Biomarker for determining severity and / or activity of atopic dermatitis

Abstract

To provide a biomarker for determining the severity and / or activity of atopic dermatitis.SOLUTION: Disclosed is a biomarker comprising at least one selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, CystatinA, FGF-18, and Ubiquitin + 1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a biomarker for determining the severity and / or activity of atopic dermatitis. [Background technology]

[0002] Atopic dermatitis (hereinafter sometimes referred to as "AD") is a disease characterized by itchy eczema that repeatedly worsens and improves, and mediators produced by epidermal cells and lymphocytes are involved in the pathogenesis. In recent years, new injectable or oral medications have been used for patients with moderate to severe AD, and the treatment of AD has made great progress.

[0003] Existing biomarkers for assessing AD activity (or progression), such as serum TARC and SCCA2 levels, are covered by health insurance. However, these require blood sampling, which is invasive. Furthermore, serum TARC levels tend to be physiologically elevated in children, making them difficult to use as indicators in the same way as in adults. Furthermore, serum SCCA2 levels are affected by new therapeutic drugs. Furthermore, serum SCCA2 levels are limited to those aged 15 years or younger.

[0004] A known minimally invasive or non-invasive method for detecting AD involves detecting at least one protein selected from a group of over 400 proteins in lipids on the skin surface collected from a subject (Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2021-175958 Summary of the Invention [Problem to be solved by the invention]

[0006] Patent Document 1 lists over 400 diverse proteins as markers for detecting AD, but does not disclose any relationship between each of these proteins and the severity and / or activity of AD.

[0007] Objects of the present invention include providing biomarkers for determining the severity and / or activity of AD. [Means for solving the problem]

[0008] As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that at least one selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1 is useful as a biomarker for determining the severity and / or activity of AD. The present invention was completed as a result of further intensive studies based on this finding.

[0009] The present invention includes aspects described in the following paragraphs. [Section 1] A biomarker for determining the severity and / or activity of atopic dermatitis, comprising at least one selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1. [Section 2] The biomarker according to item 1, comprising at least S100A6. [Section 3] A method for determining the severity of atopic dermatitis, comprising: The method comprises the step of detecting at least one biomarker selected from the group consisting of S100A6, S100A8, lipocalin-2, fibrinogen, calcitonin, VWF, cardiotrophin-1, cystatin A, FGF-18, and ubiquitin+1 in stratum corneum collected from a subject; When the biomarker detected in the step is S100A6, S100A8, lipocalin-2, or fibrinogen, the amount or concentration of the biomarker is equal to or greater than a cutoff value, which indicates high severity of atopic dermatitis; A method in which, when the biomarker detected in the step is calcitonin, VWF, cardiotrophon-1, cystatin A, FGF-18, or ubiquitin+1, the amount or concentration of the biomarker being equal to or lower than a cutoff value indicates a high severity of atopic dermatitis. [Section 4] Item 4. The method according to Item 3, wherein the cutoff value is determined based on the amount or concentration of the biomarker in stratum corneum collected from a healthy subject. [Section 5] Item 4. The method according to Item 3, wherein the stratum corneum is rash-like stratum corneum, and the cutoff value is determined based on the amount or concentration of the biomarker in rash-free stratum corneum collected from the subject. [Section 6] A method for determining the activity of atopic dermatitis, comprising: Step 1: detecting at least one biomarker selected from the group consisting of S100A6, S100A8, lipocalin-2, fibrinogen, calcitonin, VWF, cardiotrophin-1, cystatin A, FGF-18, and ubiquitin+1 in stratum corneum collected from the subject at a first time point; and a step 2 of detecting at least one biomarker selected from the group consisting of S100A6, S100A8, lipocalin-2, fibrinogen, calcitonin, VWF, cardiotrophin-1, cystatin A, FGF-18, and ubiquitin+1 in stratum corneum collected from the subject at a second time point that is later than the first time point; When the biomarker detected in steps 1 and 2 is S100A6, S100A8, lipocalin-2, or fibrinogen, a higher amount or concentration of the biomarker at the second time point than at the first time point indicates a high level of atopic dermatitis activity; A method in which, when the biomarker detected in steps 1 and 2 is calcitonin, VWF, cardiotrophon-1, cystatin A, FGF-18, or ubiquitin+1, a lower amount or concentration of the biomarker at the second time point than at the first time point indicates high activity of atopic dermatitis. [Section 7] Item 7. The method according to Item 6, wherein the first time point is day 0 and the second time point is day 28 to 56. [Section 8] Item 8. The method according to any one of Items 3 to 7, wherein the biomarker comprises at least S100A6. [Section 9] A test agent for atopic dermatitis, comprising a detection agent for at least one biomarker selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1. [Section 10] A test kit for atopic dermatitis, comprising a detection agent for at least one biomarker selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1. [Section 11] A method for screening for an active ingredient in a preventive or therapeutic agent for atopic dermatitis, using as an indicator the amount or concentration of at least one biomarker selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1 in stratum corneum collected from an animal treated with a test substance. [Section 12] A method for evaluating the induction or exacerbation of atopic dermatitis, using as an indicator the amount or concentration of at least one biomarker selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1 in stratum corneum collected from an animal treated with a test substance. [Effects of the Invention]

