KR102508680B1 - Drug loaded in situ forming hydrogel - Google Patents

Abstract

The present invention relates to a drug-carrying scaffold that can be attached to the surface of a tumor, and more specifically, to a real-time forming hydrogel loaded with a drug.

Description

Drug loaded in situ forming hydrogel {Drug loaded in situ forming hydrogel}

The present invention relates to a drug-carrying scaffold that can be attached to the surface of a tumor, and more specifically, to a real-time forming hydrogel loaded with a drug.

In general, drugs are prescribed in the form of oral or injection to treat cancer patients. This treatment method has the advantage of simple administration, but since the drug is delivered to the tumor through the whole body, it not only has side effects that adversely affect normal tissues, but also has a low drug content delivered to the tumor compared to the administered dose, resulting in an effective effect. For this reason, an excessive amount of drug is administered, and for this reason, there is a problem in that side effects (eg, systemic toxicity) appearing to patients are large. In addition, in order to continuously see the effective effect of the drug, the drug must be repeatedly administered, but this has the disadvantage of causing discomfort due to repeated administration to the patient.

In order to solve these problems, side effects caused by general chemotherapy are minimized by locally treating the tumor with a matrix containing the drug, and discomfort due to repeated administration is designed to release the drug from the support in a sustained manner. can be minimized. However, since such a drug-carrying scaffold has no adhesive force with a tumor, it can be used only in the form of subcutaneous insertion, and thus its usable range is narrow. Accordingly, it is necessary to develop an improved type of drug delivery system that can overcome the disadvantages that the conventional invention has not essentially solved.

Korean Patent Registration No. 10-1901649, "Method for producing sustained-release hydrogel composite" includes a first solution containing an alginate polymer; and a drug-releasing hydrogel containing Doxorubicin by reacting calcium ions capable of cross-linking alginate with a second solution containing a drug (Doxorubicin) in a 1:1 ratio. prepared and confirmed the sustained-release behavior of the drug over time.

However, since the hydrogel is not formed immediately, it can only be used in a pre-made form, that is, the hydrogel is not formed in real time, and the crosslinking reaction by calcium ions selectively acts only on alginate, so it does not react separately with the tissue and does not show adhesiveness. don't

Korean Patent Publication No. 10-2010-0095940, "Paclitaxel-loaded conjugated linoleic acid poloxamer hydrogel drug delivery device" is a temperature-sensitive polymer that changes from a solution (sol) state to a gel state when the temperature exceeds a certain temperature. Disclosed a hydrogel drug delivery device using liquefied linoleic acid (CLA)-conjugated poloxamer as a drug delivery system, which can form a hydrogel when sprayed onto a tissue (e.g., tumor) while carrying a drug (anti-cancer drug) are doing

However, since the hydrogel made of a temperature-sensitive polymer does not react separately with the tissue during gel formation, the adhesion to the tissue (tumor) is very low. Although the antitumor effect was confirmed through cell experiments, in reality, since the gel formation speed is slow (takes several tens of seconds), it is expected that most of it will flow out even if sprayed on the tumor, and as a result, the effective anticancer effect is expected to be low.

US registered patent No. 8,003,125, "INJECTABLE DRUG DELIVERY SYSTEMS WITH CYCLODEXTRIN-POLYMER BASED HYDROGELS" relates to an invention using a cyclodextrin-based hydrogel physically bonded with PEG as a drug support, and physical crosslinking is different from chemical crosslinking. It has a thixotropic property that temporarily changes from a gel state to a solution state when it is applied, and then changes back to a gel state when the external force disappears. The supported gel-state filled material is designed to form a gel again by applying an external force with a syringe plunger to make it into a sol state and then spraying it to the tissue.

However, since hydrogels with thixotropic properties are made of physical cross-links, their adhesion to tissues is very low.

US Patent Publication No. US 2013/0022545, "DRUG DELIVERY SYSTEM FOR TREATMENT OF LIVER CANCER BASED ON INTERVENTIONAL INJECTION OF TEMPERATURE AND PH-SENSITIVE HYDROGEL" is a poly (amino ester urethane) in which sol-gel change occurs by temperature and pH -Based block copolymer (PEG-PAEU) When the PEG-PAEU solution containing the drug is applied to the liver tumor through a catheter, it becomes a gel by changes in the temperature and pH environment and can be applied to the tumor surface. Disclosed is a drug-loading hydrogel that can be used.

However, as in the above prior literature, since a hydrogel is formed by physical cross-linking, the adhesion to tissue (tumor) is very low.

US registered patent US 9,125,814, "BIOCOMPATIBLE CROSSLINKED HYDROGELS, DRUG-LOADED HYDROGELS AND METHODS OF USING THE SAME" is a first solution in which PEG having a succinimide functional group capable of reacting with a nucleophile and a drug (pain reliever) are simultaneously dissolved; and a hydrogel carrying a drug, wherein a hydrogel is formed in real time by reacting a second solution containing PEG having a nucleophilic functional group capable of reacting with the first solution.

Korean Patent Registration No. 10-1901649, "Manufacturing Method of Sustained Release Hydrogel Composite" Korean Patent Publication No. 10-2010-0095940, "Paclitaxel-Loaded Conjugated Linoleic Acid Poloxamer Hydrogel Drug Delivery Device" US registered patent US 8,003,125, "INJECTABLE DRUG DELIVERY SYSTEMS WITH CYCLODEXTRIN-POLYMER BASED HYDROGELS" US Patent Publication No. US 2013/0022545, "DRUG DELIVERY SYSTEM FOR TREATMENT OF LIVER CANCER BASED ON INTERVENTIONAL INJECTION OF TEMPERATURE AND PH-SENSITIVE HYDROGEL" US registered patent US 9,125,814, "BIOCOMPATIBLE CROSSLINKED HYDROGELS, DRUG-LOADED HYDROGELS AND METHODS OF USING THE SAME"

As such, in the prior art, even if a hydrogel containing a drug is applied to a tissue (eg, a tumor), it is easily detached and cannot continuously deliver the drug because there is no separate adhesive force (chemical bond) with the tissue. there was

Accordingly, a technical task of the present invention is to provide a drug-carrying scaffold that has excellent tissue adhesion and can be attached to a tissue surface in situ.

The present inventors have studied to solve the above problems, and as a result (i) a first solution containing a γ-polyglutamic acid derivative of Formula 1: and

[Formula 1]

In Formula 1,

the total sum of l, m and n is an integer from 390 to 15,500, and the ratio of l, m and n is 1 : m : n = 0 to 0.5 : 0.2 to 0.5 : 0.2 to 0.8;

L is a linker;

M is each independently H, an alkali metal or an alkaline earth metal;

R is CH ₂ ;

b is 0 or 1;

c is an integer from 1 to 5;

(ii) Excellent adhesion to the tumor surface and its surrounding tissue by a hydrogel formed when a second solution containing a hydrophilic material having at least one nucleophilic functional group selected from the group consisting of an amine group, a thiol group, and a hydroxyl group is mixed It is attached to the body and is not detached even by organ movement and can deliver the drug locally while remaining attached to the tissue for a long time. In addition, some of the drug loaded in the hydrogel is not initially released but is gradually released while trapped in the hydrogel, providing a sustained release It was discovered that it could act as a drug delivery system, and the present invention was completed.

