Therapeutic drug for lipid peroxidation-induced diseases and screening method thereof

Abstract

The present invention provides a test method for detecting the lipid peroxidation inhibition activity of a test compound using a compound represented by formula (I) as a fluorescent probe molecule, a test kit using the test method, a screening method using the test method, and a method for treating a lipid peroxidation response-induced disease (e.g., a lipid peroxidation-induced disease). ), and the like. # imgabs0 #

Description

[0001] This application is a divisional application of a Chinese patent application filed on July 5, 2018, with application number 201880054837.0 and invention name “Therapeutic drugs for lipid peroxidation-induced diseases and screening methods thereof”. Technical Field

[0002] The invention provides an assay method for finding a lipid peroxidation inhibitor, an assay kit, a screening method thereof, and a therapeutic drug for lipid peroxidation-induced diseases. Background Art

[0003] Diseases in which lipid free radicals in lipid peroxidation reactions are involved involve a wide range of diseases, including cardiovascular system, central nervous system, respiratory system, and antibacterial drugs (Non-Patent Document 1) (see Figure 1 ). However, in diseases caused by lipid peroxidation, a large amount of lipid peroxides and their metabolites are involved for each disease, so it is not easy to find a useful therapeutic drug for each disease. So far, although many antioxidant substances are known, few substances are approved as pharmaceuticals. Therefore, it is necessary to provide a drug that shows the inhibitory effect of lipid peroxidation.

[0004] As a method for finding active drugs, known screening methods are provided. As a method for measuring the inhibition of lipid peroxidation, known methods include several methods (e.g., TBARs method). However, with regard to these methods, the determination object is varied, and in addition, there is a problem that the sample with different absorption wavelengths cannot be measured when utilizing fluorescence or light absorption principle, or there are problems such as pH operation, heating, etc., which are complicated. Thus, it is necessary to construct a systemic assay method that is specifically designed for the detection of lipid peroxidation and can analyze multiple samples under mild conditions close to an organism.

[0005] The inventors of the present invention have so far been developing excellent fluorescent nitroxide probe compounds capable of capturing lipid radicals (Patent Document 1).

[0006] Age-related macular degeneration (AMD) is known to be a disease with low treatment satisfaction and contribution of drugs to treatment and high unmet medical needs. Age-related macular degeneration is roughly divided into two types: atrophic (dry type) and exudative (wet type) based on its pathogenesis. In the United States, about 85 to 90% of patients suffer from atrophic (dry type), while in Japan, about 92% of patients suffer from exudative (wet type). However, effective therapeutic drugs for atrophic (dry type) diseases are not yet known. In addition, effective therapeutic drugs for the treatment and progression inhibition of age-related macular degeneration have not yet been developed from antioxidants.

[0007] Prior Art Literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application 2017-090739

[0010] Non-patent literature

[0011] Non-patent document 1: Frijhoff J et al., Antioxid. Redox Signal, 2015, 23(14), 1144-70 Summary of the invention

[0012] Problems to be solved by the invention

[0013] The present invention provides an assay method and an assay kit for detecting lipid peroxidation inhibition using a fluorescent nitroxide probe compound, and a screening method using these assay methods. In addition, a pharmaceutical composition for treating diseases induced by lipid peroxidation reactions such as age-related macular degeneration, etc., using an active drug discovered by the screening method of the present invention, is provided.

[0014] Solutions for solving problems

[0015] The present inventors have devoted themselves to studying assay methods and screening methods for detecting and evaluating lipid peroxidation inhibition, and as a result, have found that an assay method using a fluorescent nitroxide probe compound and a screening method using the assay method can easily find candidate compounds showing lipid peroxidation inhibitory activity. In addition, the present inventors have also found that these candidate compounds are useful for the treatment or prevention of diseases caused by lipid peroxidation, particularly age-related macular degeneration.

[0016] That is, the present invention provides the following aspects, but is not limited thereto.

[0017] (Test kit and test method)

[0018] Item [1]: A test kit for detecting the lipid peroxidation inhibitory activity of a test compound, the test kit comprising a compound represented by formula (I), liposomes, and at least one compound selected from the group consisting of 2,2'-azobis(2-aminopropane) dihydrochloride and a divalent iron ion supply source in a buffer solution, wherein formula (I) is as follows:

[0019] [Chemistry 1]

[0020]

[0021] Item [1-2]: In the assay kit described in Item [1],

[0022] The concentration of the compound represented by the formula (I) is 1.0 to 20.0 μM,

[0023] The liposome is prepared from phosphatidylcholine derived from egg yolk and dicetyl phosphate, wherein the concentration of the phosphatidylcholine derived from egg yolk is 5.0-10.0 mg / mL, and the concentration of the dicetyl phosphate is 0.01-1.0 mg / mL.

[0024] The concentration of the tested compound was 5 to 100 μM.

[0025] The concentration of 2,2'-azobis(2-aminopropane) dihydrochloride is 5 to 50 mM,

[0026] The concentration of the ferrous iron supply source is 0.5 to 5 mM.

[0027] Item [2]: In the assay kit described in Item [1], the divalent iron ion supply source material is iron (II) sulfate.

[0028] Item [3]: The assay kit according to claim 1 or 2, comprising additional instructions showing an activity value of the compound, wherein the compound shows an activity of inhibiting lipid peroxidation.

[0029] Item [4]: ​​A test kit for detecting the lipid peroxidation inhibitory activity of a test compound, the test kit comprising a compound represented by formula (I), cultured cells, and at least one compound selected from the group consisting of arachidonic acid and tert-butyl hydroperoxide in a buffer solution, wherein formula (I) is as follows:

[0030] [Chemistry 2]

[0031]

[0032] Item [4-2]: In the assay kit described in Item [4],

[0033] The concentration of the compound represented by the formula (I) is 1.0 to 20.0 μM,

[0034] The concentration of cultured cells was 1×10 4 ~1×10 5 Cell number,

[0035] The concentration of the tested compound was 5 to 500 μM.

[0036] The concentration of arachidonic acid is 100-400 μM.

[0037] The concentration of tert-butyl hydroperoxide is 100 to 400 μM.

[0038] Item [5]: In the assay kit described in Item [4], the cultured cells are HepG2 cells derived from human liver.

[0039] Item [6]: The assay kit of Item [4] or [5], comprising additional instructions showing an activity value of the compound, wherein the compound shows lipid peroxidation inhibitory activity.

[0040] Item [7]: A test kit comprising any two or more of the test kits described in Items [1] to [6].

[0041] Item [7-2]: The assay kit described in Item [7] comprises a combination of an assay kit in which the reaction initiator is 2,2'-azobis(2-aminopropane) dihydrochloride and an assay kit in which the reaction initiator is iron (II) sulfate.

[0042] Item [7-3]: The assay kit described in Item [7], comprising a combination of an assay kit in which the reaction initiator is arachidonic acid and an assay kit in which the reaction initiator is tert-butyl hydroperoxide.

[0043] Item [8]: A method for determining lipid peroxidation inhibitory activity, comprising:

[0044] i) preparing a buffer solution comprising the compound represented by formula (I) and liposomes;

[0045] ii) adding at least one compound selected from the group consisting of 2,2'-azobis(2-aminopropane) dihydrochloride and a divalent iron ion supply source material;

[0046] iii) adding the test compound;

[0047] iv) measuring fluorescence; and,

[0048] v) The activity value of the test compound is determined from the fluorescence measurement results.

[0049] Item [9]: A method for determining lipid peroxidation inhibitory activity, comprising:

[0050] i) preparing a buffer solution comprising a compound represented by formula (I) and cultured cells;

[0051] ii) adding at least one compound selected from the group consisting of arachidonic acid and tert-butyl hydroperoxide;

[0052] iii) adding the test compound;

[0053] iv) measuring fluorescence; and,

[0054] v) The activity value of the test compound is determined from the fluorescence measurement results.

[0055] Item

[10] : The assay method according to Item [8] or [9], comprising: vi) comparing the activity value of the compound with that of a compound that is an indicator of lipid peroxidation inhibitory activity.

[0056] Item

[11] : The assay method according to any one of Items [8] to

[10] , wherein the assay method is used in a microplate.

[0057] Item [11-2]: The test method described in Item

[11] includes:

[0058] i) dispensing a solution of the test compound into a microplate;

[0059] ii) dispensing a solution containing the compound represented by formula (I) and liposomes or cultured cells into each well;

[0060] iii) when using liposomes, dispensing a solution containing at least one compound selected from the group consisting of 2,2'-azobis(2-aminopropane) dihydrochloride and a divalent iron ion supply source substance into each well,

[0061] When using cultured cells, dispensing a solution containing at least one compound selected from the group consisting of arachidonic acid and tert-butyl hydroperoxide into each well; and,

[0062] iv) Measure the fluorescence using an enzyme-labeled instrument.

[0063] (Screening method)

[0064] Item

[12] : A screening method for selecting candidate compounds with high lipid peroxidation inhibitory activity, comprising:

[0065] i) selecting a test compound from a compound library;

[0066] ii) performing screening by the assay method using 2,2'-azobis(2-aminopropane) dihydrochloride described in item [8] using the test compound, and selecting a compound showing a high activity value; and,

[0067] iii) Next, screening is performed using the assay method for the divalent iron ion supply source substance described in Item [8] using the compound showing a high activity value in ii) to select a compound showing a high activity value.

[0068] Item

[13] : In the screening method described in Item

[12] , when the compound library is a compound library containing compounds that are not approved as food or medicine, the screening method described in Item

[12] further comprises:

[0069] i) performing screening by the assay method using arachidonic acid as described in item [9], and selecting compounds showing high activity values;

[0070] ii) performing screening by the assay method using tert-butyl hydroperoxide as described in item [9], and selecting compounds showing high activity values; and,

[0071] iii) Selecting compounds that show high activity values ​​in both the screening of i) and ii).

[0072] Item

[14] : In the screening method described in Item

[12] or

[13] , when the compound library is a compound library containing compounds that are not approved as food or medicine, the screening method described in Item

[12] further comprises:

[0073] i) performing screening by an assay method using a medium containing cultured cells, a test compound, and arachidonic acid according to the MTT method, and selecting a compound that shows a high cell viability;

[0074] ii) performing screening by an assay method using a medium containing cultured cells, a test compound, and tert-butyl hydroperoxide according to the MTT method, and selecting a compound showing a high cell viability; and,

[0075] iii) selecting candidate compounds that show high cell viability in both the screening of i) and ii).

[0076] Item

[15] : The screening method of Item

[13] or

[14] , comprising:

[0077] i) selecting a structural analogue of the compound selected by the screening method described in item

[13] or

[14] from a compound library;

[0078] ii) for the compound selected in i) and the compound appropriately selected by the screening method described in item

[13] or

[14] , performing the screening method described in item

[13] and selecting a compound showing a high activity value;

[0079] iii) for the compound selected in i) and the compound appropriately selected by the screening method described in item

[13] or

[14] , performing the screening method described in item

[14] and selecting a compound showing a high cell viability;

[0080] iv) selecting candidate compounds that show high activity values ​​and high cell viability in both screening methods ii) and iii);

[0081] v) for the compound selected in i) and the compound appropriately selected by the screening method described in item

[13] or

[14] , performing an assay method using a medium containing cultured cells, and selecting a candidate compound showing a high cell viability; and,

[0082] vi) selecting candidate compounds from the compounds selected in iv) and the compounds selected in v).

[0083] Item

[16] : In the screening method according to any one of Items

[12] to

[15] , the compound library is the Core Library of the Institute for Pharmaceutical Development, The University of Tokyo or the Prestwick Chemical Library.

[0084] Item [16-2]: The screening method described in any one of Items

[12] to

[15] is a high-throughput screening method.

[0085] (Medical Use)

[0086] Item

[17] : A pharmaceutical composition comprising an effective amount of at least one compound selected from the group consisting of the following groups, and used for preventing or treating a lipid peroxidation-induced disease in a subject or inhibiting the progression of the disease,

[0087] Wherein, the group consists of the following compounds: apomorphine ((R)-(-)-apomorphine hydrochloride), physostigmine ((-)-physostigmine fumarate), ethoxyquinoline (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline), methyldopa (methyldopa sesquihydrate), olanzapine (2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine, 3-amino-4-(phenylamino)benzoic acid methyl ester (Compound 52), 3-amino-4-((4-methoxyphenyl)amino)benzoic acid methyl ester (Compound 52-1), 3-amino-4-((3-methoxyphenyl)amino)benzoic acid methyl ester (Compound 52-3), 3-amino-4-((4-methoxyphenyl)amino)benzoic acid methyl ester (Compound 52-4), 3-amino-4-((3-methoxyphenyl)amino)benzoic acid methyl ester (Compound 52-5). Amino-4-(benzylamino)benzoic acid methyl ester (compound 52-4), 3-amino-4-((1-phenylethyl)amino)benzoic acid methyl ester (compound 52-5), 1-(4-(trifluoromethoxy)phenyl)dihydroindole-5-amine (compound 78), 1-(3,5-dimethylphenyl)-1H-indole-6-amine (compound 78-3), 1-(3,5-dimethylphenyl)dihydroindole-6-amine (compound 78-4), 1-(4-methoxyphenyl)-1H-indole-6-amine (compound 78-5), 1-(4-(methylthio)phenyl)-1H-indole-6-amine (compound 78-6), 1-(4-(trifluoromethoxy)phenyl)-1H-indole-5-amine (compound 78-8).