[0010] The present invention provides a biomarker for determining the severity and / or activity of atopic dermatitis, which comprises at least one selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1. [Brief explanation of the drawings]

[0011] [Figure 1] The figures show the results of ELISA quantification of the amounts of lipocalin-2, S100A6, and S100A8 in keratin from healthy adults, keratin from non-rash areas of AD patients, and keratin from rash areas of AD patients. [Figure 2] The results of a correlation analysis between the expression level of S100A6 protein in the keratin of rash areas of AD patients and clinical indicators of severity (EASI, rash severity, and itch score) are shown. [Figure 3]Changes in S100A6 protein levels in the keratinocytes of rash areas, clinical indices (EASI, rash severity), and hematological test results (serum TARC levels, serum IgE levels, blood eosinophil counts) in AD patients from visit 1 to visit 2 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] In this specification, the term "comprise" is used to encompass the terms "consist only of" and "consist essentially of."

[0013] In one embodiment, the biomarker for determining the severity and / or activity of AD preferably includes at least one selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1.

[0014] The severity of AD is determined by indicators such as the Eczema Area and Severity Index (EASI), Severity Scoring of Atopic Dermatitis (SCORADO), Investigator's Global Assessment (IGA), Patient-Oriented Eczema Measure (POEM), rash severity, pruritus Visual Analogue Scale (VAS), pruritus Numerical Rating Scale (NRS), pruritus Verbal Rating Scale (VRS), Peak Pruritus-Numerical Rating Scale (PP-NRS), serum TARC level, serum IgE level, blood eosinophil count, serum LDH level, serum SCCA2 level, or a combination of two or more of these.

[0015] The EASI generally divides the whole body into four regions: head and neck, trunk, upper limbs, and lower limbs. For each region, the severity of the rash (evaluated on a four-point scale (0: absent, 1: mild, 2: moderate, 3: severe) for each of the items erythema, infiltration / papules, excoriation, and lichenification) is multiplied by the area of ​​the rash (evaluated on a six-point scale (0: 0%, 1: 1-9%, 2: 10-29%, 3: 30-49%, 4: 50-69%, 5: 70-89%, 6: 90-100%)) and a region-specific coefficient (head and neck: 0.1 (0.2 for children under 7 years old), trunk: 0.3, upper limbs: 0.2, lower limbs: 0.4 (0.3 for children under 7 years old)) to calculate a score, and the scores for the four regions are then summed for evaluation.

[0016] SCORADO is generally based on the area of ​​the rash, the severity of the rash (erythema, edema / papules, exudate / crusts, excoriation, lichenification, and dry skin, each rated on a four-point scale (0: absent, 1: mild, 2: moderate, 3: severe)), and, if necessary, subjective symptoms (average degree of itching and insomnia over the past three days rated on an 11-point scale (0: absent to 10: severe)).

[0017] IGA generally evaluates the findings of eczema and dermatitis on a five-point scale (0: absent (no clinical findings or symptoms), 1: slight (very slight findings or symptoms), 2: mild (slight findings or symptoms), 3: moderate (clearly observable findings or symptoms), 4: severe (severe findings or symptoms)).

[0018] The POEM is generally assessed based on the patient's (or their guardian's) responses to seven questions about eczema over the past week: 1. How many days did your skin itch because of eczema? 2. How many days did your sleep get disrupted because of eczema? 3. How many days did your skin bleed because of eczema? 4. How many days did your skin ooze (ooze a clear liquid) because of eczema? 5. How many days did your skin crack because of eczema? 6. How many days did your skin peel off in flakes because of eczema? 7. How many days did your skin feel dry or rough because of eczema?

[0019] The severity of the rash is generally assessed on a four-point scale (0: none, 1: mild, 2: moderate, 3: severe) for each of the following: erythema, edema / papule, exudate / crust, excoriation, lichenification, and dry skin at the site where the keratin was collected, and the total score is then used to assess the rash.

