Pharmaceutical composition for preventing and / or treating autoimmune diseases

Abstract

[Problem] To find a novel pharmaceutical or method for treating autoimmune diseases. [Solution] A combination of methotrexate and 5-ALAs.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for the prevention and / or treatment of an autoimmune disease. [Background technology]

[0002] Autoimmune diseases are diseases caused by the immune system attacking normal cells and tissues of the body due to an abnormality in the immune system, and various pathologies exist depending on the type of cells and tissues that are attacked. In the treatment of autoimmune diseases, it is particularly important to suppress inflammation, and immunosuppressants, anti-inflammatory drugs, palliative care, etc. are generally used.

[0003] Among autoimmune diseases, the number of patients with rheumatoid arthritis (RA) is particularly large, with an estimated 700,000 to 800,000 in Japan and 23 million worldwide. It is known that most cases of rheumatoid arthritis are related to autoimmunity, but the cause of the disease has not been elucidated in detail, and many aspects remain unknown (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Lancet. 2017 Jun 10;389(10086):2328-2337 Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present invention is to discover new therapeutic agents or methods for treating autoimmune diseases. [Means for solving the problem]

[0006] The present inventors have found that the combined use of methotrexate (MTX) and 5-ALA analogues significantly increases the expression of anti-inflammatory proteins (e.g., HO-1) at the cellular level (i.e., in vitro) and the biological level (i.e., in vivo). Based on the discovery of this new mechanism of action, the present inventors have demonstrated that the combined use of methotrexate (MTX) and 5-ALA analogues has a therapeutic effect on autoimmune diseases. Synergistically The inventors have found that the above-mentioned properties can be improved, and have completed the present invention.

[0007] That is, in one embodiment, the present invention provides a pharmaceutical composition for preventing and / or treating an autoimmune disease, comprising: (A) A compound represented by the following formula (I): [ka] (In the formula, R 1 represents a hydrogen atom or an acyl group, R 2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. or a pharmacologically acceptable salt or ester thereof, and (B) methotrexate or a pharmacologically acceptable salt thereof, The present invention relates to a pharmaceutical composition comprising:

[0008] Another embodiment of the present invention is a pharmaceutical composition for the prevention and / or treatment of an autoimmune disease, said pharmaceutical composition comprising: A compound represented by the following formula (I): [ka] (In the formula, R 1 represents a hydrogen atom or an acyl group, R 2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. or a pharmacologically acceptable salt or ester thereof, The present invention is characterized in that it is used in combination with methotrexate or a pharmacologically acceptable salt thereof. It relates to pharmaceutical compositions.

[0009] Another embodiment of the present invention is a pharmaceutical composition for the prevention and / or treatment of an autoimmune disease, said pharmaceutical composition comprising methotrexate or a pharmacologically acceptable salt thereof, A compound represented by the following formula (I): [ka] (In the formula, R 1 represents a hydrogen atom or an acyl group, R 2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. or a pharmacologically acceptable salt or ester thereof, It relates to pharmaceutical compositions.

[0010] Another embodiment of the present invention is a combination medicine for the prevention and / or treatment of an autoimmune disease, which is administered simultaneously or at different times to a subject, comprising: (A) A compound represented by the following formula (I): [ka] (In the formula, R 1 represents a hydrogen atom or an acyl group, R 2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. or a pharmacologically acceptable salt or ester thereof (B) methotrexate or a pharmacologically acceptable salt thereof, Concerning combination medicines.

[0011] In one embodiment of the present invention, in formula (I), R 1 is selected from the group consisting of a hydrogen atom, an alkanoyl group having 1 to 8 carbon atoms, and an aroyl group having 7 to 14 carbon atoms; R 2is selected from the group consisting of a hydrogen atom, a linear or branched alkyl group having 1 to 8 carbon atoms, a cycloalkyl group having 3 to 8 carbon atoms, an aryl group having 6 to 14 carbon atoms, and an aralkyl group having 7 to 15 carbon atoms.

