Pharmaceutical composition for preventing and / or treating autoimmune diseases

Abstract

The object of the present invention is to explore a novel therapeutic drug or therapeutic method for autoimmune diseases. The solution of the present invention is a combination of methotrexate and 5-ALA.

Description

TECHNICAL FIELD

[0001] The present application relates to a pharmaceutical composition for preventing and / or treating an autoimmune disease. BACKGROUND

[0002] An autoimmune disease is a disease caused by an abnormality of the immune system, in which the immune system attacks its own normal cells or tissues, and various symptoms exist depending on the kind of cells or tissues that become the targets of the attack. In the treatment of autoimmune diseases, suppression of inflammation is particularly important, and immunosuppressants, anti-inflammatory drugs, palliative treatments, and the like are generally used.

[0003] Among autoimmune diseases, the number of patients with rheumatoid arthritis (RA) is large, and it is estimated that there are about 700,000 to 800,000 people in Japan and about 23 million people worldwide. Most rheumatoid arthritis is known to be associated with autoimmunity, but the cause has not been elucidated in detail, and there are many unknown parts (non-patent literature 1).

[0004] Prior Art Documents

[0005] Non-patent Literature

[0006] Non-patent Literature 1: Lancet. 2017 Jun 10; 389(10086): 2328-2337 SUMMARY

[0007] The present application relates to a pharmaceutical composition for preventing and / or treating an autoimmune disease.

[0008] The present inventors et al. found that by combining methotrexate (MTX) with a 5-ALA class, the expression of a protein (e.g., HO-1) that is anti-inflammatory at the cellular level (i.e., in vitro) and the biological level (i.e., in vivo) is significantly increased. Based on the discovery of this new mechanism of action, the present inventors et al. found that by combining methotrexate (MTX) with a 5-ALA class, the therapeutic effect on autoimmune diseases can be enhanced, and thus completed the present application. Synergy

[0009] That is, the present application relates to a pharmaceutical composition for preventing and / or treating an autoimmune disease, comprising:

[0010] (A) a compound represented by the following formula (I) or a pharmacologically acceptable salt or ester thereof, and

[0011] (B) methotrexate or a pharmacologically acceptable salt thereof,

[0012]

[0013] (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.

[0014] Another embodiment of the present application relates to a pharmaceutical composition for preventing and / or treating an autoimmune disease,

[0015] The above pharmaceutical composition contains a compound represented by the following formula (I) or a pharmacologically acceptable salt or ester thereof, and is used in combination with methotrexate or a pharmacologically acceptable salt thereof.

[0016]

[0017] (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.

[0018] Another embodiment of the present application relates to a pharmaceutical composition for preventing and / or treating an autoimmune disease,

[0019] The above pharmaceutical composition contains methotrexate or a pharmacologically acceptable salt thereof, and is used in combination with a compound represented by the following formula (I) or a pharmacologically acceptable salt or ester thereof.

[0020]

[0021] (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.

[0022] Another embodiment of the present application relates to a combination drug for preventing and / or treating an autoimmune disease, which is administered to a subject at the same time or different times,

[0023] is a combination drug of (A) a compound represented by the following formula (I) or a pharmacologically acceptable salt or ester thereof and (B) methotrexate or a pharmacologically acceptable salt thereof.

[0024]

[0025] (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.

[0026] In one embodiment of the present application, in the formula (I), R 1an acyl group having 1 to 8 carbon atoms, and an aroyl group having 7 to 14 carbon atoms, R 2 an 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.

[0027] In one embodiment of the present application, in the formula (I), R 1 and R 2 are hydrogen atoms.

[0028] In one embodiment of the present application, the above pharmaceutical composition or medicament further comprises an iron compound, or is further used in combination with an iron compound.

[0029] In one embodiment of the present application, the above iron compound is sodium ferrous citrate.

[0030] In one embodiment of the present application, the above autoimmune disease is rheumatoid arthritis or psoriasis.

[0031] Another embodiment of the present application relates to a kit for preventing and / or treating an autoimmune disease, comprising:

[0032] (A) a compound represented by the following formula (I) or a pharmacologically acceptable salt or ester thereof, and

[0033] (B) methotrexate or a pharmacologically acceptable salt thereof

[0034]

[0035] (in the formula, R 1 represents a hydrogen atom or an acyl group, and R 2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group).

