Biomarker for nociceptive pain and nociplastic pain

Abstract

The present invention provides: a biomarker for identifying nociceptive pain and nociplastic pain; a biomarker for determining therapeutic effects on nociceptive pain and nociplastic pain; an examination method for nociceptive pain and nociplastic pain, using the biomarkers; a method for evaluating the severity in a patient with nociceptive pain and a patient with nociplastic pain; a method for determining therapeutic effects on nociceptive pain and nociplastic pain; and a kit including a reagent for measuring the amounts of the biomarkers.

Description

Biomarkers of nociceptive and nociceptive pain

[0001] The present invention relates to biomarkers for distinguishing between nociceptive pain and nociceptive pain, biomarkers for determining the effectiveness of a treatment for nociceptive pain and nociceptive pain, methods for testing nociceptive pain and nociceptive pain using the biomarkers, methods for assessing the severity of nociceptive pain and nociceptive pain patients, methods for determining the effectiveness of a treatment for nociceptive pain and nociceptive pain, and kits comprising reagents for measuring the amount of the biomarkers.

[0002] Pain is subjective and cannot be objectively assessed. The current mainstream method for assessing pain is to ask patients to rate their pain using a numerical rating scale.

[0003] Functional brain imaging using MRI has been attempted as a method for assessing pain, but it has not yet been put to practical use, so a pain biomarker is needed.

[0004] Osteoarthritis is a typical disease accompanied by nociceptive pain, and the progression of the disease is primarily assessed by imaging diagnostics such as X-rays and MRI. Several dozen biomarkers for osteoarthritis have been investigated. Important biomarkers for osteoarthritis include cartilage oligomeric matrix protein (COMP), an extracellular matrix glycoprotein; crosslinked telopeptide of type II collagen (CTX-II), a type II collagen-derived crosslinked telopeptide of type II collagen (ARGS), aggrecanase-mediated degradation of aggrecan present in hyaline cartilage; collagen type II cleavage (C2C), a degradation product of type II collagen; and hyaluronic acid (HA).

[0005] Although still in the research stage, there are biomarkers for osteoarthritis that use non-coding RNA miRNAs (Non-Patent Documents 1-3). miRNA-136 is significantly reduced in patients with osteoarthritis (Non-Patent Document 4). Other biomarkers for osteoarthritis include those reported in blood samples (Non-Patent Document 5), miRNA-98 (Non-Patent Document 6), 140-3p (Non-Patent Document 7), 33b-3p (Non-Patent Document 7), 671-3p (Non-Patent Document 7), 675-5p (Non-Patent Document 8), 126-5p (Non-Patent Document 8), and 155-5p (Non-Patent Document 8). However, none of these have yet become widespread.

[0006] Veronesi, F., et al., Epigenetic Modifications of MiRNAs in Osteoarthritis: A Systematic Review on Their Methylation Levels and Effects on Chondrocytes, Extracellular Matrix and Joint Inflammation. Cells, 2023. 12(14).Nunez-Carro, C., et al., Epigenetics as a Therapeutic Target in Osteoarthritis. Pharmaceuticals (Basel), 2023. 16(2).Liu, H., et al., MicroRNA expression in osteoarthritis:a meta-analysis. Clin Exp Med, 2023.Wang, J., et al., EZH2 is associated with cartilage degeneration in osteoarthritis by promoting SDC1 expression via histonemethylation of the microRNA-138 promoter. Lab Invest, 2021. 101(5):p.600-611.Munjal, A., et al., Advances in Molecular biomarker for early diagnosis of Osteoarthritis. Biomol Concepts, 2019. 10(1):p.111-119.Zheng, W.D., et al., Investigation for the role of CTX-III and microRNA-98 in diagnosis and treatment of osteoarthritis.Eur RevMed Pharmacol Sci, 2018. 22(17): p.5424-5428.Ntoumou, E., et al., Serum microRNA array analysis identifies miR-140-3p, miR-33b-3p and miR-671-3p as potential osteoarthritis biomarkers involved in metabolic processes. Clin Epigenetics,2017. 9: p.127.Dong, Z., et al., Change of miRNA expression profiles in patients with knee osteoarthritis before and after celecoxib treatment. J Clin Lab Anal, 2019. 33(1): p. e22648.

[0007] Conventional pain assessment methods require patient responses and are subjective, making assessment difficult. X-rays and MRIs used to assess osteoarthritis have problems such as difficulty in early detection, lack of objectivity due to variability between assessors, and the cost of X-ray exposure and MRI imaging. The present invention aims to provide a pain biomarker that can be tested using minimally invasive blood sampling.

