KR101324647B1 - Composition for treating or preventing of multiple sclerosis and screening thereof - Google Patents

Abstract

The present invention relates to a composition for treating or preventing multiple sclerosis and a screening method thereof, and more particularly, to a composition and a screening method for effectively treating or preventing multiple sclerosis without concern of side effects or toxicity.

Description

Composition for treating or preventing multiple sclerosis and screening method thereof {COMPOSITION FOR TREATING OR PREVENTING OF MULTIPLE SCLEROSIS AND SCREENING THEREOF}

The present invention relates to a composition for treating or preventing multiple sclerosis and a screening method thereof, and more particularly, to a composition and a screening method for effectively treating or preventing multiple sclerosis without concern of side effects or toxicity.

There have been many attempts to develop various drugs to alleviate the symptoms of multiple sclerosis (MS) or to suppress the function of the immune system, but until now, no drug has been reported that can effectively treat the disease. In relation to therapeutic drugs, there have been relatively many studies to develop drugs that act on the immune system because the mechanism of expression of multiple neurosclerosis is caused by the autoimmune system.

In this regard, Betaseron (interferon (IFN) -ß), developed by Berlex Laboratories in the United States as an immunosuppressant for cytokines and proteins, is currently clinically used by FDA in 1993 (Panitch). , Mult. Scler. 1, S17-S21, 1995; Crucian et al., Clin.Diagn.Lab.Immunol. 3, 411-416, 1996; Johnson, J. Neural Transm.Suppl. 49, 111-115, 1997 ). It has been reported to be effective in the treatment of patients with relapsing-Remitting multiple neurosclerosis, by reducing T-cells secreting IFN-γ, which is involved in generating immune responses (Crucian et al., Clin. Diagn. Lab. Immunol. 3, 411-416, 1996). IFN-ß also inhibited T-cell proliferation and blocked CD25 induction in stimulated T- and B-lymphocytes. IFN-γ, tumor necrosis factor-a (TNF-a), and interleukin (IL) -13 were inhibited by IFN-ß, but IL-2 was secreted twice, and IL-10 secretion was nearly increased 4 times (Rep et al., Neuroimmunol, 67, 111-118, 1996).

Coploymer I (Cop I; myelin basic protein analogue; Copaxone), a synthetic copolymer of amino acids, has been reported to be very effective in inhibiting experimental allergic encephalomyelitis (EAE), an animal model of multiple neurosclerosis. Cop I did not induce EAE and inhibited EAE in several species of animals. This is achieved by inducing immune cross-reaction between MBP (myelin basic protein) and Cop I and suppressor of antigen specificity, and by competing with MBP binding to major histocompatibibty complex (MHC) molecules. Cop I reduced the exacerbation rate and improved the patient's condition in Relapsing-Remitting MS patients in clinical phase II and phase III (Teitelbaum et al., J. Neural. Transm. 49, 85-91, 1997 ).

Drugs belonging to immunosuppressive synthetic compounds include linomide, corticosteroids, azathioprine, cyclophosphamide, cyclosporin A, cladribine and colchicine. Linomide (quinoline-3-carboxamide) strongly inhibited EAE induction during subcutaneous injection in rats, ameliorated clinical symptoms of EAE, and inhibited myelin antigen-induced T-cell responses. It has also been reported to upregulate IL-4, IL-10 and tumor growth factor-ß (TGF-ß) and to inhibit the proinflammatory cytokines IFN-a and TNF-a (Diab et al., J. Neuroimmunol. 85, 146-154, 1998).

High doses (1000 mg, iv, 4 days) of prednisolone increased serum IL-10 concentration and IL-10 mRNA expression in clinical trials, in contrast to cytokines TNF-a and IFN- which mediate inflammation. γ reduced mRNA expression (Gayo et al., J. Neuroimmunol. 85, 122-130, 1998).

Azathioprine caused a decrease in TNF-a and an increase in suppressor-inducer lymphocytes in patients with multiple sclerosis (Salmaggi et al., J. Neurol. 244, 167-174, 1997). Azachioprin had 23 out of 1191 patients with multiple neurosclerosis who developed cancer before December 31, 1991 (Confavreux et al., Neurol. 46, 1607-1612, 1996). It has also been reported to cause a decrease in the synthesis of intrathecal IgG in patients with multiple neurosclerosis treated with azacioprine (Caputo et al., Acta. Neurol. Scand. 75, 84-86, 1987).

