the chitosan administered together in a complex state is decomposed and disappeared. In addition, ¹⁶⁶ Ho is relatively inexpensive compared to other radionuclides. However, the aqueous solution of the ¹⁶⁶ Ho-chitosan complex has the disadvantage of being an acidic solution, and there is a problem in that the radionuclides are released into normal tissues because gelation is not well formed. In addition, radionuclide therapeutics containing ¹⁶⁶ Ho or ⁹⁰ Y have a disadvantage in that they have to be manufactured on site when the therapeutic agent is manufactured with half-lives of 26.9 hours and 641 hours, respectively.

In addition, embolotherapy or embolization procedure is a treatment method characterized by selectively blocking blood vessels by injecting a specific substance (eg, balloon) into an artery through a catheter to control bleeding or treat a tumor. . The material used for such embolism treatment can block the flow of blood to the tumor tissue, thereby blocking the supply of oxygen and nutrients to the tumor cells, or controlling bleeding.

Such embolic compositions should preferably be biocompatible and hydrophilic, have a desired uniform size, be spherical, be useful for vascular occlusion, have minimal vascular irritation, and have a certain level of elasticity or It is desirable to have flexibility and to be able to swell to a certain level or more so as to occlude blood vessels by swelling after administration through a narrow tube.

Therefore, there is an urgent need for the development of a therapeutic agent using a radionuclide capable of minimizing the radionuclide liberated into a normal tissue by forming well in gelation.

In the present invention, it is an object of the present invention to provide a method for preparing a chitosan hydrogel-chelator labeled with a radionuclide.

In addition, the present invention aims to provide a chitosan hydrogel-chelator labeled with a radionuclide prepared by the above method.

In addition, the present invention aims to provide a pharmaceutical composition for cancer treatment containing the chitosan hydrogel-chelator labeled with the radionuclide.

In addition, the present invention aims to provide a pharmaceutical composition for the treatment of embolism containing the chitosan hydrogel-chelator labeled with the radionuclide.

One aspect of the present invention for achieving the above object is

1) electrospinning chitosan and a crosslinking material to prepare a chitosan hydrogel;

2) reacting the chitosan hydrogel prepared in step 1) with a chelator having a functional group capable of labeling a radionuclide to prepare a chitosan hydrogel-chelator,

The chelator is N-succinimidyl-3-(4-hydroxyphenyl)propionate, 1,4,7,10-tetraamacyclododecaine-1,4,7,10-tetraacetic acid, diethylene triamine pentaacetic acid histidine, tyrosine, and any one or more selected from the group consisting of proteins comprising tyrosine;

3) reacting the chitosan hydrogel-chelator prepared in step 2) with a radionuclide and an activator for labeling the radionuclide; will be.

Step 1) is a step of generating a chitosan hydrogel, and a hydrogel is prepared by reacting biodegradable chitosan with a crosslinking material. By controlling the concentration, radiation voltage, flow rate, radiation, etc. of the chitosan during the preparation of the hydrogel, it can be adjusted to an effective particle size for embolism treatment. In a specific embodiment, the particle size may be 60 to 100 μm.

When the hydrogel is prepared, the higher the concentration of chitosan, the higher the density of the hydrogel with respect to the particles, the stronger the particles, and the stability of the particles increases. In embolic therapy, particle size and uniformity are factors directly related to the therapeutic effect, and by increasing the concentration of chitosan, particles of uniform size can be produced.

The cross-linking material is used to form a hydrogel, and when a hydrogel is prepared using the cross-linking material, the labeling stability of the radionuclide can be increased to prevent leakage of the radionuclide into a normal tissue other than the lesion site. The crosslinking material may be at least one selected from the group consisting of tripolyphosphate, alginic acid, pectin, carboxymethyl cellulose, polyglutamic acid, protein, DNA, and RNA, but is not limited thereto. The crosslinking material may be an anionic crosslinking material, but is not limited thereto.

The electrospinning process may be to perform electrospinning at a voltage of 1 to 20 kV and a flow rate of 0.01 to 0.05 mL/min. In one specific embodiment, electrospinning may be performed at a voltage of 10 to 12 kV and a flow rate of 0.01 to 0.03 mL/min.

Step 2) is a step of preparing a chitosan hydrogel-chelator, and mixing chitosan hydrogel particles and a chelator dissolved in an organic solvent to obtain a chitosan hydrogel-chelator.

