KR20200009611A - Inflammatory peptide-loadedinjectable hydrogel by physical or chemical loading, and method of manufacturing and application - Google Patents

Abstract

The present invention relates to hydrogel as a carrier capable of carrying an anti-inflammatory peptide by containing the anti-inflammatory peptide through a physical or a chemical method, and to a method of manufacturing the hydrogel. The hydrogel according to the present invention has a structure in which a peptide having an anti-inflammatory effect is physically or chemically contained in a natural biomaterial, so that the peptide unstable in vivo can be carried to a disease site of an inflammatory reaction while having a stable and high biocompatibility. Therefore, the present invention can improve low treatment efficiency due to low biological stability of the anti-inflammatory peptide and alleviate side effects during administration which appear due to repeated administration. In addition, a pharmaceutical composition including the hydrogel according to the present invention can be applied in various ways such as injectable formulations, etc., and thus can be applied for development of a therapeutic agent for inflammatory diseases such as rheumatoid arthritis or degenerative joint diseases.

Description

Injectable formulations of physical / chemical containing anti-inflammatory peptides, preparation methods and applications thereof Inflammatory peptide-loaded injectable hydrogel by physical or chemical loading, and method of manufacturing and application

The present invention relates to a hydrogel as a carrier which can contain an inflammation inhibitory peptide in a physical or chemical manner and can deliver it, and a method for preparing the same.

Inflammatory diseases are known throughout the world as the leading cause of morbidity and mortality. Inflammatory diseases affect various organs and tissues such as blood vessels, heart, brain, nerves, joints, skin, lungs, eyes, gastrointestinal tract, kidneys, thyroid, adrenal glands, pancreas, liver and muscles, myopathy, leukocyte damage, cancer, allergies It causes diseases such as rhinitis and autoimmune diseases (rheumatic arthritis, asthma, lupus, etc.).

The treatment of inflammatory and painful diseases is of great interest from the pharmaceutical industry. Despite recent progress, current therapies for inflammatory and painful diseases have been shown to relieve symptoms with non-specific drugs, reduce inflammation and pain, reduce disease progression to disease-controlling agents, and change lifestyles. Although still improving the quality, all of these have problems with side effects and resistance to the drug. There is a need for better treatment options with less potential for side effects.

For example, rheumatism, one of the inflammatory diseases, may be caused by immune abnormalities or infections in the joints in which rheumatism occurs, infiltrating inflammatory cells in the synovial membrane, and further proliferating synovial fibroblasts with angiogenesis, which is called an inflammatory disease called pannus. Synovial granulation tissue is formed. The formation of pannus causes the destruction of bone or cartilage, leading to irreversible disorders in the joints. In the process of bone or cartilage destruction, various extracellular matrices such as collagen and proteoglycan present in large quantities are decomposed. Conventionally, the drug therapy of rheumatoid arthritis mainly uses various nonsteroidal anti-inflammatory drugs, steroids such as prednisolone, and antirheumatic drugs such as mesotrexate to alleviate joint pain and inflammation (Best Practice & Research Clinical Rheumatology). , 21, 27-42, Bernard Combe, 2007). In degenerative arthrosis, various nonsteroidal anti-inflammatory drugs or analgesics have been directly injected into the joints to remove pain and inflammation. In addition, hyaluronic acid, which inhibits cartilage destruction, is used as a cartilage protection agent as a common treatment method for joint diseases.

Drug Delivery System (DDS) refers to a formulation designed to efficiently deliver the required amount of drug by minimizing the side effects of the existing drug, maximizing the efficacy and effect. In particular, when an anti-inflammatory analgesic is administered orally when treating arthritis, the drug acts on the joints, but the other side shows side effects, so the formulation is designed to reduce such side effects and maximize the efficacy. Typical drugs that require these considerations in the drug delivery system are those used for chronic treatment that are highly toxic drugs administered once by injection, unstable drugs with cytotoxicity, easily inactivated drugs and drugs requiring topical application.

In the case of general drug injection or oral drug preparation, the drug is toxic to the body due to the high concentration of the drug in the blood. The need for is urgent.

As biomaterials, research on biodegradable materials which are excellent in biocompatibility and degraded in vivo has been continued for a long time. Specifically, research on natural biodegradable polymer materials or synthetic biodegradable polymer materials is focused. More specifically, natural biodegradable polymer materials include polysaccharides such as hyaluronic acid, carboxymethyl cellulose and alginate; Polypeptides such as collagen and gelatin; Polyamino acids such as poly-L-glutamic acid and poly-L-lysine. At this time, the polysaccharides are characterized by including a carboxyl group as a functional group.

Research into long-term sustained-release formulations of peptide drugs has focused until recently on formulation development through conjugation reactions with biocompatible biodegradable polymers. The duration of drug efficacy of such peptide drugs may be extended to several weeks, depending on the form of the formulation and the active ingredient being conjugated. In developing such formulations, consideration should be given to increasing the duration of drug efficacy and to the biocompatibility of the polymers conjugated to the active ingredient.

Therefore, recently, studies are being actively conducted to apply a biocompatible biodegradable polyethylene glycol (PEG) or hyaluronic acid (HA) and an active ingredient to a drug delivery system.

However, PEG, which is used for the conjugation of polyethylene glycol (PEG) to the active ingredient, that is, the so-called PEGylation reaction, is one of the representative biopolymer materials approved by the FDA, but is used as a drug carrier. Repeated injections of the PEG-Liposome conjugate have been reported to result in accelerated blood clearance (ABC), which leads to rapid loss of the administered drug in the body.