Cladribine has been shown to improve symptoms as a result of measuring neurological performance scores in a two-year clinical study of patients with multiple sclerosis at the Scripps Institute (Beutler et al., Proc. Natl. Acad. Sci. USA 93, 1716-1720, 1996). In vitro, colchicine treatment of antigen presenting cells and T-cell mixtures inhibited T-cell proliferation (Tateyama, Hokkaido Igaku Zasshi 63, 72-79, 1988). Other inhibitors of immune function include cyclophosphamide (Ten Berge et al., Cell. Exp. Immunol. 50, 495-502, 1982) and cyclosporin A.

In addition, baclofen (GABA agonist) is used to reduce spasticity along with tizanidine, dantrolene sodium, diazepam, and carbamazepine as an antiepiletic drug, and amantadine is used as a drug to improve the symptoms of multiple neurosclerosis. To improve fatigue among the symptoms of sclerosis patients, delta 9-tetrahydrocanabinol is used to prevent nausea and increase appetite due to spinal cord spasticity.

As described above, various drugs for treating or preventing the symptoms of multiple sclerosis have been studied, but there are concerns about side effects and toxicity, and there are still no effective materials for treating or preventing multiple sclerosis. The development of therapeutics is required.

Accordingly, the present invention has been made to solve the problems of the prior art, and an object of the present invention is to provide a pharmaceutical composition for effectively treating multiple sclerosis without fear of side effects or toxicity.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment or prevention of multiple neurosclerosis comprising LeuSH as an active ingredient.

In addition, the present invention comprises the steps of a) preparing a sample; b) adding a substrate of NADPH oxidase to the sample; c) contacting the candidate with the sample added with the substrate; And d) identifying a reaction between the sample and the candidate substance, and selecting a substance that inhibits the activity of NADPH oxidase, thereby providing a method for screening a substance for treating or preventing multiple sclerosis.

In addition, the present invention comprises the steps of: e) preparing a test animal sample induced experimental allergic encephalomyelitis (EAE) disease; f) administering a candidate to said animal sample inducing said EAE disease; g) comparing the body weight or clinical symptoms of the animal sample to which the candidate substance is administered with the animal sample to which the candidate substance has not been administered, and selecting a substance that significantly improves the weight or clinical symptoms of the sample. It provides a method for screening a material for the treatment or prophylaxis.

Hereinafter, the present invention will be described in more detail.

According to one embodiment of the invention, there is provided a pharmaceutical composition for the treatment or prevention of multiple neurosclerosis comprising LeuSH as an active ingredient.

As a result of the experiments of the present inventors, the novel use of LeuSH (L-Leucinethiol) has been found to inhibit the activity of NADPH oxidase and / or MMP (matrix metalloproteinase), due to this effect of multiple neurosclerosis The present invention has been completed by confirming that it can be usefully used for treatment or prevention.

The LeuSH (L-Leucinethiol) has the following structure, and has the characteristics of reducing the activity of NADPH oxidase and MMP, in particular MMP9.

NADPH oxidase is an enzyme involved in the production of super oxide ions (O ₂ −) from oxygen (O ₂ ). When the NADPH oxidase is activated, free radicals increase in blood vessels, thereby activating MMP. MMPs play an important role in breaking down extracellular matrix.

Since the activity of the NADPH oxidase and / or MMP9 is increased in the case of multiple neurosclerosis, when developing a substance that can reduce the activity of the NADPH oxidase and MMP9, it can be usefully used for the treatment or prevention of multiple sclerosis As a result of the study by the present inventors, the present inventors have completed the present invention by confirming that LeuSH exhibits a marked improvement in clinical symptoms and weight in the EAE-induced mouse model.

Therefore, the pharmaceutical composition comprising LeuSH of the present invention as an active ingredient can be usefully used to effectively treat or prevent multiple neurosclerosis without showing problems such as side effects or toxicity such as conventional protein drugs.

In the present invention, the term treatment refers to alleviation or amelioration of symptoms, reduction in the extent of disease, delay or alleviation of disease progression, improvement, alleviation or stabilization of disease state, partial or complete recovery, prolongation of survival, or any other beneficial treatment outcomes, etc. Used to include all of them.