The chelator is a compound having a functional group capable of labeling radionuclides, N-succinimidyl-3-(4-hydroxyphenyl)propionate, 1,4,7,10-tetraamacyclododecaine- 1,4,7,10-tetraacetic acid, diethylenetriamine pentaacetic acid histidine, tyrosine, and any one or more selected from the group consisting of proteins including tyrosine, but is not limited thereto.

The radionuclide is at least one selected from the group consisting of ¹³¹ I, ¹²⁵ I, ¹²⁴ I, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁹⁰ Y, ¹⁶⁶ Ho, ^99m Tc, and ¹⁷⁷ Lu, but is not limited thereto.

The radionuclide may vary depending on the type of chelator.

For example, when the chelator is N-succinimidyl-3-(4-hydroxyphenyl)propionate, the radionuclide is preferably at least one selected from the group consisting of ¹³¹ I, ¹²⁵ I and ¹²⁴ I. .

Further, when the chelator is 1,4,7,10-tetraamacyclododecaine-1,4,7,10-tetraacetic acid, the radionuclide is preferably ¹⁷⁷ Lu.

In addition, when the chelator is diethylenetriamine pentaacetic acid, the radionuclide is preferably at least one selected from the group consisting of ¹¹⁸⁶ Re, ¹⁸⁸ Re, and ^99m Tc.

The activator for labeling radionuclides in step 3) is preferably any one selected from the group consisting of chloramine tea, a mixture of ascorbic acid and gentisic acid, and tin (II) chloride.

In particular, step 3) is a step of preparing a chitosan hydrogel-chelator labeled with a radionuclide, and a radionuclide is added to the chitosan hydrogel-chelator and reacted with an activator for labeling the radionuclide. A chitosan hydrogel-chelator is obtained. In the chitosan hydrogel-chelator labeled with the radionuclide, the labeling rate of the radionuclide is 99%.

When the concentration of chitosan is excessively increased to increase the stability of the chitosan hydrogel, it is difficult to mix the chitosan solution with the reactant for labeling the radionuclide (e.g., radionuclide, buffer, reducing agent) due to the high viscosity of chitosan, and the precipitate can occur

In addition, when the radionuclide is labeled with the chitosan hydrogel-chelator formulated after synthesizing a chelator in chitosan, it has a low labeling efficiency of less than 5%.

Since the labeling yield of the radionuclide for the chitosan hydrogel-chelator is 20 to 40% higher than the labeling yield of the radionuclide for the chitosan-chelator, it is advantageous in terms of the labeling yield for the radionuclide.

In the case of the chitosan hydrogel-chelator, since the chitosan hydrogel particles are dispersed in a dilute reaction solution and the reactants are mixed to proceed with chelator synthesis and radionuclide labeling, it is relatively simpler than the process of labeling a high-concentration polymer solution and is radioactive. Contamination by nuclides can be reduced and the chitosan hydrogel-chelator has the advantage of having a higher labeling yield when the radionuclides are labeled.

In addition, for the production of chitosan-chelator, it takes about 10 days for the process of dialysis and freeze-drying, but when synthesizing the chelator in chitosan hydrogel, it takes less than one day, so the production time can be shortened. In the state of chitosan hydrogel, a chelator can be selected according to the radionuclide to be labeled, and the range of options for labeling the radionuclide is widened.

Therefore, the chitosan hydrogel-chelator manufacturing method can be more flexibly applied to radionuclide labeling than the chitosan-chelator manufacturing method, and since the chitosan hydrogel particles are easier to handle than the chitosan solution, highly efficient labeling of radionuclides can be expected. and safer from radiation contamination and radiation exposure.

That is, according to the present invention, after preferentially preparing a chitosan hydrogel, by performing the preparation of a chitosan hydrogel-chelator and radionuclide labeling based on this, it is possible to label radioactive confetti with high efficiency and stability as well as manufacturing It also has the advantage of significantly shortening the process time.

In addition, the manufacturing method is

4) purifying the hydrogel prepared in step 3) using a polymer network; may further include. Step 4) is a purification step of the hydrogel using a polymer network, and through this, impurities such as reaction raw materials such as unreacted crosslinking agent and impurities such as broken particles can be removed to prepare a chitosan hydrogel-chelator of high purity.

The pore size of the polymer network is smaller than the particle size of the prepared hydrogel, and it is easy to pass the reaction raw material and broken particles, and may preferably be 50 μm.

The polymer is a nylon mesh, polyester, polypropylene, polyethylene, polycarbonate, polytetra fluoroethylene, polyamide-imide (polyamide) -imide), polyethylene sulfide (polyphenylene sulfide), preferably nylon mesh.

