KR102600511B1 - Gallol-conjugated aldehyde-modified hyaluronic acid and use thereof - Google Patents

Abstract

The present application relates to aldehyde substituted hyaluronic acid derivatives modified with gallol groups and their uses.
The aldehyde-substituted hyaluronic acid derivative modified with a gallol group according to an embodiment of the present application significantly reduces the extrusion force required for injection compared to the existing gallol group-modified hyaluronic acid-based hydrogel, thereby lowering the internal pressure within the fascia during intramuscular injection. Since the change is small, the pain caused by intramuscular injection can be minimized, so the pain and stress caused by repeated drug administration in elderly patients can be minimized and the quality of life can be improved. Additionally, the delivery of extracellular matrix derived from various tissues is possible, allowing drug delivery. , It can be applied to tissue suturing and therapeutic agents.

Description

Aldehyde-substituted hyaluronic acid derivatives modified with gallol group and their uses {GALLOL-CONJUGATED ALDEHYDE-MODIFIED HYALURONIC ACID AND USE THEREOF}

The present application relates to aldehyde substituted hyaluronic acid derivatives modified with gallol groups and their uses.

The size of the global advanced drug delivery system market is expected to grow at an average annual growth rate of more than 6.1%, greatly expanding from $231 billion in 2020 to $310 billion in 2025. By region, North America is expected to show an average annual growth rate of 5.8%, and the Asia-Pacific region is expected to show an average annual growth rate of 6.7% (BBC Research, December 2020).

In this regard, various delivery systems are being developed for efficient delivery of drugs and biomolecules into the body in a local and long-term manner. Among them, unlike implantable delivery systems that require surgical procedures of incision, insertion, and suturing, injectable hydrogels can induce sustained release of the loaded material at the injection site after a relatively simple injection process. Be in the spotlight. However, the existing injectable hydrogels, which are widely used, are injected by applying force to the hydrogel that is already in a gel state to cause shear-thinning and then self-healing, so many people are required for injection. Because it requires effort, there are various problems, such as poor user convenience and difficulty in precisely controlling the injection amount. In addition, injectable hydrogels other than the above methods require mixing cross-linking accelerators, etc. when injecting, which raises concerns about reduced user convenience or side effects due to potential toxicity.

To solve this problem, self-crosslinkable hydrogels, which spontaneously form a gel in situ without the need for a separate crosslinking additive after being injected as a liquid, have recently been developed. For example, a hydrogel that induces self-crosslinking using the high natural oxidation ability of the functional group by modifying the pyrogallol group, which is known to be involved in the rapid self-healing and underwater adhesion of marine organisms such as sea squirts, to biopolymers. It was reported recently. However, even in this case, the viscosity is high due to the interaction of the pyrogallol group itself and countless hydrogen bonds between polymers, so although it is less than the existing self-recovery method after shear fluidization, there is a problem that a relatively high force is still required during injection. . Therefore, due to the high viscosity, there are limitations in that the injected hydrogel is difficult to spread and deliver evenly into the tissue, and there are also problems in that it may cause pain during injection, which may act as a factor that reduces clinical effectiveness.

In addition, most existing injectable hydrogels have the limitation of performing only the role of a simple transporter as they have no separate direct functionality. To solve this problem, tissue-specific microenvironments have recently been implemented to promote tissue regeneration. However, due to the low physical properties of decellularized matrix-based hydrogel, it fails to settle at the injection site and is lost or decomposed in the body in a short period of time. There was a problem in that it did not sufficiently induce the expected treatment effect.

Accordingly, the present inventors have discovered that aldehyde-substituted hyaluronic acid, which reduces interactions between polymers and lowers viscosity, contains a pyrogallol group through a process of substituting an aldehyde group in hyaluronic acid, a natural polymer that has excellent biocompatibility and is widely used for medical purposes. The present invention was completed by developing a novel hyaluronic acid derivative modified with the biomolecule 5-hydroxydopamine.

Korean Patent Publication No. 10-0507545

The present application relates to aldehyde substituted hyaluronic acid derivatives modified with gallol groups and their uses.

However, the problem to be solved by the present application is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

A first aspect of the present disclosure provides an aldehyde substituted hyaluronic acid derivative modified with a gallol group (AH-PG).

The second aspect of the present application provides an AH-PG based hydrogel.

The third aspect of the present application provides an AH-PG/MEM hydrogel loaded with muscle tissue-derived extracellular matrix (MEM) in an AH-PG-based hydrogel.

The fourth aspect of the present application provides a pharmaceutical composition for preventing or treating muscle disease, comprising AH-PG/MEM hydrogel as an active ingredient.

The fifth aspect of the present application provides a method for producing an aldehyde-substituted hyaluronic acid derivative (AH-PG) modified with a gallol group.

The sixth aspect of the present application provides a method for producing aldehyde-substituted hyaluronic acid (AH).

Our aldehyde-substituted hyaluronic acid derivative modified with a gallol group significantly reduces the extrusion force required for injection compared to the existing gallol group-modified hyaluronic acid-based hydrogel, so the change in internal pressure within the fascia during intramuscular injection is small, so it can be administered intramuscularly. Since pain caused by repeated drug administration can be minimized, the pain and stress caused by repeated drug administration in elderly patients can be minimized and the quality of life can be improved. It is also possible to deliver extracellular matrix derived from various tissues, which can be used for drug delivery, tissue suture, and therapeutic agents. It can be applied.