[0008] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that the expression of the miRNA let-7a-5p (also referred to as hsa-let-7a-5p) is significantly reduced in patients with preoperative hip osteoarthritis compared to controls and other patients with chronic pain, and that this let-7a-5p expression increases after hip osteoarthritis surgery is completed and pain improves. This indicates that let-7a-5p can be used as a biomarker for nociceptive pain (such as osteoarthritis) and as a biomarker for assessing the severity of nociceptive pain and osteoarthritis patients. Since let-7a-5p can be quantified using RT-PCR or other methods in blood obtained from subjects, the expression level of let-7a-5p can be measured to examine nociceptive pain (such as osteoarthritis) and to assess the severity of nociceptive pain and osteoarthritis patients.Furthermore, the inventors performed plasma RNA extraction and miRNA expression profiling using miRNA microarrays. As a result, (Group A: nociceptive pain markers): miRNA groups that were not changed by therapeutic intervention for nociceptive pain, and whose concentrations in plasma increased by therapeutic intervention for nociceptive pain: hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let-7a- 5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289 (Group B: nociceptive pain markers): miRNAs that are not changed by therapeutic intervention for nociceptive pain and whose concentrations decrease in plasma by therapeutic intervention for nociceptive pain: hsa-miR-412-3p, hsa-miR-6794-3p (Group C ... MiRNA group whose plasma concentration increases with therapeutic intervention: hsa-miR-373-5p, (Group D: nociceptive pain markers): miRNA group whose plasma concentration does not change with therapeutic intervention for nociceptive pain and whose plasma concentration decreases with therapeutic intervention for nociceptive pain: hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3p, hsa-miR-4515, hsa-miR-2113, hsa a-miR-6747-3p, hsa-miR-1288-3p, hsa-miR-574-5p, hsa-miR-4639-3p, hsa-miR-196a-1-3p, (Group E: pain marker): miRNA group whose concentration increases in plasma by therapeutic intervention for nociceptive pain and therapeutic intervention for nociceptive pain: hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p, was found. This indicates that miRNAs in groups A and B are biomarkers for identifying nociceptive pain, miRNAs in groups C and D are biomarkers for identifying nociceptive pain, and miRNAs in group E can be used as biomarkers for identifying pain related to nociceptive pain and nociceptive pain.Furthermore, it is shown that miRNAs of groups A to E can be used as markers for assessing the therapeutic effect of therapeutic intervention for nociceptive pain and / or therapeutic intervention for nociceptive pain. Specifically, when miRNAs of group A are detected whose concentration increases in plasma after therapeutic intervention for nociceptive pain, it can be determined that the therapeutic effect is present. When miRNAs of group B are detected whose concentration decreases in plasma after therapeutic intervention for nociceptive pain, it can be determined that the therapeutic effect is present. When miRNAs of group C are detected whose concentration increases in plasma after therapeutic intervention for nociceptive pain, it can be determined that the therapeutic effect is present. When miRNAs of group D are detected whose concentration decreases in plasma after therapeutic intervention for nociceptive pain, it can be determined that the therapeutic effect is present. When miRNAs of group E are detected whose concentration increases in plasma after therapeutic intervention for nociceptive pain and nociceptive pain, it can be determined that the therapeutic effect is present. Furthermore, it is shown that miRNAs of groups A and B can be used as biomarkers for assessing the severity of nociceptive pain patients. This shows that miRNAs in groups C and D can be used as biomarkers for assessing the severity of pain in patients with nociceptive pain. This shows that miRNAs in group E can be used as biomarkers for assessing the severity of pain in patients with nociceptive pain and nociceptive pain. Since miRNAs in groups A to E can be quantified by RT-PCR or the like using blood obtained from a subject, the expression levels of miRNAs in groups A to E can be measured to test for nociceptive pain (such as osteoarthritis), test for nociceptive pain, and assess the severity of nociceptive pain patients, nociceptive pain patients, and osteoarthritis patients. That is, the present invention comprises the following: 1. A biomarker for nociceptive pain consisting of let-7a-5p. 2. The biomarker according to the preceding paragraph 1, wherein the nociceptive pain is osteoarthritis pain. 3. A biomarker for assessing the severity of nociceptive pain patients consisting of let-7a-5p. 4. 4. The biomarker according to the preceding item 3, wherein the nociceptive pain patient is an osteoarthritis patient.5. A method for testing nociceptive pain in a subject, comprising: obtaining an expression level of let-7a-5p in a blood sample obtained from the subject; obtaining an expression level of a control of let-7a-5p; and comparing the expression level of let-7a-5p in the blood sample with the expression level of the control of let-7a-5p, wherein a decrease in the expression level of let-7a-5p in the blood sample obtained from the subject compared to the expression level of the control indicates the presence of nociceptive pain. 6. The method according to paragraph 5, wherein the nociceptive pain is osteoarthritis pain. 7. 7. A method for assessing the severity of nociceptive pain in a subject, comprising: obtaining an expression level of let-7a-5p in a blood sample obtained from the subject; obtaining an expression level of a control of let-7a-5p; and comparing the expression level of let-7a-5p in the blood sample with the expression level of the control of let-7a-5p, wherein a decrease in the expression level of let-7a-5p in the blood sample obtained from the subject compared to the expression level of the control is an indicator of a higher severity of the nociceptive pain patient. 8. The method according to item 7 above, wherein the nociceptive pain patient is an osteoarthritis patient. 9. The method according to any one of items 5 to 8 above, wherein the "control expression level" is obtained from a preoperative patient or another chronic pain patient. 10. A kit comprising a reagent for measuring the amount of the biomarker according to any one of items 1 to 4 above.11. Group A (hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let -7a-5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289) and group B (hsa-miR-412-3p, h Biomarkers for identifying nociceptive pain consisting of one or more of the miRNAs in group C (hsa-miR-373-5p), group D (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809- a biomarker for discriminating between nociceptive pain and nociceptive pain consisting of one or more miRNAs from group E (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p); and / or a biomarker for discriminating between nociceptive pain and nociceptive pain consisting of one or more miRNAs from group E (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p).12. Group A (hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let-7a -5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289) and group B (hsa-miR-412-3p, hsa-mi Biomarkers for assessing the therapeutic effect on nociceptive pain consisting of one or more of the miRNAs in group C (hsa-miR-373-5p), group D (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3p), and group E (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3p). A biomarker for determining the therapeutic effect on nociceptive pain consisting of one or more of the miRNAs in group E (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p), and / or a biomarker for determining the therapeutic effect on nociceptive pain and nociceptive pain consisting of one or more of the miRNAs in group E (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p).13. Group A (hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let- 7a-5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289) and group B (hsa-miR-412-3p, hs Biomarkers for assessing the severity of nociceptive pain in patients consisting of one or more of the miRNAs in group C (hsa-miR-373-5p) and group D (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3p) were used. 13. A biomarker for assessing the severity of pain patients with nociceptive pain consisting of one or more of the miRNAs in group E (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p), and / or a biomarker for assessing the severity of pain patients with nociceptive pain and nociceptive pain consisting of one or more of the miRNAs in group E (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p). 1. A method of testing for nociceptive pain in a subject, comprising: (i) obtaining, in a blood sample obtained from the subject, the expression level of one or more miRNAs selected from miRNAs in Group A (hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289); (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the miRNA expression levels obtained in (i) and (ii), wherein a decrease in the expression level of a miRNA in the blood sample obtained from the subject compared to the control expression level indicates the presence of nociceptive pain.15. A method for testing nociceptive pain in a subject, comprising: (i) obtaining the expression level of one or more miRNAs selected from miRNAs of group B (hsa-miR-412-3p, hsa-miR-6794-3p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the miRNA expression levels obtained in (i) and (ii), wherein an increase in the expression level of a miRNA in the blood sample obtained from the subject compared to the control expression level indicates the presence of nociceptive pain. 16. A method for testing for nociceptive pain in a subject, comprising: (i) obtaining the expression level of a miRNA of group C (hsa-miR-373-5p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the expression levels of the miRNA obtained in (i) and (ii), wherein a decrease in the expression level of the miRNA in the blood sample obtained from the subject compared to the control expression level is indicative of the presence of the nociceptive pain. A method for testing for nociceptively modulated pain in a subject, comprising: (i) obtaining the expression level of one or more miRNAs selected from the miRNAs of group D (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3p, hsa-miR-4515, hsa-miR-2113, hsa-miR-6747-3p, hsa-miR-1288-3p, hsa-miR-574-5p, hsa-miR-4639-3p, hsa-miR-196a-1-3p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the expression levels of the miRNA obtained in (i) and (ii), wherein an increase in the expression level of the miRNA in the blood sample obtained from the subject compared to the expression level in the control is indicative of the presence of the nociceptive pain.18. A method for testing for nociceptive pain and nociceptive pain in a subject, comprising: (i) obtaining the expression level of one or more miRNAs selected from miRNAs of Group E (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the expression levels of the miRNA obtained in (i) and (ii), wherein a decrease in the expression level of the miRNA in the blood sample obtained from the subject compared to the control expression level indicates the presence of pain related to nociceptive pain and nociceptive pain. 1. A method for assessing the severity of nociceptive pain in a subject, comprising: (i) obtaining, in a blood sample obtained from the subject, the expression level of one or more miRNAs selected from miRNAs in Group A (hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289); (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the miRNA expression levels obtained in (i) and (ii), wherein a decrease in the expression level of a miRNA in the blood sample obtained from the subject compared to the control expression level is indicative of a higher severity of the nociceptive pain in the subject.20. A method for assessing the severity of nociceptive pain in a subject, comprising: (i) obtaining the expression level of one or more miRNAs selected from miRNAs of group B (hsa-miR-412-3p, hsa-miR-6794-3p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the miRNA expression levels obtained in (i) and (ii), wherein an increase in the expression level of the miRNA in the blood sample obtained from the subject compared to the control expression level is indicative of a higher severity of the nociceptive pain in the subject. 21. A method for assessing the severity of algesic pain in a subject, comprising: (i) obtaining the expression level of a miRNA of group C (hsa-miR-373-5p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the expression levels of the miRNA obtained in (i) and (ii), wherein a decrease in the expression level of the miRNA in the blood sample obtained from the subject compared to the control expression level is an indicator of the severity of the algesic pain patient. A method for assessing the severity of dysalgia pain in a subject, comprising: (i) obtaining the expression level of one or more miRNAs selected from the miRNAs of group D (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3p, hsa-miR-4515, hsa-miR-2113, hsa-miR-6747-3p, hsa-miR-1288-3p, hsa-miR-574-5p, hsa-miR-4639-3p, hsa-miR-196a-1-3p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the expression levels of the miRNA obtained in (i) and (ii), wherein an increase in the expression level of the miRNA in the blood sample obtained from the subject compared to the expression level in the control indicates a higher severity of the pain in the patient with algesic pain.23. A method for assessing the severity of pain symptoms in a subject with respect to nociceptive pain and nociceptive pain, comprising: (i) obtaining the expression level of one or more miRNAs selected from miRNAs in group E (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the expression levels of the miRNA obtained in (i) and (ii), wherein a decrease in the expression level of the miRNA in the blood sample obtained from the subject compared to the control expression level is indicative of a higher severity of pain symptoms in the patient with respect to nociceptive pain and nociceptive pain. A method for assessing the therapeutic effect of a therapeutic intervention on nociceptive pain in a subject, comprising: (i) obtaining plasma concentrations of one or more miRNAs selected from miRNAs of Group A (hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289) from a blood sample obtained from the subject before treatment; (ii) obtaining plasma miRNA concentrations for the same miRNAs as in (i) from a blood sample obtained from the subject after treatment; and (iii) comparing the plasma miRNA concentrations obtained in (i) and (ii) before and after treatment; The method, wherein an increase in the concentration of the miRNA in the plasma after treatment is an indicator of whether or not a therapeutic intervention for nociceptive pain is effective.25. A method for determining the therapeutic effect of a therapeutic intervention on nociceptive pain in a subject, comprising: (i) obtaining the concentration of one or more miRNAs from the plasma of miRNAs in group B (hsa-miR-412-3p, hsa-miR-6794-3p) from a blood sample obtained from the subject before treatment; (ii) obtaining the plasma miRNA concentration for the same miRNA as in (i) from a blood sample obtained from the subject after treatment; and (iii) comparing the plasma miRNA concentrations obtained in (i) and (ii) before and after treatment, wherein a decrease in the plasma miRNA concentration after treatment serves as an indicator for determining that the therapeutic intervention on nociceptive pain is effective. A method for determining the therapeutic effect of a therapeutic intervention on algesia-modulated pain in a subject, comprising: (i) obtaining the concentration of miRNA of group C (hsa-miR-373-5p) in plasma from a blood sample obtained from the subject before treatment; (ii) obtaining the concentration of miRNA in plasma for the same miRNA as in (i) from a blood sample obtained from the subject after treatment; and (iii) comparing the miRNA concentrations in plasma before and after treatment obtained in (i) and (ii), wherein an increase in the miRNA concentration in plasma after treatment serves as an indicator for determining that the therapeutic intervention on algesia-modulated pain is effective.27. A method for assessing the therapeutic effect of a therapeutic intervention for algo-dysthymic pain in a subject, comprising: (i) obtaining the concentration of one or more miRNAs selected from the miRNAs of group D (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3p, hsa-miR-4515, hsa-miR-2113, hsa-miR-6747-3p, hsa-miR-1288-3p, hsa-miR-574-5p, hsa-miR-4639-3p, hsa-miR-196a-1-3p) in plasma from a blood sample obtained from the subject before treatment; 27. A method comprising: (ii) obtaining a plasma miRNA concentration for the same miRNA as in (i) from a blood sample obtained from the subject after treatment; and (iii) comparing the plasma miRNA concentrations obtained in (i) and (ii) before and after treatment, wherein a decrease in the plasma miRNA concentration after treatment is an indicator of whether a therapeutic intervention for algomodulatory pain is effective. 1. A method for determining the therapeutic effect of a therapeutic intervention on nociceptive pain and nociceptive-modulated pain in a subject, comprising: (i) obtaining the concentration of one or more miRNAs selected from the miRNAs of group E (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p) in plasma from a blood sample obtained from the subject before treatment; (ii) obtaining the miRNA concentration in plasma for the same miRNA as in (i) from a blood sample obtained from the subject after treatment; and (iii) comparing the miRNA concentrations in plasma before and after treatment obtained in (i) and (ii), wherein an increase in the miRNA concentration in plasma after treatment serves as an indicator for determining that the therapeutic intervention on nociceptive pain and nociceptive-modulated pain is effective.

