Objective assessment method for post-traumatic stress disorder (PTSD) or acute stress disorder (ASD)

Abstract

The present invention provides a method for determining whether a subject is suffering from post-traumatic stress disorder (PTSD) or acute stress disorder (ASD), the method comprising measuring, in a sample derived from the subject, the amount of at least one selected from the group consisting of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), and 13-hydroxy-9(Z),11(E)-octadecadienoic acid (13-(Z,E)-HODE).

Description

Objective assessment of post-traumatic stress disorder (PTSD) or acute stress disorder (ASD)

[0001] The present invention relates to a method for objectively assessing post-traumatic stress disorder (PTSD) or acute stress disorder (ASD), and more particularly to a method for objectively assessing post-traumatic stress disorder (PTSD) or acute stress disorder (ASD) by measuring lipid oxidation products in a sample derived from a subject.

[0002] In the first half of the 20th century, Hans Selye proposed the hypothalamus-pituitary-adrenal (HPA) axis as an organism's stress response pathway. Since then, there have been studies examining the relationship between PTSD and cortisol (glucocorticoid), which is secreted by the adrenal cortex, and adrenocorticotropic hormone, a superior hormone that enhances cortisol secretion. However, it is known that there is no consistency in the trends of cortisol levels in PTSD patients across studies (Non-Patent Document 1).

[0003] It has been reported that PTSD patients had 122% higher cortisol levels during exposure to traumatic events (Non-Patent Document 2). Baker et al. reported that adrenocorticotropic hormone levels in cerebrospinal fluid were higher in PTSD patients than in healthy controls, but that cortisol excretion in 24-hour urine samples was no different between PTSD patients and healthy controls (Non-Patent Document 3). It has also been reported that cortisol excretion in 24-hour urine samples was lower in PTSD patients than in healthy controls (Non-Patent Document 4). It has also been reported that mRNA for FK506-binding protein 5 (FKBP51 / FKBP5), an inhibitor of the glucocorticoid receptor (GR), was reduced in PTSD patients (Non-Patent Document 5).

[0004] Hyperreactivity of the sympathetic adrenal system in PTSD has also been reported, with reports of elevated urinary noradrenaline levels in PTSD patients (Non-Patent Document 6) and elevated plasma noradrenaline levels (Non-Patent Document 7). However, urinary noradrenaline measurement requires 24-hour urine collection, which is difficult to perform conveniently in an outpatient setting, and plasma noradrenaline increases acutely due to the pain of blood sampling, which is problematic.

[0005] Reduced hippocampal volume has been reported in patients with PTSD (Non-Patent Document 8), but other studies have not replicated these findings (Non-Patent Document 9).

[0006] There is increasing evidence that oxidative stress is involved in the onset and continuation of PTSD, and an increase in the lipid oxidation product malondialdehyde (MDA) (Non-Patent Document 10) and decreased activity of the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) have been observed in PTSD patients (Non-Patent Document 11). However, reactive oxygen species and antioxidant enzymes fluctuate in various diseases, so they cannot be used as specific markers for PTSD.

[0007] There has been an increase in reports on PTSD and inflammation, and the usefulness of proinflammatory cytokines, particularly interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (INF-γ), as well as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), as biomarkers are being investigated (Non-Patent Document 11). However, these markers are also increased in various inflammatory diseases, including infectious diseases, and therefore are not specific markers for PTSD.

[0008] Several mutations in genes related to neurotransmitters and other substances have also been identified as being related to PTSD. These include genes related to the hypothalamic-pituitary-adrenal axis (NR3C1, FKBP5, CRHR1, CRHR2), serotonergic nerve-related genes (SLC6A4, HTR2A, TPH2), dopaminergic nerve-related genes (DRD2 / ANKK1, DRD3, DRD4, SLC6A3, DBH, COMT), GABAergic nerve-related genes (GABRA2), endocannabinoid-related genes (SKA2, CNR1), inflammation-related genes (CRP, IL1B, TNFα), and G proteins. Mutations in various genes have been reported, including mitochondrial receptor-associated genes (RGS2, ADCYAP1, ADRB2), brain-derived neurotrophic factor (BDNF), mitochondrial-related genes (MT-ATP8, MT-ND5), and RNA regulation-related genes (LINC01090), as well as MAOB, NPY, APOE, OXTR, FAAH, PPM1F, SLC18A2, OPRL1, PRKCA, RORA, PRTFDC1, TLL1, ANKRD55, and ZNF626 (Non-Patent Document 12). Mutations in these genes can be used as constitutional susceptibility or high-risk factors for PTSD pathology when exposed to stress, but because PTSD can develop even without these gene mutations, they cannot be considered markers that can objectively evaluate the condition.

[0009] Acute Stress Disorder (ASD) is known as a stress disorder similar to PTSD. ASD also develops due to exposure to a traumatic experience. ASD typically begins immediately after the trauma and lasts for three days to one month, whereas PTSD differs in that symptoms persist for more than one month. Although ASD is a distinct disorder in itself, it is also a prodromal syndrome that occurs before the onset of PTSD.

[0010] There is also a need to establish objective evaluation methods for ASD.

