Detection method for itching

Abstract

To provide a marker for detecting itching and a detection method for itching using the detection marker.SOLUTION: A detection method for itching in a subject, comprising a step of measuring TLQP-21 levels in a biological sample taken from the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for detecting itch. [Background technology]

[0002] Itching is a pathological condition observed not only in skin diseases such as atopic dermatitis but also in internal diseases such as renal failure. Itching can also be caused by dry skin, sunburn, and friction of the skin against clothing. Scratching caused by itching physically invades the skin, further worsening symptoms, so resolving itching contributes to the prevention or amelioration of skin diseases. For example, it has been reported that in mice in which the nails of the hind legs were clipped to suppress physical damage to the skin caused by scratching, skin symptoms caused by atopic dermatitis were prevented or ameliorated (Non-Patent Document 1).

[0003] Itch sensations in peripheral tissues such as the skin are transmitted to the brain by afferent sensory nerves, which connect the periphery to the dorsal horn of the spinal cord. The cell bodies of afferent sensory nerves are located in the dorsal root ganglion (DRG), and nerve fibers extend from the cell bodies to peripheral tissues and the dorsal horn of the spinal cord. Afferent sensory nerves receive sensations in the skin and transmit them to second-order neurons in the dorsal horn of the spinal cord.

[0004] Pruritogens induce itching by binding to corresponding receptors. Pruritogens include histamine, serotonin, and chloroquine, while Th2 cytokines such as IL-4 and IL-13 have been reported as itch-enhancing substances (sensitizers). Histamine is a representative chemical prurigen. Recently, it has been suggested that histamine is essentially associated with only a few acute cases of itching, with little involvement in many chronic itching disorders (Non-Patent Document 2). Therefore, antihistamines (H1 receptor antagonists) are currently widely used to suppress itching. However, only a limited number of cases of itching can be resolved with antihistamines, and many itch conditions are intractable, with no effective solutions. Itching associated with atopic dermatitis, xerosis, renal failure, and other conditions is considered intractable. The mechanisms underlying many intractable itches remain unclear, necessitating the elucidation of these mechanisms and the development of novel target molecules.

[0005] Itching is a subjective sensation, so symptoms cannot be diagnosed at a glance. It is important for the individual to report it, but because reporting standards vary from person to person, there is a risk that the timing for early and appropriate measures and treatment may be missed. To date, there are no known clinical indicators for minimally invasive and objective evaluation of itch, and no minimally invasive method for detecting itch exists. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Hashimoto Y et al. Life Sciences. 2004 Dec 31;76(7):783-94 [Non-patent document 2] Ikoma A et al. Nature Reviews Neuroscience. 2006 Jul;7(7):535-47 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing a marker for detecting itching and a method for detecting itching using the detection marker. [Means for solving the problem]

[0008] The present inventors first isolated dorsal root ganglia (DRGs) from mouse models exhibiting xeroderma-like skin symptoms after treatment with an acetone-ether mixture and water (hereinafter referred to as AEW model mice) and performed comprehensive gene expression analysis. Vgf was identified as a gene whose expression was altered. Furthermore, quantitative expression analysis of the Vgf gene in the DRGs of AEW model mice and atopic dermatitis model mice (hereinafter referred to as AD model mice) revealed significantly increased Vgf expression in DRG tissues of both model mice compared with control mice (Example 1). Vgf encodes the neurosecretory factor VGF nerve growth factor inducible (hereinafter referred to as VGF). VGF is known to be degraded by proteases in vivo, producing several physiologically active peptides (Lewis JE et al. Front Endocrinol (Lausanne). 2015 Feb 2;6:3). We found that injection of TLQP-21, one of these proteins, into the skin of the back of the neck of healthy mice induced scratching behavior in the mice (Example 2), indicating that TLQP-21 induces itching. Further research revealed that the blood level of TLQP-21 in humans is significantly positively correlated with the severity of itching, and that TLQP-21 can be used as an indicator to detect itching.