[0009] The present invention makes it possible to objectively evaluate and visualize nociceptive pain (such as osteoarthritis pain) and pain-modulated pain. For patients whose main complaint is pain, there are times when the cause remains unknown despite various tests. However, if blood sampling reveals a decrease in the expression of miRNAs (e.g., hsa-let-7a-5p) described in Group A, the patient can be evaluated as experiencing nociceptive pain (such as osteoarthritis pain) due to actual nociceptive stimulation occurring in the body. If the expression level is not decreased, the patient can be evaluated as experiencing nociceptive pain due to actual nociceptive stimulation occurring in the body. If the expression level is not increased, the patient can be evaluated as experiencing nociceptive pain due to actual nociceptive stimulation occurring in the body. If the expression level is not increased, the patient can be evaluated as experiencing nociceptive pain due to actual nociceptive stimulation occurring in the body. If the expression level is decreased, the patient can be evaluated as experiencing nociceptive pain due to actual nociceptive stimulation occurring in the body. If the expression level is not decreased, the patient can be evaluated as experiencing pain due to other factors. If the expression level is decreased, the patient can be evaluated as experiencing nociceptive pain due to actual nociceptive stimulation occurring in the body. If the expression level is not decreased, the patient can be evaluated as experiencing pain due to other factors. Furthermore, if the expression level of the miRNAs listed in group D is elevated by blood sampling, it can be evaluated as pain-modulated pain, where pain is actually occurring in the body, and if it is not elevated, it can be evaluated as pain caused by other factors. If the expression level of the miRNAs listed in group E is reduced by blood sampling, it can be evaluated as pain caused by nociceptive pain and pain-modulated pain, where pain is actually occurring in the body, and if it is not reduced, it can be evaluated as pain caused by other factors. When used to evaluate nociceptive pain, pain-modulated pain, or osteoarthritis, it can reduce X-rays and exposure. In addition, it can provide a more objective severity diagnosis than imaging diagnosis.

[0010] Figure 1 shows the ROC curve. A: ROC analysis result 1 of let-7a-5p for OA. When 11 CPP cases and 10 control cases were considered negative (AUC 0.90110 P<0.0001). B: ROC analysis result 2 of let-7a-5p for OA. When only 10 control cases were considered negative (AUC 0.94615 P<0.0001). Figure 2 shows the results of preoperative and pretreatment blood sampling. In the OA group, let-7a-5p was clearly suppressed compared to the CPP and control groups. Figure 3 shows the results of blood sampling after treatment intervention. Comparison before and after cognitive behavioral therapy in the CPP group. In the OA group, let-7a-5p did not change before and after treatment intervention. Figure 4 shows the results of QOL and pain assessments and blood sampling before and after surgery in patients with osteoarthritis of the hip. Figure 5 shows the results of RNA extraction from plasma obtained from blood collected before and after therapeutic intervention for nociceptive pain and blood collected before and after therapeutic intervention for nociceptive pain, and miRNA expression profiling using miRNA microarrays.

[0011] According to the present invention, the miRNAs described in Groups A to E of the present specification can be used as biomarkers for distinguishing between nociceptive pain (such as osteoarthritis pain) and nociceptive-modulated pain, as biomarkers for determining the therapeutic efficacy of nociceptive pain and nociceptive-modulated pain, and as biomarkers for assessing the severity of nociceptive pain patients and nociceptive-modulated pain patients. Specifically, by collecting blood from patients with joint pain and measuring the expression levels of miRNAs in Group A (e.g., hsa-let-7a-5p) or miRNAs in Group B, it is possible to evaluate nociceptive pain (such as osteoarthritis pain), assess the severity of nociceptive pain patients and osteoarthritis patients, and assess the therapeutic efficacy of nociceptive pain. By collecting blood from patients with pain and measuring the expression levels of miRNAs in Groups C or D, it is possible to evaluate nociceptive-modulated pain, assess the severity of nociceptive pain patients, and assess the therapeutic efficacy of nociceptive pain. By taking blood samples from patients with pain and measuring the expression levels of miRNAs in group E, it is possible to evaluate nociceptive pain and nociceptive-modulated pain, evaluate the severity of nociceptive pain and nociceptive-modulated pain, and determine the effectiveness of treatment for nociceptive pain and nociceptive-modulated pain.

