Atopic dermatitis detection method

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 benefits from the priority of 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 effect of a treatment for atopic dermatitis. [Background technology]

[0003] Atopic dermatitis (AD) is an inflammatory skin disease characterized by pruritic eczema that alternates between exacerbation and remission. AD is caused by the interaction of genetic and environmental factors, but the number of AD patients has been increasing in recent years, with the prevalence rates estimated to be 9.8-13.2% in children and 2.5-9.4% in adults, respectively. It has also been reported that the onset of AD in infancy increases 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 it is known that serum TARC levels are 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 efficacy 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 lipid metabolite concentrations 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 a concentration of a lipid metabolite in a biological sample from a subject. [2] The detection or diagnosis 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 a biological sample of a healthy subject. [3] The detection or diagnostic method described in [1] or [2] above, which indicates that the subject is suffering from atopic dermatitis when the concentration of the lipid metabolite in the subject's biological sample is significantly different from the concentration of the lipid metabolite in a 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 determining the effectiveness of a treatment for 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 lipid metabolite concentration described in [5] above, further comprising a step of comparing the lipid metabolite concentration in the biological sample of the subject with the lipid metabolite concentration in the biological sample of a healthy subject. [7] The method of determining whether or not a therapeutic effect is present, as described in [5] or [6] above, wherein the concentration of a lipid metabolite in a biological sample from the subject is significantly different from the concentration of the lipid metabolite in a biological sample from the subject before treatment or from the concentration of the lipid metabolite in a biological sample from a subject suffering from atopic dermatitis, or is not significantly different from the concentration of the lipid metabolite in a biological sample from a healthy subject. [8] The method for determining whether or not atopic dermatitis has occurred according to any one of the above [5] to [7], wherein the treatment for atopic dermatitis is drug therapy or 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 or diagnosis 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 or diagnosis 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 lipid metabolite tends to have a higher concentration in a biological sample from a subject suffering from atopic dermatitis than in a biological sample from a healthy subject.

[12] The lipid metabolite is 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-PG The detection method or diagnostic method described in

[11] above, or the determination method described in

[11] above, wherein the PGF 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 or diagnosis 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 lipid metabolite tends to have a lower concentration in a biological sample from a subject suffering from atopic dermatitis than in a biological sample from a healthy subject.

[14] The detection method or diagnosis 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 described in any one of [1] to [4] and

[10] to

[14] above, or the determination method described in 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 are 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 a therapeutic or alleviating agent 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 from a subject after administration of a candidate therapeutic or alleviating agent with the concentration of the lipid metabolites in a biological sample from a healthy subject.

[21] The 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 subject's biological sample after administration of the candidate therapeutic agent or alleviator is significantly different from the concentration of the lipid metabolite in the subject's biological sample before administration of the candidate therapeutic agent or alleviator or the concentration of the lipid metabolite in a biological sample of a subject suffering from atopic dermatitis, or is not significantly different from the concentration of the lipid metabolite in a biological sample of 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, in which the lipid metabolite is shown to be an atopic dermatitis marker when the concentration of the 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.

