Pharmaceutical composition for preventing or treating skin diseases containing hyaluronic acid complex

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating skin diseases, which contains a hyaluronic acid complex as an active ingredient. The hyaluronic acid complex is a self-assembled bond between hyaluronic acid and a hydrophobic substance that provides a hydrophobic group, and has excellent resistance to hyaluronan degrading enzymes and transdermal permeability, and has excellent effects on protecting or recovering the skin barrier function, suppressing the expression of cell proliferation-related factors and inflammatory cytokines, suppressing the polarization of M1 macrophages, and blocking TLR4 signaling, and can be used as a therapeutic agent for various skin diseases.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating skin diseases, which contains a hyaluronic acid complex as an active ingredient. [Background technology]

[0002] This application was submitted at the request of the Pan-Agency National New Drug Development Corporation as part of the National New Drug Development Project's research to expand the new drug base. The research title of this application is "Securing a leading substance for the treatment of skin inflammation diseases through the optimization, profiling, and establishment of production technology for functional hyaluronic acid nanoparticles that regulate immune cell activity," and the project number is RS-2021-DD120838 (formerly HN21C0958).

[0003] Drug administration methods can be broadly divided into oral administration and transdermal administration. For local drug delivery, particularly to the skin, transdermal administration offers several advantages over oral administration. For example, oral administration requires excessive drug administration to achieve a certain level of efficacy due to significant degradation by liver metabolism, and repeated administration is required to maintain efficacy. In contrast, transdermal drug delivery eliminates the disadvantages of oral administration by delivering the administered drug directly and regulated to the affected area without liver metabolism. Transdermal administration also has the advantage of reducing the patient's pain and mental burden associated with injections and eliminating the inconvenience. Therefore, transdermal administration is considered an effective drug administration method for treating skin diseases that primarily occur locally in the skin and surrounding tissues.

[0004] Skin is large and consists of the epidermis, dermis, and subcutaneous fat. Because skin tissue is dense, drug selection is limited due to difficulty in transdermal absorption or severe skin irritation depending on the molecular weight, dose, and properties of the drug. Therefore, for effective transdermal administration, nanoparticle-formed drugs or various drug delivery systems that facilitate drug delivery from the skin surface to the interior can be used. Recently, transdermal drug delivery systems containing various biodegradable polymers, such as polylactic acid, polyglycolic acid, and hyaluronic acid, have been developed.

[0005] Hyaluronic acid is 1 x 10 5 ~1×10 7 Hyaluronan is a linear polysaccharide polymer with a molecular weight in the Da range, composed of repeating sequences of (β,1-4)-D-glucuronic acid (GlcUA) and (β,1-3)-N-acetyl-D-glucosamine (GlcNAc) units. It is found in the extracellular matrix and cell surfaces of most human tissues, particularly in synovial fluid and cartilage. Therefore, hyaluronan is biocompatible and biodegradable by hyaluronidase, a hyaluronic acid-binding enzyme present in the blood. Therefore, it is used as a biomaterial, such as a drug delivery vehicle or a tissue engineering scaffold. In particular, hyaluronan binds to CD44 and RHAMM, which are overexpressed on the surface of cancer cells and metastatic cancer cells, and is internalized by endocytosis and degraded in low pH environments such as lysosomes. Hyaluronic acid also plays an important role as a signaling molecule in cell motility, cell differentiation, wound healing, and cancer metastasis. Summary of the Invention [Problem to be solved by the invention]

[0006] Linear hyaluronic acid has too large molecular weight to penetrate the skin, so that its skin absorption rate is low, so there is no case of applying hyaluronic acid to the treatment of disease up to now, and there is a problem that it is easily degraded by hyaluronic acid degrading enzyme in the body, so that its medicinal effect is not long-lasting.Therefore, there is an urgent need to develop a drug substance or drug delivery system containing hyaluronic acid that is stable in the body and has improved transdermal permeability so that hyaluronic acid can be applied to transdermal administration by overcoming this problem. [Means for solving the problem]

[0007] An object of the present invention is to provide a pharmaceutical composition for preventing or treating skin diseases, which contains as an active ingredient a hyaluronic acid complex in which a hydrophobic substance is bound to hyaluronic acid. [Effects of the Invention]

