WO2020116831A1 - Microbeads for transarterial chemoembolization, and manufacturing method therefor - Google Patents

Abstract

Provided are microbeads for transarterial chemoembolization, comprising: microbeads comprising blend of crosslinked biocompatible polymers and anionic polymers; and a drug encapsulated in the microbeads.

Description

Microbead for hepatic artery embolization and manufacturing method thereof

The present invention relates to a microbead for hepatic artery chemoembolization and a method for manufacturing the same, and more particularly, to a microbead for hepatic artery chemoembolization and a method for manufacturing the same.

Every year, 180,000 new cancer patients occur among the 280,000 new cancer patients in Korea. Of the total 70,000 deaths, 10,000 are liver cancer, the highest mortality rate after lung cancer. When classified by pathology, 13,000 cases of hepatocellular carcinoma occur annually, accounting for about 74% of the types of liver cancer. Treatment for liver cancer with a high mortality rate is still recognized as an unresolved field, and thus, interest in various treatments for liver cancer is increasing.

There are various treatments for this type of liver cancer, one of which is a hepatic resection in which liver cancer tumors are surgically removed, and is the primary treatment used in patients with limited liver cirrhosis. Next, systemic anti-cancer therapies are widely used, such as sorafenib (Sorafenib, the first validated liver cancer molecular targeting agent VEGFR-2 and multi-tyrose kinase inhibitor targeting PDFGR, Raf-1, and c-kit receptors). Molecular target therapy is used, or other general anticancer drugs such as doxorubicin, irinotecan, tamoxifen, etc. are used to treat systemic chemotherapy.

However, liver resection can only be performed when the tumor is small or located in a specific area, and the corresponding patients are very small, 20% of all liver cancer patients. In the case of systemic chemotherapy, targeted therapies are being developed, but at the current level of targeted therapies and anticancer drugs, a significant number of patients suffer from serious side effects such as diarrhea, limb syndrome, fatigue, loss of appetite, skin rash, and hair loss. In addition, most patients with liver cancer are accompanied by chronic liver disease or cirrhosis, which adversely affects the absorption and metabolism of the anticancer drug, so it is impossible to administer a sufficient dose of the anticancer drug, and there is a limitation that the therapeutic response is insufficient.

Liver cancer needs to be supplied with nutrients in order for the cancer tissue to grow when tissue occurs. At this time, abnormal new blood vessels extend from the liver artery to the cancer cells, and the cancer cells grow. Transarterial chemoembolization (TACE) was first introduced in 1984 to overcome the limitations of existing liver cancer treatments using this mechanism of liver cancer. Hepatic arterial chemoembolization is the most preferred in Korea for patients who are unable to undergo liver resection by blocking the blood vessels extending into the tumor with embolism containing an anticancer agent, blocking the nutrition of the liver cancer tumor and administering the anticancer agent topically and directly to the cancer tumor rather than the whole body. It is in the spotlight as a treatment.

However, the embolic material used for hepatic artery embolization has a problem in that the adhesion in the blood vessels is weak, the biodegradation is poor, or it is decomposed too well, and thus it has to be repeated several times every 1 to 3 months after the procedure. In addition, there is a problem that the therapeutic effect is unstable because the loading amount of the anticancer agent is small or various anticancer agents cannot be loaded.

The present invention is based on chitosan-catechol, which has the ability to bind to various blood plasma proteins and other various plasma proteins randomly in a few seconds, and can load a lot of various anti-cancer drugs. It is an object of the present invention to provide a new microbead for hepatic artery chemoembolization (TACE) using a fucoidan having a functional group and a method for manufacturing the same.

According to an aspect of the present invention, as a microbead for hepatic artery embolization, a microbead comprising a blend of a crosslinked biocompatible polymer and an anionic polymer; And a microbead for hepatic artery chemoembolization, which includes a drug encapsulated in the microbead.

According to another aspect of the present invention, a method of manufacturing microbeads for hepatic artery chemoembolization is provided. The manufacturing method comprises the steps of (a) emulsifying a mixture of a biocompatible polymer and an anionic polymer to form microbeads; (b) crosslinking the biocompatible polymer contained in the microbead to obtain a crosslinked microbead blended with an anionic polymer; And (c) encapsulating the drug inside the crosslinked microbead.

The microbead for hepatic artery embolization of the present invention is an innovative surgical material capable of loading a large amount of various drugs, but also being able to elute the drug very well due to biodegradability, thereby improving the therapeutic performance of conventional TACE. There is an effect that can be useful.