[0012] In one embodiment of the present invention, in formula (I), R 1 and R 2 is a hydrogen atom.

[0013] In one embodiment of the present invention, the pharmaceutical composition or medicament further comprises an iron compound or is further used in combination with an iron compound.

[0014] In one embodiment of the present invention, the iron compound is sodium ferrous citrate.

[0015] In one embodiment of the present invention, the autoimmunity is rheumatoid arthritis or psoriasis.

[0016] Another embodiment of the present invention is a kit for preventing and / or treating an autoimmune disease, comprising: (A) a compound represented by formula (I): [ka] (In the formula, R 1 represents a hydrogen atom or an acyl group, R 2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. or a pharmacologically acceptable salt or ester thereof, and (B) methotrexate or a pharmacologically acceptable salt thereof, The present invention relates to a kit comprising:

[0017] In one embodiment of the present invention, the kit further comprises the compound of formula (I) and an instruction manual including instructions on the dosage and method of administration of methotrexate.

[0018] In one embodiment of the present invention, the kit further comprises an instruction manual including instructions on the dosage and method of administration of the compound represented by formula (I), methotrexate, and iron compound.

[0019] Another embodiment of the present invention relates to a compound of formula (I): [ka] (In the formula, R 1 represents a hydrogen atom or an acyl group, R 2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. or a pharmacologically acceptable salt or ester thereof, and (B) methotrexate or a pharmacologically acceptable salt thereof, Regarding the use of.

[0020] Another embodiment of the invention is a method for treating an autoimmune disease, comprising: For subjects suffering from an autoimmune disease, (A) A compound represented by the following formula (I): [ka] (In the formula, R 1 represents a hydrogen atom or an acyl group, R 2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. or a pharmacologically acceptable salt or ester thereof, and (B) methotrexate or a pharmacologically acceptable salt thereof, and applying the same or different times. It concerns the method.

[0021] Any combination of one or more of the features of the present invention described above is also included within the scope of the present invention. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 shows an overview of the experiments performed. [Diagram 2] FIG. 2 shows the change in the expression level of each gene due to the application of MTX and / or 5-ALA / SFC. [Diagram 3] FIG. 3 shows changes in the expression level of HO-1 by administration of MTX and / or 5-ALA / SFC in vivo. [Figure 4] FIG. 4 shows the results of foot volume measurement in Example 3. [Diagram 5] FIG. 5 shows the results of arthritis score measurement in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention relates to a combination of methotrexate (MTX) and 5-ALA for the prevention and / or treatment of autoimmune diseases. In the present disclosure, the autoimmune disease may refer to a disease caused by the immune system attacking normal cells or tissues of the patient's own body due to an abnormality in the immune system. Exemplary autoimmune diseases include rheumatoid arthritis and psoriasis.

[0024] Generally, the IUPAC name of methotrexate is (S)-2-(4-(((2,4-diaminopteridin-6-yl)methyl)methylamino)benzamido)pentanedioic acid, and is represented by the following formula (II). [ka]

[0025] The dose and route of administration of methotrexate to a subject are not limited, but may be the dose and route of administration of methotrexate commonly used in the treatment of autoimmune diseases (e.g., the treatment of rheumatoid arthritis or psoriasis). As a non-limiting example, 1 to 25 mg / week may be administered orally or by injection depending on the pathology of the subject. If the dose is high, the above amount may be divided and administered to the subject 2 to 5 times a week.

[0026] In the present disclosure, 5-ALA refers to 5-ALA or a derivative or a salt thereof.

[0027] In this disclosure, 5-ALA refers to 5-aminolevulinic acid, also known as δ-aminolevulinic acid, which is a type of amino acid.

[0028] An example of a 5-ALA derivative is a compound represented by the following formula (I). In formula (I), R 1 represents a hydrogen atom or an acyl group, R 2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. 1 and R 2 corresponds to the case of a hydrogen atom. [ka]

[0029] The 5-ALA derivatives may act as an active ingredient in the body in the form of 5-ALA of formula (I) or a derivative thereof, and may also be administered as a prodrug (precursor) that is decomposed by enzymes in the body.

