Therapeutic agents for PTSD and biomarkers for ptsd

Abstract

Biomarkers for post-traumatic stress disorder (PTSD) and biomarkers for fibromyalgia (FM), which is associated with a high rate of PTSD, have been identified by narrowing down biomarker genes from PTSD-related gene database on the basis of biological events related to disease conditions. In addition, Chinese herbal medicines that are potential therapeutic agents for PTSD have been identified through the examination and comparison of gene induction by Chinese herbal medicines and antipsychotics in human keratinocytes. The present method can provide: biomarkers applicable as indicators for the diagnosis and the development of therapeutic agents for PTSD and FM; effective Chinese herbal medicines for PTSD and FM; and a screening method for therapeutic agents for PTSD and FM.

Description

PTSD treatments and PTSD biomarkers

[0001] The present disclosure relates to a biomarker set for testing for posttraumatic stress disorder (PTSD), and testing and treatment methods.

[0002] Post-traumatic stress disorder (PTSD) is a debilitating mental illness that develops in some people after a major traumatic event. Following a significant emotional shock, the trauma (a psychologically stressful event) resurfaces (flashbacks), resulting in psychological after-effects (psychological trauma), such as anxiety, depression, insomnia, sadness, anger, and feelings of helplessness. Although it is a mental illness, neuroinflammation is also thought to be involved. The lifetime prevalence of PTSD is estimated to be approximately 3.9% worldwide, but in the United States, it is as high as 6-10% of the general population, with higher rates reported among war veterans and survivors of major disasters (Non-Patent Documents 1, 2).

[0003] Treatments for PTSD include prolonged exposure therapy (cognitive behavioral therapy) and anxiolytic SSRIs (selective serotonin reuptake inhibitors), but these are not sufficiently effective. There are no approved medications for flashbacks, the most worrisome symptom. Two SSRIs, paroxetine and sertraline, are approved for the treatment of anxiety in Japan and the United States. However, a significant proportion of PTSD patients do not respond adequately to these SSRIs, and a meta-analysis found that they are less effective than trauma psychotherapy, limiting their availability (Non-Patent Document 1). Furthermore, according to the package inserts for paroxetine and sertraline, the incidence of side effects, such as somnolence, nausea, diarrhea, and dizziness, is approximately 70%. There are also reports that efficacy has not been confirmed in children. Caution is advised, as severe withdrawal symptoms may occur after tapering or discontinuing administration.

[0004] Furthermore, PTSD frequently occurs in fibromyalgia (FM), a chronic pain disorder, with a report stating that the incidence reaches 72% in Europe (Non-Patent Document 3), suggesting a strong correlation. The development of new diagnostic biomarkers for PTSD (Patent Document 1) and therapeutic drugs with novel mechanisms (Patent Documents 2 and 3) has also been disclosed, but these have yet to be put into practical use.

[0005] Patent Application No. 2014-530910 No. 2015-215639 No. 2016-551703

[0006] Hori and Kim, Psychiatry and Clinical Neurosciences, 2019 73:143-153; http: / / onlinelibrary. wiley. com / doi / 10.1111 / pcn. 12820 / full. Nunez-Rios et al. , Biomedicines, 2022 10(5):1107; doi. org / 10.3390 / biomedicines10051107. Gardoki-Souto I, et al. , Pain Res Manag. ,2022 30:2022:2114451,doi: 10.1155 / 2022 / 2114451. Toshiro Sugiyama, Jpn J Psychosom Med, 2019, 59:219-224. Pinero, J. , et al. , Nucleic Acids Res. , 2020, 8; 48 (D1): D845-D855, doi: 10.1093 / nar / gkz1021. Ueno et al. , Biomed Res Int. 2015, 2015:960840. doi:10.1155 / 2015 / 960840. Zhou et al. , Nature Communication, 2019, 10:1523. doi:10.1038 / s41467-019-09234-6.

[0007] Currently, adequate treatment of PTSD is difficult, and no effective drugs that are highly safe and can be applied long-term have been found. This is due to the fact that biomarkers that can serve as diagnostic and therapeutic target molecules have not been narrowed down. There is a need to identify biomarkers related to PTSD that can serve as therapeutic targets and develop diagnostic and therapeutic technologies for them. Furthermore, there are no effective therapeutic drugs for fibromyalgia, which frequently occurs together with PTSD, and there is a need to develop diagnostic and therapeutic methods.