[0036] In one embodiment of the present application, the above kit further comprises an instruction manual including instructions on the administration amount and administration method of the above compound represented by the formula (I) and methotrexate.

[0037] In one embodiment of the present application, the above kit further comprises an instruction manual including instructions on the administration amount and administration method of the above compound represented by the formula (I), methotrexate, and an iron compound.

[0038] Another embodiment of the present application relates to the use of (A) a compound represented by the following formula (I) or a pharmacologically acceptable salt or ester thereof, and (B) methotrexate or a pharmacologically acceptable salt thereof, in the manufacture of a medicament for preventing and / or treating an autoimmune disease,

[0039]

[0040] (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).

[0041] Another embodiment of the present application relates to a method for treating an autoimmune disease, characterized by,

[0042] comprising the step of simultaneously or at different times administering (A) a compound represented by the following formula (I) or a pharmacologically acceptable salt or ester thereof, and (B) methotrexate or a pharmacologically acceptable salt thereof, to a subject having an autoimmune disease,

[0043]

[0044] (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).

[0045] Note that the application obtained by arbitrarily combining one or a plurality of features of the application described above is also included in the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 represents a summary of experiments performed.

[0047] Figure 2 represents a change in the expression amount of each gene caused by MTX and / or 5-ALA / SFC.

[0048] Figure 3 represents a change in the expression amount of HO-1 caused by MTX and / or 5-ALA / SFC in vivo.

[0049] Figure 4 represents the results of paw volume measurement in Example 3.

[0050] Figure 5 represents the results of arthritis score measurement in Example 3. DETAILED DESCRIPTION

[0051] The present application relates to a combination of methotrexate (MTX) and 5-ALA class for preventing and / or treating an autoimmune disease. The autoimmune disease in the present disclosure refers to a disease caused by an immune system attacking its own normal cells or tissues due to abnormalities in the immune system. As an example of the autoimmune disease, rheumatoid arthritis or psoriasis can be cited.

[0052] Generally, the IUPAC name of methotrexate is (S)-2-(4-(((2,4-diaminopteridin-6-yl)methyl)methylamino)benzamido)pentanedioic acid, which is represented by the following formula (II).

[0053]

[0054] The amount of methotrexate to be administered to the subject and the route of administration are not limited, and the amount and the route of administration of methotrexate generally used in the treatment of autoimmune diseases (e.g., the treatment of rheumatoid arthritis or psoriasis) can be employed. As a non-limiting example, 1 to 25 mg / week can be administered orally or by injection depending on the symptoms of the subject. It should be noted that the above amount can be administered to the subject in 2 to 5 times per week when the amount to be administered is large.

[0055] In the present disclosure, the 5-ALA group refers to 5-ALA or a derivative thereof or a salt thereof.

[0056] In the present disclosure, 5-ALA refers to 5-amino levulinic acid. 5-ALA is also called δ-amino levulinic acid, and is one type of amino acid.

[0057] As the derivative of 5-ALA, a compound represented by the following formula (I) can be exemplified. 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. It should be noted that in formula (I), 5-ALA corresponds to R 1 and R 2 are hydrogen atoms.

[0058]

[0059] The 5-ALA group can be administered as a prodrug (precursor) that is decomposed by an enzyme in the living body as long as it functions as an effective ingredient in the form of 5-ALA of formula (I) or a derivative thereof in the living body.

[0060] As the acyl group in R 1 of formula (I), a linear or branched alkanoyl group having 1 to 8 carbon atoms such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl, benzylcarbonyl, and an aroyl group having 7 to 14 carbon atoms such as benzoyl, 1-naphthoyl, and 2-naphthoyl can be exemplified.

[0061] As the alkyl group in R 2 in the formula (I), there can be mentioned a linear or branched alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, and the like.

[0062] As the cycloalkyl group in R 2 in the formula (I), there can be mentioned a cycloalkyl group having 3 to 8 carbon atoms which can have a saturated or a part of unsaturated bond, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclododecyl, 1-cyclohexenyl, and the like.

[0063] As the aryl group in R 2 in the formula (I), there can be mentioned an aryl group having 6 to 14 carbon atoms, such as phenyl, naphthyl, anthryl, phenanthryl, and the like.