[0030] R in formula (I) 1Examples of the acyl group in the formula (I) include linear or branched alkanoyl groups having 1 to 8 carbon atoms, such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl, and benzylcarbonyl groups, and aroyl groups having 7 to 14 carbon atoms, such as benzoyl, 1-naphthoyl, and 2-naphthoyl groups.

[0031] R in formula (I) 2 Examples of the alkyl group in the formula (I) include linear or branched alkyl groups having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl groups.

[0032] R in formula (I) 2 Examples of the cycloalkyl group in the formula (I) include cycloalkyl groups having 3 to 8 carbon atoms, which may have a saturated or partially unsaturated bond, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclododecyl, and 1-cyclohexenyl groups.

[0033] R in formula (I) 2 Examples of the aryl group in the formula (I) include aryl groups having 6 to 14 carbon atoms, such as phenyl, naphthyl, anthryl, and phenanthryl groups.

[0034] R in formula (I) 2 In the aralkyl group, the aryl portion can be exemplified as the above-mentioned aryl group, and the alkyl portion can be exemplified as the above-mentioned alkyl group. Specific examples include aralkyl groups having 7 to 15 carbon atoms, such as benzyl, phenethyl, phenylpropyl, phenylbutyl, benzhydryl, trityl, naphthylmethyl, and naphthylethyl groups.

[0035] Preferred 5-ALA derivatives include R 1 is a formyl, acetyl, propionyl, butyryl group, etc. Preferred 5-ALA derivatives include those in which the above R2 is a methyl, ethyl, propyl, butyl, pentyl group, etc. Also, preferred 5-ALA derivatives include compounds in which the above R 1 and R 2 Examples of the combinations of (formyl and methyl), (acetyl and methyl), (propionyl and methyl), (butyryl and methyl), (formyl and ethyl), (acetyl and ethyl), (propionyl and ethyl), and (butyryl and ethyl) include compounds in which the combinations of (formyl and methyl), (acetyl and methyl), (propionyl and ethyl), (butyryl and ethyl) are included.

[0036] Among 5-ALA, examples of salts of 5-ALA or its derivatives include pharmacologically acceptable acid addition salts, metal salts, ammonium salts, and organic amine addition salts. Examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, and sulfate, and organic acid addition salts such as formate, acetate, propionate, toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, and malate. Examples of metal salts include alkali metal salts such as lithium salt, sodium salt, and potassium salt, alkaline earth metal salts such as magnesium and calcium salt, and metal salts such as aluminum and zinc. Examples of ammonium salts include ammonium salts and alkyl ammonium salts such as tetramethylammonium salt. Examples of organic amine salts include triethylamine salts, piperidine salts, morpholine salts, and toluidine salts. These salts can also be used in the form of a solution when in use.

[0037] Among the above 5-ALA compounds, the most desirable ones are 5-ALA and various esters such as 5-ALA methyl ester, 5-ALA ethyl ester, 5-ALA propyl ester, 5-ALA butyl ester, 5-ALA pentyl ester, 5-ALA hexyl ester, 5-ALA heptyl ester, etc., as well as their hydrochlorides, phosphates, and sulfates. In particular, 5-ALA hydrochloride and 5-ALA phosphate are particularly desirable examples.

[0038] The above-mentioned 5-ALA's can be produced by known methods, such as chemical synthesis, production by microorganisms, production by enzymes, etc. The above-mentioned 5-ALA's may form a hydrate or solvate, and the 5-ALA's can be used alone or in appropriate combination of two or more kinds.

[0039] When preparing the above 5-ALA as an aqueous solution, care must be taken to prevent the aqueous solution from becoming alkaline in order to prevent the decomposition of 5-ALA. If the aqueous solution becomes alkaline, decomposition can be prevented by removing oxygen.