[0008] The present disclosure relates to biomarkers strongly associated with PTSD, and highly safe therapeutic and preventive drugs that use the biomarkers as targets for diagnosis and treatment. The present specification discloses the following: (Item 1) A therapeutic and / or preventive drug for PTSD and / or fibromyalgia, characterized by enhancing the expression of OPRL1 and PNOC and suppressing the expression of CDKN1C and KMT2A. (Item 2) The therapeutic and preventive drug according to Item 1, characterized by containing the herbal ingredients Eujubu (wheat flour), ginger, jujube (rice jujube), and ginseng. (Item 3) A biomarker set for PTSD and / or fibromyalgia, consisting of at least one gene selected from OPRL1, PNOC, CDKN1C, and KMT2A. (Item 4) A method for screening pharmaceuticals using at least one of enhanced expression of OPRL1, enhanced expression of PNOC, suppressed expression of CDKN1C, or suppressed expression of KMT2A as an index.

[0009] The present disclosure makes it possible to provide disease-related markers for diagnosis and therapeutic drugs that are both useful and safe for PTSD. Furthermore, the expression status of PTSD biomarkers makes it possible to select therapeutic drugs appropriate for individual patients and monitor their pathological condition.

[0010] Evaluation results of the suppressive effect of the Kandabashi Prescription herbal medicine on fear memory (PTSD symptoms) in a mouse fear conditioning model. Mice (n=7) were given electric shocks (eight times at 30-second intervals) in an electrical stimulation test chamber to condition fear memory, and then trained to erase the fear memory without stimulation one and two days later. On the 25th day, the mice were again placed in the electrical stimulation test chamber, and the time spent in the absence of stimulation (freezing behavior due to fear memory recall) was measured. Oral administration of the Kandabashi Prescription herbal medicine for 7 days prior to conditioning significantly reduced the time spent freezing during fear recall 25 days later, demonstrating its suppressive effect on fear memory, reflecting PTSD (flashbacks). Narrowing down PTSD-related genes. DisGeNET, a gene-disease association database, contains 418 PTSD-related genes. Based on this, we extracted biological processes of learning and memory, which are characteristic events of PTSD, and identified 72 genes as selective PTSD biomarkers. When the diseases reflected by these 72 genes were analyzed using the enrichment analysis tool Metascape / DisGeNET, PTSD ranked first, indicating that these genes can be used as selective PTSD biomarkers in the neural signaling system. Expression of 72 PTSD marker genes was induced by five herbal medicines. This scatter plot plots the 72 markers on the horizontal axis and the expression ratio (Log2 value) on the vertical axis. The herbal medicines are as follows: KDB (Kandabashi prescription), GYT (Goshuyuto), YKS (Yokukansan), NYT (Ninjinyoeito), and KKT (Kaweikihito). Focusing on the "regions where the expression ratio exceeds ±0.5 (hatched area)," which are determined to be significant, we can see that KDB and GYT show strong gene amplification. Expression induction of 72 PTSD marker genes by five psychotropic drugs (Western medicines). This is a scatter plot with the 72 markers plotted on the horizontal axis and the expression ratio (Log2 value) plotted on the vertical axis. The psychotropic drugs are as follows: PGN; pregabalin, DXN; duloxetine, SPM; escitalopram, MZP; mirtazapine, APZ; and aripiprazole. Focusing on the "region exceeding the expression ratio ±0.5 (hatched area)," which is the region determined to be a significant change, it can be seen that gene amplification is strong in PGN, DXN, and SPM.

[0011] The present invention will be described below. Terms used in this specification have the meanings commonly used in the art unless otherwise specified. [Identification of PTSD Biomarkers] Appropriate diagnosis and treatment of PTSD require biomarkers that reflect the pathological condition, and biomarkers that can be used in clinical tests in particular can serve as target molecules for monitoring the pathological condition and for treatment. At present, no highly selective PTSD biomarkers have been developed. Therefore, the present inventors have attempted to develop a PTSD biomarker that can also be used for diagnosis and the development of therapeutic drugs.

[0012] A comprehensive search for PTSD-related genes in DisGeNET (Non-Patent Document 5), a database containing disease-related genes, resulted in the extraction of 418 genes. These gene groups also showed a similar degree of selectivity for diseases such as psychiatric disorders, depression, and memory disorders, and the results indicated that the set of biomarkers was not specifically narrowed down to PTSD. From a practical standpoint, it is not realistic to test 418 genes in clinical settings, and it has become clear that the identification of genes for PTSD that will lead to new diagnoses and treatments is necessary.