1 is a schematic view showing a cross-sectional structure of an albumin/fucoidan microbead.

Figure 2 is a microbead photograph made with a microfluidic system.

FIG. 3 is a photograph of microbeads prepared with a microfluidic system according to the composition of albumin/fucoidan.

Figure 4 is a comparison graph of the size distribution of albumin / fucoidan microbeads and DC beads (DC beads).

5 is a graph showing drug adsorption of albumin/fucoidan microbeads.

6 is a graph showing the long-term dissolution behavior of albumin/fucoidan microbeads.

7 is an angiography (angiography) picture of the albumin / fucoidan microbeads performed to evaluate the embolic efficacy of hepatic artery embolization in normal pigs. A is an angiogram confirming microbeads in blood vessels by injecting microbeads, and B is an angiogram confirming clogged blood vessels by adding contrast agents again to confirm that the blood vessels are blocked by the microbeads. .

8 is a liver tissue subtest of pigs embolized by albumin/fucoidan microbeads. The part indicated by the yellow arrow is the embolized tissue.

Figure 9 is a photograph of the liver tissue of the embolized pig of Figure 8 was subjected to histopathology.

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

According to an aspect of the present invention, microbeads for hepatic artery chemoembolization are provided. The microbead for hepatic artery embolization is a microbead comprising a blend of a crosslinked biocompatible polymer and an anionic polymer; And a drug enclosed in the microbead. That is, the microbeads contain a biocompatible polymer, and may be made of a biodegradable polymer that is preferably crosslinkable so that it can decompose in the body after a certain time while having excellent mechanical properties. The biocompatible polymer may be an enzyme-degradable biodegradable polymer or a chemically hydrolyzable biodegradable polymer, and preferably may be an enzyme-degradable biodegradable polymer that is easily decomposed by enzymes present in the body's metabolic system.

Specific examples of the biocompatible polymer include polysaccharides such as hyaluronic acid, alginic acid, and chitin; Proteins such as collagen, gelatin, and albumin; Polyesters such as poly( b -hydroxybutyrate), poly(hydroxyvalerate), polyglycolic acid (PGA), poly-ε-caprolactone (PCL), and polylactic acid (PLA); Or dextran methacrylate, but is not limited thereto. The biodegradability of the biocompatible polymer may solve a problem that may cause inflammation in the body or spread to other organs through blood vessels and cause cerebral thrombus.

As a preferred example of the biocompatible polymer, it can be combined with a wide variety of drugs such as peptide or protein drugs, indole compounds, sulfonamides, fatty acids, etc., and has the characteristics of being preferentially absorbed in cancer tumors and inflammatory tissues, as well as toxicity and It may be albumin in that it has low immunogenicity and is easily decomposed in vivo and thus has excellent biocompatibility.

In one embodiment, the albumin is a simple protein that is widely distributed in living cells or body fluids, and is known to constitute a basic material of cells together with globulin, and may be divided into animal albumin and vegetable albumin. The animal albumin includes ovalbumin in eggs, serum albumin, lactoalbumin in milk, albumin in the liver and muscles (myogen), and the like, and leukocin (barley seeds), legumelin (peas), and lysine (casters) in the vegetable albumin. Seed). The albumin may include albumin modified by chemical action, and includes albumin salt.

The biocompatible polymer can be crosslinked by heat, light or a crosslinking agent. In one embodiment, in the case of crosslinking by light, an Irgacure 2959 photoinitiator that can initiate a crosslinking reaction by UV may be used. In one embodiment, a suitable crosslinking agent may be selected and used according to the type of the biocompatible polymer. For example, in the case of albumin, aldehyde crosslinking agents such as glutaraldehyde, formaldehyde, and succinic acid aldehyde may be used. In the case of dextran methacrylate, KPS (potassium peroxydisulphate), TEMED (tetramethylethylenediamine), etc. can be used as a chemical crosslinking agent, and in the case of alginic acid, divalent metal ions such as Ca ²⁺ can be used.

The anionic polymer may allow drugs such as anticancer agents to be enclosed in beads by electrostatic attraction. The anti-cancer agent is, for example, doxorubicin, daunorubicin, epirubicin, idarubicin, gemcitabine, mitosantron, pyrarubicin, valrubicin, mitosantron, cisplatin, and irinodecan. It may be more than a species.

The anionic polymer is not particularly limited as long as it is a biocompatible polymer having a sulfate group, a sulfonate group, an amine group, or a carboxyl group, and preferably a biodegradable polymer, but has a large number of sulfate functional groups, thereby providing a large amount of various drug and polymer electrolyte complexes ( polyelectrolyte complexes (PECs), fucoidan is preferable in terms of anti-cancer activity as well as anti-oxidant, anti-bacterial, anti-inflammatory and immunomodulatory activity.