[0020] The itch VAS generally involves marking the level of itch on a 100mm line, with the left end representing "0: no itch" and the right end representing "100: worst itch imaginable," and the distance (mm) from the left end to the mark represents the intensity of the itch.

[0021] The Pruritus NRS generally assesses the average severity of itching over the past 24 hours on an 11-point scale ranging from "0: no itching" to "10: worst itching imaginable."

[0022] The pruritus VRS generally lists five levels of itching intensity (none, mild, moderate, severe, very severe), and assesses the patient based on which level of itching intensity they most closely experience.

[0023] The PP-NRS generally assesses the severity of the worst itch in the past 24 hours on an 11-point scale (0: no itch at all to 10: the worst itch imaginable).

[0024] Serum TARC levels tend to increase with increasing severity of AD. For example, in adults, serum TARC levels can be classified as mild (450 pg / mL to less than 700 pg / mL) and moderate or severe (700 pg / mL or higher), while in children (especially those aged 2 years and older), levels can be classified as mild (744 pg / mL to less than 760 pg / mL) and moderate or severe (760 pg / mL or higher).

[0025] Serum IgE levels tend to increase with increasing severity of AD. Serum IgE levels can be classified as mild or severe when they are, for example, 250 IU / mL or higher, 300 IU / mL or higher, 350 IU / mL or higher, 400 IU / mL or higher, 450 IU / mL or higher, or 500 IU / mL or higher.

[0026] The blood eosinophil count tends to increase as the severity of AD increases. For example, a blood eosinophil count of 150 cells / μL or more can be classified as mild or severe.

[0027] Serum LDH levels tend to increase with increasing severity of AD. For example, serum LDH levels of 250 IU / mL or higher in adults and 270 IU / mL or higher, 300 IU / mL or higher, or 400 IU / mL or higher in children can be classified as mild or severe.

[0028] Serum SCCA2 levels tend to increase with increasing severity of AD. For example, a serum SCCA2 level of 1.6 ng / mL or higher can be classified as mild or severe.

[0029] Since the expression levels of the biomarkers (particularly the expression levels in the stratum corneum) correlate with the above-mentioned indices (particularly one, two, three, four, five, or six selected from EASI, rash severity, PP-NRS, serum TARC level, serum IgE level, and blood eosinophil count), the biomarkers can be suitably used as substitutes for the above-mentioned indices to determine the severity of AD. Furthermore, the biomarkers can also be used to estimate the severity determined by the above-mentioned indices based on their amounts or concentrations.

[0030] The activity of AD can be determined, for example, based on the change over time of the above-mentioned indicators. The change over time refers, for example, to the change between a first time point and a second time point later than the first time point. When the first time point is defined as day 0, the second time point may be, for example, day 7 or later, day 14 or later, day 21 or later, or day 28 or later, or may be day 84 or earlier, day 77 or earlier, day 70 or earlier, day 63 or earlier, or day 56 or earlier. In a preferred embodiment, the second time point is day 28 or later but earlier than day 56. If the rate of change (absolute value) is, for example, 1% or more, 2% or more, 3% or more, 4% or more, or 5% or more, the activity of AD can be considered high.

[0031] Since the rate of change in the above-mentioned indices (particularly one, two, three, four, five, or six selected from EASI, rash severity, PP-NRS, serum TARC level, serum IgE level, and blood eosinophil count) correlates with the rate of change in the expression level of the above-mentioned biomarkers (particularly the expression level in the stratum corneum), the above-mentioned biomarkers can be suitably used as substitutes for the above-mentioned indices to determine AD activity. Furthermore, the above-mentioned biomarkers can also be used to estimate activity determined by the change in the above-mentioned indices over time, based on the change in their amount or concentration over time.

[0032] The biomarker preferably includes at least one selected from the group consisting of S100A6, S100A8, and Lipocapin-2, more preferably includes at least one selected from the group consisting of S100A6 and S100A8, and even more preferably includes S100A6.

[0033] In one aspect, the present invention provides a method for assessing the severity of AD ("assessment" here may not include diagnosis by a physician), which includes a step of detecting at least one biomarker selected from the group consisting of S100A6, S100A8, lipocalin-2, fibrinogen, calcitonin, VWF, cardiotrophin-1, cystatin A, FGF-18, and ubiquitin+1 in stratum corneum collected from a subject (hereinafter, this may be simply referred to as a "detection step").

[0034] The subject is not particularly limited as long as it is a subject requiring assessment of the severity of AD. Examples of subjects include mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits. In a preferred embodiment, the subject is a human. In the case of a human, the subject may be an adult or a minor, or, due to the minimally invasive or non-invasive nature of the method, an infant or child. The subject may be, for example, a subject diagnosed with AD, or a subject suspected of having AD.