[0040] Since 5-ALA is a highly safe substance, the route of administration to the subject is not limited, and various administration routes can be selected. For example, oral administration including sublingual administration, inhalation administration, direct administration to the target tissue or organ using a catheter, intravenous administration including injection and drip infusion, subcutaneous administration using a syringe, transdermal administration using a patch, etc., suppository, or parenteral administration such as forced enteral nutrition using a nasogastric tube, nasoenteric tube, gastrostomy tube, or intestinal fistula tube, etc. The simplest administration method is oral administration.

[0041] The dosage of 5-ALA to a subject may be determined optimally by a person skilled in the art (e.g., a doctor) depending on the purpose of administration. Specifically, the dosage may be 0.1 mg to 1000 mg / day, 0.2 mg to 500 mg / day, 0.3 mg to 250 mg / day, 0.4 mg to 100 mg / day, 0.5 mg to 40 mg / day, or 0.5 mg to 20 mg / day in terms of 5-ALA per kg of the subject's body weight.

[0042] In the present disclosure, methotrexate and 5-ALA may be administered to a subject at the same time or at different times. That is, 5-ALA may be administered to a subject at substantially the same timing as methotrexate is administered to the subject, 5-ALA may be administered prior to methotrexate administration to the subject, or 5-ALA may be administered after methotrexate administration to the subject. The administration route of methotrexate to a subject and the administration route of 5-ALA to a subject may be the same or different. For example, when methotrexate and 5-ALA are prepared as a combination drug, the administration routes of both drugs are necessarily the same, and when methotrexate and 5-ALA are administered individually, the administration routes of both drugs can be appropriately selected according to the pathology and treatment policy of the subject.

[0043] 5-ALA is metabolized in vivo to become porphyrin (mainly protoporphyrin IX: PpIX). PpIX has the property of accumulating in tissues such as tumors and being excited when irradiated with light of a specific wavelength, and it is known that this property can be utilized in photodynamic therapy (PDT) and photodynamic diagnosis (PDD). However, as shown in the examples, the present invention does not require light irradiation to exert its effect. In other words, the present invention is based on a mechanism of action that is clearly different from PDT and PDD.

[0044] In the present disclosure, 5-ALA may be applied to a subject together with an iron compound. It is known that 5-ALA and an iron compound work together in a living body to exert various effects. However, since iron is a substance that is normally present in a living body, if there is sufficient iron storage in the living body of a subject or if the subject supplements iron through diet or intake of supplements, it is not necessarily necessary to use an iron compound in combination to obtain the desired effect of the present invention. Non-limiting examples of iron compounds include ferrous citrate, sodium ferrous citrate (SFC), sodium ferrous citrate, ammonium ferrous citrate, ferric pyrophosphate, ferrous heme, ferrous dextran, ferrous lactate, ferrous gluconate, ferrous DTPA, sodium ferrous diethylenetriaminepentaacetate, ammonium ferrous diethylenetriaminepentaacetate, sodium ferrous ethylenediaminetetraacetate, ammonium ferrous ethylenediaminepentaacetate, ferrous triamineferric acid, sodium ferrous dicarboxymethylglutamate, ammonium ferrous dicarboxymethylglutamate, ferrous lactoferrin, ferrous transferrin, ferric chloride, ferric oxide, sodium ferric iron, ferric chlorophyllin, ferrous ferritin, ferrous fumarate, ferrous pyrophosphate, ferrous saccharide oxide, ferrous acetate, ferrous oxalate, ferrous succinate, sodium ferrous succinate citrate, ferrous sulfate, and ferrous glycine sulfide.

[0045] The amount of the iron compound administered to the subject may be 0 to 100 times, preferably 0.01 to 10 times, and more preferably 0.05 to 5 times, the molar ratio of the amount of 5-ALA administered to the subject.

[0046] The terms used in this specification, unless otherwise defined, are used to describe particular embodiments and are not intended to limit the invention.

[0047] Furthermore, the term "comprise" as used in this specification intends that the described items (components, steps, elements, numbers, etc.) are present, unless the context clearly indicates otherwise, and does not exclude the presence of other items (components, steps, elements, numbers, etc.).