[0088] Item

[18] : In the pharmaceutical composition described in Item

[17] , the disease is selected from the group consisting of Alzheimer's disease, chronic kidney disease, diabetic neuropathy, liver damage, age-related macular degeneration, ischemic encephalopathy, vascular dementia, arteriosclerosis, Parkinson's disease, multiple sclerosis, cancer, asthma, hypertension, cardiovascular disease, and age-related eye disease.

[0089] Item

[19] : In the pharmaceutical composition described in Item

[17] , the disease is age-related macular degeneration.

[0090] Item

[20] : A method for preventing or treating a lipid peroxidation-induced disease or inhibiting the progression of a disease using at least one compound selected from the group consisting of the compounds described in Item

[17] above.

[0091] Item

[21] : Use of at least one compound selected from the group consisting of the group described in Item

[17] above in the preparation of a medicament for preventing or treating a lipid peroxidation-induced disease or inhibiting the progression of a disease.

[0092] Item

[22] : Use of at least one compound selected from the group consisting of the group described in Item

[17] above for preventing or treating a lipid peroxidation-induced disease or inhibiting the progression of a disease.

[0093] Effects of the Invention

[0094] By the test kit of the present invention and the screening method using the test kit, it is possible to easily find compounds showing lipid peroxidation inhibitory effects. In addition, the compounds found by the screening method of the present invention are useful for the treatment of lipid peroxidation-induced diseases (e.g., age-related macular degeneration), etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 This is a diagram showing the relationship between lipid peroxidation and diseases.

[0096] Figure 2 The graph shows the results of an experiment to evaluate the lipid peroxidation responsiveness of the fluorescent nitroxide probe. Liposomes (2.5 mg / mL Egg PC, 0.1 mg DCP), 5.0 μM fluorescent nitroxide, and 20 mM AAPH were mixed and the fluorescence intensity was measured after 40 minutes (NBD-TEMPO: λ Ex / λ Em =470 / 530nm, Dansyl-TEMPO: λ Ex / λ Em =300 / 500 nm). (n=3, mean+SD, **p<0.01ⅴv.s.ctrl(-)).

[0097] Figure 3 The graph shows the results of an experiment to evaluate the reactivity of the fluorescent nitroxide probe with various reducing substances. Liposomes (2.5 mg / mL Egg PC, 0.1 mg DCP), 5.0 μM fluorescent nitroxide, and 50 μM antioxidant were mixed and the fluorescence intensity (λ) was measured after 40 minutes. Ex / λ Em =470 / 530nm). The ratio of fluorescence intensity is the value of the fluorescence intensity of the probe after reaction with various antioxidants divided by the fluorescence intensity of ctrl(-) (negative control). (a) is the chemical structure of the antioxidant used in this study, and (b) is the ratio of fluorescence intensity obtained by the reaction of the probe and the antioxidant (n=3, mean+SD, *p<0.05, **p<0.01vsctrl(-)).

[0098] Figure 4The graph shows the results of an experiment for evaluating the reactivity of the fluorescent nitroxide probe with various oxidizing substances. 5.0 μM fluorescent nitroxide and various oxidizing substances were mixed and the fluorescence intensity (λ Ex / λ Em =470 / 530nm). The ratio of fluorescence intensity is the value of the fluorescence intensity of the probe after reaction with various oxidizing substances divided by the fluorescence intensity of ctrl(-). The oxidizing substances used were 0.5mM hydrogen peroxide, 0.5mM hypochlorous acid, 0.5mM potassium oxide, 0.5mM hydrogen peroxide and 5.0μM FeSO4, liposomes (2.5mg / mL Egg PC, 0.1mg DCP) and 10mM AAPH. (n=3, mean+SD, **p<0.01vsctrl(-)).

[0099] Figure 5 Graph showing the results of an experiment for evaluating the concentration-dependent lipid peroxidation reaction in an artificial lipid membrane in an AAPH system. Liposomes (2.5 mg / mL Egg PC, 0.1 mg DCP), 5.0 μM NBD-TEEPO, and AAPH were mixed and the fluorescence intensity (λ Ex / λ Em =470 / 530nm). The fluorescence response ability of NBD-TEEPO is (a) stimulus concentration dependent (AAPH is 0-20mM), and (b) antioxidant concentration dependent (AAPH is 20mM, AsA is 0-20μM). (c) Lipid peroxidation evaluation method using NBD-TEEPO (AAPH is 20mM, antioxidant is 10μM) and (d) TBARS assay (AAPH is 20mM, antioxidant is 10μM) were compared. (n=3, mean+SD, **p<0.01vsctrl(-), *p<0.05, **p<0.01vsctrl(+)).

[0100] Figure 6 It is shown that the Fe 2+ The results of the experiment on the dependence of the reaction initiator concentration on lipid peroxidation in the artificial lipid membrane in the system are shown in the figure. Liposomes (2.5 mg / mL Egg PC, 0.1 mg DCP), 5.0 μM NBD-TEEPO, and FeSO4 were mixed and the fluorescence intensity (λ Ex / λ Em=470 / 530nm). The fluorescence response ability of NBD-TEEPO is (a) stimulus concentration dependent (FeSO4 is 0-2mM), and (b) antioxidant concentration dependent (FeSO4 is 1mM, Eda is 0-20μM). (c) Lipid peroxidation evaluation method using NBD-TEEPO (FeSO4 is 1mM, antioxidant is 10μM) and (d) TBARS assay (FeSO4 is 1mM, antioxidant is 10μM) were compared. (n=3, mean+SD, **p<0.01vsctrl(-), #p<0.05, ##p<0.01vsctrl(+)).

[0101] Figure 7 This is a graph showing the results of an experiment for evaluating intracellular lipid peroxidation in a cultured cell system. 4 5.0 μM NBD-TEEPO, various oxidative substances and antioxidant substances 50 μM were added to the cells and the fluorescence intensity changes (λ Ex / λ Em =470 / 530nm). (a) is the fluorescence intensity of NBD-TEEPO after 45 minutes of adding AA (0-200μM), (b) is the fluorescence intensity of NBD-TEEPO after 45 minutes of adding 200μM AA and 50μM antioxidant, (c) is the fluorescence intensity of NBD-TEEPO after 45 minutes of adding tBHP (0-300μM), (d) is the fluorescence intensity of NBD-TEEPO after 45 minutes of adding 300μM tBHP and 50μM antioxidant, (e) is the S / B ratio, CV value, Z'-factor (AA addition system: after 45 minutes of adding 200μM AA, tBHP addition system: after 45 minutes of adding 300μM tBHP). (n=3, mean+SD, **p<0.01vsctrl(-), #p<0.05, ##p<0.01vsctrl(+)).

[0102] Figure 8 This is a graph showing the results of an experiment for evaluating lipid peroxidation-induced changes in cell viability in a cultured cell system using the MTT assay. 4 The cells were added with various oxidative substances and antioxidant substances at 50 μM and tested by MTT assay (λ max=570nm) and measured the cell survival rate after 24 hours. The cell survival rate was calculated assuming that 0μM oxidative stimulant and 0μM antioxidant were 100%. (a) is the cell survival rate after 24 hours of adding AA (0-100μM), (b) is the cell survival rate after 24 hours of adding 100μM AA and 50μM antioxidant, (c) is the cell survival rate after 24 hours of adding tBHP (0-100μM), (d) is the cell survival rate after 24 hours of adding 100μM tBHP and 50μM antioxidant, (e) is the S / B ratio, CV value, Z'-factor (AA addition system: 24 hours after adding 100μM AA, 24 hours after adding tBHP system: 24 hours after adding 100μM BHP). (n=3, mean+SD, **p<0.01vsctrl(-), #p<0.05, ##p<0.01vsctrl(+)).

[0103] Fig. 9 It is a diagram showing a schematic diagram of the screening method.

[0104] Fig.10 This is a graph showing the results of the primary screening of the Tokyo University Institute of Pharmaceutical Research in the AAPH system. Liposomes (2.5 mg / mL Egg PC, 0.1 mg DCP), 5.0 μM NBD-TEEPO, and 20 mM AAPH were mixed and the fluorescence intensity (λ Ex / λ Em =470 / 530 nm). The activity value of each compound after 40 minutes of reaction is shown.

[0105] Fig.11 It shows that Fe 2+ The results of AUC evaluation in the system are shown in FIG. Liposomes (2.5 mg / mL Egg PC, 0.1 mg DCP), 5.0 μM NBD-TEEPO, and 1.0 mM FeSO4 were mixed and the fluorescence intensity (λ Ex / λ Em =470 / 530 nm) was measured over 180 minutes. The area under the curve (AUC) was calculated from the obtained curve.

[0106] Fig.12 It shows that Fe 2+ The results of the first screening using the core library of the Tokyo University Pharmaceutical Research Institute were shown in the figure. Liposomes (2.5 mg / mL Egg PC, 0.1 mg DCP), 5.0 μM NBD-TEEPO, and 1.0 mM FeSO4 were mixed and the fluorescence intensity (λEx / λ Em =470 / 530 nm). The activity value of each compound after 180 minutes of reaction is shown.

[0107] Fig.13 This figure shows the results of secondary screening using the core library of the Tokyo Institute of Pharmaceutical Research. 4 5.0 μM NBD-TEEPO, 200 μM AA or 300 μM tBHP, and 50 μM of the compound were added to the cells and the fluorescence intensity change (λ Ex / λ Em =470 / 530nm). The dotted lines in (a) and (b) represent the activity values ​​of edaravone. (a) is the activity value of each compound after 45 minutes of adding AA, (b) is the activity value of each compound after 60 minutes of adding tBHP, (c) is a graphical representation of the activity values ​​in the AA addition system and the tBHP addition system, and (d) is an enlarged view of the portion of (c) where the activity value in the AA addition system is 30% or more and the activity value in the tBHP addition system is 50% or more.

[0108] Fig.14 This is a graph showing the results of secondary screening using the core library of the Tokyo University Pharmaceutical Research Institute based on the MTT assay. 4 100 μM AA or tBHP and 50 μM compound were added to the cells and the cell survival rate was measured by MTT assay 24 hours later. The dotted lines in (a) and (b) indicate the activity value of edaravone. (a) is the activity value of each compound after 24 hours after AA was added, (b) is the activity value of each compound after 24 hours after tBHP was added, (c) is a graphical representation of the activity values ​​in the AA addition system and the tBHP addition system, and (d) is an enlarged view of the part of (c) where the activity value in the AA addition system is 50% or more and the activity value in the tBHP addition system is 30% or more.

[0109] Fig.15 This figure shows the results of three screenings using the core library of the Tokyo Institute of Pharmaceutical Research. 4 5.0 μM NBD-TEEPO, 200 μM AA or 300 μM tBHP, and 50 μM of the compound were added to the cells and the fluorescence intensity change (λ Ex / λ Em=470 / 530nm) (white bars: original structure, black bars: structural analogs, dotted line: activity value of edaravone). (a) is the activity value of each compound after 45 minutes of adding AA, (b) is the activity value of each compound after 60 minutes of adding tBHP, (c) is a graphical representation of the activity values ​​in the AA addition system and the tBHP addition system, and (d) is an enlarged view of the portion of (c) where both the activity values ​​in the AA addition system and the activity values ​​in the tBHP addition system are 50% or more.

[0110] Fig.16 This is a graph showing the results of three screenings using the core library of the Tokyo University Pharmaceutical Research Institute based on the MTT assay. 4 100 μM AA or tBHP and 50 μM compound were added to the cells and the cell viability was measured by MTT assay after 24 hours (white bars: original structure, black bars: structural analogs, dotted line: activity value of edaravone). (a) is the activity value of each compound after 24 hours after AA addition, (b) is the activity value of each compound after 24 hours after tBHP addition, (c) is a graphical representation of the activity values ​​in the AA addition system and the tBHP addition system, and (d) is an enlarged view of the part of (c) where the activity value in the AA addition system is 0% or more and the activity value in the tBHP addition system is 40% or more.

[0111] Fig.17 The figure shows the results of the test for evaluating the cytotoxicity of the tertiary screening using the core library of the Institute of Pharmaceutical Research, The University of Tokyo. 4 Compounds were added at 50 μM to the cells and the cell viability was determined by MTT assay after 72 hours. The dotted line indicates the cell viability after 72 hours of incubation with edaravone.