[0012] As used herein, the term "biomarker" refers to an indicator for the assessment of a subject's condition or function and is used as a target for analysis of a sample obtained from the subject.

[0013] The biomarkers of the present invention are miRNAs described in Groups A to E of this specification. Group A miRNAs consist of hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7d-5p, and hsa-miR-4289. Group B miRNAs consist of hsa-miR-412-3p and hsa-miR-6794-3p. Group C miRNAs consist of hsa-miR-373-5p. Group D miRNAs consist of hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3p, hsa-miR-4515, hsa-miR-2113, hsa-miR-6747-3p, hsa-miR-1288-3p, hsa-miR-574-5p, hsa-miR-4639-3p, and hsa-miR-196a-1-3p. Group E miRNAs consist of hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, and hsa-miR-508-5p.

[0014] miRNA (microRNA) is a 15-25 base pair RNA that is transcribed as a hairpin-like RNA precursor, cleaved by a dsRNA cleaving enzyme with RNase III cleavage activity, incorporated into a protein complex called RISC, and involved in the translational repression of mRNA. miRNA is known to be involved in a variety of pathologies and to be effective as a biomarker for early disease diagnosis. Methods for diagnosing diseases by extracting and detecting miRNA from body fluids such as blood and urine, and for using miRNA for treatment, are known (see, for example, JP 2015-231376 A and JP 2014-527807 A). The contents of the documents cited herein are incorporated herein by reference in their entirety.

[0015] In one aspect, the biomarkers of the present invention are biomarkers for distinguishing between nociceptive pain (such as osteoarthritis pain) and nociceptive pain, for determining the efficacy of treatment for nociceptive pain and nociceptive pain, and for assessing the severity of nociceptive pain and nociceptive pain patients. Specifically, the biomarkers of the present invention are miRNAs (particularly, miRNAs described in Groups A to E of the present specification) contained in a sample obtained from a subject (patient). The sample can be obtained from a body fluid such as blood.

[0016] As used herein, the terms "subject" and "patient" are used interchangeably, regardless of whether the subject has received or is currently receiving any form of treatment, to which the methods of the present invention are applied. As used herein, a "subject" is a vertebrate, particularly a mammal, an amphibian, a reptile, a bird, etc., and more particularly a mammal, particularly a human (Homo sapiens).

[0017] As used herein, the term "sample" also includes whole blood, serum, plasma, saliva, urine, sputum, lymph, cells, tissues, etc., separated from an individual, and preferably refers to a sample secreted from a living body, more specifically blood, nasal discharge, saliva, urine, sputum, or lymph separated from a subject. Most preferably, it is blood.

[0018] Biomarkers can be measured using methods commonly known to those skilled in the art. Biomarkers contained in a subject sample can be measured by detecting them using methods commonly known to those skilled in the art. The measured value of a biomarker can be a value that reflects the amount or concentration in the subject.

[0019] The biomarkers of the present invention can be measured by measuring the expression level of miRNA present in a subject's sample. Measurement of miRNA expression levels can be achieved, for example, by measuring the expression level of miRNA present in the subject's blood, or by measuring the expression level of miRNA present in blood obtained from a preoperative patient or other chronic pain patient. In measuring miRNA expression levels in blood, for example, blood is drawn from the subject, the supernatant is centrifuged, and total RNA is extracted from the serum or plasma from which blood cells have been removed, and the miRNA expression level can be measured from the extracted total RNA.

[0020] Total RNA can be extracted using, for example, guanidine-cesium chloride (GT-CsCl) ultracentrifugation, AGPC, or RNA extraction columns commonly used by those skilled in the art. MiRNA expression levels can be measured using methods commonly used by those skilled in the art, such as Northern blot, microarray, QCM (Quartz Crystal Microbalance) sensor measurement, and real-time PCR including qRT-PCR.

[0021] When real-time PCR is used to measure the expression level of miRNA, expression can be normalized using an endogenous control miRNA. In this case, the endogenous control can be appropriately selected by those skilled in the art from sequences previously reported to be suitable endogenous controls, and can be one that does not change between healthy and diseased individuals.

[0022] In one embodiment, the measurement of the biomarkers of the present invention can be achieved by measuring the expression levels of miRNAs (particularly, miRNAs described in Groups A to E of the present specification). The expression levels of miRNAs described in Groups A to E of the present specification can be measured, for example, by performing real-time PCR, and can be measured using techniques known to those skilled in the art.

[0023] As used herein, the term "expression level" is essentially used interchangeably with "expression amount" and refers to the amount of polynucleotides due to gene expression in a biological sample. The term "decreased expression level" or "low expression" refers to a downward shift in the expression level compared to the expression level in a sample used as a control or the standard expression level of a normalized gene. The term "increased expression level" or "high expression" refers to an upward shift in the expression level compared to the expression level in a sample used as a control or the standard expression level of a normalized gene.

[0024] As used herein, the term "control expression level" refers to an established expression level of a biomarker for a pain-free subject, an expression level of a biomarker in a pain-free normal / healthy subject, an expression level of a biomarker in a pre-operative patient or other chronic pain patient, and / or a known expression level of a biomarker obtained from the literature, as determined by one of skill in the art using the methods described herein. In one embodiment, the control expression level can be the average or control expression level range in a population. In another embodiment, the control expression level can be the expression level of a sample previously obtained from the subject when the subject did not require treatment for pain. In a preferred embodiment, the "control expression level" refers to an expression level obtained from a pre-operative patient or other chronic pain patient.

[0025] As used herein, the term "nociceptive pain" refers to pain caused by some kind of injury or inflammation in the body, including, for example, pain associated with joint deformation. In this specification, examples of nociceptive pain include the pain of osteoarthritis (OA) (Izumi, J. et al., 8. Pain in Knee Osteoarthritis: Conservative Treatment. THE BONE 2016 30 (3) pp. 263-268; Hasegawa, M., et al., Possible Neuropathic Pain in Patients with Osteoarthritis of the Knee Before and After Total Knee Arthroplasty. J PainRes, 2021. 14: pp. 3011-3015; Sumiya, M., The Latest Treatment of Pain. Doctor Salon 2014 58 (August issue) pp. 571-575; US2005-0014847 Publication), visceral pain due to rheumatoid arthritis, fractures, cancer infiltration, and pain from irritable bowel syndrome (IBS) and fibromyalgia (Sumiya, M., The latest treatments for pain. Doctor Salon 201458 (August issue) p. 571-575.; US2005-0014847 Publication).

[0026] As used herein, "osteoarthritis (OA) pain" refers to pain associated with osteoarthritis (OA). The most common joints affected by osteoarthritis (OA) pain include heavy weight-bearing joints, including the hip and knee. Thus, osteoarthritis (OA) includes hip osteoarthritis, knee osteoarthritis, and osteoarthritis of the fingers and hands. The mechanism of osteoarthritis (OA) pain is poorly understood. Osteoarthritis (OA) pain is thought to arise from several structures within arthritic joints, including the synovial membrane, which releases prostaglandins, leukotrienes, and inflammatory mediators (Sofat et al., Rheumatology, 2011, 50(12):2157-65).

[0027] As used herein, "algesic pain" refers to pain resulting from altered nociception, occurring despite the absence of clear evidence of actual or potential tissue damage that would activate peripheral nociceptors, or evidence of pain-causing disease or injury to the somatosensory system. This corresponds to what has traditionally been called psychogenic pain or nonorganic pain. The mechanisms underlying algesic pain include central sensitization, social pain, somatization, inappropriate cognitive and behavioral responses to pain, and psychiatric disorders (Yasuno, Kozo, Jpn J Psychosom Med, 64:415-419, 2024).

[0028] As used herein, "primary chronic pain (CPP)" refers to persistent pain of unknown cause, where imaging or blood tests reveal no pathological condition that could be causing the pain. Chronic pain is understood to be pain that persists beyond the expected time frame required for treatment or pain that is due to progressive non-cancer pain. Cognitive behavior therapy (CBT) is used to treat CPP.