[0009] That is, the present invention relates to the following 1) to 3). 1) A method for detecting itch in a subject, comprising a step of measuring the TLQP-21 level in a biological sample collected from the subject. 2) A test kit for detecting itch in a subject used in the method described in 1), which contains a reagent for measuring TLQP-21 levels. 3) A detection marker consisting of TLQP-21 for detecting itch in a subject. [Effects of the Invention]

[0010] According to the present invention, it is possible to detect itching simply and minimally invasively, which allows itching to be objectively understood without being dependent on individual subjectivity, thereby enabling appropriate measures to be taken against the itching. [Brief explanation of the drawings]

[0011] [Figure 1] The results of RNA-seq analysis of AEW model DRG tissue are shown. [Figure 2] 1 shows the results of real-time PCR analysis of Vgf expression levels in the AEW model. [Figure 3] 1 shows the results of real-time PCR analysis of Vgf expression levels in an AD model. [Figure 4] The number of itch scratches in C57BL / 6J mice after administration of TLQP-21 is shown. [Figure 5] The results of a correlation analysis between blood TLQP-21 and itch VAS are shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] The method for detecting itch of the present invention comprises the step of measuring the TLQP-21 level in a biological sample collected from a subject.

[0013] In the present invention, "itch" is a subjective sensation, the cause of which is not particularly limited. The site of itching may be a wide area such as the whole body, scalp, face, back, arms, back of the hands, fingers, or legs, or a specific site. The present invention is suitable for detecting intractable itching. "Intractable itch" refers to itch that cannot be relieved by antihistamines, including, for example, itch associated with atopic dermatitis, xerosis, psoriasis, prurigo, renal failure, liver disease, etc.

[0014] In the present invention, "detection" can also be expressed in other terms such as examination, measurement, judgment, or evaluation support. Note that the terms "detection," "examination," "measurement," "judgment," or "evaluation" in the present invention do not include diagnosis by a doctor.

[0015] In the present invention, the "subject" is not particularly limited to, but includes, for example, a human or non-human mammal who desires or requires detection of itch. For example, the subject may include a person with a pruritic skin disease. Pruritic skin diseases include urticaria, atopic dermatitis, contact dermatitis, seborrheic dermatitis, cutaneous pruritus, xerosis, psoriasis, and prurigo nodularis. Of these, skin diseases other than urticaria are skin diseases accompanied by intractable itch. The subject is preferably a human, and more preferably a human who has subjective symptoms of sleep disturbance caused by the itch of a pruritic skin disease.

[0016] In the present invention, the "biological sample" may be any cell, tissue, or biological material in which the TLQP-21 level can be detected. Specific examples include body fluids such as organs, skin, blood, urine, saliva, sweat, stratum corneum, skin surface lipids (SSL), and tissue exudates, as well as serum and plasma prepared from blood, feces, and hair. Blood, serum, and plasma prepared from blood are preferred.

[0017] In the present invention, "TLQP-21" is a physiologically active peptide generated by degradation of VGF in vivo. Specific sequences of TLQP-21 include the human ( Homo sapiens ) TLQPPSALRRRHYHHALPPSR (SEQ ID NO: 1), mouse ( Mus Musculus ) TLQPPASSRRRHFHHALPPAR (SEQ ID NO: 2), rat ( Rattus norvegicus ) TLQPPASSRRRHFHHALPPAR (SEQ ID NO: 3), cynomolgus monkey ( Macaca fascicularis ) TLQPPSALRRRHYHHALPPSR (SEQ ID NO: 4), common marmoset ( Callithrix jacchus) and can be represented by a sequence such as TLQPPSASRRRHYHHALPPSR (SEQ ID NO: 5). TLQP-21 is known to bind to the complement factor C3a receptor, a GPCR-type receptor present on the cell membrane, and initiate a signal (Cero C et al. Structure. 2014 Dec 2;22(12):1744-1753). As shown in the Examples below, injection of TLQP-21 into the skin of the back of the neck of healthy mice induced scratching behavior in the mice (Figure 4). Furthermore, a positive correlation was observed between the blood TLQP-21 level and the severity of itching, particularly a significant positive correlation was observed with the severity of itching at night (Figure 5). These results clearly demonstrate that TLQP-21 induces itching and can be used as a detection marker for detecting itching. Furthermore, it is possible to use TLQP-21 as a detection marker and detect itching based on its level. The present invention is suitable for detecting itching at night, particularly during sleep. Itching varies throughout the day, and nocturnal itching is a significant issue in pruritic skin diseases such as atopic dermatitis (T Ebata et al. J Dermatol. 1996 Mar;23(3):153-5). Therefore, detecting nocturnal itching and resolving itching would be effective in preventing or ameliorating skin diseases caused by scratching due to itching, and further in ameliorating the adverse effects of itching on sleep. Note that nighttime refers to the period from sunset to sunrise (usually from 6:00 PM to 6:00 AM in a 24-hour day), and sleep time generally refers to bedtime, but is not limited to this time period.