Figure 1a is a diagram showing the process of producing a derivative (AH-PG) obtained by modifying aldehyde group-substituted hyaluronic acid with a pyrogallol group.
Figure 1b is a diagram showing the results of ¹ H-NMR analysis of the synthesized polymer derivative.
Figure 1c is a diagram showing the results of UV-vis analysis of the synthesized polymer derivative.
Figure 2a shows the addition of a chemical oxidizing agent (NaIO ₄ group _: 2% AH-PG + 1.125 mg/ml NaIO ₄ ) or alkaline pH conditions (NaOH group: 2%) to induce oxidative crosslinking of the pyrogallol group. This diagram shows that the AH-PG solution gelates through crosslinking when adjusted to (AH-PG + 0.020 M NaOH).
Figure 2b shows the elastic modulus of the hydrogel formed under two conditions (based on final concentration, NaIO ₄ group _: 2% AH-PG + 1.125 mg/ml NaIO ₄ , NaOH group: 2% AH-PG + 0.020 M NaOH). This is a drawing showing the measurement results.
Figure 2c shows the loss factor (based on final concentration, NaIO ₄ group _: 2% AH-PG + 1.125 mg/ml NaIO ₄ , NaOH group: 2% AH-PG + 0.020 M NaOH) of the hydrogel formed. This is a diagram showing the results of measuring tan delta, G'/G'').
Figure 3 is a diagram confirming the self-crosslinking ability due to natural oxidation of the pyrogallol group in the AH-PG group under three conditions.
Figure 4 is a diagram showing the results of UV-vis spectroscopy analysis for the natural oxidation group (0.2% AH-PG solution without HRP) and the body oxidation environment simulation group (0.2% AH-PG solution with 0.6 U/ml of HRP) .
Figure 5a shows the elastic modulus (elasticity) of the hydrogel formed under natural oxidation conditions (0 U/ml HRP) and in vivo simulated oxidation conditions through the addition of various concentrations of HRP (final concentration 0.06, 0.6, 6, 60 U/ml). This is a diagram comparing modulus.
Figure 5b shows the loss factor (tan) of the hydrogel formed under natural oxidation conditions (0 U/ml HRP) and in vivo simulated oxidation conditions through the addition of various concentrations of HRP (final concentration 0.06, 0.6, 6, 60 U/ml). This is a diagram comparing delta, G'/G'').
Figure 6a is a diagram in which cells were cultured in AH-PG hydrogel to evaluate the cytotoxicity of AH-PG hydrogel, and the survival rate during the culture period was analyzed through Live/Dead staining.
Figure 6b is a diagram showing the survival rate during the culture period when cells were cultured in AH-PG hydrogel to evaluate the cytotoxicity of AH-PG hydrogel.
Figure 7a is a diagram showing the results of measuring the viscosity of a 2% concentration solution of each derivative (AH-PG and HA-PG) compared to PBS using a rheometer.
Figure 7b shows the results of measuring the extrusion force of each derivative (AH-PG and HA-PG) solution at 2% concentration and PBS for each injection needle (27G, 29G) using a universal testing machine (UTM). This is the drawing shown.
Figure 7c shows the dynamic glide force (DGF) of each derivative (AH-PG and HA-PG) solution at 2% concentration and the injection needle (27G, 29G) in PBS, which maintains the movement of the plunger to eject the contents of the syringe. This is a diagram showing the results of measuring the force required to test using a UTM (universal testing machine).
Figure 8a is a diagram showing the histological form of muscle tissue before the decellularization process (Before) and muscle tissue after the decellularization process (After).
Figure 8b is a diagram showing the results of quantitative comparison of the amount of DNA remaining in muscle tissue before the decellularization process (Before) and muscle tissue after the decellularization process (After).
Figure 8c is a diagram showing the results of quantitative comparison of the remaining amount of GAG (glycosaminoglycan) in muscle tissue before the decellularization process (Before) and muscle tissue after the decellularization process (After).
Figure 8d is a diagram showing the results of confirming various types of muscle tissue-specific proteins present in MEM through mass spectrometry-based proteomics analysis. It was confirmed that it was composed of a total of 219 proteins, of which 196 proteins corresponded to skeletal muscle expressed proteins, and 54 of them were confirmed to be composed of skeletal muscle elevated proteins.
Figure 8e is a diagram showing the results of confirming various types of muscle tissue-specific proteins present in MEM through mass spectrometry-based proteomics analysis.
Figure 9a shows the extrusion force under three conditions (PBS, 2% AH-PG + 5 mg/ml MEM, 2% HA-PG + 5 mg/ml MEM solution) using a universal testing machine (UTM). This is a drawing showing the measurement results.
Figure 9b shows the dynamic glide force (DGF) of the plunger to discharge the contents of the syringe under three conditions (PBS, 2% AH-PG + 5 mg/ml MEM, 2% HA-PG + 5 mg/ml MEM solution). This diagram shows the results of measuring the force required to maintain movement using a universal testing machine (UTM).
Figure 10a shows AH-PG hydrogel (using 2% AH-PG+5 mg/ml MEM, 50 μl injection), MEM (using 5 mg/ml MEM solution, 50 μl injection), and HA-PG hydrogel (2% AH-PG+5 mg/ml MEM, 50 μl injection). This figure shows the results of analyzing the differences in MEM body distribution and stability depending on the formulation by applying % HA-PG + 5 mg/ml MEM, 50 μl injection).
Figure 10b is a diagram showing the mechanism of muscle tissue regeneration using AH-PG/MEM hydrogel.
Figure 11a is a diagram showing hydrogels of various formulations extracted after application to the TA muscle of a mouse model of disuse muscle atrophy.
Figure 11b shows each condition [HA-PG (2% HA-PG), AH-PG (2% AH-PG), HA-PG/MEM (2% HA-PG+5 mg/ml MEM), AH- This diagram shows the results confirming the muscle mass enhancement effect in [PG/MEM (2% AH-PG+5 mg/ml MEM)].
Figure 11c is a diagram showing the results of Hematoxylin & Eosin (H&E) staining on TA muscle samples collected after each hydrogel injection to confirm how well the injected hydrogel (black arrow) is fused with the surrounding muscle tissue. .
Figure 11d shows activation and proliferation of muscle stem cells by staining TA muscle samples collected after each hydrogel injection for Pax7, a marker expressed in muscle stem cells, and MyoD, a marker expressed in activated muscle stem cells. This is a drawing showing the results of confirming the degree.
Figure 11e shows the expression of each marker for histological images in which TA muscle samples collected after each hydrogel injection were stained for Pax7, a marker expressed in muscle stem cells, and MyoD, a marker expressed in activated muscle stem cells. This diagram shows the results of quantitatively comparing the degrees.
Figure 12a shows the muscle enhancement effect by injecting MEM-loaded AH-PG hydrogel (AH-PG/MEM group) into an aging sarcopenia model, showing the muscle enhancement effect under each condition [MEM (5 mg/ml MEM) ), AH-PG/MEM (2% AH-PG+5 mg/ml MEM)] This is a diagram showing the results of confirming the muscle tissue extracted after applying each hydrogel to the TA muscle of an aging mouse.
Figure 12b shows the muscle enhancement effect by injecting MEM-loaded AH-PG hydrogel (AH-PG/MEM group) into an aging sarcopenia model, showing the effect of muscle enhancement under each condition [MEM (5 mg/ml MEM) ), AH-PG/MEM (2% AH-PG+5 mg/ml MEM)] This is a diagram showing the results of H&E staining.
Figure 12c shows the muscle enhancement effect by injecting MEM-loaded AH-PG hydrogel (AH-PG/MEM group) into an aging sarcopenia model, showing the effect of muscle enhancement under each condition [MEM (5 mg/ml MEM) ), AH-PG/MEM (2% AH-PG+5 mg/ml MEM)] This is a diagram showing the results of Masson's trichrome (MT) staining.
Figure 12d shows the muscle enhancement effect by injecting MEM-loaded AH-PG hydrogel (AH-PG/MEM group) into an aging sarcopenia model. In each condition [MEM (5 mg/ml MEM) ), AH-PG/MEM (2% AH-PG+5 mg/ml MEM)] This figure shows the results of immunostaining for myosin heavy chain 1E (MYH1E) and laminin, which are muscle-specific markers.
Figure 12e shows the muscle enhancement effect by injecting MEM-loaded AH-PG hydrogel (AH-PG/MEM group) into an aging sarcopenia model, showing the effect of muscle enhancement under each condition [MEM (5 mg/ml MEM). ), AH-PG/MEM (2% AH-PG+5 mg/ml MEM)] This is a diagram showing the results of staining α-smooth muscle actin (α-SMA), a marker related to vascularization.
Figure 12f shows the muscle enhancement effect by injecting MEM-loaded AH-PG hydrogel (AH-PG/MEM group) into an aging sarcopenia model, showing the effect of muscle enhancement under each condition [MEM (5 mg/ml MEM) ), AH-PG/MEM (2% AH-PG+5 mg/ml MEM)] This is a diagram showing the results of staining von Willebrand factor (vWF), a capillary marker.
Figure 12g shows the results of immunostaining for the nerve marker nestin, acetylcholine receptor (AchR), and alpha-bungarotoxin (BTX), which is known to bind to functional AchR, to confirm the distribution and functionality of nerves present in reconstructed muscle tissue. This is a drawing showing .
Figure 13a is a diagram showing the results of weight comparison (weight difference comparison) between the tissue of the leg injected with each hydrogel and the tissue of the opposite leg without any treatment in the model of age-related sarcopenia.
Figure 13b is a diagram showing the regeneration pattern of muscle tissue by quantitatively analyzing the number of nuclei present in the muscle bundle based on histological analysis after injecting each hydrogel in an aging sarcopenia model.
Figure 13c is a diagram showing the results of confirming the effect of increasing myofiber diameter according to each hydrogel injection in an aging sarcopenia model.
Figure 13d is a diagram showing the results of confirming the myofiber diameter distribution of MYH1E-positive muscle bundles, a muscle-specific marker, based on histological analysis after injecting each hydrogel in an aging sarcopenia model.
Figure 13e is a diagram showing the results of analyzing the cross-sectional area (CSA) distribution of MYH1E-positive muscle fascicles, a muscle-specific marker, based on histological analysis after injection of each hydrogel in an aging sarcopenia model. am.
Figure 13f is a diagram showing the results of quantitative analysis of the cross-sectional area of α-SMA positive arterioles, a blood vessel-specific marker, based on histological analysis after injecting each hydrogel in an aging sarcopenia model.
Figure 13g is a diagram showing the results of quantitative analysis of the number of α-SMA positive arterioles, a blood vessel-specific marker, based on histological analysis after injection of each hydrogel in an aging sarcopenia model.
Figure 13h shows the muscle enhancement effect by injecting MEM-loaded AH-PG hydrogel (AH-PG/MEM group) into an aging sarcopenia model, showing the effect of muscle enhancement under each condition [MEM (5 mg/ml MEM). ), AH-PG/MEM (2% AH-PG+5 mg/ml MEM)] This is a diagram showing the results of quantitative evaluation by staining for von Willebrand factor (vWF), a capillary marker.
Figure 13i shows immunostaining for the nerve markers nestin, acetylcholine receptor (AchR), and alpha-bungarotoxin (BTX), which are known to bind to functional AchR, to confirm the distribution and functionality of nerves present in the reconstructed muscle tissue. This diagram shows the quantitative evaluation results.
Figure 14a is a diagram showing the expected chemical structure inside the hydrogels of the AH-PG and AH-PG+MEM groups and the adhesion mechanism to the surface of biological tissue.
Figure 14b is a diagram showing the results of comparing the adhesion strength measured as the hydrogel attached to the tissue falls off as the probe moves in the MEM, HA-PG, AH-PG, and AH-PG+MEM groups.
Figure 14c is a diagram showing the results of quantitative comparison of the maximum force measured in the MEM, HA-PG, AH-PG, and AH-PG+MEM groups.
Figure 14d is a diagram showing the results of quantitative comparison of the amount of work contributed to adhesion by the MEM, HA-PG, AH-PG, and AH-PG+MEM groups.