Therefore, the present invention provides (i) a first solution containing the γ-polyglutamic acid derivative of Formula 1: and (ii) a hydrophilic solution having at least one nucleophilic functional group selected from the group consisting of an amine group, a thiol group, and a hydroxyl group. Provided is a pharmaceutical composition for attaching to a tumor surface or surrounding tissue, including a hydrogel formed by mixing a second solution containing a substance.

In addition, the present invention provides (i) a first solution containing the γ-polyglutamic acid derivative of Formula 1: and (ii) a hydrophilic solution having at least one nucleophilic functional group selected from the group consisting of an amine group, a thiol group, and a hydroxyl group. Provided is a scaffold for drug delivery for attachment to a tumor surface or surrounding tissue, including a hydrogel formed by mixing a second solution containing a material.

In addition, the present invention a. (i) a first solution containing the γ-polyglutamic acid derivative of Formula 1: and (ii) a hydrophilic material having at least one selected from the group consisting of an amine group, a thiol group, and a hydroxyl group, which are nucleophilic functional groups. mixing a second solution to; b. applying the mixture to the tumor surface; and c. A method for inhibiting tumor growth is provided, comprising forming a hydrogel with the applied mixture in situ.

In addition, the present invention provides (i) a first solution containing the γ-polyglutamic acid derivative of Formula 1: and (ii) a hydrophilic solution having at least one nucleophilic functional group selected from the group consisting of an amine group, a thiol group, and a hydroxyl group. It relates to a use of a hydrogel formed by mixing a second solution containing a substance, and specifically provides a use for adhering to a tumor surface or surrounding tissue, inhibiting tumor growth, and as a drug delivery support.

Since the hydrogel of the present invention is fixed (adhesive) with a chemical bond, it is not detached even when an organ is moved and can locally deliver a drug while remaining attached to the tissue for a long time.

In addition, since the hydrogel of the present invention can deliver drugs locally, systemic toxicity is reduced, and effective therapeutic effects can be exhibited even when a small dose of the drug is administered.

In addition, the hydrogel of the present invention is a sustained-release drug formulation because some of the drug contained in the hydrogel is not initially released and is gradually released in a state trapped in the hydrogel, thereby reducing the inconvenience caused by repeated drug administration.

In addition, the hydrogel of the present invention can be sprayed on the surface of the tumor and used as a drug delivery vehicle. In addition, when a small amount of bleeding occurs in the area where the tumor is incised, the hydrogel formed immediately acts as a sealant to prevent bleeding. At the same time, it can be used as a surgical material that performs two functions at the same time as a treatment that removes residual tumor cells remaining in the incision.

Surprisingly, the hydrogel of the present invention itself exhibits a tumor growth inhibitory effect. Specifically, it was observed that when the hydrogel of the present invention is applied to a tumor, the growth of the tumor is inhibited by applying physical pressure during the initial growth of the tumor. Therefore, since the hydrogel of the present invention has not only the physical tumor growth inhibitory effect but also the chemical inhibitory effect of the anticancer agent, it can consequently exhibit an effective anticancer effect.

1 shows a schematic diagram showing the application of the drug-loaded hydrogel according to the present invention to the treatment of pancreatic cancer.
2 is a graph of gelation behavior of a drug-containing sealant according to Example 4.
3 is a graph showing the cumulative release of Gemza (gemcitabine) and Genexol PM (paclitaxel) in the drug-loading hydrogel over time according to Example 6.
4 shows a graph of weight change over time for each group according to Example 7.
Figure 5 shows a graph of the average initial tumor volume and average tumor growth inhibition rate of each group according to Example 7.
Figure 6 shows a graph of the average tumor metastasis grade and metastasis inhibition rate of each group according to Example 7.

The present invention relates to (i) a first solution containing a γ-polyglutamic acid derivative represented by Formula 1: and

[Formula 1]

In Formula 1,

the total sum of l, m and n is an integer from 390 to 15,500, and the ratio of l, m and n is 1 : m : n = 0 to 0.5 : 0.2 to 0.5 : 0.2 to 0.8;

L is a linker;

M is each independently H, an alkali metal or an alkaline earth metal;

R is CH ₂ ;

b is 0 or 1;

c is an integer from 1 to 5;

(ii) Formed by mixing a second solution containing a hydrophilic material having at least one selected from the group consisting of an amine group (-NH ₂ ), a thiol group (-SH), and a hydroxyl group (-OH), which are nucleophilic functional groups It relates to a pharmaceutical composition for attaching to a tumor surface or surrounding tissue, including a hydrogel to be.

In addition, the present invention provides (i) a first solution containing the γ-polyglutamic acid derivative of Formula 1: and (ii) a hydrophilic solution having at least one nucleophilic functional group selected from the group consisting of an amine group, a thiol group, and a hydroxyl group. It relates to a scaffold for drug delivery for attachment to a tumor surface or surrounding tissue, including a hydrogel formed by mixing a second solution containing a material.

In addition, the present invention a. (i) a first solution containing the γ-polyglutamic acid derivative of Formula 1: and (ii) a hydrophilic material having at least one selected from the group consisting of an amine group, a thiol group, and a hydroxyl group, which are nucleophilic functional groups. mixing a second solution to; b. applying the mixture to the tumor surface; and c. It relates to a method for inhibiting tumor growth, comprising forming a hydrogel with the applied mixture in situ.

In addition, the present invention provides (i) a first solution containing the γ-polyglutamic acid derivative of Formula 1: and (ii) a hydrophilic solution having at least one nucleophilic functional group selected from the group consisting of an amine group, a thiol group, and a hydroxyl group. It relates to the use of a hydrogel formed by mixing a second solution containing a substance, and specifically, to the use of the hydrogel for adhering to a tumor surface or surrounding tissue, inhibiting tumor growth, and as a drug delivery support.

In one embodiment, in the γ-polyglutamic acid derivative of Formula 1 contained in the first solution, L (linker) may be represented by -HN-(R)aO-, where R is CH ₂ , and a may be an integer from 1 to 5.

In one embodiment, in the γ-polyglutamic acid derivative of Formula 1 contained in the first solution, L (linker) is aminomethanol, 1-amino-2-propanol (1-amino-2-propanol) , 1-amino-3-propanol (1-amino-3-propanol), 1-amino-4-butanol (1-amino-4-butanol), 1-amino-5-pentanol (1-amino-5- pentanol) or 1-amino-2-ethanol (MEA or 1-amino-2-ethanol).

In one embodiment, in the γ-polyglutamic acid derivative of Formula 1 contained in the first solution, the -(CO)b-(R)c-CO- site is -CH ₂ CH ₂ CH ₂ CH ₂ -CO- , -CO-CH ₂ CH ₂ CH ₂ -CO-, -CH ₂ CH ₂ -CO-, -CO-CH ₂ CH ₂ -CO- or -CH ₂ -CO-.

In one embodiment, the γ-polyglutamic acid derivative of Chemical Formula 1 included in the first solution may be succinimidyl succinyl polyglutamic acid (SSPGA) having the following structure:

.

.