[0008] The hyaluronic acid complex according to the present invention is a self-assembled and bonded form of hyaluronic acid and a hydrophobic substance that provides a hydrophobic group, and has excellent resistance to hyaluronic acid degrading enzymes and transdermal permeability, and has excellent effects on protecting or restoring skin barrier function, suppressing the expression of cell proliferation-related factors and inflammatory cytokines, suppressing polarization of M1 macrophages, and blocking TLR4 signaling, so it can be used as a therapeutic agent for various skin diseases. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows the self-assembly properties of hyaluronic acid-lithocholic acid complexes (HALN-1, HALN-2, and HALN-3), where (a) is a schematic diagram of the chemical structure and self-assembly of the hyaluronic acid-lithocholic acid complex, (b) is a transmission electron microscope (TEM) photograph of the hyaluronic acid-lithocholic acid complex, (c) is the size and zeta potential of the hyaluronic acid-lithocholic acid complex, and (d) is a graph of stability obtained by measuring the changes in particle size and zeta potential over time. [Figure 2]This shows the resistance of hyaluronic acid-lithocholic acid complexes (HALN-1, HALN-2, and HALN-3) to hyaluronic acid-degrading enzymes. [Figure 3] The therapeutic effect of hyaluronic acid-lithocholic acid complexes (HALN-1, HALN-2, and HALN-3) on psoriasis is shown in (a). (b) shows the skin condition of mice with erythema and keratinization and the corresponding PASI scores. [Figure 4] This figure shows the therapeutic effect of hyaluronic acid-lithocholic acid complexes (HALN-1, HALN-2, and HALN-3) on psoriasis, and shows the skin thickness and expression levels of inflammatory cytokines (IL-1β and IL-23) measured by histological staining of mouse skin. [Figure 5] This figure shows the protective effect of hyaluronic acid-lithocholic acid complexes (HALN-1, HALN-2, and HALN-3) on the skin barrier function, and shows the expression levels of skin barrier function-related markers (ceramide, loricrin, claudin-1, and S100A9) measured by histological staining of mouse skin. [Figure 6] This shows the effect of hyaluronic acid-lithocholic acid complex (HALN-3) in restoring skin barrier function. [Figure 7] FIG. 1 shows the transdermal permeability of a hyaluronic acid-lithocholic acid complex (HALN-3), and is a graph of the fluorescence intensity of Cy5.5-HALN that has permeated a transdermal absorption cell. [Figure 8] FIG. 1 shows the transdermal penetration ability of a hyaluronic acid-lithocholic acid complex (HALN-3) in normal mice and mice with a skin disease (psoriasis), and is a fluorescence intensity image of Cy5.5-HALN in mouse skin tissue. [Figure 9] FIG. 1 is a graph showing the transdermal penetration ability of a hyaluronic acid-lithocholic acid complex (HALN-3), showing fluorescence intensity as a function of depth in mouse skin tissue. [Figure 10] 1 shows the transdermal permeability of hyaluronic acid-lithocholic acid complexes (HALN-1, HALN-2, and HALN-3). [Figure 11]This shows the therapeutic effect of psoriasis at different concentrations (0.5, 2.5, 5 mg) of hyaluronic acid-lithocholic acid complex (HALN-3) administered transdermally. (a) shows the experimental schedule, and (b) shows the skin condition of mice with erythema and keratinization and the corresponding PASI score. [Figure 12] This figure shows the therapeutic effect of psoriasis at different concentrations (0.5, 2.5, 5 mg) of hyaluronic acid-lithocholic acid complex (HALN-3) administered transdermally. The figures show the skin thickness measured by histological staining of mouse skin and the expression levels of cell proliferation-related factors (Ki-67) and inflammatory cytokines (CD68, IL-23, and IL-17). [Figure 13] This figure shows the preventive effect of psoriasis at different concentrations (2, 10 mg) of hyaluronic acid-lithocholic acid complex (HALN-3) administered transdermally, where (a) shows the experimental schedule and (b) shows the skin condition of mice with erythema and keratinization and the corresponding PASI score. [Figure 14] This figure shows the preventive effect of psoriasis at different concentrations (2, 10 mg) of hyaluronic acid-lithocholic acid complex (HALN-3) administered transdermally. The figures show the skin thickness measured by histological staining of mouse skin and the expression levels of cell proliferation-related factors (Ki-67) and inflammatory cytokines (CD68, IL-23, and IL-17). [Figure 15] This figure shows the protective effect of skin barrier function by transdermal administration of hyaluronic acid-lithocholic acid complex (HALN-3), and shows the expression levels of skin barrier function-related markers (ceramide, loricrin, claudin-1, and S100A9) measured by histological staining of mouse skin. [Figure 16] This figure shows the effect of inhibiting macrophage polarization depending on the size of HALN, where (a) is the expression level of polarization markers (TNF-α, IL-6, IL-1β, IL-12p40, and IL-23p19) of M1 macrophages by HALN-1 to 3, and (b) is the polarization level of M1 macrophages by HALN-3. [Figure 17] 1 shows a transmission electron microscope (TEM) photograph and chemical formula of hyaluronic acid-cholanic acid complex (HACN). [Figure 18] FIG. 1 shows the transdermal permeability of a hyaluronic acid-cholanic acid complex, and shows the fluorescence intensity of Cy5.5-HACN measured in mouse skin tissue. [Figure 19] This figure shows the therapeutic effect of hyaluronic acid-cholanic acid complex on macrophage-dependent psoriasis, where (a) is the experimental schedule, and (b) is the skin condition of mice with erythema and keratinization and the corresponding PASI score. [Figure 20] This figure shows the therapeutic effect of hyaluronic acid-cholanic acid complex on macrophage-dependent psoriasis, and shows (a) skin thickness and (b) expression level of skin cell proliferation-related factor (Ki-67) measured by histological staining of the skin of mice not treated with clodronate. [Figure 21] This figure shows the therapeutic effect of hyaluronic acid-cholanic acid complexes on macrophage-dependent psoriasis, and shows the expression levels of inflammatory cytokines (IL-1β, IL-23, and IL-17) measured in the skin of mice with and without clodronate treatment. [Figure 22] This figure shows the effect of HACN on inhibiting macrophage polarization depending on the size of the HACN, where (a) is the expression level of polarization markers (TNF-α, IL-6, COX-2, IL-12p40, IL-23p19, and CXCL10) in M1 macrophages induced by HACN, and (b) is the polarization level of M1 macrophages induced by HACN. [Figure 23] Surface plasmon resonance demonstrates the interaction between HACN and TLR4. [Figure 24] Fluorescence cell analysis shows the competition between LPS and HACN for binding to TLR4. [Figure 25] This is a diagram confirming the therapeutic effects of various types of hyaluronic acid complexes on skin diseases. [Figure 26] This is a diagram confirming the therapeutic effects of various types of hyaluronic acid complexes on skin diseases. DETAILED DESCRIPTION OF THE INVENTION

[0010] In order to develop a hyaluronic acid-containing drug body or drug delivery body that is stable in the body and has excellent transdermal permeability, the present inventors prepared a nano-sized hyaluronic acid complex by self-assembling low-molecular-weight hyaluronic acid, a biodegradable hydrophilic substance, with lithocholic acid, cholanic acid, or cholesterol, a hydrophobic substance, and confirmed that such a hyaluronic acid complex has excellent stability in the body and transdermal permeability, and has an effective symptom-improving or therapeutic effect on skin diseases such as psoriasis, thereby completing the present invention.

[0011] One aspect of the present invention provides a pharmaceutical composition for preventing or treating skin diseases, which comprises, as an active ingredient, a hyaluronic acid complex composed of hyaluronic acid and a hydrophobic substance.

[0012] The "hydrophobic substance" used in the present invention means a compound containing a hydrophobic substituent that imparts hydrophobicity to hydrophilic hyaluronic acid, and may be a substance with hydrophobicity that can induce the formation of nanoparticle-sized hyaluronic acid complexes.

[0013] According to one embodiment of the present invention, the hydrophobic substance may have a partition coefficient (cLogP) of 2-10, preferably 3-8.

[0014] A hydrophobic substance having such a partition coefficient value can form a nanoparticle-sized hyaluronic acid complex.

[0015] According to one embodiment of the present invention, the hydrophobic substance may induce the hyaluronic acid complex to have a particle size of 50 to 500 nm.

[0016] The hydrophobic substance according to the present invention preferably has biodegradable properties such that it is easily decomposed by enzymes produced in the living body or in the metabolic process in the living body.

[0017] More specifically, the hydrophobic substance may be hydrophobic cholanic acid, which is a type of bile acid, or a derivative thereof.

[0018] According to one embodiment of the present invention, the hydrophobic substance may be one or more selected from the group consisting of lithocholic acid, cholanic acid, cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, 7-oxo-lithocholic acid, and cholesterol.

[0019] According to one embodiment of the present invention, the hyaluronic acid complex may be one in which hyaluronic acid and a hydrophobic substance are linked together via an amide bond. According to one embodiment of the present invention, in order to bind hyaluronic acid to a hydrophobic substance, lithocholic acid or cholanic acid is reacted with ethylenediamine to produce an aminated hydrophobic substance, and the amine group of the hydrophobic substance is bound to the carboxyl group of hyaluronic acid to form a hyaluronic acid complex.

[0020] According to one embodiment of the present invention, in order to bind hyaluronic acid to a hydrophobic substance, cholesterol is reacted with DCC or the like to produce an aminated hydrophobic substance, and the amine group of this hydrophobic substance is bound to the carboxyl group of hyaluronic acid to form a hyaluronic acid complex.