Step 4) is a step of performing the purification process using a polymer network, and in steps 1) to 3), chitosan hydrogel, chitosan hydrogel-chelator and radionuclide-labeled chitosan hydrogel-chelator are excluded. It is used in the purification process to remove impurities and reactants.

Another aspect of the present invention for achieving the above object provides a chitosan hydrogel-chelator labeled with the radionuclide prepared by the above method.

Another aspect of the present invention is a chitosan hydrogel combined with a chelator having a functional group capable of labeling a radionuclide; And to provide a chitosan hydrogel-chelator labeled with a radionuclide, comprising a radionuclide linked to the functional group. Here, the radionuclide, functional group, and chelator are the same as described above. A chelator having a functional group capable of labeling a nuclide is chemically bound to the chitosan hydrogel, and the functional group allows the nuclide to be bound, thereby providing a chitosan hydrogel-chelator labeled with a radionuclide.

The binding of the chitosan hydrogel with a chelator having a functional group capable of labeling radionuclides is a chitosan hydrogel having a functional group capable of labeling radionuclides by reacting the chelator with each functional group with the chitosan hydrogel under suitable solvent conditions. There is an advantage in terms of manufacturing method and therapeutic effect over the prior method of binding the rater and binding the hydrogel.

In terms of manufacturing, since there is no need to electrospray radionuclides during manufacturing, the exposure of manufacturers to radionuclides can be reduced and contamination of manufacturing facilities can be reduced.

On the other hand, in terms of treatment, since radiochemical stability after labeling is relatively high, side effects caused by free radionuclides can be reduced, and the safety of the subject can be secured by securing the stability of radionuclides. In addition, it is possible to predict the amount of radioactivity to be labeled, and it is possible to control the amount of radioactivity, so it is possible to administer the correct amount of radioactivity to the unit chitosan hydrogel as much as desired, which is advantageous for quantification.

The hydrogel of the present invention may be administered at a time of 0.5 to 150 mCi/mass 1 cm 3 , preferably 0.5-50 mCi/mass 1 cm 3 depending on the type of disease and the size of the lesion.

When the chitosan hydrogel-chelator prepared by the above method is directly injected locally into the cancer tissue site, the hydrogel stays in the cancer tissue site and hardly leaks to the outside, and prevents the flow of blood toward the tumor tissue. By blocking the supply of oxygen and nutrients to the tumor cells, necrosis can occur in the cancerous tissue.

As described above, the chitosan hydrogel-chelator according to the present invention is excellent in labeling efficiency and labeling stability of radionuclides by directly labeling chitosan with radionuclides, thereby minimizing free radionuclides, and locally at the lesion site. It is directly injected and stays stable at the lesion site and emits radiation, which can be usefully used in the treatment of intractable diseases such as cancer. Therefore, the chitosan hydrogel-chelator according to the present invention can expect not only the embolism therapeutic effect but also the therapeutic effect by radionuclide, thereby increasing the therapeutic effect of the lesion.

The manufacturing method is a method of manufacturing a hydrogel-chelator using a high concentration of chitosan, and the density of particles is higher than that of a chitosan-chelator hydrogel prepared using a relatively low concentration of chitosan, so stability when injected into the body It is advantageous in terms of advantages and the durability of the particles is relatively high, so it has the advantage that the possibility of leakage to other organs is reduced. In addition, since the stability of the labeled radionuclide is increased, side effects such as thyroid ingestion of free I-131 are reduced. In addition, in the case of a hydrogel with a large size range of manufactured particles, relatively large particles are injected first, and blood vessels can be blocked before the particles reach the lesion. As the possibility of reaching the lesion increases, a higher therapeutic effect can be expected.

The particle size for the hydrogel is one of the most important factors in the therapeutic effect. As a result of experiments on hepatic artery of a medium animal, for example, a rabbit having a size similar to that of a human hepatic artery, the hydrogel having a particle size of 60 ~ 100 μm coincides with the location of the injected particle, whereas the location of the injected particle is 100 ~ 300 In the case of a μm hydrogel, the injected particle stays outside the tumor, so 60 ~ 100 μm hydrogel particles can enhance the therapeutic effect of the lesion. Therefore, the experiment on liver cancer heavy animals was carried out using 60 ~ 100 μm hydrogel particles, which are expected to have a high therapeutic effect.

Another aspect of the present invention for achieving the above object is to provide a pharmaceutical composition for treating cancer containing the chitosan hydrogel-chelator labeled with the radionuclide.