The amount of LeuSH, which is the active ingredient, may be a content suitable for the treatment or prevention of the subject and / or disease, which may include age, weight, general health, sex and diet, time of administration, type of disease, and severity of disease. It can be adjusted according to various factors, including the type and content of other ingredients contained in the composition, the type of formulation, the route of administration, the rate of release of the composition, the duration of treatment and the drug used concurrently. For example, if the subject is an adult, a total of 6 to 10 mg / kg (body weight), preferably 8, based on the weight of LeuSH, which is an active ingredient, when the pharmaceutical composition of the present invention is administered or taken once or several times a day. A dose of mg / kg (body weight) may be administered or taken in an effective amount, and the weight of LeuSH, an active ingredient in the total composition, may be appropriately adjusted depending on the type and effect of the desired formulation, for example, the total composition weight It may be preferably 95.0 to 99.9% by weight, more preferably 97.0 to 99.9% by weight, but is not limited thereto. However, it will be apparent to those skilled in the art that the dosage or dosage of the active ingredient should be such that the content of the active ingredient is too high so as not to cause side effects.

Another embodiment of the present invention is a method for treating or preventing multiple sclerosis in vitro or in It provides a method for screening in vivo .

The inventors have for the first time identified that by inhibiting NADPH oxidase it can treat or prevent multiple neurosclerosis.

Therefore, one embodiment of the present invention provides a method for screening a substance for the treatment or prevention of multiple neurosclerosis by identifying the NADPH oxidase inhibitory activity.

The screening method can be carried out in vitro, specifically a) preparing a sample; b) adding a substrate of NADPH oxidase to the sample; c) contacting the candidate with the sample added with the substrate; d) checking the reaction between the sample and the candidate substance may include selecting a substance that inhibits the activity of NADPH oxidase.

Selecting a substance that inhibits the activity of NADPH oxidase by confirming the reaction between the sample and the candidate substance is not limited thereto. Preferably, see fluorescence assay (Cao et al. Hypertension, 50, 663-671, 2007). ), Preferably using apocynin, a positive indicator known as a NADPH oxidase inhibitor, as a control, and the effects of the addition of each candidate and the addition of apocynin. Comparing the effects according to the method may be made by selecting a material having an equivalent or superior effect, but is not limited thereto.

The substrate of the NADPH oxidase is preferably, but not limited to, ß-NADPH. When the NADPH oxidase generates peroxide using ß-NADPH as a substrate, the generated peroxide oxidizes Dihydroethidium (DHE), for example, to produce ethidium, and accordingly, the fluorescence assay is used to determine NADPH oxidase. By measuring the activity, a substance that effectively inhibits the activity of NADPH oxidase can be selected, and the selected substance can be usefully used for the treatment or prevention of multiple neurosclerosis.

Or in vivo Screening methods can be used; in The in vivo screening method preferably comprises the steps of e) preparing a test animal sample induced with experimental allergic encephalomyelitis (EAE) disease; f) administering a candidate to said animal sample inducing said EAE disease; g) comparing the body weight or clinical symptoms of the animal sample to which the candidate substance is administered with the animal sample to which the candidate substance has not been administered may include selecting a substance that significantly improves the weight or clinical symptoms of the sample.

Since the EAE disease-induced test animal is an animal model similar to human polyneurosclerosis, when the effect on the EAE disease is recognized, the effect on multiple neurosclerosis can be expected.

Inducing the EAE disease in the test animal sample is not particularly limited and may be performed using a known method. Induction of EAE disease can preferably be confirmed by measuring body weight and / or clinical symptoms of the biological sample.

When EAE disease is induced, candidates are administered to the sample, and the body weight and / or clinical symptoms after EAE disease induction of the EAE disease induction group in which the candidate substance is administered and the candidate substance is not compared. Based on the comparative results, significant gains in treatment or prevention of multiple neurosclerosis, such as increasing the subject's weight to restore weight lost due to EAE disease and / or significantly reducing or improving undesired clinical symptoms. Substances that have an effect can be selected.

The sample is in In vitro screening may be cells, tissues, blood, etc. derived from animals, preferably mammals, including humans, rodents, etc., in For in vivo screening, it can be an animal, preferably a mammal including humans, rodents and the like.

The candidate material is intended to encompass all kinds of compounds, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, or animal tissue extracts, as well as novel substances It may be well known.

