JP7748866B2 - How to detect itch - Google Patents

Description

The present invention relates to a method for detecting itch.

Itching is a pathological condition seen 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 between the skin and clothing. Scratching caused by itching physically invades the skin, further worsening symptoms, so resolving itching contributes to the prevention or improvement of skin diseases. For example, it has been reported that in mice in which the nails of the hind legs were clipped to prevent physical damage to the skin caused by scratching, skin symptoms caused by atopic dermatitis were prevented and improved (Non-Patent Document 1).

Itch sensations in peripheral tissues such as the skin are transmitted to the brain by afferent sensory nerves that connect the periphery to the dorsal horn of the spinal cord. The cell bodies of afferent sensory nerves are located in the dorsal root ganglia (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.

Pruritogenic substances induce itching by binding to the corresponding receptors. Pruritogenic substances include histamine, serotonin, and chloroquine, while Th2 cytokines such as IL-4 and IL-13 have been reported as itch-enhancing substances (sensitizers). Among these, histamine is a representative chemical pruritic substance. Recently, it has been thought that histamine is essentially associated only with certain acute itches, with little involvement in many chronic itching disorders (Non-Patent Document 2). Therefore, antihistamines (H1 receptor antagonists) are currently widely used to suppress itching, but only a limited number of itches can be resolved with antihistamines, and many itching conditions are intractable, with no effective solutions available. Itching associated with atopic dermatitis, xerosis, renal failure, and other conditions is considered intractable. The mechanisms underlying many intractable itches remain unknown, and there is a need to clarify these mechanisms and develop new target molecules.

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.

Hashimoto Y et al. Life Sciences. 2004 Dec 31;76(7):783-94 Ikoma A et al. Nature Reviews Neuroscience. 2006 Jul;7(7):535-47

The present invention provides a marker for detecting itching and a method for detecting itching using the detection marker.

The present inventors first isolated dorsal root ganglia (DRGs) from mouse models (AEW model mice) that exhibited xeroderma-like skin symptoms after treatment with an acetone-ether mixture and water, and performed comprehensive gene expression analysis. This identified Vgf 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 (AD model mice) revealed significantly increased Vgf expression in DRG tissues in both model mice compared with control mice (Example 1). Vgf encodes the neurosecretory factor VGF nerve growth factor inducible (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). When one of these, TLQP-21, was injected into the skin of the back of the neck of healthy mice, scratching behavior was induced in the mice (Example 2), and it was found that TLQP-21 induces itching. Further research revealed that in humans, TLQP-21 levels in the blood are significantly positively correlated with the degree of itching, and that TLQP-21 can be used as an indicator to detect itching.

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

The present invention makes it possible to detect itching in a simple and minimally invasive manner. This allows itching to be objectively understood, without being dependent on individual subjectivity, making it possible to take appropriate measures against the itching.

1 shows the results of RNA-seq analysis of AEW model DRG tissue. 1 shows the results of real-time PCR analysis of Vgf expression levels in the AEW model. 1 shows the results of real-time PCR analysis of Vgf expression levels in an AD model. The number of itch scratches in C57BL/6J mice after administration of TLQP-21 is shown. 1 shows the results of a correlation analysis between blood TLQP-21 and itch VAS.

The method for detecting itch of the present invention includes a step of measuring TLQP-21 levels in a biological sample collected from a subject.

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.

In the present invention, "detection" can also be replaced with 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 physician.

In the present invention, the "subject" is not particularly limited to a human or non-human mammal who desires or requires detection of itch. Examples 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.

In the present invention, a "biological sample" may be any cell, tissue, or biological material in which TLQP-21 levels 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, and other samples such as stool and hair. Blood, and serum or plasma prepared from blood are preferred.

In the present invention, "TLQP-21" is a physiologically active peptide generated by degradation of VGF in vivo. Specific TLQP-21 sequences can be represented by sequences such as TLQPPSALRRRHYHHALPPSR (SEQ ID NO: 1) for humans ( Homo sapiens ), TLQPPASSRRRHFHHALPPAR (SEQ ID NO: 2) for mice ( Mus musculus ), TLQPPASSRRRHFHHALPPAR (SEQ ID NO: 3) for rats ( Rattus norvegicus ), TLQPPSALRRRHYHHALPPSR (SEQ ID NO: 4) for cynomolgus monkeys ( Macaca fascicularis ), and TLQPPSASRRRHYHHALPPSR (SEQ ID NO: 5) for common marmosets ( Callithrix jacchus ). TLQP-21 is known to bind to the complement factor C3a receptor, a GPCR-type receptor present on the cell membrane, and induce 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. A significant positive correlation was observed, particularly with the severity of itching at night (Figure 5). These results clearly demonstrate that TLQP-21 induces itching, and that TLQP-21 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 itching, particularly at night, and resolving the itching would be effective for preventing or ameliorating skin diseases caused by scratching due to itching, and further for ameliorating the adverse effects of itching on sleep. Note that nighttime refers to the time 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 during the night, but the time period is not limited to these.

In the present invention, the TLQP-21 level comprehensively refers to the amount and activity of TLQP-21.
TLQP-21 levels can be measured according to known methods commonly used in the art, including immunoassays (e.g., Western blot, EIA, ELISA, immunostaining, etc.), fluorescence methods, electrophoresis, protein chips, chromatography, mass spectrometry (e.g., LC-MS/MS, MALDI-TOF/MS), one-hybrid methods (PNAS 100, 12271-12276 (2003)), two-hybrid methods (Biol. Reprod. 58, 302-311 (1998)), and combinations 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. The antibody against TLQP-21 may be a polyclonal or monoclonal antibody. 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 using a protein expressed and purified in Escherichia coli or the like according to standard methods, or by synthesizing a partial polypeptide of the protein according to standard methods, and then using standard methods to obtain the antibody from the serum of the immunized animal. On the other hand, 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 the like according to standard methods, or a partial polypeptide of the protein, 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)).

