Method for detecting atopic dermatitis

Abstract

To provide a novel method detecting atopic dermatitis, and a novel determination method of a treatment effect to the atopic dermatitis.SOLUTION: According to the present invention, a detection method of atopic dermatitis is provided that includes a step of measuring concentration of lipid metabolites in a bio-sample of interest. According to the present invention, a determination method of a treatment effect to the atopic dermatitis is further provided that includes a step of measuring the concentration of the lipid metabolite in the bio-sample of interest. According to the present invention, the detection method and determination method are advantageous for enabling quantitatively detecting the atopic dermatitis or the treatment effect to the atopic dermatitis on the basis of a bio-sample of a subject of interest. The detection method and determination method are also advantageous for enabling the non-invasively collected bio-sample of the subject of interest to be used.SELECTED DRAWING: Figure 13

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from an earlier Japanese application, Patent Application No. 2019-184174 (filing date: October 4, 2019), the entire disclosure of which is incorporated herein by reference. [Technical Field]

[0002] The present invention relates to a method for detecting atopic dermatitis, and also to a method for determining the effectiveness of a treatment for atopic dermatitis. [Background technology]

[0003] Atopic dermatitis (AD) is an inflammatory skin disease characterized by repeated exacerbations and remissions of pruritic eczema. AD is caused by the interaction of genetic and environmental factors. The number of AD patients has been increasing in recent years, with the prevalence estimated at 9.8–13.2% in children and 2.5–9.4% in adults, respectively. Furthermore, the onset of AD in infancy has been reported to increase the risk of developing other allergic diseases, such as food allergies, bronchial asthma, and allergic rhinitis. In AD lesions, Th2 cells are correlated with Th2 cytokines such as IL-4 and IL-13 and chemokines such as TARC, and serum TARC levels are known to be useful as a marker of AD (Non-Patent Document 1, Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Thijs J et al., Current Opinion in Allergy and Clinical Immunology, 15(5):453-460 (2015) [Non-patent document 2] Judith L et al., J. Clin. Med, 4, 479-487(2015) Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a novel method for detecting atopic dermatitis. Another object of the present invention is to provide a novel method for determining the effectiveness of a treatment for atopic dermatitis. [Means for solving the problem]

[0006] The present inventors have now confirmed that the amounts of lipid metabolites in biological samples from atopic dermatitis model mice and atopic dermatitis patients are different from those in biological samples from healthy subjects. The present inventors have also found that atopic dermatitis can be detected by using the concentrations of the lipid metabolites as indicators. The present invention is based on these findings.

[0007] According to the present invention, the following inventions are provided. [1] A method for detecting or diagnosing atopic dermatitis, comprising the step of measuring the concentration of lipid metabolites in a biological sample from a subject. [2] The detection or diagnostic method described in [1] above, further comprising a step of comparing the concentration of the lipid metabolite in the biological sample of the subject with the concentration of the lipid metabolite in the biological sample of a healthy subject. [3] A detection or diagnostic method described in [1] or [2] above, which indicates that the subject is suffering from atopic dermatitis if the concentration of the lipid metabolite in the subject's biological sample is significantly different from the concentration of the lipid metabolite in the biological sample of a healthy subject. [4] The detection or diagnosis method according to any one of [1] to [3] above, wherein the biological sample is a body fluid. [5] A method for assessing the therapeutic effect on atopic dermatitis, comprising the step of measuring the concentration of lipid metabolites in a biological sample from a subject. [6] The method of determining the level of lipid metabolites described in [5] above, further comprising a step of comparing the concentration of the lipid metabolites in the biological sample of the subject with the concentration of the lipid metabolites in the biological sample of a healthy subject. [7] The method of determining whether or not a therapeutic effect is present is described in [5] or [6] above, wherein the concentration of a lipid metabolite in the subject's biological sample is significantly different from the concentration of the lipid metabolite in the subject's biological sample before treatment or the concentration of the lipid metabolite in the biological sample of a subject suffering from atopic dermatitis, or is not significantly different from the concentration of the lipid metabolite in the biological sample of a healthy subject. [8] The method for determining whether or not atopic dermatitis has occurred according to any one of [5] to [7] above, wherein the treatment for atopic dermatitis is a drug therapy or a proactive therapy. [9] The method according to any one of [5] to [8] above, wherein the biological sample is a body fluid.

[10] The detection method or diagnostic method described in any one of [1] to [4] above, or the determination method described in any one of [5] to [9] above, wherein the lipid metabolite is one or more selected from the group consisting of arachidonic acid metabolites, eicosapentaenoic acid metabolites, and docosahexaenoic acid metabolites.

[11] The detection method or diagnostic method described in any one of [1] to [4] and

[10] above, or the determination method described in any one of [5] to

[10] above, wherein the concentration of the lipid metabolite in a biological sample from a subject suffering from atopic dermatitis tends to be higher than the concentration in a biological sample from a healthy subject.

[12] The lipid metabolites include 13,14-dihydro-15-keto-PGJ2, tetranor-PGDM, 20-hydroxy-PGE2, 15-keto-PGE2, 13,14-dihydro-15-keto-tetranor-PGE2, tetranor-PGEM, 15-keto-PGF2α, 13,14-dihydro-15-keto-tetranor-PGF1α, tetranor-PGFM, and 6,15-diketo-13,14-dihydro-PG The detection method or diagnostic method according to

[11] above, or the determination method according to

[11] above, wherein the PGF1α is one or more selected from the group consisting of 13,14-dihydro-15-keto-PGF2α, PGK2, 13,14-dihydro-15-keto-tetranor-PGF1β, 13,14-dihydro-15-keto-PGE2, 6-keto-PGF1α and TXB2.

[13] The detection method or diagnostic method described in any of [1] to [4] and

[10] above, or the determination method described in any of [5] to

[10] above, wherein the concentration of the lipid metabolite in a biological sample from a subject suffering from atopic dermatitis tends to be lower than the concentration in a biological sample from a healthy subject.

[14] The detection method or diagnostic method described in

[13] above, or the determination method described in

[13] above, wherein the lipid metabolite is one or more selected from the group consisting of 5-HETE, arachidonic acid (AA), 11β-13,14-dihydro-15-keto-PGF2α, iPF2α-IV, EPA, 4-HDoHE, 10,17-DiHDoHE and PGD3.

[15] The detection method or diagnostic method according to any one of [1] to [4] and

[10] to

[14] above, or the determination method according to any one of [5] to

[14] above, wherein the concentration of the lipid metabolite is measured by mass spectrometry.

[16] An atopic dermatitis marker comprising a lipid metabolite.

[17] The atopic dermatitis marker described in

[16] above, wherein the lipid metabolite is one or more selected from the group consisting of arachidonic acid metabolites, eicosapentaenoic acid metabolites, and docosahexaenoic acid metabolites.

[18] The lipid metabolites include 13,14-dihydro-15-keto-PGJ2, tetranor-PGDM, 20-hydroxy-PGE2, 15-keto-PGE2, 13,14-dihydro-15-keto-tetranor-PGE2, tetranor-PGEM, 15-keto-PGF2α, 13,14-dihydro-15-keto-tetranor-PGF1α, tetranor-PGFM, 6,15-diketo-13,14-dihydro-PGF1α, 5-HpETE, 17-HETE, 11β- The atopic dermatitis marker according to

[16] or

[17] above, which is one or more selected from the group consisting of 13,14-dihydro-15-keto-PGF2α, PGK2, 13,14-dihydro-15-keto-tetranor-PGF1β, 13,14-dihydro-15-keto-PGE2, 6-keto-PGF1α, TXB2, 5-HETE, arachidonic acid (AA), iPF2α-IV, EPA, 4-HDoHE, 10,17-DiHDoHE, and PGD3.

[19] A method for screening for therapeutic or alleviating agents for atopic dermatitis, comprising the steps of administering a candidate therapeutic or alleviating agent for atopic dermatitis to a subject and measuring the concentration of lipid metabolites in a biological sample from the subject.

[20] The screening method described in

[19] above, further comprising a step of comparing the concentration of lipid metabolites in a biological sample of a subject after administration of a candidate therapeutic or alleviating agent with the concentration of the lipid metabolites in a biological sample of a healthy subject.

[21] A screening method described in

[19] or

[20] above, in which the candidate therapeutic agent or alleviator is shown to have a therapeutic effect if the concentration of a lipid metabolite in a biological sample from a subject after administration of the candidate therapeutic agent or alleviator is significantly different from the concentration of the lipid metabolite in a biological sample from a subject before administration of the candidate therapeutic agent or alleviator or the concentration of the lipid metabolite in a biological sample from a subject suffering from atopic dermatitis, or if it is not significantly different from the concentration of the lipid metabolite in a biological sample from a healthy subject.

[22] A method for identifying an atopic dermatitis marker in a lipid metabolite in a biological sample, comprising the steps of measuring the concentration of the lipid metabolite in a biological sample from a subject suffering from atopic dermatitis and the concentration of the lipid metabolite in a biological sample from a healthy subject, and comparing the two measured concentrations.

[23] The identification method described in

[22] above, wherein if the concentration of a lipid metabolite in a biological sample from a subject suffering from atopic dermatitis is significantly different from the concentration of the lipid metabolite in a biological sample from a healthy subject, the lipid metabolite is indicated to be an atopic dermatitis marker.

[24] A kit for detecting or diagnosing atopic dermatitis, comprising a means for quantifying lipid metabolites in a biological sample from a subject.

