WO2020196964A1 - Drug-loaded hydrogel formed in real time - Google Patents

Abstract

The present invention relates to a drug-loaded matrix that can attach to the surface of a tumor and, specifically, to a drug-loaded hydrogel formed in real time.

Description

Real-time formation hydrogel containing drug

The present invention relates to a drug-carrying support that can be attached to the seed surface, and more specifically, to a real-time hydrogel formed with a drug.

In general, drugs are prescribed in the form of oral or injection to treat patients with cancer. Such a therapeutic method has the advantage of being simple to administer, but since the drug is delivered to the tumor via the whole body, it has side effects that adversely affect normal tissues, and has an effective effect due to the low drug content delivered to the tumor compared to the administered dose. In order to indicate that an excessive amount of drugs is administered, there is a problem that side effects (eg systemic toxicity) appearing to patients are large for this reason. In addition, in order to continuously see the effective effect of the drug, it is necessary to repeatedly administer the drug, but this has the disadvantage of causing inconvenience to the patient due to repeated administration.

In order to solve this problem, the drug-carrying matrix is locally treated on the tumor to minimize side effects caused by general chemical use, and the drug is designed to be released from the support in a sustained manner, resulting in inconvenience due to repeated administration. Can be minimized. However, since such a drug-carrying scaffold has no adhesion to a tumor, it can be used only in a form such as subcutaneous implantation, and thus, its available range is narrow. Accordingly, there is a need for development of an improved drug delivery system capable of overcoming the disadvantages that the conventional invention has not essentially solved.

[Prior technical literature]

[Patent Literature]

(1) Korean Patent Registration No. 10-1901649, “Method of manufacturing sustained-release hydrogel composite”

A first solution containing an alginate polymer; And it relates to a drug-releasing hydrogel containing Doxorubicin by reacting a second solution containing a calcium ion capable of crosslinking alginate and a drug (Doxorubicin) in a 1:1 ratio, wherein a hydrogel for carrying a drug by mixing two solutions It is manufactured and confirmed the behavior of sustained-release drug release over time.

However, since the hydrogel is not formed immediately, it can be used only in the form already made, i.e., the hydrogel is not formed in real time, and the crosslinking reaction by calcium ions acts selectively only on alginate, so it does not react separately with the tissue and does not show adhesion. Does not.

(2) Korean Patent Publication No. 10-2010-0095940, “Paclitaxel-loaded conjugated linoleic acid poloxamer hydrogel drug delivery device”

Conjugated linoleic acid (CLA)-conjugated poloxamer, a temperature-sensitive polymer that changes from a solution (sol) state to a gel state when the temperature is above a certain temperature, is used as a drug delivery vehicle, and the tissue ( For example, a hydrogel drug delivery device capable of forming a hydrogel when sprayed on a tumor) is disclosed.

However, since hydrogels made of temperature-sensitive polymers do not react separately with tissues during gel formation, their adhesion to tissues (tumors) is very low. The antitumor effect was confirmed through cell experiments, but in fact, since the gel formation rate is slow (takes tens of seconds), it is expected that most of it will flow down even if it is sprayed on the tumor, which is expected to show low effective anticancer effect.

(3) US Patent No. 8,003,125, “INJECTABLE DRUG DELIVERY SYSTEMS WITH CYCLODEXTRIN-POLYMER BASED HYDROGELS”

It relates to the invention using a cyclodextrin-based hydrogel physically bound to PEG as a drug-bearing support. Unlike chemical crosslinking, when an external force is applied, physical crosslinking temporarily changes from a gel state to a solution (sol) state, and external force When disappears, it has a thixotropic property that changes back to a gel state.Using this structural property of a cyclodextrin-based hydrogel, a gel filled material loaded with a drug in a syringe is applied with an external force with a syringe plunger to give a solution (sol). After making it into a state, it is designed to be sprayed onto the tissue to form a gel again.

However, since the hydrogel with thixotropic properties is formed by physical crosslinking, the adhesion to the tissue is very low.

(4) 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

When a PEG-PAEU solution carrying a drug is applied to a liver tumor through a catheter using a poly(amino ester urethane)-based block copolymer (PEG-PAEU) polymer that changes sol-gel by temperature and pH Disclosed is a drug-carrying hydrogel that can be applied to a tumor surface as a gel by changes in temperature and pH environment.

However, as in the preceding literature, since the hydrogel is formed by physical crosslinking, the adhesion to the tissue (tumor) is very low.