In the present invention, the term “treatment” refers to all acts of curing cancer by administration of the composition, and treatment of a disease, a symptom of a disease, a secondary disease of the disease or disease, or a predisposition thereof , including the nanoparticles to a subject (human or animal) having a disease, a symptom of a disease, a secondary disease of a disease or disorder, or a predisposition therefor, with the purpose of alleviating, alleviating, remedy, or ameliorating It is defined as the application or administration of a composition.

The cancer is liver cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, colon cancer, breast cancer, uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma , vulvar carcinoma, esophageal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, solid tumor of childhood, differentiated lymphoma, cystitis, kidney cancer, renal cell carcinoma, kidney It may be one or more selected from the group consisting of pelvic carcinoma, first central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma, but is not limited thereto.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition, wherein the carrier includes a non-naturally occurring carrier. can do.

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. .

The "pharmaceutically acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition.

Specifically, the type of the carrier is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more. In addition, if necessary, other conventional additives such as antioxidants, buffers and/or bacteriostats may be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. It can be used by formulating it into a dosage form, pill, capsule, granule, or tablet.

The administration method of the pharmaceutical composition for the treatment of cancer according to the present invention is not particularly limited, and may follow a method commonly used in the art. As a non-limiting example of the administration method, the composition may be administered by oral administration or parenteral administration. In addition, the composition for improving or treating cancer of the present invention may be prepared in various formulations according to a desired administration method.

As used herein, the term "improvement" refers to any action that at least reduces the severity of a parameter, eg, a symptom, associated with a condition to be treated by administration of the composition of the present invention.

The composition may be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response modifiers for the treatment of cancer. Preferably, the composition is preferably in the form of an injection, but is not limited thereto. The compositions of the present invention for parenteral administration include sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders prepared immediately prior to use as sterile solutions or suspensions. Examples of suitable sterile aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, physiological saline, ethanol, polyols (eg, glycerol, propylene glycol, polyethylene glycol, etc.) and mixtures thereof, vegetable oils (eg, olive oil), and injectable organic esters (eg, ethyl oleate). For example, in the case of a dispersing agent and a suspending agent, a coating material such as lecithin may be used to maintain an appropriate specific size, and a surfactant may be used to maintain an appropriate fluidity. In addition, parenteral compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Sterilization of the injectable formulation can be used, for example, by filtration through a sterile filter or by presterilizing the components of the mixture prior to mixing, at the time of manufacture, or immediately prior to administration (as in the case of a double container syringe package).

Another aspect of the present invention for achieving the above object is to provide a composition for treating embolism containing a radionuclide-labeled chitosan hydrogel-chelator,

Embolotherapy is a treatment method that selectively blocks blood vessels by injecting a specific substance into an artery to control bleeding or treat a tumor. By blocking the flow, it can block the supply of oxygen and nutrients to the tumor cells.

The composition may be injected through the hepatic artery, and preferably used for embolism treatment of liver cancer tissue.

Another aspect of the present invention for achieving the above object is to provide a method for preventing or treating cancer in a subject comprising administering the chitosan hydrogel-chelator labeled with the radionuclide to a subject in need thereof.

Another aspect of the present invention for achieving the above object is to provide a method for preventing or treating embolism of a subject, comprising administering the chitosan hydrogel-chelator labeled with the radionuclide to a subject in need thereof.

The subject or subject refers to an animal, and may typically be a mammal capable of exhibiting a beneficial effect by treatment with the component of the present invention. Preferred examples of such subjects may include primates such as humans. Also, such subjects may include all subjects having, or at risk of having, symptoms of cancer or embolism.

The present invention also provides the use of said chitosan-hydrogel chelator in the manufacture of a medicament for the treatment of cancer.

The present invention also provides the use of said chitosan-hydrogel chelator in the manufacture of a medicament for the treatment of embolism.

The present invention also provides a composition comprising said chitosan-hydrogel chelator for use in the treatment of cancer.

The present invention also provides a composition comprising said chitosan-hydrogel chelator for use in the treatment of an embolism.

Overlapping contents are omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification have the meanings commonly used in the technical field to which the present invention pertains.

The chitosan hydrogel-chelator according to the present invention can minimize free radionuclides by directly labeling a biodegradable polymer hydrogel with radionuclides, and thus has excellent labeling efficiency, yield and labeling stability of radionuclides, and locally lesion site. It is injected directly into the lesion and emits radiation while staying stable at the lesion site, so it can be usefully used in the treatment of intractable diseases such as cancer. Therefore, the chitosan hydrogel-chelator according to the present invention can expect not only the treatment by radionuclide but also the therapeutic effect by embolism, thereby increasing the therapeutic effect of the lesion.