[0011] Biomarkers in posttraumatic stress disorder: overview and implications for future research. Schmidt U, Kaltwasser SF, Wotjak CT. Disease markers. 2013;35(1):43-54. doi: 10.1155 / 2013 / 835876.Higher cortisol levels following exposure to traumatic reminders in abuse-related PTSD. Bernet M Elzinga, Christian G Schmahl, Eric Vermetten, Richard van Dyck, J Douglas Bremner. Neuropsychopharmacology. 2003;28(9):1656-65.Serial CSF corticotropin-releasing hormone levels and adrenocortical activity in combat veterans with posttraumatic stress disorder. D G Baker, S A West, W E Nicholson, N N Ekhator, J W Kasckow, K K Hill, A B Bruce, D N Orth, T D Geracioti Jr Am J Psychiatry. 1999;156(4):585-8. doi: 10.1176 / ajp.156.4.585.Low urinary cortisol excretion in Holocaust survivors with posttraumatic stress disorder. R Yehuda, B Kahana, K Binder-Brynes, S M Southwick, J W Mason, E L Giller. Am J Psychiatry. 1995;152(7):982-6. doi: 10.1176 / ajp.152.7.982.Gene expression patterns associated with posttraumatic stress disorder following exposure to the World Trade Center attacks. Rachel Yehuda, Guiqing Cai, Julia A Golier, Casey Sarapas, Sandro Galea, Marcus Ising, Theo Rein, James Schmeidler, Bertram Muller-Myhsok, Florian Holsboer, Joseph D Buxbaum. Biol Psychiatry. 2009;66(7):708-11. doi: 10.1016 / j.biopsych.2009.02.034. Epub 2009 Apr 25.Sustained urinary norepinephrine and epinephrine elevation in post-traumatic stress disorder. T R Kosten, J W Mason, E L Giller, R B Ostroff, L Harkness. Psychoneuroendocrinology. 1987;12(1):13-20. doi: 10.1016 / 0306-4530(87)90017-5.Plasma norepinephrine and 3-methoxy-4-hydroxyphenylglycol concentrations and severity of depression in combat posttraumatic stress disorder and major depressive disorder. R Yehuda, L J Siever, M H Teicher, R A Levengood, D K Gerber, J Schmeidler, R K Yang. Biol Psychiatry. 1998;44(1):56-63. doi: 10.1016 / s0006-3223(98)80007-3.The relationship between cognitive and brain changes in posttraumatic stress disorder. J Douglas Bremner. Ann N Y Acad Sci. 2006;1071:80-6. doi: 10.1196 / annals.1364.008.A pilot longitudinal study of hippocampal volumes in pediatric maltreatment-related posttraumatic stress disorder M D De Bellis, J Hall, A M Boring, K Frustaci, G Moritz. Biol Psychiatry. 2001;50(4):305-9. doi: 10.1016 / s0006-3223(01)01105-2.Altered lipid peroxidation markers are related to post-traumatic stress disorder (PTSD) and not trauma itself in earthquake survivors. Abdullah Atli, Mahmut Bulut, Yasin Bez, Ibrahim Kaplan, Pinar Guzel Ozdemir, Cem Uysal, Hilal Selcuk, Aytekin Sir. Eur Arch Psychiatry Clin Neurosci. 2016;266(4):329-36. doi: 10.1007 / s00406-015-0638-5.Metabolic and Inflammatory Response in Post-Traumatic Stress Disorder (PTSD): A Systematic Review on Peripheral Neuroimmune Biomarkers.Valerio Dell'Oste, Sara Fantasia, Davide Gravina, Lionella Palego, Laura Betti, Liliana Dell'Osso, Gino Giannaccini, Claudia Carmassi. Int J Environ Res Public Health. 2023;20(4):2937. doi: 10.3390 / ijerph20042937.Assessing and Modelling of Post-Traumatic Stress Disorder Using Molecular and Functional Biomarkers. Skolariki K, Vrahatis AG, Krokidis MG, Exarchos TP, Vlamos P. Biology. 2023;12(8):1050. doi: 10.3390 / biology12081050.

[0012] PTSD and ASD are mental illnesses caused by exposure to strong stress (traumatic experiences), and are serious problems not only in terms of the health of the individual patient but also socioeconomically. Currently, they are diagnosed through subjective interviews based on standards such as the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V) and the International Classification of Diseases, Eleventh Edition (ICD-11), and appropriate diagnostic indicators that enable objective diagnosis have not yet been established.

[0013] In Japan, 1.195 million people suffer from depression and 650,000 people suffer from panic and anxiety disorders (Ministry of Health, Labor and Welfare 2017 Patient Survey). The lifetime prevalence of PTSD is 1.3% (WHO survey), and the lifetime prevalence of ASD is unknown. Furthermore, when Japanese people experience mental illness, they rarely go directly to a psychiatrist, but rather to an internal medicine specialist. However, general internists are not well-versed in diagnosis, and there are many cases where diagnosis and treatment are delayed. Psychiatrists are also eager to find biomarkers that can objectively and accurately evaluate mental illness.

[0014] In the United States, PTSD occurs in 7-8% of the general population and approximately 15% of veterans returning from combat zones.

[0015] It has been reported that individuals with PTSD are approximately 15 times more likely to attempt suicide than healthy individuals, and from the perspective of suicide prevention, there is a need to quickly and accurately diagnose PTSD.

[0016] As a result of extensive research into the above-mentioned problems, the present inventors have found that the amount of a specific lipid oxidation product (e.g., 12-HETE (12-hydroxy-5,8,10,14-eicosatetraenoic acid)) is significantly increased in the blood of PTSD model mice, and have carried out further research based on this finding, thereby completing the present invention. That is, the present invention is as follows.

[0017] [1] A method for determining whether a subject is suffering from post-traumatic stress disorder (PTSD) or acute stress disorder (ASD), comprising measuring the amount of at least one selected from the group consisting of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), and 13-hydroxy-9(Z),11(E)-octadecadienoic acid (13-(Z,E)-HODE) in a sample derived from the subject. [2] The method according to [1], wherein the sample derived from the subject is the subject's blood, serum, plasma, urine, or saliva. [3] The method according to [1] or [2], wherein the subject is a human. [4] Use of at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE as a biomarker for detecting PTSD or ASD. [5] A method for screening candidate substances for therapeutic agents for PTSD or ASD, comprising the following steps: (Step 1) determining the amount of at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in a sample derived from a PTSD model animal or an ASD model animal before fear conditioning; (Step 2) subjecting the PTSD model animal or ASD model animal to fear conditioning; (Step 3) administering a test substance to the PTSD model animal or ASD model animal that has been subjected to fear conditioning; and (Step 4) determining the amount of at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in a sample derived from the model animal to which the test substance has been administered.[6] A method for evaluating the therapeutic effect of a therapy applied to a patient with PTSD or ASD, comprising the following steps: (Step 1) determining the amount of at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in a sample derived from a patient suffering from PTSD or ASD using the method of the present invention; (Step 2) administering a therapy for PTSD or ASD to the patient; (Step 3) determining the amount of at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in a sample derived from the patient after the treatment; and (Step 4) comparing the amount determined in Step 1 with the amount determined in Step 3.

[0018] According to the present invention, it is possible to accurately determine whether a subject suffers from PTSD or ASD using an objective indicator. Furthermore, according to the present invention, it is possible to objectively determine the effect of a therapeutic intervention applied to a patient suffering from PTSD or ASD. In addition, according to the present invention, it is possible to efficiently screen substances that are candidates for therapeutic drugs for PTSD or ASD.