[0013] The present inventors extracted genes related to "chemical signaling" (GO:0007268) and "learning or memory" (GO:0007611), which are biological processes characteristic of PTSD, from the comprehensive 418 DisGeNET databases and identified 72 genes. When the diseases reflected by these 72 genes were analyzed using the enrichment analysis tool Metascape / DisGeNET, PTSD was ranked at the top, indicating that these 72 genes can be used as selective PTSD biomarkers (Figure 2).

[0014] Furthermore, the search was narrowed down by filtering for events related to anxiety and depression, which are the primary symptoms of PTSD. A group of 146 genes related to anxiety and depression was registered in DisGeNET (Mixed anxiety and depressive disorder). Furthermore, to focus on nervous system biomarkers, extraction was performed using the biological process of "synaptic transmission" (GO: 0099536), identifying 43 genes. These 43 genes shared 23 genes with the 72 PTSD-selective genes. Among these PTSD-related genes, the 23 genes related to the primary symptoms of anxiety and depression were ultimately identified as selective biomarker genes for PTSD.

[0015] The 23 identified genes that serve as PTSD biomarkers are as follows: PTSD biomarker genes (symbol, HGNC ID number, gene name): APOE (ID: 613, apolipoprotein E), BDNF (ID: 1033, brain-derived neurotrophic factor), COMT (ID: 2228, catechol-O-methyltransferase), CRH (ID: 2355, corticotropin-releasing hormone), DRD2 (ID: 3023, dopamine receptor D2), DRD3 (ID: 3024, dopamine receptor D3), DRD4 (ID: 3025, dopamine receptor D4), GAD (ID: 4092, glutamate decarboxylase 1), GRIN2A (ID: 4585, glutamate ionotropic receptor NMDA type subunit 2A), GRM2 (ID: 4594, glutamate receptor, metabotropic 2), HTR1A (ID: 5286, 5-hydroxytryptamine receptor 1A), HTR2A (ID: 5293, 5-hydroxytryptamine receptor 2A), HTR2C (ID: 5295, 5-hydroxytryptamine receptor 2C), NPY (ID: 7955, neuropeptide Y), NR3C1 (ID: 7978, nuclear receptor subfamily 3 group C) member 1), OPRK1 (ID: 8154, opioid receptor, kappa 1), OPRL1 (ID: 8155, opiate receptor-like 1), PNOC (ID: 9163, prepronociceptin), OXTR (ID: 8529, oxytocin receptor), P2RX7 (ID: 8537, purinergic receptor P2X7), SLC6A3 (ID: 11049, solute carrier family 6 member 3), SLC6A4 (ID: 11050,solute carrier family 6 member 4), TAC1 (ID: 11517, tachykinin precursor 1). ,

[0016] A notable feature of the individual genes selected as biomarkers was the inclusion of OPRL1 and PNOC, the precursor of its ligand, nociceptin / orphanin FQ2. The simultaneous extraction of endogenous ligands and their specific receptors suggests that they are co-expressed in vivo and regulate neuronal signaling in an autocrine manner. Therefore, it is suggested that OPRL1 and PNOC should be considered as a set, an endogenous system. Generally, therapeutic drugs are designed as agonists or antagonists of either the ligand or the receptor, and the priority of therapeutic targets is thought to vary depending on the pathological mechanism. Interestingly, this study demonstrated the importance of treating the nociceptin / OPRL1 ligand / receptor system as a set. Signal transduction by the endogenous opioid ligand nociceptin is known to be involved in pain threshold regulation, auditory modulation, and memory and learning enhancement, making it consistent with the control of traumatic fear memories through increased expression.

[0017] [Narrowing Down and Identifying Biomarkers for FM with PTSD] Fibromyalgia (FM), a chronic pain disorder, frequently co-occurs with PTSD, with reports suggesting a strong correlation, reaching 72% in Europe (Non-Patent Document 3). Therefore, 43 genes were identified by extracting 143 FM-related genes registered in DisGeNET from the biological processes of "blood flow" (GO: 0008015), "smooth muscle proliferation" (GO: 0048660), and "pain reception" (GO: 0019233), which are particularly related to FM symptoms. These 43 genes share 13 common genes with the 72 PTSD-selective genes, and these 13 genes (see Table 3) were ultimately identified as biomarkers for FM with PTSD.