Below, with reference to the attached drawings, embodiments of the present application will be described in detail so that those skilled in the art can easily implement them. However, the present application may be implemented in various different forms and is not limited to the embodiments described herein. In order to clearly explain the present application in the drawings, parts that are not related to the description are omitted, and similar reference numerals are assigned to similar parts throughout the specification.

Throughout the specification of the present application, when a member is said to be located “on” another member, this includes not only the case where the member is in contact with the other member, but also the case where another member exists between the two members.

Throughout the specification of the present application, when a part “includes” a certain component, this means that it may further include other components rather than excluding other components unless specifically stated to the contrary. As used throughout the specification, the terms “about,” “substantially,” and the like are used to mean at or close to a numerical value when manufacturing and material tolerances inherent in the stated meaning are presented, and are used to convey the understanding of the present application. Precise or absolute figures are used to assist in preventing unscrupulous infringers from taking unfair advantage of stated disclosures. The term “step of” or “step of” as used throughout the specification does not mean “step for.”

Throughout this specification, the term “combination(s) thereof” included in the Markushi format expression refers to a mixture or combination of one or more selected from the group consisting of the components described in the Markushi format expression, It means containing one or more selected from the group consisting of the above components.

Throughout this specification, references to “A and/or B” mean “A or B, or A and B.”

Hereinafter, implementation examples and examples of the present application will be described in detail with reference to the attached drawings. However, the present application may not be limited to these implementations, examples, and drawings.

A first aspect of the present disclosure provides an aldehyde substituted hyaluronic acid derivative modified with a gallol group (AH-PG).

In one embodiment of the present application, the gallol group of the present application may be a pyrogallol group, but is not limited thereto.

In one embodiment of the present application, the AH-PG derivative of the present application can induce spontaneous crosslinking.

In general, the viscosity increases due to interactions between chains due to hydrogen bonds formed by the hydroxyl groups of the sugar molecules that make up the polysaccharide chain. The AH-PG of the present invention increases the viscosity of the sugar in HA during the manufacturing process. Through a ring-opening reaction at the diol portion, the hydroxyl group is replaced with an aldehyde group, thereby reducing the interaction between hydroxyl groups within the sugar chain, increasing viscosity. It is lowered, and this has the effect of significantly improving the injection force.

In one embodiment of the present application, it can be confirmed that the AH-PG derivative of the present application exhibits a low level of viscosity. When injecting into the body, if the viscosity is high, a larger diameter needle must be used, and as the diameter of the needle increases, pain increases. Our AH-PG hydrogel has the advantage of reducing pain because it has a lower viscosity than the prior art and allows the use of injection needles with smaller diameters than before.