Therefore, the inventors of the present invention have attempted to prepare a hydrogel in which a peptide having an inflammatory inhibitory effect is physically or chemically contained in a natural biomaterial to solve the problems of the prior art. When the hydrogel is used as a peptide carrier, it can be used as an injection formulation, and it was confirmed that the problem of patient taking due to the repeated administration of the inflammatory inhibitory peptide with low biostability could be solved. In addition, compared to a single administration of a peptide having an anti-inflammatory effect, the injection-containing hydrogel containing the peptide confirmed that the anti-inflammatory effect and the duration thereof may have excellent advantages, thereby completing the present invention. .

Disclosure of Invention It is an object of the present invention to provide a hydrogel for inflammatory inhibitory peptide delivery and a method for preparing the same, for solving low therapeutic efficiency and taking side effects by repeated administration of inflammatory inhibitory peptide with low biostability.

It is also an object of the present invention to provide a pharmaceutical composition comprising the hydrogel as an injection formulation.

In order to achieve the above object, the present invention provides a method for producing a hydrogel for inflammation inhibitory peptide delivery, comprising the following steps i) to iii):

i) preparing a first reaction solution in which a natural biomaterial to which a compound having a first chemical functional group is bound and an inflammation inhibitory peptide are mixed;

ii) preparing a second reaction solution in which a natural biological material combined with a compound having a second chemical functional group and an inflammation inhibitory peptide are mixed; And

iii) mixing the first reaction solution prepared in step i) and the second reaction solution prepared in step ii).

The present invention also provides a method for preparing a hydrogel for inflammation inhibitory peptide delivery, comprising the following steps i) to iii):

i) preparing a first reaction solution comprising a natural biomaterial to which an inflammation inhibitory peptide and a compound having a first chemical functional group are bound;

ii) preparing a second reaction solution including a natural biomaterial to which an inflammation inhibitory peptide and a compound having a second chemical functional group are bound; And

iii) mixing the first reaction solution prepared in step i) and the second reaction solution prepared in step ii).

The present invention also provides a hydrogel for inflammation inhibitory peptide delivery prepared according to the above method.

In a preferred embodiment of the present invention, the first chemical functional group may be any one selected from the group consisting of tetrazine, alkane group, epoxy group and acroyl group.

In a preferred embodiment of the present invention, the second chemical functional group may be any one selected from the group consisting of cyclooctene, azide group, thiol group and amine group.

In a preferred embodiment of the present invention, the natural biomaterial may be any one selected from the group consisting of hyaluronic acid, carboxymethyl cellulose (CMC) and alginate (AGNT).

In a preferred embodiment of the present invention, the inflammation inhibitory peptide may be composed of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

In a preferred embodiment of the present invention, the bond may be made of an amide reaction induced by a condensing agent, and the condensing agent is 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) 4-methylmorpholinium chloride (4- (4,6-Dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride) (DMTMM).

In addition, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, including the hydrogel for delivering an inflammation inhibitory peptide.

In one preferred embodiment of the present invention, the inflammatory disease is insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental form uveitis, Hashimoto thyroiditis, primary mucus Edema, thyroid addiction, pernicious anemia, autoimmune atrophy gastritis, Addison's disease, early menopause, male infertility, childhood diabetes, goodpasture syndrome, common swelling, ileal swelling, sympathetic ophthalmopathy, uveitis, autoimmune hemolytic anemia, idiopathic Leukocyte reduction, primary cholangiovascular sclerosis, chronic active hepatitis Hbs-ve, latent cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis / dermatitis, discoid LE and systemic lupus erythematosus It may be one or more selected.

In one preferred embodiment of the present invention, the pharmaceutical composition may be an injection formulation.

The present invention provides a peptide for delivering a hydrogel having an inhibitory effect and a method for producing the same. The hydrogel according to the present invention has a structure in which a peptide having an inflammatory inhibitory effect on a natural biomaterial is physically or chemically contained, and thus the peptide which is unstable in vivo is delivered to a disease site of an inflammatory response while having a stable and high biocompatibility. To be possible. Accordingly, it is possible to improve the low therapeutic efficiency due to the low bio stability of the inflammation inhibitory peptide, and to alleviate the side effects at the time of administration, which is shown by repeated administration.

In addition, the pharmaceutical composition comprising a hydrogel according to the present invention can be applied in various forms, such as injection formulations, it is possible to apply to the development of therapeutic agents for inflammatory diseases such as rheumatoid arthritis or degenerative joint disease.

Hydrogel drug carriers used in the prior art should use a high concentration of hyaluronic acid in order to obtain a suitable viscosity in the use of hydrogel only without using a crosslinking agent, the hydrogel according to the present invention by forming a crosslink using a crosslinking agent Hyaluronic acid can be used at low concentrations compared to conventional drug carriers to achieve the desired viscosity, and drug trapping ability can also be significantly increased compared to the case of using high concentrations of hyaluronic acid.

1 is a schematic diagram showing a process for preparing a hydrogel according to an embodiment of the present invention, and an example of applying the hydrogel to a joint induced by rheumatoid arthritis.
Figure 2 shows the results of confirming the time-cell survival rate of the hyaluronic acid hydrogel prepared in the present invention for inflammatory cells.
FIG. 3 shows the results of Hematoxylin & Eosin staining of tissues after administration of hyaluronic acid hydrogel containing anti-inflammatory peptides through chemical bonding in an animal model induced by rheumatoid arthritis: control represents a normal control group without inducing arthritis Non treatment refers to an untreated control model of arthritis animal that did not receive anti-inflammatory peptides.
Fig. 4 shows the results of Safranin O staining of tissues after administration of hyaluronic acid hydrogel containing anti-inflammatory peptides through chemical bonding in an animal model induced by rheumatoid arthritis: control represents a normal control and non treatment is untreated Control group.
FIG. 5 shows the results of fluorescence staining of TNF-α in tissues after administration of a hyaluronic acid hydrogel comprising anti-inflammatory peptides via chemical bonding to an animal model induced by rheumatoid arthritis: control represents a normal control, Non treatment represents an untreated control.
FIG. 6 shows the result of observing the degree of swelling according to the inflammatory response after administration of a hyaluronic acid hydrogel containing an anti-inflammatory peptide through chemical bonding to an animal model induced by rheumatoid arthritis: control represents a normal control group, Non treatment represents an untreated control.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