[0018] The 13 identified FM biomarker genes are as follows: Biomarker genes for FM associated with PTSD (symbol, HGNC ID number, gene name): APOE (ID: 613, apolipoprotein E), COMT (ID: 2228, catechol-O-methyltransferase), CRH (ID: 2355, corticotropin-releasing hormone), DRD3 (ID: 3024, dopamine receptor D3), HTR2A (ID: 5293, 5-hydroxytryptamine receptor 2A), NOS1 (ID: 7872, nitric oxide synthase) 1), NPY (ID: 7955, neuropeptide Y), OPRL1 (ID: 8155, opiate receptor-like 1), PNOC (ID: 9163, prepronociceptin), PTGS (ID: 9605, prostaglandin-endoperoxide synthase 2), SLC6A4 (ID: 11050, solute carrier family 6 member 4), TAC1 (ID: 11517, tachykinin) precursor 1), TACR1 (ID: 11526, tachykinin receptor 1).

[0019] [Identification of Epigenetic Regulation Markers for PTSD] PTSD is understood to occur when an acquired (epigenetic) experience becomes excessively reinforced in memory, resulting in trauma and subsequent flashbacks. We identified two genes involved in the biological process of epigenetic memory-related genes, "epigenetic regulation of gene expression" (GO:0040029): CDKN1C (ID:1786, Cyclin Dependent Kinase Inhibitor 1C) and KMT2A (ID:7132, Lysine Methyltransferase 2A). CDKN1C has been reported to be involved in enhancing behavioral motivation, while KMT2A has been reported to be involved in memory enhancement. Since PTSD flashbacks are caused by the strengthening of acquired (epigenetic) memory circuits due to trauma, it is reasonable to assume that suppressing these genes would lead to the treatment of flashbacks. In fact, the clinically significant therapeutic effect of KDB (Kandabashi prescription) on flashbacks strongly supports this hypothesis. The expression of these two genes was strongly suppressed by KDB and GTY (Goshuyuto), as well as by some psychotropic drugs (see Table 5). It is possible that overexpression of CDKN1C and KMT2A in PTSD is an exacerbating factor, and the mechanisms controlling the overexpression of these two genes could be targets for novel therapeutic drugs.

[0020] [Development of a Chinese herbal medicine prescription for treating PTSD] Using the PTSD biomarker of the present invention, the suitability of existing compounds as PTSD therapeutic agents can be determined from the gene expression rate in cells. The suitability criteria were: (1) the presence of a gene whose expression is enhanced by at least 1.5 times or more among the 23 genes identified as PTSD markers, and (2) the expression of both the OPRL1 and PNOC genes is enhanced.

[0021] Kampo medicines are pharmaceuticals that are combinations of multiple herbal medicines and have been developed in Japan since the Edo period. They generally have few side effects and can be taken over a long period of time. They are considered to be highly suitable for treatments that require long-term medication and high safety standards, such as those for PTSD.

[0022] The so-called "Kandabashi Prescription (KDB)" is a combination of Keishikashakuyakuto (for treating intestinal spasms and abdominal pain) and Shimotsuto (for treating blood deficiency) proposed by psychiatrist Dr. Joji Kandabashi in 2007. Although the individual drugs are not applicable to PTSD, the combined formula has been known empirically to be particularly effective in treating PTSD flashbacks (Non-Patent Document 4). However, the mechanism of action on the neuropsychiatric system was completely unknown.

[0023] Therefore, we evaluated the effects of KDB in a mouse model of PTSD fear memory. After fully extinguishing the conditioned fear of electrical stimulation, we assessed fear memory recall (equivalent to flashbacks) again on day 25. We confirmed that KDB significantly suppressed "freezing behavior," which reflects the level of fear (Figure 1). Furthermore, we treated human keratinocytes with KDB lysate and analyzed the mRNA expression (transcriptome) induced by the drug. We found that five of 23 PTSD biomarker genes were upregulated with an expression ratio of 1.5 or higher, and OPRL1 and PNOC were simultaneously upregulated, indicating that KDB could be a potential therapeutic agent for PTSD. Furthermore, we also found that GYT (Goshuyuto), which is used for analgesia but not for PTSD, met the criteria (Table 1).