[0021] According to one embodiment of the present invention, the hyaluronic acid complex may be a compound represented by the following Chemical Formula 1, Chemical Formula 2, or Chemical Formula 3:

[0022] [ka]

[0023] In the above Chemical Formula 1, a and b are each independent integers, and may be the same or different, a is an integer of 1 to 750, b is an integer of 1 to 100, and b / (a+b) is in the range of 0.01 to 0.40.

[0024] More specifically, in the above Chemical Formula 1, a is an integer of 1 to 300 (preferably an integer of 1 to 150), and b is an integer of 1 to 40 (preferably an integer of 1 to 20), and in this case, b / (a+b) may be 0.01 to 0.30 (preferably 0.02 to 0.20).

[0025] [ka]

[0026] In the above Chemical Formula 2, a and b are each independent integers, and may be the same or different, a is an integer of 1 to 750, and b is an integer of 1 to 100, and b / (a+b) is in the range of 0.01 to 0.40.

[0027] More specifically, in Chemical Formula 2, a is an integer of 1 to 100 (preferably an integer of 1 to 30), and b is an integer of 1 to 10 (preferably 1 or 2), and in this case, b / (a+b) may be 0.01 to 0.20 (preferably 0.02 to 0.10).

[0028] [ka]

[0029] In the above Chemical Formula 3, a and b are each independent integers, and may be the same or different, a is an integer of 1 to 750, b is an integer of 1 to 100, and b / (a+b) is in the range of 0.01 to 0.40.

[0030] More specifically, in Chemical Formula 3, a is an integer of 1 to 300 (preferably an integer of 1 to 150), and b is an integer of 1 to 40 (preferably an integer of 1 to 20), and in this case, b / (a+b) may be 0.01 to 0.30 (preferably 0.02 to 0.20).

[0031] According to one embodiment of the present invention, the hyaluronic acid complex may have a mass ratio of hyaluronic acid to hydrophobic substance of 1:0.005 to 0.5.

[0032] More specifically, when the hydrophobic substance in the hyaluronic acid complex is lithocholic acid or cholanic acid, the mass ratio of hyaluronic acid or cholanic acid to lithocholic acid may be 1:0.005-0.5, 1:0.01-0.3, or 1:0.02-0.15.

[0033] General linear hyaluronic acid has a large molecular weight (approximately 1 x 10 5 ~1×10 7 Da), and therefore does not easily penetrate the skin. In the present invention, hyaluronic acid having a low molecular weight is preferred so that it can be easily absorbed through the skin.

[0034] According to one embodiment of the present invention, the hyaluronic acid complex may have a molecular weight of 1 to 300 kDa.

[0035] More specifically, the number average molecular weight of the hyaluronic acid complex is preferably 10 to 250 kDa, more preferably 40 to 80 kDa, in the case of Chemical Formula 1, preferably 1 to 50 kDa, more preferably 5 to 30 kDa, in the case of Chemical Formula 2. In the case of Chemical Formula 3, it is preferably 1 to 50 kDa, more preferably 5 to 30 kDa.

[0036] According to one embodiment of the present invention, the hyaluronic acid complex may form nanoparticles by self-assembly in an aqueous solution state.

[0037] According to one embodiment of the present invention, the hyaluronic acid complex may be nanoparticles having a diameter of 50 to 500 nm.

[0038] According to one embodiment of the present invention, the degree of substitution (DS: the number of hydrophobic moieties per 100 sugar residues in hyaluronic acid) varies depending on the amount of hydrophobic substance adjusted by the feed ratio (FR). The degree of substitution of the hydrophobic substance may be an integer between 1 and 50, preferably an integer between 2 and 15.

[0039] According to one embodiment of the present invention, it has been confirmed that the size of nanoparticle hyaluronic acid complexes decreases as the degree of hydrophobic group substitution (DS) of the hyaluronic acid complex increases. It has also been confirmed that the smaller the size of such nanoparticle hyaluronic acid complexes, the greater their resistance to hyaluronan degrading enzymes, their transdermal permeability, and their effectiveness in suppressing the expression of skin growth-related factors and inflammatory cytokines. Therefore, in the present invention, the diameter of the nanoparticle hyaluronic acid complexes is preferably 100 to 400 nm, and more preferably 150 to 300 nm. If the size of the nanoparticle hyaluronic acid exceeds this range, the transdermal permeability of the hyaluronic acid complex decreases, thereby reducing the effectiveness of the hyaluronic acid complex in preventing or treating skin diseases.

[0040] According to one embodiment of the present invention, the hyaluronic acid complex may be for transdermal delivery.

[0041] According to one embodiment of the present invention, the hyaluronic acid complex has excellent permeability or absorption into skin tissue, and is absorbed deeper into skin tissue than linear hyaluronic acid. Therefore, it can be seen that the hyaluronic acid complex according to the present invention is suitable for transdermal administration.

[0042] According to one embodiment of the present invention, the skin disease may be one or more selected from the group consisting of atopic dermatitis, acne, psoriasis, allergic dermatitis, inflammatory skin disease, seborrheic dermatitis, contact dermatitis, scleroderma, eczema, Behcet's disease, sarcoidosis, melanoma, vitiligo, pemphigus, corns, warts, and lichen planus.

[0043] According to one embodiment of the present invention, the hyaluronic acid complex was confirmed to improve skin condition by reducing erythema and keratinization that appeared on the skin of psoriasis-induced mice, restore damaged skin barrier function, and suppress the expression of skin growth-related factor Ki-67 and inflammatory cytokines CD68, IL-23, and IL-17.

[0044] Furthermore, the hyaluronic acid complex binds to TLR4 and blocks TLR4 signaling, thereby preventing and treating various skin diseases caused by TLR4 signaling.

[0045] Pharmaceutical compositions containing such hyaluronic acid complexes as an active ingredient may further contain pharmaceutically acceptable additives, such as, but not limited to, starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, hydroxycalcium phosphate, lactose, mannitol, candy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc.

[0046] According to one embodiment of the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, which may be commonly used in the manufacture of pharmaceuticals, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, trehalose, hyaluronic acid, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0047] According to one embodiment of the present invention, the pharmaceutical composition may additionally contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (22nd ed., 2013).

[0048] The pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms during clinical administration. Conventional diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, can be used for formulation. Solid formulations for oral administration include tablets, pills, acidulants, granules, capsules, and the like. These solid formulations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0049] The pharmaceutical composition of the present invention may be administered orally or parenterally depending on the intended method, and parenteral administration may be performed by transdermal administration, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. The dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease, etc.

[0050] The pharmaceutical compositions of the present invention are administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field. The composition according to one embodiment of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that provides maximum efficacy at the minimum dose without side effects, which can be easily determined by one skilled in the art.