[0035] The keratin collected from the subject may be keratin from a non-rash area or from a rash area, but is preferably keratin from a rash area.

[0036] When the biomarker detected in the detection step is S100A6, S100A8, lipocalin-2, or fibrinogen, if the amount or concentration of the biomarker is equal to or greater than the cutoff value, the severity of AD can be determined to be high.

[0037] When the biomarker detected in the detection step is calcitonin, VWF, cardiotrophon-1, cystatin A, FGF-18, or ubiquitin+1, if the amount or concentration of the biomarker is below the cutoff value, the severity of AD can be determined to be high.

[0038] The cutoff value can be appropriately set by a person skilled in the art from the viewpoints of measurement conditions, measurement sensitivity, measurement error, etc., and can be a value determined on a case-by-case basis or a predetermined value based on, for example, the amount or concentration (C1) of the target biomarker in keratin collected from a healthy subject (or a control sample not suffering from atopic dermatitis), the amount or concentration (C2) of the target biomarker in keratin from a rash-free area collected from a subject, etc.

[0039] When the biomarker is S100A6, S100A8, lipocalin-2, or fibrinogen, the cutoff value can be, for example, a value 1.05 times or more (e.g., 1.1 to 1.5 times) C1 (or the average value, median value, etc. if there are multiple values) and / or a value 1.05 times or more (e.g., 1.1 to 1.5 times) C2 (or the average value, median value, etc. if there are multiple values).

[0040] When the biomarker is calcitonin, VWF, cardiotrophon-1, cystatin A, FGF-18, or ubiquitin+1, the cutoff value can be, for example, a value of 0.95 times or less (e.g., 0.5 to 0.9 times) C1 (or the average value, median value, etc. if there are multiple values) and / or a value of 0.95 times or less (e.g., 0.5 to 0.95 times) C2 (or the average value, median value, etc. if there are multiple values).

[0041] In one aspect, the present invention provides a method for assessing AD activity ("assessing" herein may not include diagnosis by a physician), the method comprising: Step 1: detecting at least one biomarker selected from the group consisting of S100A6, S100A8, lipocalin-2, fibrinogen, calcitonin, VWF, cardiotrophin-1, cystatin A, FGF-18, and ubiquitin+1 in stratum corneum collected from a subject at a first time point; and Step 2: detecting at least one biomarker selected from the group consisting of S100A6, S100A8, lipocalin-2, fibrinogen, calcitonin, VWF, cardiotrophin-1, cystatin A, FGF-18, and ubiquitin+1 in stratum corneum collected from the subject at a second time point subsequent to the first time point.

[0042] The subject and keratin in the method for assessing AD activity can be selected from the same subjects and keratin in the method for assessing AD severity, respectively. The first and second time points in the method for assessing AD activity can be set in the same manner as described in the section on biomarkers.

[0043] The subject may or may not have been treated with a therapeutic agent for AD between the first and second time points. Examples of therapeutic agents for AD include nonsteroidal topical drugs, steroidal topical drugs, oral medications, and injectable drugs. Examples of nonsteroidal topical drugs include calcineurin inhibitors (e.g., tacrolimus), JAK inhibitors (e.g., delgocitinib), and PDE4 inhibitors (e.g., difamilast). Examples of topical steroid medications include strong steroids (group I) such as clobetasol propionate (or Dermovate®) and diflorasone acetate (or Diacort®); very strong steroids (group I) such as mometasone furoate (or Flumeta®), betamethasone butyrate propionate (or Antebate®), fluocinonide (or Topsym®), betamethasone dipropionate (or Rinderon DP®), difluprednate (or Myser®), amcinonide (or Visderm®), hydrocortisone butyrate propionate (or Pandel®), and diflucortolone valerate (or Nerisona®). Group II drugs include strong (III) drugs such as deprodone propionate (or Eclar®), dexamethasone propionate (or Methaderm®), dexamethasone valerate (or Boara®), betamesone valerate (or Betnetobate®, Rinderon V®), and fluocinolone acetonide (or Flucort®); medium (IV) drugs such as prednisolone valerate (or Lidomex®), alclometasone propionate (or Almeta®), clobetasone butyrate (or Kindavate®), and hydrocortisone butyrate (or Locoid®); and weak (V) drugs such as prednisolone. Oral drugs include JAK inhibitors such as baricitinib, upadacitinib, and abrocitinib. Examples of injectable drugs include dupilumab, nemolizumab, etc. The therapeutic agent for AD may be a single agent or a combination of two or more agents.