Hydrogels of natural biomaterials physically containing inflammation inhibitory peptides

The present invention provides a method for preparing a hydrogel for inflammation inhibitory peptide delivery, comprising the following steps i) to iii):

i) preparing a first reaction solution in which a natural biomaterial to which a compound having a first chemical functional group is bound and an inflammation inhibitory peptide are mixed;

ii) preparing a second reaction solution in which a natural biological material combined with a compound having a second chemical functional group and an inflammation inhibitory peptide are mixed; And

iii) mixing the first reaction solution prepared in step i) and the second reaction solution prepared in step ii).

The present invention also provides a hydrogel for inflammation inhibitory peptide delivery prepared according to the above method.

In the production method of the present invention, the first chemical functional group is preferably any one selected from the group consisting of tetrazine, alkane group, epoxy group and acroyl group, and more preferably tetrazine, but is not limited thereto. .

In the production method of the present invention, the second chemical functional group is preferably any one selected from the group consisting of a cyclooctene, an azide group, a thiol group, and an amine group, and more preferably, a cyclooctene, but is not limited thereto. It doesn't work.

More specifically, the first chemical functional group and the second chemical functional group are most preferably applied to hydrogel preparation by combining the following combinations: (tetrazin, cyclooctene), (alkaine group, azide group), ( Alkaine group, thiol group), (epoxy group, amine group), (epoxy group, thiol group), (acroyl group, amine group) or (acroyl group, thiol group).

In the production method of the present invention, the natural biomaterial preferably includes a carboxyl group. Natural biomaterials containing carboxyl groups can be combined with compounds containing chemical functional groups via amide bonds. Specifically, the natural biomaterial is more preferably one or more selected from the group consisting of hyaluronic acid, carboxymethyl cellulose (CMC), and alginate (AGNT), but is not limited thereto.

More specifically, when the natural biomaterial is hyaluronic acid, its molecular weight is preferably in the range of 500,000 Da to 6,000,000 Da. At this time, when the molecular weight of the hyaluronic acid is less than 500,000 Da, the physical properties are too low to form a hydrogel significantly, and when the molecular weight of the hyaluronic acid is greater than 6,000,000 Da, since the viscosity may rise to bring a limit to the application as an injection formulation, it is not preferable. not.

Hyaluronic acid is a basic component of extracellular matrix widely distributed in animal tissues and functions to regulate cell proliferation and differentiation in vivo. That is, it is known that hyaluronic acid is involved in important biological processes, cell mobility and tissue healing and can regulate the inflammatory response. Hyaluronic acid has been shown to be involved in tumor growth by interacting with specific receptors located on the cell surface. This may show applicability as a soluble carrier in the production of macromolecular prodrugs with modified hyaluronic acid antitumor activity, which has attracted much interest in the manufacture of drug carriers. For this reason, it is well known in the art that biomaterials based on hyaluronic acid are very suitable as tissue engineering supports useful as biocompatibility and cell growth inducing substances. Nevertheless, the use of hyaluronic acid alone is limited to its disadvantages such as generating a skeleton that is not very elastic and easily broken. In addition, the surface of these skeletons may be so hydrophilic that the adhesion and cell differentiation regulating effect may not be significant.

Therefore, a method of modifying hyaluronic acid by mixing or crosslinking hyaluronic acid with a biocompatible natural polymer such as collagen or gelatin, or by modifying hyaluronic acid with a hydrophobic group has been proposed. Similarly, by introducing functional groups on the surface of hyaluronic acid, chemical modifications in polysaccharide molecules have the goal of obtaining a drug delivery system (DDS) that slows the release of drugs introduced into hyaluronic acid. Research is ongoing. This goal means that hyaluronic acid itself can be applied by improving the physicochemical properties and changing the degree of degradation in vivo through chemical modification while maintaining the properties such as biocompatibility, biodegradability, non-immunity. Specifically, in addition to the reaction of the carboxyl group of hyaluronic acid, the polymer-drug bond for controlling the drug release mechanism, the crosslinking with hydrogel, the surface coating, and the like, a composition mixed with other materials has been discussed. In addition, the results of studies evaluating the possibility of using many hyaluronic acid derivatives (eg, self-crosslinked hyaluronic acid) at in vitro and in vivo stages have been reported. In addition, esterification of hyaluronic acid with various drugs is known to provide new physicochemical properties that can be used for drug release control and tissue engineering.

In the method for producing a hydrogel of the present invention, the fact that the inflammation inhibitory peptide is physically included in a natural biological material means that the drug is physically encapsulated by simple mixing in chemically modified hyaluronic acid, and thus the characteristics of the modified hyaluronic acid. This means that it is applied as a system to control the release mechanism of the drug.

In the production method of the present invention, the inflammation inhibitory peptide may be composed of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3. Specifically, the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 3 is a sequence known from the Republic of Korea Patent Publication No. 10-1745520, it is preferable that the peptide consisting of 18 amino acids. However, the peptide that can be applied to the production method of the present invention may be applied without limitation as long as it can be recognized by those skilled in the art as having an anti-inflammatory effect among natural protein-derived or artificially synthesized peptides.