[0051] Specifically, the effective amount of the pharmaceutical composition according to the present invention varies depending on the age, sex, and weight of the patient, and generally ranges from 0.001 mg to 1,000 mg, 0.01 mg to 100 mg, or 0.1 mg to 10 mg per kg of body weight, administered daily or every other day, or in 1 to 3 divided doses per day. However, the dosage may be increased or decreased depending on the route of administration, severity of the disease, sex, weight, age, etc., and the above dosage does not in any way limit the scope of the present invention.

[0052] Another aspect of the present invention provides a method for preventing or treating a skin disease, comprising the step of administering to an individual a composition containing, as an active ingredient, a hyaluronic acid complex composed of hyaluronic acid and a hydrophobic substance.

[0053] According to one embodiment of the present invention, the hyaluronic acid, the hydrophobic substance, the hyaluronic acid complex, and the skin disease in the method are as described above.

[0054] According to one embodiment of the present invention, the dosage of the composition may be 10 mg / kg / day to 100 mg / kg / day.

[0055] According to one embodiment of the present invention, the individual may be an individual in need of prevention or treatment of a skin disease. The individual may be a mammal, including humans, primates including monkeys, and rodents including mice and rats, or cells or tissues isolated therefrom, or cultures thereof. In this case, the patient may be a mammal other than humans.

[0056] Another aspect of the present invention provides a use of a hyaluronic acid complex composed of hyaluronic acid and a hydrophobic substance for the prevention or treatment of skin diseases.

[0057] Another aspect of the present invention provides a use of a hyaluronic acid complex composed of hyaluronic acid and a hydrophobic substance for the manufacture of a medicament for the prevention or treatment of skin diseases.

[0058] According to one embodiment of the present invention, the hyaluronic acid, the hydrophobic substance, the hyaluronic acid complex, and the skin disease in the use are as described above. [Example]

[0059] The present invention will be described in more detail below. However, such description is merely provided as an example for understanding the present invention, and the scope of the present invention is not limited by such exemplary description.

[0060] Example 1. Preparation of hyaluronic acid-lithocholic acid complex 1-1. Amination of lithocholic acid 1 g of lithocholic acid (LCA) (Sigma-Aldrich) was dissolved in 20 mL of methanol, followed by 180 μL of 12N HCl and stirring at reflux at 70°C for 8 hours. After the reaction was complete, the solvent was removed by distillation under reduced pressure. The resulting solid primary product was diluted with water, and the precipitate was collected by vacuum filtration and dried under vacuum to obtain the final methyl ester form of lithocholic acid. 920 mg of methyl ester-LCA was dissolved in 15 mL of ethylenediamine (TCI) and stirred at reflux at 130°C for 8 hours. After the reaction was complete, the ethylenediamine was removed by distillation under reduced pressure. The resulting secondary product was diluted with water, and the precipitate was collected by vacuum filtration. This was then dried to obtain the final aminated LCA.

[0061] 1-2. Hyaluronic acid-lithocholic acid complex (HALN) 120 mg of 60 kDa hyaluronic acid (HA) (Lifecore) was dissolved in 20 mL of formamide (Sigma-Aldrich). Then, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (TCI) (56.1 mg, 0.29 mmol) and N-hydroxysuccinimide (NHS) (Sigma) (33.3 mg, 0.29 mmol) were added and stirred for 30 minutes to prepare the HA mixture. An appropriate amount of aminated LCA (8–80 mg) based on the feed ratio (FR) was dissolved in 5 mL of dimethylformamide (DMF), and this solution was added dropwise to the HA mixture. The resulting mixture was stirred at room temperature for 24 hours. After the reaction was completed, the mixture was dialyzed using a 13K MWCO dialysis bag (Sigma-Aldrich) against methanol alone for 2 days, a methanol:distilled water (v:v = 1:1) mixture for 1 day, and distilled water alone for 2 days. After dialysis, the reaction mixture was sonicated for 50 seconds (10 seconds on, 1 second off). After sonication, the mixture was filtered through a 0.8 μm syringe filter. The filtrate was lyophilized to obtain the final hyaluronic acid-lithocholic acid conjugate (HALN). These conjugates are hereafter designated HALN-1, HALN-2, and HALN-3, respectively, based on the degree of hydrophobic substitution (DS) analyzed by nuclear magnetic resonance (NMR) spectroscopy.

[0062] 1-3.HALN characteristics analysis The chemical properties of HALN were analyzed by 600MHz nuclear magnetic resonance spectroscopy (NMR spectroscopy) using a solvent of deuterated methanol:deuterium oxide = 1:1 (v:v). The size and zeta potential of the generated HALN were measured using a dynamic light scattering (DLS) instrument (ELS-Z, Otsuka). HALN was dissolved in PBS at a concentration of 1 mg / mL.

[0063] As shown in Figure 1, when dried HALN was dispersed in PBS, the lithocholic acid functional groups aggregated together to form spherical particles. HALN-1, HALN-2, and HALN-3 were synthesized with increasing degrees of substitution (DS) as the feeding rate (FR) increased. The sizes of HALN-1, HALN-2, and HALN-3 were 428±20 nm, 331±2 nm, and 211±2 nm, respectively. It was confirmed that with increasing DS, the size decreased (approximately 150-440 nm) and the zeta potential increased in the order of HALN-1, HALN-2, and HALN-3.

[0064] Experimental Example 1. Analysis of resistance of HALN to hyaluronic acid degrading enzymes Hyaluronidase Type II (H2126, Sigma) was reacted with HALN-1, HALN-2, and HALN-3, which differ in size and electrochemical value, at 37°C for 0.5, 1, 2, 4, and 6 hours, respectively. The reaction was then terminated by heating to 100°C, and the reaction mixture was treated with potassium tetraborate and DMAB solution, followed by a water bath at 37°C. 200 μl of the reaction mixture was loaded into a 96-well plate, and the absorbance at 544 nm was measured using a spectrophotometer (Cytation3, BioTek Instruments Inc.).

[0065] As a result, as shown in Figure 2, it was confirmed that HALN-3 has significantly higher resistance to hyaluronan-degrading enzymes than hyaluronan alone (60 kDa linear HA) and other complexes (HALN-1 and HALN-2).

[0066] Experimental Example 2: HALN's therapeutic effect on psoriasis To generate the psoriasis animal model, female C57BL / 6N mice (OrientBio) were used (n = 5 per group). They were divided into normal mice (CON) and mice with induced psoriasis (IMQ). Before psoriasis induction, HALN-1, HALN-3, and HALN-1, HALN-3 were dissolved in PBS at a concentration of 1 mg / mL and subcutaneously injected at 20 mg / kg for 4 days using an insulin syringe. Alternatively, PBS (20 mg / kg) alone was administered instead of HALN. Two hours after drug administration, the mice's weight and PASI score were measured. After drug administration, Aldara cream (imiquimod, IMQ) 62.5 mg was applied once daily to the hairless backs of the mice for 4 days. On day 5, skin tissues from all mice were collected and analyzed.

