Anxiety relievers

Abstract

To provide an anxiolytic agent that mitigates anxiety in organisms exposed to acute stress.SOLUTION: Data on the expression levels of at least one specific microRNA is obtained, selected from the group of specific microRNAs, including miR-99b-3p, miR-136-5p, miR-140-5p, miR-199a-3p, miR-339-3p, miR-376a-3p, and miR-466f-3p, encapsulated in brain-derived exosomes taken from an organism. A higher expression level compared to the control value enables an objective assessment and diagnosis of the organism being in an acute stress state. Administering brain-derived exosomes and at least one specific microRNA, taken from the organism exposed to acute stress, to another organism in an acute stress state is anticipated to yield therapeutic effects in mitigating anxiety and preventive effects in curbing anxiety onset.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to an anxiolytic agent that can alleviate anxiety induced in a living body exposed to acute stress or prevent the onset of anxiety. [Background technology]

[0002] Subjective assessment using questionnaires is the mainstream method for assessing acute stress, and no objective method using biomarkers has yet been established. Previous attempts have been made to assess it objectively by measuring physiologically active substances in blood, urine, and saliva, but this has not yet been established. In recent years, there have been sporadic attempts to use exosomes in the blood. Specifically, a method using total exosomes (PLoS One. 2014; 9: e108748.) and a method using brain-derived exosomes during chronic stress exposure (Int J Mol Sci. 2021; 22: 9960.) have been reported. The latter method using brain-derived exosomes during chronic stress exposure is disclosed, for example, in Patent Document 1 below.

[0003] The basic method for alleviating anxiety induced in organisms exposed to acute stress is the administration of central nervous system agonists (antidepressants, etc.), but the remission rate is only approximately 20-30% (Prog Neuropsychopharmacol Biol Psychiatry. 2009; 33: 169-80.). Furthermore, conventional treatment (administration of central nervous system agonists) is a medical procedure performed after the onset of stress-related illnesses and is not used preventively in healthy individuals. On the other hand, the ingestion of exosomes is commonly carried out through food (e.g., milk), and its health-promoting effects have also been reported (Adv Nutr. 2019; 10: 711-721.). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO-A-2021 / 251656 Summary of the Invention [Problem to be solved by the invention]

[0005] Because the brain is the receptor for acute stress, biomarkers that reflect molecular pathology in the brain are desirable. However, no such biomarkers have been proposed in previous exosome research. Methods using total exosomes in blood samples contain exosomes derived from tissues other than the brain, making it difficult to detect changes in the brain (PLoS One. 2014; 9: e108748). Furthermore, the method using brain-derived exosomes disclosed in the aforementioned Patent Document 1 inflicts chronic rather than acute stress on the body. Therefore, the factors encapsulated in the exosomes likely reflect functional decline during the exhaustion phase, when the body's defense response has exceeded its limits, rather than reflecting molecular pathology in the brain caused by acute stress. On the other hand, during acute stress, brain-derived exosomes are likely to encapsulate various host defense factors, as they are resistant to external stimuli.

[0006] As mentioned above, the remission rate for stress-related disorders treated with central nervous system agonists is only approximately 20-30% (Prog Neuropsychopharmacol Biol Psychiatry. 2009; 33: 169-80.), and the development of more effective treatment methods is urgently needed. Furthermore, although the in vivo administration of whole blood exosomes from patients with depression and chronically stressed mice has been reported (Neuropsychopharmacology. 2020; 45: 1050-1058., Brain Behav Immun. 2021; 94: 225-234.), these studies did not use exosomes extracted during acute stress, and because a heterogeneous exosome population was used, it was unclear which tissue-derived exosomes were responsible for the effect.

[0007] The present invention has been made to solve the above-described problems, and is a method for treating acute stress. exposureThe object of the present invention is to provide an anxiolytic agent capable of alleviating anxiety induced in an irritated living body or preventing the induction of anxiety. [Means for solving the problem]

[0008] The anxiolytic agent according to claim 1 comprises Acute stress as a 1-hour water immersion restraint stress exposed to Mouse blood, serum, or plasma Brain-derived exosomes collected from 、 and / or As specific microRNAs, at least miR-99b-3p 、m iR-140-5p, and miR-199a-3 p It is characterized by including.

[0009] The anxiolytic agent according to claim 2 is the anxiolytic agent according to claim 1. The specific microRNA further includes at least one selected from a group of microRNAs including miR-136-5p, miR-339-3p, miR-376a-3p, and miR-466f-3p. It is characterized by the following.

