KR20110039708A - Composition comprising mycolic acid derivatives for improving immune-modulating and anti-inflammatory activity - Google Patents

Abstract

PURPOSE: A composition containing mycolic acid derivative with immune-modulating and anti-inflammation is provided to treat atopic dermatitis or asthma without side effects. CONSTITUTION: A composition for immune-modulating and anti-inflammation contains 0.01-50 weight% of mycolic acid as an active ingredient. The composition is used for treating atopic dermatitis, asthma, and allergic rhinitis. The composition is used in the form of cream, gel, lotion, paste, ointment, skin softening agent, liposome, or skin tonic.

Description

IMCOMPOSITION COMPRISING MYCOLIC ACID DERIVATIVES FOR IMPROVING IMMUNE-MODULATING AND ANTI-INFLAMMATORY ACTIVITY}

The present invention relates to a composition comprising a compound having an immunomodulatory function and an anti-inflammatory effect, and more particularly, to a composition comprising a mycolic acid derivative as an active ingredient and having in vivo immunomodulatory and anti-inflammatory activity. will be.

Mycobacterium tuberculosis was discovered in 1882 by Robert Koch. Mycobacterium tuberculosis grows very slowly and takes about 18 to 24 hours to divide from one to two. It is surrounded by a large amount of fat and can live for a long time in a dry state. It is an antioxidant that can withstand strong acids and alkalis. In the cell wall of Mycobacterium tuberculosis, unique fatty acids include phthionic acid, tuberculostearic acid, and mycolic acid, which are the main cause of nodule formation. Acids are the main cause of acid resistance. Conventional mycolic acid has been studied for its synthesis in terms of its potential as a biosurfactant. Mycolic acid has a short beta hydroxy chain and a long alpha alkyl chain of 60 to 90 carbon atoms (Takayama K, Wang C, Besra GS.Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis.Clinical microbiology Reviews. 2005, 18. 81-101)

Atopic dermatitis is a chronic inflammatory skin disease that is distinguished from allergic diseases. Atopic dermatitis has recently been on the rise, especially in prevalence and incidence in infants and young children. In the 1960s, about 3 to 4% of atopic patients occurred, but in the 1980s, about 10 to 15% of patients showed a distribution of atopic patients. The disease is characterized by causing itching, which causes damage to the skin. In the case of atopic dermatitis, Th2 cytokines dominate, resulting in an increase in immunoglobulin IgE. These results also result from hypersensitivity to house dust mites (Georgia Avgerinou, MD, Andreas V. Goules, MD, PhD, Panayiotis G. Stavropoulos, MD, and Andreas D. Katsambas, MD.Atopic dermatitis: new immunologic aspects International Journal of Dermatology . 2008.47. 219-224)

In addition, T helper2 cytokine (IL-4, IL-5, IL-13) shows superior expression to T helper1 cytokine (IFN-r) in asthma disease, an adaptive immune disease caused by foreign antigens. [Suarez CJ, Parker NJ , Finn PW. Innate immune mechanism in allergic asthma. Curr Allergy Asthma Rep. 200.8. 8451-8459]

On the other hand, when M. tuberculosis was used, it was reported that the disease was improved by lowering serum IgE value and inducing Th1 cytokine secretion in asthma and atopic patients. (Shirakawa T, Enomoto T, Shimazu S, Hopkin JM.The inverse association between tuberculin responses and atopic disorder.Science. 1997 Jan 3; 275 (5296): 77-79)

Applicant has conducted research on the synthesis of mycolic acid derivatives useful as surfactants and obtained a patent. (Korean Patent Registration No. 05423639 'Method for preparing 2-branched chain-3-hydroxy fatty acid and salts thereof') And Korean Patent Registration No. 729146 'Method for producing 2-alkyl-3-hydroxy fatty acid and its derivatives')

The present inventors completed the present invention by confirming that a particular mycolic acid derivative exhibited a result of alleviating skin damage in an atopic disease model while a study using a mycolic acid derivative was pending.

Therefore, an object of the present invention is to provide a composition containing a mycolic acid derivative as an active ingredient, which has immunomodulatory activity and exhibits anti-inflammatory action.

In addition, an object of the present invention is to provide a composition for the treatment of allergic diseases or immune diseases, including atopic dermatitis, asthma, allergic rhinitis and excellent therapeutic effects without side effects such as toxicity.

According to the present invention to achieve the above object is provided an immunomodulatory and anti-inflammatory composition comprising a mycolic acid derivative represented by the following structural formula as an active ingredient.