[0024] Furthermore, KDB enhanced three biomarker genes for FM with PTSD, and GYT enhanced seven biomarker genes for FM with PTSD at an expression rate of 1.5 or higher, and simultaneously enhanced OPRL1 and PNOC, which are expected to have analgesic effects, demonstrating their potential as effective treatments for FM with PTSD (see Table 3). GTY is easy to combine into a single prescription, and its constituent herbal ingredients are Eujubu (witch hazel) root, ginger, jujube root, and ginseng. Kampo prescriptions containing the main ingredient Eujubu (witch hazel) include Eujubu Tang, Angelica Sigyaku Jia Eujubu (witch hazel) Shojiku Tang, Sigyaku Tang, and Unkei Tang, which can also be used to treat PTSD.

[0025] The 23 PTSD marker genes of the present invention can be easily detected by PCR (Polymerase Chain Reaction) testing using cells and body fluids such as blood and saliva as materials. If developed into a kit, immediate diagnosis is possible, making it widely and easily used for PTSD testing in psychiatric departments of medical institutions. Four distinctive PTSD biomarkers are OPRL1, PNOC, CDKN1C, and KMT2A, and it is recommended that these be examined preferentially. Furthermore, the present invention provides a wide range of technologies for the discovery and selection of PTSD therapeutic compounds, including not only single and combined herbal medicines, but also novel compounds, existing drugs, supplements, food ingredients, and the like.

[0026] [Development of Novel Drug Targets for the Treatment of PTSD] According to the present invention, PTSD is understood to occur when acquired (epigenetic) experiences become excessively reinforced memories, resulting in trauma and subsequent flashbacks. Treatment options include alleviating symptoms such as flashbacks, anxiety, and depression (i.e., positive control through enhanced gene expression, i.e., amplification of OPRL1 and PNOC in the present invention), as well as mechanisms for suppressing the recall of excessively reinforced memories (i.e., negative control through gene suppression). The inventors believe that clues lie in the genes commonly suppressed by KDB and GYT, and have successfully identified CDKN1C and KMT2A as two related genes included in "Epigenetic Control of Gene Expression" (GO:0040029). These four genes, OPRL1, PNOC, CDKN1C, and KMT2A, are useful therapeutic targets for the treatment of PTSD, and can be used as indicators to screen for novel therapeutic agents.

[0027] [Development of personalized medicine for treating PTSD] Gene expression tests such as PCR tests using cells or body fluids based on the above-mentioned PTSD biomarkers can be used to diagnose the appropriateness of PTSD medications. For example, PCR tests can be performed on the blood or saliva of a PTSD patient at the time of diagnosis to identify the expression profile of 23 genes, and a suitable herbal medicine can be prescribed. If multiple candidate herbal medicines are found to be suitable, the most appropriate one can be selected by taking into account the herbal medicine diagnosis of "deficiency / excess" and "qi / blood / water." In some cases, a combination of multiple herbal medicines can also be prescribed.

[0028] Existing Western SSRI prescriptions, specifically paroxetine and sertraline, are routinely prescribed without testing for PTSD biomarkers, resulting in a high incidence of side effects and the associated problem of withdrawal symptoms when treatment is discontinued due to lack of efficacy. The PTSD biomarker test and herbal medicine selection of the present invention make it possible to prescribe a treatment that is tailored to each individual patient. Online consultations are also possible, with testers or physicians notifying test subjects of the biomarker test results and providing information on candidate drug selection via communication lines, making this a convenient method of PTSD treatment.

[0029] The present invention will be described in detail below using examples, but the present invention is not limited to the following examples in any way.

[0030] [Evaluation of Kampo Medicine in Animal Models] The mouse fear conditioning model is used as an animal model of PTSD. The suppressive effect of the Kandabashi prescription on fear memory (PTSD symptoms) was evaluated in this model. C57BL / 6J mice (male, 8 weeks old, n = 7 per group) were given electric shocks (8 mA, 1 second, 30-second intervals, 8 times) in an electrical stimulation chamber to condition fear memory. One and two days after stimulation, the mice were placed in the electrical stimulation chamber and the duration of "freezing behavior" exhibited by the mice for 6 minutes was measured as training for fear memory extinction in the absence of stimulation. Then, on the 25th day, the mice were transferred to the electrical stimulation chamber and the duration of behavior without stimulation ("freezing behavior" due to fear memory recall) was measured. In each experiment, the percentage of "freezing behavior" time relative to the experimental time was calculated and used as an index of fear memory. The test drug, Kandabashi Prescription (a 1:1 mixture of Shimotsuto and Keishikashakuyakuto, manufactured by Tsumura Co., Ltd.), was orally administered at 1.0 g / kg once daily for 7 days prior to the fear conditioning test. The control group received saline. As shown in Figure 1, after the formation of the fear stimulus, placing the animals in the same experimental box in the absence of the fear stimulus erased the fear memory, but it recurred over time. Kandabashi Prescription significantly reduced the freezing time during fear recall 25 days later, confirming its suppressive effect on fear memory, reflecting PTSD (flashbacks), even in animal models.