[0018] In the present invention, the TLQP-21 level comprehensively refers to the amount and activity of TLQP-21. TLQP-21 levels can be measured by any known method commonly used in the art, including immunoassays (e.g., Western blot, EIA, ELISA, immunostaining, etc.), fluorescence, electrophoresis, protein chips, chromatography, mass spectrometry (e.g., LC-MS / MS, MALDI-TOF / MS), one-hybrid assays (PNAS 100, 12271-12276 (2003)), two-hybrid assays (Biol. Reprod. 58, 302-311 (1998)), or a combination thereof. When measuring TLQP-21 levels using immunoassays, for example, an antibody against TLQP-21 may be contacted with a biological sample and the amount of TLQP-21 bound to the antibody may be quantified. Antibodies against TLQP-21 may be polyclonal or monoclonal. These antibodies can be produced according to known methods. Specifically, polyclonal antibodies can be obtained by immunizing a non-human animal such as a rabbit with a protein expressed and purified in Escherichia coli or a partial polypeptide of the protein according to standard methods, and then obtaining the antibody from the serum of the immunized animal according to standard methods. Meanwhile, monoclonal antibodies can be obtained from hybridoma cells prepared by immunizing a non-human animal such as a mouse with a protein expressed and purified in Escherichia coli or a partial polypeptide of the protein according to standard methods, and then fusing the resulting spleen cells with myeloma cells. Monoclonal antibodies may also be produced using phage display (Griffiths, AD; Duncan, AR, Current Opinion in Biotechnology, Volume 9, Number 1, February 1998, pp. 102-108(7)).

[0019] In the present invention, itching in a subject can be detected by comparing the TLQP-21 level with a preset reference value. Here, the "reference value" can be determined in advance based on the relationship between the degree of itching and the TLQP-21 level in a biological sample. For example, a population can be divided into multiple groups with different levels of itching based on various indices, such as the VAS score (Atopic Dermatitis Clinical Practice Guidelines 2018) or NRS, for skin itching. Then, values ​​determined based on statistical values ​​such as the mean and standard deviation of the TLQP-21 expression level in each group can be used as reference values ​​to determine whether or not a subject belongs to each group. The population is not particularly limited and may be any population, including a population with intractable itch. It may also be a population with intractable itch plus a healthy population who are not aware of itching. Furthermore, it is preferable that the population be consistent in terms of the attributes (gender, race, age, etc.) of the subjects. The specific method for setting the reference value and detecting itching based on the reference value can be appropriately carried out according to the common technical knowledge of those skilled in the art.

[0020] In the present invention, for example, when a reference value is set for the purpose of detecting the presence or absence of itching, if the TLQP-21 level is higher than the reference value, the subject can be detected as having itching; otherwise, the subject can be detected as not having itching. For example, if the TLQP-21 level in a subject is preferably 110% or more, more preferably 120% or more, and even more preferably 130% or more of the reference value, the subject can be detected as having itching; otherwise, the subject can be detected as not having itching. Alternatively, the difference between the TLQP-21 level in a subject and the reference value can be determined, for example, by whether the two are statistically significantly different. That is, if the TLQP-21 level in a subject is statistically significantly higher than the reference value, the subject can be detected as having itching; otherwise, the subject can be detected as not having itching. Furthermore, in the present invention, the degree of itching in a subject can be detected based on whether the subject belongs to a group with different degrees of itching using two or more reference values ​​determined from multiple groups with different degrees of itching. Alternatively, the degree of itching in a subject can be detected by using the expression level of TLQP-21 in the subject as an indirect indicator of the degree of itching.

[0021] Furthermore, in the present invention, biological samples are collected from a subject at least twice a year or periodically (e.g., daily, weekly, or monthly), and the TLQP-21 level in the biological sample is measured. Using the change in the level or the amount of change as an index, it is possible to detect whether or not there is a change in the subject's itching, predict the trend of change, the degree of change, the state of improvement of the itching, etc. In this case, the TLQP-21 level at the start of detection may be used as a reference value, and subsequent TLQP-21 levels may be compared with this reference value to detect whether or not there is a change in the subject's itching.