[0044] If the biomarker detected in steps 1 and 2 is S100A6, S100A8, lipocalin-2, or fibrinogen, and the amount or concentration of the biomarker is higher at the second time point than at the first time point (e.g., 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, or 1.5-fold or more), the AD activity can be determined to be high.

[0045] If the biomarker detected in steps 1 and 2 is calcitonin, VWF, cardiotrophon-1, cystatin A, FGF-18, or ubiquitin+1, and the amount or concentration of the biomarker is lower at the second time point than at the first time point (e.g., 0.95 times or less, 0.9 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, or 0.5 times or less), the activity of AD can be determined to be high.

[0046] In one aspect, the present invention encompasses a diagnostic agent or test kit for AD, which comprises a detector for at least one biomarker selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1. The diagnostic agent or test kit for AD can be suitably used to determine the severity and / or activity of AD.

[0047] The detection agent is preferably at least one detection agent selected from the group consisting of S100A6, S100A8, and Lipocapin-2, more preferably at least one detection agent selected from the group consisting of S100A6 and S100A8, and even more preferably a detection agent for S100A6.

[0048] The detection agent is not particularly limited as long as it can specifically detect the target biomarker. Examples of the detection agent include antibodies and aptamers against the target biomarker. The detection agent may be modified as long as its function is not significantly impaired. Examples of modifications include the addition of fluorescent dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, biotin, etc.

[0049] The test agent may be in the form of a composition. The composition may contain other components as needed in addition to the detection agent. Examples of other components include carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, thickeners, colorants, chelating agents, and combinations of two or more of these.

[0050] The test kit is not particularly limited as long as it contains a detection agent, and the detection agent may be included as a test drug. The detection agent or test drug is usually sealed in a container. The test kit may further contain reagents other than the detection agent (dilution reagents, sample preparation reagents, control reagents, etc.), instruments, instructions, etc., required for detection.

[0051] One aspect of the present invention includes a method for screening for an active ingredient of an agent for preventing or treating AD, using as an indicator the amount or concentration of at least one biomarker selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1 in stratum corneum collected from an animal treated with a test substance.

[0052] The animals and keratin in the screening method can be selected from the same as the subjects and keratin in the method for determining the severity of AD.

[0053] A wide range of test substances can be used, regardless of whether they are naturally occurring or artificially produced. Furthermore, not only purified compounds but also compositions containing a variety of compounds and animal and plant extracts can be used. Compounds are not limited to low-molecular-weight compounds, but also include high-molecular-weight compounds such as proteins, nucleic acids, and polysaccharides.

[0054] When the biomarker used as an indicator is S100A6, S100A8, lipocalin-2, or fibrinogen, the screening method preferably includes a step of selecting the test substance as an active ingredient of an agent for preventing or treating AD (or a candidate substance for an active ingredient of an agent for preventing or treating AD) if the value of the indicator is lower (e.g., 0.9-fold or less, 0.8-fold or less, 0.7-fold or less, 0.6-fold or less, or 0.5-fold or less) than the amount or concentration (control value) of the corresponding biomarker in keratin collected from an animal not treated with the test substance.

[0055] When the biomarker used as an indicator is calcitonin, VWF, cardiotrophon-1, cystatin A, FGF-18, or ubiquitin+1, the screening method preferably includes a step of selecting the test substance as an active ingredient of an agent for preventing or treating AD (or a candidate substance for an active ingredient of an agent for preventing or treating AD) if the value of the indicator is higher (e.g., 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, or 1.5-fold or more) than the amount or concentration (control value) of the corresponding biomarker in keratin collected from an animal not treated with the test substance.

[0056] In one aspect, the present invention includes a method for evaluating the inducibility or aggravation of AD, using as an indicator the amount or concentration of at least one biomarker selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1 in stratum corneum collected from an animal treated with a test substance.

[0057] The animals, keratin, and test substances in the method for evaluating induction or aggravation of AD can be selected from the same animals, keratin, and test substances as those in the screening method.

[0058] When the biomarker used as an indicator is S100A6, S100A8, lipocalin-2, or fibrinogen, the method for assessing the inducibility or aggravation of AD preferably includes a step of determining that the test substance has the ability to induce or aggravate AD if the value of the indicator is higher (e.g., 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, or 1.5-fold or more) than the amount or concentration (control value) of the corresponding biomarker in keratin collected from an animal not treated with the test substance.