[0031] [Narrowing down and identifying biomarkers for PTSD and FM] DisGeNET, a database of disease-related genes, can be used to comprehensively search for PTSD-related genes reported in literature. This database contains genes that have been found to be associated with disease through GWAS (genome-wide association studies) and SNP (single nucleotide polymorphism) analysis, and many of these genes have been collected based on the possibility that abnormal or decreased expression may cause the disease. However, even if hundreds of associated genes are identified for each disease, this is too many to be tested, and for practical purposes, it is necessary to narrow down the number to around several dozen genes (biomarkers).

[0032] The tool used for filtering by pathological biological process (Gene Ontology-Biological Process: GO-BP) was Metascape (Non-Patent Document 7), a bioinformatics web tool.

[0033] The inventors attempted to narrow down the 418 genes associated with PTSD (UMLS CUI: C0038436) registered with DisGeNET to several dozen genes. First, genes related to "chemical signaling" (GO: 0007268) and "learning or memory" (GO: 0007611), which are biological processes characteristic of PTSD, were extracted from the comprehensively registered 418 genes, and 72 genes were identified. When the diseases reflected by these 72 genes were analyzed using the enrichment analysis tool Metascape / DisGeNET, PTSD was displayed as the top-ranked related disease, as shown in Figure 2, indicating that these 72 genes can serve as selective PTSD biomarkers.

[0034] We decided to analyze the enhanced expression of the identified 72 genes by herbal medicines and psychotropic drugs using cultured cells. The method is as follows. [Gene expression analysis in human keratinocytes] Normal human-derived epidermal keratinocytes (NHEK-Ad, Lonza) were cultured at a density of 2 × 10 in serum-free cell culture medium (KGM-Gold™ Bullet Kit, Lonza). ５ The cells were seeded at 2 mL / well in a 6-well culture plate and incubated under CO ２ In an incubator (37°C, 5% CO ２ After confirming that the cells had reached confluence, the maintenance medium was removed and 1.8 mL of drug treatment medium was added. 200 μL of each test substance at a concentration 10 times the final concentration was added to this cell culture medium, and the cells were incubated overnight in a CO atmosphere. ２ In an incubator (37°C, 5% CO ２) overnight and then treated with drugs. Total RNA extraction from cells for transcriptome analysis was performed by removing the medium from the 6-well culture plate, washing with ice-cold D-PBS(-), and then adding 2-mercaptoethanol-containing Buffer RLT Plus (included in the RNeasy™ Plus Mini Kit) for RNA extraction. Total RNA extraction was performed according to the extraction method in the kit protocol (RNeasy™ Plus Mini Kit, QIAGEN). The extracted RNA was confirmed to have no problems with RNA degradation (RIN value 9.6 or higher) using an Agilent 2100 Bioanalyzer System. Human mRNA transcriptome analysis was performed using a microarray (3D-Gene mRNA Oligo chip, AROS (trademark) equipped with 24,460 probes, Toray Industries, Inc.) according to the method described in Non-Patent Document 6.

[0035] Figure 3 shows a scatter plot of the expression induction of 72 PTSD marker genes by five herbal medicines, plotting the 72 markers on the horizontal axis and the expression ratio (Log2 value) on the vertical axis. The herbal medicines are represented as KDB (Kandabashi prescription), GYT (Goshuyuto), YKS (Yokukansan), NYT (Ninjinyoeito), and KKT (Kamikihito). Focusing on the "region of expression ratio exceeding ±0.5 (hatched area)," which is the region determined to be significantly different, strong gene amplification was found in KDB, GYT, and YKS. As mentioned above, KDB is known to have a high clinical therapeutic effect against flashbacks, and the selected 72 genes have been shown to be useful as biomarkers for PTSD. Furthermore, strong gene amplification of GYT and YKS genes was also observed, suggesting their potential as therapeutic agents for PTSD.