Through the screening methods described above, LeuSH, an active ingredient of the pharmaceutical composition of the present invention, has the most potent inhibitory effect on NADPH oxidase activity and a marked improvement in EAE disease compared to the inhibitory effect of other substances used in the screen. It was confirmed that it can be effectively used for the treatment or prevention of multiple sclerosis.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable excipient in addition to the active ingredient, and the excipient may be a disintegrant, sweetener, binder, coating agent, swelling agent, lubricant, glidant or Solubilizers, such as a flavoring agent, can be used.

The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. When formulated in a liquid solution form, sterile and physiologically compatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol or mixtures thereof may be used as carriers. If necessary, other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added. Alternatively, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like.

In addition, the formulation and the pharmaceutically acceptable carrier of the pharmaceutical composition of the present invention is not particularly limited and may be appropriately selected according to techniques known in the art. In addition, an effective amount for the treatment or prevention of various immune-related diseases may include the type of disease, the severity of the disease, the types and amounts of the active ingredients and other ingredients contained in the composition, the type of the formulation and the age, weight, general health, and sex of the patient. And diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used concurrently. For example, if the subject is an adult, the pharmaceutical composition of the present invention may be administered in a total of 6-10 mg / kg body weight, preferably 8 mg / kg body weight, when administered or taken once to several times a day. The dose may be administered or taken in an effective amount. However, it will be apparent to those skilled in the art that the dosage or dosage of each active ingredient should be such that the content of each active ingredient is too high so as not to cause side effects.

As described above, the pharmaceutical composition of the present invention is not only effective in inhibiting the activity of NADPH oxidase and / or MMP, but also has an excellent effect in the treatment or prevention of multiple neurosclerosis, which is useful for the treatment or prevention of the disease. Can be used. In addition, by using the screening method of the present invention, in addition to the pharmaceutical composition of the present invention, there is an effect of developing a material having such an excellent effect.

Figure 1 shows the inhibitory effect of NADPH oxidase activity of the candidates according to Example 1 (n = 3 / angular concentration).
Figure 2 shows the comparison of the inhibitory effect of the candidates according to Example 1 NADPH oxidase activity represented by IC ₅₀ value.
Figure 3 shows the change in clinical score (Figure 3a) and body weight (Figure 3b) according to Example 2 (mean ± SEM (n = 6 per group), * is the statistical significance by the GLM method of SAS (* p <0.05)).

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, the following examples are merely to illustrate the present invention is not limited to the scope of the present invention.

Example  One. NADPH  Oxidase Activity Inhibitor Screening

1-1. Experimental Method

Brain tissue (whole brain) of 6-week-old female C57BL / 6 mice (20 ± 2 g body weight) was extracted and the extracted brain tissue was buffered per 50 mg of brain tissue (50 mM Tris-HCl, 10 mM CaCl ₂ , 0.25%). v / v) dissolved in 1 ml of Triton x-100), centrifuged at 6000 g for 30 minutes to obtain a supernatant, precipitated by addition of ethanol to a final concentration of 60% (v / v), and again at 10,000 g for 30 minutes. Brain tissue homogenates were prepared by centrifugation. Dihydroethidium (10 μmol / L), salmon testes DNA (Sigma) (0.5 mg / ml), ß-NADPH (0.1 mmol / L) and candidates (0-500 μM) were added to the prepared brain tissue homogenate. Reaction of NADPH oxidase activity was measured by reacting for 30 minutes in a 37 ° C. incubator in the dark. Dihydroethidium (DHE) in the added material was oxidized by peroxide to produce ethidium, and ß-NADPH was used as a substrate of NADPH oxidase to produce peroxide. 3, 4-Dichloroisocoumarin (DCC), L-Leucinethiol (LeuSH), Bestatin (BST) and 4-Amidinophenylmethanesulfonylfluoride (APMSF) were used as candidates, and the control group was a positive indicator known as an NADPH oxidase inhibitor. Apocynin was used.

The activity of NADPH oxidase is described by Cao et al. According to the method reported in Hypertension, 50, 663-671, 2007, dihydroethidium was used as a substrate and measured by fluorescence measurement. Fluorescence was measured with an luminescence spectrometer (LS 50-B, Perkin Elmer, UK) with the support of FL WinLab software (ver 4.00.02) with excitation 485 nm and emission 590 nm. Enzyme activity was expressed as nmole / minute / mg protein.