When the anionic polymer is blended with the biocompatible polymer in a microbead instead of a microbead surface, a hydrogen-bonding functional group in the crosslinked body, for example, a functional group such as -NH ₂ or -SH and anionic polymer Not only does it form a solid matrix with electrostatic attraction, it can retain a large amount of drugs by the presence of a large number of anionic functional groups inside the beads. Drugs can be delivered.

The fucoidan is a complex sulfated polysaccharide composed of L-Fucose having a sulfate group and a small amount of Galactose, Xylose, and Glucuronic acid. The kelp, seaweed and seaweed such as seaweed, abalone, and squid are abundantly contained in mollusks, such as immune enhancement function, anti-tumor function, blood sugar increase suppression function, triglyceride and cholesterol lowering function, antioxidant function, gastric ulcer healing function, etc. It is known to work.

However, in spite of the various functionalities as described above, fucoidan has a high molecular weight, and thus has high viscosity and poor water solubility, so that the human absorption rate may be low. Therefore, preferably, the fucoidan may have an average molecular weight of 400 to 20,000 Da.

When the fucoidan is blended with the biocompatible polymer to form microbeads, various drugs can be loaded in a large amount, in particular, by forming a polymer electrolyte complex with albumin and drugs, it is more robust and effective to release the drug slowly. In addition to being able to form a drug delivery system, fucoidan itself has an advantage of maximizing efficacy in hepatic artery chemoembolization targeting liver cancer because it acts as an anti-cancer agent.

The anionic polymer with respect to 100 parts by weight of the biocompatible polymer may include 1 to 80 parts by weight, preferably 1 to 50 parts by weight, more preferably 1 to 30 parts by weight, even more preferably 1 to 20 parts by weight have. If the amount of the anionic polymer is less than the above range, the encapsulation amount of the drug may be reduced, and if it is more than the above range, stable beads may be difficult to form.

Meanwhile, for 1 ml of the microbeads, the drug may be included in an amount of 0.01 to 500 mg, or 0.01 to 200 mg or 0.01 to 100 mg, based on the active ingredient. If the amount of the drug is less than the above range, the efficacy by the drug may be negligible, and if it is above the above range, the formation of microbeads may be prevented.

Meanwhile, the microbeads may be manufactured using a microfluidics system. The microfluidics system is a method of manufacturing beads by using a microstructured chip. After placing a smaller tube inside a large tube and flowing water and oil in opposite directions, beads are formed by tension of each other. Way. That is, if a solution for producing a bead (a solution containing an albumin-anionic polymer conjugate) is used as an internal fluid and the natural oil or organic solvent (collecting solution) is used as an external fluid, beads are formed by tension. The beads can be prepared again by collecting them in a collection solution and crosslinking. Alternatively, the microbeads may be prepared by crosslinking after forming microdroplets by an emulsification method in which the solution for preparing beads is dispersed by stirring in oil.

1 is a schematic diagram showing a cross-sectional structure of an albumin/fucoidan microbead manufacturing process and a microbead. The left figure of FIG. 1 shows an albumin/fucoidan microbead preparation method using a microfluidics system, and the right figure shows a microbead comprising a final crosslinked albumin/fucoidan blend.

According to another aspect of the present invention, a method of manufacturing microbeads for hepatic artery chemoembolization is provided. The manufacturing method comprises the steps of (a) emulsifying a mixture of a biocompatible polymer and an anionic polymer to form microbeads; (b) crosslinking the biocompatible polymer contained in the microbead to obtain a crosslinked microbead blended with an anionic polymer; And (c) encapsulating the drug inside the crosslinked microbead.

In step (a), the emulsification may be using a stirring or microfluidics system. For emulsification, organic solvents containing oil or viscosity-increasing agents can be used. For example, the oil is MCT oil, cottonseed oil, corn oil, almond oil, apricot oil, avocado oil, babasu palm oil, chamomile oil, canola oil, cocoa butter oil, coconut oil, cod liver oil, coffee oil, fish oil, flaxseed oil, Jojoba oil, gourd oil, grape seed oil, hazelnut oil, lavender oil, lemon oil, mango seed oil, orange oil, olive oil, mink oil, palm oil, rosemary oil, sesame oil, shea butter oil, soybean oil, sunflower oil and walnut oil And the like. Further, the organic solvent may include acetone, ethanol and butyl acetate. Cellulose-based polymers such as hydroxy methylcellulose, hydroxy propyl methylcellulose, and cellulose acetate butylate may be used as a viscosity-increasing agent for imparting appropriate viscosity. Preferred examples of the biocompatible polymer are as described above, and albumin is particularly preferable. Also, preferred examples of the anionic polymer are as described above, and fucoidan is particularly preferable.