[25] A method for treating atopic dermatitis, comprising: (A) measuring the concentration of a lipid metabolite in a biological sample from a subject; (B) comparing the concentration of the lipid metabolite in the biological sample from the subject with the concentration of the lipid metabolite in a biological sample from a healthy subject; (C) determining that the subject is suffering from atopic dermatitis if the concentration of the lipid metabolite in the biological sample from the subject is significantly different from the concentration of the lipid metabolite in the biological sample from the healthy subject; and (N) administering treatment for atopic dermatitis to the subject determined to be suffering from atopic dermatitis.

[0008] The present invention is advantageous in that it enables quantitative detection of atopic dermatitis and therapeutic effects on atopic dermatitis based on a biological sample from a subject, and also in that the biological sample from the subject can be collected non-invasively. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows the timeline of the procedure for producing a mouse model of atopic dermatitis. [Figure 2] FIG. 2 shows typical photographs of the dorsal skin of an AD model mouse before DNFB treatment and after the first, second, and third stimulations. [Figure 3]Figure 3 shows the dermatitis scores of AD model mice before DNFB treatment and after the first, second, and third stimulations. Data are shown as mean ± standard error (n = 8). **P < 0.01 indicates a significant difference compared to before treatment. [Figure 4] Figure 4 shows the number of scratches in AD model mice before DNFB treatment and after the first, second, and third stimulations. Data are shown as mean ± standard error (n = 8). *P < 0.05, **P < 0.01 indicate significant differences compared to before DNFB treatment, †P < 0.05, ††P < 0.01 indicate significant differences compared to the first stimulation. [Figure 5] Figure 5 shows the ear thickness of AD model mice before DNFB treatment and after the first, second, and third stimulations. **P<0.01 indicates a significant difference compared to before DNFB treatment. [Figure 6] Figure 6 shows the transevaporative water loss (TEWL) of the back of AD model mice before DNFB treatment and after the first, second, and third stimulations. *P<0.05, **P<0.01 indicate significant differences compared to before DNFB treatment. [Figure 7] Figure 7 shows typical examples of HE-stained images of lesional sections of the dorsal skin of an AD model mouse before DNFB treatment and after the first and third stimulations. The scale bar indicates 50 μm. [Figure 8] Figure 8 shows epidermal thickening in AD model mice measured from HE-stained images. Data are shown as mean ± standard error (n = 5-7). **P < 0.01 indicates a significant difference compared to before DNFB treatment, and †P < 0.05 indicates a significant difference compared to the first stimulation. [Figure 9] FIG. 9 shows typical images of CAE staining in AD model mice (A: mast cells, B: neutrophils). [Figure 10] Figure 10 shows the number of mast cells (A) and neutrophils (B) quantified from CAE staining images of AD model mice before DNFB treatment and after the first and third stimulations. Data are shown as mean ± standard error (n = 5-7). **P < 0.01 indicates a significant difference compared to before DNFB treatment. [Figure 11]FIG. 11 shows a typical example of an image of MGG staining in an AD model mouse (eosinophils). [Figure 12] Figure 12 shows the number of eosinophils quantified from MGG-stained images of AD model mice before DNFB treatment and after the first and third stimulations. Data are shown as mean ± standard error (n = 5-7). **P < 0.01 indicates a significant difference compared to before DNFB treatment. [Figure 13] Figures 13A-C show the amounts of AA-derived lipid mediators excreted in the urine of AD model mice via PGD2 downstream of COX. *P<0.05, **P<0.01 indicate significant differences compared to before DNFB treatment. [Figure 14] Figures 14A-E show the amounts of AA-derived lipid mediators excreted in the urine of AD model mice via PGE2 downstream of COX. *P<0.05, **P<0.01 indicate significant differences compared to before DNFB treatment, and ††P<0.01 indicate significant differences compared to the first stimulation. [Figure 15] Figure 15 shows the amount of AA-derived lipid mediators excreted in the urine of AD model mice via PGF2α downstream of COX. **P<0.01 indicates a significant difference compared to before DNFB treatment, and †P<0.05 indicates a significant difference compared to the first stimulation. [Figure 16] Figure 16 shows the amount of AA-derived lipid mediators excreted in the urine of AD model mice via PGI2 downstream of COX. *P<0.05 indicates a significant difference compared to before DNFB treatment, and †P<0.05 indicates a significant difference compared to the first stimulation. [Figure 17] 17 shows the amount of AA-derived TXB2 downstream of COX excreted in the urine of AD model mice. **P<0.01 indicates a significant difference compared to before DNFB treatment. [Figure 18] Figures 18A and 18B show the amounts of lipid mediators produced by enzyme-independent oxidation of AA excreted in the urine of AD model mice. *P<0.05 indicates a significant difference compared to before DNFB treatment, and †P<0.05 indicates a significant difference compared to the first stimulation. [Figure 19] Figures 19A and 19B show the production of n-3 fatty acid-derived lipid mediators downstream of LOX excreted in the urine of AD model mice. *P<0.05, **P<0.01 indicate significant differences compared to before DNFB treatment, and †P<0.05, ††P<0.01 indicate significant differences compared to the first stimulation. [Figure 20] Figures 20A-D show the mRNA expression levels of cox-1 (A), cox-2 (B), mpges-1 (C), Akr1b3 (D), Txs (E), H-pgds (F), mpges-2 (G), cpges (H), and L-pgds (I) extracted from the skin lesions of AD model mice after the third DNFB stimulation. Data are shown as mean ± standard error (n = 5-7 each). *P < 0.05, **P < 0.01 indicate significant differences compared to the control (vehicle treatment). [Figure 21] Figure 21 shows typical examples of immunostaining images of COX-1, COX-2, mPGES-1, AKR1B3, solvent (vehicle), normal goat serum, normal rabbit IgG, and TXS in the skin lesions of AD model mice. The scale bar indicates 50 μm. [Figure 22] Figures 22A and B show the amount of AA-derived lipid mediators excreted in urine from AD patients (n = 10-14) via PGD2 downstream of COX. Data are shown as mean ± standard error as a ratio to the internal standard. *P < 0.05 indicates a significant difference compared to controls (n = 3-4 each). [Figure 23] Figures 23A-D show the amount of AA-derived lipid mediators excreted in the urine of AD patients (n = 10-14) via PGE2 downstream of COX. Data are shown as mean ± standard error as a ratio to the internal standard. *P < 0.05 indicates a significant difference compared to controls (n = 3-4 each). [Figure 24] Figures 24A-C show the amount of AA-derived lipid mediators excreted in the urine of AD patients (n = 10-14) via PGF2α downstream of COX. Data are shown as mean ± standard error as a ratio to the internal standard. *P < 0.05 indicates a significant difference compared to controls (n = 3-4). [Figure 25]Figure 25 shows the amount of AA-derived lipid mediators excreted in the urine of AD patients (n = 10-14) via PGI2 downstream of COX. Data are shown as mean ± standard error as a ratio to the internal standard. *P < 0.05 indicates a significant difference compared to controls (n = 3-4). [Figure 26] Figures 26A and B show the amounts of AA-derived lipid mediators downstream of LOX excreted in urine from AD patients (n = 10-14). Data are shown as mean ± standard error as a ratio to the internal standard. *P < 0.05 indicates a significant difference compared to controls (n = 3-4). [Figure 27] Figure 27 shows the amount of AA-derived, CYP-dependent lipid mediators excreted in urine from AD patients (n = 10-14). Data are expressed as mean ± standard error as a ratio to the internal standard. *P < 0.05 indicates a significant difference compared to controls (n = 3-4). [Figure 28] Figure 28 shows the amounts of AA and lipid mediators produced by enzyme-independent oxidation of AA excreted in the urine of AD patients (n = 10-14). Data are expressed as mean ± standard error as a ratio to the internal standard. *P < 0.05, **P < 0.01 indicate significant differences compared to controls (n = 3-4). [Figure 29] Figure 29 shows the amount of EPA excreted in the urine of AD patients (n = 10-14). Data are expressed as mean ± standard error as a ratio to the internal standard. *P < 0.05 indicates a significant difference compared to controls (n = 3-4 each). [Figure 30] Figures 30A and B show the amounts of DHA-derived lipid mediators downstream of LOX excreted in the urine of AD patients (n = 10-14). Data are shown as mean ± standard error as a ratio to the internal standard. *P < 0.05 indicates a significant difference compared to controls (n = 3-4 each). [Figure 31]Figure 31A shows a time series of experiments using mice (non-allergic dermatitis model mice) treated with tape stripping. Figure 31B shows a typical example of HE-stained images of dorsal skin sections from a non-allergic dermatitis model mouse. The scale bar indicates 50 μm. Figure 31C shows epidermal thickening in a non-allergic dermatitis model mouse measured from HE-stained images. Data are shown as mean ± standard error (n = 4-7). Figure 31D shows the mRNA expression levels of Tslp, Il-4, Il-13, and Ccl17 extracted from the skin of a non-allergic dermatitis model mouse. Data are shown as mean ± standard error (n = 4-7). Figure 31E shows the amount of AA-derived lipid mediators downstream of COX excreted in the urine of a non-allergic dermatitis model mouse. Data are shown as a ratio to the internal standard and as mean ± standard error (n = 4-7). *P<0.05 indicates a significant difference compared to the day of tape stripping (day 0). Figure 31F shows the mRNA expression levels of Cox-2, mPGES-2, Akr1B3, and H-pgds extracted from the skin of a non-allergic dermatitis model mouse. Data are shown as mean ± standard error (n=4-7). Figure 31G shows a typical example of immunostaining images of the skin of a non-allergic dermatitis model mouse (left: ISO (negative control using isotype antibody); right: AKR1B3 antibody). The scale bar indicates 50 μm. [Figure 32] Figure 32A shows the number of eosinophils (left), neutrophils (middle), and mast cells (right) quantified from MGG-stained images of non-allergic dermatitis model mice. Data are shown as mean ± standard error (n = 4-7). *P < 0.05, **P < 0.01 indicate significant differences compared to controls (n = 4-7). The figures show the amounts of lipid mediators mediated by PGF2α (left) and PGE2 (middle and right) excreted in the urine of non-allergic dermatitis model mice. Specific Description of the Invention

[0010] In the present invention, the term "lipid metabolite" refers to a lipid degradation product produced by enzyme-dependent or enzyme-independent oxidation (hereinafter sometimes referred to as "OX") in vivo, and includes lipid mediators with physiological functions that regulate inflammatory responses. Enzyme-dependent oxidation is promoted by lipid metabolic enzymes present in vivo. Examples of such enzymes include lipid metabolic enzymes associated with the onset and progression of atopic dermatitis (preferably lipid metabolic enzymes activated by the onset and progression of atopic dermatitis), such as cyclooxygenase (hereinafter sometimes referred to as "COX"), lipoxygenase (hereinafter sometimes referred to as "LOX") (e.g., 5-LOX, 15-LOX), and cytochrome p450 (hereinafter sometimes referred to as "CYP"). Examples of lipids decomposed by enzyme-dependent or enzyme-independent oxidation include arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid.