[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 the steps of: (A) measuring a 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 atopic dermatitis or a therapeutic effect against atopic dermatitis can be quantitatively detected based on a biological sample from a subject, and also advantageous in that the biological sample from the subject can be non-invasively collected. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows a timeline of the procedure for producing an atopic dermatitis model mouse. [Diagram 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. [Diagram 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. [Diagram 5] Figure 5 shows the thickness of the ears 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 staining images of lesional skin sections from the back of AD model mice before DNFB treatment, and after the first and third stimulations. The scale bar indicates 50 μm. [Figure 8] Figure 8 shows the epidermal thickening of AD model mice measured from HE staining images. Data are shown as mean ± SEM (n = 5-7). **P < 0.01 indicates significant difference compared to before DNFB treatment, †P < 0.05 indicates 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 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 13] 13A to 13C show the amount of lipid mediators derived from AA and mediated by PGD2 downstream of COX excreted in the urine of AD model mice. *P<0.05, **P<0.01 indicate significant differences compared to before DNFB treatment. [Figure 14] Figures 14A-E show the amount of lipid mediators derived from AA and mediated by PGE2 downstream of COX excreted in the urine of AD model mice. *P<0.05, **P<0.01 indicate significant differences compared to before DNFB treatment, and ††P<0.01 indicate significant differences compared to the first stimulation. [Figure 15] Figure 15 shows the amount of lipid mediators excreted in the urine of AD model mice via AA-derived 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 lipid mediators derived from AA and mediated by PGI2 downstream of COX excreted in the urine of AD model mice. *P<0.05 indicates a significant difference compared to before DNFB treatment, †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 B show the amount of lipid mediators produced by enzyme-independent oxidation derived from AA excreted in the urine of AD model mice. *P<0.05 indicates a significant difference compared to before DNFB treatment, †P<0.05 indicates a significant difference compared to the first stimulation. [Figure 19] Figures 19A and B show the production of lipid mediators downstream of LOX derived from n-3 fatty acids excreted in the urine of AD model mice. *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 20] Figure 20A-D shows 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 (solvent 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 ± SEM as a ratio to the internal standard. *P<0.05 indicates a significant difference compared to the control (n=3-4 each). [Figure 23] Figures 23A-D show the amount of lipid mediators derived from AA and mediated by PGE2 downstream of COX excreted in urine from AD patients (n=10-14). Data are shown as ratios to the internal standard and are expressed as mean ± standard error. *P<0.05 indicates a significant difference compared to the control (n=3-4 each). [Figure 24] Figures 24A-C show the amount of lipid mediators derived from AA and mediated by PGF2α downstream of COX excreted in urine from AD patients (n=10-14). Data are shown as ratios to the internal standard and are expressed as mean ± standard error. *P<0.05 indicates a significant difference compared to controls (n=3-4). [Diagram 25]Figure 25 shows the amount of lipid mediators derived from AA and mediated by PGI2 downstream of COX 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 the control (n = 3-4). [Figure 26] Figures 26A and B show the amount of AA-derived lipid mediators downstream of LOX excreted in urine from AD patients (n=10-14). Data are shown as mean ± SEM 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-downstream lipid mediators 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 the control (n=3-4). [Figure 28] Figure 28 shows the amount of AA and lipid mediators generated by enzyme-independent oxidation of AA 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, **P<0.01 indicate significant differences compared to controls (n=3-4). [Figure 29] Figure 29 shows the amount of EPA excreted in urine from AD patients (n=10-14). Data are expressed as mean ± SEM as a ratio to the internal standard. *P<0.05 indicates a significant difference compared to the control (n=3-4 each). [Diagram 30] Figures 30A and B show the amount of DHA-derived lipid mediators downstream of LOX excreted in urine from AD patients (n=10-14). Data are shown as mean ± SEM as a ratio to the internal standard. *P<0.05 indicates a significant difference compared to the control (n=3-4 each). [Diagram 31]FIG. 31A shows a time series of experiments using mice (non-allergic dermatitis model mice) that were subjected to tape stripping treatment. FIG. 31B shows a typical example of HE staining images of dorsal skin fragments of non-allergic dermatitis model mice. The scale bar indicates 50 μm. FIG. 31C shows epidermal thickening of non-allergic dermatitis model mice measured from HE staining images. Data are shown as mean ± standard error (n = 4-7). FIG. 31D shows the mRNA expression levels of Tslp, Il-4, Il-13 and Ccl17 extracted from the skin of non-allergic dermatitis model mice. Data are shown as mean ± standard error (n = 4-7). FIG. 31E shows the amount of AA-derived, COX-downstream lipid mediators excreted in the urine of non-allergic dermatitis model mice. Data are the ratio to the internal standard and shown as mean ± standard error (n = 4-7). *P<0.05 indicates a significant difference compared to the day when tape stripping was performed (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 an immunostained image 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. [Diagram 32] Figure 32A shows the number of eosinophils (left), neutrophils (middle), and mast cells (right) quantified from MGG staining 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 the control (n = 4-7). The amount of lipid mediators mediated by PGF2α (left) and lipid mediators mediated by PGE2 (middle, right) excreted in the urine of non-allergic dermatitis model mice is shown. Description of the Invention

[0010] In the present invention, the term "lipid metabolite" refers to a lipid decomposition product generated by decomposition by enzyme-dependent or enzyme-independent oxidation (sometimes referred to as "OX" in the present specification) in a living body, and includes lipid mediators having a physiological effect of controlling inflammatory reactions. Enzyme-dependent oxidation proceeds by lipid metabolic enzymes present in the living body. Examples of such enzymes include lipid metabolic enzymes related to the onset and progression of atopic dermatitis (preferably lipid metabolic enzymes activated by the onset and progression of atopic dermatitis), such as cyclooxygenase (sometimes referred to as "COX" in the present specification), lipoxygenase (sometimes referred to as "LOX" in the present specification) (e.g., 5-LOX, 15-LOX), and cytochrome p450 (sometimes referred to as "CYP" in the present specification). 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 concentration in the biological sample of a subject suffering from atopic dermatitis tends to be higher (or higher) than that in the biological sample of a healthy subject (lipid metabolite X) and those whose concentration in the biological sample of a subject suffering from atopic dermatitis tends to be lower (or lower) than that in the biological sample of a healthy subject (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) (in the present specification, "tetranor-PGEM" 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) (in the present specification, "tetranor-PGFM" 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, PGK2, 13,14-dihydro-15-keto-tetranor-PGF1β, 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), and preferably those characterized in that their concentrations in biological samples from subjects suffering from atopic dermatitis are significantly higher than their concentrations 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 in that the concentration in a biological sample from a subject suffering from atopic dermatitis is significantly lower than the concentration in a biological sample from a healthy subject.