[0011] Claim 3 The anxiety relievers listed in As specific microRNAs, at least miR-99b-3p 、m iR-140-5p, and miR-199a-3 p An anxiolytic comprising: It is extracted from the body fluids of living organisms that have not been exposed to acute stress and is artificially synthesized. Before Brain-derived exosomes containing the specific microRNA, It is extracted from living nerve cells that have not been exposed to acute stress and is artificially synthesized. Before Brain-derived exosomes containing the specific microRNA, Extracted from a neuronal cell line and artificially synthesized Before The composition is characterized by containing one or more brain-derived exosomes selected from brain-derived exosomes encapsulating the specific microRNA.

[0012] Claim 4 The anxiety relievers listed in As specific microRNAs, at leastmiR-99b-3p 、m iR-140-5p, and miR-199a-3 p An anxiolytic comprising: taken from organisms not exposed to acute stress 、 The specific microRNA Forced expression Brain-derived exosomes extracted from neural cells modified to The specific microRNA Forced expression The exosomes are characterized by containing either one or both of brain-derived exosomes collected from a neural cell line modified to induce mitochondrial dysfunction. [Effects of the Invention]

[0013] The anxiolytic agent of the present invention contains brain-derived exosomes collected from a living organism exposed to acute stress and / or at least one specific microRNA selected from miR-99b-3p, miR-136-5p, miR-140-5p, miR-199a-3p, miR-339-3p, miR-376a-3p, and miR-466f-3p, which are highly reliable markers of acute stress. Alternatively, the agent contains one or both of brain-derived exosomes collected from neurons collected from a living organism not exposed to acute stress and given a stimulus equivalent to acute stress in a culture medium, and brain-derived exosomes collected from a neuronal cell line given a stimulus equivalent to acute stress in a culture medium. Alternatively, the agent may contain one or more brain-derived exosomes selected from brain-derived exosomes collected from body fluids of a living organism not exposed to acute stress and artificially synthesized, encapsulating at least one specific microRNA; brain-derived exosomes collected from neurons of a living organism not exposed to acute stress and artificially synthesized, encapsulating at least one specific microRNA; and brain-derived exosomes collected from a neuronal cell line and artificially synthesized, encapsulating at least one specific microRNA. Alternatively, the agent may contain one or both of brain-derived exosomes collected from neurons collected from a living organism not exposed to acute stress and modified to produce the specific microRNA, and brain-derived exosomes collected from a neuronal cell line modified to produce the specific microRNA. Therefore, when this anxiety-relieving agent is administered to a living organism exposed to acute stress, it can be expected to have a therapeutic effect of alleviating anxiety induced in the living organism or a preventive effect of suppressing the onset of anxiety in the living organism. Furthermore, since exosomes encapsulate multiple substances, unlike the conventional concept of medication in which a single compound acts on a single target molecule, multiple substances are delivered to the target tissue, and their combined effect acts on tissues and cells, so a synergistic effect greater than that of conventional medication can be expected. Furthermore, if brain-derived exosomes that exhibit anxiolytic effects and / or the above-mentioned highly accurate markers of acute stress are ingested from food, it is possible to alleviate the symptoms of acute stress without medical intervention. exposure This is expected to have a therapeutic or preventive effect on anxiety induced in the living body. [Brief explanation of the drawings]

[0014] [Figure 1] MicroRNAs contained in brain-derived exosomes obtained from mice exposed to acute stress and control mice were extracted and their expression levels were analyzed using microarrays. Of the microRNAs whose expression levels were altered, only those with homologs in humans are listed in this table. [Figure 2] FIG. 2 shows the results of reanalysis by real-time RT-PCR of the expression ratios of the 27 types of microRNAs shown in FIG. 1. [Figure 3] Brain-derived exosomes obtained from mice exposed to acute stress, brain-derived exosomes obtained from control mice, and vehicle were each administered (A: intravenous administration; B: intracerebroventricular administration) to other mice exposed to acute stress. The graphs show the results of measuring anxiety behavior in the open field test and elevated plus maze test for each individual. [Figure 4] This graph shows the results of measuring anxiety behavior in an elevated plus maze test conducted on mice exposed to acute stress after intravenous administration of three types of microRNAs whose expression was increased by acute stress and a control microRNA that does not exhibit physiological effects. [Figure 5] In addition to the three types of mouse groups shown in Figure 3, a group of mice exposed to acute stress but not administered intravenous drugs, and a group of mice not exposed to acute stress and not administered intravenous drugs were also included. This graph shows the results of measuring anxiety behavior in an open field test for each individual. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present inventors have conducted various experiments, including those described below, and as a result of extensive research, have reached the following findings: Specifically, because the brain is the organ that perceives acute stress, extracting brain-derived exosomes from body fluids (e.g., blood) of a living organism exposed to acute stress and further extracting the microRNAs contained within the brain-derived exosomes can serve as novel and objective markers for acute stress. Furthermore, by obtaining data indicating the expression levels of the markers and using the data for acute stress assessment and diagnosis, the presence or absence of an acute stress state can be objectively assessed. Furthermore, because the acute stress period is a period of resistance to external stimuli, brain-derived exosomes during this period are likely to contain various biological defense factors. Therefore, the inventors have discovered that such brain-derived exosomes and specific microRNAs contained within the brain-derived exosomes can serve as anxiolytic agents that can alleviate or prevent the onset of anxiety in other living organisms exposed to acute stress.