[0019] Figure 1 shows an outline of the mouse fear conditioning experiment. First day (Day 0): A shock generator (electrical stimulation device) was connected to the stimulation cage (a cage consisting of an acrylic box with a grid-like floor covering and black plastic screens on three sides except for the floor and top). Electrical stimulation was controlled by a PC. The behavior of the mice in the stimulation cage was observed. Each mouse was placed in the stimulation cage and allowed to move freely. Their behavior was observed for 8 minutes. At the same time, a 0.3 mA current was passed through the electric wires on the floor three times: for 2 seconds starting 2 minutes after entry, 2 seconds starting 4 minutes after entry, and 2 seconds starting 6 minutes after entry. The mice were then returned to their previous cages for rearing. Control mice were placed in the same stimulation cage and their free-movement behavior was observed for 8 minutes before being returned to the stimulation cage. The time it took for the mice to exhibit freezing during the 8 minutes of free movement was measured. The next day (Day 1): Each electrically stimulated mouse and control mouse was placed in the same stimulation cage as Day 0. No electrical stimulation was applied to either group, and free behavior was observed for 5 minutes. After that, the mice were returned to their previous cages and kept in their previous cages. The time it took for the mice to exhibit a freezing response (freezing) during the 5 minutes of free behavior was measured. The mice that received electrical stimulation on Day 0 had remembered the association (context) between the stimulation cage and the electrical stimulation (fear), and therefore recalled the fear memory and exhibited a freezing response (freezing). By placing the mice in the stimulation cage for these 5 minutes, the establishment of a fear memory that associated (context) the stimulation cage with the electrical stimulation (fear) was confirmed. The day before sampling (Day 2, 6, 13, 27): Each electrically stimulated mouse and control mouse was placed in the same stimulation cage as Day 0. No electrical stimulation was applied to either group, and free behavior was observed for 3 minutes. After that, the mice were returned to their previous cages and kept in their previous cages. The time spent freezing during a 3-minute period of free movement was measured. Sampling (Days 3, 7, 14, and 28): After 4 hours of fasting, blood and brain tissue samples were collected under anesthesia. Figure 2 shows the results of measuring the percentage of time spent freezing per minute in mice during a fear conditioning experiment.The day of electrical stimulation is designated Day 0. Mice sampled on Day 3, Day 7, Day 14, and Day 28 exhibited freezing responses (freezing) on ​​Day 0, Day 1, and the day before sampling. The percentage of freezing responses (freezing) per minute after the stimulation cage is shown. ○: Control group, ■: Fear-conditioned group (PTSD group). Statistical analysis was performed using two-way ANOVA (Bonferroni). P<0.05 was considered significant. Figure 2-1 shows the results of Day 3 and Day 7. Figure 2 shows the results of measuring the percentage of time per minute spent freezing (freezing) by mice in a fear-conditioning experiment. The day of electrical stimulation was designated Day 0. Mice sampled on Days 3, 7, 14, and 28 showed freezing on Day 0, Day 1, and the day before sampling. The percentage of freezing per minute after the stimulation cage is shown. ◯: Control group; ■: Fear-conditioned group (PTSD group). Statistical analysis was performed using two-way ANOVA (Bonferroni). P<0.05 was considered significant. Figure 2-2 shows the results for Days 14 and 28. Figure 3 shows the plasma 12-HETE concentration and the ratio of 12-HETE to arachidonic acid (AA) (12-HETE / AA) in mice subjected to electrical stimulation. □: Control group; ■: Fear conditioning group (PTSD group). *P<0.05 was considered significant. Figure 4 is a schematic diagram illustrating the synthesis pathways of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in mice and humans. Mouse 12 / 15-lipoxygenase (12 / 15-LOX) has the activity of oxidizing arachidonic acid to 12-HETE, but also has the activity of partially producing 15-HETE and oxidizing linoleic acid to 13-(Z,E)-HODE. Meanwhile, enzymes with the same enzymatic activity as mouse 12 / 15-LOX exist in humans as 12-lipoxygenase (12-LOX) and 15-lipoxygenase (15-LOX).12-HETE oxidizes arachidonic acid to 12-HETE, and 15-LOX oxidizes arachidonic acid and linoleic acid to 15-HETE and 13-(Z,E)-HODE, respectively. Figure 5 shows plasma corticosterone concentrations in mice subjected to electrical stimulation. Corticosterone was measured by enzyme-linked immunosorbent assay (ELISA). The day of electrical stimulation was designated Day 0, and plasma corticosterone concentrations were measured for mice sampled on Days 3, 7, 14, and 28. □: Control group; ■: Fear-conditioned group (PTSD group). Figure 6 is a schematic diagram illustrating AMPA-type glutamate receptors (AMPA receptors) and neuroexcitation transmission. When glutamate released from presynaptic terminals binds to AMPA receptors, neuronal excitation occurs in postsynaptic cells, and neuronal excitation is transmitted. Palmitoylation of membrane-resident AMPA receptors results in their intracellular migration and attenuation of excitation transmission. Depalmitoylation re-expresses them on the membrane. Synaptic plasticity and learning and memory are controlled by the increase or decrease of AMPA receptors at synapses. Figure 7 shows the freezing response of AMPA receptor-modified mice (GluA1C811S mice) in which the cysteine ​​at the palmitoylation site of the AMPA receptor was replaced with serine after fear conditioning. In GluA1C811S mice, the formed fear memory was strongly maintained for a long period (4 months) even after repeated exposure to a stimulating environment to extinction learning of context-dependent fear memory. ○: Wild-type mice; ●: GluA1C811S mice. Figure 8 shows a comparison of plasma 12-HETE levels in GluA1C811S and wild-type mice one year after electrical stimulation in a fear conditioning experiment. ○: Wild-type mice; ●: GluA1C811S mice (mutant mice). Figure 9 shows the results of measuring the percentage of time (%) spent freezing during a 3-minute period of free movement in a fear conditioning experiment. The graph shows time-course changes on Day 1 and Day 25 for mice that had not undergone extinction learning (the four left columns) and on Days 1, 2, 3, 4, 5, 7, 14, 21, and 25 for mice that had undergone extinction learning.(□Control, EXT-): Group without fear conditioning and without extinction learning, (□Control, EXT+): Group without fear conditioning and with extinction learning, (■Stress, EXT-): Group with fear conditioning and without extinction learning, (■Stress, EXT+): Group with fear conditioning and with extinction learning. Figure 10 shows the results of measuring the percentage (%) of the freezing response (freezing) time shown by mice on Day 1 and Day 25 (Day 1: 5 minutes stay, Day 25: 3 minutes stay). (□Control, EXT-): Group without fear conditioning and without extinction learning, (■Stress, EXT-): Group with fear conditioning and without extinction learning, (□Control, EXT+): Group without fear conditioning and with extinction learning, (■Stress, EXT+): Group with fear conditioning and with extinction learning. Statistical analysis was performed using ANOVA. P<0.05 was considered significant. Figure 11 shows the plasma 12-HETE concentration in mice and the ratio of 12-HETE to arachidonic acid (AA) (12-HETE / AA). (□Control, no extinction learning): group without fear conditioning and no extinction learning, (■Stress, no extinction learning): group with fear conditioning and no extinction learning, (□Control, with extinction learning): group without fear conditioning and with extinction learning, (■Stress, with extinction learning): group with fear conditioning and with extinction learning. Statistical analysis was performed using ANOVA. P<0.05 was considered significant.

[0020] The present invention will be described in detail below.

[0021] 1. Method for Determining Whether a Subject Has Post-Traumatic Stress Disorder or Acute Stress Disorder The present invention provides a method for determining whether a subject has post-traumatic stress disorder (PTSD) or acute stress disorder (ASD) (hereinafter, sometimes referred to as the "method of the present invention"), which comprises measuring the amount of at least one selected from the group consisting of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), and 13-hydroxy-9(Z),11(E)-octadecadienoic acid (13-(Z,E)-HODE) in a sample derived from the subject.

[0022] As used herein, Post-Traumatic Stress Disorder (PTSD) and Acute Stress Disorder (ASD) refer to a distinctive set of stress symptoms resulting from exposure to a traumatic event that results in psychological shock, such as actual or threatened death, serious injury, or sexual violence. Examples of traumatic events include disasters, violence, serious sexual assault, severe accidents, combat, and abuse. Witnessing someone else involved in such an event or learning that a family member or loved one has been involved can also be a traumatic experience. The experiences of disaster relief personnel can also be traumatic.