Therefore, it can be seen that the AH-PG derivative herein has a structural difference (ring-opening of the diol portion of the sugar in HA) and an effective difference (viscosity is lowered and injection force is significantly improved) compared to the prior art.

The second aspect of the present application provides an AH-PG based hydrogel. Content that overlaps with the first aspect also applies to the hydrogel of the second aspect.

In one embodiment of the present application, the hydrogel can be used for tissue repair, drug delivery, tissue suturing, cell transplantation, wound treatment, tissue regeneration, hemostatic agents, and fillers.

The third aspect of the present application provides an AH-PG/MEM hydrogel loaded with muscle tissue-derived extracellular matrix (MEM) in an AH-PG-based hydrogel. The overlapping contents of the first and second aspects also apply to the AH-PG/MEM hydrogel of the third aspect.

The term "MEM" used throughout the specification herein refers to muscle tissue-derived extracellular matrix (MEM).

The fourth aspect of the present application provides a pharmaceutical composition for preventing or treating muscle disease, comprising AH-PG/MEM hydrogel as an active ingredient. Contents that overlap with the first to third aspects also apply to the pharmaceutical composition of the fourth aspect.

In one embodiment of the present application, the muscle disease is one or more selected from the group consisting of muscular dystrophy, age-related sarcopenia, traumatic muscle injury, muscular dystrophy, myasthenia gravis, inflammatory myopathy, polymyositis, and myotonia, but is not limited thereto. .

In one embodiment of the present application, the pharmaceutical composition for preventing or treating muscle disease may include other pharmaceutical compositions in addition to the AH-PG/MEM hydrogel.

In one embodiment of the present application, the pharmaceutical composition is administered in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. It may be formulated and used, but may not be limited thereto.

In one embodiment of the present application, when formulating the pharmaceutical composition, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, or surfactants, but is limited thereto. It may not work.

In one embodiment of the present application, solid preparations for oral administration include tablets, pills, powders, granules, or capsules, and such solid preparations include dead cells of the above-mentioned strain with at least one excipient, for example, It can be prepared by mixing starch, calcium carbonate, sucrose, lactose, or gelatin. Additionally, for example, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used, but may not be limited thereto.

In one embodiment of the present application, liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, Sweeteners, fragrances, preservatives, etc. may be included, but may not be limited thereto.

In one embodiment of the present application, preparations for parenteral administration may include, but are not limited to, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. For example, the non-aqueous solvent or suspension may be propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc., but may not be limited thereto. For example, the suppository may include witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc., but may not be limited thereto.

The pharmaceutical composition according to one embodiment of the present application may be a pharmaceutical composition or a quasi-drug composition.

The term "quasi-drugs" used throughout the specification herein refers to products with a milder effect than pharmaceuticals among products used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases in humans or animals. For example, the Pharmaceutical Affairs Act According to the Act, quasi-drugs exclude products used for medicinal purposes and include products used to treat or prevent diseases in humans and animals, and products that have a mild or no direct effect on the human body.

The quasi-drug composition of the present application consists of body cleanser, disinfectant cleaner, detergent, kitchen cleaner, cleaning cleaner, toothpaste, mouthwash, wet tissue, detergent, soap, hand wash, hair cleaner, hair softener, humidifier filler, mask, ointment, and filter filler. It can be manufactured in a formulation selected from the group, but is not limited thereto.

In one embodiment of the present application, the pharmaceutical composition may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" herein refers to the treatment of diseases with a reasonable benefit/risk ratio applicable to medical treatment or prevention. Or it means an amount sufficient for prevention, and the effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, gender, the patient's sensitivity to the drug, the administration time of the composition of the present invention used, and the administration route. and excretion rate can be determined based on factors including treatment duration, drugs used in combination or concurrently with the compositions of the invention used, and other factors well known in the medical field. The pharmaceutical composition of the present application can be administered alone or in combination with ingredients known to exhibit therapeutic effects on known intestinal diseases. It is important to consider all of the above factors and administer the amount that will achieve the maximum effect with the minimum amount without side effects.

In one embodiment of the present application, the dosage of the pharmaceutical composition can be determined by a person skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the patient's age, weight, gender, antecedent history, or the type of substance used as an active ingredient. there is. For example, the pharmaceutical composition of the present invention can be administered at about 0.1 ng to about 1,000 mg/kg, preferably 1 ng to about 100 mg/kg per adult, and the frequency of administration of the composition of the present invention is specifically limited thereto. However, it can be administered once a day, or the dose can be divided and administered several times. The above dosage or frequency of administration does not limit the scope of the present application in any way.

The pharmaceutical composition herein is not particularly limited, but depending on the purpose, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. It can be administered through the route. However, when administered orally, it can also be administered in an unformulated form, and since the Lactobacillus paracasei LM1014 strain may be denatured or decomposed by stomach acid, the oral composition may be coated with the active agent or protected from decomposition in the stomach. It can also be administered orally in a protective form or in the form of an oral patch. Additionally, the composition can be administered by any device that allows the active substance to move to target cells.

The fifth aspect of the present application provides a method for producing an aldehyde-substituted hyaluronic acid derivative (AH-PG) modified with a gallol group. Contents that overlap with the first to fourth aspects also apply to the manufacturing method of the fifth aspect.

In one embodiment of the present application, the present application includes the steps of (a) reacting hyaluronic acid (HA) and sodium periodate (NaIO ₄ ); (b) treatment with ethylene glycol, dialysis, and freeze-drying; And (c) subjecting the aldehyde group-substituted hyaluronic acid (AH), EDC, NHS, and 5-hydroxydopamine produced through steps (a) and (b) to an EDC/NHS reaction. A method for producing an aldehyde group-substituted pyrogallol-modified hyaluronic acid derivative (AH-PG) is provided.

The sixth aspect of the present application provides a method for producing aldehyde-substituted hyaluronic acid (AH). Contents that overlap with the first to fifth aspects also apply to the manufacturing method of the sixth aspect.

In one embodiment of the present application, the present application includes the steps of (a) reacting hyaluronic acid (HA) and sodium periodate (NaIO ₄ ); and (b) treating with ethylene glycol, dialysis, and then freeze-drying; It provides a method for producing aldehyde group-substituted hyaluronic acid (AH) comprising.

Hereinafter, the present invention will be described in more detail through examples of the present application. However, the following examples are merely illustrative to aid understanding of the present application, and the content of the present application is not limited to the following examples.