(Wherein R ₁ and R ₂ are each a straight or branched alkyl group having 1 to 90 carbon atoms)

In the above structural formula, R ₁ , R ₂ are each a compound having 6 carbon atoms, or R ₁ , R ₂ is a linear alkyl group having 16 or 18 carbon atoms, respectively, excellent in immunomodulatory activity and anti-inflammatory activity.

In the composition of the present invention, the mycolic acid derivative as an active ingredient is preferably contained in an amount of 0.01 to 50% by weight based on the total weight of the composition. When included in less than 0.01% by weight it is not possible to obtain the desired activity, when it exceeds 50% by weight there is a problem in the stability of the formulation.

The composition of the present invention is particularly suitable for use in the treatment of allergic diseases or immune diseases including atopic dermatitis, asthma, allergic rhinitis. The composition of the present invention can be applied in the form of external skin preparations, injections, or inhalants or oral drugs having a formulation such as cream, gel, lotion, paste, ointment, emollient, liposome, skin tonic.

The daily dosage of the composition of the present invention is preferably in the range of 1 to 10 mg / kg. However, the specific dosage may vary depending on the weight, age, sex, health condition, administration method, administration time and disease state of the patient.

Compositions containing mycolic acid derivatives according to the present invention can be usefully used as a composition for the treatment of immune diseases such as atopic dermatitis or asthma disease because it shows excellent immunomodulatory activity and anti-inflammatory activity without side effects such as toxicity.

Hereinafter, an immunomodulatory and anti-inflammatory composition according to the present invention will be described in detail.

The immunomodulatory and anti-inflammatory composition according to the present invention contains a mycolic acid derivative represented by the following formula as an active ingredient.

(Wherein R ₁ and R ₂ are each a straight or branched alkyl group having 1 to 90 carbon atoms)

In the above structural formula, R ₁ , R ₂ are each 6 carbon atoms, or R ₁ , R ₂ is a linear alkyl group having 16 or 18 carbon atoms, respectively, is excellent in immunomodulatory activity and anti-inflammatory activity.

In the composition of the present invention, the mycolic acid derivative as an active ingredient is preferably contained in an amount of 0.01 to 50% by weight based on the total weight of the composition. When included in less than 0.01% by weight it is not possible to obtain the desired activity, when it exceeds 50% by weight there is a problem in the stability of the formulation.

The composition of the present invention is particularly suitable for use in the treatment of allergic diseases or immune diseases including atopic dermatitis, asthma, allergic rhinitis. The composition of the present invention can be applied as a skin external preparation having a formulation of creams, gels, lotions, pastes, ointments, emollients, liposomes, skin tonics, and the like, and injections or formulations of inhalants or oral drugs.

The composition of the present invention is involved in regulating immune function by regulating Th1 and Th2 cytokines in vivo.

Patients suffering from atopic dermatitis, asthma, or allergic rhinitis usually exhibit an inflammatory response, and the Th2 cytokine level is overexpressed compared to Th1 cytokine, indicating that the immune regulation is abnormal. By administering the mycolic acid derivative composition according to the present invention it is possible to control the inflammatory response and the immunomodulatory response. When the topical agent of mycolic acid derivatives was applied to atopic model mice, the expression of CRAMP, an antimicrobial peptide, was increased, and the loss of transdermal moisture and the water content were increased, which was effective in repairing damaged skin barrier.

In addition, when the injection of the mycolic acid derivative according to the present invention was applied to Th2 cytokine overexpressing mice, immunoglobulin G1 (IgG1) was decreased and the expression of Th1 and Th2 cytokines was also reduced. Mycolic acid derivatives are ingested by macrophages and form foam in macrophages.

[Example]

Hereinafter, the present invention will be described in detail with reference to the following examples and test examples, but the present invention is not limited to these examples.

Example  One: Mycolic acid  Synthesis of Derivative Compounds

Applicant's registered patent (Korean Patent Registration No. 05423639 'Method for preparing 2-branched chain 3-hydroxy fatty acid and its salt' or Korean Patent Registration No. 729146 '2-Alkyl-3-hydroxy fatty acid and its derivatives) In the above formula, R ₁ and R ₂ are linear alkyl having 6 carbon atoms, respectively, through the ring-opening reaction of the alkylketene dimer under the basic conditions and the reduction reaction of the beta-witch ketone group. Phosphorus mycolic acid derivative compounds were prepared.

Example  2: Mycolic acid  Synthesis of Derivative Compounds

Mycolic acid derivative compounds (C1618, ie C16 / 16, C16 / 18, C18 / 16, C18, wherein R ₁ and R ₂ are linear alkyl groups having 16 or 18 carbon atoms, respectively) as in the method of Example 1 above. / 18 mixture of compounds).