[0011] In the present invention, examples of lipid metabolites include arachidonic acid metabolites, eicosapentaenoic acid metabolites, and docosahexaenoic acid metabolites. Lipid metabolites can be classified into those whose concentrations in biological samples from subjects with atopic dermatitis tend to be higher (or higher) than those in biological samples from healthy subjects (lipid metabolite X) and those whose concentrations in biological samples from subjects with atopic dermatitis tend to be lower (or lower) than those in biological samples from healthy subjects (lipid metabolite Y). Examples of lipid metabolite X include COX metabolites of arachidonic acid (e.g., 11β-13,14-dihydro-15-keto-PGF2α, 13,14-dihydro-15-keto-PGJ2), tetranor-PGDM (tetranor-Prostaglandin D Metabolite) (herein, "tetranor-PGDM" refers to 9-hydroxy-11,15-dioxo-13,14-dihydro-2,3,4,5-tetranor-prostan-1,20-dioic acid). acid), 20-hydroxy-PGE2, 15-keto-PGE2, 13,14-dihydro-15-keto-tetranor-PGE2, tetranor-PGEM (tetranor-Prostaglandin E Metabolite) ("tetranor-PGEM" herein refers to 9,15-dioxo-11-hydroxy-13,14-dihydro-2,3,4,5-tetranor-prostan-1,20-dioic acid). acid), 15-keto-PGF2α (15-keto-PGF2α), 13,14-dihydro-15-keto-tetranor-PGF1α (13,14-dihydro-15-keto-tetranor-PGF1α), tetranor-PGFM (tetranor-Prostaglandin F Metabolite) ("tetranor-PGFM" in this specification refers to 9α,11-dihydroxy-15-oxo-13,14-dihydro-2,3,4,5-tetranor-prostan-1,20-dioic acid acid), 6,15-diketo-13,14-dihydro-PGF1α (6,15-diketo-13,14-dihydro-PGF1α), PGK2, 13,14-dihydro-15-keto-tetranor-PGF1β (13,14-dihydro-15-keto-tetranor-PGF1β), 13,14-dihydro-15-keto-PGE2 (13,14-dihydro-15-keto-PGE2), 6-keto Examples of such metabolites include o-PGF1α, TXB2, LOX metabolites of arachidonic acid (e.g., 5-LOX metabolites of arachidonic acid such as 5-HpETE), CYP metabolites of arachidonic acid (e.g., 17-HETE), and OX metabolites of arachidonic acid (e.g., 8-iso-15(R)-PGF2α), preferably those characterized in that their concentrations in biological samples from subjects suffering from atopic dermatitis are significantly higher than those in biological samples from healthy subjects. Examples of lipid metabolite Y include AA, COX metabolites of arachidonic acid (e.g., 11β-13,14-dihydro-15-keto-PGF2α), LOX metabolites of arachidonic acid (e.g., 5-LOX metabolites of arachidonic acid such as 5-HETE), OX metabolites of arachidonic acid (e.g., iPF2α-IV), LOX metabolites of eicosapentaenoic acid and docosahexaenoic acid (e.g., 5-LOX metabolites of docosahexaenoic acid such as 4-HDoHE and 15-LOX metabolites of docosahexaenoic acid such as 10,17-DiHDoHE), and COX metabolites of eicosapentaenoic acid (e.g., COX metabolites of eicosapentaenoic acid such as PGD3), preferably characterized by a significantly lower concentration in biological samples from subjects with atopic dermatitis than in biological samples from healthy subjects.

[0012] In this invention, "atopic dermatitis" refers to a disease characterized by itchy eczema as the main lesion, which repeatedly worsens and improves, and most patients have an atopic predisposition (allergic constitution). It is an eczematous disease with a characteristic bilaterally symmetrical distribution, with the predilection site varying with age, and it begins in infancy or early childhood, sometimes remits during childhood, and sometimes recurs repeatedly without remission, with characteristic eczema lesions chronically observed, with symptoms persisting into adulthood (Atopic Dermatitis Clinical Practice Guidelines 2018).

[0013] In the present invention, the term "biological sample" refers to a sample isolated from a living organism, such as a body fluid such as urine, blood, saliva, nasal discharge, sweat, tears, feces, etc. The method for collecting a biological sample may be invasive or non-invasive, and can be selected depending on the subject.

[0014] In the present invention, the term "subject" is used to mean not only humans but also non-human mammals (for example, monkeys, mice, rats, dogs, cats, rabbits, horses, cows, pigs, and sheep).

[0015] According to a first aspect of the present invention, there is provided a method for detecting atopic dermatitis, which enables the detection of atopic dermatitis using lipid metabolites in a biological sample as an indicator.

[0016] The detection method of the present invention first involves (A) measuring the concentration of lipid metabolites in a biological sample from a subject. Measurement of lipid metabolite concentrations can be performed by known methods, such as mass spectrometry or immunoassays such as ELISA and immunochromatography. Examples of mass spectrometry include liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MSMS), high-performance liquid chromatography-mass spectrometry (HPLC-MS), and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MSMS). Immunoassays are analytical methods that use detectably labeled anti-lipid metabolite antibodies or detectably labeled antibodies (secondary antibodies) against the anti-lipid metabolite antibodies. Depending on the antibody labeling method, immunoassays are classified into enzyme immunoassays (EIA or ELISA), radioimmunoassays (RIA), fluorescence immunoassays (FIA), fluorescence polarization immunoassays (FPIA), chemiluminescence immunoassays (CLIA), etc., and any of these can be used in the present invention. From the viewpoint of accurately measuring the concentrations of structurally similar lipid metabolites, measurement by mass spectrometry (particularly LC-MSMS and HPLC-MSMS) is preferred.

[0017] The detection method of the present invention can further include a step of determining whether or not the subject from whom the biological sample was collected has atopic dermatitis based on the lipid metabolite concentration measured in step (A). This step may include a step (B) of comparing the lipid metabolite concentration in the subject's biological sample with the concentration of the lipid metabolite in a biological sample from a healthy subject. Here, if the lipid metabolite concentration in the subject's biological sample is significantly different from the concentration of the lipid metabolite in a biological sample from a healthy subject, the subject is indicated to have atopic dermatitis. That is, the detection method of the present invention may further include a step (C) of determining that the subject has atopic dermatitis if the lipid metabolite concentration in the subject's biological sample is significantly different from the concentration of the lipid metabolite in a biological sample from a healthy subject. The term "significantly different" in step (C) means that the concentration of the lipid metabolite is either higher or lower than that of a healthy subject, depending on the lipid metabolite. For example, when the lipid metabolite is lipid metabolite X, the concentration of the lipid metabolite in the biological sample of the subject is higher, preferably significantly higher, than the concentration of the lipid metabolite in the biological sample of the healthy subject (for example, the concentration of the lipid metabolite in the biological sample of the subject is statistically significantly higher than the concentration of the lipid metabolite in the biological sample of the healthy subject, or is about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more, about 2.0 times or more, about 2.1 times or more, about 2.2 times or more, about 2.3 times or more, about 2.4 times or more, about 2.5 times or more, about 2.6 times or more, about 2.7 times or more, about 2.8 times or more, about 2.9 times or more, about 3.0 times or more, about 3.1 times or more, about 3.2 times or more, about 3.3 times or more, about 3.4 times or more, about 3.5 times or more, about 3.6 times or more, about 3.7 times or more, about 3.8 times or more, about 3.9 ... "Likely a lipid metabolite concentration in a biological sample from a healthy subject is about 0.8 times or more, about 1.9 times or more, about 2.0 times or more, about 2.5 times or more, or about 3 times or more." In the case of lipid metabolite Y, "atopic dermatitis" refers to a condition in which the concentration of the lipid metabolite in a biological sample from a healthy subject is lower, preferably significantly lower, than the concentration of the lipid metabolite in a biological sample from a healthy subject (e.g., the concentration of the lipid metabolite in a biological sample from a subject is statistically significantly lower than the concentration of the lipid metabolite in a biological sample from a healthy subject, or is about 0.9 times or less, about 0.8 times or less, about 0.7 times or less, about 0.6 times or less, about 0.5 times or less, about 0.4 times or less, or about 0.3 times or less). The lipid metabolite concentration in a biological sample from a healthy subject can be calculated by measuring the lipid metabolite concentrations in biological samples collected from multiple healthy subjects in advance. Furthermore, "suffering from atopic dermatitis" is also used to mean cases in which there is a possibility of the subject being affected.