[Example]

Example 1. AH-PG production

To replace some diols of the hyaluronic acid polymer with aldehyde groups, NaIO ₄ is treated (molar ratio of HA: NaIO ₄ = 1 : 1), and to remove remaining NaIO ₄ , ethylene glycol treatment and dialysis are performed. After going through this process, it was freeze-dried (see Figure 1a). As a result of quantitative confirmation using hydroxylamine hydrochloride and an indicator, it was confirmed that about 45% of the diol groups were substituted with aldehydes (see Figure 1a).

Afterwards, an EDC/NHS chemical reaction was performed to modify the freeze-dried aldehyde group-substituted hyaluronic acid (AH) with a Pyrogallol group (molar ratio of AH: EDC: NHS: 5-hydroxydopamine = 1 : 1.5 : 2). : 5-hydroxyDopamine was chemically introduced into AH using 1) (see Figure 1a).

Through ¹ H-NMR analysis of the synthesized polymer derivative, it was confirmed that HA was substituted with an aldehyde group and simultaneously modified with a pyrogallol group (see Figures 1b and 1c).

Afterwards, through UV-vis spectroscopy, it was confirmed again that hyaluronic acid was modified with a pyrogallol group, and the degree of substitution (DS) of the pyrogallol group to hyaluronic acid was about 10%. This was confirmed (see Figures 1b and 1c).

Hereinafter, aldehyde-substituted hyaluronic acid derivatives modified with a pyrogallol group are referred to as 'AH-PG derivatives'.

Example 2. Qualitative confirmation of pyrogallol group modification in AH-PG derivatives

In order to further confirm the modification of the pyrogallol group in the AH-PG derivative, it was confirmed whether it operates in the same manner as the crosslinking method of the previously reported pyrogallol group-modified-hyaluronic acid derivative-based hydrogel.

As in the conventional pyrogallol group-modified hydrogel, when a chemical oxidizing agent (NaIO ₄ ) is added to induce oxidative crosslinking of the pyrogallol group or when adjusted to alkaline pH conditions (using NaOH), the AH-PG solution Gelation was confirmed through cross-linking (see Figure 2a). Another characteristic of the pyrogallol group-modified hydrogel is that the two cross-linking methods are different, and through this, it was confirmed once again that the pyrogallol group was successfully modified into AH (based on final concentration, NaIO ₄ group: 2% AH-PG) + 1.125 mg/ml NaIO ₄ , NaOH group: 2% AH-PG + 0.020 M NaOH)

The loss factor (tan delta, G'/G'') values related to the elastic modulus and ductility of the hydrogel formed under the above two conditions (NaIO ₄ group, NaOH group) are also similar to those previously reported. It was confirmed that the trend was similar to that of the galol group-modified hydrogel. In addition, to measure the elastic modulus, it was measured in frequency sweep mode (0.1 ~ 10 Hz range) using a rheometer, and the elastic modulus and loss modulus were measured using the storage modulus and loss modulus at 1 Hz. The loss factor was calculated (see Figures 2b and 2c).

Example 3. Confirmation of self-crosslinking ability of AH-PG derivative-based hydrogel

The self-crosslinking ability was confirmed due to natural oxidation of the pyrogallol group in the AH-PG derivative. 2% AH-PG solution (in PBS) can induce gelation by cross-linking between polymers over time without any additional additives [(1) spontaneous oxidation (in vitro) group, see Figure 3], and can be used in the body After injection into the mouse's subcutaneous tissue, the tissue at the injection site was collected from the mouse and confirmed that a gel was stably formed in the body [(3) in situ self-crosslinking (in vivo) group. , see Figure 3]. In this case, a gel with different color and physical properties is formed than when natural oxidation occurs in vitro. Based on this, it was predicted that the in vivo self-crosslinking reaction of AH-PG derivatives induces additional chemical crosslinking in addition to the in vitro natural oxidation reaction. To mimic this in vitro, peroxidase (horseradish peroxidase (HRP used here)), which is thought to play a major role in the oxidative environment in the body, is added to the AH-PG solution to induce gelation, resulting in a product similar to the result of cross-linking in the body. It was confirmed that a gel with good color and physical properties was formed [(2) oxidation using peroxidase (in vitro) group, see Figure 3]. The crosslinking conditions at this time were a final concentration of 2% AH-PG + 6 U/ml HRP.

Example 4. Confirmation of crosslinking mechanism of AH-PG derivative-based hydrogel

To confirm the crosslinking reaction of AH-PG derivatives, UV-vis was performed on the natural oxidation group (0.2% AH-PG solution without HRP) and the group simulating an oxidation environment in the body (0.2% AH-PG solution with 0.6 U/ml of HRP). Spectroscopy analysis was performed.

In both groups, an increase in absorbance was observed in the 280-300 nm range, indicating various oxidation products of the pyrogallol group. In addition, an increase in the absorbance of the peak (330 nm, 420-440 nm) corresponding to purpurogallin formed by the bond between pyrogallols was observed in both groups, indicating that it was mainly formed by purpurogallin rather than biphenol type cross-linking. It was found that cross-linking occurred in the form of purpurogallin. However, looking at the rate and degree of increase in absorbance of the two groups, it is significantly increased in the presence of peroxidase, so it is expected that this increased crosslinking rate and physical properties will be shown even under oxidizing conditions where peroxidase is present in the body. It is predictable. In particular, an increase in the peak at 500-570 nm was observed prominently in the group to which peroxidase was added. This was due to the fact that oxidized pyrogallol, produced in large quantities under the oxidation conditions enhanced by peroxidase, was present in the peroxidase protein. It is thought to be a peak produced by a product bound to a functional group such as an amine group (see Figure 4).

Through this, it is predicted that AH-PG derivatives can effectively crosslink and gel at the injected site through various oxidation reactions and interactions with biological substances such as body proteins when exposed to the body's oxidizing environment after injection in vivo.

Example 5. Comparison of AH-PG hydrogel physical properties according to oxidation conditions

To confirm the physical properties of the AH-PG hydrogel formed under natural oxidation and simulated oxidation conditions in the body, measurements were made in frequency sweep mode (0.1 to 10 Hz range) using a rheometer, and the storage modulus at 1 Hz was measured. The elastic modulus and loss factor were calculated using the (storage modulus) value and the loss modulus.

Comparison of the elastic modulus of hydrogels formed under natural oxidation conditions (0 U/ml HRP) and under simulated oxidation conditions in the body through the addition of various concentrations of HRP (final concentrations of 0.06, 0.6, 6, 60 U/ml). As seen, it was confirmed that a hydrogel with a higher elastic modulus was formed under enhanced oxidation conditions due to the addition of HRP (see Figure 5).

Additionally, when comparing the loss factor (tan delta, G'/G'') values, it was confirmed that the hydrogels formed under simple natural oxidation conditions and in a body-simulating environment with the addition of HRP showed different physical properties. This means that under oxidation conditions in the body, AH-PG derivatives can form hydrogels with physical properties that are distinct from naturally oxidized hydrogels through additional cross-linking promotion reactions in addition to reactions caused by natural oxidation. The concentration of AH-PG derivative was analyzed at the final 2% condition (see Figure 5).