In one embodiment, the hydrophilic material included in the second solution may have two or more nucleophilic functional groups selected from the group consisting of an amine group, a thiol group, and a hydroxyl group.

In one embodiment, the hydrophilic material included in the second solution is a multi-arm poly (ethylene glycol) (PEG) derivative, poly (vinyl alcohol) (PVA), poly (ethylene imine) (PEI), poly(lysine) (PL), trilysine amine, and poly(allylamine) (PAA). Preferably, the hydrophilic material included in the second solution is multi-arm PEG, PEI or trilysine amine.

In one embodiment, the hydrophilic material included in the second solution is a polyethylene glycol-based polymer, and the polyethylene glycol-based polymer may be represented by Formula 2 below:

[Formula 2]

In Formula 2,

I is a radical derived from a di- to 12-hydric polyhydric alcohol;

X is an amine group, a thiol group or a hydroxy group;

n is 19 to 170;

m is an integer of 2 to 12, equal to the number of hydroxyl groups of the polyhydric alcohol from which I is derived.

In one embodiment, the hydrophilic material included in the second solution is a polyethylene glycol-based polymer, and the polyethylene glycol-based polymer may be represented by Formula 3 or 4 below:

[Formula 3]

[Formula 4]

In Formulas 3 and 4, X represents an amine group, a thiol group or a hydroxyl group, and n is 19 to 170.

In one embodiment, the first solution and the second solution may be a buffer solution. Specifically, the pH of the first solution may be in the range of 5-6, and the pH of the second solution may be in the range of 10-11. In addition, after mixing the first solution and the second solution, the pH may be in the range of 5 to 9.

In one embodiment, the buffer solution is distilled water, NaCl (sodium chloride), KCl (potassium chloride), NaH ₂ PO ₄ (Monosodium phosphate), Na ₂ HPO ₄ (Disodium phosphate), KH ₂ PO ₄ (Monopotassium phosphate), It may be an aqueous solution in which at least one selected from the group consisting of Na ₂ CO ₃ (sodium carbonate), HCl (hydrochloric acid), Borate, MES, Tris, and HEPES is dissolved.

In one embodiment, the buffer of the first solution may be a mixed buffer of NaH ₂ PO ₄ (Monosodium phosphate) and Na ₂ HPO ₄ (Disodium phosphate). Specifically, the buffer of the first solution may be a mixture of NaH ₂ PO ₄ and Na ₂ HPO ₄ in a volume ratio of 9:1 to 1:9, preferably 9:1 to 5:5.

The buffer of the second solution may be a mixed buffer of Na ₂ HPO ₄ (Disodium phosphate) and Na ₂ CO ₃ (Sodium carbonate). Specifically, the buffer of the second solution may be a mixture of Na ₂ HPO ₄ and Na ₂ CO ₃ at a volume ratio of 9:1 to 1:9, preferably 5:5 to 1:9.

In one embodiment, the pharmaceutical composition of the present invention can be used for tumor growth inhibition. Surprisingly, the hydrogel of the present invention itself has an anticancer effect and exhibits a tumor growth inhibitory effect. Specifically, it was observed that when the hydrogel of the present invention is applied to a tumor, the growth of the tumor is inhibited by applying physical pressure during the initial growth of the tumor. Therefore, the hydrogel of the present invention has not only such a physical tumor growth inhibitory effect, but also a chemical inhibitory effect of an anticancer agent by additionally including a drug, and as a result, can exhibit an effective anticancer effect.

In addition, the hydrogel of the present invention can be attached to the surrounding tissue of the tumor. In one embodiment, the hydrogel of the present invention may be used in the form of a sealant at a site where a tumor is incised.

In one embodiment, the pharmaceutical composition of the present invention may additionally contain a drug. Specifically, the first solution and/or the second solution may further contain a drug, and more specifically, both the first solution and the second solution may further contain a drug.

Specifically, the drug may be one or more selected from the group consisting of anticancer agents, antibiotics and analgesics. Specifically, the anticancer agent is at least one selected from the group consisting of Paclitaxel, Gemcitabine, Docetaxel, Doxorubicin and Cyclophosphamide; the antibiotic is at least one selected from the group consisting of Streptomycin, Gentamicin, Amoxicillin, Doxycycline, Cephalexin, and Ciprofloxacin; The analgesic may be at least one selected from the group consisting of Acetaminophen, Bupivacaine, Levobupivacaine, Aspirin Diphenhydramine, Ropivacaine, Lidocaine, Mepivacaine, Prilocaine, Cinchocaine, Etidocaine and Articaine.

In one embodiment, in the pharmaceutical composition of the present invention, each of the first solution and the second solution may further contain an anticancer agent. Specifically, the first solution may include Paclitaxel, Docetaxel, or a mixture thereof, and the second solution may include Gemcitabine.

Hereinafter, the pharmaceutical composition of the present invention will be described in detail.

(i) the polymer of the first solution

The first solution of the present invention contains the γ-polyglutamic acid derivative of Formula 1 above. The γ-polyglutamic acid derivative of Formula 1 is disclosed in Korean Patent Publication No. 10-2013-0078549, which is a prior patent document of the present applicant.

The γ-polyglutamic acid derivative of Chemical Formula 1 is obtained by (a) a first step of reacting at least a part of the carboxy group of γ-polyglutamic acid with a lower alkanolamine having 1 to 5 carbon atoms to form γ-polyglutamic acid-alkanolamine; (b) the hydroxy group of the γ-polyglutamic acid-alkanolamine is composed of an anhydride of an acid selected from the group consisting of glutaric acid and succinic acid, or 1-halo valeric acid, 1-halo propionic acid and 1-halo methyl carboxylic acid A second step of reacting with 1-halo alkanoic acid selected from the group to form a carboxy terminal into which an alkyl group is introduced; and (c) in a third step of forming a γ-polyglutamic acid derivative in which at least some of the carboxyl groups are activated by reacting the formed carboxy terminal with N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide. can be produced by

The γ-polyglutamic acid derivative of Formula 1 is succinimidyl valerate (Succinimidyl Valerate, SVA) depending on the type of introduced alkyl group, that is, according to the substituent definition of the -(CO)b-(R)c-CO- site. : -CH ₂ CH ₂ CH ₂ CH ₂ -CO-O-Succinimide), Succinimidyl Glutarate (SG: -CO-CH ₂ CH ₂ CH ₂ -CO-O-Succinimide) , Succinimdyl Propionate (SPA: -CH ₂ CH ₂ -CO-O-succinimide), Succinimidyl Succinate (SS: -CO-CH ₂ CH ₂ -CO-O- succinimide), and succinimidyl methyl carbonate (Succinimidyl Carboxymethylated, SCM: -CH ₂ -CO-O-succinimide).

In one embodiment, the γ-polyglutamic acid derivative of Chemical Formula 1 included in the first solution may be succinimidyl succinyl polyglutamic acid (SSPGA) having the following structure:

.

.

In one embodiment, when the first solution contains the SSPGA, the first solution is also referred to as "SSPGA solution" or "SSPGA buffer". In Examples to be described later, a case in which an SSPGA solution is used as the first solution is exemplified.