[0112] Fig.18 This is a graph showing the results of a primary screening using the Prestwick chemical library. Liposomes (2.5 mg / mL Egg PC, 0.1 mg DCP), 5.0 μM NBD-TEEPO, and a reaction initiator were mixed and the change in fluorescence intensity (λ Ex / λ Em =470 / 530nm). (a) is the activity value of each compound 40 minutes after the start of the AAPH system measurement, (b) is the Fe 2+ The system measures the activity value of each compound 180 minutes after the start.

[0113] Fig.19 The graphs show the results of a primary screening using the Prestwick chemical library and the relationship between candidate compounds and target disease areas. (a) is a graph showing the relationship between the AAPH system and Fe in the primary screening. 2+ Graph showing the activity values ​​of the additive system, (b) is the target disease areas of the first 16 compounds, (c) is the activity value of the AAPH system in (a) is 0.7 or more and Fe 2+ This is an enlarged view of the portion having an activity value of 0.8 or more in the addition system.

[0114] Fig. 20 The figure shows the method of preparing age-related macular degeneration (AMD) model mice. (a) is the timeline of preparing AMD model mice, and (b) is the ONL measurement method: a total of 27 points (A-center-Z) were measured for each 180 μm thickness of the ONL (left: observation field is 4 times, right: observation field is 60 times).

[0115] Fig.21 The figure shows the results of evaluating the thickness of the outer nuclear layer (ONL). (a) is a bright field image of ONL (observation magnification: 60 times), and (b) is the thickness of ONL (model compound: compound W). The left side shows the lower hemisphere of the eyeball, and the right side shows the upper hemisphere. (c) is the average thickness of ONL at points Q to T (n=3-5, mean+SD, *p<0.05, **p<0.01vsctrl(-), #p<0.05, ##p<0.01vsctrl(+)).

[0116] Fig. 22 It shows the main effects / action points, median lethal dose (LD50) of active index compounds and candidate compounds. 50 ) and a table of animal dosing examples. For edaravone and the five compounds used in this study, the main action / action site, LD 50 , Animal Administration Examples. Among them, oral means oral administration, sc means subcutaneous injection, and iv means intravenous injection. DETAILED DESCRIPTION

[0117] (Test method and test kit)

[0118] The present inventors provide an assay method and an assay kit for the purpose of finding lipid peroxidation inhibitors, wherein the assay method and the assay kit are used to develop a screening method (eg, a high-throughput screening method) capable of testing and evaluating a plurality of compounds at one time.

[0119] The present invention provides an assay kit for detecting the lipid peroxidation inhibition activity of a test compound, wherein the assay kit comprises a compound represented by formula (I), a liposome, and at least one compound selected from the group consisting of 2,2'-azobis(2-aminopropane) dihydrochloride and a divalent iron ion supply source in a buffer, wherein formula (I) is as follows:

[0120] [Chemistry 3]

[0121]

[0122] The liposome used in the assay kit of the present invention comprises liposomes prepared from phosphatidylcholine from egg yolk (egg yolk-derived phosphatidylcholine (Egg PC)) and dihexadecyl phosphate (DCP) as lipid sources.

[0123] The radical reaction initiator includes at least one compound selected from the group consisting of 2,2′-azobis(2-aminopropane) dihydrochloride (hereinafter referred to as “APPH”) or a divalent iron ion supply source substance (eg, FeSO 4 ).

[0124] The assay kit of the present invention comprises a solution of a buffer (eg, phosphate buffer).

[0125] Fluorescent nitroxides

[0126] As the lipid radical scavenger used in the assay method and assay kit of the present invention, a fluorescent nitroxide compound represented by formula (I) is used. The fluorescent nitroxide compound is described in the literature proposed by the present inventors (for example, Japanese Patent Application No. 2017-090739).

[0127] Here, nitroxide (NO·) is a stable free radical with paramagnetism, which has the property of attenuating fluorescence by intersystem crossing caused by photoinduced electron transfer or electron-spin exchange in a charge separation state. Fluorescent nitroxide covalently bound to a fluorescent chromophore is in an intramolecular quenching state. However, it is confirmed that once nitroxide reacts with a free radical and loses paramagnetism, it is in a fluorescent luminescent state. That is, for fluorescent nitroxide, it can be evaluated as a probe useful for detecting the capture of lipid free radicals by fluorescence observation and measuring fluorescence intensity.

[0128] Most of the lipid molecules as the detection object are present in the biomembrane and form a hydrophobic environment. Therefore, environmental responsive fluorescent chromophores are most suitable, although the fluorescence decays in a hydrophilic environment, it selectively emits high fluorescence in a hydrophobic environment. As fluorescent chromophores, fluorescent chromophores (for example, nitrobenzofurazan (nitrobenzofurazan, hereinafter referred to as "NBD") and 5-(dimethylamino)naphthalene-1-sulfonyl chloride (hereinafter referred to as "dansyl (Dansyl)") widely used in lipid fields such as phase transfer, membrane fusion or intracellular lipid metabolism of biomembrane can be enumerated.

[0129] In addition, as for the probe molecules that are α-substituents of fluorescent nitroxide compounds, for example, 2,2,6,6-tetramethylpiperidin-1-oxyl (hereinafter referred to as "TEMPO"), 2,2,6,6-tetraethylpiperidin-1-oxyl (hereinafter referred to as "TEEPO") and 2,2,6-trimethyl-6-pentyl-piperidin-1-oxyl (hereinafter referred to as "Pen").

[0130] In order to select fluorescent nitroxide compounds suitable for the assay method and assay kit of the present invention, the above-mentioned fluorophore molecules and probe molecules are optimized respectively. The optimization of fluorophore molecules is carried out by testing the responsiveness of lipid peroxidation reactions.

[0131] In addition, the α-substituent of the fluorescent nitroxide compound, i.e., the probe molecule, is optimized by a test for evaluating the reactivity with a reducing substance and a test for evaluating the reactivity with an oxidizing substance. Here, in the assay method of the present invention, the inhibition of lipid peroxidation by means of a reducing substance (i.e., an antioxidant) is used as the evaluation of inhibition, that is, whether the increase in fluorescence intensity caused by the lipid peroxidation reaction that has occurred can be reduced by means of an antioxidant (e.g., a test compound) is used as the evaluation of inhibition. Therefore, the increase in fluorescence intensity caused by the direct reaction of the probe molecule with the antioxidant involves the detection of a false negative. Therefore, the reactivity of the antioxidant with the probe molecule is first checked.

[0132] Next, the responsiveness of the probe molecule to lipid peroxidation was evaluated by a test for evaluating the reactivity of the probe molecule in the fluorescent nitroxide compound with an oxidative substance. 2+ ·OH as active oxygen is generated. In addition, lipid peroxidation reaction occurs by liposomes and AAPH.

[0133] From the results of the above tests, the NBD-TEEPO compound showing the highest resistance to reduction and high responsiveness to lipid peroxidation was selected as the fluorescent nitroxide compound.

[0134] Establishment of assay methods and assay kits

[0135] The present invention provides an assay method and an assay kit using an NBD-TEEPO compound represented by formula (I) that can be applied to a screening method.

[0136] Cell-free system assays

[0137] The present invention provides an assay method in a cell-free system for detecting lipid peroxidation inhibitory activity using an NBD-TEEPO compound represented by formula (I).

[0138] As in the above method for optimizing fluorescent nitroxides, liposomes were used as lipids, and AAPH and FeSO4 were used as free radical reaction initiators. 2+ In any system, the fluorescence intensity of the probe also increased in a concentration-dependent manner. In addition, when a water-soluble antioxidant (e.g., ascorbic acid (AsA)) was used in a system using AAPH, the fluorescence intensity of the probe increased in a concentration-dependent manner. 2+ When lipid antioxidants (e.g., edaravone (EDA)) are used in the system, the increase in fluorescence is suppressed in a concentration-dependent manner. In the two assay methods, by using a variety of known antioxidants, it can be seen that the assay method of the present invention is an assay method that can evaluate lipid peroxidation and its inhibitory effect brought about by antioxidants.

[0139] In addition, for the 2-thiobarbituric acid reactive substance (hereinafter referred to as "TBARS") method for measuring the inhibitory effect of lipid peroxidation, the same known antioxidant was used to test and compared with the results of the assay method of the present invention, thereby obtaining the same results. It can be seen that the assay method of the present invention is an assay method that can evaluate lipid peroxidation and its inhibitory effect brought about by antioxidants. In addition, the assay method of the present invention does not require complicated operations such as the TBARS method.

[0140] In one embodiment, a specific assay method includes the following steps.

[0141] i) preparing a buffer solution comprising the compound represented by formula (I) and liposomes;

[0142] ii) adding at least one compound selected from the group consisting of 2,2'-azobis(2-aminopropane) dihydrochloride and a divalent iron ion supply source material;

[0143] iii) adding the test compound;

[0144] iv) measuring fluorescence; and,

[0145] v) The activity value of the test compound is determined from the fluorescence measurement results.

[0146] In one embodiment, the cell-free system assay kit of the present invention comprises a combination of an assay kit in which the reaction initiator is 2,2'-azobis(2-aminopropane) dihydrochloride and an assay kit in which the reaction initiator is iron (II) sulfate.

[0147] Composition of the Cell-Free System Assay Kit

[0148] With respect to the cell-free system assay kit of the present invention,

[0149] The concentration of the compound represented by formula (I) is 1.0 to 20.0 μM (eg, 5.0 μM to 20.0 μM, typically 5.0 μM),

[0150] Liposomes are prepared from egg yolk-derived phosphatidylcholine and dicetyl phosphate, wherein the concentration of the egg yolk-derived phosphatidylcholine is 5.0 to 10.0 mg / mL (e.g., 2.5 mg / mL), and the concentration of the dicetyl phosphate is 0.01 to 1.0 mg / mL (e.g., 0.1 mg / mL).

[0151] The concentration of the test compound is 5 to 100 μM (e.g., 10 μM).

[0152] The concentration of 2,2'-azobis(2-aminopropane) dihydrochloride is 5 to 50 mM (for example, 20 mM),

[0153] The concentration of the ferrous iron supply source (eg, FeSO 4 ) is 0.5 to 50 mM (eg, 1 mM).

[0154] In addition, the cell-free system assay method of the present invention can be applied to a screening method because the indices (eg, S / B ratio, CV value, Z'-factor) representing the characteristics of the screening system are sufficient values.

[0155] Cell System Assays

[0156] In addition to providing the above-mentioned cell-free system assay method, the present invention also provides a cell-based system assay method for detecting lipid peroxidation inhibitory activity using the NBD-TEEPO compound represented by formula (I).

[0157] The cell system assay method is performed by using cultured cells (e.g., HepG2 cells from human liver cancer) instead of liposomes used in the cell-free system, and using either arachidonic acid (hereinafter referred to as "AA") or tert-butyl hydroperoxide (hereinafter referred to as "tBHP") as a free radical reaction initiator instead of AAPH and a divalent iron supply source.

[0158] In one embodiment, a specific assay method includes the following steps.

[0159] i) preparing a buffer solution comprising a compound represented by formula (I) and cultured cells;

[0160] ii) adding at least one compound selected from the group consisting of arachidonic acid and tert-butyl hydroperoxide;

[0161] iii) adding the test compound;

[0162] iv) measuring fluorescence; and,

[0163] v) The activity value of the test compound is determined from the fluorescence measurement results.

[0164] In one embodiment, the cell system assay kit of the present invention comprises a combination of an assay kit in which the reaction initiator is arachidonic acid and an assay kit in which the reaction initiator is tert-butyl hydroperoxide.

[0165] Composition of the Cell System Assay Kit

[0166] With respect to the cell system assay kit of the present invention,

[0167] The concentration of the compound represented by formula (I) is 1.0 to 20.0 μM (e.g., 5.0 μM),

[0168] The concentration of cultured cells was 1×10 4 ~1×10 5 The number of cells (e.g. 1×10 4 Cell number),

[0169] The concentration of the test compound is 5 to 500 μM (e.g., 50 μM),

[0170] The concentration of arachidonic acid is 100 to 400 μM (e.g., 200 μM),

[0171] The concentration of tert-butyl hydroperoxide is 100 to 400 μM (eg, 300 μM).

[0172] In addition, the cell system assay method of the present invention can be applied to a screening method because the indices (eg, S / B ratio, CV value, Z'-factor) representing the characteristics of the screening system are sufficient values.

[0173] The test kit of the present invention may include additional instructions showing the activity value of the compound, wherein the compound shows the activity of inhibiting lipid peroxidation. The lipid peroxidation inhibitory activity of the test compound can be evaluated by comparing the activity value of the test compound obtained by using the test kit or the test method of the present invention with the activity value of the index compound described in the additional instructions.

[0174] In one embodiment, the assay method of the present invention comprises a combination of at least two assay methods selected from the group consisting of an assay method for a cell-free system in which the reaction initiator is 2,2'-azobis(2-aminopropane) dihydrochloride, an assay method for a cell-free system in which the reaction initiator is iron (II) sulfate, an assay method for a cell-free system in which the reaction initiator is arachidonic acid, and an assay method for a cell-based system in which the reaction initiator is tert-butyl hydroperoxide.