[0036] Figure 4 shows a scatter plot of the expression induction of PTSD72 marker genes for five psychotropic drugs (Western medicines). As mentioned above, the approved drugs for PTSD are the SSRIs paroxetine and sertraline, but these drugs are known to be not very effective. Therefore, five SSRIs other than the approved drugs and psychotropic drugs with antidepressant effects were selected. The psychotropic drugs are represented by PGN (neuropathic pain treatment pregabalin), DXN (antidepressant SNRI duloxetine), SPM (antidepressant SSRI escitalopram), MZP (antidepressant NaSSA mirtazapine), and APZ (atypical antipsychotic aripiprazole). Focusing on the "region exceeding the expression rate ±0.5 (hatched area)," which is determined to be a significant change, strong gene amplification was found in PGN, DXN, and SPM. PGN and DXN are treatments for FM, which is consistent with their use for conditions including PTSD.

[0037] Furthermore, the search was narrowed down by filtering for events related to anxiety and depression, which are the primary symptoms of PTSD. 146 related genes were registered in DisGeNET's Mixed anxiety and depressive disorder (UMLS CUI: C033890). Furthermore, to focus on nervous system biomarkers, extraction was performed using the biological process of "synaptic transmission" (GO: 0099536), identifying 43 genes. These 43 genes shared 23 genes with the 72 PTSD-selective genes, and these 23 genes were ultimately identified as selective biomarker genes for PTSD.

[0038] Examination of individual genes revealed that PNOC, the precursor of OPRL1 and its ligand, nociceptin / orphanin FQ2, was included. The simultaneous extraction of endogenous ligands and their specific receptors suggests that they are co-expressed in vivo and regulate neuronal signaling in an autocrine manner. Therefore, it is suggested that OPRL1 and PNOC should be considered as a set, an endogenous system. While therapeutic drugs are generally designed as agonists or antagonists of either the ligand or receptor, the priority of therapeutic targets is thought to vary depending on the pathological mechanism. Interestingly, this study demonstrated the importance of treating the nociceptin / OPRL1 ligand / receptor system as a set. Signal transduction by the endogenous opioid ligand nociceptin is known to be involved in pain threshold regulation, auditory modulation, and memory and learning enhancement, making it consistent with its involvement in events that control traumatic events such as fear memory.

[0039] The expression induction of 23 PTSD biomarkers in human keratinocytes by five herbal medicines is shown in Table 1. When "expression induction rate ≥ 1.5" and "OPRL1 + PNOC expression" were set as the criteria, KDB and GYT were found to be relevant. KDB has been reported to be a potential cure for PTSD / flashbacks (Non-Patent Document 4), suggesting that GYT may also be effective.

[0040]

[0041] The expression induction of 23 PTSD biomarkers by five psychotropic drugs in human keratinocytes is shown in Table 2. PGN, DXN, and SPM showed high induction ability at an "expression induction rate ≥ 1.5," but did not meet the criterion of "OPRL1 + PNOC expression."

[0042]

[0043] [Narrowing Down and Identifying Biomarkers for PTSD-Associated FM] Fibromyalgia (FM), a chronic pain disorder, frequently co-occurs with PTSD, with reports suggesting a strong correlation, reaching 72% in Europe (Non-Patent Document 3). Therefore, we extracted 143 FM-related genes (UMLS CUI: C0016053) registered in DisGeNET based on the biological processes of "blood flow" (GO: 0008015), "smooth muscle proliferation" (GO: 0048660), and "pain reception" (GO: 0019233), which are particularly related to FM symptoms, and ultimately identified 43 genes. These 43 genes shared 13 genes with the 72 PTSD-selective genes, and these 13 genes were ultimately identified as selective biomarker genes for FM-associated with PTSD.

[0044] The expression induction of 13 biomarkers for FM with PTSD by five herbal medicines in human keratinocytes is shown in Table 3. When the criteria for "expression induction rate ≥ 1.5" and "OPRL1 + PNOC expression" were set, KDB and GYT were included. This suggests that KDB and GYT also have therapeutic effects on FM with PTSD.

[0045]

[0046] Table 4 shows the expression induction of 13 biomarkers for FM with PTSD by five psychotropic drugs in human keratinocytes. PGN and SPM showed high induction potential with an "expression induction rate ≥ 1.5," but did not meet the criterion of "OPRL1 + PNOC expression." Although PGN and DXN are approved as therapeutic agents for FM, their efficacy in FM with PTSD was suggested to be weak.