[0067] The PASI score was based on the area and severity of psoriasis: 0; none, 1; slight, 2; moderate, 3; extensive, and 4; very obvious.

[0068] For histological staining of mouse skin, each tissue was fixed in 4% paraformaldehyde (PFA), then paraffin blocks were prepared and 0.4 μm sections were prepared for slides. Nuclei and cytoplasm were stained with hematoxylin and eosin. Subsequently, IL-1β and IL-23 antibodies were used to confirm the expression of inflammatory cytokines.

[0069] Skin thickness was automatically analyzed using the "MRI Skin Tool" function of the Image J program. The average image values ​​of three sections for each tissue were used.

[0070] As a result, as shown in Figures 3 and 4, administration of HALN-1 to 3 reduced erythema, keratinocytes, and skin thickness compared to administration of PBS, with HALN-3, which exhibited the smallest reduction in size, demonstrating superior efficacy. Furthermore, HALN-3 was found to more effectively suppress the expression of IL-1β and IL-23, inflammatory cytokines important in psoriasis, compared to HALN-1 and HALN-2. In conclusion, HALN-3 demonstrated superior efficacy in preventing and treating psoriasis compared to HALN-1 and HALN-2.

[0071] Experimental Example 3: Protective effect of HALN on skin barrier function To create a psoriasis animal model, female C57BL / 6N mice (5 per group) were used. They were divided into normal mice (CON) and mice with induced psoriasis (IMQ). Prior to psoriasis induction, HALN-1, HALN-3, and HALN-2 were dissolved in PBS at a concentration of 1 mg / mL and subcutaneously injected at 20 mg / kg using an insulin syringe into the backs of hairless mice. Alternatively, PBS was administered at 20 mg / kg instead of HALN. Three hours after drug administration, Aldara cream (62.5 mg) was applied for four days. On the final day of administration, skin tissues were collected from all mice and immunohistochemically stained for markers of skin barrier function (ceramide, loricrin, claudin-1, and S100A9).

[0072] As a result, as shown in FIG. 5, it was confirmed that HALN-3 was most effective in restoring the skin barrier function destroyed by psoriasis induction compared to HALN-1 and HALN-2.

[0073] As in the previous experiment, PBS or HALN-3 was administered to the psoriasis animal model, and then Cy5.5-HALN-3 was administered subcutaneously to all mice on the final day. Four hours later, the skin tissue was observed under a fluorescence microscope.

[0074] As a result, as shown in Figure 6, it was revealed that the percutaneous absorption of Cy5.5-HALN-3 was lower in the psoriasis animal model injected with HALN-3 than in the psoriasis animal model injected with PBS, indicating that HALN-3 restored the skin barrier function that had been destroyed by psoriasis induction.

[0075] Experimental Example 4: Analysis of HALN transdermal permeability 4-1.Transdermal absorption cell After fixing skin tissue from a micropig in a Franz diffusion cell, 0.5 ml of Cy5.5-HALN-3 was applied to the skin tissue. At 0, 1, 2, 4, 6, 12, and 24 hours after application, 20 μl of the solution was extracted from the receiver chamber and loaded into a black 96-well plate. Fluorescence (absorption wavelength: 670 nm, emission wavelength: 700 nm) was measured using a spectrophotometer (Cytation3, BioTek Instruments Inc.). Cy5.5 was used as a control.

[0076] As a result, as shown in FIG. 7, it became clear that the amount of Cy5.5-HALN that permeated the skin tissue increased with the passage of time, demonstrating that HALN has the ability to permeate through the skin. Additionally, back skin samples were removed from normal mice treated with PBS and from mice with psoriasis induced as in Experimental Example 3. The samples were then mounted in a transdermal absorption cell, and Cy5.5-HALN (0.5 ml) was applied to the skin tissue. Skin tissue samples were collected 1, 2, 4, and 12 hours after application and fixed in 4% PFA. Paraffin blocks were then prepared, and 0.4 μm sections were cut into slides for histological staining. After DAPI staining, the skin tissue samples were observed under a fluorescence microscope.

[0077] As a result, as shown in Figure 8, the fluorescence intensity was stronger in psoriasis-induced mice (IMQ) than in normal mice under the same time conditions, confirming that HALN has superior transdermal penetration ability in psoriatic skin.

[0078] Then, back skin excised from mice with psoriasis induced as in Experimental Example 3 was fixed in a transdermal absorption cell, and PBS, Cy5.5 (0.5 ml), Cy5.5-HA (0.5 ml), or Cy5.5-HALN (0.5 ml) was applied to the skin tissue. After 4 hours, the skin tissue was collected and stained, and the fluorescence was observed using a two-photon microscope.

[0079] As a result, as shown in Figure 9, HALN had stronger fluorescence intensity than Cy5.5 and free HA at the same depth in skin tissue, and fluorescence was observed at deeper depths. These results suggest that HALN has excellent transdermal penetration ability.

[0080] 4-2. Comparison of transdermal penetration ability of HALN-1 to 3 Cy5.5-HALN-1 to 3 were applied to the back skin of normal mice that had been treated with PBS and mice that had been induced with psoriasis as in Experimental Example 3. After 4 hours, skin tissues were collected and observed under a fluorescence microscope.

[0081] As a result, as shown in Figure 10, it was confirmed that the transdermal permeability of all HALNs was increased in the psoriasis animal model compared to normal mice, and that HALN-3 in particular had the highest transdermal permeability.

[0082] Experimental Example 5: Psoriasis treatment and prevention effects of different concentrations of HALN administered transdermally Female C57BL / 6N mice (5 per group) were used to create the psoriasis animal model. They were divided into normal mice (CON) and mice with induced psoriasis (IMQ). To induce psoriasis, Aldara cream (62.5 mg) was applied to the backs of hairless mice once daily for 4 days. Starting on the second day of application, PBS (20 mg / kg) or HALN-3 (0.5, 2.5, or 5 mg) was applied with a brush once daily for 3 days, 3 hours before administration of Aldara cream. Skin tissues and spleens were collected from all mice on the fifth day.

[0083] PASI scores were assessed in the same manner as in Experimental Example 2, and tissue staining was performed to measure skin thickness and the expression of cell proliferation-related factors (Ki-67 and CD68) and inflammatory cytokines (IL-23 and IL-17).

[0084] As a result, as shown in Figures 11 and 12, it was confirmed that when HALN was administered, the skin condition improved in a concentration-dependent manner, and cell proliferation-related factors and inflammatory cytokines were effectively suppressed.

[0085] These results suggest that HALN may be effective in treating inflammatory skin diseases such as psoriasis.

[0086] To create a psoriasis animal model, female C57BL / 6N mice (5 per group) were used. They were divided into normal mice (CON) and mice with psoriasis-induced disease (IMQ). Before psoriasis induction, PBS (20 mg / kg) or HALN-3 (2 or 10 mg) was applied to the backs of hairless mice with a brush twice daily at 3-hour intervals for 4 days. Three hours after drug application, Aldara cream (62.5 mg) was applied once daily for 4 days. Skin tissues were collected from all mice on the 5th day.