[0059] When the biomarker used as an indicator is calcitonin, VWF, cardiotrophon-1, cystatin A, FGF-18, or ubiquitin+1, the method for assessing the inducibility or aggravation of AD preferably includes a step of determining that the test substance has the ability to induce or aggravate AD if the value of the indicator is lower (e.g., 0.9-fold or less, 0.8-fold or less, 0.7-fold or less, 0.6-fold or less, or 0.5-fold or less) than the amount or concentration (control value) of the corresponding biomarker in keratin collected from an animal not treated with the test substance. [Example]

[0060] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0061] Example 1. Expression analysis of proteins characteristic of atopic dermatitis (AD) 1-1. Subjects and samples The subjects were (i) 10 male patients (A01-A10) with atopic dermatitis (AD) who were diagnosed by a dermatologist and met the diagnostic criteria of the Japanese Dermatological Association, aged between 20 and 80 years, and (ii) 5 healthy adult males (C01-C05) with no history of AD, bronchial asthma, or chronic skin disease, and no family history of AD. Subjects who had been taking immunosuppressants within the past 4 weeks were excluded.

[0062] Healthy adults underwent keratinocyte and blood samples and clinical evaluations on the initial evaluation day (Visit 1), while AD patients underwent keratinocyte and blood samples and clinical evaluations on the initial evaluation day and on the evaluation day 4 to 8 weeks later (Visit 2) (Table 1). Between Visits 1 and 2, 10 AD patients were using topical atopic dermatitis medication (tacrolimus ointment for Patient A06, and very strong steroid ointment for the other 9 patients). No significant changes were observed in general laboratory values ​​between Visits 1 and 2, and no adverse events were observed during the study period.

[0063] [Table 1]

[0064] 1-2. Sample collection Stratum corneum: After confirming that no topical agents had been applied to the rash and non-rash areas (back) since the previous night, adhesive tape (Nichiban Co., Ltd., 24 mm wide) approximately 13 cm long was applied, and a constant amount of pressure was applied and gently removed. The tape was then reapplied to the same area, and the tape was removed from the opposite side to ensure uniform collection of stratum corneum, a maximum of 22 times. If the research participant expressed pain or reluctance, collection was terminated. Tapes up to the second time were discarded, and tapes from the third through 22nd times were collected. Each tape was attached to the front and back of a glass slide, placed in a Ziploc bag, and stored at -30°C. The tape was kept on ice until storage at -30°C.

[0065] Plasma: 6 mL of collected blood was poured into a blood collection tube (Terumo Corporation VP-NA070, anticoagulant EDTA-2Na) and mixed by inverting several times. The tube was centrifuged at 3500 rpm (2300 g) for 10 minutes at room temperature. After centrifugation, 100 μL of the blood was dispensed into two 0.5 mL loop screw-cap tubes (WATSON Corporation, freestanding type), and the remaining blood was dispensed into 1.5 mL aliquots in 2 mL microtubes and immediately stored at -80°C.

[0066] Serum: 4 mL of collected blood was poured into a blood collection tube (Terumo Corporation VP-AS054K50) and mixed by inverting several times. After leaving it at room temperature for 30 minutes, it was centrifuged at 3500 rpm (2300 g) for 10 minutes at room temperature. After centrifugation, 1.5 mL aliquots were dispensed into 2 mL microtubes and immediately stored at -80°C.

[0067] 1-3. Protein extraction from keratin samples A 6 mL Petri dish was filled with 0.2% Tween 20 / PBS, and a tape sample, cut into approximately 5 mm cubes, was immersed in the solution. The soaked tape and the 0.2% Tween 20 / PBS solution on the Petri dish were transferred to a 15 mL centrifuge tube, sonicated on ice for 30 seconds, and then centrifuged (3000 rpm, 4°C, 15 minutes). Half of the sample supernatant was transferred to an Amicon Ultra-4 mL (Merck #UFC800324) and half to a Vivaspin Turbo 4 (Sartorius #VS04T91) and centrifuged (3000 rpm, 4°C, 30 minutes) to concentrate the protein solution to a concentration of approximately 1.0 mg / mL. The concentration of the protein solution was measured by the BCA method (Nacalai Tesque, Inc. Protein Assay BCA Kit #06385-00), and 0.5 mL (for microarray) and the remainder (for ELISA) were transferred to 1.0 mL microtubes and promptly stored at -80°C. The obtained protein samples were subjected to antibody array analysis and ELISA.