[0048] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in the present specification and related technical fields, and should not be interpreted in an idealized or overly formal sense, unless otherwise defined.

[0049] In the following, the present invention will be described in more detail with reference to examples. However, the present invention can be embodied in various forms and should not be construed as being limited to the examples set forth herein. EXAMPLES

[0050] Example 1: In vitro experiments 8-week-old female C57BL / 6NCrSlc (B6 / N) mice were intraperitoneally administered 1 ml of 4% thioglycollate (70157-100G, Sigma-Aldrich). After 72 hours, peritoneal macrophages were collected and cultured in MEMα medium supplemented with 10% FBS at 37°C under 5% CO2 and 95% air for 24 hours. Stimulation with different concentrations of MTX (M8407-100MG / Sigma-Aldrich Co. LLC) (100 nM) and / or 5-ALA hydrochloride / SFC (Komatsuya Co., Ltd.) (1 mM / 0.5 mM) was performed for 24 hours. These were cultured in 2 ml of 6-well tissue culture plates (Greiner Bio-One) at 5 × 10 6 cells / well (Figure 1).

[0051] RNA samples were then extracted from the cultured cells and reverse transcribed into cDNA using oligo(dT) primers and Super Script reverse transcriptase (Invitrogen). Gene expression was analyzed by qRT-PCR using the TaqMan system (18S and Arg1) and the SYBR Green system (18s, HO-1, CD206, IL-10) on an Applied Biosystem PRISM7700 instrument (Applied Biosystems). The threshold cycle (Ct) values ​​of the target genes were normalized to the Ct value of 18S rRNA. The final results were analyzed by the △△Ct method. The sequences of the target-specific primers and probes are shown in Tables 1 and 2. [Table 1] [Table 2]

[0052] Statistical analysis of experimental results Results are expressed as the mean and standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism (version 7.0, GraphPad Software, San Diego, CA). Comparisons between two unpaired groups were performed using a one-tailed Student's t test. Comparisons between multiple groups were performed using one-way ANOVA with Tukey's multiple comparison test. *, **, ***, **** in the figure have the following meanings. *: p<0.05 **: p<0.01 ***: p<0.001 ****:p<0.0001

[0053] result The results of qRT-PCR gene analysis are shown in Figure 2. By administering both MTX and 5-ALA / SFC, the expression level of heme oxygenase 1 (HO-1) was significantly increased compared to the control group, the 5-ALA / SFC only administration group, and the MTX only administration group.

[0054] HO-1 is an enzyme that catalyzes the first rate-limiting step in heme degradation, and in most tissues, expression of this enzyme is low or absent under normal conditions. However, it is known that HO-1 is expressed at high levels in inflamed tissues and has a strong anti-inflammatory effect. Therefore, the results of this experiment suggest that the combination of MTX and 5-ALA / SFC exerts a synergistic anti-inflammatory effect.

[0055] Furthermore, as shown in Figure 2, the application of both MTX and 5-ALA / SFC significantly increased the expression of M2 macrophage-related factors IL-10, CD206, and Arg1. M2 macrophages are known to be factors that mainly act to control inflammation. Therefore, this result further supports the idea that the combination of MTX and 5-ALA / SFC exerts a synergistic anti-inflammatory effect.

[0056] Example 2: In vivo experiments Eight-week-old female C57BL / 6NCrSlc (B6 / N) mice were intraperitoneally administered 1 ml of 4% thioglycollate (70157-100G, Sigma-Aldrich). The mice were then divided into a control group, a 5-ALA / SFC group, an MTX group, and a 5-ALA / SFC+MTX group. The control group received no treatment. The 5-ALA / SFC group received oral administration of 100 mg / kg 5-ALA hydrochloride and 157 mg / kg SFC (Komatsuya Co., Ltd.) immediately after administration of 4% thioglycollate, 24 hours later, and 48 hours later. The MTX group received a subcutaneous injection of 10 mg / kg MTX (M8407-100MG / Sigma-Aldrich Co. LLC) immediately after administration of 4% thioglycollate. The 5-ALA / SFC+MTX group received both oral administration of 5-ALA / SFC and subcutaneous injection of MTX. The group that did not receive any of thioglycollate, 5-ALA hydrochloride, SFC, or MTX was designated as the naive group.