In the production method of the present invention, the bonding is preferably performed by inducing a condensation reaction, preferably an amide reaction, by further adding a condensing agent to a solution containing a natural biomaterial. The condensing agent is 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (4- (4,6-Dimethoxy-1,3,5 -triazin-2-yl) -4-methylmorpholinium chloride) (DMTMM) is preferred, but is not limited thereto, and any condensing agent can be understood by those skilled in the art to induce condensation reactions against natural biomaterials. Can be applied without. The amount of the condensing agent can be arbitrarily adjusted by those skilled in the art to control the degree of condensation reaction.

In the production method of the present invention, after mixing an inhibitory peptide to a natural concentration to which a compound containing a chemical functional group is bound (condensed) at a predetermined concentration, if the vortexing is enough for 5 to 10 minutes, the peptide is natural It can be uniformly mixed with the biomaterial, thereby producing a natural biomaterial that physically includes a peptide having an anti-inflammatory effect.

In the preparation method of the present invention, in the "mixing of the first reaction solution and the second reaction solution" of step iii), the physical properties of the hydrogel for inflammation inhibitory peptide delivery can be adjusted according to the mixing ratio. Specifically, assuming that the "compound having a first chemical functional group" and the "compound having a second chemical functional group" are prepared by including the same wt% concentration in each reaction liquid, the first reaction liquid and the The second reaction solution is preferably mixed in a molar ratio of 1: 0.1 to 1:10, but is not limited thereto. More specifically, the first reaction liquid and the second reaction liquid are more preferably mixed at a molar ratio of 1: 0.5 to 1: 5, and most preferably at a molar ratio of 1: 0.5 to 1: 2. Do. In the case where the first reaction solution and the second reaction solution are mixed at a lower ratio than the above ratio, the hydrogel is not formed significantly and thus cannot effectively contain TAP2, which is not effective as a drug carrier and is higher than the above ratio. When mixed with, the viscosity of the hydrogel prepared by the high crosslinking degree increases and the mechanical strength becomes stronger, so that TAP2 cannot be significantly introduced and / or released, and thus cannot show a significant effect as a drug carrier.

Hydrogels of natural biological substances chemically containing inflammation inhibitory peptides

The present invention also provides a method for preparing a hydrogel for inflammation inhibitory peptide delivery, comprising the following steps i) to iii):

i) preparing a first reaction solution comprising a natural biomaterial to which an inflammation inhibitory peptide and a compound having a first chemical functional group are bound;

ii) preparing a second reaction solution including a natural biomaterial to which an inflammation inhibitory peptide and a compound having a second chemical functional group are bound; And

iii) mixing the first reaction solution prepared in step i) and the second reaction solution prepared in step ii).

The present invention also provides a hydrogel for inflammation inhibitory peptide delivery prepared according to the above method.

In the manufacturing method of the present invention, the first chemical functional group and / or the second chemical functional group have been described in the "hydrogel of a natural biological substance physically containing an inflammation inhibitory peptide", and thus, the overlapping descriptions thereof are repeated. Will be omitted.

In the manufacturing method of the present invention, the natural biomaterial has been described in the "hydrogel of a natural biomaterial physically including an inflammation inhibitory peptide", and thus, redundant description will be omitted.

In the present invention, in preparing a hydrogel in which an inflammation inhibitory peptide is chemically bound to a natural biomaterial, when the first chemical functional group is used as tetrazine, as a first chemical functional compound-containing compound, methyltetrazine-PEG4-amine It is more preferable to use (methyltetrazine-PEG4-amine), and the specific reaction scheme is as shown in the following [Scheme 1]:

Scheme 1

.

.

In the present invention, in preparing a hydrogel in which an inflammation inhibitory peptide is chemically bonded to a natural biomaterial, when the second chemical functional group is used as a cyclooctene, as a second chemical functional compound-containing compound, a trans-cyclooctene-amine ( It is more preferable to use trans-cyclooctene-amine, and the specific reaction scheme is as shown in [Scheme 2]:

Scheme 2

.

.

In the production method of the present invention, it can be chemically included an inflammation inhibitory peptide, the inflammation inhibitory peptide (for example TAP2 peptide) according to the present invention is a covalent bond to a natural biological material (for example, hyaluronic acid) Is applied in such a way that it can be directly chemically bonded to and transported to the site of action and its bioavailability can be increased, and / or physically encapsulated in a chemically modified natural biomaterial (eg hyaluronic acid) The modified mechanism of the hyaluronic acid means that the release mechanism of the drug is controlled.

In the preparation method of the present invention, since the inflammation inhibitory peptides have been described in the "hydrogels of natural biological substances physically including the inflammation inhibitory peptides," duplicated descriptions will be omitted.

In the preparation method of the present invention, the condensation and condensing agents are described in the "hydrogel of a natural biological material physically containing an inflammation inhibitory peptide", and thus redundant description will be omitted.

In the manufacturing method of the present invention, "mixing the first reaction liquid and the second reaction liquid" in the step iii) may be understood as a step for inducing chemical bonding of the first chemical functional group and the second chemical functional group. . Such chemical bonding can be achieved within a short time, in particular, within a few seconds, and has the advantage that gelation is possible in a short time through the mixing of the first reaction liquid and the second reaction liquid.

In preparing the hydrogel in the present invention, when using a combination of (tetrazine, cyclooctene) as a combination of the first chemical group and the second chemical group, the specific reaction scheme is as shown in [Scheme 3]:

Scheme 3

.

.

Application of Natural Biomaterials Containing Peptides with Inhibitory Effects

In addition, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, including the hydrogel for delivering an inflammation inhibitory peptide.