[0018] According to the detection method of the present invention, atopic dermatitis can be detected in a subject. Therefore, the detection method of the present invention can be used as an auxiliary method for diagnosing atopic dermatitis, and the determination of whether a subject has atopic dermatitis can ultimately be made by a physician or veterinarian, optionally in combination with other findings.

[0019] According to the detection method of the present invention, atopic dermatitis can be quantitatively detected based on a biological sample collected from a test subject. That is, the detection method of the present invention is advantageous in that it can detect atopic dermatitis simply and accurately while reducing the burden on the patient. The detection method of the present invention is also advantageous in that it can be used to detect atopic dermatitis from a biological sample collected by a non-invasive method such as urine collection, and therefore can be applied to subjects from whom atopic dermatitis is difficult to detect by an invasive method such as blood collection, for example, children (including infants) from whom it is difficult to collect blood.

[0020] Another aspect of the present invention provides a method for diagnosing atopic dermatitis. According to the diagnostic method of the present invention, it is possible to diagnose whether a subject has atopic dermatitis using lipid metabolites in a biological sample as an indicator. The diagnostic method of the present invention, like the detection method of the present invention, involves (A') measuring the concentration of a lipid metabolite in the biological sample of a test subject. The diagnostic method of the present invention may further include (B') comparing the concentration of the lipid metabolite in the biological sample of the subject with the concentration of the same lipid metabolite in a biological sample of a healthy subject, and may further include (C') determining that the subject has atopic dermatitis if the concentration of the lipid metabolite in the biological sample of the subject is significantly different from the concentration of the same lipid metabolite in the biological sample of the healthy subject. Steps (A'), (B'), and (C') correspond to steps (A), (B), and (C), respectively, and can be carried out according to the description of the detection method of the present invention.

[0021] According to a second aspect of the present invention, there is provided a method for determining the therapeutic effect on atopic dermatitis, which can determine the therapeutic effect on atopic dermatitis using lipid metabolites in a biological sample as an indicator.

[0022] In the determination method of the present invention, similarly to the detection method of the present invention, the step (D) of measuring the concentration of lipid metabolites in a biological sample of a subject is carried out. The measurement of the concentration of lipid metabolites can be carried out in the same manner as in the detection method of the present invention.

[0023] The method of the present invention can further include a step of determining the therapeutic effect of atopic dermatitis on the treated subject based on the lipid metabolite concentration measured in step (D). This step may include a step (E) of comparing the lipid metabolite concentration in a biological sample from a subject treated for atopic dermatitis with the lipid metabolite concentration in a biological sample from a healthy subject. A therapeutic effect is indicated when the lipid metabolite concentration in a biological sample from a subject treated for atopic dermatitis is significantly different from the lipid metabolite concentration in a biological sample from a subject before treatment or from a biological sample from a subject with atopic dermatitis, or when the lipid metabolite concentration is not significantly different from the lipid metabolite concentration in a biological sample from a healthy subject. That is, the determination method of the present invention may further include (F) a step of determining that there is a therapeutic effect when the concentration of a lipid metabolite in a biological sample of a subject receiving treatment for atopic dermatitis is significantly different from the concentration of the lipid metabolite in a biological sample of the subject before treatment or the concentration of the lipid metabolite in a biological sample of a subject suffering from atopic dermatitis (preferably, when it is significantly different in a direction approaching the concentration of the lipid metabolite in a biological sample of a healthy subject), or when it is not significantly different from the concentration of the lipid metabolite in a biological sample of a healthy subject.The term "significantly different" in step (F) means that the concentration is either higher or lower than that of the subject before treatment or that of the subject suffering from atopic dermatitis, depending on the lipid metabolite. For example, when the lipid metabolite is lipid metabolite X, the concentration of the lipid metabolite in the subject's biological sample is lower, preferably significantly lower, than the concentration of the lipid metabolite in the biological sample of the subject before treatment or that of the subject suffering from atopic dermatitis (for example, the concentration of the lipid metabolite in the subject's biological sample is statistically significantly lower than the concentration of the lipid metabolite in the biological sample of the subject before treatment or that of the subject suffering from atopic dermatitis, or is about 0.9 times or less, about 0.8 times or less, about 0.7 times or less, about 0.6 times or less, about 0.5 times or less, about 0.4 times or less). or less than about 0.3 times), and when the lipid metabolite is lipid metabolite Y, it means that the concentration of the lipid metabolite in the subject's biological sample is higher, preferably significantly higher, than the concentration of the lipid metabolite in the biological sample of the subject before treatment or the subject with atopic dermatitis (for example, the concentration of the lipid metabolite in the subject's biological sample is statistically significantly higher than the concentration of the lipid metabolite in the biological sample of the subject before treatment or the subject with atopic dermatitis, or is about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more, about 2.0 times or more, about 2.5 times or more, or about 3 times or more). Furthermore, "not significantly different" in step (F) means that, for both lipid metabolite X and lipid metabolite Y, the concentration of the lipid metabolite in the subject's biological sample is equivalent to the concentration of the lipid metabolite in the biological sample from a healthy subject (for example, the concentration of the lipid metabolite in the subject's biological sample is not statistically significantly different from the concentration of the lipid metabolite in the biological sample from a healthy subject, or is greater than about 0.8-fold and less than about 1.2-fold, or greater than about 0.9-fold and less than about 1.1-fold). The concentration of the lipid metabolite in the subject's biological sample before treatment can be determined by measuring the concentration of the lipid metabolite in the subject's biological sample before treatment. The concentration of the lipid metabolite in the biological sample from a subject with atopic dermatitis can be determined by measuring the lipid metabolite concentrations in biological samples collected from multiple subjects with atopic dermatitis in advance.Furthermore, the concentration of lipid metabolites in a biological sample from a healthy subject can be an average value calculated by measuring the lipid metabolite concentrations of biological samples collected in advance from multiple healthy subjects. The subject to be treated in the determination method of the present invention is preferably a subject suffering from atopic dermatitis. Furthermore, in the present invention, the "subject suffering from atopic dermatitis" may be any subject whose results from other testing methods indicate atopic dermatitis, and preferably a subject diagnosed by a doctor or veterinarian as having atopic dermatitis.

[0024] Therapies for atopic dermatitis whose therapeutic effects can be determined by the determination method of the present invention include, for example, drug therapy and proactive therapy. Drug therapy includes treatment with therapeutic drugs for atopic dermatitis, and examples of such therapeutic drugs include anti-inflammatory topical drugs (topical steroids, tacrolimus, nonsteroidal anti-inflammatory drugs), antihistamines, cyclosporine, oral steroids, herbal medicines, antibody drugs, and other pharmaceuticals.

[0025] According to the assessment method of the present invention, the therapeutic effect of a subject who has received treatment for atopic dermatitis can be assessed, thereby verifying the effectiveness of the treatment for atopic dermatitis administered to the subject. Furthermore, if no therapeutic effect is observed, the treatment can be immediately discontinued and an alternative treatment plan can be developed. Therefore, the assessment method of the present invention is advantageous in that it can prevent unnecessary medication, thereby contributing to reducing medical costs and the burden on patients. Furthermore, according to the assessment method of the present invention, detection can be performed based on biological samples collected by non-invasive methods such as urine collection, and therefore it is advantageous in that it can be applied to subjects from which it is difficult to assess the therapeutic effect using invasive methods such as blood collection, such as children (including infants) and animals (e.g., pets such as dogs and cats), from which blood cannot be easily collected.

[0026] According to a third aspect of the present invention, there are provided an atopic dermatitis marker comprising a lipid metabolite, and use of the lipid metabolite as an atopic dermatitis marker. In the present invention, an "atopic dermatitis marker" refers to a substance whose presence or amount serves as an indicator of the presence or absence of atopic dermatitis and the severity of its symptoms, and can be used as a marker for detecting, identifying, evaluating, etc. atopic dermatitis. That is, according to the present invention, a lipid metabolite can be used as a disease identification marker for atopic dermatitis, and can also be used to evaluate the severity of atopic dermatitis.