[0022] The test kit for detecting itch in a subject of the present invention contains a test reagent for measuring TLQP-21 levels in a biological sample isolated from the subject. Specifically, the kit includes a reagent for measuring TLQP-21 levels (e.g., a reagent for immunological measurement containing an antibody that recognizes TLQP-21). The antibody or the like included in the kit can be obtained by known methods as described above. In addition to the above-mentioned antibodies, the test kit may also include labeling reagents, buffer solutions, color-developing substrates, secondary antibodies, blocking agents, equipment necessary for the test, control reagents used as positive and negative controls, and tools for collecting biological samples.

[0023] In relation to the above-described embodiment, the present invention further discloses the following aspects.

[0024] <1> A method for detecting itch in a subject, comprising measuring a TLQP-21 level in a biological sample collected from the subject.

[0025] <2> The itching is preferably intractable itching, more preferably itching associated with atopic dermatitis, xerosis, psoriasis, prurigo, renal failure, or liver disease. <1> The method described. <3> The itch is preferably sleep-related <1> or <2> The method described. <4> The biological sample is preferably a body fluid such as an organ, skin, blood, urine, saliva, sweat, stratum corneum, skin surface lipids (SSL), or tissue exudate, serum prepared from blood, plasma, stool, or hair of a subject, and more preferably blood, serum prepared from blood, or plasma. <1> ~ <3> A method according to any one of the preceding claims. <5> and detecting itch in a subject based on TLQP-21 levels. <1> ~ <4> A method according to any one of the preceding claims. <6> The subject is a human suffering from a pruritic skin disease, preferably a human suffering from itching caused by a pruritic skin disease, more preferably a human suffering from subjective symptoms of sleep disturbance caused by itching caused by a pruritic skin disease. <1> ~ <5> A method according to any one of the preceding claims. <7> containing a reagent for measuring TLQP-21 levels, <1> ~ <6> A test kit for detecting itching in a subject, which is used in any one of the methods above. <8> A detection marker for detecting itch in a subject, comprising TLQP-21. <9> TLQP-21 is a peptide having a sequence selected from TLQPPSALRRRHYHHALPPSR (SEQ ID NOs: 1 and 4), TLQPPASSRRRHFHHALPPAR (SEQ ID NOs: 2 and 3), and TLQPPSASRRRHYHHALPPSR (SEQ ID NO: 5), <1> ~ <6> Either of the following methods: <7> Test kits or <8> Detection marker. [Example]

[0026] Example 1 Comprehensive gene expression analysis 1. Experimental Animals Male C57BL / 6J mice and male NC / Nga mice were used. Xeroderma model (AEW model) A cotton ball soaked in a 1:1 mixture of acetone and diethyl ether was applied to the shaved skin of a C57BL / 6J mouse and left to stand for 15 seconds. Immediately afterwards, a cotton ball soaked in pure water was applied for 30 seconds. This treatment was repeated twice a day (morning and evening) for 7 days to create an AEW model mouse. Control mice were generated by applying only water-soaked cotton balls for 30 seconds. Seven mice per group (n=7) were used in this analysis.

[0027] Atopic dermatitis model (AD model) NC / Nga mice were infested with M. musculi to induce spontaneous dermatitis, creating an AD model. Control mice were the same strain, raised in a SPF environment free of mite infestation. Eight mice per group were used for this analysis.

[0028] 2. Purification of total RNA Dorsal root ganglia were excised from the cervical spine of mice. The excised tissue was homogenized using a Polytron homogenizer, and total RNA was purified using the QIAGEN RNeasy Mini Kit.

[0029] 3. Gene Expression Analysis RNA-seq Total RNA purified from AEW model mice and their control mice was used. Reverse transcription was performed using SuperScript VILO (Thermo Fisher Scientific), and subsequent processing followed Thermo Fisher Scientific's Ion AmpliSeq standard protocol. Sequencing was performed using the Ion S5 system. The results are shown in Figure 1. As shown in Figure 1, Vgf expression per given sequence amount was significantly increased in the AEW model compared to control mice (p<0.05 by Student's t-test).