[0064] As the aralkyl group in R 2 in the formula (I), the aryl group portion can be exemplified as the same as the above-mentioned aryl group, and the alkyl group portion can be exemplified as the same as the above-mentioned alkyl group, and specifically, there can be mentioned an aralkyl group having 7 to 15 carbon atoms, such as benzyl, phenethyl, phenylpropyl, phenylbutyl, benzhydryl, trityl, naphthylmethyl, naphthylethyl, and the like.

[0065] As the preferred 5-ALA derivative, there can be mentioned a compound in which R 1 is a formyl group, an acetyl group, a propionyl group, a butyryl group, or the like. Further, as the preferred 5-ALA derivative, there can be mentioned a compound in which R 2 is a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, or the like. Further, as the preferred 5-ALA derivative, there can be mentioned a compound in which the combination of R 1 and R 2 is each combination of (formyl group and methyl group), (acetyl group and methyl group), (propionyl group and methyl group), (butyryl group and methyl group), (formyl group and ethyl group), (acetyl group and ethyl group), (propionyl group and ethyl group), (butyryl group and ethyl group).

[0066] Among the 5-ALA group, as the salt of 5-ALA or its derivative, pharmacologically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts, and the like can be given. As the acid addition salts, for example, each of the inorganic acid salts of hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, and the like, each of the organic acid addition salts of formate, acetate, propionate, tosylate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, and the like can be given. As the metal salts, each of the alkali metal salts of lithium, sodium, potassium, and the like, each of the alkaline earth metal salts of magnesium, calcium, and the like, each of the metal salts of aluminum, zinc, and the like can be given. As the ammonium salts, ammonium salt, alkylammonium salts such as tetramethylammonium salt, and the like can be given. As the organic amine salts, each of the salts of triethylamine salt, piperidine salt, morpholine salt, toluidine salt, and the like can be given. It should be noted that these salts can be used in the form of a solution at the time of use.

[0067] Among the above 5-ALA group, the most preferable are: 5-ALA, and each of the ester groups of 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, and the like, and each of the hydrochloride, phosphate, and sulfate salts thereof. As particularly preferable examples, 5-ALA hydrochloride and 5-ALA phosphate can be given.

[0068] The above 5-ALA group can be produced by, for example, a publicly known method such as chemical synthesis, production using microorganisms, production using enzymes, and the like. In addition, the above 5-ALA group can form a hydrate or a solvate, and the 5-ALA group can be used alone or in combination of two or more as appropriate.

[0069] When the above 5-ALA group is prepared into an aqueous solution, in order to prevent decomposition of the 5-ALA group, care is taken so that the aqueous solution does not become alkaline. When it becomes alkaline, decomposition can be prevented by removing oxygen.

[0070] Since the 5-ALA group is a substance with high safety, the administration route 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 a target tissue or an organ using a catheter, intravenous administration including injection or drip infusion, subcutaneous administration using a syringe, transdermal administration using a patch or the like, suppositories, or non-oral administration based on forced enteral nutrition therapy using a nasogastric tube, a nasoenteral tube, a gastrostomy tube, or an enterostomy tube, and the like can be given. As the simplest administration method, oral administration can be given.

[0071] The amount of the 5-ALA derivative to be administered to the subject can be determined by a person skilled in the art (e.g., a physician) depending on the purpose of administration, and the optimal amount can be determined. Specifically, the amount of the 5-ALA derivative to be administered can be 0.1 mg to 1000 mg per 1 kg of the body weight of the subject per day, 0.2 mg to 500 mg per 1 kg of the body weight of the subject per day, 0.3 mg to 250 mg per 1 kg of the body weight of the subject per day, 0.4 mg to 100 mg per 1 kg of the body weight of the subject per day, 0.5 mg to 40 mg per 1 kg of the body weight of the subject per day, or 0.5 mg to 20 mg per 1 kg of the body weight of the subject per day, in terms of 5-ALA.

[0072] In the present disclosure, methotrexate and the 5-ALA derivative can be administered to the subject at the same time or at different times. That is, the 5-ALA derivative can be administered at substantially the same time as when methotrexate is administered to the subject, before methotrexate is administered to the subject, or after methotrexate is administered to the subject. It should be noted that the administration route of methotrexate to the subject can be the same as or different from the administration route of the 5-ALA derivative to the subject. For example, in the case where methotrexate and the 5-ALA derivative are prepared as a combined preparation, the administration routes of the two agents are necessarily the same, and in the case where methotrexate and the 5-ALA derivative are administered separately, the administration routes of the two agents can be appropriately selected depending on the symptoms of the subject and the treatment plan.