[0175] In addition, in one embodiment, the assay kit of the present invention comprises a combination of at least two assay kits selected from the group consisting of an assay kit for a cell-free system in which the reaction initiator is 2,2'-azobis(2-aminopropane) dihydrochloride, an assay kit for a cell-free system in which the reaction initiator is iron (II) sulfate, an assay kit for a cell-free system in which the reaction initiator is arachidonic acid, and an assay kit for a cell-based system in which the reaction initiator is tert-butyl hydroperoxide.

[0176] Lipid peroxidation-induced cell death occurs in the process where lipid peroxidation promotes the onset and progression of diseases (Reference: Uchida K., Prog. Lipid Res., 2003, 42 (4), 318-43.). The method using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (hereinafter referred to as "MTT") is used to evaluate cell survival rate. It is also known that the MTT method can be used for plate assay.

[0177] In one embodiment, the assay method of the present invention includes an assay method according to the MTT method and comprising the following steps.

[0178] i) performing an assay using a medium containing cultured cells, a test compound, and arachidonic acid according to the MTT method, and selecting a compound that shows a high cell viability; and / or

[0179] ii) An assay method using a culture medium containing cultured cells, a test compound, and tert-butyl hydroperoxide is performed according to the MTT method, and a compound showing a high cell viability is selected.

[0180] The assay method and assay kit of the present invention may be combined with the above-mentioned assay method and assay kit for a cell-free system and / or a cell system of the present invention to include an assay method and assay kit in accordance with the MTT method.

[0181] The MTT method can be applied to a screening method because the indices (eg, S / B ratio, CV value, Z'-factor) representing the characteristics of the screening system are sufficient values.

[0182] Any of the assay methods of the present invention can also be performed using a microplate. Here, the microplate includes multi-well plates (e.g., 96-well plates and 384-well plates), but is not limited to these well plates. The microplate can be, for example, a commercially available microplate.

[0183] In one embodiment, the assay method of the present invention using a microplate includes the following steps.

[0184] i) dispensing a solution of the test compound into a microplate;

[0185] ii) dispensing a solution comprising the compound represented by formula (I) and liposomes or cultured cells into each well;

[0186] iii) when using liposomes, dispensing a solution containing at least one compound selected from the group consisting of 2,2'-azobis(2-aminopropane) dihydrochloride and a divalent iron ion supply source substance into each well,

[0187] When using cultured cells, dispensing a solution containing at least one compound selected from the group consisting of arachidonic acid and tert-butyl hydroperoxide into each well; and,

[0188] iv) Measure the fluorescence using a microplate reader.

[0189] (Screening method)

[0190] The present invention provides a screening method for finding a compound showing lipid peroxidation inhibitory activity, the screening method comprising a screening step of using the assay method of the present invention on a certain compound library.

[0191] Here, compound library can be a known compound library, or it may not be a known compound library. As a known compound library, compound libraries (e.g., Prestwick compound library (PRESTWICK CHEMICAL LIBRARY)) (compounds that the compound library collects are compounds that have expired in the patent term) and compound libraries (e.g., core libraries (Core library) in the comprehensive library (General library) of the Tokyo University's drug creation agency) that have not yet obtained the approval of compounds of those foods or pharmaceuticals can be enumerated. Fig. 9 A schematic diagram showing the screening method of the present invention.

[0192] 1 screening

[0193] In the screening using the liposome-AAPH system assay method, compounds with free radical capture ability can be detected, ranging from highly water-soluble antioxidants that inhibit free radical species derived from water-soluble AAPH to highly fat-soluble antioxidants that inhibit lipid peroxidation chain reactions. 2+ In the screening of the system assay method, compounds with higher lipid solubility are easily detected among compounds with free radical scavenging ability, but on the other hand, there is a possibility that iron chelators without free radical scavenging ability will be detected. Therefore, in this first screening, first, candidate compounds with free radical scavenging ability in the AAPH system screening are widely selected from the test compounds, and then, for the compounds narrowed in the liposome-AAPH system screening, the liposome-Fe chelator is screened. 2+ The range of candidate lipid-soluble compounds was further narrowed in systematic screening.

[0194] The screening method using the assay method of the present invention includes screening by a cell-free system assay method using liposomes as a primary screening.

[0195] The primary screening method of the present invention includes the following method. That is, the screening method for selecting candidate compounds with high lipid peroxidation inhibitory activity includes:

[0196] i) selecting a test compound from a compound library;

[0197] ii) performing screening by a cell-free assay method (hereinafter referred to as "liposome-AAPH system") using 2,2'-azobis(2-aminopropane) dihydrochloride (hereinafter referred to as "AAPH") using a test compound, and selecting a compound showing a high activity value; and,

[0198] iii) Next, a cell-free assay method using a divalent iron ion supply source substance (hereinafter referred to as "liposome-Fe 2+ System") and select compounds showing high activity values.

[0199] The activity value of each test compound was calculated based on the following formula from the fluorescence intensity measured in the above screening ii).

[0200] Activity value = 1-(Flu Sample -Flu Background ) / (Flu Control -Flu Background )

[0201] in,

[0202] Flu Sample : Fluorescence intensity in the presence of AAPH and each compound (n=1),

[0203] Flu Background : Fluorescence intensity without AAPH,

[0204] Flu Control : Fluorescence intensity in the presence of AAPH but without each compound.

[0205] The activity value of each test compound was calculated based on the following formula from the fluorescence intensity measured in the screening in iii) above.

[0206] Activity value = 1-(AUC Sample / AUC Control )

[0207] in,

[0208] AUC Sample :According to Fe 2+ The area under the curve calculated from the fluorescence intensity of each compound (n=1), AUC Control :According to Fe 2+ The area under the curve was calculated from the fluorescence intensity in the absence of each compound.

[0209] The candidate compounds are narrowed down by comparing the activity values ​​of known compounds (hereinafter referred to as "active index compounds") known to have high lipid peroxidation inhibitory activity. Examples of the active index compounds include edaravone, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol) and (-)-epicatechin.

[0210] 2nd screening

[0211] In the case where a compound library (e.g., a core library in the General Library of the Tokyo University Pharmaceutical Research Institute) that has been collected and has not been approved as a food or pharmaceutical product is used as a compound library, for the purpose of examining the lipid peroxidation inhibitory activity and cell death inhibitory activity in the cell system, screening by means of the cell system assay method of the present invention using cultured cells (e.g., HepG2 cells derived from human liver cancer) is performed as a higher level screening (e.g., secondary screening). On the other hand, in the case of using a compound library that has been collected and has been approved as a food or pharmaceutical product, it is not necessary to examine the lipid peroxidation inhibitory activity and cell death inhibitory activity in the cell system, so the secondary screening can be appropriately omitted.

[0212] As a screening for examining lipid peroxidation inhibitory activity in a cell system, screening using the cell system assay method of the present invention is performed, and activity values ​​are obtained based on the results of fluorescence intensity measurement, and then compared with the activity values ​​of activity index compounds to narrow down the range of candidate compounds.

[0213] The secondary screening method of the present invention includes the following methods. That is, it includes:

[0214] i) performing screening by a cell system assay method using arachidonic acid and selecting compounds showing high activity values;

[0215] ii) performing a cell system assay method using tert-butyl hydroperoxide and selecting compounds showing high activity values; and,

[0216] iii) Selecting compounds which show high activity values ​​in both the assays of i) and ii).

[0217] In the screening using the above-mentioned cell system assay method, the activity value was determined based on the following formula from the fluorescence intensity value measured when the measurement was performed using AA or tBHP as a reaction initiator.

[0218] Activity value = 100 (AUC Sample -AUC Background ) / (AUC Control -AUC Background )

[0219] in,

[0220] AUC Sample : The area under the curve calculated from the fluorescence intensity when a reaction initiator and each compound (n=1) are present,

[0221] AUC Control: The area under the curve calculated from the fluorescence intensity when there is a reaction initiator but no compound,

[0222] AUC Background : The area under the curve calculated from the fluorescence intensity in the absence of a reaction initiator and each compound.

[0223] Next, the candidate compounds were narrowed down by comparison with the values ​​of the activity index compounds. The activity values ​​obtained in each screening using the obtained AA or tBHP are shown in the figure. In both screenings, candidate compounds showing high activity values ​​for inhibiting lipid peroxidation were selected.

[0224] In addition, compounds showing high activity in suppressing cell death induced by lipid peroxidation were searched.

[0225] As a screening for examining the cell death inhibitory activity in a cell system, HepG2 cells derived from human liver cancer were used to perform an assay using AA and tBHP as free radical initiators to measure the cell survival rate. The activity value was calculated based on the following formula from the cell survival rate value.

[0226] Activity value = 100 (survival rate Sample -Survival rate Control(+) ) / (survival rate Control(-) -Survival rate Control(+) )

[0227] in,

[0228] Survival rate Sample : Cell survival rate in the presence of reaction initiator and each compound (n=1),

[0229] Survival rate Control(+) : Cell survival rate with reaction initiator and each compound,

[0230] Survival rate Control(-) : Cell viability in the absence of reaction initiator and each compound.

[0231] Then, the candidate compounds were narrowed down by comparing with the values ​​of the activity index compounds. The above activity values ​​obtained in each assay system using the obtained AA or tBHP are shown in the figure. In both assay systems, candidate compounds showing high cell death inhibition (cell death prevention) activity values ​​were selected.

[0232] In one embodiment, the secondary screening method of the present invention further comprises the following method. That is, it comprises:

[0233] i) performing screening by an assay method using a medium containing cultured cells, a test compound, and arachidonic acid according to the MTT method, and selecting a compound that shows a high cell viability;

[0234] ii) performing screening by an assay method using a medium containing cultured cells, a test compound, and tert-butyl hydroperoxide according to the MTT method, and selecting a compound showing a high cell viability; and,

[0235] iii) selecting candidate compounds that show high cell viability in both the assays of i) and ii).

[0236] 3 screenings

[0237] In the compound library used (e.g., the core library of the Tokyo University Institute for Pharmaceutical Research), there are cases where structural analogs related to the candidate compound showing a high activity value in the low-level screening assay (e.g., secondary screening) described above are included. Therefore, for such structural analogs, a higher-level screening (e.g., tertiary screening) is appropriately performed for lipid peroxidation inhibitory activity together with the candidate compound to sort the candidate compound.

[0238] In addition, these candidate compounds were further screened using an assay for examining cell death inhibitory activity.

[0239] Finally, candidate compounds are selected by comprehensively considering the final high-level screening results (eg, 3rd screening) and the screening results for cell death inhibitory activity.

[0240] In one embodiment, the screening method of the present invention comprises:

[0241] i) selecting from the compound library a structural analogue of the compound selected by the screening method based on the cell system assay of the present invention;

[0242] ii) performing a screening method using the cell system assay of the present invention on the compound selected in i) and the previously appropriately selected compound, and selecting a compound showing a high activity value;

[0243] iii) performing a screening method using a cell assay according to the MTT method of the present invention on the compound selected in i) and the previously appropriately selected compound, and selecting a compound showing a high cell viability;

[0244] iv) selecting candidate compounds showing high activity values ​​and high cell viability in both screening methods ii) and iii);

[0245] v) for the compound selected in i) and the compound appropriately selected by screening based on a cell system assay or screening based on a cell assay according to the MTT method, screening by an assay method using a medium containing cultured cells is performed, and a candidate compound showing a high cell viability is selected; and,

[0246] vi) selecting candidate compounds from the compounds selected in iv) and the compounds selected in v).

[0247] The screening method of the present invention can be used as a high-throughput screening method.

[0248] (Application of filter results)

[0249] Diseases in which lipid free radicals in lipid peroxidation reactions participate involve a wide range of disease areas. Therefore, for each target disease, it is also possible to narrow the scope of candidate compounds based on the knowledge of its mechanism of action and considering other factors. For example, in the case of a disease that requires permeability to the blood-brain barrier as the target, the scope of candidate compounds can be further narrowed by considering fat solubility. Specifically, in the case of cerebral infarction and retinal diseases (e.g., age-related macular degeneration) as target diseases, compounds with permeability to the blood-brain barrier are advantageous, and on the other hand, in the case of liver cancer and arteriosclerosis as target diseases, permeability to the blood-brain barrier is not required.

[0250] (Medical Use)

[0251] As used in this specification, "treatment" or "prevention" of a disease caused by lipid peroxidation means one or more of (1) eliminating the disease; (2) alleviating or minimizing the severity of the disease; (3) delaying the progression or onset of the disease; and (4) reducing, minimizing or eliminating the occurrence or frequency of the disease.

[0252] For example, Figure 1 As shown in the figure, the "disease caused by lipid peroxidation" or "lipid peroxidation-induced disease" used in this specification includes diseases for which the relationship between lipid peroxidation and the disease is known. For example, it includes one or more diseases selected from the group consisting of Alzheimer's dementia, chronic kidney disease, diabetic neuropathy, liver damage, age-related macular degeneration, ischemic encephalopathy, vascular dementia, arteriosclerosis, Parkinson's disease, multiple sclerosis, cancer, asthma, hypertension, cardiovascular disease, and age-related eye disease.