(5) US registered patent US 9,125,814, “BIOCOMPATIBLE CROSSLINKED HYDROGELS, DRUG-LOADED HYDROGELS AND METHODS OF USING THE SAME”

A first solution in which PEG having a succinimide functional group capable of reacting with a nucleophile and a drug (analgesic) are simultaneously dissolved; And a hydrogel carrying a drug, which reacts with the first solution and a second solution containing PEG having a reactive nucleophilic functional group to form a hydrogel in real time.

As such, the conventional technology is a problem in that even if a hydrogel containing a drug is applied to a tissue (for example, a tumor), since there is no separate adhesion (chemical bond) with the tissue, it is easily detached and cannot continuously deliver the drug. There was this.

Accordingly, the present invention has a technical problem to provide a drug-carrying support that can be attached to a tissue surface in real time (in situ) with excellent adhesion to the tissue.

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 the following formula (1): and

[Formula 1]

In Formula 1,

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

L is a linker;

Each M is 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) When a second solution containing a hydrophilic material having at least one nucleophilic functional group selected from the group consisting of amine, thiol and hydroxy groups is mixed, the hydrogel formed has excellent adhesion to the tumor surface and surrounding tissues. As it is attached to the body, it is not detached from the movement of the organs and can deliver the drug locally while attached to the tissue for a long time. Also, a part of the drug carried on the hydrogel is not released initially, but is gradually released while trapped in the hydrogel. It was discovered that it can act as a drug delivery system, and the present invention was completed.

Accordingly, the present invention provides a hydrophilicity having at least one nucleophilic functional group selected from the group consisting of (i) a γ-polyglutamic acid derivative of Formula 1 above: and (ii) an amine group, a thiol group, and a hydroxy group. It provides a pharmaceutical composition for adhering to a tumor surface or surrounding tissues, comprising a hydrogel formed by mixing a second solution containing a substance.

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

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 hydroxy group, which are nucleophilic functional groups. Mixing the second solution; b. Applying the mixture to the tumor surface; And c. It provides a method for inhibiting tumor growth, comprising the step of forming a hydrogel in which the applied mixture is in situ.

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

Since the hydrogel of the present invention is fixed (adhered) by a chemical bond, the drug can be delivered locally while being attached to the tissue for a long time without being detached from the movement of the organ.

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

In addition, in the hydrogel of the present invention, some of the drugs carried on the hydrogel are not initially released, but are slowly released while trapped in the hydrogel, so that discomfort caused by repeated drug administration as a sustained-release drug formulation can be alleviated.

In addition, the hydrogel of the present invention can be used as a drug delivery vehicle by spraying it on the surface of the tumor, and when a small amount of bleeding occurs in the incision site of the tumor, the hydrogel formed immediately serves to prevent bleeding as a sealant. At the same time, it can be used as a surgical material that performs two functions at the same time as a treatment to remove the remaining tumor cells remaining in the incision.

In addition, 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, physical pressure is applied during the initial growth of the tumor to suppress the growth of the tumor. Therefore, the hydrogel of the present invention can exhibit effective anti-cancer effect as a result, since it has both the physical tumor growth inhibitory effect as well as the chemical inhibitory effect of the anticancer agent.

1 shows a schematic diagram of the application of a drug-loaded hydrogel according to the present invention to treat pancreatic cancer.

2 is a graph of gelation behavior of the drug-containing sealant according to Example 4.

3 is a graph of cumulative release of gemza (gemcitabine) and genexol PM (paclitaxel) in a drug-supported hydrogel over time according to Example 6.

4 is a graph showing a change in weight over time for each group according to Example 7.

5 shows a graph of an average initial vs. tumor volume and an average tumor growth inhibition rate of each group according to Example 7.

6 is a graph showing the average tumor metastasis grade and metastasis inhibition rate of each group according to Example 7.

The present invention (i) a first solution containing a γ-polyglutamic acid derivative represented by the following formula (1): and

[Formula 1]

In Formula 1,

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

L is a linker;

Each M is 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 substance having at least one selected from the group consisting of an amine group (-NH ₂ ), a thiol group (-SH), and a hydroxy group (-OH), which are nucleophilic functional groups. It relates to a pharmaceutical composition for attaching to the tumor surface or surrounding tissues, including a hydrogel.

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

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 hydroxy group, which are nucleophilic functional groups. Mixing the second solution; b. Applying the mixture to the tumor surface; And c. The applied mixture relates to a method for inhibiting tumor growth, comprising the step of forming a hydrogel in situ.