[0047]

[0048] [Identification of Epigenetic Regulation Markers for PTSD] PTSD is understood to occur when an acquired (epigenetic) experience becomes excessively reinforced in memory, resulting in trauma and subsequent flashback. Focusing on the biological process of genes involved in epigenetic memory, "epigenetic regulation of gene expression" (GO: 0040029), we identified two genes involved in this process: CDKN1C (ID: 1786, Cyclin Dependent Kinase Inhibitor 1C) and KMT2A (ID: 7132, Lysine Methyltransferase 2A). CDKN1C has been reported to be involved in enhancing behavioral motivation, and KMT2A has been reported to be involved in memory enhancement.

[0049] If we hypothesize that PTSD flashbacks are caused by the strengthening of epigenetic memory circuits following trauma, leading to the overexpression of these genes, it is reasonable to assume that suppressing these genes could lead to the treatment of flashbacks. Indeed, the clinical efficacy of KDB in treating flashbacks strongly supports this hypothesis. Expression of these two genes was suppressed by KDB, GTY, and some psychotropic drugs (Table 5). It is possible that overexpression of CDKN1C and KMT2A in PTSD is an exacerbating factor, and the mechanisms by which they are inhibited could provide novel therapeutic targets.

[0050]

[0051] From the above, GYT showed a favorable gene expression profile common to both KDB, which is an effective drug for treating PTSD / flashbacks as an agent that suppresses excessive memory, and PGN and DXN, which are effective in treating FM as original analgesics, indicating that it is an effective treatment for both PTSD without FM and PTSD with FM. Gene expression analysis suggested that its mechanism of action involves two types of regulation: enhanced production of adrenal cortical hormones and autocrine activation of the endogenous NOP system (positive control by enhanced gene expression), as well as suppression of excessive enhancement of acquired memory (negative control by suppressed gene expression).

[0052] [Evaluation of Kampo Medicine in Patients with Anxiety Disorders] A male subject in his 70s experienced anxiety symptoms accompanied by headaches several times a month, so he took the Kampo medicine "Goshuyuto (GYT)" (Kracie Pharmaceuticals) six tablets per day (360 mg per tablet, two tablets per dose, three times a day). As a result, headache and anxiety symptoms improved within the day of taking the medicine. With continued administration, the frequency of headache and anxiety symptoms significantly decreased or disappeared, and symptoms at the time of onset were alleviated to a level that did not interfere with daily life. Goshuyuto is a Kampo medicine prescribed for headaches and vomiting, and is not intended for anxiety symptoms. The subject's improvement and resolution of not only headaches but also anxiety symptoms were demonstrated, demonstrating that Goshuyuto is also effective against anxiety.

[0053] A female subject in her 90s suffered from anxiety without headache and a decrease in daily activity. She took six tablets of the herbal medicine "Goshuyuto" (Kracie Pharmaceutical) daily (360 mg per tablet, two tablets three times a day) for one week. Around the third day of medication, her mood improved, and she began to have more everyday conversations. One week after medication, her anxiety had subsided to the point where it no longer interfered with her daily life. As mentioned above, Goshuyuto is not effective for anxiety symptoms. This subject did not suffer from headaches, but only complained of anxiety symptoms. However, taking Goshuyuto relieved her anxiety. The effectiveness of Goshuyuto for anxiety without headaches is in good agreement with the results of gene expression analysis.

[0054] As described above, biomarkers for PTSD and FM, which frequently co-occurs with PTSD, have been discovered. These biomarkers can be used not only for diagnosis and treatment, but also as indicators for the development of new treatments and therapeutic drugs. To develop therapeutic drugs, human cultured cells can be treated with candidate compounds / drugs and the expression of genes identified as biomarkers for PTSD or FM can be analyzed. Furthermore, the effects of candidate compounds / drugs can be analyzed using animal models such as mice. Effective therapeutic drugs can be screened for PTSD and FM co-occurring with PTSD, for which no effective therapeutic drugs have been previously considered.

Claims

1. A therapeutic and / or prophylactic agent for PTSD and / or fibromyalgia, characterized in that the expression of OPRL1 and PNOC is enhanced and the expression of CDKN1C and KMT2A is suppressed.

2. The therapeutic and prophylactic drug according to claim 1, characterized in that it contains the herbal ingredients Eujubu, ginger, jujube, and ginseng.

3. A biomarker set for PTSD and / or fibromyalgia consisting of at least one of the following genes: OPRL1, PNOC, CDKN1C, and KMT2A.

4. A method for screening a pharmaceutical product using at least one of enhanced expression of OPRL1, enhanced expression of PNOC, suppression of expression of CDKN1C, or suppression of expression of KMT2A as an index.

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