[0073] The 5-ALA derivative is metabolized in vivo to porphyrin (mainly protoporphyrin IX: PpIX). PpIX has both the property of accumulating in tissues such as tumors and the property of being excited when irradiated with light of a prescribed wavelength, and is known to be useful for photodynamic therapy (PDT) and photodynamic diagnosis (PDD) that utilize this property. However, as shown in the examples, the present application exerts its effects without irradiation with light. That is, the present application is based on a mechanism of action that is significantly different from PDT or PDD.

[0074] In the present disclosure, the 5-ALA derivative can be administered to the subject together with an iron compound. It is known that the 5-ALA derivative cooperates with the iron compound in the living body to exert various effects. However, since iron is a substance that is normally present in the living body, in the case where sufficient storage iron is present in the living body of the subject, or in the case where the subject replenishes iron by eating or taking a nutritional supplement, it is not necessarily required to use the iron compound in order to obtain the desired effect of the present application. As non-limiting examples of the iron compound, mention can be made of ferrous citrate, sodium ferrous citrate (SFC), sodium ferric citrate, ferric ammonium citrate, ferric pyrophosphate, heme iron, dextran iron, ferric lactate, ferrous gluconate, DTPA iron, sodium iron diethylenetriaminepentaacetate, ferric ammonium diethylenetriaminepentaacetate, sodium iron ethylenediaminetetraacetate, ferric ammonium ethylenediaminepentaacetate, iron triethylenetetramine, sodium iron dicarboxymethyl glutamate, ferric ammonium dicarboxymethyl glutamate, lactoferrin iron, transferrin iron, ferric chloride, ferric oxide, sodium chlorophyllin iron, ferritin iron, ferrous fumarate, ferrous pyrophosphate, sugar-containing oxidized iron, ferric acetate, ferric oxalate, ferrous succinate, succinate-citrate ferric sodium, ferric sulfate, ferric glycine sulfate.

[0075] The amount of the iron compound administered to the subject can be 0 to 100 times, preferably 0.01 times to 10 times, and more preferably 0.05 times to 5 times, the amount of the 5-ALA derivative administered to the subject, in terms of molar ratio.

[0076] Unless specifically defined otherwise, the terms used in the specification are used only for the purpose of describing particular embodiments and are not intended to limit the present application.

[0077] In addition, unless clearly understood otherwise in the context, the term "comprising" used in the specification means that the described matter (component, step, element, number, etc.) is present, and does not exclude the presence of matters (component, step, element, number, etc.) other than the described matter.

[0078] Unless defined otherwise, all terms used in the present text (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Unless explicitly defined otherwise, the terms used in the present text are to be interpreted as having a meaning that is consistent with the meaning in the context of the specification and the relevant art, and are not to be interpreted in an idealized or overly formal sense.

[0079] The present application will be described in more detail by referring to the following examples. However, the present application can be embodied in many aspects, and should not be construed as being limited to the examples described herein.

[0080] Example

[0081] Example 1 : Experiment in vitro

[0082] Eight-week-old female C57BL / 6NCrSlc (B6 / N) mice were intraperitoneally administered 4% mercaptoacetate (70157-100G, Sigma-Aldrich) 1 ml. Peritoneal macrophages were recovered 72 hours later and cultured for 24 hours at 37°C in a 5% CO2, 95% air atmosphere using MEMa medium added with 10% FBS. Stimulation was performed for 24 hours using different concentrations of MTX (M8407-100MG / Sigma-Aldrich Co. LLC) (100 nM) and / or 5-ALA hydrochloride / SFC (Shimadzu Corporation) (1 mM / 0.5 mM). They were plated at 5 x 105cells / well on 2 ml of 6-well tissue culture plates (Greiner Bio-One) (Greiner Bio-One) (Fig. 1). 6 Cells / well were plated on 2 ml of 6-well tissue culture plates (Greiner Bio-One) (Greiner Bio-One) (Fig. 1). Figure 1 ) (Fig. 1).