In addition, the present invention is (i) a first solution containing the γ-polyglutamic acid derivative of Formula 1: and (ii) a hydrophilicity having at least one nucleophilic functional group selected from the group consisting of an amine group, a thiol group, and a hydroxy 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 to adhere to the tumor surface or surrounding tissues, to inhibit tumor growth, and to use 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 of 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-3-propanol, 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- moiety 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 Formula 1 contained in the first solution may be succinimidyl succinyl polyglutamic acid (SSPGA) having the following structure:

.

In one embodiment, the hydrophilic material contained 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 hydroxy group.

In one embodiment, the hydrophilic material contained in the second solution is a poly(ethylene glycol) (PEG) derivative, poly(vinyl alcohol) (PVA), poly(ethylene imine) having two or more arms (multi-arm) (PEI), poly (lysine) (PL), trilysine amine (Trilysine amine), and may be one or more selected from the group consisting of poly (allylamine) (PAA). Preferably, the hydrophilic material contained in the second solution is multi-arm PEG, PEI or trilysine amine.

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

[Formula 2]

In Chemical Formula 2,

I is a radical derived from a dihydric 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, and is the same as the number of hydroxy groups of the polyhydric alcohol from which I is derived.

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

[Formula 3]

[Formula 4]

In Formulas 3 and 4, X represents an amine group, a thiol group, or a hydroxy 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 in a volume ratio of 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 in a volume ratio of 9:1 to 1:9, preferably in a volume ratio of 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, physical pressure is applied during the initial growth of the tumor to suppress the growth of the tumor. Therefore, since the hydrogel of the present invention has a chemical inhibitory effect of an anticancer agent by adding a drug as well as such a physical tumor growth inhibitory effect, as a result, it can exhibit an effective anticancer effect.

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

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

Specifically, the drug may be at least one 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, the first solution and the second solution may each further contain an anticancer agent. Specifically, the first solution may contain Paclitaxel, Docetaxel, or a mixture thereof, and the second solution may contain Gemcitabine.

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

(i) Polymer in the first solution

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

The γ-polyglutamic acid derivative of Formula 1 includes (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 a γ-polyglutamic acid-alkanolamine; (b) the hydroxy group of the γ-polyglutamic acid-alkanolamine, consisting of an anhydride of an acid selected from the group consisting of glutaric acid and succinic acid, or 1-halo valeric acid, 1-halopropionic acid, and 1-halo methylcarboxylic acid A second step of forming a carboxy terminal into which an alkyl group is introduced by reacting with 1-halo alkanoic acid selected from the group; And (c) reacting the formed carboxy terminal with N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide to form a γ-polyglutamic acid derivative in which at least some carboxyl groups are activated. Can be manufactured by

The γ-polyglutamic acid derivative of Formula 1 is, depending on the type of the introduced alkyl group, that is, according to the definition of the substituent at the -(CO)b-(R)c-CO- site, succinimidyl valerate (SVA : -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 Carboxymethylated (SCM: -CH ₂ -CO-O-succinimide).

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

.

In one embodiment, when the SSPGA is included in the first solution, the first solution is also referred to as “SSPGA solution” or “SSPGA buffer”. In the examples to be described later, the case where the SSPGA solution is used as the first solution is illustrated.

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) Polymer in the second solution

The second solution of the present invention contains a hydrophilic substance having at least one selected from the group consisting of an amine group (-NH ₂ ), a thiol group (-SH), and a hydroxy 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 hydroxy group. Specifically, the hydrophilic material is a poly(ethylene glycol) (PEG) derivative having two or more arms (multi-arm), poly(vinyl alcohol) (PVA), poly(ethylene imine) (PEI), poly(lysine) It may be one or more selected from the group consisting of (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 is a polyethylene glycol repeating unit is bonded to each hydroxy group of a polyhydric alcohol having a dihydric or higher, preferably 2 to 12, amine group at its end. , A thiol group or a hydroxy group may have a bonded structure. More specifically, such a polyethylene glycol-based polymer may be represented by the following formula (2).

[Formula 2]

In Chemical Formula 2,

I is a radical derived from a dihydric 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, and is the same as the number of hydroxy 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 hexanediol; Or glycerol, erythritol, thritol, pentaerythritol, xylitol, adonitol, sorbitol, mannitol palatinose (palatinose), maltose monohydrate (maltose monohydrate) or maltitol (maltitol), such as disaccharides and trisaccharides such as D-raffinose pentahydrate (D-raffinose pentahydrate) selected from 3 to 12-valent polyol Can be mentioned. More specifically, I may be a radical derived from a 4 to 12 polyhydric alcohol.