In one embodiment, the concentration of the γ-polyglutamic acid derivative of Formula 1 in the first solution may be 3 to 20% (w/v), preferably 5 to 15% (w/v).

(ii) the polymer of the second solution

The second solution of the present invention includes a hydrophilic material having at least one selected from the group consisting of an amine group (-NH ₂ ), a thiol group (-SH), and a hydroxyl group (-OH), which are nucleophilic functional groups.

In one embodiment, the hydrophilic material may have two or more nucleophilic functional groups selected from the group consisting of an amine group, a thiol group, and a hydroxyl group. Specifically, the hydrophilic material is a multi-arm poly (ethylene glycol) (PEG) derivative, poly (vinyl alcohol) (PVA), poly (ethylene imine) (PEI), poly (lysine) (PL), trilysine amine, and poly(allylamine) (PAA). Preferably, the hydrophilic material is multi-arm PEG, PEI or trilysine amine.

For example, such a hydrophilic material is a polyethylene glycol-based polymer, and the polyethylene glycol-based polymer has a polyethylene glycol repeating unit bonded to each hydroxyl group of a divalent or more, preferably 2 to 12, polyhydric alcohol, and an amine group at its terminal. , may have a structure in which a thiol group or a hydroxy group is bonded. More specifically, the polyethylene glycol-based polymer may be represented by Formula 2 below.

[Formula 2]

In Formula 2,

I is a radical derived from a di- to 12-hydric polyhydric alcohol;

X is an amine group, a thiol group or a hydroxy group;

n is 19 to 170;

m is an integer of 2 to 12, equal to the number of hydroxyl groups of the polyhydric alcohol from which I is derived.

Specific examples of I in Formula 2 include diols such as ethylene glycol, propandiol, butandiol, pentandiol, and hexandiol; or glycerol, erythritol, threitol, pentaerythritol, xylitol, adonitol, sorbitol, mannitol palatinose A radical derived from a tri- to 12-valent polyol selected from disaccharides such as palatinose, maltose monohydrate, or maltitol, or trisaccharides such as D-raffinose pentahydrate. can be heard More specifically, I may be a radical derived from a 4- to 12-hydric polyhydric alcohol.

An example of the polyethylene glycol-based polymer belonging to the category of Formula 2 is a polymer in which a polyethylene glycol repeating unit having a terminal nucleophilic functional group is bonded to a radical derived from pentaerythritol or sorbitol, for example, the following Formula 3 Or the polymer of 4 can be mentioned.

[Formula 3]

[Formula 4]

In Formulas 3 and 4, X represents an amine group, a thiol group or a hydroxyl group, and n is 19 to 170.

As these polyethylene glycol-based polymers contain a plurality of nucleophilic functional groups such as the amine group, thiol group, or hydroxyl group, they can form an amide bond, a thioamide bond, or an ester bond with the activated γ-polyglutamic acid derivative of Formula 1. From this, a crosslinked structure can be formed to form a crosslinked product and a hydrogel having a three-dimensional network structure. In particular, such a polyethylene glycol-based polymer causes a rapid cross-linking reaction with the activated γ-polyglutamic acid derivative, enabling the provision of a hydrogel having a short gelation time.

In one embodiment, the polyethylene glycol-based polymer may be 4arm-PEG-thiol (4PEGSH), which has four arms and has a thiol group at each end.

In one embodiment, when the 4PEGSH is included in the second solution, the second solution is also referred to as "4PEGSH solution" or "4PEGSH buffer". In Examples to be described later, a case in which a 4PEGSH solution is used as the second solution is exemplified.

In one embodiment, the concentration of the hydrophilic material in the second solution may be 3 to 30% (w/v), preferably 10 to 20% (w/v).

(iii) the solvent of the first solution and the second solution

As the solvent for preparing the first solution and the second solution, distilled water, physiological saline, sodium bicarbonate (NaHCO ₃ ), boric acid, phosphoric acid, or the like, buffers having no toxicity may be used.

The mixing ratio of the first solution and the second solution may be 9: 1 to 1: 9, but may be prepared by mixing in a ratio of 1: 2 to 2: 1. In order to rapidly form a hydrogel by mixing the two solutions, the pH after mixing is important, and the reactivity can be adjusted by changing the concentration of the buffer to buffer when the final pH is changed according to the mixing ratio.

In one embodiment, the first solution and the second solution may be a buffer (buffer). Specifically, the pH of the first solution may be in the range of 5-6, and the pH of the second solution may be in the range of 10-11. In addition, after mixing the first solution and the second solution, the pH may be in the range of 5 to 9.

In one embodiment, the buffer is distilled water, NaCl (sodium chloride), KCl (potassium chloride), NaH ₂ PO ₄ (Monosodium phosphate), Na ₂ HPO ₄ (Disodium phosphate), KH ₂ PO ₄ (Monopotassium phosphate), Na ₂ CO ₃ (sodium carbonate), HCl (hydrochloric acid), borate, MES, Tris, and at least one selected from the group consisting of HEPES may be dissolved in an aqueous solution.

The buffer in the solvent affects the gelation time. That is, the gelation reaction may or may not occur depending on the type of buffer, and the gelation time may be adjusted quickly or slowly. Buffers should be made using salts with pKas similar to the pH of each polymer in the first solution and the second solution, at which time the buffering effect is maximized to help reduce the decrease in activity of the γ-polyglutamic acid derivative activated in the aqueous solution. can give

Specifically, a sodium phosphate buffer may be used as a buffer to prepare the first solution, or a mixture of different types of sodium phosphate buffer may be used. A mixed buffer of sodium phosphate and sodium carbonate may be used to prepare the second solution.

In one embodiment, the buffer of the first solution may be a mixed buffer of NaH ₂ PO ₄ (Monosodium phosphate) and Na ₂ HPO ₄ (Disodium phosphate). Specifically, the buffer of the first solution may be a mixture of NaH ₂ PO ₄ and Na ₂ HPO ₄ at a volume ratio of 9:1 to 1:9, preferably 9:1 to 5:5.

In one embodiment, the buffer of the second solution may be a mixed buffer of Na ₂ HPO ₄ (Disodium phosphate) and Na ₂ CO ₃ (Sodium carbonate). Specifically, the buffer of the second solution may be a mixture of Na ₂ HPO ₄ and Na ₂ CO ₃ at a volume ratio of 9:1 to 1:9, preferably 5:5 to 1:9.

The buffer of the first solution may be used at a concentration of preferably 0.01 mol to 0.3 mol, more preferably 0.05 mol to 0.2 mol, and the buffer of the second solution is preferably 0.01 mol to 0.5 mol, more preferably. It can be used at a concentration of 0.1 mol to 0.3 mol.

At this time, the drug that can be mixed can be used by selecting a drug that is not reactive with substances dissolved in the first solution or the second solution, and a surfactant (eg, mPEG-PLA, sodium dodecyl sulfate, Tween 80) and the like can be used.

In spite of using a drug that is not reactive with the materials of the first solution and the second solution, when the pH of the buffer is changed when dissolved, the reaction rate of the hydrogel may increase or decrease. In this case, by changing the buffer composition of the first solution or the second solution to adjust the buffer effect, it is possible to minimize the change in gel formation time of the hydrogel by the drug.