[0023] The four main symptoms of PTSD and ASD are:

[0024] 1. Intrusion Symptoms: Unpleasant and distressing memories related to the traumatic event suddenly resurface and / or recur as nightmares, accompanied by emotional upset and / or physiological reactions (e.g., palpitations and sweating).

[0025] 2. Avoidance Symptoms: Trying to avoid remembering or thinking about the traumatic event and / or avoiding people, things, situations, or conversations that remind you of the traumatic event.

[0026] 3. Negative changes in cognition and mood: Negative cognition, loss of interest and concern, feelings of alienation and isolation from those around you, and an inability to experience positive emotions (happiness, love, etc.).

[0027] 4. Marked changes in alertness and reactivity, including irritability, reckless or self-destructive behavior, hypervigilance, exaggerated startle response, difficulty concentrating, and sleep disturbances.

[0028] PTSD is medically diagnosed when the above symptoms persist for more than four weeks and cause significant distress or impairment in social or daily functioning. ASD is medically diagnosed when the onset and resolution of symptoms occur within four weeks of the traumatic event.

[0029] In the method of the present invention, at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE is measured in a sample derived from a subject to determine whether the subject is suffering from PTSD or ASD. The measurement of 12-HETE, 15-HETE, and 13-(Z,E)-HODE may be performed using a method known per se. For example, measurement can be performed using a liquid chromatograph (HPLC), a liquid chromatograph mass spectrometer (LC-MS), a liquid chromatograph tandem mass spectrometer (LC-MS / MS), a gas chromatograph mass spectrometer (GC-MS), or an enzyme-linked immunosorbent assay (ELISA). In a preferred embodiment, the measurement of 12-HETE, 15-HETE, and 13-(Z,E)-HODE can be performed using LC-MS / MS.

[0030] The method for preparing a sample derived from a subject is not particularly limited, and a person skilled in the art can prepare the sample using an appropriate method depending on the measurement method. As a non-limiting example, a sample derived from a subject can be prepared by subjecting plasma (or tissue homogenate, serum, urine, etc.) derived from a subject to addition of an antioxidant, nitrogen substitution, reduction treatment, saponification treatment, and filtration.

[0031] When liquid chromatography (HPLC) is used as the measurement method, for example, a C18 HPLC column (e.g., an octadecyl-silica (ODS) column (e.g., Hypersil Gold, 3.0 μm, 100 × 2.1 mm; manufactured by Thermo Fisher Scientific)) can be used as the separation column. Alternatively, in addition to the column, a chiral column for the purpose of separating optical isomers can also be used in combination. The detector can be appropriately selected from ultraviolet / visible absorbance detectors, multi-wavelength detectors, differential refractive index detectors, fluorescence detectors, photodiode array detectors, electrochemical detectors, and the like. The sample pretreatment conditions can be the same as those described above.

[0032] When a liquid chromatograph mass spectrometer (LC-MS) is used as the measurement method, the same separation column as that described for HPLC can be used. A mass spectrometer can be used as the detector. The sample pretreatment conditions can be the same as those described above.

[0033] When liquid chromatography tandem mass spectrometry (LC-MS / MS) is used as the measurement method, the conditions shown in the Examples of the present specification can be used as a specific example. As with the case of using HPLC as the measurement method, measurement by LC-MS / MS can also be performed using a C18 HPLC column or a combination of such a column and a chiral column.

[0034] When a gas chromatograph mass spectrometer (GC-MS) is used as a measurement method, the conditions reported in a previous paper (Invest Ophthalmol Vis Sci. 2001; 42(2): 328-32. Detection of endogenous 12-hydroxyeicosatrienoic acid in human tear film. P A Mieyal et al.) can be used, but are not limited to these. A brief explanation is given below. The sample pretreatment conditions can be the same as those described above. The pretreated sample can be separated and purified by reverse-phase high-performance liquid chromatography using, for example, an ODS column (e.g., 4.6 x 200 mm; ODS-Hypersil; manufactured by Hewlett-Packard) and a solvent consisting of 80% acetonitrile-water-acetic acid (50:50:0.1 (vol / vol)) and 20% acetonitrile / acetic acid (100:0.1 (vol / vol)) at a flow rate of 1 ml / min for 25 minutes, followed by 100% acetonitrile / acetic acid (100:0.1 (vol / vol)) for 10 minutes. The purified sample can be derivatized to pentafluorobenzyl ester or trimethylsilyl ether. GC-MS can be performed using a capillary gas chromatography column (e.g., DB-1 fused silica, 10 m, inner diameter 0.25 mm, film thickness 0.25 μm; manufactured by J&W Scientific) with helium as the carrier gas at a rate of 25°C / min from 180°C to 300°C.

[0035] When enzyme-linked immunosorbent assay (ELISA) is used as the measurement method, it can be performed according to standard methods using antibodies specific to 12-HETE, 15-HETE, or 13-(Z,E)-HODE. ELISA kits for measuring 12-HETE, 13-(Z,E)-HODE, etc. are commercially available from Enzo Life Sciences, Inc., and these commercially available products may also be used. Furthermore, when using ELISA as the measurement method, an open sandwich system or an aptamer system can also be used.

[0036] The subject in the method of the present invention is not particularly limited as long as it is a mammal that can suffer from PTSD or ASD. Examples include, but are not limited to, humans, monkeys, gorillas, chimpanzees, orangutans, cows, horses, pigs, apes, dogs, cats, mice, rats, rabbits, hamsters, elephants, etc. In a preferred embodiment, the subject is a human.

[0037] The subject in the method of the present invention is a subject who has experienced a traumatic event that may lead to the development of PTSD or ASD. In one embodiment, the method of the present invention can be performed on a subject typically 1 day or more, preferably 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more, 16 days or more, 17 days or more, 18 days or more, 19 days or more, 20 days or more, 21 days or more, 22 days or more, 23 days or more, 24 days or more, 25 days or more, 26 days or more, 27 days or more, 28 days or more, 29 days or more, 30 days or more, 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more after the subject experienced the most recent traumatic event. Additionally, the methods of the present invention can be performed on a subject typically 50 years or less, preferably 45 years or less, 40 years or less, 35 years or less, 30 years or less, 25 years or less, 24 years or less, 23 years or less, 22 years or less, 21 years or less, 20 years or less, 10 years or less, 9 years or less, 8 years or less, 7 years or less, 6 years or less, 5 years or less, 4 years or less, 3 years or less, 2 years or less, or 1 year or less since the subject experienced their most recent traumatic event.

[0038] In one aspect of the method of the present invention, the subject may be a subject who has been determined by a physician or veterinarian to be likely to suffer from PTSD or ASD. Alternatively, in one aspect of the method of the present invention, the subject may be a subject who has been diagnosed by a physician or veterinarian as suffering from PTSD or ASD. In this embodiment, the method of the present invention can be used for the purpose of objectively confirming the diagnosis made by the physician or veterinarian.