1 is a view of a chitosan hydrogel prepared with 1% or 2% chitosan under an optical microscope.
Figure 2 is an optical microscope observation of the chitosan hydrogel prepared according to the change of electrospinning conditions.
Figure 3 shows the labeling efficiency of the chitosan hydrogel-SHPP labeled with I-131 chitosan hydrogel.
Figure 4 shows the labeling efficiency of chitosan hydrogel-DOTA with Lu-177 labeled chitosan hydrogel.
5 shows the labeling efficiency of chitosan hydrogel-DTPA labeled with Tc-99m.
6 shows the labeling stability of I-131 for chitosan hydrogel-SHPP measured through a radiochemical foreign body test.
7 shows the label stability of I-131 to chitosan hydrogel-SHPP measured through a leaching test.
8 is a particle distribution evaluation of chitosan hydrogel-DTPA labeled with Tc-99m for a rabbit liver cancer model.
Figure 9 shows the labeling yield of I-131 for chitosan-SHPP (A) and chitosan hydrogel-SHPP (B).
Figure 10 shows a CT scan image of a liver cancer middle animal model treated with chitosan hydrogel-SHPP prepared with a particle size of 60 to 100 μm.
11 is a graph showing the results of measuring the tumor volume in a middle animal liver cancer model for 4 weeks.
12 is a graph showing the results of processing the chitosan hydrogel-SHPP prepared with a particle size of 60 to 100 μm in a liver cancer medium animal model and measuring the weight of the tumor.
13 is a graph showing a correlation by comparing the result values of tumor weight and volume.

Examples and Experimental Examples

Hereinafter, the present invention will be described in more detail through examples. However, the examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Preparation of chitosan hydrogel

(1) Preparation of 1% chitosan hydrogel

After dissolving 25 mg of chitosan in 2.5 mL of 0.1 M acetic acid (1%), a solution in which 5 g of tripolyphosphate (TPP) was dissolved through a pump was electrospun under a voltage of 1 to 20 kV to prepare a hydrogel.

(2) Preparation of 2% chitosan hydrogel

After dissolving 50 mg of chitosan in 2.5 mL of 0.1 M acetic acid (2%), a solution in which 5 g of tripolyphosphate (TPP) was dissolved through a pump was electrospun under a voltage of 1 to 20 kV to prepare a hydrogel.

Example 2. Preparation of chitosan hydrogel-chelator

(1) Synthesis of chitosan hydrogel-SHPP

After purifying the chitosan hydrogel using a polymer network, N-succinimidyl-3-(4-hydroxyphenyl)propionate dissolved in DMF or DMSO (N-ssucinimidyl-3-(4-hydroxyphenyl)propionate, Hereinafter, 10 mg of SHPP) was added and reacted for at least 6 hours. The prepared chitosan hydrogel-SHPP was purified using a polymer network.

(2) Synthesis of chitosan hydrogel-DOTA

After purifying the chitosan hydrogel using a polymer network, 1,4,7,10-tetraamacyclododecaine-1,4,7,10-tetraacetic acid (1,4,7,10) dissolved in DMF or DMSO -tetraazacyclododecane- 1,4,7,10-tetraacetic acid or less (DOTA) 10 mg was added and reacted for more than 6 hours. The prepared chitosan hydrogel-DOTA was purified using a polymer network.

(3) Synthesis of chitosan hydrogel-DPTA

After the chitosan hydrogel was purified using a polymer network, 10 mg of diethylenetriamine penta acetic acid (DTPA) dissolved in distilled water and methanol were added and reacted for more than 6 hours. The prepared chitosan hydrogel-DTPA was purified using a polymer network.

Example 3. Preparation of chitosan hydrogel-chelator labeled with radionuclides

(1) Labeling of radioactive iodine (I-131) in chitosan hydrogel-SHPP

Radioactive oxo (I-131) and chloramine-T (2 mg/0.1 mL phosphate buffer (pH 7.5)) were added to the chitosan hydrogel-SHPP prepared in (1) of Example 2, and reacted for 1 hour. After the reaction was completed, it was purified with a polymer network to obtain a chitosan hydrogel-SHPP labeled with radioactive iodine (I-131).