[0057] 72 hours after administration of 4% thioglycollate, cells were collected from the ascites and analyzed by a flow cytometer (FACSymphony TM A3; Becton Dickinson) was used, and the cells were stained with Live / Dead stain (L34957, Thermo Fisher Scientific), anti-CD11b-FITC antibody (BioLegend), and anti-HO-1-PE antibody (ab83214; Abcam), and analyzed using FlowJo software (Version 10.5.0; BD Biosciences). After gating out dead cells, HO-1 expression in each group was compared. The statistical analysis method of the experimental results was the same as that used in Example 1.

[0058] result The results of the mean fluorescent intensity (MFI) by flow cytometry are shown in FIG. 3. The results show that the use of MTX, 5-ALA, and SFC significantly increased the expression of HO-1 compared to the control group administered only 4% thioglycollate. Surprisingly, the combination of MTX, 5-ALA, and SFC resulted in an increase in the expression of HO-1 that exceeded the additive level. This indicates that the combination of MTX, 5-ALA, and SFC synergistically increases the expression of HO-1. Therefore, this result further supports the idea that the combination of MTX and 5-ALA / SFC exerts a synergistic anti-inflammatory effect.

[0059] Consideration MTX is an immunosuppressant with a folate metabolic antagonistic mechanism, and is a drug that is widely used worldwide as a treatment for autoimmune diseases (especially rheumatoid arthritis and psoriasis). As shown in the results of this example, it was shown that the combination of MTX and 5-ALA / SFC exerts a synergistic anti-inflammatory effect. In other words, the combination of MTX and 5-ALA / SFC is expected to have a synergistically enhanced therapeutic effect against autoimmune diseases.

[0060] Example 3: Experiments using collagen-induced arthritis (CIA) model Nine-week-old female DA / Slc rats were anesthetized with isoflurane using a simple animal inhalation anesthesia device (model: NARCOBIT-E (II type), Natsume Seisakusho Co., Ltd.), and the tail, which was the administration site, was shaved with clippers. Using a syringe (Terumo Corporation) and a needle (Terumo Corporation), 0.05 mL was administered intradermally to the left and right sides of the tail of all animals, for a total of 0.1 mL of symptom-inducing substance administration solution. The symptom-inducing substance administration solution was prepared by adding bovine type II collagen and an equal amount of Adjuvant Incomplete Freund to an interchangeable syringe (Top Co., Ltd.) and mixing well.

[0061] The symptom-inducing substance was administered only on Day 0, and on the first day, each test substance was administered before administration of the symptom-inducing substance. MTX was suspended in 0.5 w / v% methylcellulose 400 solution (MC). ALA was used in the form of aminolevulinic acid hydrochloride, and the weights shown below are those of aminolevulinic acid hydrochloride.

[0062] The rats were divided into groups 1 to 5 (n=10 per group), and each test substance was orally administered once a day for 18 consecutive days using an oral probe (Fuchigami Instruments, Ltd.) and a syringe (Terumo Corporation). The daily doses were as follows: [Group 1] MTX: 0.02mg / kg [Group 2] MTX:0.02mg / kg+ALA:100mg / kg+SFC:157mg / kg [Group 3] ALA: 100mg / kg + SFC: 157mg / kg [Group 4] ALA: 100mg / kg [Group 5 (Control group)] MC: 10ml / kg

[0063] Symptoms were assessed using two measures: paw volume increase and a score for the degree of gross arthritis.