[0039] Examples of samples derived from a subject in the method of the present invention include blood (including plasma, serum, and blood cells (red blood cells and white blood cells)), urine, tears, semen, saliva, and lymph. In a preferred embodiment, the sample derived from a subject is blood (including plasma, serum, and blood cells) or urine, and in a particularly preferred embodiment, the sample derived from a subject may be plasma or serum. Only one type of sample may be used, or two or more types may be used to increase the accuracy of the determination.

[0040] In the methods of the present invention, whether a subject suffers from PTSD or ASD can be determined by comparing the amounts of 12-HETE, 15-HETE, and / or 13-(Z,E)-HODE in a sample derived from the subject with those in a sample derived from a healthy individual. If the amounts of 12-HETE, 15-HETE, and / or 13-(Z,E)-HODE in a sample derived from the subject are higher than those in a sample derived from a healthy individual, the subject can be determined to have PTSD. Assessment may also be made by comparing the amounts of 12-HETE, 15-HETE, and / or 13-(Z,E)-HODE with other lipid oxidation products produced by reactive oxygen species. For example, the amounts of 12-HETE, 15-HETE, etc. can be substituted with the ratio to arachidonic acid (12-HETE / AA or 15-HETE / AA), but are not limited thereto. The method of the present invention may be carried out by determining the amount of any one of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in a sample derived from a subject; by determining the amount of a combination of any two of 12-HETE, 15-HETE, and 13-(Z,E)-HODE (e.g., 12-HETE and 15-HETE, 15-HETE and 13-(Z,E)-HODE, or 12-HETE and 13-(Z,E)-HODE); or by determining the amount of all three of 12-HETE, 15-HETE, and 13-(Z,E)-HODE.

[0041] In mammals, the enzymes that produce 12-HETE, 15-HETE, and 13-(Z,E)-HODE are distributed as follows:

[0042] 12-lipoxygenase (*12 / 15-lipoxygenase does not exist in humans) ・Platelet-type 12-lipoxygenase Arachidonic acid → 12-HETE (Humans only have the platelet type) ・Leukocyte-type 12-lipoxygenase Arachidonic acid → 12-HETE, 15-HETE (12-lipoxygenase in leukocytes of pigs, horses, and mice is highly homologous to 15-lipoxygenase and is said to be 12 / 15-lipoxygenase.) 15-lipoxygenase Found in various tissues. Present in humans and rabbits ・15-lipoxygenase-1: Linoleic acid → 13-(Z,E)-HODE ・15-lipoxygenase-2: Arachidonic acid → 15-HETE

[0043] From the viewpoint of enzymatic oxidation, three biomarkers of PTSD or ASD in humans are considered to be 12-HETE, 15-HETE, and 13-(Z,E)-HODE.

[0044] It has been explained above that measuring the amount of 12-HETE or the like in a sample from a subject can determine whether the subject is suffering from PTSD or ASD. In other words, 12-HETE, 15-HETE, and 13-(Z,E)-HODE can be used as biomarkers for detecting PTSD. Therefore, the method of the present invention can be said to provide an invention relating to "use of at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE as a biomarker for detecting PTSD or ASD."

[0045] 2. Method for Treating PTSD or ASD The present invention also provides a method for treating a patient suffering from PTSD or ASD (hereinafter, may be referred to as "the treatment method of the present invention"), which comprises the following steps: identifying a patient suffering from PTSD or ASD using the method of the present invention, and administering a PTSD or ASD treatment method to the patient.

[0046] In the treatment method of the present invention, the PTSD or ASD treatment method is not particularly limited as long as it can improve or alleviate the symptoms of PTSD or ASD. Examples of PTSD or ASD treatment methods include, but are not limited to, stress management, exposure therapy, and drug therapy.

[0047] Stress management is a treatment that helps relieve symptoms of PTSD or ASD by engaging in exercises (e.g., breathing exercises, yoga, meditation, etc.) that reduce and / or control anxiety.

[0048] Prolonged exposure therapy, or exposure therapy, is a type of behavioral cognitive therapy that attempts to eliminate fears that linger from traumatic events in patients with PTSD or ASD. Eye Movement Desensitization and Reprocessing (EMDR) is also an effective psychotherapy for patients with PTSD or ASD.

[0049] Pharmacotherapy is a treatment that attempts to treat the symptoms of PTSD or ASD by administering drugs to a patient with PTSD or ASD that may improve and / or alleviate the symptoms of PTSD or ASD. Examples of therapeutic agents for PTSD or ASD include, but are not limited to, selective serotonin reuptake inhibitors (escitalopram, sertraline, paroxetine, fluvoxamine, fluoxetine, etc.), serotonin-norepinephrine reuptake inhibitors (venlafaxine, etc.), antidepressants (mirtazapine, etc.), atypical antipsychotics (amitriptyline, olanzapine, risperidone, aripiprazole, quetiapine, etc.), mood stabilizers (lamotrigine, valproic acid, topiramate, etc.), sympatholytics (prazosin, etc.), and Chinese herbal medicines (Saiko-ka-ryukku-bouki-to, Saiko-keishi-kankyo-to, Kan-mai-da-zao-to, Kei-shi-ka-ryukku-bouki-to, Kamishyo-yao-san, Hange-kouboku-to, Kamishyo-hi-to, etc.).

[0050] Furthermore, in one embodiment of the treatment method of the present invention, after the step of administering a PTSD or ASD treatment to the PTSD or ASD patient, the determination method of the present invention may be further applied to the PTSD or ASD patient to determine the amount of 12-HETE or the like in the PTSD or ASD patient, and whether the PTSD or ASD treatment administered to the PTSD or ASD patient was effective may be determined. That is, the present invention also provides a method for evaluating the therapeutic effect of a treatment administered to a PTSD or ASD patient (hereinafter, sometimes referred to as the "evaluation method of the present invention"), which comprises the following steps: (Step 1) identifying a patient suffering from PTSD or ASD using the method of the present invention, (Step 2) administering a PTSD or ASD treatment to the patient, and (Step 3) evaluating whether the patient is suffering from PTSD or ASD using the method of the present invention.

[0051] If the patient is still evaluated as having PTSD or ASD in step 3, the PTSD or ASD treatment can be continued, or another PTSD or ASD treatment can be selected. If the patient is evaluated as not having PTSD or ASD in step 3, the PTSD or ASD treatment can be terminated in step 2. In another aspect, the evaluation method of the present invention evaluates the therapeutic effect of a PTSD or ASD treatment administered to a patient. In this aspect, the present invention provides a method for evaluating the therapeutic effect of a therapy applied to a patient with PTSD or ASD, the method comprising the following steps: (Step 1) determining the amount of at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in a sample derived from a patient suffering from PTSD or ASD using the method of the present invention, (Step 2) administering a therapy for PTSD or ASD to the patient, (Step 3) determining the amount of at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in a sample derived from the patient after the treatment, and (Step 4) comparing the amount determined in Step 1 with the amount determined in Step 3. When the amount determined in Step 3 is less than the amount determined in Step 1, the therapy applied to the patient in Step 2 is evaluated to be effective for the patient. Furthermore, by comparing the amount determined in step 1 with the amount determined in step 3, if the amount determined in step 3 is greater than or equal to the amount determined in step 1, it is evaluated that the treatment applied to the patient in step 2 is ineffective for treating the patient.