[0012] In the present invention, "atopic dermatitis" refers to a disease whose main lesion is an itchy eczema that repeatedly worsens and improves, and many patients have an atopic predisposition (allergy constitution). It is an eczematous disease that shows a characteristic symmetrical distribution, and the predilection site varies depending on age. It begins in infancy or early childhood and goes into remission during childhood, or recurs repeatedly without remission, and symptoms persist into adulthood, resulting in characteristic chronic eczema lesions (Atopic Dermatitis Guidelines 2018).

[0013] In the present invention, the term "biological sample" refers to a sample separated from a living body, such as a body fluid such as urine, blood, saliva, nasal mucus, sweat, tears, feces, etc. The method of collecting the 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 (eg, 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 makes it possible to detect atopic dermatitis using lipid metabolites in a biological sample as an indicator.

[0016] In the detection method of the present invention, first, a step (A) of measuring the concentration of lipid metabolites in a biological sample of a subject is carried out. The concentration of lipid metabolites can be measured by a known method, for example, 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). Immunoassay is an analytical method using a detectably labeled anti-lipid metabolite antibody or a detectably labeled antibody (secondary antibody) against the anti-lipid metabolite antibody. Depending on the antibody labeling method, they are classified into enzyme immunoassay (EIA or ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), fluorescence polarization immunoassay (FPIA), chemiluminescence immunoassay (CLIA), etc., and any of these can be used in the present invention. From the viewpoint of accurately measuring the concentration of lipid metabolites having similar structures, measurement by mass spectrometry (especially LC-MSMS and HPLC-MSMS) is preferred.

[0017] In the detection method of the present invention, a step of determining the presence or absence of atopic dermatitis for the subject from whom the biological sample was collected can be further carried out based on the concentration of the lipid metabolite measured in the step (A). This step may include a step (B) 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 the healthy subject. Here, if the concentration of the lipid metabolite in the biological sample of the subject is significantly different from the concentration of the lipid metabolite in the biological sample of the healthy subject, it is indicated that the subject is suffering from atopic dermatitis. That is, the detection method of the present invention may further include a step (C) of determining that the subject is suffering from atopic dermatitis if the concentration of the lipid metabolite in the biological sample of the subject is significantly different from the concentration of the lipid metabolite in the biological sample of the healthy subject. The term "significantly different" in step (C) 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 subject's biological sample is higher than the concentration of the lipid metabolite in the biological sample of a healthy subject, preferably significantly higher (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 a 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 ... 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 when the lipid metabolite is lipid metabolite Y, the concentration of the lipid metabolite in the biological sample of the subject is lower than the concentration of the lipid metabolite in the biological sample of a healthy subject, preferably significantly lower (for example, the concentration of the lipid metabolite in the biological sample of the subject 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 concentration of the lipid metabolite in the biological sample of a healthy subject can be an average value calculated by measuring the lipid metabolite concentration by collecting biological samples from multiple healthy subjects in advance. In addition, "suffering from atopic dermatitis" is used to mean a case where there is a possibility of being afflicted.

[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 for the diagnosis of atopic dermatitis, and the judgment of whether a subject has atopic dermatitis can be finally made by a doctor or a veterinarian, possibly 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 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. According to the detection method of the present invention, since detection can be performed based on a biological sample collected by a non-invasive method such as urine collection, it is also advantageous in that it can be applied to subjects from which atopic dermatitis is difficult to detect by an invasive method such as blood collection, for example, subjects from which it is difficult to collect blood, such as children (including infants).