[0029] In this specification, "QOL" is an abbreviation for quality of life, and is used to mean "quality of life" or "quality of life." QOL is influenced by an individual's subjective views, values, outlook on life, etc., and even in the same environment, the level of QOL can vary depending on how the individual perceives it. QOL can vary based on individual subjectivity, and it is difficult to measure its level. However, evaluation scales that analyze responses to a large number of questions have emerged as relatively objective measures, and known examples of such evaluation scales include the SF-36 (registered trademark) (MOS Short-Form 36-Item Health Survey) and the EQ-5D.

[0030] In one embodiment, the present invention relates to a method for testing nociceptive pain (such as osteoarthritis pain) in a subject. The method includes obtaining an expression level of let-7a-5p in a blood sample obtained from the subject, obtaining an expression level of a control of let-7a-5p, and comparing the expression level of let-7a-5p in the blood sample with the expression level of the control of let-7a-5p. In the method, a decrease in the expression level of let-7a-5p in the blood sample obtained from the subject, compared to the expression level of the control, is an indicator of the presence of nociceptive pain (such as osteoarthritis pain). That is, when the expression level of let-7a-5p in the blood sample obtained from the subject is lower, compared to the expression level of the control, nociceptive pain (such as osteoarthritis pain) is assessed to be present.

[0031] The methods of the present invention utilize a blood sample obtained from a subject and are performed in vitro.

[0032] In one embodiment, the present invention provides a method for testing nociceptive pain in a subject, comprising: (i) obtaining the expression level of one or more miRNAs selected from Group A miRNAs (hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the miRNA expression levels obtained in (i) and (ii). In the method, a decrease in the expression level of the miRNA in the blood sample obtained from the subject compared to the control expression level is indicative of the presence of nociceptive pain. That is, if the expression level of Group A miRNA in a blood sample obtained from the subject is lower compared to the expression level of a control, it is assessed as indicating the presence of nociceptive pain (such as osteoarthritis pain).

[0033] In one embodiment, the present invention provides a method for testing nociceptive pain in a subject, comprising: (i) obtaining the expression level of one or more miRNAs selected from group B miRNAs (hsa-miR-412-3p, hsa-miR-6794-3p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the miRNA expression levels obtained in (i) and (ii). In the method, an increase in the expression level of the miRNA in the blood sample obtained from the subject, compared to the control expression level, is an indicator of the presence of nociceptive pain. That is, when the expression level of group B miRNA in the blood sample obtained from the subject is high, compared to the control expression level, nociceptive pain (such as osteoarthritis pain) is assessed to be present.

[0034] In one embodiment, the present invention provides a method for testing for nociceptive pain in a subject, comprising: (i) obtaining the expression level of a group C (hsa-miR-373-5p) miRNA in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the miRNA expression levels obtained in (i) and (ii). In the method, a decrease in the expression level of the miRNA in the blood sample obtained from the subject compared to the control expression level is an indicator of the presence of nociceptive pain. That is, when the expression level of group C miRNA in the blood sample obtained from the subject is lower compared to the control expression level, nociceptive pain is assessed as being present.

[0035] In one embodiment, the present invention provides a method for testing nociceptive pain in a subject, comprising: (i) detecting mimics of group D (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3p, hsa-miR-4515, hsa-miR-2113, hsa-miR-6747-3p, hsa-miR-1 The method includes (i) obtaining the expression level of one or more miRNAs selected from the group D miRNAs (hsa-miR-288-3p, hsa-miR-574-5p, hsa-miR-4639-3p, hsa-miR-196a-1-3p), (ii) obtaining a control expression level for the same miRNA as in (i), and (iii) comparing the miRNA expression levels obtained in (i) and (ii). In the method, a decrease in the expression level of the miRNA in a blood sample obtained from the subject, compared to the control expression level, is an indicator of the presence of nociceptive pain. That is, if the expression level of the Group D miRNA in a blood sample obtained from the subject is high, compared to the control expression level, nociceptive pain is assessed to be present.

[0036] In one embodiment, the present invention provides a method for testing for nociceptive pain and nociceptive-mediated pain in a subject, comprising: (i) obtaining the expression level of one or more miRNAs selected from group E miRNAs (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the miRNA expression levels obtained in (i) and (ii). In the method, a decrease in the expression level of the miRNA in the blood sample obtained from the subject, compared to the control expression level, is an indicator of the presence of pain related to nociceptive pain and nociceptive-mediated pain. That is, when the expression level of group E miRNA in the blood sample obtained from the subject is low, compared to the control expression level, it is assessed that nociceptive pain and nociceptive-mediated pain are present.

[0037] In one embodiment, the present invention relates to a method for assessing the therapeutic effect of a therapeutic intervention on nociceptive pain, comprising: (i) obtaining plasma concentrations of one or more miRNAs selected from group A miRNAs (hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289) from a blood sample obtained from a subject before treatment; (ii) obtaining plasma miRNA concentrations for the same miRNAs as in (i) from a blood sample obtained from the subject after treatment; and (iii) comparing the plasma miRNA concentrations obtained in (i) and (ii) before and after treatment. In the method, an increase in the concentration of miRNA in plasma after treatment serves as an indicator for determining whether a therapeutic intervention for nociceptive pain has a therapeutic effect.

[0038] In one embodiment, the present invention relates to a method for assessing the therapeutic effect of a therapeutic intervention for nociceptive pain, comprising: (i) obtaining plasma concentrations of one or more miRNAs from a blood sample obtained from a subject before treatment, (ii) obtaining plasma concentrations of the same miRNAs as in (i) from a blood sample obtained from the subject after treatment, and (iii) comparing the plasma miRNA concentrations obtained in (i) and (ii) before and after treatment. In this method, a decrease in plasma miRNA concentration after treatment serves as an indicator for determining the therapeutic effect of the therapeutic intervention for nociceptive pain.

[0039] In one embodiment, the present invention relates to a method for assessing the therapeutic effect of a therapeutic intervention for nociceptive pain, comprising: (i) obtaining the concentration of a group C (hsa-miR-373-5p) miRNA in plasma from a blood sample obtained from a subject before treatment; (ii) obtaining the concentration of the same miRNA in plasma from a blood sample obtained from the subject after treatment; and (iii) comparing the miRNA concentrations in plasma obtained in (i) and (ii) before and after treatment. In this method, an increase in the miRNA concentration in plasma after treatment serves as an indicator for determining the therapeutic effect of the therapeutic intervention for nociceptive pain.

[0040] In one embodiment, the present invention relates to a method for assessing the therapeutic effect of a therapeutic intervention on nociceptive pain, wherein the method comprises: (i) detecting group D mitochondria (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3p, hsa-miR-4515, hsa-miR-2113, hsa-miR-6747-3p, hsa-miR-1288-3p, hsa-miR-574-5 ... (i) obtaining the concentration of one or more miRNAs selected from the miRNAs (hsa-miR-4639-3p, hsa-miR-196a-1-3p), (ii) obtaining the plasma miRNA concentration for the same miRNA as in (i) from a blood sample obtained from the subject after treatment, and (iii) comparing the plasma miRNA concentrations obtained in (i) and (ii) before and after treatment. In this method, a decrease in the plasma miRNA concentration after treatment serves as an indicator for determining whether a therapeutic intervention for algesia-modulated pain has a therapeutic effect.

[0041] In one embodiment, the present invention relates to a method for assessing the therapeutic effect of a therapeutic intervention for nociceptive pain and nociceptive pain, comprising: (i) obtaining plasma concentrations of one or more miRNAs selected from group E miRNAs (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p) from a blood sample obtained from a subject before treatment; (ii) obtaining plasma miRNA concentrations for the same miRNAs as in (i) from a blood sample obtained from the subject after treatment; and (iii) comparing the plasma miRNA concentrations obtained in (i) and (ii) before and after treatment. In this method, an increase in plasma miRNA concentration after treatment serves as an indicator for determining the therapeutic effect of the therapeutic intervention for nociceptive pain and nociceptive pain.