Examples of polyethylene glycol-based polymers falling within the scope of Formula 2 include polymers 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 hydroxy group, and n is 19 to 170.

As such polyethylene glycol-based polymer contains a plurality of nucleophilic functional groups such as the amine group, thiol group, or hydroxy group, the activated γ-polyglutamic acid derivative of Formula 1 can form an amide bond, a thioamide bond, or an ester bond. And, by forming a crosslinked structure from this, a crosslinked product and a hydrogel having a three-dimensional network structure can be formed. In particular, this polyethylene glycol-based polymer causes a rapid crosslinking reaction with the activated γ-polyglutamic acid derivative, thereby 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 a thiol group at each end.

In one embodiment, when 4PEGSH is included in the second solution, the second solution is also referred to as “4PEGSH solution” or “4PEGSH buffer”. In the examples to be described later, a case where 4PEGSH solution is used as the second solution is illustrated.

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

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

As a solvent for preparing the first solution and the second solution, non-toxic ones such as distilled water, physiological saline, sodium hydrogen carbonate (NaHCO ₃ ), boric acid, and a buffer such as phosphoric acid may be used.

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

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 It} may be an aqueous solution in which at least one selected from the group consisting of CO ₃ (Sodium carbonate), HCl (Hydrochloric acid), Borate, MES, Tris, and HEPES is dissolved.

The buffer in the solvent affects the gelation time. That is, depending on the type of buffer, the gelation reaction may or may not occur, and the gelation time can be adjusted quickly or slowly. The buffer should be made using a salt having a pKa similar to the pH of each polymer of the first solution and the second solution, and the buffering effect is maximized at that time, helping to reduce the decrease in the 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, and may be a mixture of different types of sodium phosphate buffers. To prepare the second solution, a mixing buffer of sodium phosphate and sodium carbonate may be used.

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 in a volume ratio of 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 in a volume ratio of 9:1 to 1:9, preferably in a volume ratio of 5:5 to 1:9.

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

At this time, a drug that can be mixed can be used by selecting a drug that is not reactive with a substance dissolved in the first solution or the second solution. To improve the solubility of the drug, a surfactant (e.g. mPEG-PLA, Sodium dodecyl sulfate, Tween 80), etc. may be used.

When the pH of the buffer is changed when dissolved even though a drug that is not reactive with the first solution and the second solution substance is used, 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 control the buffering effect of the buffer, it is possible to minimize the change in the gel formation time of the hydrogel by the drug.

(iv) Hydrogel formation

The term “hydrogel” as used herein may be defined to mean a swellable polymer matrix (support) containing water, and may have a crosslinked structure including a covalent bond or a non-covalent bond. The hydrogel absorbs water through a three-dimensional network structure composed of the crosslinked structure, and may exhibit elasticity according to the degree of crosslinking of the three-dimensional network.

In the present specification, “sealant” is used in the same meaning as the “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 support the drug in the hydrogel formation system, the drug is mixed in the step of preparing the first solution and the second solution. When one or more drugs are dissolved in the first solution, the second solution, or each of the two solutions, and then the two solutions are mixed as described above, a hydrogel carrying 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 becomes a crosslinking point. A hydrogel having a three-dimensional network structure may 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) are chemically reacted to prepare a hydrogel. . Since such a reaction reacts rapidly under an appropriate pH condition, it is possible to prepare a hydrogel that is formed in situ in real time. Since the number of NHS functional groups introduced into SSPGA is more than the number of thiol functional groups of 4PEGSH, the residual NHS functional groups of SSPGA that did not participate in the hydrogel reaction even if the hydrogel formation reaction occurs are functional groups (Amine groups, Thiol Group, Hydroxyl group) and chemically form a covalent bond (see FIG. 1). Since the hydrogel formed by this covalent bond shows strong adhesion to the tissue, spraying a mixed solution of two polymers on the tissue surface can serve as a sealant capable of performing a sealing function.

In order to generate an adhesive hydrogel, it is preferable that cross-linking occurs by bonding between mutually reacting polymer chains 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 cross-linking reaction.