[0020] According to another aspect of the present invention, a method for diagnosing atopic dermatitis is also provided. According to the method for diagnosis of the present invention, it is possible to diagnose whether or not a subject suffers from atopic dermatitis by using lipid metabolites in a biological sample as an indicator. In the method for diagnosis of the present invention, a step (A') of measuring the concentration of lipid metabolites in a biological sample of a subject is carried out, as in the detection method of the present invention. In the method for diagnosis of the present invention, a step (B') of comparing the concentration of lipid metabolites in the biological sample of the subject with the concentration of the lipid metabolites in the biological sample of a healthy subject may be further included, and a step (C') of determining that the subject suffers from atopic dermatitis when the concentration of lipid metabolites in the biological sample of the subject is significantly different from the concentration of the lipid metabolites in the biological sample of the healthy subject may be further included. The steps (A'), (B') and (C') correspond to the 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, the step (D) of measuring the concentration of lipid metabolites in a biological sample of a subject is carried out in the same manner as in the detection method of the present invention. 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] In the method of the present invention, a step of determining the therapeutic effect against atopic dermatitis for the treated subject can be further carried out based on the concentration of the lipid metabolite measured in the step (D). This step may include a step (E) of comparing the concentration of the lipid metabolite in the biological sample of the subject treated for atopic dermatitis with the concentration of the lipid metabolite in the biological sample of the healthy subject. Here, when the concentration of the lipid metabolite in the biological sample of the subject treated for atopic dermatitis is significantly different from the concentration of the lipid metabolite in the biological sample of the subject before treatment or the concentration of the lipid metabolite in the biological sample of the subject suffering from atopic dermatitis, or when the concentration of the lipid metabolite in the biological sample of the healthy subject is not significantly different from the concentration of the lipid metabolite in the biological sample of the subject suffering from atopic dermatitis, it is shown that there is a therapeutic effect. That is, the determination method of the present invention may further include a step of (F) 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 is about 0.3-fold or less), 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 treatment 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 treatment 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). In addition, "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 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 concentration of the lipid metabolite in the biological sample of the subject before treatment can be a value obtained by measuring the concentration of the lipid metabolite in the biological sample of the subject before treatment. The concentration of the lipid metabolite in the biological sample of a subject suffering from atopic dermatitis can be an average value calculated by measuring the lipid metabolite concentration by collecting biological samples from multiple subjects suffering from atopic dermatitis in advance.In addition, 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 taken from multiple healthy subjects in advance. The subject to be treated in the determination method of the present invention is preferably a subject suffering from atopic dermatitis. In addition, in the present invention, the "subject suffering from atopic dermatitis" may be any subject whose results of other test methods indicate atopic dermatitis, and is preferably a subject diagnosed by a doctor or veterinarian as having atopic dermatitis.

[0024] The treatment for atopic dermatitis, the effect of which can be evaluated by the evaluation method of the present invention, can be, for example, drug therapy or proactive therapy. Drug therapy can be, for example, treatment with a drug for treating atopic dermatitis, and examples of such a drug include anti-inflammatory drugs for external use (steroid external use, tacrolimus, nonsteroidal anti-inflammatory drugs), antihistamines, cyclosporine, steroid internal use, herbal medicine, antibody medicine, and other medicines.

[0025] According to the method of the present invention, the therapeutic effect can be evaluated in a subject who has received a treatment for atopic dermatitis, and therefore the effectiveness of the treatment for atopic dermatitis performed on the subject can be verified. If the therapeutic effect is not observed, the treatment can be immediately stopped and another treatment plan can be made. Therefore, the method of the present invention is advantageous in that it can suppress unnecessary medication, which in turn contributes to reducing medical expenses and reducing the burden on patients. According to the method of the present invention, since detection can be performed based on a biological sample collected by a non-invasive method such as urine collection, it is also advantageous in that it can be applied to subjects from which it is difficult to evaluate the therapeutic effect by an invasive method such as blood collection, for example, children (including infants) and animals (for example, pets such as dogs and cats) from which it is difficult to collect blood.