In the step (b), the crosslinking process may be performed by crosslinking by ultraviolet light or heat, and crosslinking may be performed by additionally including a crosslinking agent in a solution for preparing beads and applying ultraviolet light or heat to increase reactivity. If a chemical crosslinking agent is not used, the toxicity is low and the crosslinking agent removal process may be omitted. When the biocompatible polymer is albumin and the anionic polymer is fucoidan, crosslinking microbeads may be preferably obtained by crosslinking by heat. According to an embodiment of the present invention, the heat crosslinking temperature may be 60°C or higher, for example, 60 to 160°C, and the heat crosslinking time may be 1 to 4 hours. Sufficient crosslinking does not occur below the temperature and crosslinking time, and denaturation of the microbeads may occur above the temperature and time.

In the step (c), the drug is first prepared at a concentration of 0.1 to 10 mg/mL, and then mixed and stirred in an amount of 0.1 to 50 parts by weight with respect to 100 parts by weight of the microbeads to be sufficiently enclosed in the microbeads. have. For sufficient encapsulation, the mixing and stirring conditions can be set to 1 minute to 24 hours at room temperature to 4 to 60°C.

As described above, the microbead for hepatic arterial chemoembolization of the present invention can be used for hepatic arterial chemoembolization because various anticancer agents can be loaded in a large amount. Therefore, it can be useful as an innovative surgical material that can improve the therapeutic performance of conventional TACE.

Hereinafter, the present invention will be described with reference to specific examples, and the present invention may be modified in various other forms. These embodiments are only for helping to understand the technical idea of the present invention, and the scope of the present invention is not limited to the embodiments described below.

[Example]

Example 1: Preparation of microbeads using a microfluidics system

As a self-designed system as shown in FIG. 1, mineral oil was flowed at a flow rate of 0.1 to 0.5 mL/min, and the flow rate of albumin was adjusted to a flow rate of 10 μL/min to form microbeads. After that, for crosslinking, the PTFE tube was wound in a coil form to perform heat denaturation at 100°C. The microbeads made according to FIG. 1 are shown in FIG. 2.

Example 2: Composition of microbeads

The anion compositions of albumin and fucoidan for the production of microbeads are shown in Table 1 below, and the microbead photographs according to each composition are shown in FIG. 3.

Weight ratio ratio Composition 1 Composition 2 Composition 3 Composition 4 Composition 5 Albumin: Fucoidan 10:0 10:0.5 10:1 10:2.5 10:5

As can be seen in Figure 3, as a microbead in the form of a complex of albumin and fucoidan, it was stabilized at a weight ratio of albumin:fucoidan=10:1, but when the concentration of fucoidan is relatively higher than 10:2.5, the bead structure is generally increased. It was confirmed that it collapsed.

Example 3: Size distribution diagram of albumin/fucoidan microbeads

Microbeads were made through the microfluidic system shown in FIG. 1 with the composition 3 albumin/fucoidan solution of Table 1 above. Fig. 4 shows a comparison photograph of the produced microbead and commercially available microbead embolizer DC bead.

As can be seen in Figure 4, it was confirmed that the size distribution of the microbeads made through the microfluidic system is more uniform.

Example 4: Drug adsorption

The drug was placed in doxorubicin (DOX), mitosantron (MX), and irinotecan (IRI) to perform adsorption experiments as follows. First, the drug was dissolved at a concentration of 2 mg/mL, and 10 mL of the drug was added to 500 μL of the microbeads and DC bead made in the ratio of the composition 3 and mixed well. After standing at room temperature for 1 hour, the supernatant was taken and measured at 480 nm, 608 nm, or 370/426 nm through an ultraviolet spectrometer or a fluorescence reader. The amount of the drug adsorbed on the microbeads is shown in FIG. 5 by calculating the concentration against the previously prepared calibration curve.

5, adsorption of the drug showed that the albumin/fucoidan micro beads adsorbed more drugs than the commercially available DC bead. This shows that the albumin/fucoidan microbeads can adsorb a lot of various drugs by the sulfate structure of albumin and fucoidan, while the PVA structure of DC bead can adsorb relatively fewer drugs.