[0042] In one embodiment, the present invention relates to a method for assessing the severity of nociceptive pain and osteoarthritis in a subject. The method comprises obtaining an expression level of let-7a-5p in a blood sample obtained from the subject, obtaining an expression level of a control of let-7a-5p, and comparing the expression level of let-7a-5p in the blood sample with the expression level of the control of let-7a-5p. In the method, a decrease in the expression level of let-7a-5p in the blood sample obtained from the subject, compared to the expression level of the control, is an indicator of the severity of the nociceptive pain and osteoarthritis patient. That is, a lower expression level of let-7a-5p in the blood sample obtained from the subject, compared to the expression level of the control, indicates a higher severity of the nociceptive pain and osteoarthritis patient.

[0043] In one embodiment, the present invention relates to a method for assessing the severity of nociceptive pain and osteoarthritis in a subject, the method comprising: (i) obtaining the expression level of one or more miRNAs selected from group A miRNAs (hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the miRNA expression levels obtained in (i) and (ii). A decrease in the miRNA expression level in the blood sample obtained from the subject compared to the control expression level is an indicator of the severity of the nociceptive pain in the subject. That is, when the expression level of group A miRNA in a blood sample obtained from the subject is lower compared to the expression level of a control, the severity of the nociceptive pain patient and osteoarthritis patient is assessed to be high.

[0044] In one embodiment, the present invention relates to a method for assessing the severity of nociceptive pain and osteoarthritis in a subject. The method comprises: (i) obtaining the expression level of one or more miRNAs selected from group B miRNAs (hsa-miR-412-3p, hsa-miR-6794-3p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the miRNA expression levels obtained in (i) and (ii). An increase in the expression level of the miRNA in the blood sample obtained from the subject compared to the control expression level is an indicator of the severity of the nociceptive pain patient. In other words, a higher expression level of group B miRNA in the blood sample obtained from the subject compared to the control expression level indicates a higher severity of the nociceptive pain patient and osteoarthritis patient.

[0045] In one embodiment, the present invention relates to a method for assessing the severity of an allopathic pain patient in a subject. The method comprises: (i) obtaining the expression level of a group C (hsa-miR-373-5p) miRNA in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the miRNA expression levels obtained in (i) and (ii). A decrease in the expression level of the miRNA in the blood sample obtained from the subject compared to the control expression level is an indicator of the severity of the allopathic pain patient. That is, if the expression level of group C miRNA in the blood sample obtained from the subject is lower compared to the control expression level, the severity of the allopathic pain patient is assessed as being high.

[0046] In one embodiment, the present invention relates to a method for assessing the severity of algesic pain in a subject. The method includes (i) obtaining the expression level of one or more miRNAs selected from group D miRNAs (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3p, hsa-miR-4515, hsa-miR-2113, hsa-miR-6747-3p, hsa-miR-1288-3p, hsa-miR-574-5p, hsa-miR-4639-3p, hsa-miR-196a-1-3p) in a blood sample obtained from the subject, (ii) obtaining a control expression level for the same miRNA as in (i), and (iii) comparing the miRNA expression levels obtained in (i) and (ii). An increase in the expression level of the miRNA in the blood sample obtained from the subject, compared to the expression level of the control, is an indicator of the severity of the pain in the patient with algesia-modulated pain. That is, when the expression level of the miRNA in group D in the blood sample obtained from the subject is high, compared to the expression level of the control, the severity of the pain in the patient with algesia-modulated pain is evaluated as being high.

[0047] In one embodiment, the present invention relates to a method for assessing the severity of nociceptive and nociceptive pain in a subject. The method includes: (i) obtaining the expression level of one or more miRNAs selected from group E miRNAs (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the miRNA expression levels obtained in (i) and (ii). A decrease in the expression level of the miRNA in the blood sample obtained from the subject compared to the control expression level is an indicator of the severity of the nociceptive and nociceptive pain in the patient. That is, a lower expression level of the group E miRNA in the blood sample obtained from the subject compared to the control expression level indicates a higher severity of the nociceptive and nociceptive pain in the patient.

[0048] In this specification, the severity of nociceptive pain patients specifically refers to the severity of patients who visit the hospital due to joint deformity and pain, but not severe enough to require surgery. The severity of nociceptive pain patients and osteoarthritis patients correlates with low expression levels of miRNAs described in Group A herein, and correlates with high expression levels of miRNAs described in Group B herein. The severity of osteoarthritis patients is described as early, early, advanced, and end stages as explained below. Specifically, osteoarthritis of the hip joint is divided into early, early, advanced, and end stages based on plain frontal X-ray images of the hip joint: I. Early hip joint osteoarthritis: There is no narrowing of the joint space, and no sclerosis of the subchondral bone is observed. II. Early hip joint osteoarthritis: The joint space is slightly narrowed, and sclerosis is observed in the subchondral bone. III. Advanced hip joint osteoarthritis: In addition to sclerosis of the subchondral bone, the joint space is clearly narrowed, and osteophytes and bone cysts appear. IV. End-stage hip joint osteoarthritis: In addition to sclerosis of the subchondral bone, osteophytes, and bone cysts, the joint space disappears. The severity of osteoarthritis increases as the stage progresses, from early to early, advanced to end-stage.

[0049] In the present specification, the severity of algesia-modulated pain is measured as follows: for example, in the case of a disease in which central sensitization is thought to be involved and whose primary symptom is pain, the more comorbid fibromyalgia, temporomandibular joint disorder, migraine / tension-type headache, irritable bowel syndrome, nonspecific lower back pain, or chronic pelvic pain is, the more severe the pain becomes. The severity of algesia-modulated pain in patients is correlated with low expression levels of the miRNAs described in Group C herein and with high expression levels of the miRNAs described in Group D herein.

[0050] Currently, pain assessment methods include the Numerical Rating Scale (NRS), in which patients answer by giving a pain score. Widely used pain assessment methods are the visual analogue scale (VAS), the visual rating scale (VRS), and the NRS.

[0051] In one embodiment, the present invention relates to a kit comprising a reagent for measuring the amount of a biomarker of the present invention. As used herein, the term "kit" refers to a unit in which the components to be provided (e.g., test agent, diagnostic agent, reagent, instruction manual, etc.) are provided, typically divided into two or more compartments. Reagents for measuring the amount of a biomarker of the present invention include reagents for measuring miRNA, particularly reagents for measuring miRNAs described in Groups A to E of the present specification. Reagents for measuring miRNA include those compatible with detection methods such as Northern blot, microarray, QCM sensor measurement, and real-time PCR. Examples of reagents include probes and primers for detecting miRNA, particularly miRNAs described in Groups A to E of the present specification, various enzymes, buffers, washing solutions, lysis solutions, etc. In addition, materials, equipment, etc. for detecting miRNA may also be included. The kit of the present invention can be used to evaluate the severity of nociceptive pain (e.g., pain from osteoarthritis (OA)) and nociceptive pain, or the severity of nociceptive pain, osteoarthritis, and nociceptive pain. The kit of the present invention can be used to determine the therapeutic effect of a therapeutic intervention on nociceptive and / or nociceptive pain.

[0052] In one embodiment, the present invention relates to a method for diagnosing or aiding in the diagnosis of nociceptive pain (such as osteoarthritis pain) and nociceptive pain by detecting the expression levels of miRNAs described in Groups A to E herein.

[0053] As used herein, the term "diagnosis" refers to determining the presence or characteristics of a pathological condition in a subject, including determining pain. By using the biomarkers or methods of the present invention, conditions within the body can be examined, and such information can be used to select various parameters, such as the condition in the subject, and the treatment or prophylactic formulation or method to be administered.