The preferred time until the two mixed solutions form a gel may vary slightly depending on the application, but preferably within 10 minutes, for example, within 2 minutes, within 1 minute, within 30 seconds, and 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, smooth application may be difficult due to clogging of the injection needle or spray tip, and since there is not enough time for each component to be mixed, it is difficult to form a non-uniform gel or to form a covalent bond with the tissue surface. There is a risk of this lowering. On the other hand, if the gelation time is too long, it may flow into a solution state before the gel is formed at the application site, and thus the usability is insufficient and it is not easy to accurately apply the desired amount. Therefore, it is preferable that it is 1 second or longer, particularly 1 second or more and 2 seconds or less, until it penetrates into living tissues and maintains high adhesion 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 it touches the tissue surface, and if it exceeds 2 seconds, even if it is sprayed on the tissue, it flows down, making it impossible to apply a quantitative 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, the 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 against long-term tumors.

(v) supported drugs

The drug contained 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. Hydrophilic drugs are convenient to use because they have good solubility in nature, but depending on circumstances, poorly soluble drugs and surfactants capable of dissolving them can be used after dissolving in the first solution.

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

The dissolution concentration of the drug in the first solution should be set to a concentration capable of exhibiting an effective drug effect and a concentration not exhibiting toxicity. It can be used at a concentration of 1-30% (w/v) of the first solution, and preferably dissolved in 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 may be adjusted to buffer such changes.

The dissolution concentration of the drug in the second solution should be set to a concentration that can exhibit an effective drug effect and a concentration that does not exhibit toxicity. It can be used at a concentration of 1-30% (w/v) of the second solution, and preferably dissolved in 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 may be adjusted to buffer such changes.

Specifically, the drug may be at least one 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, the first solution and the second solution may each further contain an anticancer agent. Specifically, the first solution may contain Paclitaxel, Docetaxel, or a mixture thereof, and the second solution may contain Gemcitabine.

In one embodiment, mixing and using of the first solution and the second solution may be carried out by various methods. For example, mixing can be carried out by coating one of the undiluted solutions of the first solution and the second solution on the surface of the adherend and then applying the other solution, or the first solution and the second solution are mixed with a double barrel syringe ( It can also be applied 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 spray-attached connector and sprayed onto a tissue surface such as a tumor, the two solutions immediately react at the tissue surface, and the drug is loaded in a form attached to the tissue. A hydrogel is formed.

Hereinafter, in order to describe the present invention in detail, it will be described in detail with reference to examples. However, the embodiments according to the present invention may be modified in various 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 describe the present invention to those of ordinary skill in the art.

Example One. Genexol PM Preparation of containing sealant and Gelation characteristic

1-1. Preparation of sealant containing Genexol PM

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

[Table 1]

1-2. Gelling Characteristics of Sealant Containing Genexol PM

An experiment was conducted to confirm the gelling properties 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 gelling properties are shown in Table 2 below.

Specifically, 200 μL of the first solution was added to a 96 well plate, and then 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. After the second solution was added, the time taken until the magnetic spin bar could no longer rotate was defined as the gelation time.

[Table 2]

*P.O: precipitation occurs

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

Example 2. Nanoxel M Preparation of containing sealant and Gelation characteristic

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 (Docetaxel's raw material drug) in 0.15 M SSPGA buffer. Next, 12% of 4PEGSH was dissolved in 0.3M PEG buffer to prepare a second solution, and a sealant containing Nanoxel M was prepared by mixing the first solution and the second solution in a volume of 1:1.

2-2. Gelling properties of sealant containing Nanoxel M

The experiment was performed in the same manner as in Example 1-2 in order to confirm the gelling properties 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 gelling properties are shown in Table 3 below.

[Table 3]

*P.O: occurrence of sediment

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

Example 3. Gemja Preparation of containing sealant and Gelation characteristic

Example 3-1. Manufacture of sealant containing gemja

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

Example 3-2. Gelling properties of sealants containing gemja

The experiment was performed in the same manner as in Example 1-2 in order to confirm the gelling properties of the sealant according to the content of gemza, PEG buffer, or SSPGA of the second solution. 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 Gemja exists in an opaquely dispersed form under all PEG buffer concentration conditions. In addition, it was confirmed that the gel formation time increased as the amount of gemja increased in the same PEG buffer condition, but the gelation time could be shortened by buffering acidification by the gemza by increasing the SSPGA polymer content or increasing the concentration of the PEG buffer.

Example 4. Genexol PM Department Gemja Preparation of containing sealant and Gelation Characteristics (drug content change)

4-1. Preparation of sealant containing Genexol PM and Gemja

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

4-2. Gelation Characteristics of Sealant Containing Genexol PM and Gemja

The experiment was performed in the same manner as in Example 1-2 in order 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 content of each component used in the first solution and the second solution and the measured gelling properties are shown in Table 5 below and FIG. 2.