[0253] The term “subject” used in this specification includes human beings and non-human animals.

[0254] The active drug of the present invention includes the form of its pharmaceutically acceptable salt. In addition, the active drug of the present invention or its pharmaceutically acceptable salt includes its hydrate or solvate with a solvent, etc. In addition, the present invention includes all forms of crystals of the active drug of the present invention.

[0255] As the pharmaceutically acceptable salt, for example, salts with organic bases (eg, diethanolamine salts, ethylenediamine salts) and salts with inorganic bases (eg, alkali metals (eg, sodium, potassium) and alkaline earth metals (eg, calcium or magnesium) are included.

[0256] When the active drug of the present invention is used for treatment or prevention, it can be administered orally or parenterally as a pharmaceutical composition (for example, intravenous, subcutaneous, or intramuscular injection, topical, rectal, percutaneous, intraspinal or nasal administration). As a composition for oral administration, for example, tablets, capsules, pills, granules, powders, solutions, suspensions, etc. can be mentioned, and as a composition for parenteral administration, for example, aqueous preparations or oily preparations for injection, ointments, creams, lotions, aerosols, suppositories, patches, etc. can be mentioned. These preparations can be prepared using existing well-known techniques and contain non-toxic and inert carriers or additives commonly used in the medical field (hereinafter referred to as "pharmaceutically acceptable carriers").

[0257] As used in this specification, "pharmaceutically acceptable carriers" may contain, in addition to effective active ingredients, various active ingredients or medicinal ingredients (including pharmacologically active ingredients and physiologically active ingredients) and combinations of additives (e.g., buffers, isotonic agents, pH adjusters, preservatives / stabilizers, thickeners, chelating agents, surfactants, fragrances, etc.) according to various uses, as long as they do not hinder the pharmacological effects. Such ingredients can be appropriately formulated within a concentration range that does not cause irritation and the like, and the types of ingredients are not particularly limited, and examples thereof include buffers (e.g., sodium phosphate), isotonic agents (e.g., sodium chloride), pH adjusters (e.g., boric acid), preservatives / stabilizers (e.g., benzalkonium chloride), stabilizers (e.g., mannitol), thickeners (e.g., sodium alginate), chelating agents (e.g., sodium ethylenediaminetetraacetate), surfactants (e.g., polyoxyethylene sorbitan monooleate), fragrances (e.g., menthol), and the like.

[0258] The term "administering" used in this specification includes: providing and / or prescribing an active drug or a pharmaceutical composition containing the same to a subject; or, the subject receiving the active drug or pharmaceutical composition of the present invention, etc. The administration route of the active drug or pharmaceutical composition of the present invention may be any administration route, and may vary depending on, for example, the disease, symptoms, or age, weight, sex, etc. of the subject.

[0259] The "effective amount" used in this specification refers to the amount of the active drug sufficient to provide the indicated effect, i.e., the treatment or prevention of the lipid peroxidation-induced disease described in this specification. In addition, for the indicated disease, the active drug or pharmaceutical composition of the present invention can be used in combination with a known active drug or pharmaceutical composition.

[0260] As for the dosage of the active drug of the present invention, although it varies according to each active drug or pharmaceutical composition and the disease, age, weight, sex, symptom, route of administration, etc. of the test subject, in the case of parenteral administration, it is usually 0.001 to 100 mg / kg per day, preferably 0.01 to 100 mg / kg. In the case of oral administration, it is usually 0.01 to 1000 mg / kg per day, preferably 0.1 to 100 mg / kg. The active drug of the present invention is administered once or several times a day (or 2 to 3 times). In addition, it can also be administered once every few days to weeks.

[0261] Example

[0262] Although examples of the present invention are described below as examples, the present invention is not limited to these examples.

[0263] Reagents, cell culture-related reagents, and fluorescent nitroxide compounds were commercially available products or substances prepared according to known methods. Core library compounds and Prestwick chemical library compounds were provided by the Tokyo University Institute for Drug Discovery and the Kyushu University Center for Advanced Research and Education in Chemical Library Drug Discovery, respectively. In addition, various measuring instruments used commonly used instruments.

[0264] (Reference Example 1)

[0265] Preparation of NBD-TEMPO compounds

[0266] 2,2,6,6-Tetramethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)piperidin-1-oxyl (NBD-TEMPO) was prepared according to the following sequence. Specifically, 366 mg (2.0 mmol) of 4-fluoro-7-nitro-2,1,3-benzoxadiazole was dissolved in 10 mL of AcOEt (ethyl acetate) and 342 mg (2.0 mmol) of 4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl was added. After stirring at room temperature for 3 hours, saturated brine was added, extracted with AcOEt, the organic layer was dried with Na2SO4, and the solvent was completely distilled off. Thereafter, separation and purification were carried out by silica gel column chromatography (CHCl3), and 574 mg of orange-yellow crystals were obtained (yield: 86%). HRMS (ESI + )Calculated value (cald for) C 15 H 20 N5NaO4[M+Na] + :357.1413, measured value(found):357.1415.

[0267] (Reference Example 2)

[0268] Preparation of Dansyl-TEMPO compounds

[0269] 2,2,6,6-tetramethyl-4-(5-(dimethylamino)naphthalene-1-sulfonylamino)piperidin-1-oxyl (Dansyl-TEMPO) was prepared according to the method described in the literature (e.g., Lozinsky et al., Lozinsky, E., et.al., J. Biolchem. Biophys., Methods, 1999, 38, 29-42). Specifically, 1.03 g (6.0 mmol) of 4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl was dissolved in 5 ml of acetone, and 1.35 g (5.0 mmol) of 5-(dimethylamino)naphthalene-1-sulfonyl chloride and 0.483 ml of pyridine were added in an ice bath. After stirring at room temperature overnight, saturated brine was added, extracted with diethyl ether, the organic layer was dried with Na2SO4, and the solvent was completely distilled off. Thereafter, the product was separated and purified by silica gel column chromatography (CHCl3:MeOH=99:1) to obtain 396 mg (yield: 20%). HRMS (ESI + )Calculated value (cald for) C 21 H 30 N3NaO3S[M+Na] + :427.1906, Measured value(found):427.1900.

[0270] (Reference Example 3)

[0271] Preparation of NBD-TEEPO compounds

[0272] 2,2,6,6-tetraethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)piperidin-1-oxyl (NBD-TEEPO) was prepared according to the method described in the literature (e.g., Bognar et al., Bognar, B., et al., J. Heterocycl. Chem., 2006, 43, 81-86). Specifically, 87.6 mg (0.44 mmol) of 4-chloro-7-nitro-2,1,3-benzoxadiazole and 61 μL of Et3N (triethylamine) were dissolved in 10 mL of AcOEt (ethyl acetate) and 100 mg (0.44 mmol) of 4-amino-2,2,6,6-tetraethylpiperidin-1-oxyl was added. After stirring at room temperature for 6 hours, saturated brine was added, extracted with AcOEt, the organic layer was dried with Na2SO4, and the solvent was completely distilled off. Thereafter, separation and purification were performed by silica gel column chromatography (Hexane:AcOEt=100:0 to 70:30) to obtain 83 mg of orange-yellow crystals (yield: 6%). HRMS (ESI + )Calculated value (cald for) C 19 H 28 N5NaO4[M+Na] + :413.2034, Found:413.2024

[0273] (Reference Example 4)

[0274] Preparation of NBD-Pen compounds

[0275] The reaction was carried out in the same manner as the synthesis of 2,2,6,6-tetraethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)piperidin-1-oxyl (NBD-TEEPO), except that 100 mg (0.44 mmol) of 4-amino-2,2,6-trimethyl-6-pentylpiperidin-1-oxyl (8) was substituted for 100 mg (0.44 mmol) of 4-amino-2,2,6,6-tetraethylpiperidin-1-oxyl (4). The product was separated and purified by silica gel column chromatography (hexane:AcOEt=100:0 to 70:30) to obtain 83 mg of orange-yellow crystals (yield: 48%). HRMS (ESI + )Calculated value (cald for) C 19 H 28 N5NaO4[M+Na] + :413.2034, Found:413.2056

[0276] (Example 1)

[0277] Evaluation of the responsiveness of fluorescent nitroxide probes to lipid peroxidation

[0278] Fluorescent nitroxide (NBD-TEMPO compound or Dansyl-TEMPO compound) (5.0 μM) and liposomes (2.5 mg / mL Egg PC, 0.1 mg / mL DCP) were mixed in phosphate buffer (10 mM, pH 7.4, 0.5% DMSO, 0.5% acetonitrile) at 37°C. AAPH (20 mM) was added to start the lipid peroxidation reaction. After 40 minutes, the fluorescence intensity at an excitation wavelength of 470 nm and a fluorescence wavelength of 530 nm was measured for NBD-TEMPO, and the fluorescence intensity at an excitation wavelength of 300 nm and a fluorescence wavelength of 500 nm was measured for Dansyl-TEMPO.

[0279] The results are shown in Figure 2 .

[0280] When the conditioned liposomes were stimulated with AAPH, the fluorescence intensity in NBD-TEMPO increased by 8.2 times compared to when AAPH was not added. On the other hand, the increase in fluorescence intensity in Dansyl-TEMPO remained at 1.4 times. Therefore, it was decided to use the NBD group as a fluorophore.

[0281] (Example 2)

[0282] Evaluation of the reactivity of fluorescent nitroxide probes with various reducing substances

[0283] Fluorescent nitroxide (5.0 μM) (NBD-TEMPO compound, NBD-Pen compound, or NBD-TEEPO compound) and 50 μM of various reducing substances (AsA, UA, TPL, Eda, Catechin, Trolox) were mixed in phosphate buffer (10 mM, pH 7.4, 0.5% DMSO, 0.5% acetonitrile) containing liposomes (2.5 mg / mL Egg PC, 0.1 mg / mL DCP) at 37°C. AA (0.5 mM) and LOX (25 μg / mL) were mixed to perform lipid peroxidation. After 40 minutes, the fluorescence intensity was measured at an excitation wavelength of 470 nm and a fluorescence wavelength of 530 nm. Here, AsA refers to ascorbic acid, UA refers to uric acid, TPL refers to 2,2,6,6-tetramethylpiperidin-1-oxyl, Eda refers to edaravone, Catechin refers to (-)-epicatechin, and Trolox refers to 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

[0284] The results are shown in Figure 3 .

[0285] Six substances were selected as model antioxidants (Figure (3a)). These six model antioxidants are AsA, which acts as a water-soluble antioxidant in the human body and is also contained in many foods, etc., uric acid, which is the antioxidant with the highest content in human blood, and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxy (Tempol), which is widely used in studies using experimental animal models due to its low toxicity among nitroxides, edaravone (Edaravone: Eda), which is approved as a free radical scavenger, catechin ((-)-epicatechin), which is contained in tea / wine, etc. and is usually ingested, and Trolox, a similar compound of VitE (vitamin E). It is known that the water-octanol partition coefficient (LogPo / w) of these six model antioxidants increases in the order of AsA < UA < TPL < Eda < Catechin < Trolox.

[0286] In the NBD-TEMPO compound, the fluorescence intensity increased by approximately 7.7 times through the reaction with AsA, and in the NBD-Pen compound, the fluorescence intensity increased by approximately 2.9 times through the reaction with Eda. However, in the NBD-TEEPO compound, the fluorescence intensity hardly increased (Figure (3b)).

[0287] (Example 3)

[0288] Reactivity evaluation of fluorescent nitroxide probes with various oxidizing substances

[0289] At 37 °C, a fluorescent nitroxide (NBD-TEMPO compound, NBD-Pen compound, or NBD-TEEPO compound) (5.0 μM) and various oxidizing substances were mixed in a phosphate buffer (10 mM, pH 7.4, 0.5% DMSO, 0.5% acetonitrile). Hydrogen peroxide, hypochlorous acid, and potassium oxide (each 0.5 mM) were used as oxidizing substances. ·OH was generated from hydrogen peroxide (0.5 mM) and FeSO4 (5.0 μM). Lipid radicals were generated from liposomes (2.5 mg / mL of Egg PC, 0.1 mg / mL of DCP) and AAPH (10 mM). After 30 minutes, the fluorescence intensity was measured at an excitation wavelength of 470 nm and a fluorescence wavelength of 530 nm.

[0290] The results are shown in Figure 4 .

[0291] In any probe molecule, the fluorescence intensity hardly increased in the reaction with ROS. It can be seen that the NBD-TEMPO compound has the lowest responsiveness to lipid peroxidation, while the NBD-TEEPO compound and the NBD-Pen compound have excellent responsiveness to lipid peroxidation.

[0292] Based on the above results, it was decided to use the NBD-TEEPO compound which showed the highest reduction resistance and high responsiveness to lipid peroxidation.