(iv) formation of hydrogel

The term "hydrogel" used herein may be defined as meaning a polymer matrix (support) containing water and capable of swelling, and may have a cross-linked structure including covalent bonds or non-covalent bonds. The hydrogel absorbs water through the three-dimensional network structure composed of the cross-linked structure, and may exhibit elasticity according to the degree of cross-linking of the three-dimensional network.

In this specification, "sealant" is used as the same meaning as the above "hydrogel".

When the first solution and the second solution are mixed, the polymer of the first solution and the polymer of the second solution react to form a hydrogel in real time. In order to carry the drug in the hydrogel forming system, the drug is mixed in the step of preparing the first solution and the second solution. After dissolving one or more drugs in the first solution, the second solution, or each of the two solutions, when the two solutions are mixed as described above, a hydrogel containing the drug can be formed.

Specifically, when the first solution and the second solution are mixed, an amide, thioamide or ester bond is formed between the activated carboxyl group (succinimide ester group, etc.) of the γ polyglutamic acid derivative and the nucleophilic functional group, which serves as a crosslinking point. A hydrogel having a three-dimensional network structure can be formed.

In one embodiment of the present invention, when the SSPGA solution and the 4arm-PEG-thiol (4PEGSH) solution are mixed, the NHS functional group of SSPGA and the thiol functional group of 4arm-PEG-thiol (4PEGSH) chemically react to prepare a hydrogel. . Since this reaction reacts rapidly under appropriate pH conditions, an in situ forming hydrogel can be prepared. Since the number of NHS functional groups introduced into SSPGA is greater than the number of thiol functional groups in 4PEGSH, even if the hydrogel formation reaction occurs, the remaining NHS functional groups of SSPGA that do not participate in the hydrogel reaction are functional groups (Amine group, Thiol group, Hydroxyl group) to chemically form a covalent bond (see FIG. 1). Since the hydrogel formed by this covalent bond exhibits strong adhesion to tissues, spraying a mixed solution of the two polymers on the tissue surface can serve as a sealant capable of performing a sealing function.

In order to produce an adhesive hydrogel, it is preferable that crosslinking occurs by bonding between polymer chains that react with each other having at least two or more functional groups, and at least one of the polymers forms a covalent bond with the tissue surface at the same time as the crosslinking reaction.

Although the preferred time for the two mixed solutions to form a gel may vary slightly depending on the application, it is preferably within 10 minutes, for example, within 2 minutes, within 1 minute, within 30 seconds, within 20 seconds. , Within 10 seconds, within 5 seconds, within 3 seconds, preferably within 1 second to 3 seconds. If the gelation time is less than 1 second, the injection needle or spray tip may be clogged, making it difficult to apply smoothly, and since there is not enough time for each component to be mixed, an uneven gel is formed or it is difficult to form a covalent bond with the tissue surface, resulting in adhesive strength. There is a risk that this will decrease. On the other hand, if the gelation time is excessively long, it may flow in the form of a solution before the gel is formed at the application site, resulting in insufficient ease of use and difficulty in accurately applying the desired amount. Therefore, it is preferably 1 second or more, particularly 1 second or more and 2 seconds or less, until it penetrates into living tissue and maintains high adhesive strength and burst strength.

Specifically, the hydrogel of the present invention may have the following physical property conditions.

(1) Gelation time: within 10 seconds, for example, 1-2 seconds. If the gelation time is too fast, it hardens before reaching the tissue surface, and if it exceeds 2 seconds, it flows down even if sprayed on the cotton tissue, making it impossible to apply the amount.

(2) Burst Pressure: 25 to 500 mmHg, more preferably 50 to 250 mmHg. If it is lower than 50mmHg, it is easily crushed by external damage, and if it is higher than 250mmHg, flexibility is greatly reduced.

(3) Sustained-release drug release period: 7 days or more, more preferably 14 days or more. If the drug release period is short, within 7 days, it is difficult to show an effective drug effect on tumors for a long time.

(v) supported drug

The drug included in the first solution is a drug that is not reactive with the polymer (eg, SSPGA) of the first solution, and a drug that does not have an amine group, a thiol group, or a hydroxyl group, which is a nucleophilic functional group, may be used. Although hydrophilic drugs have good solubility in nature and are convenient to use, depending on circumstances, they can be used after dissolving them in the first solution using poorly soluble drugs and surfactants capable of dissolving them.

The drug included in the second solution should be a drug that does not cause an oxidation reaction with the polymer (eg, 4PEGSH) of the second solution. In the case of 4PEGSH, since it causes a crosslinking reaction by itself by an oxidation reaction, the stability of the second solution is lowered.

The dissolution concentration of the drug in the first solution should be set to a concentration that can exhibit effective drug effects and does not exhibit toxicity. It may be used at a concentration of 1-30% (w/v) of the first solution, and preferably dissolved at a concentration of 5-15% (w/v). When the drug contained in the second solution changes the pH of the buffer, the concentration of the buffer can be adjusted to buffer this change.

The dissolved concentration of the drug in the second solution should be set to a concentration capable of exhibiting an effective drug effect and not exhibiting toxicity. It may be used at a concentration of 1-30% (w/v) of the second solution, and preferably dissolved at a concentration of 5-15% (w/v). When the drug contained in the second solution changes the pH of the buffer, the concentration of the buffer can be adjusted to buffer this change.

Specifically, the drug may be one or more selected from the group consisting of anticancer agents, antibiotics and analgesics. Specifically, the anticancer agent is at least one selected from the group consisting of Paclitaxel, Gemcitabine, Docetaxel, Doxorubicin and Cyclophosphamide; the antibiotic is at least one selected from the group consisting of Streptomycin, Gentamicin, Amoxicillin, Doxycycline, Cephalexin, and Ciprofloxacin; The analgesic may be at least one selected from the group consisting of Acetaminophen, Bupivacaine, Levobupivacaine, Aspirin Diphenhydramine, Ropivacaine, Lidocaine, Mepivacaine, Prilocaine, Cinchocaine, Etidocaine and Articaine.

In one embodiment, in the pharmaceutical composition of the present invention, each of the first solution and the second solution may further contain an anticancer agent. Specifically, the first solution may include Paclitaxel, Docetaxel, or a mixture thereof, and the second solution may include Gemcitabine.

In one embodiment, the mixing and use of the first solution and the second solution can be carried out by various methods. For example, mixing can be performed by applying one of the stock solutions of the first solution and the second solution to the surface of the adherend, and then applying the other one, or the first solution and the second solution can be mixed with a double barrel syringe ( Application may be performed by mixing in an applicator such as a double barrel syringe). In one embodiment, when the first solution and the second solution are mixed through a connector to which a spray is attached and sprayed on a tissue surface such as a tumor, the two solutions react immediately on the tissue surface to carry the drug in a form attached to the tissue form a hydrogel.