[Table 5]

*P.O: occurrence of sediment

As shown in Table 5, since Genexol PM contained in the first solution did not affect the pH change of the buffer, it was confirmed that it did not affect the gelation time regardless of the drug content. In addition, in the case of the second solution, it was confirmed that the gel formation time increased as the amount of gemja increased in the same PEG buffer condition, but the gelation time could be shortened by buffering acidification by the 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 decreased significantly, resulting in precipitation and thus not forming a gel.

4-3. Rheological Gelation Characteristics of Sealant Containing Genexol PM and Gemja

In order to confirm the rheological gelation properties of the sealant containing the drug, the storage modulus (G') and loss modulus (G'') changes were confirmed by setting the conditions of the rotational viscometer analysis as follows. At this time, the first solution was first loaded onto the geometry and analysis was started.After about 100 seconds, the second solution was injected to check the numerical change of G'and G''. It was defined as the time point of sol-gel change (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

Figure 2 shows a first solution mixed with 8 w/v% of SSPGA 7 w/v%/Genexol PM (paclitaxel), 12 w/v% of 4PEGSH, and 10 w/v% of Gemja reacted with each other for several seconds ( 1~2 seconds) shows that hydrogel is formed. The results shown in FIG. 2 indicate that the 0.5M 4PEGSH buffer sufficiently exhibited a buffering effect under the current drug content condition to sufficiently buffer the pH change caused by the gemza. This shows that when sprayed from the tumor surface with the composition, a hydrogel can be formed quickly.

Example 5. Genexol PM Department Gemja Preparation of containing sealant and measurement of gel properties (change of polymer content)

5-1. Preparation of sealant containing Genexol PM and Gemja

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

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

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

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

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 with distilled water and ethanol, respectively, to remove glycerin on the surface and used as a tissue replacement. After drilling a 3 mm diameter hole, the first solution and the second solution were put into a double barrel syringe, and then sprayed to a thickness of about 2 mm through a spray tip in the casing hole. After coating, it was allowed to stand for 5 minutes, hardened, and fixed to a burst strength meter, and water was flowed at a rate of 2 mL/min to measure the water pressure applied at this time. At this time, the highest hydraulic pressure when the cured gel breaks or falls from the collagen casing was defined as the burst pressure.

The disintegration time was measured as follows. First, 0.3 g of hydrogel was put in 10 mL PBS buffer (pH 7.4), 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 swelling degree (%) 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 a point in time when no more samples were recovered (ie, all samples were decomposed), and this was defined as the decomposition 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 polymer (SSPGA, 4PEGSH) concentration of the sealant did not affect the gelation time, but the decomposition time was prolonged and the burst pressure was also increased.

Example 6. Genexol PM Department Gemja In vitro drug release profile of containing sealant

In order to confirm the drug release behavior over time of the hydrogels loaded with the two drugs Genexol PM and Gemza, and through this, an HPLC experiment was conducted to confirm whether the drug supported on the hydrogel was released sustainedly. 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, and acetonitrile was added to dissolve it, and this solution was dissolved in 80% acetonitrile for 1, 2, 5, 10 50 , 100, 200, 600 μg/mL to prepare a standard solution. Subsequently, HPLC analysis was performed under the following conditions. Column (Poroshell 120 PFP column (2.7 µm, 4.6 x 150 mm, Agilent, USA)), detector (λnm), flow rate (0.6 mL/min), injection volume (10 uL), column temperature (25 °C, mobile phase (0~ 25 minutes: water/ACN=65/35(v/v) →45/55(v/v), 25~28 minutes: water/ACN=45/55(v/v), 25~30 minutes: water/ ACN=45/55(v/v)) →65/35(v/v), 30~35 minutes: water/ACN=65/35(v/v))

In order to analyze the drug release behavior of Gemza, first 100 mg of gemcitabine was precisely weighed, placed in a 100 mL volumetric flask, and dissolved in acetonitrile (1,000 μg/mL). 100 mg of gemcitabine was precisely weighed, placed in a 100 mL volumetric flask, and dissolved by adding acetonitrile (1,000 μg/mL). The two standard solutions were mixed at 1:1 to prepare a standard solution (500 μg/mL). Then, HPLC analysis was performed under the following conditions. Zorbax SB-C8 (5 µm, 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), 5~10min: 0.1% Phosphoric acid/ACN =95/5(v/v) →20/80(v/v), 10~15min: 0.1% Phosphoric acid/ACN =20/80(v/v), 15~18 min: 0.1% Phosphoric acid/ACN =20/80(v/v) →95/5(v/v), 18~25 min: 0.1% Phosphoric acid/ACN =95/5(v/v))