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 level of itching, for example, the VAS score (Atopic Dermatitis Clinical Practice Guidelines 2018) or NRS, related to skin itching. Values determined based on statistical values such as the average value and standard deviation of the TLQP-21 expression level in each group can then be used as reference values for determining 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.

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 or not 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 index representing the degree of itching.

In addition, 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 levels in the biological samples are measured. Changes in the levels or the amount of change can be used as indicators to detect whether or not there has been a change in the subject's itching, predict the trend of change, the degree of change, and the extent of improvement in itching. In this case, the TLQP-21 level at the start of detection can be used as a reference value, and subsequent TLQP-21 levels can be compared with this reference value to detect whether or not there has been a change in the subject's itching.

The test kit of the present invention for detecting itch in a subject contains a test reagent for measuring TLQP-21 levels in a biological sample isolated from the subject. Specific examples include reagents for measuring TLQP-21 levels (such as reagents for immunological measurements containing antibodies that recognize TLQP-21). The antibodies and other components 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.

With respect to the above-described embodiment, the present invention further discloses the following aspects:

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

<2> The method according to <1>, wherein the itching is preferably intractable itching, more preferably itching associated with atopic dermatitis, xerosis, psoriasis, prurigo, renal failure, or liver disease.
<3> The method according to <1> or <2>, wherein the itching is preferably itching during sleep.
<4> The method according to any one of <1> to <3>, wherein 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, feces, or hair of the subject, and more preferably blood, or serum or plasma prepared from blood.
<5> The method according to any one of <1> to <4>, comprising detecting itch in a subject based on a TLQP-21 level.
<6> The method according to any one of <1> to <5>, wherein the subject is a human having a pruritic skin disease, preferably a human having itching caused by a pruritic skin disease, more preferably a human having subjective symptoms of sleep disturbance caused by itching caused by a pruritic skin disease.
<7> A test kit for detecting itching in a subject, which is used in any one of the methods <1> to <6>, and which contains a reagent for measuring TLQP-21 levels.
<8> A detection marker for detecting itching in a subject, comprising TLQP-21.
<9> The method of any one of <1> to <6>, the testing kit of <7>, or the detection marker of <8>, wherein 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).

Example 1 Comprehensive Gene Expression Analysis 1. Experimental Animals Male C57BL/6J mice and male NC/Nga mice were used.
Xeroderma model (AEW model) : Absorbent cotton soaked in a 1:1 mixture of acetone and diethyl ether was applied to the shaved skin of C57BL/6J mice and left to stand for 15 seconds. Immediately afterwards, absorbent cotton soaked in pure water was applied for 30 seconds. This treatment was repeated twice a day (morning and evening) for 7 days to create AEW model mice. Control mice were created by applying only absorbent cotton soaked in pure water for 30 seconds. Each group (n = 7) was used in this analysis.

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 of mice raised in SPF without mite infestation. Each group (n = 8) was used for this analysis.

2. Purification of total RNA Dorsal root ganglia were excised from mouse cervical vertebrae. The excised tissue was homogenized using a Polytron homogenizer, and total RNA was purified using QIAGEN's RNeasy Mini Kit.

3. Gene expression analysis
RNA-seq : Total RNA purified from AEW model mice and their control mice was used. SuperScript VILO (Thermo Fisher Scientific) was used for reverse transcription, 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).

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, with Vgf as the target gene. Rplp0 was used as the 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.

As shown in Figure 2, Vgf expression levels in DRG tissue of AEW model mice were significantly increased compared to control mice. Furthermore, as shown in Figure 3, Vgf expression levels in DRG tissue of AD model mice were also significantly increased compared to control mice.

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

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, measurement of the mice's scratching behavior was initiated, and measurements taken for 30 minutes after the start were analyzed.

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

As shown in Figure 4, after administration of TLQP-21, 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.

Example 3: Correlation analysis between blood TLQP-21 and pruritus VAS 1. Serum separation Blood was 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 had given consent to participate in the study. The blood was collected in a blood collection tube containing a coagulation-promoting film and centrifuged at 1,200 × g for 15 minutes at 4°C. The supernatant was then separated and stored at -80°C until analysis.

TLQP-21 EIA
TLQP-21 in serum was quantified using the TLQP-21 Human EIA Kit from Phoenix Pharmaceuticals. 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.

2. statistical analysis
Survey analysis
The subjects who had their blood drawn were asked to rate their subjective symptoms of itching using a visual analog scale (VAS), with a maximum value of 100. The questionnaire items were as follows:
[For the last 7 days]
- Degree of skin itching (VAS1)
- Itching interferes with sleep (VAS 2)
[About the past day (present)]
- Degree of skin itching (VAS1)
- Itching interferes with sleep (VAS 2)

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-sided) of <0.05 was determined to be a significant correlation. GraphPad Prism was used as the analysis software. The results are shown in Figure 5.

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, particularly at night.

Claims (3)

A method for detecting itch in a subject, comprising a step of measuring a TLQP-21 level in a biological sample collected from the subject , wherein the biological sample is the subject's blood, or serum or plasma prepared from the blood, and the itch is itch associated with atopic dermatitis or xerosis . A test kit for detecting itch in a subject, which is used in the method of claim 1 , comprising a reagent for measuring TLQP-21 levels. A detection marker for detecting itching associated with atopic dermatitis or xerosis in a subject, comprising TLQP-21.