[0027] Because the assessment method of the present invention can be used to determine the effectiveness of a therapeutic agent for atopic dermatitis, the present invention also provides a method for screening for a therapeutic agent or alleviator for atopic dermatitis. Specifically, a fourth aspect of the present invention provides a method for screening for a therapeutic agent or alleviator for atopic dermatitis, comprising the steps of (G) administering a candidate therapeutic agent or alleviator for atopic dermatitis to a subject, and (H) measuring the concentration of lipid metabolites in a biological sample from the subject. The screening method of the present invention can further include a step of determining whether or not the candidate therapeutic agent or alleviator has a therapeutic effect based on the concentration of lipid metabolites measured in step (H). This step may include the step of (I) comparing the concentration of lipid metabolites in the subject's biological sample after administration of the candidate therapeutic agent or alleviator with the concentration of the lipid metabolite in a biological sample from a healthy subject. Here, if the concentration of a lipid metabolite in a subject's biological sample after administration of a candidate therapeutic drug or alleviator is significantly different from the concentration of the lipid metabolite in the subject's biological sample before administration of the candidate therapeutic drug or alleviator or the concentration of the lipid metabolite in the biological sample of a subject with atopic dermatitis, or if it is not significantly different from the concentration of the lipid metabolite in the biological sample of a healthy subject, the therapeutic drug or alleviator candidate is indicated to have a therapeutic effect. That is, the screening method of the present invention may further include (J) a step of determining that a therapeutic effect is present if the concentration of a lipid metabolite in a subject's biological sample after administration of a candidate therapeutic drug or alleviator is significantly different from the concentration of the lipid metabolite in the subject's biological sample before administration of the candidate therapeutic drug or alleviator or the concentration of the lipid metabolite in the biological sample of a subject with atopic dermatitis (preferably, if it is significantly different in the direction approaching the concentration of the lipid metabolite in the biological sample of a healthy subject), or if it is not significantly different from the concentration of the lipid metabolite in the biological sample of a healthy subject, thereby allowing the selection of a candidate therapeutic drug or alleviator candidate.The term "significantly different" in step (J) means that the concentration of the lipid metabolite is either higher or lower than the concentration in the subject or the subject suffering from atopic dermatitis before administration of the candidate therapeutic or alleviating agent, depending on the lipid metabolite. For example, when the lipid metabolite is lipid metabolite X, the concentration of the lipid metabolite in the subject's biological sample is lower, preferably significantly lower, than the concentration of the lipid metabolite in the subject's biological sample before administration of the candidate therapeutic or alleviating agent or the subject suffering from atopic dermatitis (for example, the concentration of the lipid metabolite in the subject's biological sample is statistically significantly lower than the concentration of the lipid metabolite in the subject or the subject suffering from atopic dermatitis before administration of the candidate therapeutic or alleviating agent, or is about 0.9-fold or less, about 0.8-fold or less, about 0.7-fold or less, about 0.6-fold or less, about 0.5-fold or less, or about 0.9-fold or less. or less than about 0.4-fold, or less than about 0.3-fold), and in the case of lipid metabolite Y, it means that the concentration of the lipid metabolite in the subject's biological sample is higher, preferably significantly higher, than the concentration of the lipid metabolite in the biological sample of the subject before administration of the candidate therapeutic or alleviating agent or of the subject suffering from atopic dermatitis (for example, the concentration of the lipid metabolite in the subject's biological sample is statistically significantly higher than the concentration of the lipid metabolite in the biological sample of the subject before administration of the candidate therapeutic or alleviating agent or of the subject suffering from atopic dermatitis, or is about 1.1-fold or more, about 1.2-fold or more, about 1.3-fold or more, about 1.4-fold or more, about 1.5-fold or more, about 1.6-fold or more, about 1.7-fold or more, about 1.8-fold or more, about 1.9-fold or more, about 2.0-fold or more, about 2.5-fold or more, or about 3-fold or more). Furthermore, "not significantly different" in step (J) means that for both lipid metabolite X and lipid metabolite Y, the concentration of the lipid metabolite in the subject's biological sample is equivalent to the concentration of the lipid metabolite in the biological sample of a healthy subject (for example, the concentration of the lipid metabolite in the subject's biological sample is not statistically significantly different from the concentration of the lipid metabolite in the biological sample of a healthy subject, or is more than about 0.8 times and less than about 1.2 times, or more than about 0.9 times and less than about 1.1 times). The screening method of the present invention can be carried out according to the description of the detection method and determination method of the present invention.In the screening method of the present invention, the subject to which a candidate therapeutic or alleviating agent is administered is preferably a subject suffering from atopic dermatitis. When carrying out the screening method of the present invention, mammals other than humans can be used as the subject. The therapeutic agents for atopic dermatitis that are the subject of the screening method of the present invention are the same as those described for the determination method of the present invention. In addition, alleviating agents for atopic dermatitis that are the subject of the screening method of the present invention include foods (e.g., food compositions), quasi-drugs (e.g., medicated cosmetics), feeds (e.g., pet foods), cosmetics (e.g., cosmetic compositions), and skin care compositions that have the function of alleviating the symptoms of atopic dermatitis, and such foods include supplements, foods for specified health uses, and foods with functional claims. In addition, the term "alleviation" is used to mean improvement as well.

[0028] According to a fifth aspect of the present invention, there is provided a method for identifying an atopic dermatitis marker in a lipid metabolite in a biological sample, comprising the steps of (K) measuring the concentration of a lipid metabolite in a biological sample from a subject suffering from atopic dermatitis and the concentration of a lipid metabolite in a biological sample from a healthy subject, and (L) comparing the two measured concentrations. The identification method of the present invention can further include the step of determining the lipid metabolite as an atopic dermatitis marker based on the results of the concentration comparison performed in step (L). In this step, if the concentration of the lipid metabolite in the biological sample from the subject suffering from atopic dermatitis is significantly different from the concentration of the lipid metabolite in the biological sample from the healthy subject, the lipid metabolite is indicated as an atopic dermatitis marker. That is, the identification method of the present invention may further include the step of (M) determining that the lipid metabolite is an atopic dermatitis marker if the concentration of the lipid metabolite in the biological sample from the subject suffering from atopic dermatitis is significantly different from the concentration of the lipid metabolite in the biological sample from the healthy subject. The term "significantly different" in step (M) refers to a case where the concentration is higher or lower than that of a healthy subject, depending on the lipid metabolite. For example, when the lipid metabolite is lipid metabolite X, the concentration of the lipid metabolite in the biological sample of the subject is higher than the concentration of the lipid metabolite in the biological sample of the healthy subject, preferably significantly higher (for example, the concentration of the lipid metabolite in the biological sample of the subject is statistically significantly higher than the concentration of the lipid metabolite in the biological sample of the healthy subject, or is about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more, about 2.0 times or more, about 2.1 times or more, about 2.2 times or more, about 2.3 times or more, about 2.4 times or more, about 2.5 times or more, about 2.6 times or more, about 2.7 times or more, about 2.8 times or more, about 2.9 times or more, about 3.0 times or more, about 3.1 times or more, about 3.2 times or more, about 3.3 times or more, about 3.4 times or more, about 3.5 times or more, about 3.6 times or more, about 3.7 times or more, about 3.8 times or more, about 3.9 ... or about 0.8 times or more, about 1.9 times or more, about 2.0 times or more, about 2.5 times or more, or about 3 times or more), and in the case of lipid metabolite Y, it means that the concentration of the lipid metabolite in the subject's biological sample is lower, preferably significantly lower, than the concentration of the lipid metabolite in the biological sample of a healthy subject (for example, the concentration of the lipid metabolite in the subject's biological sample is statistically significantly lower than the concentration of the lipid metabolite in the biological sample of a healthy subject, or is about 0.9 times or less, about 0.8 times or less, about 0.7 times or less, about 0.6 times or less, about 0.5 times or less, about 0.4 times or less, or about 0.3 times or less).The identification method of the present invention can be carried out according to the description of the detection method and determination method of the present invention.

[0029] A subject in whom atopic dermatitis has been detected by the detection method of the present invention or diagnosed by the diagnostic method of the present invention can be treated for atopic dermatitis. Thus, according to a sixth aspect of the present invention, there is provided a method for treating atopic dermatitis, comprising the steps of: (A) measuring the concentration of a lipid metabolite in a biological sample from the subject; (B) comparing the concentration of the lipid metabolite in the biological sample from the subject with the concentration of the lipid metabolite in a biological sample from a healthy subject; (C) determining that the subject has atopic dermatitis if the concentration of the lipid metabolite in the biological sample from the subject is significantly different from the concentration of the lipid metabolite in the biological sample from the healthy subject; and (N) administering treatment for atopic dermatitis to the subject determined to have atopic dermatitis. The atopic dermatitis detection and determination steps (i.e., steps (A), (B), and (C)) of the treatment method of the present invention can be carried out according to the description of the detection method of the present invention and the diagnostic method of the present invention. Furthermore, treatment for atopic dermatitis can be carried out according to the description of the assessment method of the present invention.

[0030] According to a seventh aspect of the present invention, there is provided a kit for detecting atopic dermatitis, comprising a means for quantifying lipid metabolites in a biological sample of a subject. The kit of the present invention is typically a kit for detecting atopic dermatitis according to the detection method of the present invention. The means for quantifying lipid metabolites includes, for example, a substance that specifically binds to the lipid metabolite, typically an antibody against the lipid metabolite. The means for quantifying lipid metabolites also includes a mass spectrometer used in the above-mentioned mass spectrometry.

[0031] In the kit of the present invention, when the means for quantifying lipid metabolites is an antibody, the kit of the present invention includes the reagents (and, in some cases, equipment) necessary to measure the concentration of lipid metabolites by immunoassay using the antibody.

[0032] An example of a kit of the present invention is a kit for measuring the concentration of lipid metabolites by the sandwich method, which may include a microtiter plate, an anti-lipid metabolite antibody for capture, an anti-lipid metabolite antibody labeled with alkaline phosphatase or peroxidase, and a substrate for alkaline phosphatase or a substrate for peroxidase.

[0033] Another example of a kit of the present invention is a kit for measuring the concentration of lipid metabolites by a sandwich method using a secondary antibody, which may include a microtiter plate, an anti-lipid metabolite antibody for capture, the anti-lipid metabolite antibody as a primary antibody, an antibody against the anti-lipid metabolite antibody labeled with alkaline phosphatase or peroxidase as a secondary antibody, and an alkaline phosphatase substrate or a peroxidase substrate.

[0034] The kit can be used, for example, as follows. First, a capture antibody is immobilized on a microtiter plate, and a target biological sample is added after appropriate dilution, followed by incubation. The sample is then removed and washed. Next, a primary antibody is added, incubated, and washed. An enzyme-labeled secondary antibody is added and incubated, and then a substrate is added to develop color. The concentration of lipid metabolites can be determined by measuring the color development using a microtiter plate reader or the like.

[0035] In the kits of the present invention, the labeled antibody is not limited to an enzyme-labeled antibody, but may also be an antibody labeled with a radioactive substance (25I, 131I, 35S, 3H, etc.), a fluorescent substance (fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, near-infrared fluorescent material, etc.), a luminescent substance (luciferase, luciferin, aequorin, etc.), nanoparticles (gold colloid, quantum dots), etc. Alternatively, a biotinylated antibody may be used as the labeled antibody, and labeled avidin or streptavidin may be added to the kit.