As shown in FIG. 3, unlike paclitaxel, gemcitabine, a water-soluble drug, was released most (67%) at the beginning, and additional drugs were released over time, but the amount was not large (71%). In the case of Genexol PM, although paclitaxel was a poorly soluble drug, it was dissolved by mPEG-PLA, a surfactant, and was rapidly released by 20% for 1 day. However, after the surfactant mPEG-PLA completely escaped, it was confirmed that paclitaxel was released in a sustained release state while trapped in the hydrogel. From the above results, it was confirmed that by loading a water-soluble drug (gemcitabine) and a poorly soluble drug (paclitaxel) together in a hydration gel, treatment with a water-soluble drug is possible at the beginning, and anticancer treatment with a poorly soluble drug is possible over time.

Example 7. In of drug-carrying sealant vivo Anticancer efficacy evaluation

7-1. Induction of stereotactic transplantation mouse model for pancreatic cancer and treatment with drug-laden sealant

An experiment was performed to evaluate the anticancer effect of the drug-carrying sealant on tumors through a pancreatic cancer orthotopic mouse model. First, a stereotactic pancreatic cancer transplant mouse model was derived. Specifically, human-derived pancreatic cancer cell line (AsPC-1) was cultured and prepared at a concentration of 6 × 10 ⁶ /mL. After 45 6-week-old female nude mice were anesthetized with ketamine and lumpun, the left abdomen of the mouse was incised about 2 cm vertically, and the spleen and pancreas were exposed. After 0.1 mL of the prepared pancreatic cancer cell line was injected to the exposed pancreatic extremity using a 30 G needle to avoid pancreatic blood vessels, the pancreas and spleen were placed in place and abdominal suture was performed using 5-0 sutures.

Next, about 2 weeks after the pancreatic cancer cell line was transplanted, drug treatment was performed when the diameter of the tumor became more than about 5 mm when touched by hand. The composition of each experimental group, drug administration, number of individuals, and administration route were set as shown in Table 7 below. Subsequently, the mouse was anesthetized with ketamine and lumpun, the left abdomen of the mouse was incised about 2 cm vertically, and the tumor and pancreas were exposed, and then the tumor volume (TV0) on the day of drug administration was measured using a vernier caliper. The untreated group in Table 7 did not treat anything, and in the positive control group, Genexol PM was injected once intravenously 2-3 days after the tumor volume measurement day, and gemza was injected intravenously 3 times at 3-4 days intervals. . In test group G2, only sealant was applied to the tumor, and in test group G3, a sealant loaded with Gemza and Genexol PM was applied to the tumor. After the sealant was hardened, the pancreas and tumor were placed in place, and abdominal sutures were performed using 5-0 sutures.

[Table 7]

7-2. In vivo anticancer efficacy evaluation

The clinical symptoms and death of the mice in the untreated group, test group G1, test group G2, and positive control group of 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 balance twice a week including the day of drug administration, and the results are shown in FIG. 4.

(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 diameter of tumor / B: diameter perpendicular to tumor long diameter

(3) 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.

[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 experimental group / RTVC: RTV of non-treatment group)

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

[Table 8]

As a result of the in vivo anticancer efficacy evaluation, 11 animals were administered per group, but subjects G2-3, G3-3, G3-4, and G4-10 died the day after drug-administered laparotomy. No moribund, death, or specific clinical symptoms were observed. Initially, it was planned to administer Gemza a total of 6 times, but on the scheduled day of the 4th administration, a sudden decrease in body weight was observed and the administration was stopped.

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

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

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

From the above results, it was confirmed that the drug-carrying sealant exhibited excellent tumor growth inhibitory effect even with a single administration as well as low systemic toxicity (no weight loss phenomenon) compared to intravenous administration. Furthermore, it was confirmed that the sealant alone group also exhibited a certain level of tumor growth inhibitory effect, and thus tumor growth was inhibited by simply applying the sealant to the tumor, and anticancer effect was present.

It can be seen that the drug-carrying sealant has not only a physical tumor growth inhibitory effect, but also a chemical inhibitory effect of an anticancer agent, resulting in an effective anticancer effect.