In the pharmaceutical composition of the present invention, the inflammatory disease is insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental form uveitis, Hashimoto thyroiditis, primary mucus Edema, thyroid addiction, pernicious anemia, autoimmune atrophy gastritis, Addison's disease, early menopause, male infertility, childhood diabetes, goodpasture syndrome, common swelling, ileal swelling, sympathetic ophthalmopathy, uveitis, autoimmune hemolytic anemia, idiopathic One selected from the group consisting of leukopenia, primary cholangiovascular sclerosis, chronic active hepatitis Hbs-ve, latent cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis / dermatitis, discoid LE and systemic lupus erythematosus It is preferable that it is above.

In the pharmaceutical composition of the present invention, the pharmaceutical composition is preferably an injection formulation, but is not limited thereto, and may be formulated and administered in the following oral or parenteral dosage forms.

Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, etc. These formulations may contain, in addition to the active ingredients, diluents (e.g., lactose, dexter). Rose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols. Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and optionally such as starch, agar, alginic acid or its sodium salt. Disintegrant or boiling mixtures and / or absorbents, colorants, flavors, and sweeteners.

The pharmaceutical composition of the present invention may be administered parenterally, wherein the parenteral administration is by a method of injecting subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. In this case, in order to formulate into a parenteral formulation, a hydrogel including an inflammation inhibitory peptide may be mixed with water with a stabilizer or buffer to prepare a solution or suspension, which may be prepared in an ampule or vial unit dosage form. The composition may contain sterile and / or preservatives, stabilizers, hydrating or emulsifying accelerators, auxiliaries such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, and conventional methods of mixing, granulating It may be formulated according to the formulation or coating method.

In addition, the dosage of the pharmaceutical composition of the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and based on an adult patient having a weight of 60 kg, Generally, it is 0.001 to 1,000 mg / day, preferably 0.01 to 500 mg / day, and may be dividedly administered once a day to several times at regular time intervals according to the judgment of a doctor or a pharmacist.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the following Examples and Experimental Examples.

EXAMPLE

Example 1 Preparation of Hyaluronic Acid Hydrogel Comprising Anti-Inflammatory Peptides Through Physical Bonding

(1) 100 mg of hyaluronic acid (HA) (molecular weight: 1,000, 000 Da) was added to 10 ml of tertiary distilled water, followed by stirring at 25 ° C. for 24 hours to prepare a hyaluronic acid solution. The prepared hyaluronic acid solution was dispensed 5 ml each.

(2) To the hyaluronic acid solution dispensed in step (1), condensate 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholinium chloride (4 8.3 mg of (4, 6-Dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholinium chloride; DMTMM) and 9.93 mg of methyltetrazine-PEG4-amine (TET) It was added, and stirred at 25 ° C. for 24 hours to react. Thereafter, the solution was concentrated by dialysis for 72 hours, and then lyophilized at -80 ° C to prepare HA-TET in which TET was introduced into hyaluronic acid.

(3) 8.3 mg of DMTMM and 7.7 mg of trans-cyclooctene-amine (TCO) were added to the hyaluronic acid solution dispensed in step (1), followed by stirring at 25 ° C. for 24 hours. I was. Then, the solution was concentrated by dialysis for 72 hours, and then lyophilized at -80 ° C to prepare HA-TCO in which TCO was introduced into hyaluronic acid.

(4) HA-TET prepared in step (2) and HA-TCO prepared in step (3) were each dissolved in 2% physiological saline to prepare a 5 ml solution, and the amino acid sequence of SEQ ID NO: 2 was prepared in each solution. Eggplant was added 0.207 mg anti-inflammatory peptide TAP2. After stirring for 1 hour, each stirred mixture was mixed through a dual syringe to finally obtain a hyaluronic acid hydrogel solution (Cx-HA + TAP2) containing physically an anti-inflammatory peptide.

Example 2 Preparation of Hyaluronic Acid Hydrogel Comprising Anti-Inflammatory Peptides Through Chemical Bonding

 (1) 100 mg of hyaluronic acid (HA) (molecular weight: 1,000, 000 Da) was added to 10 ml of tertiary distilled water, followed by stirring at 25 ° C. for 24 hours to prepare a hyaluronic acid solution. The prepared hyaluronic acid solution was dispensed 5 ml each.

(2) To the hyaluronic acid solution dispensed in step (1), 0.066 mg of DMTMM as a condensate was added. Then, this was slowly added dropwise to 5 ml of tertiary distilled water containing 0.207 mg of TAP2 having the amino acid sequence of SEQ ID NO: 2, and stirred at 25 ° C. for 24 hours. After stirring, the solution was concentrated by dialysis for 72 hours and then lyophilized at -80 ° C to prepare hyaluronic acid into which TAP2 was introduced.

(3) The hyaluronic acid to which the TAP2 prepared in step (2) was introduced was introduced into 5 ml of tertiary distilled water so as to have a concentration of 1 wt%, and stirred at 25 ° C. for 24 hours to prepare a hyaluronic acid solution. 8.3 mg of DMTMM and 9.93 mg of TET were added thereto, followed by stirring at 25 ° C. for 24 hours. Then, the solution was concentrated by dialysis for 72 hours, and then lyophilized at -80 ° C to prepare HA-TET (HA-TET-TAP2) into which TAP2 was introduced. The amount of TAP2 introduced into HA-TET was analyzed by elemental analysis (Table 1).

(4) The same method as step (3) was performed, but instead of TET, 7.7 mg of TCO was added to the hyaluronic acid solution to prepare HA-TCO (HA-TCO-TAP2) into which TAP2 was introduced. The amount of TAP2 introduced into HA-TCO was analyzed by elemental analysis (Table 1).