[0068] 1-4. Acquisition of AD clinical evaluation indicators (1) EASI (Entire Body Severity Index) Physicians assessed erythema, edema / papules, excoriation, and lichenification on a scale of 0 to 3 (0: none, 1: mild, 2: moderate, 3: severe) for each of the areas on the top of the head, trunk, upper limbs, and lower limbs, and calculated a total score for each area. The total score for each area was multiplied by the area score based on the eczema area (0: 0%, 1: 1-9%, 2: 10-29%, 3: 30-49%, 4: 50-69%, 5: 70-89%, 6: 90-100%) to determine the overall severity of the condition.

[0069] (2) Skin rash severity (local severity) The severity of the keratin-collected area was assessed by a physician on a scale of 0 to 3 (0: none, 1: mild, 2: moderate, 3: severe) for erythema, edema / papule, exudate / crust, excoriation, lichenification, and dry skin. The total score was used to indicate the severity of the area where the keratin was collected.

[0070] (3) PP-NRS (itchiness severity) Patients were asked to rate the severity of their worst itching in the past 24 hours on a scale of 0 to 10 (0 = no itching at all, 10 = the worst itching imaginable).

[0071] (4) Blood tests (indicators of AD disease progression) Serum TARC levels were measured using the AIA-Pack CL reagent and the AIA-CL2400 automated enzyme assay system (LA / Tosoh High-Tech Corporation). Serum IgE and LDH levels were measured using the LABOSPECT 008 α (Hitachi High-Tech Corporation). Peripheral blood eosinophil counts in serum were measured using the XN-3000 (Sysmex Corporation).

[0072] 1-5. Comprehensive antibody array analysis (1) Analysis method Seventy protein samples extracted from plasma and stratum corneum (rash and non-rash areas) of healthy adults and AD patients at visits 1 and 2 were analyzed for 1,000 biomarkers (RayBio™ Label-Based Human Antibody Array L-1000, RayBiotech, Inc.).

[0073] (2) Heat map analysis There were no biomarkers with zero variance across all samples. Biomarker values ​​were normalized (centered and scaled) by subtracting the mean and dividing by the standard deviation. The normalized data were plotted on a heatmap using Euclidean distance. After scaling and centering, 1,000 biomarkers were clustered into 10 groups using Euclidean distance. Protein expression was analyzed between healthy adults and AD patients (visit 1) and between visits 1 and 2 in AD patients. Analysis was performed using the R programming language V3.6.3 (R Core Team 2017).

[0074] (3) Expression analysis Each data point was normalized by the signal intensity of the positive control. Proteins whose average measured values ​​for each group were below the background (average signal intensity of the negative control) were excluded from the analysis.

[0075] [Table 2]

[0076] <step1> (A) The ratio of the expression of each protein in keratin samples from the rash at Visit 1 of AD patients (n = 8; Table 2) whose rash severity improved between Visit 1 and Visit 2 to that of keratin samples from healthy adults (HC; n = 5) (AD v1 / HC ratio) was calculated. (B) The ratio of the expression of each protein in keratin samples from the rash at Visit 2 of the above AD patients (n = 8) to that of keratin samples from the rash at Visit 1 (AD v2 / AD v1 ratio) was calculated. Proteins that showed a change of 50% or more (1.5-fold or more or 0.5-fold or less) in both (A) and (B) were identified as candidate biomarkers for AD diagnosis (Table 3).

[0077] [Table 3]

[0078] <step2> (A) The ratio of the expression of each protein in keratin samples from the rash at Visit 1 of AD patients (n = 4; Table 2) whose rash severity improved by 50% or more between Visit 1 and Visit 2 to that of keratin samples from healthy adults (HC; n = 5) (AD v1 / HC ratio) was calculated. (B) The ratio of the expression of each protein in keratin samples from the rash at Visit 2 of the above AD patients (n = 4) to that of keratin samples from the rash at Visit 1 (AD v2 / AD v1 ratio) was calculated. Proteins that showed a change of 50% or more (1.5-fold or more or 0.5-fold or less) in both (A) and (B) were identified as candidate biomarkers for AD diagnosis (Table 4).

[0079] [Table 4]

[0080] 1-6. Quantitative analysis using ELISA Based on the results of comprehensive antibody array analysis, keratin samples from rash and non-rash areas of AD patients (visit 1, n = 10) and keratin samples from healthy adults (n = 5) were analyzed. Proteins considered to be potential biomarkers were identified using ELISA kits (S100A6: S100A6 / Calcyclin ELISA Kit, HumanLifeSpan Biosciences, LS-F50028-1; S100A8: Human S100A8 DuoSet ELISA, 5 Plates, R&D Systems, DY4570-05; Lipocalin-2: Human Lipocalin-2 / NGAL DuoSet Kit ELISA, 5 Plates, R&D Systems, DY1757-05) to measure the protein levels in the keratin. Significantly increased expression of S100A6, S100A8, and lipocalin-2 was confirmed in the keratin of rash-affected AD patients compared with that of healthy adults and that of unaffected AD patients (Figure 1).