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

[0027] Since the determination 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 a therapeutic agent or alleviator for atopic dermatitis. That is, according to a fourth aspect of the present invention, a method for screening a therapeutic agent or alleviator for atopic dermatitis is provided, comprising: (G) administering a candidate therapeutic agent or alleviator for atopic dermatitis to a subject; and (H) measuring the concentration of a lipid metabolite in a biological sample from the subject. In the screening method of the present invention, a step of determining whether or not the candidate therapeutic agent or alleviator has a therapeutic effect based on the concentration of the lipid metabolite measured in the step (H) can be further carried out. This step may include a step of (I) comparing the concentration of the lipid metabolite in the biological sample from the subject 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, when the concentration of the lipid metabolite in the subject's biological sample after administration of the candidate therapeutic agent or alleviator is significantly different from the concentration of the lipid metabolite in the subject's biological sample before administration of the candidate therapeutic agent or alleviator or the concentration of the lipid metabolite in the biological sample of the subject suffering from atopic dermatitis, or when it is not significantly different from the concentration of the lipid metabolite in the biological sample of the healthy subject, it is shown that the candidate therapeutic agent or alleviator has a therapeutic effect. That is, the screening method of the present invention may further include a step of determining that the therapeutic effect is present when the concentration of the lipid metabolite in the subject's biological sample after administration of the candidate therapeutic agent or alleviator is significantly different from the concentration of the lipid metabolite in the subject's biological sample before administration of the candidate therapeutic agent or alleviator or the concentration of the lipid metabolite in the biological sample of the subject suffering from atopic dermatitis (preferably, when it is significantly different in the direction approaching the concentration of the lipid metabolite in the biological sample of the healthy subject), or when it is not significantly different from the concentration of the lipid metabolite in the biological sample of the healthy subject, thereby making it possible to select a candidate having a therapeutic effect.The term "significantly different" in step (J) means that the concentration of the lipid metabolite is either higher or lower than the concentration of the lipid metabolite in the subject before administration of the candidate therapeutic or alleviating agent or in the subject suffering from atopic dermatitis, 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 administration of the candidate therapeutic or alleviating agent or in 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 administration of the candidate therapeutic or alleviating agent or in 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, ...8 times or less, about 0.8 times or less, about 0.8 times or less, about 0.8 times or less, about 0.8 times 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 a candidate therapeutic or alleviating agent or of a 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 a candidate therapeutic or alleviating agent or of a 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). In addition, "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 agent or alleviating agent is administered is preferably a subject suffering from atopic dermatitis. When carrying out the screening method of the present invention, a mammal other than a human can be used as the subject. The therapeutic agent for atopic dermatitis that is the subject of the screening method of the present invention is the same as that described in the determination method of the present invention. In addition, the alleviating agent for atopic dermatitis that is the subject of the screening method of the present invention includes food (e.g., food composition), quasi-drug (e.g., medicated cosmetic), feed (e.g., pet food), cosmetics (e.g., cosmetic composition), and skin care composition that have the function of alleviating the symptoms of atopic dermatitis, and the food includes supplements, foods for specified health uses, and foods with functional claims. In addition, the term "alleviation" is used in a sense that includes improvement.

[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: (K) measuring the concentration of a lipid metabolite in a biological sample of a subject suffering from atopic dermatitis and the concentration of a lipid metabolite in a biological sample of a healthy subject; and (L) comparing the two measured concentrations. In the identification method of the present invention, a step of determining the lipid metabolite as an atopic dermatitis marker based on the result of the comparison of the concentrations performed in the step (L) can be further carried out. In this step, when the concentration of the lipid metabolite in the biological sample of the subject suffering from atopic dermatitis is significantly different from the concentration of the lipid metabolite in the biological sample of the healthy subject, the lipid metabolite is shown to be an atopic dermatitis marker. That is, the identification method of the present invention may further comprise a step of (M) determining that the lipid metabolite is an atopic dermatitis marker when the concentration of the lipid metabolite in the biological sample of the subject suffering from atopic dermatitis is significantly different from the concentration of the lipid metabolite in the biological sample of 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 subject's biological sample is higher than the concentration of the lipid metabolite in the biological sample of a healthy subject, preferably significantly higher (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 a 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 ... 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 which atopic dermatitis is detected by the detection method of the present invention or diagnosed with atopic dermatitis by the diagnostic method of the present invention can be treated for atopic dermatitis. Thus, according to the sixth aspect of the present invention, a method for treating atopic dermatitis is provided, comprising: (A) measuring the concentration of a lipid metabolite in a biological sample of the subject; (B) 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; (C) determining that the subject suffers from atopic dermatitis when the concentration of the lipid metabolite in the biological sample of the subject is significantly different from the concentration of the lipid metabolite in the biological sample of the healthy subject; and (N) administering treatment for atopic dermatitis to the subject determined to suffer from atopic dermatitis. The detection and determination steps of atopic dermatitis (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. Treatment for atopic dermatitis can also be carried out according to the description of the determination method of the present invention.

[0030] According to the seventh aspect of the present invention, a kit for detecting atopic dermatitis is provided, 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 contains the reagents (and, in some cases, equipment) necessary for measuring 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 a lipid metabolite 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 a lipid metabolite 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 above kit can be used, for example, as follows. First, a capture antibody is fixed to a microtiter plate, a target biological sample is appropriately diluted and added thereto, then incubated, the sample is removed, and washed. Next, a primary antibody is added, incubated and washed, and 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 kit of the present invention, the labeled antibody is not limited to an enzyme-labeled antibody, but may 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. Also, a biotinylated antibody can be used as the labeled antibody, and labeled avidin or streptavidin can be added to the kit.

