KR20150057503A - composition for preventing or treating neuromyelitis optica, comprising glycyrrhizic acid or pharmaceutical acceptable salts thereof - Google Patents

Abstract

The present invention relates to: a pharmaceutical composition including glycyrrhizic acid or pharmaceutically acceptable salts thereof for preventing or treating neuromyelitis optica (NMO); a food composition for preventing or treating NMO; and a method for treating NMO including a step of injecting the pharmaceutical composition into an object.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating optic neuritis, comprising glycyrrhizin acid or a pharmaceutically acceptable salt thereof as an active ingredient,

The present invention relates to a pharmaceutical composition for the prevention or treatment of neuromyelitis optica (NMO), which comprises glycyrrhizic acid or a pharmaceutically acceptable salt thereof as an active ingredient, a pharmaceutical composition for preventing or ameliorating optic neuritis A food composition and a method of treating optic neuropathy comprising administering the composition to a subject.

Neuromyelitis optica (NMO) is one of the central nervous system dehydrator diseases called Devic disease. It is characterized by optic neuritis of unilateral or bilateral and longitudinally extensive myelitis involving more than three segments of the vertebra Lt; / RTI > In the past, NMO was thought to be a subtype or a variant of MS due to its clinical features similar to multiple sclerosis (MS). However, recently, several animal studies and clinical studies have shown that NMO pathology is aquaporin- , AQP4) were identified as being mediated by antibodies to the water channel, NMO was recognized as a separate disease from multiple sclerosis.

The anti-aquaporin 4-autologous antibody is observed in 90% of patients with optic neuritis, but not in other diseases. Aqua purin 4 is present in the astrocytes, which are the main members of the blood brain barrier (BBB), which acts as a barrier between blood vessels and the brain. In optic neuritis, autoantibodies against aquaporin 4 bind to aquaporin 4, (Int J Biochem Cell Biol. 2012 Sep; 44 (9): 1519-30). Inhibition of BBB may be induced by stimulation of macrophages and macrophages.

Glycyrrhizic acid is a sweet tasting compound that is predominantly present in licorice root, also called glycyrrhizin. It does not dissolve well in water and is used as an artificial sweetener for beverages because of its sweet taste. Glycyrrhizin is composed of glycyrrhetinic acid and two glucuronic acids. Glycyrrhizin is a triterpenoid saponin substance in chemical structure. It is 30-50 times more sweet than sugar. And has a sweetness unique to licorice. Also, in the United States, it is generally considered to be Generally Recognized as Safe (GRAS), and is used for candy, medicines and tobacco.

It is also known that glycyrrhizic acid inhibits the development of cancer and tumors, reduces cholesterol, improves atherosclerosis, and has a liver protective effect. However, the effect of optic neuritis is still unknown.

Under these circumstances, the present inventors have made intensive efforts to identify a substance capable of effectively inhibiting the CDC response of an autoantibody to aquaporin 4 and thereby bring about the therapeutic effect of optic neuritis. As a result, it has been found that glycyrrhizin acid is an autoantibody to aquaporin 4 The present inventors completed the present invention based on the findings that it is possible to effectively inhibit the CDC response of optic nerve spinal cord,

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of neuromyelitis optica (NMO), which comprises glycyrrhizic acid or a pharmaceutically acceptable salt thereof as an active ingredient .

Another object of the present invention is to provide a food composition for preventing or ameliorating optic neuritis, which comprises glycyrrhizin acid or a pharmaceutically acceptable salt thereof as an active ingredient.

It is another object of the present invention to provide a method for treating optic neuropathy which comprises the step of administering the pharmaceutical composition to a subject.

In one aspect of the present invention, the present invention provides a method of preventing or treating neuromyelitis optica (NMO), which comprises glycyrrhizic acid or a pharmaceutically acceptable salt thereof as an active ingredient, A pharmaceutical composition is provided.

The term "glycyrrhizic acid" in the present invention means a compound represented by the following formula (1). The glycyrrhizic acid may be used in combination with Glycyrrhizin or Glycyrrhizinic acid.

The glycyrrhizic acid may be obtained by extracting and purifying from plants containing it, synthesized by a method of synthesizing a known compound, or purchased commercially and used.