[0083] Next, RNA samples were extracted from the cultured cells and reverse-transcribed into cDNA using oligo(dT) primers and Super Script reverse transcriptase (Invitrogen). Gene expression was resolved by qRT-PCR using the TaqMan system (18S and Arg1) and the SYBR Green system (18s, HO-1, CD206, IL-10) with the Applied Biosystems PRISM 7700 device (Applied Biosystems). The cycle threshold (Ct) of the target genes was normalized with the Ct value of 18S rRNA. The final results were resolved with the ΔΔCt method. The sequences of the target-specific primers and probes are shown in Table 1 and Table 2.

[0084] [Table 1]

[0085] Table 1: Sequences of primers and probes used in the TaqMan system

[0086]

[0087] [Table 2]

[0088] Table 2: Sequences of primers used in the SYBR Green system

[0089]

[0090] Statistical resolution method of experimental results

[0091] Results are expressed as mean and standard deviation (mean ± SD). Statistical analysis was analyzed using GraphPad Prism (version 7.0, GraphPad Software, San Diego, CA). Comparison of two groups without correspondence was analyzed using one-tailed Student's t-test. Comparison of multiple groups was analyzed using one-way ANOVA with Tukey's multiple comparison test.

[0092] In the figures, *, **, ***, **** respectively indicate the following meanings.

[0093] *: p < 0.05

[0094] **: p < 0.01

[0095] ***: p < 0.001

[0096] ****: p < 0.0001

[0097] Results

[0098] The results of qRT-PCR gene analysis are shown in Figure 2 By using both MTX and 5-ALA / SFC, the expression amount of Heme oxygenase 1 (HO-1) was significantly increased compared to the control group / 5-ALA / SFC alone administration group / MTX alone administration group.

[0099] HO-1 is an enzyme that catalyzes the initial rate-limiting stage of heme degradation, and in most tissues, the enzyme is expressed at low or not expressed under normal conditions. However, it is known that HO-1 is expressed at a high level in inflamed tissues, and shows a strong anti-inflammatory effect. Therefore, according to the results of the present experiment, it was shown that by combining MTX with 5-ALA / SFC, a synergistic anti-inflammatory effect can be exerted.

[0100] In addition, as shown in Figure 2 , by using both MTX and 5-ALA / SFC, the expression of IL-10, CD206, Arg1, which are M2 macrophage-related factors, was also significantly increased. It is known that M2 macrophages are mainly factors that control inflammation. Therefore, according to this result, it was further confirmed that by combining MTX with 5-ALA / SFC, a synergistic anti-inflammatory effect can be exerted.

[0101] Example 2: Experiment in vivo

[0102] To 8-week-old female C57BL / 6NCrSlc (B6 / N) mice, 4% mercaptoacetate (70157-100G, Sigma-Aldrich) 1 ml was administered intraperitoneally. Thereafter, the mice were divided into a control group, a 5-ALA / SFC group, an MTX group, and a 5-ALA / SFC + MTX group. The control group was not administered any substance. For the 5-ALA / SFC group, 5-ALA hydrochloride 100 mg / kg and SFC (Shokuhomu Co., Ltd.) 157 mg / kg were administered orally immediately, 24 hours later, and 48 hours later after administration of 4% mercaptoacetate. For the MTX group, MTX (M8407-100MG / Sigma-Aldrich Co. LLC) 10 mg / kg was injected subcutaneously immediately after administration of 4% mercaptoacetate. For the 5-ALA / SFC + MTX group, both the above-described oral administration of 5-ALA / SFC and the above-described subcutaneous injection of MTX were performed. Note that a group to which none of mercaptoacetate, 5-ALA hydrochloride, SFC, and MTX was administered was used as a blank group.

[0103] After recovering ascites cells 72 hours after administration of 4% mercaptoacetate, the cells were stained with Live / Dead stain (L34957, Thermo Fisher Scientific), anti-CD11b-FITC antibody (BioLegend), and anti-HO-1-PE antibody (ab83214; Abeam) using a flow cytometer (FACSymphony TM A3; Becton Dickinson), and analyzed using FlowJo software (version 10.5.0; BD Biosciences). After excluding dead cells, the expression of HO-1 was compared among the groups. The statistical analysis method for the experimental results was the same as that used in Example 1.