Hereinafter, an example will be described in detail in order to explain the present invention in detail. However, embodiments according to the present invention may be modified in many different forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Example 1. Preparation and gelation characteristics of sealant containing Genexol PM

1-1. Preparation of sealant containing Genexol PM

A first solution was prepared by dissolving 7 w/v% of SSPGA and 14 to 32 w/v% of Genexol PM (Paclitaxel drug product) in 0.15M SSPGA buffer. Next, a second solution was prepared by dissolving 12 w/v% of 4PEGSH in 0.3M PEG buffer. A sealant containing Genexol PM was prepared by mixing the first solution and the second solution in a 1:1 volume. Table 1 below shows the composition and buffer concentration of SSPGA and 4PEGSH buffer.

[Table 1]

1-2. Gelation characteristics of sealant containing Genexol PM

An experiment was performed to confirm the gelation characteristics of the sealant according to the content of Genexol PM in the first solution. The contents of each component used in the first solution and the second solution and the measured gelation properties are shown in Table 2 below.

Specifically, after putting 200 μL of the first solution into a 96 well plate, a magnetic spin bar was added, and 200 μL of the second solution was added while the magnetic spin bar was rotated at 200 rpm. The time taken from the introduction of the second solution to the point at which the magnetic spin bar no longer rotates was defined as the gelation time.

[Table 2]

As shown in Table 2, Genexol PM was transparently dissolved in 0.15M SSPGA buffer up to 24% concentration, but was not completely dissolved at higher concentrations, resulting in precipitates. In addition, since Genexol PM does not affect the pH change of the buffer, it was confirmed that the gelation time was about 1 second to form a hydrogel in all conditions regardless of the drug content.

Example 2. Preparation and gelation characteristics of sealant containing Nanoxel M

2-1. Preparation of sealant containing Nanoxel M

A first solution was prepared by dissolving 7 w/v% of SSPGA and 20-30 w/v% of Nanoxel M (raw material of Docetaxel) in 0.15M SSPGA buffer. Next, a second solution was prepared by dissolving 12% of 4PEGSH in 0.3M PEG buffer, and a sealant containing Nanoxel M was prepared by mixing the first solution and the second solution in a 1:1 volume.

2-2. Gelation characteristics of sealant containing Nanoxel M

Experiments were conducted in the same manner as in Example 1-2 to confirm the gelation characteristics of the sealant according to the content of Nanoxel M in the first solution. The contents of each component used in the first solution and the second solution and the measured gelation properties are shown in Table 3 below.

[Table 3]

As shown in Table 3, Nanoxel M was transparently dissolved in 0.15M SSPGA buffer up to 24% concentration, but was not completely dissolved at higher concentrations, resulting in precipitates. In addition, since Nanoxel M does not affect the pH change of the buffer, it was confirmed that the gelation time was about 1 second in all conditions, regardless of the drug content, forming a hydrogel quickly.

Example 3. Preparation and gelling properties of sealants containing Gemza

Example 3-1. Manufacture of sealants containing Gemza

A first solution was prepared by dissolving 7 w/v% of SSPGA in 0.15M SSPGA buffer. Subsequently, a second solution was prepared by dissolving 12 w/v% of 4PEGSH and 6-24 w/v% of Gemzar (Gemcitabine drug product) in 0.3-0.6M PEG buffer. Next, a sealant containing Gemza was prepared by mixing the first solution and the second solution in a 1:1 volume.

Example 3-2. Gelling properties of sealants containing Gemza

Experiments were performed in the same manner as in Example 1-2 to confirm the gelation characteristics of the sealant according to the Gemza content of the second solution and the content of PEG buffer or SSPGA. The contents of each component used in the first solution and the second solution and the measured gelation properties are shown in Table 4 below.

[Table 4]

As shown in Table 4, it was confirmed that Gemza was present in an opaque dispersed form under all PEG buffer concentration conditions. In addition, it was confirmed that the gel formation time increased as the Gemza content increased under the same PEG buffer conditions, but the gelation time could be shortened by buffering the acidification by Gemza by increasing the SSPGA polymer content or increasing the concentration of the PEG buffer.

Example 4. Preparation and gelation characteristics of sealant containing Genexol PM and Gemza (drug content change)

4-1. Manufacture of sealant containing Genexol PM and Gemza

A first solution was prepared by dissolving SSPGA 7-10 w/v% and Genexol PM 8-24 w/v% in 0.15M SSPGA buffer. Subsequently, a second solution was prepared by dissolving 4PEGSH 12-18 w/v% and Gemza 6-30 w/v% in 0.3-0.7M PEG buffer, and the first solution and the second solution were mixed in a 1:1 volume By mixing, a sealant containing both Genexol PM and Gemza was prepared.

4-2. Gelation characteristics of sealant containing Genexol PM and Gemza

Experiments were performed in the same manner as in Example 1-2 to confirm the gelation characteristics of the sealant according to the content of Genexol PM in the first solution, the content of Gemza in the second solution, and the content of PEG buffer or SSPGA. The contents of each component used in the first solution and the second solution and the measured gelation characteristics are shown in Table 5 and FIG. 2 below.

[Table 5]

As shown in Table 5, it was confirmed that Genexol PM contained in the first solution did not affect the gelation time regardless of the drug content because it did not affect the pH change of the buffer. In addition, in the case of the second solution, it was confirmed that the gel formation time increased as the Gemza content increased under the same PEG buffer conditions, but the gelation time could be shortened by buffering the acidification by Gemza by increasing the concentration of the PEG buffer. When the concentration of the PEG buffer was 0.7M or more, the solubility of 4PEGSH was greatly reduced and precipitated and the gel was not formed.

4-3. Rheological Gelling Characteristics of Sealant Containing Genexol PM and Gemza

In order to confirm the rheological gelation characteristics of the drug-containing sealant, the change in storage modulus (G') and loss modulus (G'') was confirmed by setting the rotational viscometer analysis conditions as follows. At this time, the analysis was started after loading the first solution into the geometry first, and after about 100 seconds, the second solution was injected to check the numerical change of G' and G'', and the point at which the G' value became greater than the G'' value was determined. It was defined as the sol-gel change point (gelation time).

(1) Mode: Time-sweep oscillatory mode

(2) Measuring geometry: 40 mm, Frequency: 1 Hz, Temperature: 22±2°C, Time: 10 min, Strain: 5%, Measuring gap: 0.1 mm

2 shows that the first solution in which SSPGA 7 w / v% / Genexol PM (paclitaxel) 8 w / v% was mixed and the solution in which 4PEGSH 12 w / v% and Gemza 10 w / v% were mixed reacted with each other, resulting in several seconds ( 1-2 seconds) shows that a hydrogel is formed. The results shown in FIG. 2 indicate that the 0.5M 4PEGSH buffer sufficiently buffered the pH change caused by Gemza under the current drug content condition. This shows that the composition can rapidly form a hydrogel when sprayed on the tumor surface.

Example 5. Preparation of Sealant Containing Genexol PM and Gemza and Measurement of Gel Properties (Polymer Content Change)

5-1. Manufacture of sealant containing Genexol PM and Gemza

A first solution was prepared by dissolving SSPGA 5-9 w/v% and Genexol PM 8 w/v% in 0.15M SSPGA buffer. Subsequently, 8-16 w/v% of 4PEGSH and 10 w/v% of Gemza were dissolved in 0.5M PEG buffer to prepare a second solution, and the first solution and the second solution were mixed in a 1:1 volume to obtain Genexol PM. A sealant containing and Gemza was prepared.