(5) HA-TET-TAP2 and HA-TCO-TAP2 in which TAP2 was introduced were dissolved in 2% physiological saline, respectively, and mixed through a dual syringe to introduce TAP2 hyaluronic acid hydrogel solution (Cx-HA-TAP2). Was prepared final.

sample C (%) H (%) N (%) C / N molar ratio Degree of substitution (%) HA 34.7 5.2 2.9 14.0 - HA-TCO 45.1 6.2 4.6 9.7 24.9 HA-TET 40.2 6.0 5.4 7.4 20.7 HA-TCO-TAP2 40.2 6.3 5.0 8.0 12.9 HA-TET-TAP2 45.1 6.2 5.2 9.2 10.8

Example 3: Preparation of CMC and CMC-hydrogel comprising Anti-Inflammatory Peptides Through Chemical Bonding

(1) CMC-TET (CMC-TET-TAP2) and CMC-TCO in which TAP2 was introduced as a physical bond by using carboxymethyl cellulose (CMC), followed by the same method as in [Example 1]. (CMC-TCO-TAP2) was prepared respectively.

(2) CMC-TET-TAP2 and CMC-TCO-TAP2 prepared in step (1), respectively, dissolved in 2% physiological saline, and mixed through a dual syringe CMC-hydrogel solution (Cx- CMC-TAP2) was finally prepared. Similarly, the amount of introduction was analyzed through elemental analysis.

sample C (%) H (%) N (%) C / N molar ratio Degree of substitution (%) CMC 43.6 5.45 - - - CMC-TCO-TAP2 20.2 5.3 3.2 8.0 9.9 CMC-TET-TAP2 25.1 6.2 5.2 9.2 11.7

Example 4 Preparation of AGNTs and AGNT-hydrogels Containing Anti-Inflammatory Peptides Through Chemical Bonding

(1) Using alginate (AGNT) and proceeding in the same manner as in [Example 1], AGNT-TET (AGNT-TET-TAP2) and AGNT-TCO (AGNT-) in which TAP2 was introduced as a physical bond; TCO-TAP2) was prepared respectively.

(2) AGNT-TET-TAP2 and AGNT-TCO-TAP2 prepared in step (1), respectively, were dissolved in 2% physiological saline, and mixed through a dual syringe AGNT-hydrogel solution (Cx- AGNT-TAP2) was final prepared. Similarly, the amount of introduction was analyzed through elemental analysis.

sample C (%) H (%) N (%) C / N molar ratio Degree of substitution (%) AGNT 441.1 4.0 - - - AGNT-TCO-TAP2 18.2 6.3 8.2 8.0 9.9 AGNT-TET-TAP2 22.1 7.2 4.2 9.2 11.7

[Comparative Example]

Comparative Example 1: Preparation of TAP2 Repeated Dosing Solution

A solution of TAP2 dissolved in physiological saline at a concentration of 5 μg / uL was prepared.

Comparative Example 2: Preparation of TAP2 Incorporated with HA

In order to use the hyaluronic acid carrier used according to the prior art as a comparative example, the TAP2 peptide was simply mixed with hyaluronic acid (HA) without introducing a crosslinking agent of TCO or TET used in the present invention.

(1) 100 mg of hyaluronic acid (HA) (molecular weight: 1,000, 000 Da) was added to 5 ml of tertiary distilled water, followed by stirring at 25 ° C. for 24 hours to prepare a 2 wt% hyaluronic acid solution. The prepared hyaluronic acid solution was dispensed 5 ml each.

(2) 0.207 mg of the anti-inflammatory peptide TAP2 having the amino acid sequence of SEQ ID NO: 2 was added to the hyaluronic acid solution prepared in step (1). Stirring for 1 hour yielded an uncrosslinked hyaluronic acid hydrogel solution (HA + TAP2) that physically contained anti-inflammatory peptides.

Experimental Example

Experimental Example 1: Evaluation of the viscosity according to whether or not the chemical functional group

In order to compare the mechanical properties of the biomaterial hydrogel for peptide delivery prepared in the present invention, the biomaterial according to whether the chemical functional group 1 or the chemical functional group 2 is included by the method of the following steps (1) to (4) The viscosity of the hydrogel was checked.

(1) Preparation of biological material containing chemical functional group 1: HA-TCO prepared in step (3) of [Example 1] was prepared by dissolving it in distilled water at a concentration of 2 wt%. In addition, CMC-TCO and ALGN-TCO were prepared using the same method, and then dissolved in distilled water at a concentration of 2 wt%, respectively.

(2) Preparation of biological material containing chemical functional group 2: HA-TET prepared in step (2) of [Example 1] was prepared by dissolving it in distilled water at a concentration of 2 wt%. In addition, CMC-TET and ALGN-TET were prepared using the same method, and then dissolved in distilled water at a concentration of 2 wt%, respectively.

(3) Formation of the crosslinked hydrogel: The HA-TCO solution prepared in step (1) and the HA-TET solution prepared in step (2) have a molar ratio of 1: 1 as described in Table 4 below. Each was mixed to induce the formation of crosslinked hydrogel (Cx-HA). The same method was used to mix CMC-TCO and CMC-TET and ALGN-TCO and ALGN-TET were mixed to induce the formation of Cx-CMC and Cx-AGLN, respectively.

(4) Viscosity Measurement: The viscosity of Cx-HA, Cx-CMC and Cx-AGLN prepared in step (3) was measured using a rheometer.