[0036] Another example of a kit of the present invention is a kit for measuring the concentration of lipid metabolites by immunochromatography, which can be configured to have an antibody storage section containing a first anti-lipid metabolite antibody labeled with gold colloid or the like, and a determination section in which a second anti-lipid metabolite antibody (preferably an antibody that recognizes a different epitope of a lipid metabolite) is immobilized in a line on a cellulose membrane or the like, connected by a thin groove.

[0037] The kit can be used, for example, as follows: First, a biological sample is added to the antibody storage section or the biological sample receiving section adjacent to the antibody storage section. The labeled antibody and lipid metabolite in the antibody storage section bind to form a lipid metabolite-labeled antibody complex, which then migrates by capillary action through the groove to the test section. Next, when the complex is captured by the immobilized second anti-lipid metabolite antibody, a red line appears in the test section due to the plasmon effect of the gold colloid, allowing the presence of lipid metabolite to be detected. In this case, the kit can be provided in the form of a stick, such as a test stick, which may further include absorbent paper for absorbing the biological sample, a desiccant, etc.

[0038] In the kit of the present invention, when the means for quantifying lipid metabolites is a mass spectrometer, the kit of the present invention may include an internal standard device in addition to the mass spectrometer. By using an internal standard, the extraction efficiency and ionization efficiency for each analysis can be corrected during measurement by a mass spectrometer. The internal standard used in mass spectrometry includes deuterated lipid metabolites.

[0039] In addition to the above, the kit of the present invention can be carried out according to the description of the detection method and determination method of the present invention. [Example]

[0040] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0041] Example 1: Creation of atopic dermatitis model mice The procedure is shown in Figure 1. BALB / C mice (male, 7-8 weeks old) (CLEA Japan) were anesthetized and their dorsal hair was removed using clippers and depilatory cream. The mice were then acclimated for 3 days. After this, 25 μL of a 0.5% DNFB (2,4-dinitrofluorobenzene, Nacalai Tesque) mixture (acetone (Fujifilm Wako Pure Chemical):olive (Fujifilm Wako Pure Chemical), 4:1) was applied dropwise to the upper back of the mice for sensitization (day 1). Four days later, 20 μL of a 0.2% DNFB mixture (acetone:olive, 4:1) was applied dropwise to the right ear and 100 μL to the lower back of the mice for the first stimulation. This stimulation procedure was repeated three times every 3 days (first stimulation: day 5, second stimulation: day 8, third stimulation: day 11) to generate atopic dermatitis (AD) model mice.

[0042] Example 2: Evaluation of atopic dermatitis symptoms The symptoms of the AD model mice prepared in Example 1 were evaluated.

[0043] (1) Observation of skin pathology The changes in the skin condition of the AD model mice over time are shown in Figure 2. It was confirmed that the inflammation on the back of the AD model mice worsened from the first to the third stimulation.

[0044] (2) Pathological score The results are shown in Figure 3. Using the Severity Scoring of Atopic Dermatitis (SCORAD), a diagnostic index for human AD, as a standard, pathology scores were calculated for the dorsal skin lesions of AD model mice before DNFB treatment and after the first, second, and third stimulations. SCORAD is composed of indicators such as erythema, hemorrhage, edema, papules, epidermal peeling, erosions, scratch marks, dryness, and lichenification. A score (0: none, 1: mild, 2: moderate, 3: severe) was calculated according to the severity of each symptom, with a maximum score of 12 points. Significant increases in scores were confirmed after the second and third stimulations compared to before DNFB treatment.

[0045] (3) Number of scratches The results are shown in Figure 4. The number of scratches made by AD model mice was visually counted for 10 minutes before DNFB treatment and 1 hour after each of the first, second, and third stimulations. Scratches were counted as scratches made with the hind paws. Compared to before DNFB treatment, the scores were significantly increased after each of the first, second, and third stimulations.

[0046] (4) Thickness of the ears The results are shown in Figure 5. The ear thickness of the AD model mice was measured using an ABS solar-powered Digimatic caliper (Mitutoyo), and the values ​​in the graph were calculated from the difference in ear thickness between before DNFB treatment and 24 hours after each stimulation.

[0047] (5) Water evaporation rate (TEWL g / (m 2 h)) The results are shown in Figure 6. The amount of water loss from the back of the AD model mice was measured using the multi-display device MDD4 (Courage+Khazaka).

[0048] (6) Statistical processing In Examples 2 to 7, the measured values ​​are expressed as mean ± standard error, and all experiments were performed at least three times. Statistical analysis was performed using BellCurve software for Excel 2015 (Social Information Services). Significant differences were tested by Student's t test or Mann-Whitney U test for two-group comparisons. Multi-group comparisons were performed by a combination of one-way analysis of variance (ANOVA) and Tukey's test, or a combination of Kruskal-Wallis test and Steel-Dwass test. Statistically significant differences were * P<0.05, ** P was defined as <0.01.

[0049] Example 3: Histopathological evaluation of atopic dermatitis The AD model mice prepared in Example 1 were subjected to histopathological evaluation of the skin lesions. (1) Procedure Excised skin lesions were fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and 4-μm-thick tissue sections were prepared. The tissue sections were stained with hematoxylin and eosin (HE), chloroacetate esterase (CAE), or May-Grünwald-Giemsa (MGG) according to standard methods. The specific staining procedures are described below. The stained skin lesion tissues were observed and photographed using a BZ-X710 microscope (Keyence). The numbers of CAE-positive mast cells, neutrophils, and MGG-positive eosinophils were counted in 10 randomly selected sections per field (magnification ×400).

[0050] (2) Chloroacetate esterase (CAE) staining CAE staining is a staining method that uses naphthol AS-D chloroacetate as a substrate to stain esterase specific to mast cells and neutrophils red. The tissue section specimens prepared in (1) above were deparaffinized and then used for staining. 4% sodium nitrite solution and New Fuchsin solution were mixed in a 1:1 ratio, and then this solution was mixed with naphthol solution in a 1:9 ratio. This solution was mixed with phosphate buffer (0.2M NaH2PO4, 0.2M Na2HPO4, pH 7.6) in a 1:20 ratio, and the sections were immersed in the solution for 10 minutes. The sections were washed with distilled water and then counterstained with hematoxylin solution. 1mm 2 The number of mast cells and neutrophils in the blood was measured, and the mean and standard deviation were calculated.

[0051] (3) May-Grünwald-Giemsa (MGG) staining May-Grünwald-Giemsa staining stains eosinophil-specific acidophilic granules red. After deparaffinizing the tissue sections prepared in (1) above, the sections were immersed in May-Grünwald staining solution for 6 minutes. Next, the sections were washed with 1 / 15M phosphate buffer (x10) (0.2M NaH2PO4, 0.2M Na2HPO4, pH 6.4-6.8) and then immersed in a solution of Giemsa staining solution and distilled water in a 1:20 ratio for 5 minutes. 2 The number of eosinophils in the stool was counted, and the mean and standard deviation were calculated.

[0052] (4) Results The results of the histopathological evaluations (2) and (3) performed on the AD model mice prepared in Example 1 are shown in FIGS.

[0053] The HE stained images were as shown in Figure 7. The changes in acanthosis measured from the HE stained images were as shown in Figure 8 (data are mean ± standard error (n = 5-7)).

[0054] The CAE staining images are shown in Figure 9 (A: mast cells, B: neutrophils). The changes in the numbers of mast cells and neutrophils quantified from the CAE staining images are shown in Figure 10 (A: mast cell numbers, B: neutrophil numbers) (data are mean ± standard error (n = 5-7)).

[0055] The MGG stained images were as shown in Figure 11. The changes in the number of eosinophils quantified from the MGG stained images were as shown in Figure 12 (data are mean ± standard error (n = 5-7)).

[0056] The results of Examples 2 and 3 confirmed that the atopic dermatitis model mice in Example 1 exhibited symptoms of atopic dermatitis.

[0057] Example 4: Analysis of urinary lipid mediators in AD model mice The urinary lipid mediators of the AD model mice (n=8) prepared in Example 1 were analyzed.

[0058] (1) Method A. Urine collection Urine samples from AD model mice were collected for 24 hours each day before DNFB treatment (day 0), and on the first (day 5) and third (day 11) stimulations (see Table 1). Urine samples were stored at -80°C until analysis.

[0059] B. Urinary regulation The urine prepared in step A above was centrifuged at 15,000 rpm for 10 minutes at 4°C. To 200 μL of the supernatant, 300 μL of 0.1% formic acid solution and 10 μL of the internal standard solution shown in Table 2 were added to prepare a sample solution. This sample solution was loaded onto a solid phase extraction cartridge (OASIS HLB μElute, Waters), and the cartridge was washed with 200 μL of distilled water and 200 μL of hexane. Then, the lipid mediator adhered to the cartridge was eluted with 100 μL of 100% methanol.

[0060] Measurement of lipid mediator The eluate prepared in step I above was injected into LCMS-8060, and the lipid mediator was measured according to the following procedure.

[0061] <LC conditions> The conditions used for liquid chromatography-mass spectrometry were as follows. Analysis column: Kinetex C8 (2.1 mm I.D. × 150 mm, 2.6 μm, Phenomenex) Mobile phase A: 0.1% formic acid Mobile phase B: acetonitrile Flow rate: 0.4 mL / min Injection volume: 5 μL Column temperature: 40°C Gradient program: as shown in Table 1.

[0062]

Table 1

[0063] Mass spectrometer: LCMS-8060 (Shimadzu) Measurement program; LC / MS / MS method package lipid mediator ver.2 (Shimadzu) Nebulizer gas flow rate: 3 L / min Heating gas flow rate: 10 L / min Interface temperature: 300°C Drying gas flow rate: 10 L / min DL temperature: 250°C Heat block temperature: 400 °C Ionization mode: ESI+ / - Internal standard solution: The composition was as shown in Table 2.