[0036] Further examples of kits of the present invention include kits for measuring the concentration of lipid metabolites by immunochromatography, which can be configured to include an antibody storage section in which a first anti-lipid metabolite antibody labeled with gold colloid or the like is stored, 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 shape on a cellulose membrane or the like, connected by a thin groove.

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

[0038] In the kit of the present invention, when the quantitative measurement means for the 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 the internal standard, the extraction efficiency and ionization efficiency for each analysis can be corrected during measurement by the mass spectrometer. The internal standard used for 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. EXAMPLES

[0040] The present invention will be described in more detail with reference to 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 as shown in Figure 1. BALB / C mice (male, 7-8 weeks old) (Japan CLEA) were anesthetized and the hair on the back of the mouse was removed using clippers and hair removal cream, and the mice were acclimated for 3 days. Then, 25 μL of 0.5% DNFB (2,4-dinitrofluorobenzene, Nacalai Tesque) mixed solution (acetone (Fujifilm Wako Pure Chemical): olive (Fujifilm Wako Pure Chemical), 4:1) was dropped onto the upper back of the mouse for sensitization (day 1). Four days later, 20 μL of 0.2% DNFB mixed solution (acetone: olive, 4:1) was dropped onto the right ear of the mouse and 100 μL onto the lower back of the mouse for the first stimulation. This stimulation procedure was repeated three times at three-day intervals (first time: day 5, second time: day 8, third time: day 11) to create 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 over time in the skin pathology of the AD model mice are shown in Figure 2. It was confirmed that the inflammation in the back of the AD model mice worsened from the first stimulation to the third stimulation.

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

[0046] (4) Thickness of ears The results are shown in Figure 5. The ear thickness of the AD model mice was measured using an ABS solar-type digital caliper (Mitutoyo), and the values ​​in the graph were calculated from the difference in ear thickness 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 the 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. Furthermore, one-way analysis of variance (ANOVA) and Tukey's test in combination, or Kruskal-Wallis test and Steel-Dwass test in combination, for multigroup comparisons. Statistically significant differences were * P < 0.05, ** P was defined as <0.01.

[0049] Example 3: Histopathological evaluation of atopic dermatitis The skin lesions of the AD model mice prepared in Example 1 were subjected to histopathological evaluation. (1) Procedure The 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-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 used for staining after being deparaffinized. A 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 counterstained with hematoxylin solution. 1mm 2 The numbers of mast cells and neutrophils in the blood were counted, and the average values ​​and standard deviations 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 cells 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 images of HE staining were as shown in Figure 7. Furthermore, the changes in acanthosis measured from the images of HE staining were as shown in Figure 8 (data are mean ± standard error (n = 5-7)).

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

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

[0056] From the results of Examples 2 and 3, it was confirmed that the atopic dermatitis model mouse in Example 1 exhibited symptoms of atopic dermatitis.

[0057] Example 4: Analysis of urinary lipid mediators in AD model mice The lipid mediators in the urine 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 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 water 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, and then the lipid mediator attached to the cartridge was eluted with 100 μL of 100% methanol.

[0060] Measurement of lipid mediator The eluate prepared in 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 Corporation) Measurement program; LC / MS / MS method package lipid mediator ver. 2 (Shimadzu Corporation) 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 their 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 values calculated from the chromatograms of each lipid mediator were divided by the peak area values of the corresponding internal standard substances above to correct for quantitative errors and the like that occurred during lipid extraction. The concentration of the lipid mediator (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 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 series fatty acids> Lipid mediators downstream of COX As shown in Figures 13 to 17, it was confirmed that 10 lipid mediators derived from arachidonic acid (hereinafter sometimes referred to as "AA"), an n-6 fatty acid, downstream of cyclooxygenase (COX) were significantly increased. Specifically, those metabolized from prostaglandin D2 (hereinafter sometimes referred to as "PGD2" or "PGD2") were 11β-13,14-dihydro-15-keto-PGF2α (Figure 13A), 13,14-dihydro-15-keto-PGJ2 (Figure 13B), and prostaglandin K2 (hereinafter sometimes referred to as "PGK2" or "PGK2") (Figure 13C). The metabolites of prostaglandin E2 (sometimes referred to as "PGE2" or "PGE2" in this specification) were 15-keto-PGE2 (Figure 14A), 13,14-dihydro-15-keto-tetranor-PGF1β (Figure 14B), 13,14-dihydro-15-keto-PGE2 (Figure 14C), 13,14-dihydro-15-keto-tetranor-PGE2 (Figure 14D), and PGK2 (Figure 14E (same as Figure 13C)). Note that PGK2 is metabolized from either PGD2 or PGE2. PGJ2 is prostaglandin J2 (sometimes referred to as "PGJ2" in this specification), and PGF1β is prostaglandin F1β (sometimes referred to as "PGF 1β Prostaglandin F2α (PGF 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 known as 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 acid used to produce lipid mediators in the urine of AD model mice is 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 13A-C), five PGE2-derived lipid mediators (15-keto-PGE2, 13,14-dihydro-15-keto-tetranor-PGF1β, 13,14-dihydro-15-keto-tetranor-PGF1β, and ... keto-PGE2, 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 urine stimulated with DNFB. PGK2 (Fig. 13C and Fig. 14E are the same figures) is metabolized from both PGD2 and PGE2.