[0030] Real-time PCR Quantitative PCR analysis of the Vgf gene was performed using total RNA purified from AEW model mice, AD model mice, and their control mice using a Real-Time PCR System with specific TaqMan Gene Expression Assays. Rplp0 was used as an internal standard. Figures 2 and 3 show the relative expression levels of the Vgf gene, with the expression level of the Vgf gene in control mice set at 1.

[0031] As shown in Figure 2, Vgf expression levels in DRG tissues of AEW model mice were significantly increased compared to control mice. Also, as shown in Figure 3, Vgf expression levels in DRG tissues of AD model mice were significantly increased compared to control mice.

[0032] Example 2: Itching caused by TLQP-21 1. Experimental Animals Male C57BL / 6J mice were used.

[0033] 2. Administration of candidate mediator molecules TLQP-21 was prepared using a peptide identical to the mouse sequence (Tocris Bioscience, sequence: TLQPPASSRRRHFHHALPPAR (SEQ ID NO: 6)) at 15 nmol / 20 μL and 30 nmol / 20 μL. Physiological saline was used as the solvent. 20 μL of this solution was administered intradermally to the hind neck of shaved C57BL / 6J mice. Immediately after injection, the mice began measuring their scratching behavior, and measurements were analyzed for 30 minutes after the start of the injection.

[0034] 3. Measurement of Scratching Behavior MicroAct (Neuroscience) was used to measure scratching behavior after TLQP-21 administration. The results are shown in Figure 4. A series of consecutive scratching actions was counted as one, and the total number of these actions was expressed as Events. The total number of all scratching actions was expressed as Beats.

[0035] As shown in Figure 4, after TLQP-21 administration, an increase in scratching behavior in mice was observed as a significant increase in Events and Beats. The onset of scratching behavior was an immediate response within 30 minutes.

[0036] Example 3: Correlation analysis between blood TLQP-21 and itch VAS 1.Serum separation Blood samples were collected from male and female subjects aged 22 to 80 years (4 healthy subjects, 6 subjects with atopic dermatitis, and 1 subject with prurigo) who agreed to participate in the study. The blood was collected in blood collection tubes containing a coagulation-promoting film and centrifuged at 1,200 x g for 15 minutes at 4°C. The supernatant was then separated and stored at -80°C until analysis.

[0037] TLQP-21 EIA TLQP-21 in serum was quantified using the Phoenix Pharmaceuticals TLQP-21 Human EIA Kit. After thawing, the stored serum was diluted 16-fold with the 1x Assay Buffer provided with the kit, and the experiment was performed according to the protocol provided with the kit. Absorbance was measured using a Molecular Devices SpectraMax 340PC384, and TLQP-21 concentrations were calculated using SoftMax Pro software.

[0038] 2.Statistical analysis Survey analysis For subjects who had blood samples taken, subjective symptoms of itching were quantified using a VAS (Visual Analog Scale), with a maximum value of 100. The questionnaire items were as follows: [For the last 7 days] ·Skin itching level (VAS1) · Sleep disturbance due to itching (VAS2) [About the past day (present)] ·Skin itching level (VAS1) · Sleep disturbance due to itching (VAS2)

[0039] statistical analysis Pearson's correlation coefficient (r) was calculated between serum TLQP-21 concentrations and each VAS index from the questionnaire. A positive correlation coefficient and a p-value (two-tailed) of <0.05 was considered to be a significant correlation. GraphPad Prism software was used for analysis. The results are shown in Figure 5.

[0040] As shown in Figure 5, a significant positive correlation was observed between blood TLQP-21 and the itch VAS. In other words, TLQP-21 was considered to be an index for detecting itch. Furthermore, a stronger correlation was observed with sleep disturbance due to itching (VAS2), suggesting that TLQP-21 is a preferable indicator for detecting itching, especially at night.

Claims

1. A method for detecting itch in a subject, comprising the step of measuring a TLQP-21 level in a biological sample collected from the subject.

2. The method according to claim 1, wherein the itching is intractable itching.

3. 3. The method according to claim 1, wherein the biological sample is blood from a subject, or serum or plasma prepared from blood.

4. A test kit for detecting itch in a subject, which is used in the method according to any one of claims 1 to 3, comprising a reagent for measuring TLQP-21 level.

5. A detection marker for detecting itching in a subject, comprising TLQP-21.

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