Radioactive oxo (I-131) and chloramine-T (2 mg/0.1 mL phosphate buffer (pH 7.5)) were added to the chitosan hydrogel-SHPP prepared in Example 2 1, and reacted for 1 hour. After the reaction was completed, it was purified with a polymer network to obtain a chitosan hydrogel-SHPP labeled with radioactive iodine (I-131) as shown in Scheme 1 below.

[Scheme 1]

(2) Labeling of Lu-177 in chitosan hydrogel-DOTA

Lu-177, ascorbic acid and gentisic acid were added to the chitosan hydrogel-DOTA prepared in (2) of Example 2, and reacted at 80° C. for 30 minutes. After the reaction was completed, it was purified with a polymer network to obtain Lu-177-labeled chitosan hydrogel-DOTA as shown in Scheme 2 below.

(3) Labeling of Tc-99m in chitosan hydrogel-DTPA

To the chitosan hydrogel-DTPA prepared in (3) of Example 2, Tc-99m and tin(II) chloride dissolved in distilled water from which dissolved oxygen was removed was added and reacted for 30 minutes. After the reaction was completed, it was purified with a polymer network to obtain Tc-99m-labeled chitosan hydrogel-DTPA as shown in Scheme 3 below. In addition, ¹⁸⁶ Re and ¹⁸⁸ Re can be labeled on chitosan hydrogel-DTPA in the same manner.

Experimental Example 1. Particle size and shape comparison experiment according to chitosan concentration

In order to compare and analyze the particle size and shape of the chitosan hydrogel prepared in Example 1 (1) and Example 1 (2), the following experiment was performed. Specifically, each chitosan hydrogel was observed and evaluated at 40 magnification using an optical microscope, and the results are shown in FIG. 1 .

As shown in Figure 1, when prepared with 2% chitosan hydrogel (right) rather than a hydrogel prepared with 1% chitosan (left), the particle shape is a smooth spherical shape, the particle density is high, and more uniform particles are produced it has been confirmed to be

Experimental Example 2. Control of particle size of chitosan hydrogel by changing electrospinning conditions

In order to control the particles of the hydrogel for 2% chitosan as in (2) of Example 1, the following experiment was performed. Specifically, the particle size was evaluated by changing the voltage control and the flow rate control. At a voltage of 10 kV and a flow rate of 0.03 mL/min, particles with an average particle size of 150 (left) were generated, and by adjusting the voltage to 11 kV and a flow rate of 0.01 mL/min, it was confirmed that an average particle size of 70 μm (right) was generated. 2 is shown.

Experimental Example 3. Measurement of labeling efficiency of chitosan hydrogel-chelator labeled with radionuclides

As in Example 3, chitosan hydrogel-SHPP was labeled with I-131, chitosan hydrogel-DOTA was labeled with Lu-177, and chitosan hydrogel-DTPA was labeled with Tc-99m, followed by radioactive-TLC scanning using ITLC. After measuring the labeling efficiency through , the results are shown in FIGS. 3, 4 and 5, respectively.

The labeling efficiency of I-131-labeled chitosan hydrogel-SHPP (top) was 99.8%, the labeling efficiency (middle) of Lu-177-labeled chitosan hydrogel-DOTA was 98.0%, and Tc-99m-labeled chitosan hydrogel was 99.8%. It was confirmed that the labeling efficiency of gel-DTPA (bottom) was 99.5%.

Experimental Example 4. Measurement of label stability through radiochemical foreign matter of chitosan hydrogel-chelator labeled with radionuclides

The radioactive iodine (I-131)-labeled chitosan hydrogel-SHPP prepared as in (1) of Example 3 was measured for 8 days in a radiochemical foreign body test as in Experimental Example 3, and I for chitosan hydrogel-SHPP The label stability of -131 was evaluated and the results are shown in FIG. 6 .

As a result of measuring the radiochemical foreign body test, it was confirmed that the labeled radioactive iodine (I-131) was stably maintained at 97.88% or more in the chitosan hydrogel-SHPP for 8 days.

Experimental Example 5. Measurement of label stability through leaching test of chitosan hydrogel-chelator labeled with radionuclides

The chitosan hydrogel-SHPP labeled with radioactive iodine prepared as in (1) of Example 3 was evaluated for labeling stability of I-131 for chitosan hydrogel-SHPP through an 8-day leaching test, and the results are shown. 7 is shown.

As a result of measurement through the leaching test, it was confirmed that the labeled radioactive iodine (I-131) was stably maintained at 95.22% or more in the chitosan hydrogel-SHPP for 8 days.