[0052] 3. Method for screening candidate substances for therapeutic agents for PTSD or ASD The present invention also provides a method for screening candidate substances for therapeutic agents for PTSD or ASD (hereinafter, may be referred to as the "screening method of the present invention"), which comprises the following steps: (Step 1) determining the amount of at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in a sample derived from a PTSD model animal or an ASD model animal before fear conditioning, (Step 2) subjecting the PTSD model animal or ASD model animal to fear conditioning, (Step 3) administering a test substance to the PTSD model animal or ASD model animal that has been subjected to fear conditioning, and (Step 4) determining the amount of at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in a sample derived from the model animal to which the test substance has been administered.

[0053] In the screening method of the present invention, PTSD, ASD, 12-HETE, 15-HETE, and 13-(Z,E)-HODE, the measurement methods therefor, samples, animals, etc. are the same as those explained in the method of the present invention.

[0054] In a preferred embodiment, the model animal may be a mouse model. PTSD and ASD model animals can be prepared by methods known per se. As an example, as described in detail in the Examples below, Hayashi et al., one of the inventors of the present application, have reported that cysteine ​​at the palmitoylation modification site of the AMPA receptor, a major receptor for the excitatory neurotransmitter glutamate, is substituted with serine, thereby making the AMPA receptor-modified mouse (GluA1C811S mouse) non-palmitoylable. This can be used as a PTSD model mouse, but is not limited to this.

[0055] Fear conditioning of model animals can also be performed by methods known per se. As used herein, fear conditioning refers to a process in which a model animal learns to associate a conditioned stimulus (CS) that does not induce a fear response with an unconditioned stimulus (US) that induces a fear response, so that the model animal will exhibit a fear response even when only the conditioned stimulus is presented. The conditioned and unconditioned stimuli that can be used in fear conditioning are not particularly limited as long as they can achieve the desired conditioning in the model animal, and any conditions may be used. Examples of conditioned stimuli include, but are not limited to, sound, light, location, etc. Examples of unconditioned stimuli include, but are not limited to, means of inflicting pain on the model animal (e.g., aversive stimuli such as electric shock, startle sounds, pungent-smelling chemicals, and the smell of a predator).

[0056] In the screening method of the present invention, the method of administering a test substance to a model animal is not particularly limited, and either oral or parenteral administration may be used. When administering a test substance to a model animal orally, the test substance may be mixed with the feed or water of the model animal, or the test substance itself may be administered orally. When administering a test substance to a model animal parenterally, intravenous administration, intraarterial administration, subcutaneous administration, intraperitoneal administration, intramuscular administration, intrathecal administration, etc. may be used, but are not limited to these.

[0057] As mentioned above, it is known that the enzymes that produce 12-HETE, 15-HETE, and 13-(Z,E)-HODE in mammals differ depending on the animal species. Therefore, the optimal assay for measuring 12-HETE, 15-HETE, or 13-(Z,E)-HODE can be selected depending on the model animal species.

[0058] In step 3, the test substance is administered to the model animal. The test substance may be administered once or multiple times. When the test substance is administered multiple times, the number of administrations is not particularly limited, but is usually two or more, preferably three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, twenty-five or more, thirty or more, thirty-five or more, forty or more, forty-five or more, or fifty or more. The number of administrations is usually 100 or less, preferably 95 or less, 90 or less, 85 or less, 80 or less, 75 or less, 70 or less, 65 or less, 60 or less, or 55 or less, but is not limited thereto. When the administration is performed multiple times, the frequency of administration is not particularly limited, but may be, but is not limited to, typically daily, preferably once every two days, once every three days, once every four days, once every five days, once every six days, once every week, once every two weeks, once every three weeks, or once every month. The period from performing step 3 to collecting a sample from the model animal in step 4 is not particularly limited. This period is typically 30 minutes or more after the final administration of the test substance, and may be, but is not limited to, 40 minutes or more, 50 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 24 hours or more, or 48 hours or more. Furthermore, this period is usually within one week after the final administration of the test substance, and may preferably be within 6 days, 5 days, 4 days, 3 days, 48 ​​hours, or 24 hours, but is not limited to these.

[0059] The amount of 12-HETE, 15-HETE, and / or 13-(Z,E)-HODE determined in step 1 is compared with the amount thereof determined in step 4. When the amount determined in step 4 is decreased compared to the amount determined in step 1, the test substance is determined to be a candidate substance for a therapeutic agent for PTSD or ASD.

[0060] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.

[0061] Quantitative Methods In the following examples, lipid oxidation products such as 12-HETE, 15-HETE, and 13-(Z,E)-HODE, arachidonic acid, and linoleic acid were measured by the following methods.

[0062] 1) Add 450 μL of saline or PBS to 50 μL of plasma (or tissue homogenate, serum, or urine) to make a total volume of 500 μL.

[0063] 2) To one sample from 1), add 510 μL of a methanol solution containing internal standards (50 ng of 13-(Z,E)-HODE-d4 and 50 ng of 8-iso-prostaglandin F2α-d4), 1 mM triphenylphosphine, and 100 μM 2,6-di-tert-butyl-4-methylphenol (BHT).

[0064] 3) After replacing the atmosphere with nitrogen, close the lid and let stand at room temperature for 30 minutes while stirring (reduction treatment).

[0065] 4) 500 μL of a 1 M potassium hydroxide-containing methanol solution was added, the mixture was purged with nitrogen again, and treated at 40° C. for 30 minutes (saponification treatment).

[0066] 5) Add 2 ml of 10% aqueous acetic acid solution.

[0067] 6) Add 5 ml of chloroform / ethyl acetate (chloroform:ethyl acetate=4:1) and stir vigorously for 1 minute.

[0068] 7) Centrifuge at 3,000 G for 10 minutes at 4°C.

[0069] 8) Remove the top layer and protein layer by aspiration.

[0070] 9) The lower layer (approximately 5 ml) was completely dried with nitrogen gas.

[0071] 10) Add 200 μl of 70% aqueous methanol solution and stir vigorously.

[0072] 11) Remove impurities using a filter (centrifuge at 14,000 G for 10 minutes at 4°C using a filter with a pore size of 0.22 μm).

[0073] 12) 10 μl of the filtered sample was measured using a liquid chromatograph tandem mass spectrometer (LC-MS / MS).

[0074] 13) Using a liquid chromatograph tandem mass spectrometer (LC-MS / MS), 12-HETE, 15-HETE, 13-(Z,E)-HODE, as well as 9-(E,Z)-HODE (9-hydroxyoctadeca-10(E),12(Z)-dienoic acid), 9-(E,E)-HODE (9-hydroxyoctadeca-10(E),12(E)-dienoic acid), 13-(E,E)-HODE (13-hydroxyoctadeca-9(E),11(E)-dienoic acid), 5-HETE (5-hydroxyeicosa-6,8,11,14-tetraenoic acid), 8-isoprostaglandin F2α, arachidonic acid, and linoleic acid were simultaneously measured.