Example 6. Cytotoxicity evaluation of AH-PG hydrogel

To evaluate the cytotoxicity of AH-PG hydrogel, cells were 3D cultured within the hydrogel and the survival rate was analyzed during the culture period.

In the AH-PG hydrogel, crosslinking was induced under conditions simulating the oxidizing environment in the body by adding HRP at two concentrations (0.6 U/ml and 6 U/ml) to the AH-PG solution (final AH-PG concentration 2%). C2C12 cells, a mouse-derived myogenic cell line, were encapsulated at a concentration of 1.0 x 10 ⁶ cells per 100 μl hydrogel and subjected to three-dimensional culture.

When the cell survival rate was analyzed using Live/Dead staining at 1, 4, 7, and 14 days after starting the culture, it was confirmed that the cell survival rate was more than 90% at all analysis time points, which showed that the AH-PG hydrogel It was verified that there was almost no cytotoxicity (see Figure 6).

Example 7. Evaluation of viscosity and injection force of AH-PG hydrogel

The viscosity and injection force of the AH-PG hydrogel of the present invention and the conventional technology (HA-PG hydrogel) were measured and compared.

The viscosity of each derivative (AH-PG and HA-PG) solution at 2% concentration compared to PBS was measured using a rheometer. When measuring, the strain rate was fixed at 1 s ^-1 and the viscosity was calculated as the average of the repeatedly measured values. Compared to the previously reported HA-PG derivative solution, the AH-PG derivative solution of the present invention showed a very low level of viscosity and was confirmed to have a viscosity almost similar to that of PBS. This not only affects the injection force during injection, but can also serve as a great advantage in that the solution can easily permeate into the desired tissue area after injection into the body (see Figure 7a).

In addition, the force required for injection was tested universally by loading PBS, 2% AH-PG, and 2% HA-PG solutions into a 1 ml syringe and attaching needles of different sizes (using 27 G and 29 G). It was measured using a machine (UTM). Similar to the tendency in the viscosity analysis, it was confirmed that the AH-PG derivative solution of the present invention required a much lower injection force. It is believed that economic feasibility and clinical utility can be improved by applying AH-PG hydrogel in that a precise amount can be injected with less force (see Figures 7b and 7c).

Example 8. Preparation and analysis of muscle tissue-derived extracellular matrix (MEM)

Muscle tissue-derived cells were obtained through a decellularization process in which pig leg muscles were treated with 1% SDS (sodium dodecyl sulfate) for 2 days and then treated with 1% Triton X-100 + 0.1% NH ₄ OH (ammonium hydroxide) solution for 2 hours. Muscle extracellular matrix (MEM) was obtained.

The histological form (see Figure 8a), DNA (see Figure 8b), and GAG (glycosaminoglycan) residual amount (see Figure 8c) of muscle tissue before decellularization (Before) and muscle tissue after decellularization (After) were quantitatively compared. 95.6% of genetic material (DNA) is removed through the decellularization process to minimize the immune response that may occur when applied to the body, while GAG, a representative effective extracellular matrix component, is maintained at the original tissue level to facilitate the decellularization process of muscle tissue. It was confirmed that the protocol was successfully built.

Various types of muscle tissue-specific proteins present in MEM were identified through mass spectrometry-based proteomics analysis. MEM is composed of collagen, glycoprotein, proteoglycan, ECM regulators, and ECM-affiliated proteins. It was confirmed that it is composed of a total of 219 proteins, of which 196 proteins are skeletal muscle expressed protein (Skeletal muscle elevated protein: human tissue). This is information provided by “https://www.proteinatlas.org/” that analyzes the protein expression patterns of, and refers to proteins with an average expression level of 4 times more in muscle tissue compared to other tissues). Among them, 54 were confirmed to be composed of skeletal muscle elevated proteins (see Figures 8d and 8e).

Through a series of analyses, it was confirmed that various tissue-specific proteins present in actual muscles were present in MEM.

Example 9. Evaluation of injection power of AH-PG/MEM hydrogel

The injection force of AH-PG/MEM hydrogel and the conventional technology (HA-PG hydrogel) was measured and compared.

PBS, 2% AH-PG+5 mg/ml MEM, and 2% HA-PG+5 mg/ml MEM solutions were loaded onto an insulin syringe equipped with a 31G needle, and the force required for injection was measured using a universal testing machine (UTM). It was measured through. Similar to the tendency of the viscosity and injection force measurement results for the previously measured AH-PG and HA-PG derivative solutions, the AH-PG/MEM derivative solution was much more stable when applied using the clinically widely used insulin syringe. It was confirmed that injection was possible even with low injection force (see FIGS. 9A and 9B).

Therefore, not only can a precise amount be injected with less force, improving economic efficiency and clinical effectiveness, but when applied as personalized medicine for the elderly population, this feature is expected to serve as a great advantage over the prior art. It is understandable.

Example 10. Evaluation of body distribution and stability of AH-PG/MEM hydrogel

MEM-loaded AH-PG hydrogel (using 2% AH-PG + 5 mg/ml MEM, AH-PG/MEM group) was injected intramuscularly into the tibialis anterior (TA) muscle of mice to evaluate its stability in vivo.

Tissues at days 1, 14, and 28 after injection of MEM only (MEM group), injection of MEM with existing HA-PG (HA-PG/MEM group), and injection of MEM with AH-PG (AH-PG/MEM group) was collected and its stability in the body was confirmed through histological analysis (see Figure 10a). At this time, in order to check the remaining amount of the injected MEM component in the body, tetramethylrhodamine (TAMRA) was modified into MEM to enable tracking, and AH-PG hydrogel (using 2% AH-PG + 5 mg/ml MEM, 50 μl injection) ) as a control group, MEM (using 5 mg/ml MEM solution, 50 μl injection) and HA-PG hydrogel (using 2% HA-PG+5 mg/ml MEM, 50 μl injection) were applied together to obtain MEM according to formulation. Differences in body distribution and stability were analyzed.

As a result, it was confirmed that MEM existed in all groups one day after hydrogel injection. However, almost no MEM was observed in the MEM group 14 days after injection. On the other hand, the HA-PG/MEM group and the AH-PG/MEM group confirmed that MEM remained stable even after 28 days, confirming that MEM can be stably maintained in the body by HA hydrogel.

In addition, the MEM group and the AH-PG/MEM group with relatively low viscosity were suitable for intramuscular injection, and the MEM component was evenly penetrated and distributed between muscle fibers. However, in the HA-PG/MEM group, the MEM component was distributed evenly between muscle fibers. It does not completely penetrate into the tissue, but due to the high viscosity of the HA-PG solution and the relatively hard physical properties of the formed HA-PG hydrogel, it may be excessively filled between muscle fibers or escape out of the fascia (see white dotted line in Figure 10a) after injection. The pattern was confirmed.