[0104] Results

[0105] The results of the mean fluorescent intensity (MFI) obtained using the flow cytometer are shown in Figure 3 According to the results, it was found that the expression of HO-1 was significantly increased by using MTX with 5-ALA and SFC, as compared with the group to which only 4% mercaptoacetate was administered. Surprisingly, the use of MTX with 5-ALA and SFC brought about an increase in the expression of HO-1 exceeding the degree of synergy. This indicates that the use of MTX with 5-ALA and SFC synergistically increases the expression of HO-1. Therefore, according to the results, it was further confirmed that the combination of MTX with 5-ALA / SFC enables the exertion of a synergistic anti-inflammatory effect.

[0106] Discussion

[0107] MTX is an immunosuppressant having a mechanism of antagonizing folic acid metabolism, and is a drug agent widely used as a therapeutic drug for autoimmune diseases (particularly rheumatoid arthritis, psoriasis) in the world. Here, as shown by the results of the present example, it was shown that a synergistic anti-inflammatory effect can be exerted by the combination of MTX with 5-ALA / SFC. That is, by the combination of MTX with 5-ALA / SFC, a synergistically enhanced therapeutic effect on autoimmune diseases is expected.

[0108] Example 3: Experiment using a collagen-induced arthritis (CIA) model Results

[0109] Nine-week-old female DA / Slc rats were shaved with a pusher on the dorsal part as a drug administration site under isoflurane anesthesia using an animal simple inhalation anesthesia device (Model: NARCOBIT-E (Type II), NARCO Corporation) and 0.05 mL of a symptom-inducing drug solution was intradermally administered to both left and right sides of the dorsal part of all animals using a syringe (TERUMO Corporation) and a needle (TERUMO Corporation) in a total of 0.1 mL. The symptom-inducing drug solution was prepared by adding bovine type II collagen and an equal amount of Freund's incomplete adjuvant (Adjuvant Incomplete Freund) to an interchangeable syringe (TOP Corporation) and thoroughly mixing.

[0110] The symptom-inducing substance was administered only on day 0, and each test substance was administered on day 1 before the administration of the symptom-inducing substance. MTX was suspended in a 0.5 w / v% methylcellulose 400 solution (MC). ALA used amino levulinic acid hydrochloride, and the weight indicated below is the weight based on amino levulinic acid hydrochloride.

[0111] The rats were divided into groups 1 to 5 (n = 10 for each group), and each test substance was orally administered once a day for 18 days using a gavage needle (Fuchigami Kikai Co., Ltd.) and a syringe (TERUMO Corporation). The administration amount on day 1 was as follows.

[0112] [Group 1] MTX: 0.02 mg / kg

[0113] [Group 2] MTX: 0.02 mg / kg + ALA: 100 mg / kg + SFC: 157 mg / kg

[0114] [Group 3] ALA: 100 mg / kg + SFC: 157 mg / kg

[0115] [Group 4] ALA: 100 mg / kg

[0116] [Group 5 (control group)] MC: 10 ml / kg

[0117] As the evaluation of the symptoms, both the increase in the paw volume and the score of the degree of the arthritic condition were used.

[0118] The paw volume of the left and right hind paws was measured using a plethysmometer (Model: MK-101P, Murakami Engineering) at 0 days (before grouping), 4 days, 7 days, 11 days, 14 days, and 18 days. The paw volume was measured 3 times for each paw, and the average of the 3 measurements was calculated. The paw volume was also measured for the other paw in the same manner. The paw volume measurement value for one individual was calculated by the following mathematical formula.

[0119] [ Paw volume measurement value = (average of right paw + average of left paw) / 2]

[0120] The measurement value before grouping was set as the Pre value, and the measurement value at each measurement point after grouping was set as the Post value. The increase in the paw volume (Δ mL) for each individual was calculated from the Post value - the Pre value.

[0121] The arthritic score measurement was performed at 0 days (before grouping), 4 days, 7 days, 11 days, 14 days, and 18 days. The states of the left and right hind paws and forepaws were observed, and the score was 0 when there was no symptom, 1 when swelling of one small joint such as a finger was observed, 2 when swelling of two or more small joints or a large joint was observed, 3 when swelling of one paw was observed, and 4 when swelling of the entire one paw was observed. It should be noted that the sum of the arthritic scores of the left and right was calculated as the score for the individual.