[0064] Paw volume was measured on both the left and right hind paws using a Plethysmometer (model: MK-101P, Muromachi Kikai Co., Ltd.) on days 0 (before grouping), 4, 7, 11, 14, and 18. Paw volume was measured three times for each paw, and the average of the three measurements was calculated. Paw volume was also measured for the other paw in the same manner. The measured paw volume for each individual was calculated using the following formula. [Foot volume measurement value = (average right foot value + average left foot value) / 2] The measurement values ​​before grouping were defined as Pre values, and the measurement values ​​at each measurement point after grouping were defined as Post values. The increase in paw volume (ΔmL) for each individual was calculated by subtracting the Pre value from the Post value.

[0065] Arthritis scores were measured on days 0 (before grouping), 4, 7, 11, 14, and 18. The condition of the hind and front legs was observed on both the left and right sides, and the score was evaluated as follows: no symptoms was scored as 0, swelling of one small joint such as a finger was scored as 1, swelling of two or more small joints or a large joint was scored as 2, swelling of one limb was scored as 3, and maximum swelling of the entire limb was scored as 4. The sum of the arthritis scores of the left and right sides was calculated as the score of the individual.

[0066] In addition, the mean ± standard error (Mean ± SE) of the foot volume and score were calculated using spreadsheet software Excel (Microsoft Corporation) and statistical processing was performed. Intergroup comparison was performed using Student's t-test with a one-sided significance level of 5%. In addition, synergistic effects were judged by whether the intergroup difference in the mean values ​​of groups 1 and 5 exceeded the combined value of the intergroup difference between groups 1 and 5 and the intergroup difference between groups 3 and 5, and by using Colby's formula (theoretical value E = A + B - (A × B) / 100), whether the intergroup difference ratio of the mean values ​​of groups 2 and 5 exceeded the theoretical value E calculated from the intergroup difference ratio A between groups 1 and 5 and the intergroup difference ratio B between groups 3 and 5.

[0067] result (1) Increase in foot volume The results of foot volume measurement are shown in Figure 4. On Day 18, the final day of measurement, the difference between groups 2 and 5 (0.42 mL) was greater than the sum of the difference between groups 1 and 5 (0.16 mL) and the difference between groups 3 and 5 (0.09 mL), which was 0.25 mL. In addition, the reduction rate of group 2 relative to group 5 (40.8%) was greater than the theoretical value of 24.2% calculated by Colby's formula. In addition, when comparing groups 2 and 5, and groups 2 and 3 on Day 18, group 2 was significantly lower in both cases. In addition, the difference between groups 1 and 2 was p=0.08, and group 2 tended to be lower. In other words, the combination of MTX and 5-ALA / SFC produced a surprising synergistic symptom suppression effect that exceeded the additive effect predicted from the effects of each drug alone.

[0068] (2) Arthritis score The results of the arthritis score measurement are shown in Figure 5. On Day 18, the final day of measurement, the intergroup difference between Groups 2 and 5 (3.6) was greater than the sum of the intergroup difference between Groups 1 and 5 (1.5) and the intergroup difference between Groups 3 and 5 (0.0 (worse than control)), which was 1.5. In addition, the reduction rate of Group 2 relative to Group 5 (30.5%) was greater than the theoretical value of 12.7% calculated by Colby's formula. In addition, when comparing Groups 2 and 5, Groups 1 and 2, and Groups 2 and 3 on Day 18, Group 2 was significantly lower in all cases. In other words, the combination of MTX and 5-ALA / SFC showed a surprising synergistic arthritis score reduction effect that exceeded the additive effect predicted from the effects of each drug alone.

[0069] Consideration In this example, the combination of MTX and 5-ALA / SFC was also effective in experiments using collagen-induced arthritis (CIA) model rats. Synergistic It was confirmed that MTX exerted an anti-inflammatory effect. As mentioned above, MTX is an immunosuppressant with a folate metabolic antagonistic mechanism, and is a drug that is widely used worldwide as a treatment for autoimmune diseases (especially rheumatoid arthritis and psoriasis). In this example, a certain anti-inflammatory effect was observed in the group (group 1) administered with only MTX, and this result indicates that this experimental system is an appropriate experimental system.