[0087] PASI scores were assessed in the same manner as in Experimental Example 2, and tissue staining was performed to measure skin thickness and the expression of cell proliferation-related factors (Ki-67 and CD68) and inflammatory cytokines (IL-23 and IL-17).

[0088] As a result, as shown in Figures 13 and 14, it was confirmed that when HALN was administered, the skin condition improved in a concentration-dependent manner, and cell proliferation-related factors and inflammatory cytokines were effectively suppressed.

[0089] These results suggest that HALN is effective in preventing inflammatory skin diseases such as psoriasis.

[0090] Experimental Example 6: Protective effect of skin barrier function by transdermal administration of HALN Mice were divided into normal (CON) and psoriasis-induced (IMQ) mice. Before psoriasis induction, PBS (20 mg / kg) or HALN-3 (2 or 10 mg) was applied twice daily with a brush to the hairless backs of mice at 3-hour intervals for 4 days. Three hours after drug application, Aldara cream (62.5 mg) was applied once daily for 4 days. Skin tissues were collected from all mice on day 5. Skin tissues were immunostained for markers of skin barrier function (ceramide, loricrin, claudin-1, and S100A9).

[0091] As a result, as shown in FIG. 15, it was confirmed that administration of HALN-3 concentration-dependently restored the skin barrier function that had been destroyed by psoriasis induction.

[0092] Experimental Example 7. Macrophage polarization suppression effect depending on HALN size Human-derived mononuclear cells, THP-1 cells, were plated in a 6-well plate (SPL) at 5 × 10 5 The cells were dispensed at 100°C / well and simultaneously treated with 50ng / ml of PMA (phorbol 12-myristate 13-acetate) and cultured for 24 hours in an incubator at 37°C and 5% CO. The medium was then replaced with RPMI-1640 containing 10% fetal bovine serum (FBS), and further cultured for 24 hours in an incubator at 37°C and 5% CO.

[0093] Each HALN was diluted in serum-free medium (RPMI-1640) containing LPS and IFNγ to a concentration of 50 μg / ml, and 1 ml of the solution was applied to THP-1 cells. Cells were then cultured at 37°C in a 5% CO2 incubator for 4 hours. After the experiment, RNA was isolated from the cells using Trizol reagent (Molecular Research Center). Samples with a yield of 260 / 280 or greater using Nanodrop were used for analysis. The isolated RNA was then used with reverse transcriptase to synthesize cDNA templates. The cDNA templates were amplified using gene-specific nucleotide primers (see Table 1 below) with SYBR-GREEN using Real-Time Multiplexing Amplification (CFX connect™, Bio-Rad) and qPCR was performed.

[0094] [Table 1]

[0095] As a result, as shown in Figure 16a, HALN-1 to 3 reduced the expression of all M1 macrophage-specific genes that were increased by LPS and IFNγ, and it was confirmed that HALN-3, which has the smallest size, most effectively suppressed polarization into M1 macrophages.

[0096] HALN-3 was diluted with serum-free medium (RPMI-1640) containing LPS and IFNγ to concentrations of 0, 25, 50, and 100 μg / ml, and 1 ml of the solution was applied to THP-1 cells, which were then cultured at 37°C in a 5% CO2 incubator for 48 hours. After the treatment was complete, the cells were washed once with PBS, detached from the cell culture dish using cell detachment solution (Accutase), and then centrifuged to collect the collected cells, which were then collected at 5 x 10 per FACS tube. 5After adding each cell, the cells were washed once with a reaction solution (containing PBS, 5 mM EDTA, 2% FBS, and 2% BSA). Then, a reaction solution containing CD86, a marker for M1 macrophages, was added and incubated in the dark at 4°C for 1 hour. After washing the cells three times with the reaction solution, 300 μl of the reaction solution was added to disperse the cells, and APC fluorescence was measured using a flow cytometer (Novocyte, Agilent).

[0097] As a result, as shown in Figure 16b, the CD86 expression level of M1 macrophages, which was increased by LPS and IFNγ, was reduced in a HALN-3 concentration-dependent manner, suggesting that HALN-3 effectively inhibits the polarization of M1 macrophages.

[0098] Example 2. Preparation of hyaluronic acid-cholanic acid complex (HACN) 2-1. Amination of cholanic acid 1 g of 5β-cholanic acid (CA) (Sigma Aldrich) was dissolved in 20 mL of methanol, followed by the addition of 180 μL of HCl (12N) and stirring at reflux at 70°C for 8 hours. Upon completion of the reaction, the solvent was removed by distillation under reduced pressure. The resulting solid primary product was diluted with water, and the precipitate was collected by filtration under reduced pressure and dried under vacuum to obtain the final methyl ester form of CA. 925 mg of methyl ester-CA was dissolved in 15 mL of ethylenediamine (TCI) and stirred at reflux at 130°C for 8 hours. Upon completion of the reaction, the ethylenediamine was removed by distillation under reduced pressure. The resulting secondary product was diluted with water, and the precipitate was collected by filtration under reduced pressure. This was then dried to obtain the final aminated CA.

[0099] 2-2. Hyaluronic acid-cholanic acid complex (HACN) 120 mg of 10 kDa hyaluronic acid (HA) (Lifecore) was dissolved in 20 mL of formamide (Sigma-Aldrich). Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (TCI) and N-hydrosuccinimide were added and stirred for 30 minutes to prepare the HA mixture. Aminated CA (29.4 mg, 0.073 mmol) with a feed ratio of 3 was dissolved in 5 mL of dimethylformamide, and this solution was then added intravenously to the HA mixture. The resulting mixture was stirred at room temperature for 24 hours. After the reaction was complete, the mixture was dialyzed using a 35K MWCO snakeskin dialysis bag (Thermo Scientific) against methanol for 2 days, a methanol:distilled water mixture (v:v=1:1) for 1 day, and distilled water for 2 days. After dialysis, the reaction mixture was sonicated for 50 seconds (10 seconds on, 1 second off). After sonication, the mixture was filtered through a 0.8 μm syringe filter. The filtered solution was lyophilized to obtain the final hyaluronic acid-cholanic acid complex (HACN).

[0100] 2-3. HACN characteristic analysis The chemical properties of HACN were analyzed by 600MHz nuclear magnetic resonance (NMR) spectroscopy using a 1:1 (v:v) deuterated methanol:deuterium oxide solvent. The size and zeta potential of the HACN were measured using a dynamic light scattering (DLS) instrument (ELS-Z, Otsuka). HACN was dissolved in PBS at a concentration of 1 mg / mL.

[0101] As a result, when dried HACN was dispersed in PBS, the functional groups of cholanic acid aggregated together to form spheres, as shown in Figure 17. The DS (degree of substitution of CA), size, and zeta potential (ζ) of such HACN are shown in Table 2 below.