1-2. Experiment result

As a result of measuring the degree of inhibition of NADPH oxidase activity, it was confirmed that LeuSH inhibited the activity of NADPH oxidase most strongly compared to other substances used in the screen (Fig. 1 and Fig. 2).

Specifically, IC ₅₀ values were calculated to compare the effects of each material and the results are shown in FIG. 2. Compared with the inhibitory effect of NADPH oxidase on the use of the control aposinin in an amount of 25 μM, the candidates were 22.11 μM for DCC, 36.1 μM for LeuSH, 115.07 μM for BST, and APMSF showed an effect equivalent to that of 25 μM of aposinin at 284.30 μM, respectively. As can be seen from these results, DCC and LeuSH of the candidates showed a better inhibitory effect of NADPH oxidase activity, especially in the amount of 500μM LeuSH was confirmed to have the strongest inhibitory effect.

Example  2. In vivo EAE  For induced mouse models LeuSH Evaluation of effectiveness of

2-1. Experimental Method

Mix 3 mg of H37Ra Mycobacterium tuberculosis (Difco Laboratories, Detroit, MI, USA) with 1 mg / ml of complete Freund's adjuvant (Sigma) to make a 4 m / ml Mycobacterium tuberculosis-CFA solution. This solution was mixed well with a solution of 5.1 mg MOG _{35 -55} (Takara, Chungnam) dissolved in 3.4 ml of PBS. The mixture solution 200 ul (containing 400 ug Mycobacterium tuberculosis and 150ug MOG _{35 -55)} and then injected subcutaneously in female C57BL / 6 mice of 6-week-old, intraperitoneal administration of pertussis toxin (pertussis toxin) (Sigma) and 200 ng of the instant, After 2 days, the same amount of pertussis toxin was re-administered to induce EAE disease in mice, and body weight and clinical symptoms were measured to confirm that the disease was induced. Candidate LeuSH was administered intraperitoneally at doses of 1 and 8 mg / kg, respectively, once every two days, starting at day 17 after induction of EAE disease in mice, and the clinical score of mice after EAE was induced. (clinical score) (Table 1 and FIG. 3A), body weight (Table 2 and FIG. 3B), and organ weight (Table 3) were measured at the same time every day to determine the change with administration of LeuSH (mean ± SEM (n = 6 per group), * indicates statistical significance by GLM method of SAS (* p <0.05)). Clinical symptoms were divided into six stages: 0, no clinical sign; 1, flaccid tail; 2, one hind limb weakness; 3, two hind limbs weakness; 4, hind limb paralysis; 5, tetraplegia; 6, moribund / death.

2-2. Experiment result

(1) clinical score

Maximum value Cumulative value medium EAE 3.00 ± 0.258 34.17 ± 1.96 1.102 ± 0.064 LeuSH (1 mg / kg) 2.66 ± 0.211 30.33 ± 1.61 0.980 ± 0.051 LeuSH (8 mg / kg) 1.40 ± 0.245 * 16.20 ± 1.39 * 0.522 ± 0.046 *

In Table 1, * indicates that the clinical score of the high concentration of LeuSH group was significantly reduced compared to the EAE induction group (n = 6 per group, * p <0.05).

In the case of clinical scores, as shown in Table 1 and FIG. 3A, the group with the LeuSH 8 mg / kg showed a significant improvement in EAE symptoms. In the control group of EAE animals not receiving LeuSH, the clinical score reached a maximum of about 3.0 by the 21st day, but the mean value of the LeuSH-treated group was about 2.7 (1 mg / kg) and 1.4 (8 mg, respectively). / kg administration group), the clinical score was significantly reduced concentration-dependently. Statistical processing for significance test was calculated using GLM of SAS.

(2) weight

Start date (0 day) Day 10 Day 20 Day 30 Control 16.80 ± 0.151 19.14 ± 0.193 18.72 ± 0.930 20.23 ± 0.651 EAE 17.00 ± 0.415 18.26 ± 0.067 16.11 ± 0.035 * 16.60 ± 0.119 * LeuSH (1mg / kg) 16.46 ± 0.402 18.27 ± 0.307 16.31 ± 0.349 * 17.06 ± 0.240 * LeuSH (8mg / kg) 16.39 ± 0.298 17.58 ± 0.512 * 17.47 ± 0.168 ^# 18.13 ± 0.093 * ^#

As shown in Table 2 and Figure 3b, the EAE disease-induced group showed a significant weight loss on both the 20 and 30 days compared to the normal control group, the low concentration of LeuSH (1 mg / kg) The dose group also showed a similar weight loss, whereas the group receiving high concentrations of LeuSH (8 mg / kg) had a mean weight of 0, 10, 20 and 30 days compared to the normal control group. Although there was no significant change in body weight, compared to the EAE induction group, the body weight was significantly increased especially on the 20th and 30th days, and it was confirmed that the weight lost by EAE disease was recovered by the administration of high concentrations of LeuSH.