[0016] 1. First experiment Figure 1 shows the results of the first experiment. Brain-derived exosomes were collected from mice exposed to acute stress and control mice, and the microRNAs contained within them were extracted and their expression levels were analyzed using the microarray method. Of the microRNAs whose expression levels were altered, only those with homologs in humans were listed.

[0017] Experimental Method Brain-derived exosomes were extracted from the blood of mice subjected to one hour of acute stress by water immersion restraint stress, and from the blood of control mice not subjected to water immersion restraint stress. Next, all microRNAs contained within the exosomes were extracted, and the expression level of each microRNA was analyzed using microarray analysis. Of the microRNAs whose expression was altered by acute stress, only those with human homologs (corresponding microRNAs) were extracted and listed as shown in Figure 1. The relative values ​​in the figure indicate the relative expression level of the acute stress group, with the expression level in the control group set to 1.

[0018] 《Explanation of the results》 Figure 1 shows that the expression of 27 types of microRNAs changes, suggesting that these microRNAs could be candidates for assessing acute stress in humans. However, the expression ratios obtained by the microarray method are generally not quantitative, and the reliability of the values ​​is considered low. Therefore, a more accurate method for quantification is required, and a second experiment, the results of which are shown in Figure 2, was conducted.

[0019] 2. Second experiment Figure 2 shows the results of a second experiment in which the expression ratios of the 27 types of microRNAs shown in Figure 1 were reanalyzed by real-time RT-PCR.

[0020] Experimental Method Brain-derived exosomes were extracted from the blood of mice that had been subjected to acute stress by water immersion and restraint stress for one hour (stress group, black bars in Figure 2) and from the blood of control mice that had not been subjected to water immersion and restraint stress (control group, gray bars in Figure 2). Furthermore, microRNAs contained within the exosomes were extracted, and the expression levels of 27 types of microRNA were analyzed using real-time RT-PCR.

[0021] 《Explanation of the results》 The vertical axis of the graph in Figure 2 shows the expression levels of the 27 target microRNAs arranged along the horizontal axis, expressed as relative expression levels to the endogenous control, miR-16. In Figure 2, the stress group is shown as a black bar, and the control group is shown as a gray bar. Among the black bars in the stress group, those that fall within the p-value range of the t-test shown in the upper right of Figure 2 are marked with the symbol assigned to that p-value range.

[0022] As shown in Figure 2, when a p-value of <5% was used as the criterion for statistical significance, six microRNAs (miR-99b-3p, miR-136-5p, miR-140-5p, miR-199a-3p, miR-339-3p, and miR-376a-3p) were significantly increased (p<0.05, p<0.01), and one microRNA (miR-466f-3p) tended to increase (p<0.1). Among the remaining 20 microRNAs, one microRNA (miR-323-3p) remained unchanged in both groups, and the remaining 19 microRNAs were undetectable by real-time RT-PCR.

[0023] miR-16 was used as an endogenous control because this microRNA is stably expressed in neural tissue and has also been used as an endogenous control in other reports (Front Neurosci. 2019; 13: 1208.).