Example 5: Drug release experiment

The release of the drug proceeded through the micro beads adsorbed in Example 4 above. The experimental method is as follows. The adsorbed micro-beads were placed in a 50 mL conical tube, and 30 mL of a discharge solution (PBS + Lysozyme, pH 7.4) was filled, followed by release at 150 rpm in a 37°C shake incubator. The release solution was replaced with a new release solution as much as it was reduced at the time the sample was made, and the release curve was accumulated with the sample collected to calculate the amount of drug released. Analysis of the released drug was measured through an ultraviolet spectrometer or a fluorescence reader in the same manner as the adsorption experiment. The results of the release are shown in Figure 6.

6, the release of the drug was expressed as a ratio of the release amount to the adsorption amount, and as a whole, the release of the drug showed higher albumin/fucoidan microbeads than DC bead. This not only adsorbs many drugs to the sulfate structure of albumin and fucoidan, but initially releases the ionic adsorbed drug slowly, and gradually degrades the albumin/fucoidan microbead itself by enzymes such as lysozyme in the body. Appeared to release. On the other hand, DC bead, which has a strong anionic property in structure, does not release the drug as well as the strong binding force with the drug, and it also proves that the drug release rate is low because it does not biodegrade well with an enzyme such as lysozyme. As described above, albumin/fucoidan microbeads not only adsorb many drugs, but also gradually release more drugs due to the better biodegradable composition than DC beads, thereby making albumin/fucoidan microbeads a substance for hepatic artery chemoembolization. It proves that the performance is excellent.

Example 6: Evaluation of hepatic artery embolization (TACE) efficacy of albumin/fucoidan microbeads in normal pigs

To evaluate the embolic efficacy of albumin/fucoidan microbeads, hepatic artery embolization (TACE) using microbeads of about 200 μm size was performed on the left hepatic artery of normal pigs. Healthy normal pigs (approximately 60 kg) weighing similar to humans were selected. The pig's right femoral artery was cut-down and a 5 Fr sheath was connected. Angiography was performed using 2.7 Fr catheter, 0.014 in guide wire. 1 mL of the microbeads developed by approaching the left hepatic artery basin of the pig was injected very slowly (it took more than 10 minutes) until the microbeads were settled in the blood vessel while checking the angiogram. The evaluation results were as shown in FIG. 7. As shown in FIG. 7A, it was confirmed that the microbead was injected into the blood vessel on the angiogram.

Angiogram confirmed that the blood vessels embolized by hepatic artery embolization, and after 3 hours, pigs were sacrificed and autopsy was performed with the naked eye. The autopsy results were as shown in FIG. 8. 8, necrosis of the tissue due to embolism was observed in part of the liver. To verify whether the necrosis was caused by microbeads, a histopathological examination was performed on the necrotic liver tissue. Figure 9 is a photograph of the liver tissue of the embolized pig of Figure 8 was subjected to histopathology. Referring to FIG. 9, microbeads injected from arteries in liver tissue were observed. As can be seen in FIG. 9, microbeads in the hepatic artery were observed and it was proved that the necrotic site of liver tissue observed by the naked eye on autopsy was due to albumin/fucoidan microbeads.

Claims (9)

As a microbead for hepatic artery embolization, A microbead comprising a blend of a crosslinked biocompatible polymer and an anionic polymer; And A microbead for hepatic artery chemoembolization, which includes a drug encapsulated within the microbead. According to claim 1, The biocompatible polymer is albumin, which is a microbead for hepatic artery chemoembolization. According to claim 1, The anionic polymer is a microbead for hepatic artery chemoembolization, which is fucoidan. According to claim 1, The anionic polymer is 1 to 80 parts by weight based on 100 parts by weight of the biocompatible polymer Microbeads for hepatic artery embolization. According to claim 1, For 1 ml of the microbeads, the drug is a microbead for hepatic artery chemoembolization, which is 0.01 to 500 mg based on active ingredient. According to claim 1, The drug is encapsulated inside the microbead by electrostatic attraction with the anionic polymer microbead for hepatic artery embolization. (a) emulsifying a mixture of a biocompatible polymer and an anionic polymer to form microbeads; (b) crosslinking the biocompatible polymer contained in the microbead to obtain a crosslinked microbead blended with an anionic polymer; And (c) A method of manufacturing microbeads for hepatic artery embolization, comprising encapsulating a drug inside the crosslinked microbeads. The method of claim 7, The biocompatible polymer is a method of manufacturing microbeads for hepatic artery embolization, which is albumin. The method of claim 7, The anionic polymer is a method for producing microbeads for hepatic artery embolization, which is fucoidan.

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