5-2. Measurement of gel properties of sealant containing Genexol PM and Gemza

When the content of Genexol PM and Gemza are the same, the gel properties of the sealant according to the content of SSPGA in the first solution and the 4PEGSH polymer content in the second solution Gelation time, burst pressure, decomposition time ( Degradation time) was measured. The contents of each component used in the first and second solutions and the measured gelation characteristics of the three types are shown in Table 6 below.

Specifically, the gelation time was measured in the same manner as in Example 1-2 above.

Burst strength was measured according to ASTM2392-04 (Standard Test Method for Burst Strength of Surgical Sealants). The collagen casing was cut into a size of 3 cm × 3 cm, washed twice in distilled water and ethanol, respectively, to remove glycerin attached to the surface, and used as a tissue substitute. After drilling a hole with a diameter of 3 mm, the first solution and the second solution were put into a double barrel syringe, and spraying was performed to a thickness of about 2 mm through a spray tip into the casing hole. After coating, it was allowed to stand for 5 minutes to harden, and then fixed to a bursting strength measuring instrument, and water pressure applied at this time was measured by flowing water at a rate of 2 mL/min. At this time, the highest water pressure when the cured gel was broken or dropped from the collagen casing was defined as the burst pressure.

Decomposition time was measured as follows. First, 0.3 g of hydrogel was put in 10 mL PBS buffer (pH 7.4) and immersed in a constant temperature bath at 37 ° C and 50 rpm for 24 hours, and then lightly wiped off the moisture on the surface and the degree of swelling (%) after immersion was calculated as follows. Samples were taken at regular time intervals to calculate the degree of swelling, and the experiment was stopped at the point at which the sample was no longer recovered (that is, the point at which all samples were decomposed), and this was defined as the degradation time.

[Equation 1]

Swelling degree (%) = (Weight of hydrogel after immersion) / (Weight of initial hydrogel) * 100%

[Table 6]

As shown in Table 6, the increase in the concentration of the polymer (SSPGA, 4PEGSH) of the sealant did not affect the gelation time, but the decomposition time was prolonged and the burst pressure also tended to be improved.

Example 6. In vitro drug release profile of sealant containing Genexol PM and Gemza

In order to confirm the drug release behavior over time of the hydrogel loaded with the two drugs, Genexol PM and Gemza, and to confirm whether the drug loaded in the hydrogel was released in a sustained release, an HPLC experiment was conducted. The experimental results are shown in FIG. 3 .

Specifically, in order to analyze the drug release behavior of Genexol PM, 100 mg of paclitaxel was precisely weighed and placed in a 100 mL volumetric flask, dissolved in acetonitrile, and this solution was dissolved in 80% acetonitrile at 1, 2, 5, 10 50 , 100, 200, and 600 μg/mL were diluted to prepare standard solutions. Subsequently, HPLC analysis was performed under the following conditions. Column (Poroshell 120 PFP column (2.7 ㎛, 4.6 x 150 mm, Agilent, USA)), detector (λnm), flow rate (0.6 mL/min), injection amount (10 uL), column temperature (25 ℃, mobile phase (0~ 25 minutes: water/ACN=65/35 (v/v) → 45/55 (v/v), 25 to 28 minutes: water/ACN=45/55 (v/v), 25 to 30 minutes: water/ ACN=45/55 (v/v)) → 65/35 (v/v), 30 to 35 minutes: water/ACN=65/35 (v/v))

To analyze the drug release behavior of Gemza, first, 100 mg of gemcitabine was precisely weighed and placed in a 100 mL volumetric flask, and dissolved in acetonitrile (1,000 μg/mL). 100 mg of gemcitabine was precisely weighed into a 100 mL volumetric flask and dissolved in acetonitrile (1,000 μg/mL). A standard solution was prepared by mixing the two standard solutions at a ratio of 1:1 (500 μg/mL). Subsequently, HPLC analysis was performed under the following conditions. Zorbax SB-C8 (5 ㎛, 4.6 x 250 mm), detector (λnm), flow rate (1.0 mL/min), injection volume (20 uL), column temperature (25 ℃, mobile phase (0-5 min: 0.1% Phosphoric acid) /ACN =95/5(v/v), 5~10 minutes: 0.1% Phosphoric acid/ACN =95/5(v/v) → 20/80(v/v), 10~15 minutes: 0.1% Phosphoric acid acid/ACN =20/80 (v/v), 15 to 18 minutes: 0.1% Phosphoric acid/ACN =20/80 (v/v) → 95/5 (v/v), 18 to 25 minutes: 0.1% Phosphoric acid/ACN =95/5(v/v))

As shown in FIG. 3, unlike paclitaxel, gemcitabine, a water-soluble drug, was mostly released at the beginning (67%), and additionally the drug was released over time, but the amount was not large (71%). In the case of Genexol PM, although paclitaxel is a poorly soluble drug, it was dissolved by the surfactant mPEG-PLA and was rapidly released by about 20% in one day. However, it was confirmed that paclitaxel was released in a sustained release form while trapped in the hydrogel after the surfactant, mPEG-PLA, was completely released. From the above results, it was confirmed that by loading a water-soluble drug (gemcitabine) and a poorly soluble drug (paclitaxel) together in a hydrogel, treatment with a water-soluble drug is possible at the beginning, and anti-cancer treatment with a poorly soluble drug is possible over time.

Example 7. In vivo anticancer efficacy evaluation of drug-bearing sealants

7-1. Induction of pancreatic cancer orthotopic mouse model and treatment with drug-bearing sealant

An experiment was performed to evaluate the anticancer effect of the drug-loaded sealant on tumors through an orthotopic mouse model of pancreatic cancer. First, a pancreatic cancer orthotopic mouse model was induced. Specifically, a human-derived pancreatic cancer cell line (AsPC-1) was cultured and prepared at a concentration of 6 × 10 ⁶ /mL. After anesthetizing 45 6-week-old female nude mice using ketamine and rhumpun, a 2 cm incision was made in the left abdomen of the mouse to expose the spleen and pancreas. After injecting 0.1 mL of the pancreatic cancer cell line prepared by avoiding the pancreatic blood vessels using a 30 G injection needle into the exposed end of the pancreas, the pancreas and spleen were placed in place and the abdomen was sutured using a 5-0 suture.

Next, drug treatment was performed when the diameter of the tumor reached about 5 mm or more when touched by hand about 2 weeks after the transplantation of the pancreatic cancer cell line. The composition of each experimental group, the administered drug, the number of subjects, and the route of administration were set as shown in Table 7 below. Subsequently, after the mouse was anesthetized using ketamine and rumpun, the left abdomen of the mouse was vertically incised about 2 cm, and the tumor and pancreas were exposed, and the tumor volume (TV0) on the drug administration day was measured using a vernier caliper. The untreated group in Table 7 was not treated with anything, and the positive control group received an intravenous injection of Genexol PM once 2-3 days after the tumor volume measurement day and an intravenous injection of Gemza 3 times at 3-4 day intervals. . In test group G2, only the sealant was applied to the tumor, and in test group G3, the sealant loaded with Gemza and Genexol PM was applied to the tumor. After the sealant hardened, the pancreas and tumor were placed in place, and the abdomen was sutured using a 5-0 suture.