[0293] (Example 4)

[0294] Evaluation of the Initiator Concentration Dependence of Lipid Peroxidation in Artificial Lipid Membranes (NBD-TEEPO Assay)

[0295] The probe (NBD-TEEPO compound) (5.0 μM) and liposomes (2.5 mg / mL Egg PC, 0.1 mg / mL DCP) were mixed in phosphate buffer (10 mM, pH 7.4, 0.5% DMSO, 0.5% acetonitrile) at 37°C. AAPH (0-20 mM) or FeSO4 (0-2.0 mM) was added to start the lipid peroxidation reaction. After 40 minutes in the AAPH system, the lipid peroxidation reaction was started in the FeSO4 system. 2+ After 60 minutes in the system, the fluorescence intensity was measured at an excitation wavelength of 470 nm and a fluorescence wavelength of 530 nm.

[0296] The results are shown in Figure 5 (AAPH) and Figure 6 (FeSO4).

[0297] In both the AAPH system and the Fe 2+ The fluorescence intensity of the probes in the system increased in a concentration-dependent manner (Figure (5a) and Figure (6a)). 2+ When the fat-soluble antioxidant Eda was added to the system, the fluorescence rise was suppressed in a concentration-dependent manner (Figure (5b) and Figure (6b)). Therefore, the lipid peroxidation inhibitory effects of 6 representative antioxidants were measured. The results were compared with the results of the conventional method, namely the TBARS method shown in Example 5 (Figure (5d) and Figure (6d)), and the results showed the same tendency (Figure (5c) and Figure (6c)).

[0298] For AAPH using NBD-TEEPO compounds or using Fe 2+ The cell-free system was tested for the S / B ratio, CV value, and Z'-factor, which are characteristics of the test system. The results are shown in the table below. All indicators exceeded the target value (Table 1).

[0299] [Table 1]

[0300] Table 1

[0301] index# AAPH System# <![CDATA[Fe 2+ System#]]> Target Value S / B ratio# 18.7# 8.7# 2 or more# CV value of Background (%)# 4.9# 3.2# Below 10%# CV value of Control (%)# 5.4# 3.2# Below 10%# Z'-factor# 0.82# 0.88# 0.5 or more#

[0302] (Example 5)

[0303] Evaluation of lipid peroxidation inhibition using antioxidant substances in artificial lipid membranes (TBARS assay)

[0304] At 37°C, various reducing substances (10 μM) and liposomes (2.5 mg / mL Egg PC, 0.1 mg / mL DCP) were mixed in phosphate buffer (10 mM, pH 7.4, 0.5% DMSO, 0.5% acetonitrile). AAPH (20 mM) or FeSO4 (1.0 mM) was mixed to start lipid peroxidation. After 60 minutes, BHT (10 mM) was used to stop lipid peroxidation. Acetic acid (5.7%), TBA (0.56%), and SDS (1.07%) were added and stirred, and then reacted at 60°C for 60 minutes. After centrifugation (2000 rpm, 4°C, 15 minutes), the fluorescence intensity was measured at an excitation wavelength of 512 nm and a fluorescence wavelength of 553 nm.

[0305] In the same manner as in Example 4, the lipid peroxidation inhibitory effects of six representative antioxidant substances were measured.

[0306] The results are shown in Figure 5 (AAPH) and Figure 6 (FeSO4). The results of Example 4 showed the same tendency as the lipid peroxidation inhibitory effect.

[0307] (Example 6)

[0308] Cell culture

[0309] Human hepatoma cells (HepG2 cells) were cultured in a CO2 incubator (37°C, 5% CO2) using DMEM medium (containing 10% FBS, 1% penicillin-streptomycin, and 1×MEM non-essential amino acids). The cells were passaged when they reached a 60-70% subconfluent state. In addition, DMEM medium (containing no phenol red and 1% penicillin-streptomycin) was used for various measurements.

[0310] (Example 7)

[0311] Evaluation of intracellular lipid peroxidation in response to initiator concentration dependence (NBD-TEEPO assay)

[0312] HepG2 cells were seeded in 96-well plates at 10,000 cells / well. Incubated and adhered for 24 hours. AA (0-200 μM) or tBHP (0-300 μM) and probe (5.0 μM) were added to DMEM medium (DMSO 0.5%, acetonitrile 0.5%), and after 45 minutes, the fluorescence intensity was measured at an excitation wavelength of 470 nm and a fluorescence wavelength of 530 nm.

[0313] (Example 8)

[0314] Evaluation of Inhibition of Intracellular Lipid Peroxidation Using Antioxidants (NBD-TEEPO Assay)

[0315] HepG2 cells were seeded in 96-well plates at 10,000 cells / well. Incubated and adhered for 24 hours. AA (200 μM) or tBHP (0-300 μM), antioxidants (50 μM) and probes (5.0 μM) were added to DMEM medium (DMSO 0.5%, acetonitrile 0.5%), and after 45 minutes, the fluorescence intensity was measured at an excitation wavelength of 470 nm and a fluorescence wavelength of 530 nm.

[0316] The results are shown in Figure 7 .

[0317] Concentration-dependent fluorescence increases were observed for AA and tBHP (Figures (7a) and (7c)). In addition, inhibition was achieved by adding antioxidants (Figures (7b) and (7d)). Catechin showed the strongest inhibitory effect, and the same tendency was observed regardless of the reaction initiator.

[0318] (Example 9)

[0319] Evaluation of changes in cell viability in response to initiator concentration (MTT assay)

[0320] HepG2 cells were seeded in 96-well plates at 10,000 cells / well. Incubated and adhered for 24 hours. AA (0-100 μM) or tBHP (0-100 μM) was added to DMEM medium (DMSO was 0.5%, acetonitrile was 0.5%), and the medium was replaced after 24 hours. After adding MTT solution (0.5 mg / mL, 0.5% DMSO), incubated for 4 hours and the solution was eliminated. 100 μL of DMSO was added and the absorbance at 630 nm was measured. The cell survival rate was calculated with no addition of AA or tBHP as 100%.

[0321] (Example 10)

[0322] Evaluation of changes in cell viability using antioxidant substances (MTT assay)

[0323] HepG2 cells were seeded in 96-well plates at 10,000 cells / well. Incubated and adhered for 24 hours. AA (200 μM) or tBHP (0-300 μM) and antioxidants (50 μM) were added to DMEM medium (DMSO was 0.5%, acetonitrile was 0.5%), and the medium was replaced after 24 hours. After adding MTT solution (0.5 mg / mL, 0.5% DMSO), incubated for 4 hours and the solution was eliminated. 100 μL of DMSO was added and the absorbance at 630 nm was measured. The cell survival rate was calculated with no addition of AA or tBHP as 100%.

[0324] The results are shown in Figure 8 .

[0325] The cell viability was reduced in a concentration-dependent manner by the addition of AA and tBHP (Figures (8a) and (8c)). In addition, the effect was inhibited by antioxidants (Figures (8b) and (8d)). In addition, different antioxidants showed a different tendency to show a high inhibitory effect depending on the reaction initiator used.

[0326] The S / B ratio, CV value, and Z'-factor, which are characteristics of the assay system, were examined for the assay system using NBD-TEEPO compound or MTT, and the cell system using AA or tBHP. The results are shown in Tables 2 and 3 below. All indicators exceeded the target value.

[0327] [Table 2]

[0328] Table 2: NBD-TEEPO compounds

[0329] index# AA System# tBHP system# Target Value S / B ratio# 4.1# 2.2# 2 or more# CV value of Background (%)# 6.5# 3.8# Below 10%# CV value of Control (%)# 2.8# 5.0# Below 10%# Z'-factor# 0.83# 0.62# 0.5 or more#

[0330] [Table 3]

[0331] Table 3: MTT assay

[0332] index# AA System# tBHP system# Target Value S / B ratio# 25.0# 6.3# 2 or more# CV value of Background (%)# 4.7# 6.9# Below 10%# CV value of Control (%)# 4.8# 6.9# Below 10%# Z'-factor# 0.85# 0.71# 0.5 or more#

[0333] (Example 11)

[0334] The University of Tokyo Institute of Pharmaceutical Research 1st screening (AAPH system)

[0335] A 2mM 100% DMSO solution of the compound (dispensed at 0.125 μL / well) was obtained from the Institute of Pharmacy and Chemical Engineering, University of Tokyo. Solution A containing liposomes (5.0 mg / mL Egg PC, 0.2 mg / mL DCP) and the probe (10 μM) in phosphate buffer (10 mM, pH 7.4, 1.0% acetonitrile) and solution B containing AAPH (40 mM) in phosphate buffer (10 mM, pH 7.4) were prepared. 12.5 μL of solution A and solution B were dispensed by an automatic dispenser (Multidrop Combi). The final concentration was 5.0 μM of liposomes (2.5 mg / mL Egg PC, 0.1 mg / mL DCP) and NBD-TEEPO compound, 50 μM of the test compound, and 20 mM AAPH in phosphate buffer (10 mM, pH 7.4, 0.5% acetonitrile, 0.5% DMSO). The reaction mixture was mixed at 37° C. for 40 minutes, and then the fluorescence intensity was measured at an excitation wavelength of 470 nm and a fluorescence wavelength of 530 nm. In addition, the activity value of each test compound was calculated according to the formula described in the specification.

[0336] The results are shown in Fig.10 .

[0337] Among the 9600 compounds, 1858 compounds had activity values ​​lower than 0, i.e., they did not inhibit lipid peroxidation. On the other hand, 7711 compounds inhibited lipid peroxidation, of which 836 compounds showed higher activity values ​​than the known compound edaravone. It was decided to use these 836 compounds as hit compounds (candidate compounds) in the primary screening and enter the Fe 2+ Evaluation in the system.

[0338] (Example 12)

[0339] The University of Tokyo Institute of Pharmaceutical Research 1st screening (FeSO4 system)

[0340] Next, 836 compounds that showed a higher lipid peroxidation inhibitory effect than edaravone in the AAPH system were analyzed by Fe 2+The system was evaluated. A 2mM 100% DMSO solution of the compound (dispensed at 0.2μL / well) was obtained from the Institute of Pharmacy and Chemical Engineering, University of Tokyo. Solution A containing liposomes (2.78mg / mL Egg PC, 0.11mg / mL DCP) and 5.6μM of the probe in phosphate buffer (10mM, pH7.4, 0.56% acetonitrile) and solution B containing 10mM FeSO4 in distilled water were prepared. 36μL of solution A was dispensed by an automatic dispenser (Multidrop Combi). 4μL of solution B was dispensed by an automated pipetting workstation (Biomek NXP). The final concentration was liposomes (2.5mg / mL Egg PC, 0.1mg / mL DCP) and 5.0μM of the probe, 50μM of the test compound, and 1.0mM FeSO4 in phosphate buffer (10mM, pH7.4, 0.5% acetonitrile, 0.5% DMSO). The reaction mixture was mixed at 37°C and the fluorescence intensity at an excitation wavelength of 470 nm and a fluorescence wavelength of 530 nm was measured every 3 minutes. AUC was calculated from the fluorescence intensity at 180 minutes, and the activity value of each test compound was calculated according to the formula described in the manual ( Fig.11 ).

[0341] The results are shown in Fig.12 .

[0342] Among the 836 compounds, 268 compounds had activity values ​​lower than 0, i.e., they did not inhibit lipid peroxidation. On the other hand, 568 compounds inhibited lipid peroxidation, of which 197 compounds showed activity higher than that of edaravone. It was decided to use the top 80 compounds with good inhibitory effects as lead compounds (candidate compounds) and enter the second screening.

[0343] (Example 13)

[0344] Secondary screening by the Institute for Pharmaceutical Innovation, University of Tokyo (NBD-TEEPO assay)

[0345] A second screening was performed for the 80 compounds selected in the first screening.

[0346] A 10mM 100% DMSO solution of the compound (distributed as 5.0 μL / well) was obtained from the Institute of Pharmaceutical Research, University of Tokyo. 495 μL of DMEM medium for measurement was added to adjust it to a 100 μM 1.0% DMSO solution of the compound. It was distributed in 80 μL aliquots to the assay plate in which cells were pre-seeded by an automated pipetting workstation (Biomek NXP). 64 μL of DMEM medium (1.25% acetonitrile) containing 12.5 μM of NBD-TEEPO compound, 16 μL of AA (2000 μM, 5.0% ethanol) or tBHP (3000 μM) dissolved in PBS were manually distributed in the test compound (100 μM 1.0% DMSO). The final concentration is 50 μM of the test compound, 200 μM of AA or 300 μM of tBHP in DMEM medium (0.5% DMSO, 0.5% acetonitrile).

[0347] The reaction mixture was mixed at 37°C and the fluorescence intensity at an excitation wavelength of 470 nm and a fluorescence wavelength of 530 nm was measured every 3 minutes. The AUC was calculated from the fluorescence intensity at 45 minutes in the AA addition system and from the fluorescence intensity at 60 minutes in the tBHP addition system, and the activity value of each test compound was calculated according to the formula described in the instructions.

[0348] The results are shown in Fig.13 .