Experimental Example 6. Evaluation of distribution of lesion sites according to chitosan particle size

As in Experimental Example 2, DTPA, a chelator, was synthesized in the chitosan hydrogel prepared at 60 ~ 100 μm (average 70 μm) and 100 ~ 300 μm (average 150 μm), and Tc-99m, a radioisotope, was labeled to Tc- 99m-labeled chitosan hydrogel-DTPA was prepared. Then, two types of Tc-99m-labeled chitosan hydrogel-DTPA (average 70 μm and 150 μm) with different particle sizes were injected into a middle-sized animal (rabbit) liver cancer model, and particle distribution evaluation was performed for the medium-sized animal liver cancer model. and the results are shown in FIG. 8 .

It can be seen that particles with an average size of 150 μm cannot access the tumor lesion due to the size of blood vessels outside the tumor and are distributed outside the tumor. It was confirmed that it was distributed in a very effective position. In Experimental Example 6, when the particles stay in the outer blood vessels of the tumor, the embolic effect can be confirmed to some extent, but the tumor treatment through the effect of radiation from I-131 is insignificant. The embolism and radiation effects can be seen at the same time only when evenly distributed around the outside of the lesion, and these results can be confirmed through the modified particles.

Experimental Example 7. Evaluation of labeling yield of radioiodine (I-131) for chitosan-SHPP and chitosan hydrogel-SHPP

The labeling yield of chitosan-SHPP (A) labeled with radioiodine and chitosan hydrogel-SHPP (B) labeled with radioiodine (I-131) prepared as in (1) of Example 3 is shown in FIG. It was.

The labeling yield of group A was 26.93 ± 7.70%, and the labeling yield of group B was 65.50 ± 9.64%, so group B obtained a labeling yield that was 38.57% higher than that of group A. It was confirmed that it was more effective in terms of yield than the I-131 labeling method for chitosan-SHPP.

Experimental Example 8. Evaluation of the therapeutic effect of chitosan hydrogel-SHPP in a medium-sized animal liver cancer model through CT image

In Experimental Example 2, chitosan hydrogel-SHPP prepared with a particle size of 60 to 100 μm was treated in a liver cancer medium animal model, and the treatment effect was evaluated through a CT image, and the results are shown in FIG. 10 .

In the control group that was not treated with anything (top), it was confirmed that the contrast was enhanced and the size of the tumor was increased for 2 weeks, and in the group treated with chitosan hydrogel-SHPP (bottom), the contrast of the edge of the tumor was increased for 2 weeks. I was able to see this thanks to the decrease in size. Therefore, it was possible to confirm the therapeutic effect of chitosan hydrogel-SHPP in the liver cancer model of a medium-sized animal.

Experimental Example 9. Evaluation of the therapeutic effect of chitosan hydrogel-SHPP in a medium-sized animal liver cancer model through tumor volume measurement

In the CT image of the middle animal liver cancer model of Experimental Example 8, the tumor volume was measured for 4 weeks, and the results are shown in FIG. 11 .

In the control group, the tumor volume increased linearly, whereas in the group treated with chitosan hydrogel-SHPP, it was confirmed that the tumor size decreased with time.

Experimental Example 10. Evaluation of the therapeutic effect of chitosan hydrogel-SHPP in a medium-sized animal liver cancer model through tumor weight measurement

In Experimental Example 2, chitosan hydrogel-SHPP prepared with a particle size of 60 to 100 μm was treated in a liver cancer medium animal model, and the weight of the tumor was measured to evaluate the therapeutic effect, and the results are shown in FIG. 12 .

The weight of the tumor in the control group was 20.8 ± 3.8 g, and the weight of the tumor in the group treated with chitosan hydrogel-SHPP was 2.2 ± 0.6 g, confirming the therapeutic effect by reducing the tumor weight.

Experimental Example 11. Evaluation of correlation between tumor weight and volume

The correlation was evaluated by comparing the result values of the weight and volume of the tumor measured in Experimental Example 9 and Experimental Example 10, and the results are shown in FIG. 13 .

In the case of the control group, it was confirmed that the tumor was growing with a linear relationship between the weight and volume of the tumor, and in the case of chitosan hydrogel-SHPP, it was confirmed that the correlation between the weight and the volume of the tumor disappeared as the tumor was treated.

As described above, the present invention has been described through examples. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the following claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be construed as being included in the scope of the present invention.