[0064]

Table 2

[0065] Data processing According to the above measurement program, the detected lipid mediators (158 types) were divided into 16 groups based on physical properties, and an internal standard substance was set for each group (see Table 2). Using LabSolutions LCMS (Version 5.65, Shimadzu Corporation), the peak area value calculated from the chromatogram of each lipid mediator was divided by the peak area value of the corresponding internal standard substance above to correct for quantitative errors and the like that occurred during lipid extraction. The concentration of the lipid mediator (the vertical axis in Figures 13 to 19 and Figures 21 to 30) was shown as the value obtained by further dividing the peak area value of the lipid mediator by the peak area value of the internal standard substance and then dividing by the value of urinary creatinine in each sample. The concentration of urinary creatinine was measured using Lab Assay Creatinine (FUJIFILM Wako Pure Chemical Corporation).

[0066] (2) Results <n-6 fatty acid> Lipid mediators downstream of COX As shown in Figures 13-17, significant increases in 10 lipid mediators derived from the n-6 fatty acid arachidonic acid (AA) downstream of cyclooxygenase (COX) were confirmed. Specifically, metabolites of prostaglandin D2 (PGD2) were 11β-13,14-dihydro-15-keto-PGF2α (Figure 13A), 13,14-dihydro-15-keto-PGJ2 (Figure 13B), and prostaglandin K2 (PGK2) (Figure 13C). The metabolites of prostaglandin E2 (sometimes referred to herein as "PGE2" or "PGE2") were 15-keto-PGE2 (Fig. 14A), 13,14-dihydro-15-keto-tetranor-PGF1β (Fig. 14B), 13,14-dihydro-15-keto-PGE2 (Fig. 14C), 13,14-dihydro-15-keto-tetranor-PGE2 (Fig. 14D), and PGK2 (Fig. 14E (same as Fig. 13C)). PGK2 is metabolized from either PGD2 or PGE2. PGJ2 is prostaglandin J2 (sometimes referred to herein as "PGJ2"), and PGF1β is prostaglandin F1β (sometimes referred to herein as "PGF 1β Prostaglandin F2α (hereinafter referred to as "PGF") is a prostaglandin that acts as a stimulant for the production of steroid hormones. 2α The compound metabolized from prostaglandin I2 (sometimes referred to as "PGI2" in this specification) was 13,14-dihydro-15-keto-tetranor-PGF1α (Figure 15). The compound metabolized from prostaglandin I2 (sometimes referred to as "PGI2" in this specification) was 6-keto-PGF1α (Figure 16). PGF1α is also metabolized from prostaglandin F1α (sometimes referred to as "PGF 1αThere is such a thing as "). And it was thromboxane B2 (thromboxane B2, sometimes referred to as "TXB2" in this specification) (Figure 17). The metabolic pathway of lipid mediators downstream of COX (derived from AA) is as shown in Table 3.

[0067]

Table 3

[0068] Lipid mediators downstream of COX As shown in Figure 16, isoprostaglandin F2α-IV (isoprostaglandin F2α-IV, sometimes referred to as "iPF2α-IV" or "iPF 2α -IV" in this specification) (Figure 18A) and 8-iso-15(R)-PGF2α (Figure 18B), which are lipid mediators downstream of enzyme-independent oxidation (OX) derived from AA, were confirmed to increase significantly.

[0069] <n-3 polyunsaturated fatty acids> Lipid mediators derived from n-3 polyunsaturated fatty acids As shown in Figure 19, prostaglandin D3 (prostaglandin D3, sometimes referred to as "PGD3" or "PGD3" in this specification) (Figure 19A) and resolvin D1 (Figure 19B), which are lipid mediators downstream of their respective lipoxygenases (LOX) and are derived from eicosapentaenoic acid (eicosapentaenoic acid, sometimes referred to as "EPA" in this specification) and docosahexaenoic acid (docosahexaenoic acid, sometimes referred to as "DHA" in this specification), which are n-3 polyunsaturated fatty acids, were confirmed to decrease significantly after the first stimulation.

[0070] These results suggest that the main type of fatty acids used to produce lipid mediators in the urine of AD model mice are n-6 fatty acids. Among the lipid mediators downstream of COX, three PGD2-derived lipid mediators (11β-13,14-dihydro-15-keto-PGF2α, 13,14-dihydro-15-keto-PGJ2, and PGK2) (Figure ​(Figure13A-C)), five PGE2-derived lipid mediators (15-keto-PGE2, 13,14-dihydro-15-keto-tetranor-PGF1β, 13,14-dihydro-15-keto-tetranor-PGF1β, and PGK2) were identified. The levels of PGD2, 13,14-dihydro-15-keto-tetranor-PGE2, and PGK2 (Fig. 14A-E), one PGF2α-derived lipid mediator (13,14-dihydro-15-keto-tetranor-PGF1α) (Fig. 15), one PGI2-derived lipid mediator (6-keto-PGF1α) (Fig. 16), and TXB2 (Fig. 17) were significantly increased in DNFB-stimulated urine. PGK2 (Fig. 13C and Fig. 14E are the same panel) is metabolized from both PGD2 and PGE2.

[0071] Example 5: Gene expression analysis of lipid metabolic enzymes and lipid synthesis enzymes in skin lesions Gene expression analysis of lipid metabolic enzymes and lipid synthetic enzymes was carried out in the skin lesions of the AD model mice prepared in Example 1.

[0072] (1) Quantitative RT-PCR Total RNA was isolated from the skin using Trizol Reagent (Molecular Research) and reverse transcribed to cDNA using ReverTra Ace (Toyobo). Quantitative RT-PCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) and the AriaMx Real-Time PCR System (Agilent Technologies) under the conditions listed in Tables 3–5. Quantification was performed using the delta-delta Ct method.

[0073] [Table 4]

[0074] [Table 5]

[0075] [Table 6]

[0076] (2) Results The results are shown in Figure 20. PGD2 is produced by the activity of COX (COX-1, COX-2) and PGD synthase (H-PGDS, L-PGDS), PGE2 by PGE synthase (mPGES-1, mPGES-2, and cPGES), PGF2α by PGF synthase (PGFS), and TXA2 by thromboxane synthase (TXS). Mouse aldo-keto reductase (AKR) 1B3 correlates with PGFS activity. 24 hours after the third stimulation, DNFB-treated skin showed significant increases in mRNA for Cox-1 (Figure 20A), Cox-2 (Figure 20B), mPGES-1 (Figure 20C), AKR1B3 (Figure 20D), Txs (Figure 20E), and H-PGDS (Figure 20F). On the other hand, the expression levels of mPges-2 (Fig. 20G), cPges (Fig. 20H), and L-pgds (Fig. 20I) mRNA were not affected by DNFB treatment.

[0077] Example 6: Immunohistochemical analysis of skin lesions The skin lesions collected from the AD model mice prepared in Example 1 were subjected to immunohistochemical analysis.

[0078] (1) Immunostaining Dissected tissues were fixed in 4% paraformaldehyde and embedded in paraffin or OCT compound (Sakura Finetech Japan). 4-μm-thick sections were incubated in 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature. For staining of COX-1, COX-2, and mPGES-1, sections were immersed in 50 mM Tris buffer containing 0.1% trypsin and 0.1% calcium chloride for 15 minutes at 37°C. For staining of AKR1B3 and TBXAS1, sections were incubated in antigen retrieval buffer (10 mM Tris, 1 mM EDTA, pH 9.0) for 10 minutes at 95°C. After incubation in PBS containing 0.1% Triton X-100 and 5% normal goat serum for 30 min at room temperature, the sections were incubated overnight at 4°C with goat anti-mPGES-1 antibody (Santa Cruz Biotechnology), rabbit anti-TBXAS1 antibody (Abcam), rabbit anti-AKR1B3 antibody (Osaka Bioscience Institute), rabbit anti-COX-1 antibody (Cayman Chemical), and rabbit anti-COX-2 antibody (Cayman Chemical) at a ratio of 1:200. mPGES-1 was incubated with biotinylated horse anti-goat antibody (VECTOR), and COX-1, COX-2, AKR1B3, and TBXAS1 were incubated with goat anti-rabbit antibody (VECTOR) at a ratio of 1:500 for 2 h at room temperature. After incubation with avidin-biotin complex (VECTASTAIN) for 30 min at room temperature, the sections were stained by incubation in 50 mM Tris buffer containing 200 μg / ml DAB and 0.03% hydrogen peroxide. Images were captured using a BZ-X710 microscope (Keyence).

[0079] (2) Results The results are shown in Figure 21. Immunostaining revealed staining for COX-2, mPGES-1, AKR1B3, and TXS in the epidermal layer (Figure 21). COX-2 and mPGES-1 were also observed in some infiltrating cells. COX-1 stained weakly. No positive staining was observed in normal serum or control (solvent) skin (Figure 21). These results suggest that PGE2 and PGF2α-derived lipid mediators are the major lipid mediators in the urine of AD model mice.

[0080] Example 7: Analysis of urinary lipid mediators in patients with atopic dermatitis Lipid mediators in urine samples collected from a group of atopic dermatitis (AD) patients (13 patients) and a control group (4 patients) were analyzed.

[0081] (1) Method A Urine samples Urine samples collected from 17 allergic patients who regularly visited the allergy department of the National Center for Child Health and Development were used. Among the allergic patients, 13 patients clinically diagnosed with atopic dermatitis were classified as the AD patient group, and 4 patients without clinical symptoms (such as eczema) were classified as the control group. The clinical characteristics of the subjects are as shown in Table 6. The collected urine was stored at -80 °C until analysis.