[0349] In the AA addition system, the activity values ​​of 40 compounds out of 80 compounds were less than 0%, that is, lipid peroxidation was not inhibited in the cultured cell system. On the other hand, 40 compounds inhibited lipid peroxidation. Among them, 32 compounds showed activity higher than that of edaravone (Figure (13a)). In addition, in the tBHP addition system, the activity values ​​of 17 compounds out of 80 compounds were less than 0%, while 63 compounds inhibited lipid peroxidation. Among them, 31 compounds showed activity higher than that of edaravone (Figure (13b)). It was decided to illustrate the activity values ​​in the two systems (Figure (13c)), and 4 compounds that showed high activity values ​​in either system: Compound No. 7, 48, 64, and 80 were selected as lead compounds (Figure (13d)) and entered the third screening.

[0350] Compound 7:

[0351] 2-((4-(phenylamino)phenyl)amino)-N-(4-sulfamoylphenyl)propionamide

[0352] Compound 48:

[0353] 2,6-Dimethoxy-4-(2-(8-nitroquinolin-2-yl)vinyl)phenol

[0354] Compound 64:

[0355] N 2 ,N 2 -Dimethyl-9H-fluorene-2,3-diamine

[0356] Compound 80:

[0357] N-(3-methoxy-4-((3-methyl-1-10H-indolo[3,2-b]quinolin-11-yl)amino)phenyl)methanesulfonamide

[0358] [Chemistry 4]

[0359]

[0360] (Example 14)

[0361] Secondary screening by the Institute for Pharmaceutical Innovation, University of Tokyo (MTT assay)

[0362] A second screening was performed for the 80 compounds selected in the first screening.

[0363] A 10mM 100% DMSO solution of the compound (distributed as 5.0 μL / well) was obtained from the Institute of Pharmaceutical Research, University of Tokyo. 495 μL of DMEM medium for measurement was added to adjust it to a 100 μM 1.0% DMSO solution of the compound. It was distributed in 80 μL aliquots to the assay plate in which cells were pre-seeded by an automated pipetting workstation (Biomek NXP). 64 μL of DMEM medium, 16 μL of AA (1000 μM, 5.0% ethanol) or tBHP (1000 μM) dissolved in PBS were manually distributed to the test compound (100 μM 1.0% DMSO). The final concentration is 5.0 μM of NBD-TEEPO compound, 50 μM of test compound, and 100 μM of AA or tBHP in DMEM medium (0.5% DMSO, 0.5% acetonitrile). After 24 hours, the medium was replaced, and MTT solution (0.5 mg / mL, 0.5% DMSO) was added and incubated for 24 hours to eliminate the solution. 100 μL of DMSO was added and the absorbance at 630 nm was measured. The cell survival rate was calculated according to the formula described in the manual, with no addition of AA or tBHP as 100%.

[0364] The results are shown in Fig.14 .

[0365] In the AA addition system, the activity values ​​of 6 compounds out of 80 compounds were less than 0%, that is, they did not inhibit cell death stimulated by AA. On the other hand, 74 compounds inhibited cell death when stimulated by AA. Among them, 64 compounds showed activity higher than that of edaravone (Figure (14a)). In addition, in the tBHP addition system, the activity values ​​of 8 compounds out of 80 compounds were less than 0%, while 73 compounds inhibited cell death stimulated by tBHP. Among them, 14 compounds showed activity higher than that of edaravone (Figure (14b)). It was decided to show the inhibition rate in the two systems (Figure (14c)), and 5 compounds that showed high inhibition rates in either system: Compound No. 19, 39, 52, 73, and 78 were selected as lead compounds (Figure (14d)) and entered the third screening.

[0366] Compound 19:

[0367] N-(2-chlorophenyl)-5-(2-(1-pyridin-2-yl)ethylidene)hydrazino)-1,3,4-thiadiazol-2-amine

[0368] Compound 39:

[0369] 1-(7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-yl)-N-(1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)-N-methylmethanesulfonamide

[0370] Compound 52:

[0371] 3-Amino-4-(phenylamino)benzoic acid methyl ester

[0372] Compound 73:

[0373] 3-(2-(3-(2-hydroxyethoxy)phenyl)-3-((2-morpholinoethyl)amino)imidazo[1,2-a]pyridin-6-yl)benzamide

[0374] Compound 78:

[0375] 1-(4-(Trifluoromethoxy)phenyl)indolin-5-amine

[0376] [Chemistry 5]

[0377]

[0378] (Example 15)

[0379] The University of Tokyo Institute of Pharmaceutical Research 3rd screening (NBD-TEEPO test)

[0380] The Tokyo University Core Library contains structural analogs for each compound, so structural analogs of each of the candidate compounds were screened twice as in Example 13 and their activity values ​​were evaluated.

[0381] Specifically, for the 9 lead compounds No. 7, 19, 39, 48, 52, 64, 73, 78, and 80 in the above two screenings, 5, 2, 4, 6, 6, 5, 5, 10, and 2 structural analogs were obtained, respectively, that is, a total of 45 structural analogs were obtained. In addition, a total of 54 compounds after adding the original 9 compounds were screened three times and the activity values ​​were calculated in the same way.

[0382] The results are shown in Fig.15 , Fig.16 as well as Fig.17 .

[0383] 1) First, in the assay using NBD-TEEPO, the activity values ​​of 15 out of 54 compounds in the AA addition system were less than 0%, i.e., lipid peroxidation was not inhibited. On the other hand, 39 compounds inhibited lipid peroxidation. Among them, 33 compounds showed activity higher than that of edaravone (Figure (15a)). In the tBHP addition system, the activity values ​​of 9 out of 54 compounds were less than 0%, while 45 compounds inhibited lipid peroxidation. Among them, 41 compounds showed activity higher than that of edaravone (Figure (15b)).

[0384] 2) Next, in the MTT assay, the activity values ​​of 7 out of 54 compounds in the AA addition system were less than 0%, that is, they did not inhibit cell death stimulated by AA. On the other hand, 47 compounds inhibited cell death stimulated by AA. Among them, 31 compounds showed an activity higher than that of edaravone (Figure (16a)). In the tBHP addition system, the activity values ​​of 17 out of 54 compounds were less than 0%, while 37 compounds inhibited cell death stimulated by tBHP. Among them, 37 compounds showed an activity higher than that of edaravone (Figure (16b)).

[0385] 3) Furthermore, the test compound alone was incubated for 72 hours and the cytotoxicity was evaluated. For cytotoxicity, 50 μM of antioxidants were added to HepG2 cells in DMEM medium (DMSO 0.5%) and incubated at 37° C. for 72 hours, and then the cell survival rate was measured.

[0386] 4) For each assay using the above-mentioned NBD-TEEPO assay and MTT assay, the activity values ​​in the two addition systems are shown (Figure (15c) and Figure (16c)). The analogs of compound No. 52 (Compound No. 52, 52-1, 52-3, 52-4, 52-5) and the analogs of compound No. 78 (Compound No. 78, 78-3, 78-4, 78-5, 78-6, 78-8) showed high lipid peroxidation inhibitory effects and cell death inhibitory effects (Figure (15d) and Figure (16d)).

[0387] In addition, although the analogs of Compound No. 80 (Compound Nos. 80 and 80-2) showed the highest lipid peroxidation inhibitory effect, they were extremely cytotoxic ( FIG. ( 17 )).

[0388] Among these Compound No. 52 and its analogs (Compound Nos. 52-1 to 52-6), the compounds that exert lipid peroxidation inhibitory effects have a skeleton (bone) A represented by the following structure as a common structure. Skeleton A is reported to have antioxidant activity (Hu ML., et al., Nutr. Biochem., 1995, 6, 504-508).

[0389] In addition, among Compound No. 78 and its analogs (Compound Nos. 78-1 to 78-10), the compounds that exert lipid peroxidation inhibitory effects have a skeleton (bone) B represented by the following structure as a common structure.

[0390] [Chemistry 6]

[0391]

[0392] The above results suggest that the compounds found as candidate compounds through the screening of the present invention have skeleton A and skeleton B as common structures, and that compounds having skeleton A and skeleton B are highly likely to be extremely useful as lipid peroxidation inhibitors.

[0393] Compound 52-1:

[0394] 3-Amino-4-((4-methoxyphenyl)amino)benzoic acid methyl ester

[0395] Compound 52-2:

[0396] Methyl 3-amino-4-((2-methoxyphenyl)amino)benzoate

[0397] Compound 52-3:

[0398] 3-Amino-4-((3-methoxyphenyl)amino)benzoic acid methyl ester

[0399] Compound 52-4:

[0400] 3-Amino-4-(benzylamino)benzoic acid methyl ester

[0401] Compound 52-5:

[0402] 3-Amino-4-((1-phenylethyl)amino)benzoic acid methyl ester

[0403] Compound 52-6:

[0404] N-(2-(Phenylamino)phenyl)acetamide

[0405] [Chemistry 7]

[0406]

[0407] Compound 78-1:

[0408] 1-(3,5-Dimethylphenyl)indoline-2,3-dione

[0409] Compound 78-2:

[0410] 1-(3,5-Dimethylphenyl)-3,3-difluoroindolin-2-one

[0411] Compound 78-3:

[0412] 1-(3,5-Dimethylphenyl)-1H-indol-6-amine

[0413] Compound 78-4:

[0414] 1-(3,5-Dimethylphenyl)indolin-6-amine

[0415] Compound 78-5:

[0416] 1-(4-Methoxyphenyl)-1H-indol-5-amine

[0417] Compound 78-6:

[0418] 1-(4-(Methylthio)phenyl)-1H-indol-5-amine

[0419] Compound 78-7:

[0420] 1-(4-(Trifluoromethyl)phenyl)-1H-indol-5-amine

[0421] Compound 78-8:

[0422] 1-(4-(Trifluoromethoxy)phenyl)-1H-indol-5-amine

[0423] Compound 78-9:

[0424] 1-(4-(Methylthio)phenyl)indolin-5-amine

[0425] Compound 78-10:

[0426] 1-(4-(Trifluoromethyl)phenyl)indolin-5-amine

[0427] [Chemistry 8]

[0428]

[0429] (Example 16)

[0430] Prestwick chemical library 1st screening (AAPH system)

[0431] In this screening, in the AAPH system and Fe 2+ 1280 compounds were measured as test compounds in the system. The experimental method and activity value calculation method used were the same as the above method, and the compound library used was the same as the core library of the Tokyo University Institute for Pharmaceutical Research and Education. The test compounds were phosphate buffer (10mM, pH7.4, 2% DMSO) and 20μM diluent (distributed as 20μL / well) provided by the Kyushu University Center for Advanced Research and Education in Pharmaceutical Research.

[0432] First, in the case of using the AAPH system assay, solution A containing liposomes (10 mg / mL Egg PC, 0.4 mg / mL DCP) and NBD-TEEPO compound (20 μM) in phosphate buffer (10 mM, pH 7.4, 2.0% acetonitrile) and solution B containing 80 mM AAPH in phosphate buffer (10 mM, pH 7.4) were prepared. 10 μL of solution A and solution B were dispensed by an automatic dispenser (Multidrop Combi). The final concentration was 5.0 μM of liposomes (2.5 mg / mL Egg PC, 0.1 mg / mL DCP) and NBD-TEEPO compound, 10 μM of the test compound, and 20 mM AAPH in phosphate buffer (10 mM, pH 7.4, 0.5% acetonitrile, 1% DMSO). After 40 minutes at 37°C, the fluorescence intensity was measured at an excitation wavelength of 470 nm and a fluorescence wavelength of 530 nm.

[0433] (Example 17)

[0434] Prestwick chemical library 1st screening (FeSO4 system)

[0435] Next, using Fe 2+ In the case of system testing, solution A containing liposomes (10 mg / mL Egg PC, 0.4 mg / mL DCP) and 20 μM NBD-TEEPO compound in phosphate buffer (10 mM, pH 7.4, 2.0% acetonitrile) and solution B containing 4.0 mM FeSO4 in distilled water were prepared. 10 μL of solution A and solution B were dispensed by an automatic dispenser (Multidrop Combi). The final concentration was 5.0 μM liposomes (2.5 mg / mL Egg PC, 0.1 mg / mL DCP) and NBD-TEEPO compound, 10 μM test compound, and 1.0 mM FeSO4 in phosphate buffer (10 mM, pH 7.4, 0.5% acetonitrile, 1% DMSO). After 180 minutes at 37°C, the fluorescence intensity was measured at an excitation wavelength of 470 nm and a fluorescence wavelength of 530 nm.

[0436] The results are shown in Fig.18 and Fig.19 .

[0437] 1) As a result, in the AAPH system, 330 out of 1280 compounds had activity values ​​lower than 0, that is, they did not inhibit lipid peroxidation. On the other hand, 950 compounds inhibited lipid peroxidation, of which 190 compounds showed higher activity values ​​than the known compound edaravone (Figure (18a)). 2+ In the system, 434 compounds among 1280 compounds had activity values ​​lower than 0, while 846 compounds inhibited lipid peroxidation, of which 19 compounds showed higher activity values ​​than the known compound edaravone (Fig. (18b)).