Claims (22)

1) electrospinning chitosan and a crosslinking material to prepare a chitosan hydrogel;
2) reacting the chitosan hydrogel prepared in step 1) with a chelator having a functional group capable of labeling a radionuclide to prepare a chitosan hydrogel-chelator,
The chelator is N-succinimidyl-3-(4-hydroxyphenyl)propionate, 1,4,7,10-tetraamacyclododecaine-1,4,7,10-tetraacetic acid, diethylene triamine pentaacetic acid histidine, tyrosine, and any one or more selected from the group consisting of proteins comprising tyrosine;
3) reacting the chitosan hydrogel-chelator prepared in step 2) with a radionuclide and an activator for labeling the radionuclide; According to claim 1, wherein the crosslinking material is tripolyphosphate, alginic acid, pectin, carboxymethyl cellulose, polyglutamic acid, protein, characterized in that at least one selected from the group consisting of DNA and RNA, radionuclide-labeled chitosan hydrogel - A method of manufacturing a chelator. The method of claim 1, wherein the electrospinning is performed at a voltage of 1 to 20 kV and a flow rate of 0.01 to 0.05 mL/min. The method of claim 1, further comprising: 4) purifying the hydrogel prepared in step 3) using a polymer network; According to claim 1, wherein the radionuclide is characterized in that at least one selected from the group consisting of ¹³¹ I, ¹²⁵ I, ¹²⁴ I, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁹⁰ Y, ¹⁶⁶ Ho, ^99m Tc and ¹⁷⁷ Lu, radioactive material A method for preparing a chitosan hydrogel-chelator labeled with a nuclide. According to claim 1, wherein the activator for labeling the radionuclide is any one selected from the group consisting of chloramine tea, a mixture of ascorbic acid and gentisic acid and tin (II) chloride. Chitosan hydrogel-method for producing a chelator. The method according to claim 1, wherein when the chelator is N-succinimidyl-3-(4-hydroxyphenyl)propionate, the radionuclide is at least one selected from the group consisting of ¹³¹ I, ¹²⁵ I and ¹²⁴ I. Characterized, radionuclide-labeled chitosan hydrogel-method for producing a chelator. The radionuclide according to claim 1, wherein when the chelator is 1,4,7,10-tetraamacyclododecaine-1,4,7,10-tetraacetic acid, the radionuclide is ¹⁷⁷ Lu. A method for preparing a labeled chitosan hydrogel-chelator. According to claim 1, wherein when the chelator is diethylenetriamine pentaacetic acid, the radionuclide is at least one selected from the group consisting of ¹¹⁸⁶ Re, ¹⁸⁸ Re and ^99m Tc, radionuclide-labeled chitosan hydrogel - Method of manufacturing a chelator. The chitosan hydrogel-chelator prepared by the method of any one of claims 1 to 9, labeled with a radionuclide. A composition for treating cancer containing the chitosan hydrogel-chelator labeled with the radionuclide according to claim 10. 12. The method of claim 11, wherein the cancer is liver cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, colon cancer, breast cancer, uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, Cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, solid tumors of childhood, differentiated lymphoma, cystitis, kidney cancer , Renal cell carcinoma, renal pelvic carcinoma, first central nervous system lymphoma, spinal cord tumor, brainstem glioma, and a composition for preventing or treating cancer, characterized in that at least one selected from the group consisting of adenoma of the pituitary gland. The composition for treating cancer according to claim 11, wherein the composition is an injection formulation. A pharmaceutical composition for treating embolism comprising the chitosan hydrogel-chelator labeled with the radionuclide according to claim 10 as an active ingredient. The pharmaceutical composition for treating embolism according to claim 14, wherein the composition is injected through the hepatic artery. A method for preventing or treating cancer in a subject, including; treating a subject in need thereof with the chitosan hydrogel-chelator labeled with the radionuclide according to claim 10. The method of preventing or treating embolism of a subject, including; treating a subject in need thereof with the chitosan hydrogel-chelator labeled with the radionuclide according to claim 10. Use of the radionuclide-labeled chitosan hydrogel-chelator according to claim 10 in the manufacture of a medicament for the treatment of embolism. Use of the radionuclide-labeled chitosan hydrogel-chelator according to claim 10 in the manufacture of a medicament for the treatment of cancer. A composition comprising a chitosan hydrogel-chelator labeled with the radionuclide according to claim 10 for use in the treatment of cancer. A composition comprising a chitosan hydrogel-chelator labeled with the radionuclide according to claim 10 for use in the treatment of embolism. a chitosan hydrogel combined with a chelator having a functional group capable of labeling radionuclides; and a radionuclide linked to the functional group, a radionuclide-labeled chitosan hydrogel-chelator.

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