Claims (23)

1. (i) a first solution containing a γ-polyglutamic acid derivative represented by the following formula (1): and

   [Formula 1]

   

   In Formula 1,

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

   L is a linker;

   Each M is 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) A hydrogel formed by mixing 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 hydroxy group, including a hydrogel formed, for attaching to the tumor surface or surrounding tissues Pharmaceutical composition.
2. The method of claim 1, wherein the linker of the γ-polyglutamic acid derivative of Formula 1 in the first solution is -HN-(R)aO- (wherein R is CH ₂ and a is an integer of 1 to 5). Pharmaceutical composition.
3. The method of claim 2, wherein the linker is aminomethanol, 1-amino-2-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 A pharmaceutical composition, characterized in that derived from).
4. The method of claim 1, wherein the -(CO)b-(R)c-CO- moiety of the γ-polyglutamic acid derivative of Formula 1 in the first solution 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-, characterized in that the pharmaceutical composition.
5. The pharmaceutical composition according to claim 1, wherein the γ-polyglutamic acid derivative of Formula 1 in the first solution is succinimidyl succinyl polyglutamic acid (SSPGA) having the following structure:

   

   .
6. The pharmaceutical composition according to claim 1, wherein the hydrophilic material in the second solution has two or more nucleophilic functional groups selected from the group consisting of an amine group, a thiol group, and a hydroxy group.
7. The method of claim 1, wherein the hydrophilic material in the second solution is a poly(ethylene glycol) (PEG) derivative, poly(vinyl alcohol) (PVA), poly(ethylene imine) (PEI) having two or more arms (multi-arm). ), poly (lysine) (PL), trilysine amine (Trilysine amine), and a pharmaceutical composition, characterized in that at least one selected from the group consisting of poly (allylamine) (PAA).
8. The pharmaceutical composition according to claim 1, wherein the hydrophilic substance in the second solution is multi-arm PEG, PEI or trilysine amine.
9. The pharmaceutical composition according to claim 1, wherein the hydrophilic material in the second solution is a polyethylene glycol-based polymer, and the polyethylene glycol-based polymer is represented by the following formula (2):

   [Formula 2]

   

   In Chemical Formula 2,

   I is a radical derived from a dihydric 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, and is the same as the number of hydroxy groups of the polyhydric alcohol from which I is derived.
10. The pharmaceutical composition according to claim 9, wherein the hydrophilic material in the second solution is a polyethylene glycol-based polymer, and the polyethylene glycol-based polymer is represented by the following Chemical Formula 3 or 4:

    [Formula 3]

    

    [Formula 4]

    

    In Formulas 3 and 4, X represents an amine group, a thiol group, or a hydroxy group, and n is 19 to 170.
11. The pharmaceutical composition according to claim 1, wherein the first solution and the second solution are buffer solutions.
12. The pharmaceutical composition according to claim 11, wherein the pH of the first solution is in the range of 5-6, and the pH of the second solution is in the range of 10-11.
13. The pharmaceutical composition according to claim 11, wherein after mixing the first solution and the second solution, the pH is in the range of 5 to 9.
14. The method of claim 11, 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), A pharmaceutical composition, characterized in that it is 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.
15. The method of claim 14, 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 Pharmaceutical composition, characterized in that the mixing buffer of ₂ CO ₃ (Sodium carbonate).
16. The method of claim 15, 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.
17. The pharmaceutical composition according to any one of claims 1 to 16, which is used for inhibiting tumor growth.
18. The pharmaceutical composition according to any one of claims 1 to 16, wherein the surrounding tissue is a tumor incision site.
19. The pharmaceutical composition according to any one of claims 1 to 16, characterized in that the first solution and/or the second solution further comprise a drug.
20. The pharmaceutical composition according to claim 19, wherein the drug is at least one selected from the group consisting of anticancer agents, antibiotics and analgesics.
21. The method of claim 20, 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 agent is at least one selected from the group consisting of Acetaminophen, Bupivacaine, Levobupivacaine, Aspirin Diphenhydramine, Ropivacaine, Lidocaine, Mepivacaine, Prilocaine, Cinchocaine, Etidocaine and Articaine.
22. The pharmaceutical composition according to claim 19, wherein the first solution and the second solution each further contain an anticancer agent.
23. The pharmaceutical composition according to claim 22, wherein the first solution comprises Paclitaxel, Decetaxel, or a mixture thereof, and the second solution comprises Gemcitabine.

Images