Example  3: injection preparation

0.05%, 0.1% mycolic acid synthesized in Example 1 was dissolved in propylene glycol to prepare an injection.

Example  4: Liposome  Produce

In order to overcome the properties of poorly soluble mycolic acid, liposome type mycolic acid was prepared as follows. 20 mg of phosphatidylcholine (Phosphatidylcholine) and 180 mg of chloroform (chloroform) are mixed with 15 mg each of mycolic acid (C6, C1618), TDM, and mock and sonicated for 5 minutes. Using a vacuum pump, the solvent was evaporated for 6 hours or more so that no residual solvent remained, and then 15 ml of PBS was added and sonicated for 30 seconds to prepare a liposome type mycolic acid.

After applying the mycolic acid derivative compound to the atopic dermatitis model as an external preparation, the anti-inflammatory function of the external preparation containing the mycolic acid derivative as an active ingredient was observed by observing the expression level of the antimicrobial peptide and the recovery of the damaged skin. . To this end, mycolic acid derivatives were used as external preparations and applied to atopic dermatitis model mice, and the expression level of CRAMP, an antimicrobial peptide, was measured. In addition, the degree of percutaneous moisture loss and moisture content were measured to observe the skin damage recovery of the mice.

Furthermore, OVA / Alum was injected into normal mice to make a mouse model overexpressing Th2 cytokine level, and then injected with a mycolic acid derivative compound as an injection, and then the Th1 and Th2 cytokine levels were measured to include mycolic acid derivatives. The immunomodulatory function of the injection was observed.

To this end, injections were made with mycolic acid derivatives and injected into Th2 cytokine overexpressing mice to measure the expression levels of IgG1, IL-4 and IFN-r.

Test Example  1: Efficacy Evaluation Trials for Atopic Dermatitis Animal Models

After percutaneous sensitization by applying 5% Oxazolone solution once to the abdomen of 6-8 weeks old hairless mice, 0.1% Oxazolone solution was applied 6 times every other day from 1 week, and 1% Oxazolone ) Was applied four times every other day to build an atopic dermatitis animal model. In animal models of atopic dermatitis using oxazolone, reduction of antimicrobial peptides in the epidermis has been reported (Man MQ, Hatano Y, Lee SH, et al . Characterization of a hepten-induced, murine model with multiple features of atopic dermatitis:. structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges J Invest Dermatol (2008) 128, 79~86). A substance (mycolic acid) synthesized according to Examples 1 and 2 diluted in the vehicle at the concentration of vehicle (PEG: EtOH = 7: 3) and 0.1% on the skin of the animal model of atopic dermatitis constructed as described above was am Apply once a afternoon for a total of four days. The hydration of the skin and the thickness of the skin were measured on the last day.

The obtained result is shown to FIG. 1A. From these results, it was confirmed that the mycolic acid derivatives synthesized according to the example had an anti-inflammatory effect in an atopic dermatitis animal model.

In addition, after applying the compound synthesized according to Examples 1 and 2 for 4 days, skin tissue was biopsied to make a paraffin block. Tissue was attached on slides using a paraffin cutter. Peroxides blocking reagent was reacted for 30 minutes after loading 500ul. PBS solvent was washed three times at 5 minute intervals. Protein blocking reagent was reacted for 15 minutes after loading 500ul. Primary goat anti-mouse CRAMP was reacted at 25 ° C. for 30 minutes each. As a secondary antibody, donkey anti-goat IgG-HRP was reacted at 25 ° C. for 30 minutes. DAB, a color developing reagent, was reacted for 10 minutes. After the reaction, the expression of CRAMP was measured in the stratum corneum of the skin through a microscope, and the obtained result is shown in FIG. 1B.

From these results, it was confirmed that the mycolic acid derivative has an anti-inflammatory effect.

Test Example  2: Immunomodulatory Action Evaluation Test 1

In the abdomen of 8-week-old C57BL / 6 A mice, 50 ug of Ovalbumin was dissolved in PBS buffer and injected. As in Example 3, 0.05% and 0.1% Mycolic acid was dissolved in propylene glycol. Ovalbumin was added to mycolic acid and mixed. After 14 days, a second injection was performed. After a week, blood was drawn from each group of mice. After centrifugation at 10,000 rpm for 10 minutes, the serum was stored at -70 ° C. (Kurosaka K, Chen Q et al . Mouse cathelin-related antimicrobial peptide chemoattracts leukocytes using formyl peptide receptor-like 1 / mouse formyl peptide receptor-like 2 as the receptor and acts as an immune adjuvant. J Immunol . 174: 6257-65.