In addition, the glycyrrhizic acid may be in the form of a pharmaceutically acceptable salt.

The term " pharmaceutically acceptable salt " of the present invention means salts in which the cation and anion are pharmaceutically usable among the salts in which they are bound by electrostatic attraction, Salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), an aluminum salt and the like; Examples of salts with organic bases include salts with organic bases such as triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, And the like; The salt with inorganic acid may be a salt with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like; Salts with organic acids may be salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like; Salts with basic amino acids may be salts with arginine, lysine, ornithine and the like; The salt with an acidic amino acid may be a salt with aspartic acid, glutamic acid and the like. The glycyrrhic acid compound according to the present invention can be converted into its salt by a conventional method.

Such glycyrrhizic acid or a pharmaceutically acceptable salt thereof may inhibit complement-dependent cytotoxicity (CDC) by an anti-aquaporin4 (AQP4) autoantibody, resulting in the prevention or therapeutic effect of optic neuritis.

In the present invention, the term "optic neuritis" refers to a disease characterized by the manifestation of optic neuritis in monocular or bilateral and longitudinally extensive myelitis involving more than three segments of the vertebra. The optic nerve spondylitis is characterized by the fact that the anti-aquaporin 4 autoantibody binds to aquaporin 4 present in astrocytes and induces complement-dependent cytotoxicity, thereby causing destruction of the blood brain barrier (BBB) . Thus, the glycyrrhizic acid of the present invention, which can prevent or inhibit complement-dependent cytotoxicity by anti-aquaporin 4 autoantibodies, can be used as a therapeutic agent for optic neuritis.

The term "prevention" in the present invention means any action which inhibits or delays the onset of a disease by the administration of the composition according to the present invention. The term "treatment" in the present invention means all the actions of improving or alleviating the symptoms of the disease upon administration of the composition according to the present invention.

The composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories May have one formulation, and may be various forms of oral or parenteral administration. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The composition of the present invention may be administered in a pharmaceutically effective amount.

The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level will vary depending on the species and severity, age, sex, Activity, sensitivity to the drug, time of administration, route and rate of excretion, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition 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. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. The preferred dosage of the composition of the present invention may vary depending on the condition and the weight of the patient, the degree of the disease, the drug form, the administration route and the period. The administration may be carried out once a day or divided into several times. The composition may be administered to various mammals such as rats, livestock, humans, and the like in a variety of routes. The mode of administration is not limited as long as it is a conventional method in the art. For example, oral, rectal or intravenous, Subcutaneous, intra-uterine dural or intracerebral injection.

In one embodiment of the present invention, when a cell line overexpressing aquaporin 4 was established and when the cell line was treated with IgG isolated from the serum of patients with optic nerve schizophrenia, (Figs. 1 to 3). From this, optic neuritis in vitro Model. Then, the above-mentioned aquaporin-4 over-expressing cell line was directly treated with the separated plasma of the optic nerve spinal cord patient, treated with glycyrrhizin acid by concentration, and then the secretion of LDH (lactate dehydrogenase) The results showed that glycyrrhizin acid suppressed LDH secretion in a concentration dependent manner and glycyrrhizic acid could be used as a therapeutic agent for optic neuritis by inhibiting CDC by anti - (Fig. 5). In addition, using the optic nerve spinal mimic model established through FIGS. 1 to 3, it was confirmed that the complement-dependent cytotoxic response by IgG isolated from optic neuritis patients could be inhibited by glycyrrhizin acid As a result, it was confirmed that the glycyrrhizic acid could be a therapeutic agent for optic neuritis (FIG. 6).

In another aspect, the present invention provides a food composition for preventing or ameliorating optic neuritis, comprising glycyrrhizin acid or a pharmaceutically acceptable salt thereof as an active ingredient.

The glycyrrhizic acid, its pharmaceutically acceptable salt and optic nerve spondylitis are as described above.

The glycyrrhizic acid or a pharmaceutically acceptable salt thereof may be added to the food composition for the purpose of preventing or ameliorating optic neuritis. When the compound or a pharmaceutically acceptable salt thereof is used as a food additive, it may be added as it is or may be added together with other foods or ingredients. The addition amount thereof can be appropriately determined depending on the purpose of use.