[0024] As described above, the data obtained from this experiment indicate that when an organism is exposed to acute stress, the expression levels of at least the following microRNAs encapsulated in brain-derived exosomes collected from the organism's body fluids show a statistically significant increase (or a tendency toward an increase) when evaluated relative to the endogenous control: miR-99b-3p, miR-136-5p, miR-140-5p, miR-199a-3p, miR-339-3p, miR-376a-3p, and miR-466f-3p. Therefore, by collecting brain-derived exosomes from an organism's body fluids and obtaining data indicating the relative expression levels of specific microRNAs encapsulated within them, this data can be used as data for assessing acute stress. In other words, by determining whether specific microRNAs are increased in this acute stress assessment data, it is possible to objectively evaluate and diagnose whether the organism has been exposed to acute stress with a high degree of accuracy.

[0025] In other words, by collecting brain-derived exosomes from the body fluids of a living organism and evaluating whether the relative expression level of a specific microRNA contained therein is increased, it is possible to provide an auxiliary means or method for a doctor's final judgment (diagnosis) of whether the organism has been exposed to acute stress. Note that in this experiment, the relative expression level of a specific microRNA was evaluated, but the absolute amount may also be evaluated.

[0026] 3. Third Experiment Figure 3 shows the results of the third experiment, which is a graph showing the results of measurements of anxiety behavior in the open field test and elevated plus maze test for each individual mouse after administering brain-derived exosomes obtained from mice exposed to acute stress, brain-derived exosomes obtained from control mice, and vehicle to other mice exposed to acute stress. The experimental results shown in Figure 3 demonstrate that administration of brain-derived exosomes obtained from an individual exposed to acute stress attenuated anxiety behavior.

[0027] Experimental Method Brain-derived exosomes extracted from the blood of mice exposed to acute stress (stress BDE group) for one hour, brain-derived exosomes extracted from the blood of control mice (control BDE group), or vehicle alone (vehicle group) were administered (A; intravenous administration; B; intracerebroventricular administration) to mice exposed to acute stress. The administered mice then performed an open-field test and an elevated plus-maze test to measure anxiety-related behavior. In Figure 3, the black bars show the measurement results for mice administered brain-derived exosomes from mice exposed to acute stress (stress BDE group), the gray bars show the measurement results for mice administered brain-derived exosomes from control mice (control BDE group), and the white bars show the measurement results for mice administered vehicle alone (vehicle group).

[0028] The preparation and formulation of brain-derived exosomes for administration was carried out according to the following procedure. 1) The collected serum was processed by ultracentrifugation to recover exosomes. 2) The collected exosomes were subjected to immunoprecipitation using anti-CD171 antibody, and only CD171 antibody-positive exosomes were extracted. 3) CD171 antibody-positive exosomes were suspended in Tris-glycine buffer (for intravenous administration) or phosphate-buffered saline (for intracerebroventricular administration) and formulated.

[0029] Details of the administration method are as follows. A: Intravenous administration: CD171 antibody-positive exosomes / Tris-glycine buffer solution was injected into the tail vein of mice. B; Intraventricular administration: CD171 antibody-positive exosomes / phosphate-buffered saline were injected into the lateral ventricle of mice using a microsyringe pump.

[0030] 《Explanation of the results》 In the intravenous administration experiment (Fig. 3A), the time spent in the central compartment (vertical axis) in the open field test and the ratio of movement in the open arms to the total movement in the entire area (vertical axis) in the elevated plus maze test were both significantly increased in the stress BDE group (black bars) compared to the other groups.

[0031] In the intracerebroventricular administration experiment (Fig. 3B), the ratio of movement in the open arms to movement in the entire area (vertical axis) in the elevated plus maze test in the stress BDE group (black bars) was significantly increased compared to the other groups.

[0032] In Figures 3A and 3B, the black bars for the stress BDE group and the bars for the other two groups are marked with symbols representing the range of p-values ​​in the Tukey-Kramer test performed as a post-hoc test for the one-way ANOVA shown in the upper right of Figure 3.

[0033] As explained above, this experiment detected a statistically clear difference between when brain-derived exosomes obtained from individuals exposed to acute stress (stress BDE group) were administered to other individuals exposed to acute stress and when brain-derived exosomes obtained from the control group (control BDE group) were administered to other individuals exposed to acute stress. Therefore, brain-derived exosomes obtained from individuals exposed to acute stress, or the specific microRNAs they encapsulate, are considered to be effective as anxiolytic agents that induce anxiolytic effects in other individuals, and to have the ability to alleviate stress in other individuals exposed to acute stress.