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

[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 described in Tables 3 to 5. Quantification was performed by 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 is produced by the activity of PGE synthase (mPGES-1, mPGES-2 and cPGES), PGF2α is produced by the activity of PGF synthase (PGFS), and TXA2 is produced by the activity of thromboxane synthase (TXS). Mouse aldo-keto reductase (AKR) 1B3 is correlated with PGFS activity. The mRNA of Cox-1 (Figure 20A), Cox-2 (Figure 20B), mPges-1 (Figure 20C), Akr1B3 (Figure 20D), Txs (Figure 20E), and H-pgds (Figure 20F) was significantly increased in the skin treated with DNFB 24 hours after the third stimulation. 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). Sections 4 μm thick were incubated in methanol containing 0.3% hydrogen peroxide for 30 min at room temperature. Staining for COX-1, COX-2, and mPGES-1 was performed by immersing sections in 50 mM Tris buffer containing 0.1% trypsin and 0.1% calcium chloride for 15 min at 37°C. Staining for AKR1B3 and TBXAS1 was performed by incubating sections in antigen retrieval buffer (10 mM Tris, 1 mM EDTA, pH 9.0) at 95°C for 10 min. After incubation in PBS containing 0.1% TritonX-100 and 5% normal goat serum for 30 minutes at room temperature, the sections were immersed in 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 overnight at 4°C. 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 hours at room temperature. After incubation with avidin-biotin complex (VECTASTAIN) for 30 min at room temperature, 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. In immunostaining, staining of COX-2, mPGES-1, AKR1B3, and TXS was observed in the epidermal layer (Figure 21). COX-2 and mPGES-1 were also observed in some infiltrating cells. COX-1 was weakly stained. 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 13 patients with atopic dermatitis (AD) and a control group of 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 It was performed in the same manner as described in Example 4(1)B.

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

[0086] D Data processing It was 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 to 25, the production of seven lipid mediators derived from AA and downstream of COX was significantly higher in the AD patient group than in the control group. Specifically, the metabolites derived from PGD2 were 13,14-dihydro-15-keto-PGJ2 (Figure 22A) and tetranor-PGDM (Figure 22B). The metabolites derived from PGE2 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). The metabolites derived from PGF2α were 15-keto-PGF2α (Figure 24A), 13,14-dihydro-15-keto-tetranor-PGF1α (Figure 24B), and tetranor-PGFM (Figure 24C). The product metabolized from PGI2 was 6,15-diketo-13,14-dihydro-PGF1α (FIG. 25). The metabolic pathway of lipid mediators downstream of COX (derived from AA) is shown in Table 8.

[0088] [Table 8]

[0089] Lipid mediators downstream of LOX As shown in Figure 26, the production amount of 5-hydroperoxyeicosatetraenoic acid (hereinafter sometimes referred to as "HpETE"), an AA-derived lipid mediator downstream of LOX (Figure 26A), was significantly higher in the AD patient group than in the control group, whereas the production amount of its metabolite, 5-hydroxyeicosatetraenoic acid (hereinafter sometimes referred to as "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 and 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 are lipid mediators downstream of AA-derived and 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) and 10,17-dihydroxy docosahexaenoic acid (dihydroxy docosahexaenoic acid, sometimes referred to as "DiHDoHE" in this specification) (Fig. 30B), which are lipid mediators downstream of DHA and LOX, 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 been induced with non-allergic dermatitis 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), 1 day after treatment, and 9 days 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. The changes in acanthosis measured from HE staining images (Figure 31B) confirmed an increase in acanthosis 9 days after tape stripping (Figure 31C). Quantitative RT-PCR confirmed that the expression levels of Tslp mRNA tended to increase, whereas interleukin 4 (Il-4), interleukin 13 (Il-13) and Ccl17, cytokines mainly 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) activates NF-κB and increases TSLP levels in the skin (Redhu D, et al., Br J Dermatol 2020; 182:119-29.), suggesting that tape stripping induces Th1-type inflammation mediated mainly by IL-1 in the skin (Sanmiguel JC, et al., Cell Signal 2009; 21:685-94.). Neutrophils and mast cells infiltrated the dermis 9 days after tape stripping, but eosinophils did not (Figure 32A).