The kind of the food of the present invention is not particularly limited. Examples of the food to which the compound or a pharmaceutically acceptable salt thereof can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, Soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include all foods in a conventional sense, and foods used as feedstuffs for animals.

In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , A carbonating agent used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. The food may also be prepared in the form of tablets, granules, powders, capsules, solutions in liquid form, and the like according to known production methods. There are no particular restrictions on the other ingredients other than the glycyrrhizic acid or its pharmaceutically acceptable salt of the present invention as an active ingredient, and it may contain various conventional flavors or natural carbohydrates as an additional ingredient.

In another aspect, the present invention provides a method for treating optic neuropathy comprising the step of administering the pharmaceutical composition to a subject.

The glycyrrhizic acid, its pharmaceutically acceptable salt and optic nerve spondylitis are as described above.

Specifically, the method of treatment of the present invention comprises administering a pharmaceutical composition comprising the glycyrrhinic acid, or a pharmaceutically acceptable salt thereof, in a suspected optic neuropathic subject. The term refers to whole mammals including dogs, cows, horses, rabbits, mice, rats, chickens or humans, but the mammal of the present invention is not limited by these examples. The pharmaceutical composition may be administered non-oral, subcutaneous, intraperitoneal, intrapulmonary or intranasal, and may be administered by a suitable method, including localized administration, if necessary, for local treatment. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the individual, the severity of the disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art.

The composition comprising the glycyrrhizic acid of the present invention effectively inhibits cytotoxicity by the aquaporin 4-autologous antibody, which is regarded as a major therapeutic target in optic nerve spondylitis, and thus can be effectively used for prevention or treatment of optic neuritis.

Brief Description of the Drawings Fig. 1 is a diagram (B) showing the sequence of the pIRES2-AQP4-DsRed2 vector sequence and the overexpression of aquaporin 4 (APQ4) in the U87MG cell line into which it is introduced.
FIG. 2 shows the immunocytochemical analysis of binding between IgG and APQ4 by treating IgG isolated from patients with optic neuritis with U87MG AQP4 and HEK293T AQP4.
FIG. 3 shows a cytotoxic effect induced by APQ4 autoantibodies in astrocytes of optic neuritis patients (A) and a cytotoxic response induced by IgG isolated from patients with optic neuritis (FIG. 3) B).
FIG. 4 shows the results of confirming the degree of cytotoxicity when plasma proteins of optic nerve spinal cord patients were directly treated with AP87 over-expressing U87MG cells.
FIG. 5 is a graph showing the degree of cytotoxicity after treatment of platelets of patients with optic neuritis in APQ4-overexpressing U87MG cell line, treatment of glycyrrhizic acid by concentration, and the like.
FIG. 6 is a graph showing the degree of cytotoxicity after treatment of IgG isolated from patients with optic neuritis in APQ4 over-expressing U87MG cell line, treatment of glycyrrhizic acid concentration by concentration.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the scope of the present invention is not limited by the following examples.

Example 1: Optic neuropathy in vitro Establishment of experimental model

(1) Establishment of aquaporin 4 (AQP4) overexpressing cell line

The present inventors used a cell line expressing human AQP4 to establish a live cell based AQP4-antibody binding assay and to establish an in vitro model using NMO for patient NMO IgG. human glioblastoma cell line was transfected with human M23 AQP4-DsRed2 to establish an AQP4 over-expressing cell line.

Specifically, pIRES2-AQP4-DsRed2 (Ds-Red2 tagged AQP4 Vector: kind donation form Dr. Waters and Professor Vincent in Oxford University) was transfected into U87MG cell line using Lipofectamine 2000 (Invitrogen) AQP4 over-expressing cell line was established (Fig. 1)

(2) IgG purification in the serum of the collected patients

Plasma proteins were collected from patients who underwent acute phase plasmapheresis and stored in a -80 ° C freezer. The stored plasma proteins were purified by IgG purification using Montage IgG purification kit (Millipore, Billerica, Mass.) After 4 hours of thawing at 4 ° C for IgG separation.

Then, IgG (30 μg / μl) of the separated patient was subjected to SDS-PAGE gel to confirm that the patient's IgG was separated by a coma blue color.