[0034] The dose of the anxiolytic agent required to achieve the anxiolytic effect was as follows: In the third experiment, brain-derived exosomes were collected from approximately 1.4 mL of serum using a collection method combining ultracentrifugation and immunoprecipitation, and administered to one mouse. In terms of weight, 6 μg of brain-derived exosomes (measured by the BCA method) was administered to a mouse weighing approximately 25 g. Converting this to a human dose, this would equate to brain-derived exosomes collected from 3.58 L of serum, or 14.4 mg of brain-derived exosomes administered to a human weighing 60 kg. This dose is sufficient to achieve the anxiolytic effect described above.

[0035] In the third experiment, an anxiolytic effect was obtained by administering brain-derived exosomes from mice that had been subjected to acute stress (stress BDE group) to other mice that had been exposed to acute stress. It is believed that if at least one of the seven microRNAs identified in the second experiment contained in these brain-derived exosomes is administered, it will be possible to obtain a corresponding anxiolytic effect as an anxiety-relieving agent.

[0036] 4. Fourth Experiment Figure 4 is a graph showing the results of measuring anxiety behavior in an elevated plus maze test for each individual mouse after intravenous administration of three microRNAs whose expression was upregulated by acute stress and a control microRNA that does not exhibit physiological effects to other mice exposed to acute stress. The experimental results shown in Figure 4 demonstrate that administration of microRNAs whose expression was upregulated by acute stress reduces anxiety behavior.

[0037] Experimental Method Three microRNAs (miR-99b-3p, miR-140-5p, and miR-199a-3p) whose expression was upregulated in brain-derived exosomes extracted from the blood of mice exposed to acute stress (1 hour of water immersion restraint stress) were intravenously administered to other mice exposed to acute stress (the three-type mixed microRNA group, shown by the black bars in Figure 4). A control microRNA that exhibits no physiological effect was also intravenously administered to other mice (the control microRNA group, shown by the gray bars in Figure 4). These mice were then subjected to an elevated plus maze test to measure the degree of anxiety behavior.

[0038] The preparation and formulation of microRNA for administration was carried out according to the following procedure. 1) MicroRNA for administration was synthesized. 2) The synthesized microRNA was mixed with a transfection reagent (a reagent for efficient introduction into cells). Specifically, 60 μg of synthesized microRNA, 9.6 μL of polyethylenimine (PEI) solution, 100 μL of 10% glucose solution, and 100 μL of sterile water were thoroughly mixed and reacted to form a microRNA-PEI complex.

[0039] The administration method was intravenous administration, and the synthetic microRNA / transfection reagent mixture was injected into the tail vein of the mice.

[0040] 《Explanation of the results》 The ratio of entries into the open arm area to the total number of entries into all areas in the elevated plus-maze test (vertical axis) for the three-type mixed microRNA group (black bars) was significantly increased compared to the control microRNA group (gray bars), with a p-value of less than 0.05 in the t-test, as shown in Figure 2.

[0041] As explained above, the fourth experiment revealed that when brain-derived exosome-encapsulated microRNA whose expression was increased by acute stress was synthesized and administered to a living body, it induced an anxiolytic effect, similar to the results of the third experiment.

[0042] 5. Fifth Experiment Figure 5 shows the results of the fifth experiment. In addition to the three types of mouse groups shown in Figure 3, a group of mice exposed to stress but not administered intravenously and a group of mice not exposed to stress and not administered intravenously were also included. Open field tests were conducted on each individual mouse. For experimentation The experimental method, preparation and formulation of brain-derived exosomes for administration, administration method, statistical evaluation method, etc. were the same as those in the third experiment described above.

[0043] As shown in Figure 5, the results of the fifth experiment showed that the actions of needle puncture, administration of the liquid dissolving brain-derived exosomes, and administration of brain-derived exosomes did not affect anxiety behavior.

[0044] Furthermore, because exosomes are generally derived from living organisms, it is well known that exosomes themselves are not immunogenic (the property of foreign substances from outside the body to induce an immune response in the human body) (Curr Gene Ther. 2012; 12; 262), and it is thought that even if the animal species is different (for example, if exosomes collected from mice are administered to humans), no immune response will be evoked (J Extracell Vesicles. 2018 Feb 21;7(1):1440132.).

[0045] In each of the experiments described above, blood was used as an example of the body fluid from which brain-derived exosomes were collected, but brain-derived exosomes can also be collected from other body fluids, such as serum, plasma, saliva, urine, sweat, breast milk, cerebrospinal fluid, etc. Brain-derived exosomes can also be collected from culture media of neurons and neuronal cell lines collected from living organisms exposed to acute stress.