[Table 7]

7-2. In vivo anticancer efficacy evaluation

Clinical symptoms and death of the untreated group, test group G1, test group G2, and positive control group mice in Table 6 were checked every day, and weight, tumor volume (TV), relative tumor volume (RV), Tumor Growth Inhibition rate (TGI) and tumor metastasis criteria were measured, and the results are shown in FIGS. 4 to 6.

(1) The weight of the mouse was measured using an electronic scale twice a week, including the day of drug administration, and the results are shown in FIG.

(2) The tumor volume (TV) was measured using Equation 2 below, and the results are shown in FIG. 5 .

[Equation 2]

TV (mm ³ )= A × A × B/2

(A: long axis of tumor / B: diameter perpendicular to long axis of tumor)

(3) The relative tumor volume (RV) was measured using Equation 3 below, and the tumor growth inhibition rate (TGI) was measured using Equation 4, and the results are shown in FIG. 5 .

[Equation 3]

RV = TVn/TV0

(TVn: tumor volume at the end of the experiment / TV0: tumor volume at the day of drug administration)

[Equation 4]

TGI (%) = (1-RTVT/RTVC) × 100

(RTVT: RTV of the experimental group / RTVC: RTV of the untreated group)

(4) The degree of tumor metastasis was scored based on the following Table 8, and shown in FIG. 6.

[Table 8]

As a result of the in vivo anticancer efficacy evaluation, 11 animals in each group were administered the drug, but individuals G2-3, G3-3, G3-4, and G4-10 died the day after laparotomy after drug administration. Moribundity, death, and specific clinical symptoms were not observed. Initially, it was planned to administer Gemza a total of 6 times, but on the scheduled date of the 4th administration, a rapid decrease in body weight was observed, so administration was discontinued.

As shown in Figure 4, the body weight was recovered from 20 days after drug administration, and in the case of G3, there was a temporary weight loss from the next day of drug administration, but all were recovered within 10 days.

As shown in FIG. 5, test group G3 showed a significant increase in tumor growth inhibition rate by about 57% compared to untreated group G1 (P<0.01), which was similar to positive control group G4. The tumor weight-to-weight ratio was also significantly decreased in test group G3 compared to untreated group G1 (P<0.05).

As shown in Figure 6, it was confirmed that the transition grade of test group G3 also decreased. On the other hand, G2 treated with only the sealant also showed a tendency to increase the tumor growth inhibition rate compared to the untreated group, and also showed a tendency to decrease metastasis.

From the above results, it was confirmed that the drug-bearing sealant exhibits excellent tumor growth inhibitory effect even with a single administration as well as lower systemic toxicity (no weight loss) compared to intravenous administration. Furthermore, as the sealant alone group also showed a certain level of tumor growth inhibitory effect, it was confirmed that the tumor growth was suppressed and the anticancer effect was present just by applying the sealant to the tumor.

It can be seen that the drug-bearing sealant exhibits an effective anticancer effect as a result of having a chemical inhibitory effect of an anticancer agent as well as a physical tumor growth inhibitory effect.

Claims (13)

(i) a first solution containing a γ-polyglutamic acid derivative of Formula 1 below and a buffer solution having a pH in the range of 5-6; and
(ii) a second solution, which is a buffer solution having a pH in the range of 10-11, including a hydrophilic material that is a polyethylene glycol-based polymer represented by Formula 3; and a hydrogel formed by mixing,
Pharmaceutical composition for inhibiting tumor growth by adhering to the tumor surface or surrounding tissue:
[Formula 1]

In Formula 1,
the total sum of l, m and n is an integer from 390 to 15,500, and the ratio of l, m and n is 1 : m : n = 0 to 0.5 : 0.2 to 0.5 : 0.2 to 0.8;
L is a linker that is -HN-(CH ₂ ) ₂ -O-;
M is each independently H, an alkali metal or an alkaline earth metal;
R is CH ₂ ;
b is 1;
c is an integer from 1 to 5;
[Formula 3]

In Formula 3, X represents an amine group, a thiol group or a hydroxyl group, and n is 19 to 170. The pharmaceutical composition according to claim 1, characterized in that the linker is -HN-(CH ₂ ) ₂ -O- derived from 1-amino-2-ethanol (MEA or 1-amino-2-ethanol). According to claim 1, in formula 1

go

A pharmaceutical composition, characterized in that. The pharmaceutical composition according to claim 1, wherein the pH after mixing of the first solution and the second solution is in the range of 5 to 9. The method of claim 1, wherein the buffer solution is distilled water, NaCl (sodium chloride), KCl (potassium chloride), NaH ₂ PO ₄ (Monosodium phosphate), Na ₂ HPO ₄ (Disodium phosphate), KH ₂ PO ₄ (Monopotassium phosphate), Na ₂ CO ₃ (Sodium carbonate), HCl (Hydrochloric acid), Borate, MES, Tris, and a pharmaceutical composition, characterized in that at least one selected from the group consisting of HEPES is dissolved in an aqueous solution. The method of claim 5, wherein the buffer of the first solution is a mixed buffer of NaH ₂ PO ₄ (Monosodium phosphate) and Na ₂ HPO ₄ (Disodium phosphate), and the buffer of the second solution is Na ₂ HPO ₄ (Disodium phosphate) and Na ₂ CO ₃ (Sodium carbonate) pharmaceutical composition, characterized in that the mixing buffer. The method of claim 6, wherein the buffer of the first solution is a mixture of NaH ₂ PO ₄ and Na ₂ HPO ₄ in a volume ratio of 9:1 to 1:9, and the buffer of the second solution is Na ₂ HPO ₄ and Na ₂ CO A pharmaceutical composition, characterized in that ₃ is mixed in a volume ratio of 9: 1 to 1: 9. The pharmaceutical composition according to any one of claims 1 to 7, wherein the surrounding tissue is a tumor incision site. The method according to any one of claims 1 to 7, wherein the first solution further contains a drug, the second solution further contains a drug, or both the first and second solutions further contain a drug. A pharmaceutical composition comprising a. 10. The pharmaceutical composition according to claim 9, wherein the drug is at least one selected from the group consisting of anticancer agents, antibiotics and analgesics. The method of claim 10, wherein the anticancer agent is at least one selected from the group consisting of Paclitaxel, Gemcitabine, Docetaxel, Doxorubicin and Cyclophosphamide; the antibiotic is at least one selected from the group consisting of Streptomycin, Gentamicin, Amoxicillin, Doxycycline, Cephalexin, and Ciprofloxacin; Or a pharmaceutical composition, characterized in that the analgesic is at least one selected from the group consisting of Acetaminophen, Bupivacaine, Levobupivacaine, Aspirin Diphenhydramine, Ropivacaine, Lidocaine, Mepivacaine, Prilocaine, Cinchocaine, Etidocaine and Articaine. 10. The pharmaceutical composition according to claim 9, wherein each of the first solution and the second solution further contains an anticancer agent. 13. The pharmaceutical composition according to claim 12, wherein the first solution contains Paclitaxel, Decetaxel or a mixture thereof, and the second solution contains Gemcitabine.

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