Serum was used to measure IgG1. First, the capture antibody was diluted and dissolved in a coating buffer, and then put into a 96well plate and stored at -4 ° C for one day. The next day, Tween-20 was added 0.05% to PBS buffer, and the plate was washed three times. Blocking buffer containing 10% FBS was put into each well 200ul and reacted at room temperature for 1 hour. 300ul of 0.05% PBS-Tween20 (Washing buffer) was washed three times. 100ul each sample diluted 1: 1000 with a standard sample, and reacted at -4 ℃ for one day. Next day, wash plate was added and plate 3 was washed. Biotin Rat anti-mouse IgG1 was diluted 1: 1000 and 100ul was added to each well. 300ul of 0.05% PBS-Tween20 (Washing buffer) was washed three times. After diluting Steptavidin-HRP 1: 1000, 100ul was added to each well. 300ul of 0.05% PBS-Tween20 (Washing buffer) was washed three times. 100 μl of the coloring reagent, TMB solution, was added to each well and reacted at room temperature for 30 minutes. The reaction is stopped by adding 50ul of 2N sulfuric acid. After the reaction, the absorbance was measured at 450 nm, and the obtained result is shown in FIG. 2.

From these results, it was confirmed that the mycolic acid derivative has an immunomodulatory action.

Test Example  3: Intake observation test by macrophage

Mycolic acid liposomes, TDM liposomes, and liposomes prepared in Example 4 were injected into the abdomen of 4-6 week-old C57BL / 6 mice, each 100 ug. After 48 hours of injection, ascites fluid was collected from the mice to isolate and grow macrophages only, plated onto plates, stained with Giemsa, and observed with an optical microscope. The results are shown in FIG. 3.

From these results, it was confirmed that the mycolic acid derivative is ingested by macrophages.

Test Example  4: immunomodulatory activity evaluation test 2

In the abdomen of 4-6 week-old C57BL / 6 mice, a first injection of 20 μg and 2 mg of OVA and Alum, respectively, was injected. Two weeks later, 100 μg of mycolic acid (C6, C1618) liposomes, TDM liposomes, and liposomes prepared according to Examples 1 and 2 together with the same concentration of OVA / Alum were injected in a second dose of 100 ug. Two days later, the ascites fluid and spleen were collected from them, and splenocyte mononuclear cells of macrophages were isolated and grown. At this time, OVA (10ug / ml) and LPS (0.1ug / ml) were added to each of the media together with the control group which did not put anything in each sample media. The samples were grown for two days. After two days, each media was walked at -70 ℃. Stored in.

The media obtained from macrophages was measured using IL-4 ELISA kit (BD biosciences) to measure the expression level of IL-4, and the results are shown in FIG. 4A. The media obtained from splenocytes were shown in IFN-r ELISA kit (BD biosciences). ) To measure the expression level of IFN-r is shown in Figure 4b.

From these results, it was confirmed that the mycolic acid derivative has an immunomodulatory effect by Th1 and Th2 cytokines.

Figure 1a is a graph showing the degree of damaged skin recovery in the atopic animal model of the synthetic material of the present invention.

Figure 1b is a photograph showing the degree of expression of the antimicrobial peptide CRAMP in the stratum corneum of the skin when the synthetic material of the present invention is applied to an atopy animal model.

Figure 2 is the result of measuring the expression level of IgG1 when injected into the mouse compound of the present invention.

3 is a photograph of macrophages recovered and stained after injecting the synthetic material of the present invention into the mouse.

Figures 4a and 4b is a graph showing the results of measuring the expression level of IL-4 and IFN-r with macrophages and splenocytes, respectively, after injecting the compound of the present invention into the mouse.

Claims (5)

An immunomodulatory and anti-inflammatory composition comprising a mycolic acid derivative represented by the following structural formula as an active ingredient.

(Wherein R ₁ and R ₂ are each a straight or branched alkyl group having 1 to 90 carbon atoms) The immunomodulatory and anti-inflammatory agent according to claim 1, wherein R ₁ and R ₂ of the structural formulas each have 6 carbon atoms, or R ₁ and R ₂ are linear alkyl groups each having 16 or 18 carbon atoms. Composition. The immunomodulatory and anti-inflammatory composition according to claim 1 or 2, wherein the mycolic acid derivative is contained in an amount of 0.01 to 50% by weight based on the total weight of the composition. The immunomodulatory and anti-inflammatory composition according to claim 1 or 2, wherein the composition is for treating allergic diseases or immune diseases including atopic dermatitis, asthma and allergic rhinitis. According to claim 1 or claim 2, wherein the composition is a cream, gel, lotion, paste, ointment, emollients, liposomes, skin tonic, such as external skin or injection, or inhalation or oral drug formulations, characterized in that Immunomodulatory and anti-inflammatory compositions.

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