As a result, as shown in FIG. 2, the viscosity of the natural biomaterial hydrogel into which the chemical functional group was introduced was confirmed to increase the mechanical rigidity. In addition, as the introduction content of the chemical functional group increased as shown in Figure 3, it was confirmed that the viscosity of the prepared hydrogel also increased. Through this, it was confirmed that the biomaterial hydrogel of the present invention can control the mechanical rigidity by controlling the introduction content of the chemical functional group.

cxHA 500 cxHA750 HA (without crosslinking agent) DMTMN 15.7 mg (0.059 mmol) 23.7 mg
(0.09 mmol) 0.0 mg TCO 13.8 mg (0.050 mmol) 20.8 mg
(0.075 mmol) 0.0 mg TET 18.5 mg (0.050 mmol) 27.7 mg
(0.075 mmol) 0.0 mg

Experimental Example 2 Evaluation of Inflammatory Inhibitory Effect and Cell Growth Rate in Inflammatory Cells and Synovial Cells

<2-1> Confirmation of Inflammation-related Cell Proliferation Inhibitory Effect

In order to determine whether an injection preparation containing an anti-inflammatory peptide prepared in the present invention can have an effect as a carrier showing anti-inflammatory activity, first it was to determine whether it can affect inflammatory cell proliferation in vitro.

First, the cells were cultured. Synovial cells, SW 982 cells were dispensed into 24-transwell plates at a density of 3 × 10 ⁴ cells / well. Leibovitz 'L15 medium containing 10% FBS and 1% penicillin-streptomycin (PS) was added for cultivation and cultured in a culture environment without a supply of CO ₂ . RAW 264.7 cells were cultured as inflammatory cells. RAW 264.7 cells were aliquoted into 24-transwell plates at 1 × 10 ⁴ cells / well density, then added DMEM medium containing 10% FBS and 1% PS and incubated in a humidified environment of 5% CO ₂ .

Then, TAP2 carrier solution was prepared. As an experimental group, 2% (w / v) of Cx-HA + TAP2 chemically containing TAP2 prepared in [Example 1] and Cx-HA-TAP2 chemically containing TAP2 prepared in [Example 2], respectively. ) Was dissolved in physiological saline, and injected into a dual syringe to prepare. In addition, as a control, a TAP2 repeated dose solution prepared in [Comparative Example 1] and a solution containing HA + TAP2 prepared in [Comparative Example 2] were prepared. Prepared experimental or control groups were treated with cultured RAW 264.7 cells and SW 982 cells, respectively. After treatment with TAP2 for 1 day, 4 days and 7 days, the survival rate of each cell was confirmed by Alamar blue analysis.

As a result, as shown in FIG. 4, it was confirmed that the SW982 cells, which are synovial cells, showed cell proliferation with time in all experimental groups and control groups. Through this, it was confirmed that TAP2 and HA did not significantly affect the growth of synovial cells, which are normal cells in the joint (FIG. 4B). In contrast, in the case of inflammatory cells, RAW 264.7 cells, the growth rate of cells in the experimental group treated with Cx-HA + TAP2 (including physical) or Cx-HA-TAP2 (including chemical) together with the control group treated with HA with TAP2 was introduced. It was found to show a significantly reduced level. Through this, it was confirmed that the anti-inflammatory effect of HA itself showed the effect of inhibiting the proliferation of inflammatory cells (Fig. 4a).

<2-2> Confirmation of TNF-a Expression Level Changes in Inflammatory Cells

In order to confirm whether the hydrogel complex prepared in the present invention can have an effect as a carrier showing anti-inflammatory activity, the expression level of TNF-a expressed in inflammatory cells was confirmed.

Specifically, RAW264.7 cells, which are inflammatory cells, were cultured in the same manner as in Example <2-1>, and TAP2 transporters were respectively treated. After 1, 4 and 7 days of TAP2 treatment, cell cultures were obtained and the concentration of TNF-a in the cultures was measured by ELISA method.

As a result, as shown in FIG. 5, it was confirmed that TNF-a expression was suppressed in all the control and experimental groups treated with the TAP2-containing sample as compared to the TAP2 untreated control. In particular, in the case of Cx-HA containing TAP2 physically or chemically, it was confirmed that the TAP2 is continuously released from the hydrogel as a biological material, thereby gradually decreasing the expression level of TNF-a. It was confirmed that the prepared hydrogel complex acts as a carrier of the anti-inflammatory peptide and can effectively inhibit inflammation.

Experimental Example 3: Histological Evaluation of Inflammation Effect of Joint Tissues by Staining

In order to confirm whether the injection preparation containing the anti-inflammatory peptide prepared in the present invention can have an effect as a carrier showing anti-inflammatory activity, first confirmed the inhibitory effect on the cells at the in vivo stage.

Specifically, Cx-HA-TAP2 chemically containing TAP2 prepared in Example 2 as an experimental group was dissolved in physiological saline at 2% (w / v), respectively, and then injected into a dual syringe to be administered in hydrogel form. Ready to do it. As an animal model, the Lewisette was prepared by inducing rheumatoid arthritis by the CIA method. Then, 100 μl of Cx-HA-TAP2 was injected into the kneelet of Lewis let, and after 1 week, 3 weeks, and 6 weeks, the joints were removed. As a control, the TAP2 repeat solution prepared in [Comparative Example 1] was prepared, injected into the rheumatoid arthritis model in the same manner, and the knee joints were extracted.

The extracted tissue was fixed in 10% formalin for 3 days, and then made into paraffin blocks. This was cut to 8μm thickness, fixed on slides and subjected to H & E staining, safranin-o staining and TNF-α fluorescence staining for histological evaluation.