[0102] [Table 2]

[0103] Experimental Example 8. Analysis of HACN percutaneous permeability The back skin of normal mice and mice with psoriasis induced after PBS application was fixed in a transdermal absorption cell, and then Cy5.5-HACN (0.3 ml) was applied to the skin tissue. Psoriasis was induced by applying 62.5 mg of Aldara cream to the backs of hairless mice once daily for four days. Skin tissue was collected 1, 4, and 12 hours after Cy5.5-HACN application and fixed in 4% PFA. Paraffin blocks were prepared, and 0.4 μm sections were cut into slides and prepared for histological staining. The skin tissue was then stained with DAPI and observed under a fluorescence microscope.

[0104] As a result, as shown in Figure 18, in the skin of normal mice (PBS), Cy5.5-HACN was observed to penetrate the skin more as time passed. In the psoriasis-induced mice (IMQ), the fluorescence intensity was stronger than that of normal mice over the same time period, confirming that HACN has a superior transdermal penetration ability in psoriatic skin.

[0105] Experimental Example 9. Therapeutic effect of HACN on macrophage-dependent psoriasis Seven- to eight-week-old female C57BL / 6N mice received a primary intraperitoneal injection of 200 μl / mouse (1.4 mg) of clodronate liposomes, followed by a secondary intraperitoneal injection of 100 μl / mouse (0.7 mg) on ​​day 4 to induce macrophage depletion. The day after the primary injection, the backs were shaved, and after a one-day delay, 62.5 mg of Aldara cream was applied to the backs once daily for five days to induce psoriasis. HACN or PBS was administered via tail vein injection 2 hours before Aldara cream application for the same period (five mice per group). Photographs of the psoriasis-induced areas were taken on the first and final days of psoriasis induction, and the PASI score for each mouse was measured daily and compared. The dorsal skin was dissected and fixed in 4% PFA. Paraffin blocks were prepared and cut into 4 μm-thick sections to prepare skin tissue slides. Epidermal thickness was measured by H&E staining, skin cell proliferation was measured by Ki-67 staining, and the degree of inflammatory response was measured by staining for the inflammatory cytokines IL-1β, IL-23, and IL-17.

[0106] As shown in Figures 19-21, in the group not treated with clodronate (Vehicle), HACN reduced the PASI score and epidermal thickness, and inhibited skin cell proliferation and inflammatory responses, confirming its therapeutic effect on psoriasis. However, in the group treated with clodronate, the therapeutic effect of HACN on psoriasis was reduced due to a lack of macrophages. These results suggest that the therapeutic effect of HACN on psoriasis is dependent on macrophages.

[0107] Experimental Example 10. Inhibitory effect of HACN on macrophage polarization qPCR was performed in the same manner as in Experimental Example 7, except that 0, 10, 25, and 50 μg / ml of HACN was used instead of HALN, and the primers in Table 3 below were used instead of the primers in Table 1.

[0108] [Table 3]

[0109] As a result, as shown in Figure 22a, HACN concentration-dependently reduced the expression of all M1 macrophage-specific genes that were increased by LPS and IFNγ, confirming that HACN inhibits the polarization of THP-1 macrophages.

[0110] Furthermore, APC fluorescence was measured in the same manner as in Experimental Example 7, except that HACN at 0, 10, 25, and 50 μg / ml was used instead of HALN.

[0111] As a result, as shown in Figure 22b, the CD86 expression level of M1 macrophages, which was increased by LPS and IFNγ, was reduced in a HACN concentration-dependent manner, suggesting that HACN effectively inhibits the polarization of M1 macrophages.

[0112] Experimental Example 11. Analysis of the interaction between HACN and TLR4 11-1.Surface plasmon resonance (SPR) To immobilize the target receptor on the analysis chip, EDC / NHS solvent was injected simultaneously with recombinant TLR4, allowing the receptor to be immobilized on the chip. Subsequently, ethanolamine was injected to remove receptors that were weakly or not immobilized on the chip. PBS, HACN in PBS, and 10 kDa free HA were injected at concentrations of 0, 25, 50, 100, 200, 400, and 800 μg / ml, respectively, at 10 μl / min over the completed recombinant TLR4-bound chip, and the change in RU (Response Unit) value was monitored for 120 seconds.

[0113] As a result, as shown in Figure 23, it was confirmed that the RU value increased with increasing HACN concentration, confirming the mutual interaction between TLR4 and HACN. In contrast, the control groups injected with PBS or 10 kDa free HA alone did not show the increase in RU value observed in the HACN group. Therefore, it is clear that binding to TLR4 is a unique characteristic of HACN.

[0114] 11-2.Fluorescent cell analysis THP-1 cells were cultured in a 35 mm cell culture dish (SPL) at a density of 5 × 10 5 The cells were then aliquoted to 100 μg / ml and simultaneously treated with 50 ng / ml PMA and cultured at 37°C in a 5% CO2 incubator for 24 hours. The medium was then replaced with 10% FBS-containing RPMI-1640 and cultured for an additional 24 hours at 37°C in a 5% CO2 incubator. HACN and 10 kDa free HA were diluted in serum-free medium (RPMI-1640) to prepare concentrations of 0, 10, 25, and 50 μg / ml, respectively. 1 ml of the diluted solution was added to the cells and cultured for 1 hour at 37°C in a 5% CO2 incubator. The cell culture medium was then treated with an additional 1 ml of Cy5.5-labeled LPS and HACN, and cultured for an additional 30 minutes at 37°C in a 5% CO2 incubator. After the experiment, the cells were washed with cold PBS and fixed with 4% PFA. After fixing, the cells were analyzed for color under a fluorescent microscope.

[0115] As a result, as shown in Figure 24, the binding of Cy5.5-labeled LPS to THP-1 macrophages was reduced in a concentration-dependent manner by HACN, but not by 10 kDa free HA. These results suggest that HACN binds to TLR4 competitively with LPS and inhibits LPS-induced TLR4 activation.

[0116] Example 3. Preparation of hyaluronic acid-cholesterol complex (HACHN) 3-1. Amination of cholesterol 1 g of cholesterol (cholesterol, Sigma-Aldrich), 544 mg of N-Boc-glycine (Alfa Aesar), 640 mg of N,N'-dicyclohexylcarbodiimide (DCC, Sigma-Aldrich), and 47.4 mg of 4-(dimethylamino)pyridine (DMAP, Alfa Aesar) were dissolved in 25 mL of dichloromethane and stirred at 25 °C for 24 hours. After the reaction was completed, the mixture was dried under vacuum and separated by liquid phase chromatography (hexane:ethyl acetate = 1:10). The resulting mixture was dried to obtain the final Boc-Gly-cholesterol. Next, 1.09 g of Boc-Gly-cholesterol was dissolved in 20 mL of dichloromethane, and 5 mL of 4N hydrochloric acid in 1,4-dioxane was added and stirred at 25 °C for 1 hour. After the reaction was completed, the mixture was dried under vacuum to obtain the final aminated cholesterol.