As such, it can be seen that the administration of LeuSH has an effect of offsetting the weight loss due to EAE disease. * Indicates statistical significance compared to the normal control group (* p <0.05; n = 6 mice per group) and # indicates statistical significance compared to the EAE induction group (#p <0.05; n = 6 mice per group). Statistical processing for significance test was calculated using GLM of SAS.

(3) long-term weight

Unit (g) Normal control EAE LeuSH (1 mg / kg) LeuSH (8 mg / kg) brain 0.410 ± 0.006 0.395 ± 0.002 * 0.407 ± 0.002 # 0.408 ± 0.004 # spleen 0.184 ± 0.007 0.133 ± 0.003 * 0.180 ± 0.009 # 0.182 ± 0.009 # lungs 0.206 ± 0.012 0.172 ± 0.012 * 0.182 ± 0.005 * 0.204 ± 0.005 # liver 1.096 ± 0.056 0.882 ± 0.023 * 1.092 ± 0.029 # 1.092 ± 0.055 # Thymus 0.046 ± 0.005 0.033 ± 0.002 * 0.040 ± 0.000 * 0.044 ± 0.002 # Heart 0.098 ± 0.004 0.092 ± 0.003 0.097 ± 0.002 0.098 ± 0.002 kidney 0.136 ± 0.005 0.122 ± 0.007 0.132 ± 0.005 0.128 ± 0.006

In Table 3, * indicates that the statistical significance is shown in comparison with the normal control group (* p <0.05; n = 6 mice per group), # is indicating that the statistical significance compared to the EAE induction group (#P <0.05; n = 6 mice per group).

As shown in Table 3, a significant decrease in organ weight was observed in the EAE-induced group, but there was no significant difference in the LeuSH-treated group compared to the normal control group, indicating that LeuSH reduced the toxicity induced by EAE. Could. In the EAE-induced group treated with LeuSH, the clinical symptoms of EAE were significantly improved in a concentration-dependent manner.

Specifically, in the 1 mg / kg LeuSH group, the lung and thymus weights were decreased compared to the normal control group, but the brain, spleen and liver weights did not differ from the normal control group. All of the weights increased significantly.

In particular, in the group of 8 mg / kg LeuSH, the weight of the brain, spleen, lung, liver and thymus showed no significant difference compared to the normal control group, and the weight of the organs was significantly increased compared to the EAE induction group. It was shown. Statistical processing for significance test was calculated using GLM of SAS.

Therefore, LeuSH, which acts to inhibit MMP and NADPH oxidase, suggests the possibility of development as a candidate for treatment of EAE disease, ie multiple neurosclerosis.

Claims (6)

L-leucine thiol (L-Leucinethiol, LeuSH) as an active ingredient comprising a pharmaceutical composition for the treatment or prevention of multiple neurosclerosis. The pharmaceutical composition of claim 1, wherein the LeuSH has an oxidase inhibitory activity of nicotinamide adenine dinucleotide phosphate (reduced form, NADPH). The pharmaceutical composition of claim 1, wherein the LeuSH has a matrix metalloproteinase 9 (MMP9) inhibitory activity. The pharmaceutical composition of claim 1, wherein the LeuSH is contained in an amount of 95.0% to 99.9% by weight based on the total weight of the composition. The pharmaceutical composition of claim 1, wherein the LeuSH is applied at a dose of 6 to 10 mg / kg. a) preparing a sample;
b) adding a substrate of NADPH oxidase to the sample;
c) contacting the candidate with the sample added with the substrate; And
d) when the reaction between the sample and the candidate substance is confirmed and the activity of NADPH oxidase is inhibited, determining the candidate substance as a substance for treating or preventing multiple sclerosis,
A method of screening for the treatment or prophylaxis of multiple neurosclerosis.

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