[0054] Examples of the present invention are shown below, but the present invention is not limited to the following examples. Those skilled in the art can make many variations and modifications based on the description in this specification without departing from the technical scope of the present invention.

[0055] Example 1

[0056] In this example, the following three groups were analyzed: Osteoarthritis of the hip (OA) group, Chronic primary pain (pain of unknown cause) (CPP) group, and Control group (people without pain).

[0057] OA Group: The OA group consisted of patients scheduled for surgery for hip osteoarthritis. OA group: (1) Patients scheduled for total hip replacement who were in good general condition with an ASA (American Society of Anesthesiologists) classification of 1 or 2; (2) Patients aged 20 to 80 years; (3) Patients who understood the purpose and content of this study and voluntarily provided written consent to participate in the study. Excluded were: (1) patients with severe pain in areas other than the hip joint; (2) patients with severe cognitive impairment (delirium, dementia, intellectual disability, or other mental impairment) at the time of pre-examination; (3) patients with difficulty communicating, reading, or writing in Japanese; (4) patients with contraindications for MRI imaging (because this study was conducted simultaneously with a head MRI); and (5) patients deemed inappropriate for the study by the principal investigator. All patients completed a medical questionnaire before surgery. An initial blood sample was drawn in the operating room. A second blood sample was drawn at the 6-month follow-up visit, and a medical questionnaire was completed.

[0058] CPP group: The CPP group consisted of patients who had persistent pain and were scheduled for cognitive behavioral therapy, even though imaging and blood tests showed no pathological conditions that could be causing pain. The inclusion criteria were as follows: (1) patients with pain lasting for more than three months, (2) patients aged 20 to 80 years, and (3) patients who understood the purpose and content of the study and could submit and confirm written consent to participate in the study. The following patients were excluded: (1) those with a medical condition requiring immediate treatment, (2) those with a history of alcohol or drug abuse or those currently hospitalized, (3) those with a history of manic or psychotic states or those currently hospitalized, (4) those attempting suicide, (5) those with severe cognitive impairment, (6) those with difficulty communicating, reading, or writing in Japanese, (7) those with ongoing pain due to an accident or surgery, (8) those seeking compensation (workers' compensation, welfare assistance) or litigation for pain, (9) those with a current pain level of 10 out of 10, (10) those with contraindications to MRI imaging (because this study was conducted simultaneously with a head MRI), and (11) those deemed inappropriate for participation by the principal investigator. Patients in the CPP group received a total of eight sessions of cognitive behavioral therapy administered by a clinical psychologist. Post-treatment evaluations were conducted 3 months after the completion of cognitive behavioral therapy (approximately 6 months after the start of cognitive behavioral therapy). Blood samples were taken before and six months after the start of cognitive behavioral therapy.

[0059] The control group was defined as patients who met the following criteria: (1) no pain lasting for more than 3 months, (2) were aged 20 to 80 years, and (3) understood the purpose and content of the study and provided written informed consent. The exclusion criteria for the control group were the same as those for the CPP group. After consent was obtained, blood samples were taken and a medical questionnaire was completed on the same day.

[0060] In the OA and CPP groups, blood samples were obtained before and after treatment for evaluation.

[0061] Blood samples were centrifuged at 3000 rpm for 10 minutes at 4°C within 1 hour of collection. Plasma was then aliquoted and centrifuged at 3400 rpm (2000 G) for 10 minutes at 4°C. Plasma was then stored at -30°C or -80°C.

[0062] RNA extraction from plasma was performed using the mirVana PARIS system (Thermo Fisher Scientific Inc.). Caenorhabditis Elegans miR-39-3p (cel-miR-39-3p) was added. Total RNA was extracted from plasma, and miRNA was purified.

[0063] Reverse transcription was performed using the TaqMan™ MicroRNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using miRNA-specific primers. Primers were purchased from Thermo Fisher Scientific Inc. (TaqMan MicroRNA Assay Catalog Number: 4427975 ID: 000377).

[0064] Quantification by real-time PCR PCR was performed using a TaqMan Fast Advanced Master Mix kit (Thermo Fisher Scientific Inc.) on a StepOne® Plus real-time PCR system (Thermo Fisher Scientific Inc.).

[0065] Statistical Analysis: The primary endpoint was to identify miRNAs with significant differences between the CPP and OA groups when comparing pre- and post-treatment. OA treatment consisted of surgery, while CPP treatment consisted of cognitive behavioral therapy. Sample size was calculated using G*Power software version 3.1, with a minimum significance level (α) of 0.05 and a statistical power (1-β) of 0.80. Assuming an effect size of 0.8 for the paired t-test, a significance level of 0.05, a statistical power of 0.80, and a two-sided test, 12 patients were required. Considering the possibility of withdrawals and ineffectiveness, the sample size was set at 15 for the CPP group and 15 for the OA group. The control group consisted of 10 patients, due to the cost of miRNA measurement. In the OA group, 13 patients were evaluated before surgery and 11 before and after surgery. In the CPP group, 11 patients were evaluated before cognitive behavioral therapy and 8 after cognitive behavioral therapy. All statistical and correlation analyses were performed using JMP Statistical Discovery™ software version 14.2 (SAS Institute, Cary, North Carolina, USA). Differences between two groups were identified by analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparison test. For longitudinal data, a paired t-test was used to determine significance between two groups. All tests were two-sided, and a P < 0.05 was considered statistically significant. Receiver operating characteristic curves (ROC) were plotted, and the area under the curve (AUC) was estimated with a 95% confidence interval (CI).

[0066] The ROC curve in Figure 1A shows the OA group as positive and the CPP + control group as negative. The cutoff value calculated using Youden's index was 0.204201. The sensitivity was 86.4% and the specificity was 95.2%. In other words, if the cutoff for let-7a-5p is set to 0.204201, the probability of identifying OA patients as having OA is 86.4%, and the probability of identifying normal individuals (those without OA) as normal is 95.2%. AUC is the area under the ROC curve (AUC), and the larger this area, the higher the diagnostic ability. Specifically, an AUC of 0.9 or higher is considered to have high diagnostic ability.

[0067] The ROC curve in Figure 1B shows the OA group as positive and the control as negative. When the cutoff value of let-7a-5p was set at 0.204201, the sensitivity was 86.4% and the specificity was 100%.

[0068] Figure 2 shows the expression levels of let-7a-5p in blood samples taken before treatment in the OA group (OA), CPP group (CPP), and control group (Cont). In the OA group, let-7a-5p was clearly suppressed compared to the CPP group and the control group.

[0069] 3 shows the results of let-7a-5p expression levels in blood samples taken after treatment. In the CPP group, let-7a-5p did not decrease before and after treatment.

[0070] Figure 4 shows the results of QOL and pain assessments and blood sampling before and after surgery in patients with osteoarthritis of the hip.

[0071] Table 1 shows the changes in QOL and pain before and after surgery in patients with osteoarthritis of the hip.

[0072] Postoperative quality of life and pain in patients with osteoarthritis of the hip were significantly improved. Among miRNAs, only let-7a-5p significantly increased after surgery (Figure 4). Preoperative let-7a-5p levels were lower compared to the control group (Figure 4). Therefore, the pain in the CPP group and the pain in the OA group are thought to be of different types.

[0073] The above results indicate that the expression of let-7a-5p is significantly reduced in patients before hip osteoarthritis surgery compared to controls and other patients with chronic pain, and that the expression of let-7a-5p increases after hip osteoarthritis surgery is completed and pain improves.

[0074] Example 2

[0075] Plasma RNA extraction and miRNA expression profiling using miRNA microarray

[0076] Blood samples taken before and after intervention for nociceptive pain were obtained from two patients undergoing total hip arthroplasty (THA), while blood samples taken before and after intervention for nociceptive pain were obtained from two patients with chronic pain syndrome (CPP) undergoing cognitive behavioral therapy (CBT).