[0117] 3-2. Hyaluronic acid-cholesterol complex (HACHN) 120 mg of 10 kDa hyaluronic acid (HA) (Lifecore Biomedical) was dissolved in 20 mL of formamide (Sigma-Aldrich). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (TCI) and N-hydroxysuccinimide (Sigma-Aldrich) were then added and stirred for 20 minutes to prepare the HA mixture. Aminated cholesterol (70.1 mg, 0.146 mmol) with a feed ratio of 2 (DS value) was dissolved in 5 mL of dimethylformamide with triethylamine (TCI), and this solution was then added intravenously to the HA mixture. The resulting mixture was stirred at room temperature for 24 hours. After the reaction was completed, the mixture was dialyzed using a 3.5K MWCO snakeskin dialysis bag (Thermo Scientific) for two days against methanol alone, one day against a methanol:distilled water mixture (v:v=1:1), and two days against distilled water alone. After dialysis, the reaction mixture was sonicated for 50 seconds (10 seconds on, 1 second off). After sonication, the mixture was filtered through a 0.8 μm syringe filter. The filtered solution was lyophilized to obtain the final hyaluronic acid-cholesterol complex (HA-Cholesterol, HACHN).

[0118] 3-3.HACHN characteristics analysis The 1H NMR (Nuclear Magnetic Resonance) spectrum of HACHN was analyzed using a 600 MHz nuclear magnetic resonance spectrometer (JNM-ECZ 600R, JEOL) in a 1:1 (v:v) solvent mixture of deuterated methanol (METHANOL-D6):deuterium oxide (DEUTERIUM OXIDE). The size and zeta potential of the resulting HACHN were measured using a dynamic light scattering instrument (ELS-Z, Otsuka). HACHN was dissolved in PBS at a concentration of 1 mg / mL. When dried HACHN was dispersed in PBS, the cholesterol functional groups aggregated together to form spherical particles. The DS (degree of cholesterol substitution), size, and zeta potential (ζ) of the resulting HACHN are shown in Table 4.

[0119] [Table 4]

[0120] Experimental Example 12: Analysis of the therapeutic effects of various types of hyaluronic acid complexes, including HACHN, on skin diseases Twenty-eight female C57BL / 6N mice were divided into normal mice (CON) and mice with psoriasis-induced disease (IMQ). The following reagents were prepared: Aldara Cream, PBS, HALN, 10kJ-HALN, 10kJ-HACN, and 10kJ-HACHN. HALN: Hyaluronic acid with a molecular weight of 60 kDa + lithocholic acid (lithocholic acid) 10k-HALN: 10kDa hyaluronic acid + lithocholic acid 10k-HACN: 10kDa hyaluronic acid + cholanic acid 10k-HACHN: 10kDa hyaluronic acid + cholesterol

[0121] To induce psoriasis, mice in each group (except the control group) were given 62.5 mg of Aldara cream once daily for four days. Six hours before applying Aldara cream, the drug was applied to the back skin of each group of mice using a brush, and 2 mg of the HA nanoparticle complex was dissolved in PBS and applied. On the fifth day, skin tissue was collected from all mice and analyzed.

[0122] The analysis method followed the Psoriasis Area and Severity Index (PASI) scoring system, with scores of 0 (none); 1 (slight); 2 (moderate); 3 (substantial); and 4 (very well pronounced). The tissues were fixed, embedded, and sectioned at 0.4 μm, then stained with hematoxylin, eosin, and DAB staining kits and antibodies. Intensity measurements were performed using the Image J program and a Leica DMi8 fluorescence microscope. The average image values ​​from three sections of each tissue were used.

[0123] As a result, as shown in Figures 25 and 26, in all groups to which hyaluronic acid complexes bound to other types of hydrophobic substances were applied, PASI score, skin thickness, skin cell proliferation, and inflammatory response were reduced, confirming the effectiveness of these complexes against skin diseases.

[0124] In summary, the hyaluronic acid complex of the present invention, in which hyaluronic acid is bound to a hydrophobic substance, has strong resistance to hyaluronan-degrading enzymes and transdermal permeability. In particular, the smaller the size of the complex, the better the transdermal permeability. It is found to be effective in preventing or treating various skin diseases such as psoriasis, malignant melanoma, bacterial and fungal inflammation, and autoimmune diseases due to its excellent effects on protecting or restoring skin barrier function, suppressing the expression of cell proliferation-related factors and inflammatory cytokines, and inhibiting the polarization of M1 macrophages, as well as its action of blocking TLR4 signaling.

[0125] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention can be embodied in various modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims, not the above description, and all variations within the scope of the claims should be construed as being within the scope of the present invention. [Industrial Applicability]

[0126] The present invention relates to a pharmaceutical composition for preventing or treating skin diseases, which contains a hyaluronic acid complex as an active ingredient, and can be used as a therapeutic agent for various skin diseases, and has industrial applicability.

Claims

1. A pharmaceutical composition for preventing or treating skin diseases, comprising a hyaluronic acid complex composed of hyaluronic acid and a hydrophobic substance as an active ingredient, the hydrophobic substance is hydrophobic cholanic acid, lithocholic acid, or cholesterol; The hyaluronic acid complex is for transdermal administration, The skin diseases include atopic dermatitis, acne, psoriasis, allergic dermatitis, inflammatory skin disease, seborrheic dermatitis, contact dermatitis, scleroderma, eczema, Behcet's disease, and the like. disease), sarcoidosis, melanoma, vitiligo, pemphigus, corns, warts and lichen planus.

2. 2. The pharmaceutical composition of claim 1, wherein the hydrophobic substance has a partition coefficient (cLogP) in the range of 2 to 10.

3. The pharmaceutical composition according to claim 1, wherein the hyaluronic acid complex is a compound represented by the following formula 1, 2, or 3: 【Chemical 1】 In Formula 1, a and b are each independently an integer and may be the same or different, a is an integer from 1 to 750, b is an integer from 1 to 100, and b / (a+b) is in the range of 0.01 to 0.

40. 【Chemistry 2】 In Formula 2, a and b are each independently an integer and may be the same or different, a is an integer from 1 to 750, b is an integer from 1 to 100, and b / (a+b) is in the range of 0.01 to 0.

40. 【Chemistry 3】 In Formula 3, a and b are each independently an integer and may be the same or different, a is an integer from 1 to 750, b is an integer from 1 to 100, and b / (a+b) is in the range of 0.01 to 0.

40.

4. The pharmaceutical composition according to claim 1, wherein the hyaluronic acid complex has a mass ratio of hyaluronic acid to hydrophobic substance of 1:0.005-0.

5.

5. The pharmaceutical composition according to claim 1, wherein the hyaluronic acid complex is formed by self-assembly in an aqueous solution.

6. The pharmaceutical composition according to claim 1, wherein the hyaluronic acid complex has a diameter of 50 to 500 nm.

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