Therefore, it was found that AH-PG hydrogel can stably deliver MEM, the active ingredient to be delivered into the muscle, throughout the muscle for a long period of time, and is also a suitable material as an intramuscular injection formulation.

Figure 10b below shows the mechanism of muscle tissue regeneration using AH-PG/MEM hydrogel. Due to the excellent injectability of AH-PG, the MEM-loaded AH-PG pre-gel solution can penetrate between fine muscle fibers when injected, and then self-crosslinking occurs due to natural oxidation, forming a stable hydrogel. As a result, MEM, an active ingredient for muscle regeneration, is maintained for a long period of time and can continuously activate muscle stem cells existing between muscle fibers, leading to effective muscle regeneration.

Example 11. Verification of muscle mass enhancement effect of AH-PG/MEM hydrogel in insoluble muscular atrophy animal model

The MEM-loaded AH-PG hydrogel (AH-PG/MEM group) was applied to a mouse model of disuse muscle atrophy to evaluate its effect on improving muscle mass. As a control for the new AH-PG hydrogel, the existing HA-PG hydrogel was used together to compare differences depending on the type of hydrogel.

After inducing insoluble skeletal muscle atrophy, which reduces the weight of the TA muscle by about 20%, by fixing the mouse's legs with surgical staples for 2 weeks, hydrogels of various formulations were injected intramuscularly into the TA muscle, and the TA muscle was extracted in the 2nd week. Weights were compared with normal tissue (using the contralateral uninjured leg from the same subject) (see FIGS. 11A and 11B). HA-PG (2% HA-PG), AH-PG (2% AH-PG), HA-PG/MEM (2% HA-PG+5 mg/ml MEM), AH-PG/MEM (2% AH In the group injected with -PG+5 mg/ml MEM), the effect of increasing muscle mass at the level of normal tissue was observed, and among these, the group injected with AH-PG/MEM was the only group that received intramuscular injection of PBS (PBS). By comparison, it was confirmed that there was a statistically significant weight increase effect (see Figure 11b), and this feature could be confirmed with the naked eye (see Figure 11a). In all groups, 50 μl of hydrogel was injected.

After each hydrogel injection, Hematoxylin & Eosin (H&E) staining was performed on TA muscle samples collected at week 2 to determine how well the injected hydrogel (black arrow) was fused with the surrounding muscle tissue. In the groups injected with HA-PG hydrogel (HA-PG, HA-PG/MEM), which has high viscosity and relatively hard properties, few cells were observed within the injected hydrogel, whereas in the group injected only MEM In all groups (AH-PG, AH-PG/MEM) injected with AH-PG, it was confirmed that many cells grew and distributed within the injected hydrogel, indicating active interaction with surrounding muscle tissue. This was confirmed (see Figure 11c).

In addition, muscle fiber bundles were smaller in the group administered AH-PG hydrogel (AH-PG, AH-PG/MEM group) compared to the group administered HA-PG hydrogel (HA-PG, HA-PG/MEM group). By confirming that the hydrogel injected into the space was evenly distributed, the efficient functionality of AH-PG hydrogel as an injectable agent for muscle reconstruction was confirmed once again.

Similarly, immunostaining for Pax7, a marker expressed in muscle stem cells, and MyoD, a marker expressed in activated muscle stem cells, was performed on TA muscle samples collected at week 2. It was confirmed that the number of Pax7+ muscle stem cells increased in the group injected with MEM, and the number of muscle stem cells also increased in the AH-PG group, which penetrated well between muscle fibers and provided appropriate stimulation. Among them, it was confirmed that the proliferation and activity of muscle stem cells were most improved when AH-PG hydrogel loaded with MEM was injected (AH-PG/MEM group) (see Figures 11d and 11e).

These results showed that AH-PG/MEM hydrogel can induce excellent muscle tissue regeneration effects due to the excellent injectability of AH-PG and the advantage of continuously providing MEM, the active ingredient.

Example 12. Confirmation of muscle mass improvement effect of AH-PG/MEM hydrogel in aging sarcopenia model

The muscle enhancement effect was confirmed by injecting MEM-loaded AH-PG hydrogel (AH-PG/MEM group) into an aging sarcopenia model.

In the case of mice, it is known that muscle mass begins to decrease at 15 months of age. Therefore, hydrogel under various conditions for each group was applied to the TA muscles of 18-month-old mice expected to have age-related sarcopenia (MEM: 5 mg/ml MEM solution/AH-PG/MEM: 2% AH-PG + 5 mg/ml After injection of MEM (50 μl each), tissue was collected at 8 weeks and the weight was compared with the untreated leg of the other side.

As a result of the analysis, only the group injected with AH-PG/MEM had a significant weight difference from the group injected intramuscularly with PBS (PBS), and this feature could be confirmed with the naked eye (see FIGS. 12A and 13A).

TA muscle samples collected 8 weeks after hydrogel injection were subjected to histological analysis to determine how much muscle mass was improved (see FIGS. 12B to 12E), and quantitative analysis was performed for each indicator based on the histological analysis ( 13B to 13G).

A significant myofiber diameter enhancement effect was observed in the group injected with MEM through H&E (see Figure 12b) and Masson's trichrome (MT) (see Figure 12c) staining (see Figure 13c). In addition, even 8 weeks after injection of the hydrogel, nuclei were present inside 47.3% of the myofibers in the AH-PG/MEM group, showing that myofiber proliferation observed during the regeneration process continues (Figure 13b).

As a result of immunostaining for muscle-specific markers myosin heavy chain 1E (MYH1E) and laminin, the number of MYH1E+ myofibers increased and correct laminin deposition was observed in all groups injected with MEM (MEM, AH-PG/MEM) (Figure 12d). In addition, when analyzing the minimum Feret diameter (see Figure 13d) and cross-sectional area (CSA) (see Figure 13e) of MYH1E+ myofibers in the AH-PG/MEM group, it was confirmed that the myofiber size was increased the most, showing that muscle mass improvement was the greatest. It was confirmed that it was successfully induced.

Meanwhile, improvement in muscle mass can be maximized only when oxygen and nutrients are supplied smoothly through blood vessels that exist in the tissue. As a result of staining α-smooth muscle actin (α-SMA), a marker related to vascularization (see Figure 12e), the number and size of α-SMA+ arterioles were significantly increased in the AH-PG/MEM group compared to the PBS group. This was confirmed (see Figures 13f and 13g). In addition, the expression of von Willebrand factor (vWF), a capillary marker, and the number of vWF+ capillaries were confirmed to be significantly increased in the AH-PG/MEM group (see Figures 12f and 13h).