[0077] Plasma RNA was extracted from plasma using 3D-Gene RNA extraction reagent from liquid sample (Toray Industries, Kamakura, Japan) according to the manufacturer's instructions. The extracted total RNA was analyzed using a bioanalyzer (Agilent, CA, USA) and labeled with the 3D-Gene miRNA labeling kit (Toray Industries, Kamakura, Japan). Half of the labeled RNA was hybridized to a 3D-Gene miRNA Oligo chip (Toray Industries, Kamakura, Japan). The probe annotation and oligonucleotide sequence were based on the miRBase miRNA database (http: / / microrna.sanger.ac.uk / sequences / ). After washing, the fluorescent signals were scanned using a 3D-Gene Scanner (Toray Industries, Inc.) and analyzed using 3D-Gene Extraction software (Toray Industries, Inc.).

[0078] The raw data for each spot was normalized by replacing it with the average background signal intensity, determined by the signal intensity of all blank spots at a 95% confidence interval. Spot measurements with signal intensities greater than two standard deviations (SD) above the background signal intensity were considered valid. Relative miRNA expression levels were calculated by comparing the signal intensities of valid spots across the entire microarray experiment. Normalized data were globally normalized for each array so that the median signal intensity was 25.

[0079] Figure 5 shows the results of RNA extraction from plasma obtained from blood samples taken before and after therapeutic intervention for nociceptive pain and blood samples taken before and after therapeutic intervention for nociceptive pain, and miRNA expression profiling using miRNA microarrays.

[0080] Types of pain that can be distinguished in groups A to E and their rationale

[0081] Groups A and B (nociceptive pain markers)

[0082] Group A was extracted from subjects whose miRNA log2 ratios varied within -0.5 to 0.5 before and after therapeutic intervention for nociceptive pain (i.e., miRNAs whose expression levels remained unchanged after therapeutic intervention for nociceptive pain), and whose miRNA log2 ratios were 0.5 or higher before and after therapeutic intervention for nociceptive pain (i.e., miRNAs whose expression levels were clearly elevated after therapeutic intervention for nociceptive pain). Thus, Group A represents a miRNA group whose plasma concentration increases with improvement in nociceptive pain (a miRNA group whose expression levels are suppressed in the presence of nociceptive pain). Conversely, Group B represents a miRNA group whose plasma concentration decreases with improvement in nociceptive pain (a miRNA group whose expression levels are elevated in the presence of nociceptive pain).

[0083] Groups C and D (nociceptive pain markers)

[0084] Group C is a miRNA group that does not change with nociceptive pain therapeutic intervention, but clearly increases in plasma concentration with therapeutic intervention for nociceptive pain (a miRNA group that is suppressed in the presence of nociceptive pain). Therefore, it is thought that nociceptive pain can be differentiated. Conversely, Group D is a miRNA group that does not change with nociceptive pain therapeutic intervention, but clearly decreases in plasma concentration with therapeutic intervention for nociceptive pain (a miRNA group whose expression is elevated in the presence of nociceptive pain).

[0085] Group E (pain marker)

[0086] Group E is a group of miRNAs whose plasma concentrations clearly increase with therapeutic intervention for nociceptive pain and nociceptive pain (a group of miRNAs that are suppressed in the presence of nociceptive pain and nociceptive pain, and whose expression is thought to increase with therapeutic intervention). In Group E, it is thought that it is not possible to distinguish between nociceptive pain and nociceptive pain.

[0087] According to the present invention, by collecting blood from a patient suffering from pain and measuring the expression levels of the miRNAs described in Groups A to E of the present specification, the burden on the patient can be minimized, and nociceptive pain (particularly osteoarthritis pain) and nociceptive-modulated pain can be objectively evaluated and visualized in a minimally invasive, simple, and economical manner. Similarly, the severity of nociceptive pain (particularly osteoarthritis pain) and nociceptive-modulated pain can be evaluated. Furthermore, the therapeutic effect of nociceptive pain and nociceptive-modulated pain can be assessed.

Claims

1. Group A (hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let -7a-5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289) and group B (hsa-miR-412-3p, h Biomarkers for identifying nociceptive pain consisting of one or more of the miRNAs in group C (hsa-miR-373-5p) and group D (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809- and / or a biomarker for discriminating pain related to nociceptive pain and nociceptive pain consisting of one or more of the miRNAs of group E (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p).

2. Group A (hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let-7a -5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289) and group B (hsa-miR-412-3p, hsa-mi Biomarkers for assessing the therapeutic effect on nociceptive pain consisting of one or more of the miRNAs in group C (hsa-miR-373-5p) and group D (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3 A biomarker for determining the therapeutic effect on nociceptive pain consisting of one or more of the miRNAs in group A (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p), and / or a biomarker for determining the therapeutic effect on nociceptive pain and nociceptive pain consisting of one or more of the miRNAs in group B (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p).

3. Group A (hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let- 7a-5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289) and group B (hsa-miR-412-3p, hs Biomarkers for assessing the severity of nociceptive pain patients consisting of one or more of the miRNAs in group A (hsa-miR-373-5p) and group D (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3 A biomarker for evaluating the severity of pain patients with nociceptive pain consisting of one or more of the miRNAs in group E (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p).

4. A method of testing nociceptive pain in a subject, comprising: (i) obtaining an expression level of one or more miRNAs selected from miRNAs of Group A (hsa-miR-26a-5p, hsa-miR-22-5p, hsa-miR-3680-3p, hsa-miR-16-5p, hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-4289) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the expression levels of the miRNA obtained in (i) and (ii), wherein a decrease in the expression level of a miRNA in the blood sample obtained from the subject compared to the control expression level is indicative of the presence of the nociceptive pain.

5. A method of testing nociceptive pain in a subject, comprising: (i) obtaining an expression level of one or more miRNAs of group B (hsa-miR-412-3p, hsa-miR-6794-3p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the expression levels of the miRNA obtained in (i) and (ii), wherein an increase in the expression level of a miRNA in the blood sample obtained from the subject compared to the control expression level is indicative of the presence of nociceptive pain.

6. A method of testing for nociceptive pain in a subject, comprising: (i) obtaining an expression level of miRNA of group C (hsa-miR-373-5p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the expression levels of the miRNA obtained in (i) and (ii), wherein a decrease in the expression level of the miRNA in the blood sample obtained from the subject compared to the control expression level is indicative of the presence of the nociceptive pain.

7. A method for testing nociceptive pain in a subject, comprising: (i) obtaining an expression level of one or more miRNAs selected from the miRNAs of group D (hsa-miR-4305, hsa-miR-7152-5p, hsa-miR-1914-5p, hsa-miR-6809-3p, hsa-miR-4515, hsa-miR-2113, hsa-miR-6747-3p, hsa-miR-1288-3p, hsa-miR-574-5p, hsa-miR-4639-3p, hsa-miR-196a-1-3p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the expression levels of the miRNA obtained in (i) and (ii), wherein an increase in the expression level of the miRNA in the blood sample obtained from the subject compared to the expression level of the control is indicative of the presence of said nociceptive pain.

8. A method for testing for nociceptive and nociceptive pain in a subject, comprising: (i) obtaining an expression level of one or more miRNAs selected from miRNAs of group E (hsa-let-7f-5p, hsa-let-7e-5p, hsa-let-7i-5p, hsa-miR-508-5p) in a blood sample obtained from the subject; (ii) obtaining a control expression level for the same miRNA as in (i); and (iii) comparing the expression levels of the miRNA obtained in (i) and (ii), wherein a decrease in the expression level of a miRNA in the blood sample obtained from the subject compared to the control expression level is indicative of the presence of pain related to nociceptive and nociceptive pain.

9. The method of any one of claims 4 to 8, wherein the "control expression level" is obtained from a pre-operative patient or other chronic pain patient.

10. A kit comprising a reagent for measuring the amount of a biomarker according to any one of claims 1 to 3.

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