[0438] The AAPH system and Fe 2+ The results of the system were added together to obtain 16 hit compounds (Figure (19a) and Figure (19c)).

[0439] 2) The Prestwick chemical library is a compound library that collects compounds with known pharmacological activity. Therefore, information related to the site of action, pharmacokinetics, safety, etc. can be obtained from the database and literature information provided by the library. Therefore, based on this information, the scope of the 16 lead compounds was narrowed down. These lead compounds include therapeutic drugs in a wide range of disease areas such as the cardiovascular system, central nervous system, respiratory system, and antibacterial drugs (Figure (19b)).

[0440] (Pharmacological test)

[0441] The pharmacological activity against age-related macular degeneration (AMD) was examined.

[0442] The light irradiation model widely used as atrophic AMD model mouse was used as an experimental model. As for the test compound, 16 candidate compounds sorted by screening related to the above-mentioned Prestwick chemical library were further narrowed down to the following 5 compounds (Compound V, Compound W, Compound X, Compound Y, Compound Z) reported to have high permeability of the blood retinal barrier (BRB) and the pharmacological activity was examined.

[0443] Compound V:

[0444] Apomorphine ((R)-(-)-Apomorphine hydrochloride)

[0445] It is known as an anti-Parkinson's disease drug acting on dopamine D1D2 receptors.

[0446] Compound W:

[0447] Physostigmine ((-)-physostigmine fumarate)

[0448] It is known to have analgesic effects acting on opioid receptors.

[0449] Compound X:

[0450] Ethoxyquinoline (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline)

[0451] It is known to have antioxidant effects.

[0452] Compound Y:

[0453] Methyldopa (methyldopa sesquihydrate)

[0454] It is known as a hypotensive drug acting on adrenergic α2 receptors.

[0455] Compound Z:

[0456] Olanzapine (2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine)

[0457] It is known as an antipsychotic drug that acts on multiple receptors

[0458] [Chemistry 9]

[0459]

[0460] (operate)

[0461] First, AMD model mice were prepared according to the following schedule.

[0462] Experimental animals

[0463] Male BALB / c mice (4 weeks old) were purchased from SLC Co., Ltd., Japan, and used for experiments after acclimating for one week. They were fed with solid feed for experimental animals (CLEA Rodent Diet CE-2, CREAJAPAN, INC.) and tap water as drinking water, and were raised under a 12-hour light-dark cycle. All animal experiments were performed with the approval of the Animal Experiment Committee of Kyushu University.

[0464] Preparation of light-induced AMD model mice

[0465] BALB / c mice were intraperitoneally injected with 5 mL / kg of a 10 mM compound dissolved in PBS containing 10% polyethylene glycol (PEG) 300. 30 minutes later, one drop of Mydrin P (5 mg / mL tropicamide, 5 mg / mL phenylephrine hydrochloride, manufactured by Santen Pharmaceutical Co., Ltd.) was dripped into each eye as a mydriatic. The mice were irradiated with 8000 lux of white light for 10 hours and returned to the normal light-dark cycle for 6 days. On the 7th day, the mice were euthanized by cervical dislocation and the eyeballs were removed (Figure (20a)).

[0466] Preparation of eyeball cryosections

[0467] The test compound was dissolved in PBS containing 10% PEG300 at 10 mM and injected intraperitoneally into male BALB / c mice at 5 mL / kg once. 30 minutes after administration, the mice were irradiated with 8000 lux of white light for 10 hours. After that, the mice were raised for 6 days under normal light-dark cycle, and the eyeballs were removed after euthanasia on the 7th day. 8 μm thick frozen sections were prepared and stained with hematoxylin-eosin (HE), and the thickness of the outer nuclear layer (ONL) was measured at 27 points every 180 μm (Figure (20b)).

[0468] HE staining

[0469] The preparation was air-dried for 1 hour, and fixed with acetone for 15 minutes at room temperature, then immersed in 99.5% EtOH, 80% EtOH, 70% EtOH, and purified water for 3 minutes each, and stained with hematoxylin for 10 minutes. Thereafter, it was washed with running water for 10 minutes, soaked in warm water for 1 minute, and stained with eosin for 1.5 minutes. After washing with purified water, it was immersed in 70% EtOH, 80% EtOH, and 99.5% EtOH for 3 minutes each, then washed 3 times with xylene, and sealed with VectaMount (trademark) mounting medium after drying. Observation and photography were performed using a Keyence fluorescence microscope (BZ-9000).

[0470] Statistical analysis

[0471] The results are shown as mean + standard deviation. Dunnett's test was used as a multiple group test method.

[0472] (result)

[0473] The imaging results are shown in Fig.21 In the figure, there is an inner nuclear layer (INL) at the top, an outer nuclear layer (ONL) in the middle, and a retinal pigment epithelium (RPE) at the bottom. When cell death occurs due to lipid peroxidation, the thickness of the outer nuclear layer in the middle becomes thinner.

[0474] First, the thickness of ONL was significantly reduced by light exposure (refer to Fig.21 (a) Negative control). ONL damage is particularly severe on the superior side of the eyeball ( Fig.21(b)), these results are consistent with those known in the literature (eg, Tanito M., et al., Invest. Ophthalmol. Vis. Sci., 2007, 48(4), 1900-5.).

[0475] On the other hand, in the case of the five test compounds used in this experiment, there was almost no difference in the thickness of ONL in any case compared with the case of the positive control, and even in the case of the same dosage of 50 μmol / kg, a significant thickness was observed compared with the case of edaravone or OT-551 as the control compound ( Fig.21 (c)).

[0476] Here, it is reported that OT-551, which is known to have a high retinal protective effect, requires about 100 mg / kg (360 μmol / kg) in the light irradiation model mouse. Therefore, 50 μmol / kg in this test is about one-seventh of the amount and is a very low dose.

[0477] In addition, 50 μmol / kg is the median lethal dose (LD50) of each of the five tested compounds. 50 ) is less than one tenth of the Fig. 22 ).

[0478] The above results suggest that the compounds selected by the screening of the present invention are useful compounds for age-related macular degeneration.

[0479] Industrial Applicability

[0480] According to the assay method, assay kit, and screening reaction using the fluorescent compound of the present invention, it is easy to find compounds showing lipid peroxidation inhibitory activity. In addition, the candidate compounds using the screening method of the present invention are useful for treating lipid peroxidation-induced diseases such as age-related eye diseases.

Claims

1. A test kit for detecting the lipid peroxidation inhibition activity of a test compound, wherein the test kit is characterized in that: The buffer contains a compound represented by formula (I), liposomes, and at least one compound selected from the group consisting of 2,2'-azobis(2-aminopropane) dihydrochloride and a divalent iron ion supply source substance, in, Formula (I) is as follows: [Chemistry 1] 2. The assay kit according to claim 1, characterized in that The source material of the divalent iron ion supply is iron (II) sulfate.

3. The assay kit according to claim 1 or 2, characterized in that: Additional instructions are included showing the activity value of the compound, wherein the compound shows lipid peroxidation inhibitory activity.

4. A test kit for detecting the lipid peroxidation inhibition activity of a test compound, wherein the test kit is characterized in that: The buffer contains a compound represented by formula (I), cultured cells, and at least one compound selected from the group consisting of arachidonic acid and tert-butyl hydroperoxide, wherein Formula (I) is as follows: [Chemistry 2] 5. The assay kit according to claim 4, characterized in that: The cultured cells are HepG2 cells derived from human liver.

6. The assay kit according to claim 4 or 5, characterized in that: Additional instructions are included showing the activity value of the compound, wherein the compound shows lipid peroxidation inhibitory activity.

7. A test kit, characterized in that: A test kit comprising any two or more of claims 1 to 6.

8. A method for determining the activity of lipid peroxidation inhibition, characterized in that: include: i) preparing a buffer solution comprising the compound represented by formula (I) and liposomes; ii) adding at least one compound selected from the group consisting of 2,2'-azobis(2-aminopropane) dihydrochloride and a divalent iron ion supply source material; iii) adding the test compound; iv) measuring fluorescence; as well as, v) The activity value of the test compound is determined from the fluorescence measurement results.

9. A method for determining the activity of lipid peroxidation inhibition, characterized in that: include: i) preparing a buffer solution comprising a compound represented by formula (I) and cultured cells; ii) adding at least one compound selected from the group consisting of arachidonic acid and tert-butyl hydroperoxide; iii) adding the test compound; iv) measuring fluorescence; as well as, v) The activity value of the test compound is determined from the fluorescence measurement results.

10. The testing method according to claim 8 or 9, characterized in that: include: vi) Comparison with the activity value of the compound serving as an index of lipid peroxidation inhibitory activity.

11. The testing method according to any one of claims 8 to 10, characterized in that: The assay was used in microtiter plates.

12. A screening method for selecting candidate compounds with high lipid peroxidation inhibitory activity, characterized in that: include: i) selecting a test compound from a compound library; ii) performing screening by the assay method using 2,2'-azobis(2-aminopropane) dihydrochloride as claimed in claim 8 using the test compound to select a compound showing a high activity value; and, iii) Next, screening is performed using the assay method for the divalent iron ion supply source substance according to claim 8 using the compound showing a high activity value in ii) to select a compound showing a high activity value.

13. The screening method according to claim 12, characterized in that In the case where the compound library is a compound library containing compounds that are not approved as food or medicine, the screening method according to claim 12 further comprises: i) performing screening by the assay method using arachidonic acid as claimed in claim 9 to select compounds showing high activity values; ii) performing screening by means of the assay method using tert-butyl hydroperoxide as described in claim 9 to select compounds showing high activity values; and, iii) Selecting compounds that show high activity values ​​in both the screening of i) and ii).

14. The screening method according to claim 12 or 13, characterized in that In the case where the compound library is a compound library containing compounds that are not approved as food or medicine, the screening method according to claim 12 further comprises: i) performing screening by an assay method using a medium containing cultured cells, a test compound, and arachidonic acid according to the MTT method, and selecting a compound that shows a high cell viability; ii) performing screening by an assay method using a medium containing cultured cells, a test compound, and tert-butyl hydroperoxide according to the MTT method, and selecting a compound showing a high cell viability; and, iii) selecting candidate compounds that show high cell viability in both the screening of i) and ii).

15. The screening method according to claim 13 or 14, characterized in that include: i) selecting a structural analogue of the compound selected by the screening method according to claim 13 or 14 from a compound library; ii) for the compound selected in i) and the compound appropriately selected by the screening method according to claim 13 or 14, performing the screening method according to claim 13 to select a compound showing a high activity value; iii) for the compound selected in i) and the compound appropriately selected by the screening method according to claim 13 or 14, performing the screening method according to claim 14 to select a compound showing a high cell viability; iv) selecting candidate compounds that show high activity values ​​and high cell viability in both screening methods ii) and iii); v) for the compound selected in i) and the compound appropriately selected by the screening method according to claim 13 or 14, performing an assay method using a medium containing cultured cells to select a candidate compound showing a high cell viability; and, vi) selecting candidate compounds from the compounds selected in iv) and the compounds selected in v).

16. The screening method according to any one of claims 12 to 15, characterized in that The compound library is the core library of the Institute for Pharmaceutical Development, The University of Tokyo or the Prestwick Compound Library.

17. A pharmaceutical composition, characterized in that A method for preventing or treating a lipid peroxidation-induced disease in a subject or inhibiting the progression of a disease comprising an effective amount of at least one compound selected from the group consisting of the following groups, wherein the group consists of the following compounds: apomorphine ((R)-(-)-apomorphine hydrochloride), physostigmine ((-)-physostigmine fumarate), ethoxyquinoline (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline), methyldopa (methyldopa sesquihydrate), olanzapine (2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine, 3-amino-4-(phenylamino)benzoic acid methyl ester, 3-amino-4-((4-methoxyphenyl)amino)benzoic acid methyl ester, Methyl 4-((3-methoxyphenyl)amino)benzoate, methyl 3-amino-4-(benzylamino)benzoate, methyl 3-amino-4-((1-phenylethyl)amino)benzoate, 1-(4-(trifluoromethoxy)phenyl)indolin-5-amine, 1-(3,5-dimethylphenyl)-1H-indolin-6-amine, 1-(3,5-dimethylphenyl)indolin-6-amine, 1-(4-methoxyphenyl)-1H-indolin-6-amine, 1-(4-(methylthio)phenyl)-1H-indolin-6-amine, 1-(4-(trifluoromethoxy)phenyl)-1H-indolin-5-amine.

18. The pharmaceutical composition according to claim 17, characterized in that The disease is selected from the group consisting of Alzheimer's dementia, chronic kidney disease, diabetic neuropathy, liver damage, age-related macular degeneration, ischemic encephalopathy, vascular dementia, arteriosclerosis, Parkinson's disease, multiple sclerosis, cancer, asthma, hypertension, cardiovascular disease, and age-related eye disease.

19. The pharmaceutical composition according to claim 17, characterized in that The disease is age-related macular degeneration.

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