[0122] In addition, with respect to the paw volume and the score, the group mean ± standard error (Mean ± S.E) was calculated using the table calculation software Excel (Microsoft Corporation), and statistical processing was performed. Comparison between groups was performed by Student's t-test at a one-tailed significance level of 0.05. In addition, the synergistic effect can be determined based on whether the difference between the average values of Group 1 and Group 5 exceeds the sum of the difference between Group 1 and Group 5 and the difference between Group 3 and Group 5, and in addition, the Colby formula (theoretical value E = A + B - (A x B) / 100) can be used to determine whether the proportion of the difference between the average values of Group 2 and Group 5 exceeds the theoretical value E calculated from the proportion of the difference between Group 1 and Group 5 A and the proportion of the difference between Group 3 and Group 5 B.

[0123] Figure 4

[0124] (1) Increase in the paw volume

[0125] Results of the measurement of paw volume are shown in Table 1 Figure 5 On day 18 of the final day of measurement, the inter-group difference of 0.42 mL between Group 2 and Group 5 was greater than the sum of the inter-group difference of 0.16 mL between Group 1 and Group 5 and the inter-group difference of 0.09 mL between Group 3 and Group 5, which was 0.25 mL. In addition, the reduction ratio of Group 2 relative to Group 5 was 40.8%, which was greater than the theoretical value of 24.2% calculated by the Colby formula. In addition, when comparing Group 2 with Group 5 and Group 2 with Group 3 on day 18, the results were significantly lower in Group 2. In addition, the inter-group difference between Group 1 and Group 2 was p = 0.08, which had a tendency for Group 2 to be lower. That is, a surprising synergistic inhibitory effect on symptoms was observed by the combination of MTX and 5-ALA / SFC, which exceeded the synergistic effect predicted from the individual effects of each agent.

[0126] (2) Arthritis score

[0127] Results of the measurement of arthritis score are shown in Table 2 Discussion On day 18 of the final day of measurement, the inter-group difference of 3.6 between Group 2 and Group 5 was greater than the sum of the inter-group difference of 1.5 between Group 1 and Group 5 and the inter-group difference of 0.0 (worse than the control) between Group 3 and Group 5, which was 1.5. In addition, the reduction ratio of Group 2 relative to Group 5 was 30.5%, which was greater than the theoretical value of 12.7% calculated by the Colby formula. In addition, when comparing Group 2 with Group 5, Group 1 with Group 2, and Group 2 with Group 3 on day 18, the results were significantly lower in Group 2. That is, a surprising synergistic effect on the reduction of arthritis score was observed by the combination of MTX and 5-ALA / SFC, which exceeded the synergistic effect predicted from the individual effects of each agent.

[0128] Synergistic

[0129] In the present example, even in the experiment using a collagen-induced arthritis (CIA) model rat, it was confirmed that by the combination of MTX and 5-ALA / SFC, it was possible to exert ​ anti-inflammatory effects. Note that, as described above, MTX is an immunosuppressant having a folic acid metabolism antagonizing mechanism, and is a drug that is widely used as a therapeutic drug for autoimmune diseases (particularly rheumatoid arthritis, psoriasis) in the world. In the present example, a certain anti-inflammatory effect was also observed in the group to which only MTX was administered (Group 1), and this result indicates that the present experimental system is a suitable experimental system.

Claims

1. Use of a composition comprising (A) 5-aminolevulinic acid (5-ALA) or a pharmacologically acceptable salt thereof, (B) methotrexate or a pharmacologically acceptable salt thereof, and (C) an iron compound for the manufacture of a medicament for the prophylaxis and / or treatment of rheumatoid arthritis.

2. Use according to claim 1, characterized in that, The iron compound is sodium ferrous citrate.

3. Use of (A) 5-aminolevulinic acid (5-ALA) or a pharmacologically acceptable salt thereof, (B) methotrexate or a pharmacologically acceptable salt thereof, and (C) an iron compound for the manufacture of a kit for the prophylaxis and / or treatment of rheumatoid arthritis, the kit comprising (A) 5-aminolevulinic acid (5-ALA) or a pharmacologically acceptable salt thereof, (B) methotrexate or a pharmacologically acceptable salt thereof, and (C) an iron compound.

4. Use according to claim 3, characterized in that, The iron compound is sodium ferrous citrate.

4. A composition comprising (A) 5-aminolevulinic acid (5-ALA) or a pharmacologically acceptable salt thereof, (B) methotrexate or a pharmacologically acceptable salt thereof, and (C) an iron compound for the prophylaxis and / or treatment of rheumatoid arthritis. The iron compound is sodium ferrous citrate.

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