Claims

1. A pharmaceutical composition for the prevention and / or treatment of rheumatoid arthritis, comprising: (A) 5-aminolevulinic acid (5-ALA), 5-ALA methyl ester, 5-ALA ethyl ester, 5-ALA propyl ester, 5-ALA butyl ester, 5-ALA pentyl ester, 5-ALA hexyl ester, or a pharmacologically acceptable salt of any of these, and (B) methotrexate or a pharmacologically acceptable salt thereof, Characterized in that it comprises Pharmaceutical compositions.

2. A pharmaceutical composition for the prevention and / or treatment of rheumatoid arthritis, comprising: The pharmaceutical composition comprises: 5-aminolevulinic acid (5-ALA), 5-ALA methyl ester, 5-ALA ethyl ester, 5-ALA propyl ester, 5-ALA butyl ester, 5-ALA pentyl ester, 5-ALA hexyl ester, or a pharmacologically acceptable salt of any of these; The present invention is characterized in that it is used in combination with methotrexate or a pharmacologically acceptable salt thereof. Pharmaceutical compositions.

3. A pharmaceutical composition for the prevention and / or treatment of rheumatoid arthritis, comprising: The pharmaceutical composition comprises methotrexate or a pharmacologically acceptable salt thereof, The composition is characterized in that it is used in combination with 5-aminolevulinic acid (5-ALA), 5-ALA methyl ester, 5-ALA ethyl ester, 5-ALA propyl ester, 5-ALA butyl ester, 5-ALA pentyl ester, 5-ALA hexyl ester, or a pharmacologically acceptable salt thereof; Pharmaceutical compositions.

4. A combination medicine for the prevention and / or treatment of rheumatoid arthritis, which is administered simultaneously or at different times to a subject, (A) 5-aminolevulinic acid (5-ALA), 5-ALA methyl ester, 5-ALA ethyl ester, 5-ALA propyl ester, 5-ALA butyl ester, 5-ALA pentyl ester, 5-ALA hexyl ester, or a pharmacologically acceptable salt thereof; (B) methotrexate or a pharmacologically acceptable salt thereof, Combination medicines.

5. A pharmaceutical composition or medicament according to any one of claims 1 to 4, The composition further comprises an iron compound or is further used in combination with an iron compound. A pharmaceutical composition or medicament.

6. 6. A pharmaceutical composition or medicament according to claim 5, The iron compound is sodium ferrous citrate. A pharmaceutical composition or medicament.

7. A kit for preventing and / or treating rheumatoid arthritis, comprising: (A) 5-aminolevulinic acid (5-ALA), 5-ALA methyl ester, 5-ALA ethyl ester, 5-ALA propyl ester, 5-ALA butyl ester, 5-ALA pentyl ester, 5-ALA hexyl ester, or a pharmacologically acceptable salt of any of these, and (B) methotrexate or a pharmacologically acceptable salt thereof, Characterized in that it comprises kit.

8. For the manufacture of a medicament for the prevention and / or treatment of rheumatoid arthritis, (A) 5-aminolevulinic acid (5-ALA), 5-ALA methyl ester, 5-ALA ethyl ester, 5-ALA propyl ester, 5-ALA butyl ester, 5-ALA pentyl ester, 5-ALA hexyl ester, or a pharmacologically acceptable salt of any of these, and (B) methotrexate or a pharmacologically acceptable salt thereof, Use of.

9. 1. A method for treating rheumatoid arthritis, comprising: In a non-human subject suffering from rheumatoid arthritis, (A) 5-aminolevulinic acid (5-ALA), 5-ALA methyl ester, 5-ALA ethyl ester, 5-ALA propyl ester, 5-ALA butyl ester, 5-ALA pentyl ester, 5-ALA hexyl ester, or a pharmacologically acceptable salt of any of these, and (B) methotrexate or a pharmacologically acceptable salt thereof, and applying the same or different times. method.

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