[0075] 14) An octadecyl-silica (ODS) column (Hypersil Gold, 3.0 μm, 100 × 2.1 mm; Thermo Fisher Scientific) was used for liquid chromatography, and the column temperature was set to 40°C. The mobile phases used were mobile phase A (ultrapure water containing 2 mM ammonium acetate) and mobile phase B (acetonitrile:methanol = 95:5), and the flow rate was set to 0.2 mL / min. For the measurement of 5-HETE, 12-HETE, 15-HETE, 13-(Z,E)-HODE, 13-(E,E)-HODE, 9-(E,Z)-HODE, 9-(E,E)-HODE, and 8-isoprostaglandin F2, the mobile phase composition was maintained at 80%:20% mobile phase A:B for 2 minutes at the start of the measurement, and then the composition was changed to 50%:50% by 45 minutes at a constant rate. For the measurement of arachidonic acid and linoleic acid, the mobile phase composition was maintained at 50%:50% mobile phase A:B for 2 minutes at the start of the measurement, and then the composition was changed to 10%:90% by 12 minutes at a constant rate. An electrospray ionization (ESI) probe is used and the capillary is heated to 270° C. Argon gas is used as the collision gas.

[0076] 15) Each parameter was quantitatively analyzed using the following m / z values ​​by MS / MS.

[0077] 13-(Z,E)-HODE and 13-(E,E)-HODE: m / z=295.0 194.6-195.6 9-(E,Z)-HODE and 9-(E,E)-HODE: m / z=295.0170.5-171.5 13-HODE-d4: m / z=299.0 197.6-198.6 5-HETE: m / z=319.0 114.5-115.5 12-HETE: m / z=319.3 162.8-163.8 15-HETE: m / z=319.3 202.5-203.5 8-isoprostaglandin F2: m / z=353.5 192.6-193.6 8-isoprostaglandin F2-d4: m / z = 357 196.5-197.5 Arachidonic acid: m / z = 303 258.8-259.8 Linoleic acid: m / z = 279.2 278.7-279.7

[0078] Example 1: Preparation of PTSD Model Mice PTSD model mice were prepared by fear conditioning using a stimulation cage combined with electrical stimulation. An overview of this experiment is shown in Figure 1. Specifically, on the first day (Day 0), a shock generator (electrical stimulation device) was connected to the stimulation cage (a cage consisting of an acrylic box with a grid-like floor covering and black plastic screened on three sides except for the floor and top) and the electrical stimulation was controlled by a PC. The behavior of the mice in the stimulation cage was observed. Each mouse was placed in the stimulation cage and allowed to behave freely. Their behavior was observed for 8 minutes. At the same time, a 0.3 mA current was applied to the electric wires on the floor three times: for 2 seconds starting 2 minutes after entry, for 2 seconds starting 4 minutes, and for 2 seconds starting 6 minutes after entry. The mice were then returned to their previous cages for rearing. Control mice were placed in the same stimulation cage and their behavior was observed for 8 minutes before being returned to the stimulation cage. The time required for the mice to exhibit a freezing response (freezing) during 8 minutes of free movement was measured. The next day (Day 1), each of the electrically stimulated mice and the control mice was placed in the same stimulation cage as on Day 0, and no electrical stimulation was administered to either group. Free movement was observed for 5 minutes. The mice were then returned to their previous cages and kept there. The time required for the mice to exhibit a freezing response (freezing) during 5 minutes of free movement was measured. The mice that received electrical stimulation on Day 0 had remembered the association (context) between the stimulation cage and the electrical stimulation (fear), and therefore recalled the fear memory and exhibited a freezing response (freezing). By placing the mice in the stimulation cage for these 5 minutes, the establishment of a fear memory that associated (context) the stimulation cage with the electrical stimulation (fear) was confirmed. On the day before sampling (Day 2, 6, 13, 27), each electrically stimulated mouse and control mouse were placed in the same stimulation cage as Day 0. Neither group received electrical stimulation, and free behavior was observed for 3 minutes. The mice were then returned to their previous cages and kept in their previous cages. The time required for freezing during the 3 minutes of free behavior was measured. Sampling was performed on Days 3, 7, 14, and 28. Specifically, blood and brain tissue samples were collected under anesthesia after 4 hours of fasting. The results are shown in Figure 2-1 (Day 3, 7) and Figure 2-2 (Day 14, 28).

[0079] PTSD is a pathological condition caused by fear memories, and the diagnostic criteria for PTSD (DSM-V) include "hypervigilance and excessive startle response" as symptoms. The mice produced in this example exhibit a freezing response upon fear recall in fear conditioning experiments, and can therefore be used as a PTSD model mouse (Anja Siegmund et al., J Psychiatr Res. 2007 Nov;41(10):848-60.).

[0080] Example 2: Measurement of 12-HETE in Plasma of PTSD Model Mice. Plasma samples were collected 3, 7, 14, and 28 days after electrical stimulation and analyzed for 12-HETE levels and the ratio of 12-HETE to arachidonic acid (AA) in plasma (12-HETE / AA). The results are shown in Figure 3. On Day 14 and Day 28, significant increases in 12-HETE and 12-HETE / AA were observed in the fear-conditioned group (PTSD group) compared with the control group (Control group). On Day 3 and Day 7, no statistically significant differences were observed in 12-HETE and 12-HETE / AA in the fear-conditioned group (PTSD group) compared with the control group (Control group), but a tendency for increases was observed.

[0081] 12-HETE is produced by the lipid-oxidizing enzyme 12 / 15-lipoxygenase (12 / 15-LOX) present in mouse leukocytes. Mouse 12 / 15-LOX is known to oxidize arachidonic acid, converting most of it to 12-HETE and a portion to 15-HETE (Figure 4). Mouse 12 / 15-LOX is also known to have a weak activity to oxidize linoleic acid to produce 13-(Z,E)-HODE. In this fear conditioning experiment, significant increases in 12-HETE and 12-HETE / AA were observed on Day 14 and Day 28, whereas no significant changes were observed in 15-HETE, 15-HETE / AA, 13-(Z,E)-HODE, or 13-(Z,E)-HODE / LA. Specific values ​​are shown in Table 1 below. In Table 1, the day of electrical stimulation is designated Day 0, and plasma samples were taken on Days 3, 7, 14, and 28. The table shows the ratio of 12-HETE to 12-HETE and arachidonic acid (AA) (12-HETE / AA), the ratio of 15-HETE to 15-HETE and arachidonic acid (AA) (15-HETE / AA), and the ratio of 13-(Z,E)-HODE to 13-(Z,E)-HODE and linoleic acid (LA) (13-(Z,E)-HODE / LA). *P<0.05 was considered significant.