[0104] In addition, analysis of urine on days 1 and 9 after tape stripping showed that urinary PGE2 metabolites 13,14-dihydro-15-keto-tetranor-PGF1β and PGF2α increased on day 1 after tape stripping (Fig. 31E, left, center), whereas PGF2α and PGE2 metabolites did not increase on day 9 after tape stripping (Fig. 32B). In contrast, the level of PGF3α, a metabolite of EPA, was significantly decreased on day 9 after tape stripping (Fig. 31E, right). It was also confirmed that tape stripping did not alter the mRNA expression of Cox-2, Akr1b3, and H-pgds, except for mPges-1 (Fig. 31F). Tape stripping slightly increased the expression level of AKR1B3 protein (Fig. 31G), but the degree of increase was much smaller than that 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 a step of measuring the concentration of lipid metabolites in a biological sample from a subject.

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

3. The detection method described in claim 1 or 2, which indicates that the subject is suffering from atopic dermatitis when the concentration of a lipid metabolite in the subject's biological sample is significantly different from the concentration of the lipid metabolite in a 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 determining the effectiveness of a treatment for atopic dermatitis, comprising a step of measuring the concentration of lipid metabolites in a biological sample from a subject.

6. The method of claim 5, further comprising a step of comparing the concentration of the lipid metabolite in the subject's biological sample with the concentration of the lipid metabolite in a biological sample from a healthy subject.

7. The method of 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 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 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 1 to 4 or the determination method according to any one of claims 5 to 9, 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 according to any one of claims 1 to 4 and 10, or the determination method according to any one of claims 5 to 10, wherein the lipid metabolite tends to have a higher concentration in a biological sample from a subject suffering from atopic dermatitis than in a biological sample from a healthy subject.

12. The lipid metabolites are 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 The detection method according to claim 11 or the determination method according to claim 11, wherein the protein is one or more selected from the group consisting of 13,14-dihydro-15-keto-tetranor-PGF1β, 13,14-dihydro-15-keto-PGE2, 6-keto-PGF1α and TXB2.

13. The detection method according to any one of claims 1 to 4 and 10, or the determination method according to any one of claims 5 to 10, wherein the lipid metabolite tends to have a lower concentration in a biological sample from a subject suffering from atopic dermatitis than in a biological sample from a healthy subject.

14. The detection method according to claim 13 or the determination method according to claim 13, 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 according to any one of claims 1 to 4 and 10 to 14, or the determination method according to any one of claims 5 to 14, 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 according to claim 16, 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 are 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-1 The atopic dermatitis marker according to claim 16 or 17, which is one or more selected from the group consisting of 3,14-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 a therapeutic or alleviating agent 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 of claim 19, further comprising a step of comparing the concentration of the lipid metabolite in the subject's biological sample after administration of a candidate therapeutic or alleviating agent with the concentration of the lipid metabolite in a biological sample from a healthy subject.

21. The screening method of claim 19 or 20, wherein the candidate therapeutic or alleviating agent is indicated to have a therapeutic effect if the concentration of a lipid metabolite in a subject's biological sample after administration of the candidate therapeutic or alleviating agent is significantly different from the concentration of the lipid metabolite in the subject's biological sample before administration of the candidate therapeutic or alleviating agent or the concentration of the lipid metabolite in a biological sample of a subject suffering from atopic dermatitis, or is not significantly different from the concentration of the lipid metabolite in a biological sample of a healthy subject.

22. A method for identifying an atopic dermatitis marker in a lipid metabolite in a biological sample, the method 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 claim 22, wherein the lipid metabolite is indicated as an atopic dermatitis marker when the concentration of the lipid metabolite in a biological sample of a subject suffering from atopic dermatitis is significantly different from the concentration of the lipid metabolite in a biological sample of a healthy subject.

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

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

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