The immunohistochemical staining of AQP4 overexpressing AQP4 and AQP4 (AQP4) was performed using 4% PFA (paraformaldehyde) and reacted with IgG for 30 min. As a result, the binding of isolated IgG to the patient was increased and the concentration of the IgG was increased in a dose-dependent manner (FIG. 2).

(3) NMO in vitro  Establishment of model - AQP4 overexpressing cell line

In vitro NMO model of complement dependent cytotoxicity (CDC) based on binding between AQP4 and NMO IgG was constructed using U87MG AQP4 cells and primary astrocyte as AQP4 overexpressing cell line model.

This method is based on the observation that complement dependent cytotoxicity is induced by the anti-aquaporin 4 antibody in optic nerve spondylitis patients, specifically, that the anti-aquaporin 4 antibody binds to aquaporin and binds to C1q, leading to apoptosis 3A).

Specifically, CDC was assayed using a LDH (Lactate Dehydrogenase) assay using IgG and human complement (Innovative Research) isolated from patient plasma proteins in U87MG AQP4 cells. More specifically, U87MG AQP4 cells were treated with U87MG cells for 1 hour by adding the patient's IgG and complement to a HEPES control salt salt solution, and the amount of LDH released was measured. The results are shown in FIG. 3B .

As a result, as shown in Fig. 3B, the amount of LDH secretion increased with the amount of IgG. In addition, since the degree of binding of IgG is different depending on the patient, the degree of cytotoxicity is different in each patient

In addition, the degree of cytotoxicity occurred when the plasma protein of the patient was directly treated with the cells was examined, and the result is shown in FIG.

As a result, as shown in FIG. 4, the increase in plasma protein binding resulted in an increase in the degree of cytotoxicity, so that the results were correlated with plasma binding activity as shown in FIG.

Example 2: Effect of glycyrrhizic acid on optic neuritis

The NMO in vitro model established in Example 1 above was used to confirm the effect of glycyrrhizin on optic neuritis.

First, the plasma isolated from the optic nerve spinal cord patient was directly treated with U87 cell line overexpressing AQP4, and the degree of secretion of LDH was measured after glycyrrhizic acid (Sigma) was treated in a concentration-dependent manner. Respectively.

As a result, as shown in Fig. 5, glycyrrhizin acid effectively inhibited cytotoxicity induced from plasma of patients with optic neuritis, in a concentration-dependent manner.

In addition, since various inflammatory mediators (IgG, NK cell, complement, cytokine, etc.) are present in serum, confirmation of cytotoxicity using whole plasma may not be specific to complement dependent cytotoxicity, which is the main pathogenesis of optic neuritis , IgG isolated from patients with optic neuritis was treated with U87 cell line overexpressing AQP4, and glycyrrhizic acid was treated in a concentration-dependent manner, and the level of LDH secretion was measured. The results are shown in FIG.

As a result, the glycyrrhizic acid inhibited cytotoxicity by IgG isolated from patients with optic neuritis, in a concentration-dependent manner, and the result was consistent with FIG. 5.

All of the above results suggest that glycyrrhizin acid effectively inhibits optic neuropathy induced by AQP4 autoantibody, and thus may have an effect of treating optic neuritis.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (6)

A pharmaceutical composition for the prevention or treatment of optic neuromyelitis optica (NMO) comprising glycyrrhizic acid or a pharmaceutically acceptable salt thereof as an active ingredient.
2. The composition of claim 1, wherein the glycyrrhizin acid or a pharmaceutically acceptable salt thereof inhibits complement-dependent cytotoxicity (CDC) by an anti-aquaporin4 (AQP4) autoantibody.
The composition of claim 1, wherein the composition comprises a pharmaceutically acceptable carrier.
The composition of claim 1, wherein the composition is in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, nonaqueous solutions, suspensions, emulsions, lyophilized preparations, ≪ / RTI > wherein the composition has at least one pharmaceutically acceptable carrier.
A food composition for the prevention or amelioration of optic neuromyelitis optica (NMO), comprising glycyrrhizic acid or a pharmaceutically acceptable salt thereof as an active ingredient.
A method for treating optic neuropathy, comprising administering the pharmaceutical composition of any one of claims 1 to 4 to an animal other than a human.

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