To confirm the distribution and functionality of the nerves present in the reconstructed muscle tissue, immunostaining analysis was performed for the nerve markers nestin, acetylcholine receptor (AchR), and alpha-bungarotoxin (BTX), which is known to bind to functional AchR. It was confirmed that the expression of nestin and AchR markers was detected at the same location in the MEM and AH-PG/MEM groups, showing that the neuromuscular junction was well formed. In particular, BTX was stained the most at the neuromuscular junction location in the AH-PG/MEM group. It was found that functional nerve regeneration was most effectively induced (see Figure 12g). In addition, when quantitative analysis of the expression level of AchR marker in muscle tissue by group was confirmed, it was confirmed to be nearly two-fold improved in the AH-PG/MEM group compared to the PBS group (see Figure 13i).

Through this, it was confirmed that AH-PG/MEM hydrogel can induce the regeneration of not only muscles but also surrounding tissues such as blood vessels and nerves, leading to the reconstruction of functional muscle tissue.

Through this, it was confirmed that AH-PG/MEM hydrogel can induce the regeneration of not only muscles but also surrounding tissues such as blood vessels and nerves, leading to an improved tissue regeneration effect.

Example 13. Evaluation of tissue adhesion of AH-PG and AH-PG/MEM hydrogels

Tissue of AH-PG hydrogel (2% AH-PG, AH-PG group) and MEM-loaded AH-PG hydrogel (2% AH-PG+5 mg/ml MEM, AH-PG+MEM group) Adhesion was evaluated. As a comparison group, the conventional HA-PG hydrogel (2% HA-PG, HA-PG group) and MEM hydrogel (5 mg/ml) were compared as the control group. AH-PG hydrogel and HA-PG hydrogel were mixed with HRP (horseradish peroxidase, final HRP concentration: 6 U/ml) to induce hydrogel formation to simulate the oxidizing environment in the body.

In the case of hydrogels containing AH-PG, the aldehyde group present in AH derivatives along with the gallol group (PG), which is an adhesive functional group, can form amine groups and Schiff bases present on the tissue surface. It was predicted that it would show improved adhesion compared to HA-PG hydrogel (see Figure 14a).

To measure tissue adhesion, pig muscle tissue is attached to the plate and probe of the rheometer, and each material is applied between them to induce hydrogel formation and adhesion to the tissue. The force that detaches it from the tissue by pulling the probe. was measured (see Figure 14b). It was confirmed that AH-PG hydrogel showed significantly improved adhesion compared to MEM and HA-PG, a conventional adhesive hydrogel (see FIGS. 14b and 14c). It was confirmed that the AH-PG+MEM group also showed superior adhesion compared to the MEM and HA-PG groups. At this time, the AH-PG+MEM group showed slightly reduced adhesion compared to the AH-PG group. This is because the aldehyde group present in AH combines with the amine group present in MEM, and the reactivity to the amine group on the tissue surface was partially reduced. This is presumed to be because of this, but on the other hand, it can also be said to be a result that indirectly shows that AH-PG can effectively contain MEM, an active ingredient for muscle regeneration treatment.

When comparing the total amount of work contributed to adhesion, it was confirmed that both the AH-PG and AH-PG+MEM groups were higher than MEM and HA-PG, but the two groups showed similar levels (see Figure 14d). As AH-PG mixes with MEM, it forms an additional network between polymers internally and can contribute to energy dissipation. Therefore, although the peak force of AH-PG+MEM is slightly reduced compared to AH-PG, the performance contributing to adhesion itself is almost similar. It means.

The description of the present application described above is for illustrative purposes, and those skilled in the art will understand that the present application can be easily modified into other specific forms without changing its technical idea or essential features. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as unitary may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present application is indicated by the claims described below rather than the detailed description above, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present application.

Claims (13)

A derivative (AH-PG) obtained by modifying an aldehyde group-substituted hyaluronic acid with a pyrogallol group,
The derivative of the aldehyde group-substituted hyaluronic acid modified with a pyrogallol group (AH-PG) is capable of inducing spontaneous crosslinking.
A derivative of aldehyde group-substituted hyaluronic acid modified with a pyrogallol group (AH-PG).
According to paragraph 1,
In the derivative (AH-PG) obtained by modifying the aldehyde group-substituted hyaluronic acid with a pyrogallol group, the vicinal diol structure in hyaluronic acid is replaced with an aldehyde group, thereby interacting with the hydroxyl group. Characterized by a decrease in viscosity level due to,
A derivative of aldehyde group-substituted hyaluronic acid modified with a pyrogallol group (AH-PG).
delete According to clause 1,
The derivative (AH-PG) obtained by modifying the aldehyde group-substituted hyaluronic acid with a pyrogallol group is capable of penetrating into the space between muscle fibers.
A derivative of aldehyde group-substituted hyaluronic acid modified with a pyrogallol group (AH-PG).
delete Comprising the AH-PG of any one of paragraphs 1, 2, and 4,
AH-PG based hydrogel.
The AH-PG-based hydrogel of item 6 is loaded with muscle tissue-derived extracellular matrix (MEM),
AH-PG/MEM hydrogel.
In clause 7,
The AH-PG/MEM hydrogel has the effect of promoting or improving muscle mass,
AH-PG/MEM hydrogel.
Contains the AH-PG/MEM hydrogel of item 7,
Used for the prevention or treatment of one or more muscle diseases selected from the group consisting of muscular dystrophy, age-related sarcopenia, traumatic muscle injury, muscular dystrophy, myasthenia gravis, inflammatory myopathy, polymyositis and myotonia,
Pharmaceutical composition for preventing or treating muscle disease
delete In the method for producing aldehyde group-substituted pyrogallol-modified hyaluronic acid derivative (AH-PG),
(a) reacting hyaluronic acid (HA) and sodium periodate (NaIO ₄ );
(b) treatment with ethylene glycol, dialysis, and freeze-drying; and
(c) comprising the step of subjecting aldehyde group-substituted hyaluronic acid (AH), EDC, NHS, and 5-hydroxydopamine (5-hydroxydopamine) produced through steps (a) and (b) to EDC/NHS reaction,
Method for producing aldehyde group-substituted pyrogallol-modified hyaluronic acid derivative (AH-PG).
In the method for producing aldehyde group-substituted hyaluronic acid (AH),
(a) reacting hyaluronic acid (HA) with sodium periodate (NaIO ₄ ); and
(b) treatment with ethylene glycol, dialysis, and freeze-drying; which includes,
Method for producing aldehyde group-substituted hyaluronic acid (AH).
Manufactured by the manufacturing method of paragraph 12,
Aldehyde group substituted hyaluronic acid (AH).