[0082]

[0083] Comparative Example 1: Measurement of corticosterone in plasma from PTSD model mice. Corticosterone (a glucocorticoid secreted by mice, a stress-related steroid hormone secreted from the adrenal cortex in response to stress) was analyzed in samples collected from the same mice in which plasma 12-HETE was analyzed. The results are shown in Figure 5. No significant increase in plasma corticosterone was observed in the fear-conditioned group (PTSD group) compared to the control group at any sampling time, including days 14 and 28. This result suggests that 12-HETE has advantages in the objective evaluation of PTSD compared to glucocorticoids, which are known as candidate marker molecules for PTSD.

[0084] Example 3: Analysis of changes in plasma 12-HETE levels in AMPA receptor-modified mice with long-term PTSD-like symptoms. Using mice that are genetically prone to developing PTSD symptoms, changes in plasma 12-HETE levels were analyzed. Hayashi et al., one of the inventors of this application, generated AMPA receptor-modified mice (GluA1C811S mice) that are not palmitoylated by substituting serine for cysteine ​​at the palmitoylation site of the AMPA receptor, the main receptor for the excitatory neurotransmitter glutamate (Figure 6). GluA1C811S mice are prone to hyperexcitability due to increased membrane expression of AMPA receptors (Deficiency of AMPAR-Palmitoylation Aggravates Seizure Susceptibility. Masayuki Itoh et al., J Neurosci. 2018;38(47):10220-10235. and Reduced Effect of Anticonvulsants on AMPA Receptor Palmitoylation-Deficient Mice. Madoka Iizumi et al., Front Pharmacol. 2021;12:711737.). When these GluA1C811S mice were subjected to fear conditioning experiments, they showed a prolonged freezing response compared to wild-type mice, and reported that PTSD-like symptoms persisted for a long period of time (Figure 7 and Prolonged contextual fear memory in AMPA receptor palmitoylation-deficient mice. Akiko Oota-Ishigaki et al., Neuropsychopharmacology. 2022;47(12):2150-2159.).

[0085] One year after PTSD was induced in GluA1C811S mice by fear stimulation, blood 12-HETE levels tended to increase, although this was not statistically significant (Figure 8). This result also suggests that blood 12-HETE levels are elevated over a long period in mice exhibiting PTSD symptoms.

[0086] Example 4: Evaluation of PTSD or ASD Treatment Using the Present Invention To examine the relationship between the therapeutic intervention effect on PTSD or ASD and blood 12-HETE, a fear memory extinction learning experiment was conducted. Similar to exposure therapy, a type of cognitive behavioral therapy used to treat PTSD in humans, extinction learning experiments reduce fear by exposing the subject to a feared traumatic event without any risk. Mice undergoing extinction learning were subjected to a fear conditioning experiment using electrical stimulation on the first day (Day 0) under the same conditions as in Example 1 and shown in Figure 1. Then, on Days 1, 2, 3, 4, 5, 7, 14, and 21, the mice were placed in the same stimulation cage as Day 0 for extinction learning. The percentage of freezing responses during 10 minutes of free movement was observed without electrical stimulation. The mice were then returned to their previous cages for rearing. On the day before sampling (Day 25), the rats were placed in the same stimulation cage as Day 0, and free movement was observed for 3 minutes without electrical stimulation. The freezing response (freezing time) was measured every minute. For sampling on Day 26, blood and brain tissue samples were taken under anesthesia after 4 hours of fasting. The results are shown in Figures 9 to 11.

[0087] As shown in Figure 9, in mice that received electrical stimulation on Day 0 and did not undergo subsequent extinction learning, the percentage of mice showing a freezing response (freezing) on ​​the day before sampling (Day 25) was significantly higher than in mice that did not receive electrical stimulation. On the other hand, in mice that received electrical stimulation on Day 0 and then underwent extinction learning on Days 1, 2, 3, 4, 5, 7, 14, and 21, the percentage of mice showing a freezing response (freezing) gradually decreased.

[0088] 10, there was no significant difference between the groups without extinction learning and those with extinction learning in the percentage of mice exhibiting freezing on Day 1. On the other hand, the percentage of mice exhibiting freezing on the day before sampling (Day 25) was reduced to the same level as mice that had undergone extinction learning alone without electrical stimulation on Day 0, and no statistically significant difference was observed between the two groups.

[0089] Furthermore, as shown in FIG. 11, the plasma 12-HETE concentration in mice and the ratio of 12-HETE to arachidonic acid (AA) (12-HETE / AA) were significantly reduced by extinction learning.

[0090] These results demonstrate that PTSD symptoms are improved by the therapeutic intervention of extinction learning after fear conditioning, and that plasma 12-HETE and 12-HETE / AA levels are reduced accordingly. In other words, this example demonstrates that the present invention is useful for objectively evaluating the effects of therapeutic intervention for PTSD and / or ASD, and for screening therapeutic drugs for PTSD and / or ASD.

[0091] According to the present invention, it is possible to accurately determine whether a subject suffers from PTSD or ASD using an objective indicator. Furthermore, according to the present invention, it is possible to objectively determine the effectiveness of a therapeutic intervention applied to a patient suffering from PTSD or ASD. In addition, according to the present invention, it is possible to efficiently screen substances that are candidates for treating PTSD or ASD. Therefore, the present invention is extremely useful in the medical field related to the prevention and treatment of PTSD or ASD.

[0092] This application is based on patent application No. 2024-006258 filed in Japan (filing date: January 18, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for determining whether a subject suffers from post-traumatic stress disorder (PTSD) or acute stress disorder (ASD), comprising measuring the amount of at least one selected from the group consisting of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), and 13-hydroxy-9(Z),11(E)-octadecadienoic acid (13-(Z,E)-HODE) in a sample derived from the subject.

2. The method according to claim 1, wherein the sample derived from the subject is blood, serum, plasma, urine, or saliva of the subject.

3. The method according to claim 1 or 2, wherein the subject is a human.

4. Use of at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE as a biomarker for detecting PTSD or ASD.

5. A method for screening a candidate substance for a therapeutic agent for PTSD or ASD, comprising the following steps: (Step 1) determining the amount of at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in a sample derived from a PTSD model animal or an ASD model animal before fear conditioning; (Step 2) subjecting the PTSD model animal or the ASD model animal to fear conditioning; (Step 3) administering a test substance to the PTSD model animal or the ASD model animal that has been subjected to fear conditioning; and (Step 4) determining the amount of at least one selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in a sample derived from the model animal to which the test substance has been administered.

6. A method for evaluating the therapeutic effect of a treatment method applied to a patient with PTSD or ASD, comprising the following steps: (Step 1) determining at least one quantity selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in a sample derived from a patient suffering from PTSD or ASD using the method of the present invention; (Step 2) applying a PTSD or ASD treatment method to the patient; (Step 3) determining at least one quantity selected from the group consisting of 12-HETE, 15-HETE, and 13-(Z,E)-HODE in a sample derived from the patient after treatment; and (Step 4) comparing the quantity determined in Step 1 with the quantity determined in Step 3.

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