[0082]

Table 7

[0083] All subjects gave informed consent, and the research protocol was approved by the ethics committees of the University of Tokyo and the National Center for Child Health and Development. All experiments were conducted in accordance with the approved guidelines.

[0084] B Adjustment of urine Performed in the same manner as described in Example 4(1) B.

[0085] C Measurement of lipid mediators Performed in the same manner as described in Example 4(1) C.

[0086] D Data processing Performed in the same manner as described in Example 4(1) D.

[0087] (2) Results <n-6 series fatty acids> Lipid mediators downstream of COX As shown in Figures 22-25, the production of seven AA-derived lipid mediators downstream of COX was significantly higher in the AD patient group compared with the control group. Specifically, PGD2-derived metabolites were 13,14-dihydro-15-keto-PGJ2 (Figure 22A) and tetranor-PGDM (Figure 22B). PGE2-derived metabolites were 20-hydroxy-PGE2 (Figure 23A), 15-keto-PGE2 (Figure 23B), 13,14-dihydro-15-keto-tetranor-PGE2 (Figure 23C), and tetranor-PGEM (Figure 23D). PGF2α-derived metabolites were 15-keto-PGF2α (Figure 24A), 13,14-dihydro-15-keto-tetranor-PGF1α (Figure 24B), and tetranor-PGFM (Figure 24C). The substance metabolized from PGI2 was 6,15-diketo-13,14-dihydro-PGF1α (FIG. 25). The metabolic pathways of lipid mediators downstream of COX (derived from AA) are shown in Table 8.

[0088] [Table 8]

[0089] B. Lipid mediators downstream of LOX As shown in Figure 26, the production of 5-hydroperoxyeicosatetraenoic acid (HpETE) (Figure 26A), an AA-derived lipid mediator downstream of LOX, was significantly higher in the AD patient group than in the control group, whereas the production of its metabolite, 5-hydroxyeicosatetraenoic acid (HETE) (Figure 26B), was significantly lower.

[0090] C. CYP downstream lipid mediators As shown in Fig. 27, in the AD patient group, the production amount of 17-HETE, a lipid mediator downstream of AA-derived cytochrome p450 (CYP), was significantly higher compared to the control group.

[0091] AA and lipid mediators downstream of AA-derived OX As shown in Fig. 26, in the AD patient group, the production amounts of AA (Fig. 28A) and iPF2α-IV (Fig. 28B), which is a lipid mediator downstream of AA-derived enzyme-independent oxidation (OX), were significantly lower compared to the control group.

[0092] <n-3 fatty acids> EPA and EPA-derived lipid mediators As shown in Fig. 29, in the AD patient group, the production amount of EPA (Fig. 29) was significantly lower compared to the control group.

[0093] Lipid mediators derived from DHA As shown in Fig. 30, in the AD patient group, the production amounts of 4-hydroxy docosahexaenoic acid (hydroxy Docosahexaenoic Acid, sometimes referred to as "HDoHE" in this specification) (Fig. 30A), which is a lipid mediator downstream of DHA-derived LOX, and 10,17-dihydroxy docosahexaenoic acid (dihydroxy docosahexaenoic acid, sometimes referred to as "DiHDoHE" in this specification) (Fig. 30B) were significantly lower compared to the control group.

[0094] Example 8: Study using non-allergic dermatitis model mice In Example 8, in order to verify the specificity of lipid metabolism in AD model mice, a study was conducted using non-allergic dermatitis model mice that had non-allergic dermatitis induced by tape stripping.

[0095] (1) Method a Tape stripping BALB / C mice (male, 7-8 weeks old) (CLEA Japan) were tape-stripped 20 times to induce non-allergic dermatitis (see FIG. 31A for the experimental timeline).

[0096] B. HE staining The procedure was carried out in the same manner as described in Example 3(1).

[0097] C. Measurement of eosinophils, neutrophils, and mast cells The procedure was carried out in the same manner as described in Example 3(2).

[0098] D. Urine collection and preparation The procedure was the same as described in Example 4(1) a and b, except that urine was collected on the day of tape stripping (day 0), day 1 after treatment, and day 9 after treatment.

[0099] Measurement of lipid mediators The procedure was carried out in the same manner as described in Example 4(1) (c).

[0100] F. Data Processing The procedure was carried out in the same manner as described in Example 4(1)E.

[0101] G Quantitative RT-PCR The procedure was carried out in the same manner as described in Example 5(1).

[0102] Q Immunostaining The procedure was carried out in the same manner as described in Example 6(1).

[0103] (2) Results The results are shown in Figures 31 and 32. Changes in acanthosis measured from HE staining images (Figure 31B) confirmed increased acanthosis 9 days after tape stripping (Figure 31C). Quantitative RT-PCR confirmed that the expression levels of Tslp mRNA tended to increase, whereas those of interleukin-4 (Il-4), interleukin-13 (Il-13), and Ccl17, cytokines primarily produced by Th2 cells, did not (Figure 31D). It has been reported that 6 hours after tape stripping, activation of IL-1 and protease-activated receptor 2 (PAR-2) leads to NF-κB activation and elevated TSLP levels in the skin (Redhu D, et al., Br J Dermatol 2020;182:119-29). Therefore, tape-stripping may induce Th1-type inflammation mediated primarily by IL-1 (Sanmiguel JC, et al., Cell Signal 2009;21:685-94). Neutrophils and mast cells, but not eosinophils, infiltrated the dermis 9 days after tape stripping (Figure 32A).

[0104] Furthermore, analysis of urine on days 1 and 9 after tape stripping revealed that urinary PGE2 metabolites, 13,14-dihydro-15-keto-tetranor-PGF1β and PGF2α, increased on day 1 after tape stripping (Figure 31E, left, center), whereas PGF2α and PGE2 metabolites did not increase on day 9 after tape stripping (Figure 32B). In contrast, the level of PGF3α, a metabolite of EPA, significantly decreased on day 9 after tape stripping (Figure 31E, right). Tape stripping did not alter the mRNA expression of Cox-2, Akr1b3, or H-pgds, except for mPges-1 (Figure 31F). Tape stripping slightly increased AKR1B3 protein expression (Figure 31G), but the magnitude was much smaller than that observed in DNFB-stimulated skin.

[0105] These results indicate that, unlike non-allergic dermatitis model mice, hypertrophied keratinocytes associated with allergic (Th2-type) inflammation in AD model mice produce PGD2, PGE2, PGF2α, and PGI2, and that these metabolites are excreted in the urine of AD model mice.

Claims

1. A method for detecting atopic dermatitis, comprising the step of measuring the concentration of a lipid metabolite in a biological sample from a subject, wherein the lipid metabolite is a COX metabolite of arachidonic acid, and the COX metabolite is one or more selected from the group consisting of 13,14-dihydro-15-keto-tetranor-PGF1α, 13,14-dihydro-15-keto-tetranor-PGE2, and 13,14-dihydro-15-keto-PGJ2.

2. The detection method of claim 1, further comprising a step of comparing the concentration of the lipid metabolite in the biological sample of the subject with the concentration of the lipid metabolite in a biological sample of a healthy subject.

3. The detection method described in claim 1 or 2, wherein the subject is indicated to be suffering from atopic dermatitis if the concentration of the lipid metabolite in the subject's biological sample is significantly different from the concentration of the lipid metabolite in the biological sample of a healthy subject.

4. The detection method according to any one of claims 1 to 3, wherein the biological sample is a body fluid.

5. A method for detecting a therapeutic effect on atopic dermatitis, comprising a step of measuring the concentration of a lipid metabolite in a biological sample from a subject, wherein the lipid metabolite is a COX metabolite of arachidonic acid, and the COX metabolite is one or more selected from the group consisting of 13,14-dihydro-15-keto-tetranor-PGF1α, 13,14-dihydro-15-keto-tetranor-PGE2, and 13,14-dihydro-15-keto-PGJ2.

6. The detection method according to claim 5 , further comprising a step of comparing the concentration of the lipid metabolite in the biological sample of the subject with the concentration of the lipid metabolite in a biological sample of a healthy subject.

7. The detection method described in claim 5 or 6, wherein a therapeutic effect is indicated when the concentration of the lipid metabolite in the subject's biological sample is significantly different from the concentration of the lipid metabolite in the subject's biological sample before treatment or the concentration of the lipid metabolite in the biological sample of a subject suffering from atopic dermatitis, or when the concentration of the lipid metabolite is not significantly different from the concentration of the lipid metabolite in the biological sample of a healthy subject.

8. The detection method according to any one of claims 5 to 7, wherein the treatment for atopic dermatitis is a drug therapy or a proactive therapy.

9. The detection method according to any one of claims 5 to 8, wherein the biological sample is a body fluid.

10. The detection method according to any one of claims 2 to 4 or any one of claims 6 to 9, wherein the concentration of the lipid metabolite in a biological sample from a subject suffering from atopic dermatitis tends to be higher than the concentration of the lipid metabolite in a biological sample from a healthy subject.

11. The detection method according to any one of claims 1 to 4 and 10 or the detection method according to any one of claims 5 to 10, wherein the concentration of the lipid metabolite is measured by mass spectrometry.

12. A kit for detecting atopic dermatitis, comprising a means for quantifying lipid metabolites in a biological sample from a subject, wherein the lipid metabolites are COX metabolites of arachidonic acid, and the COX metabolites are one or more selected from the group consisting of 13,14-dihydro-15-keto-tetranor-PGF1α, 13,14-dihydro-15-keto-tetranor-PGE2, and 13,14-dihydro-15-keto-PGJ2.

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