[0081] Example 2. Search for biomarkers of AD severity Of the biomarker candidates obtained in Example 1, for S100A6, we calculated the Spearman rank correlation coefficient between clinical indices of severity (EASI, rash severity, itch score) and protein amount in keratin of the rash (quantitative results by ELISA) of AD patients (n=10) at Visit 1. The correlation coefficient of S100A6 protein amount in keratin of the rash with each clinical indices was 0.3 or higher, indicating a tendency for correlation (Figure 2).

[0082] Furthermore, the amount of S100A6 in the stratum corneum of rash-affected AD patients (n = 10) was measured by ELISA at two time points, Visit 1 and Visit 2, and compared with the changes in the severity index (EASI, rash severity) and hematological test results (serum TARC level, serum IgE level, blood eosinophil count). EASI score and rash severity improved from Visit 1 to Visit 2, and the amount of S100A6 in the stratum corneum also decreased over time, along with the improvement in hematological test values ​​(Figure 3).

Claims

1. A biomarker for determining the severity and / or activity of atopic dermatitis, comprising at least one selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1.

2. The biomarker of claim 1, comprising at least S100A6.

3. A method for determining the severity of atopic dermatitis, comprising: The method comprises the step of detecting at least one biomarker selected from the group consisting of S100A6, S100A8, lipocalin-2, fibrinogen, calcitonin, VWF, cardiotrophin-1, cystatin A, FGF-18, and ubiquitin+1 in stratum corneum collected from a subject; When the biomarker detected in the step is S100A6, S100A8, lipocalin-2, or fibrinogen, the amount or concentration of the biomarker is equal to or greater than a cutoff value, which indicates high severity of atopic dermatitis; A method in which, when the biomarker detected in the step is calcitonin, VWF, cardiotrophon-1, cystatin A, FGF-18, or ubiquitin+1, the amount or concentration of the biomarker being equal to or lower than a cutoff value indicates a high severity of atopic dermatitis.

4. The method of claim 3, wherein the cutoff value is determined based on the amount or concentration of the biomarker in stratum corneum collected from a healthy subject.

5. The method of claim 3, wherein the stratum corneum is rash-like stratum corneum, and the cutoff value is determined based on the amount or concentration of the biomarker in non-rash-like stratum corneum collected from the subject.

6. A method for determining the activity of atopic dermatitis, comprising: Step 1: detecting at least one biomarker selected from the group consisting of S100A6, S100A8, lipocalin-2, fibrinogen, calcitonin, VWF, cardiotrophin-1, cystatin A, FGF-18, and ubiquitin+1 in stratum corneum collected from the subject at a first time point; and a step 2 of detecting at least one biomarker selected from the group consisting of S100A6, S100A8, lipocalin-2, fibrinogen, calcitonin, VWF, cardiotrophin-1, cystatin A, FGF-18, and ubiquitin+1 in stratum corneum collected from the subject at a second time point that is later than the first time point; When the biomarker detected in steps 1 and 2 is S100A6, S100A8, lipocalin-2, or fibrinogen, a higher amount or concentration of the biomarker at the second time point than at the first time point indicates a high level of atopic dermatitis activity; A method in which, when the biomarker detected in steps 1 and 2 is calcitonin, VWF, cardiotrophon-1, cystatin A, FGF-18, or ubiquitin+1, a lower amount or concentration of the biomarker at the second time point than at the first time point indicates high activity of atopic dermatitis.

7. The method of claim 6, wherein the second time point is from day 28 to day 56 when the first time point is day 0.

8. The method of any one of claims 3 to 7, wherein the biomarker comprises at least S100A6.

9. A test agent for atopic dermatitis, comprising a detection agent for at least one biomarker selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1.

10. A test kit for atopic dermatitis, comprising a detection agent for at least one biomarker selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1.

11. A method for screening for an active ingredient in a preventive or therapeutic agent for atopic dermatitis, using as an indicator the amount or concentration of at least one biomarker selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1 in stratum corneum collected from an animal treated with a test substance.

12. A method for evaluating the induction or exacerbation of atopic dermatitis, using as an indicator the amount or concentration of at least one biomarker selected from the group consisting of S100A6, S100A8, Lipocapin-2, Fibrinogen, Calcitonin, VWF, Cardiotrophin-1, Cystatin A, FGF-18, and Ubiquitin+1 in stratum corneum collected from an animal treated with a test substance.

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