As a result, the nucleus and cytoplasm of the cells were stained and observed through H & E staining (FIG. 6), and glycosaminoglycans of cartilage were stained by safranin-o staining to confirm the function and regeneration of cartilage (FIG. 7). . In addition, the degree of inflammatory response was confirmed through TNF-α fluorescence staining (FIG. 8). Through this, both hyaluronic acid hydrogel containing TAP2 and hyaluronic acid hydrogel in which TAP2 is chemically introduced are highly effective in the treatment of inflammatory disease rheumatoid arthritis by reducing the inflammatory response in the joint and restoring the function of cartilage. Confirmed. In other words, it was confirmed that the hydrogel of the injection type prepared as a TAP2 transporter can significantly exhibit the anti-inflammatory effect of TAP2.

Experimental Example 4 Observation of Feet and Soles of Letts for Evaluating Inflammation

Physiologically, 2% (w / v) of Cx-HA + TAP2 physically containing TAP2 prepared in Example 1 and Cx-HA-TAP2 chemically containing TAP2 prepared in Example 2, respectively. After dissolving in saline, it was prepared to be administered in the form of a hydrogel by injection into a dual syringe. As an animal model, the Lewisette was prepared by inducing rheumatoid arthritis by the CIA method. Then, 100 μl of Cx-HA-TAP2 was injected into the Lewisett's knee joint, followed by 1, 3, and 6 weeks to photograph the sides and the soles of the experimental animal feet to determine the degree of swelling due to inflammation. Confirmed.

As a result, as shown in Figure 9 it was confirmed that the swelling also decreases as the inflammation level decreases over time in the experimental group administered a hydrogel containing TAP2 (Fig. 9). Compared with the control group of the comparative example in which the TAP2 repeat dose solution was injected, it was confirmed that the swelling reduction effect appeared in the experimental group injected with TAP2 in the form of hydrogel.

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Inflammatory peptide-loaded injectable hydrogel by physical or          chemical loading, and method of manufacturing and application <130> 1063848 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> TAP1 <400> 1 Ala Ser Ala Asn Lys Asn Leu Leu Phe Lys Ile Arg Tyr Ser Thr Ala   1 5 10 15 Arg Gly Gly Ser              20 <210> 2 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> TAP2 <400> 2 Ala Met Ala Leu Asp Cys Phe Arg Trp Gly Trp Arg Met Trp Cys Ser   1 5 10 15 Ser gly         <210> 3 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> TAP3 <400> 3 Ala Met Ala Tyr Glu Ile Arg Cys Trp Trp Arg Trp Cys Tyr Thr Ser   1 5 10 15 Ser gly        

Claims (12)

i) preparing a first reaction solution in which a natural biomaterial to which a compound having a first chemical functional group is bound and an inflammation inhibitory peptide are mixed;
ii) preparing a second reaction solution in which a natural biological material combined with a compound having a second chemical functional group and an inflammation inhibitory peptide are mixed; And
iii) mixing the first reaction solution prepared in step i) and the second reaction solution prepared in step ii);
Method for preparing a hydrogel for inflammation inhibitory peptide delivery.
i) preparing a first reaction solution comprising a natural biomaterial to which an inflammation inhibitory peptide and a compound having a first chemical functional group are bound;
ii) preparing a second reaction solution including a natural biomaterial to which an inflammation inhibitory peptide and a compound having a second chemical functional group are bound; And
iii) mixing the first reaction solution prepared in step i) and the second reaction solution prepared in step ii);
Method for preparing a hydrogel for inflammation inhibitory peptide delivery.
The method of claim 1 or 2, wherein the first chemical functional group is any one selected from the group consisting of tetrazine, alkane group, epoxy group, and acroyl group. .
According to claim 1 or claim 2, wherein the second chemical functional group is characterized in that any one selected from the group consisting of cyclooctene, azide group, thiol group and amine group, the production of hydrogel for the delivery of inflammation inhibitory peptides Way.
The method of claim 1 or 2, wherein the natural biomaterial is any one selected from the group consisting of hyaluronic acid, carboxymethyl cellulose (CMC) and alginate (AGNT), inflammation inhibition Method for preparing a hydrogel for peptide delivery.
According to claim 1 or 2, wherein the inflammation inhibitory peptide is characterized in that consisting of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3, the preparation of a hydrogel for delivery of inflammation inhibitory peptides. Way.
According to claim 1 or 2, wherein the binding is characterized in that the amide reaction induced by the condensing agent, characterized in that the method for producing a hydrogel for inhibiting peptide delivery.
The method of claim 7, wherein the condensing agent is 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (4- (4,6-Dimethoxy -1,3,5-triazin-2-yl) -4-methylmorpholinium chloride) (DMTMM), a method for producing a hydrogel for inflammation inhibitory peptide delivery.
A hydrogel for inflammation inhibitory peptide delivery, prepared by the method of claim 1 or 2.
A pharmaceutical composition for preventing or treating an inflammatory disease, comprising the hydrogel of claim 9.
The method of claim 10, wherein the inflammatory disease is insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental form uveitis, Hashimoto thyroiditis, primary myxedema, thyroid gland Addiction, pernicious anemia, autoimmune atrophy gastritis, Addison's disease, premature menopause, male infertility, childhood diabetes, Goodpasture syndrome, normal pemphigus, leiomyelitis, sympathetic ophthalmitis, lens uveitis, autoimmune hemolytic anemia, idiopathic leukocyte reduction, At least one selected from the group consisting of primary cholangiocytosis, chronic active hepatitis Hbs-ve, latent cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis / skin myositis, discoid LE and systemic lupus erythematosus Characterized in that for the prevention or treatment of inflammatory diseases.
The pharmaceutical composition for preventing or treating inflammatory disease according to claim 10, wherein the pharmaceutical composition is an injection formulation.

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