[0046] 6. Other Embodiments of Anxiety Relief Agents The experimental embodiments described above relate to a method for generating acute stress assessment data used to evaluate the effects of acute stress on a living organism, and an anxiety-relieving agent containing brain-derived exosomes or specific microRNA collected from a living organism that has experienced acute stress. However, the anxiety-relieving agent of the present invention can also be produced by methods other than those described in the experimental examples. That is, the anxiety-relieving agent of the present invention can also be produced by using body fluids or neurons collected from a living organism that has not been exposed to acute stress, or by using a commonly available neuronal cell line and subjecting it to a predetermined treatment to obtain brain-derived exosomes containing the specific microRNA.

[0047] 6-1 Anxiety Relief Agent A according to Other Embodiments This anxiety-relieving agent A is collected from a living organism that has not been exposed to acute stress and contains one or both of brain-derived exosomes collected from neurons that have been given a stimulus equivalent to acute stress in a culture medium, and brain-derived exosomes collected from a neuronal cell line that has been given a stimulus equivalent to acute stress in a culture medium.

[0048] Anxiolytic agent A can be produced by collecting neural tissue (such as the brain) from a living body that has not been exposed to acute stress, and then isolating neural cells from the neural tissue by enzymatic digestion and mechanical disruption, or by using a commonly available neural cell line.

[0049] A stimulus equivalent to acute stress applied to neurons or neuronal cell lines in culture medium may be any stimulus that increases at least one of the seven miRNAs, such as the addition of physiologically active substances such as lipopolysaccharide or hormones.

[0050] Brain-derived exosomes can then be extracted from the culture supernatant using techniques such as ultracentrifugation, affinity purification, size exclusion chromatography, antibody capture, polymer precipitation, and microfluidics, and formulated as an anxiolytic. The formulation methods are as described in the experimental examples above.

[0051] 6-2 Anxiety Relief Agent B in Other Embodiments This anxiety-relieving agent B contains at least one specific microRNA selected from a group of microRNAs including miR-99b-3p, miR-136-5p, miR-140-5p, miR-199a-3p, miR-339-3p, miR-376a-3p, and miR-466f-3p. Anxiety-relieving agent B contains one or more brain-derived exosomes selected from brain-derived exosomes collected from the body fluid of a living organism not exposed to acute stress and encapsulating at least one artificially synthesized specific microRNA, brain-derived exosomes collected from neurons of a living organism not exposed to acute stress and encapsulating at least one artificially synthesized specific microRNA, and brain-derived exosomes collected from a neuronal cell line and encapsulating at least one artificially synthesized specific microRNA.

[0052] The body fluids that are the raw materials for anxiolytic B include blood, serum, plasma, saliva, urine, sweat, breast milk, and cerebrospinal fluid collected from living organisms.

[0053] Alternatively, anxiolytic agent B can be produced by collecting neural tissue (such as the brain) from a living body that has not been exposed to acute stress, and then isolating the neural tissue by enzymatic digestion and mechanical disruption, or by using a commonly available neural cell line.

[0054] Brain-derived exosomes can be extracted from the body fluids by combining exosome purification and immunoprecipitation. Because the body fluids contain exosomes that are not derived from the brain, immunoprecipitation is effective in removing them.

[0055] Brain-derived exosomes are extracted from the culture supernatant using ultracentrifugation, affinity purification, size exclusion chromatography, antibody capture, polymer precipitation, microfluidic systems, etc. Here, since only exosomes derived from neurons (brain cells) are present in the culture supernatant, immunoprecipitation is not necessary.

[0056] Then, specific artificially synthesized microRNAs can be encapsulated into brain-derived exosomes using techniques such as electroporation, ultrasound, membrane permeabilization, freeze-thawing, microRNA modification (linking a vector or sequence to be incorporated into exosomes to the specific microRNA sequence to be encapsulated), transfection, etc., and then formulated as an anxiety-relieving agent. The formulation techniques are as shown in the experimental examples described above.

[0057] 6-3 Anxiety Relief Agent C According to Another Embodiment This anxiety-relieving agent C contains at least one specific microRNA selected from a group of microRNAs including miR-99b-3p, miR-136-5p, miR-140-5p, miR-199a-3p, miR-339-3p, miR-376a-3p, and miR-466f-3p, and contains either or both of brain-derived exosomes collected from neurons modified to produce the specific microRNA and brain-derived exosomes collected from a neuronal cell line modified to produce the specific microRNA, both of which are collected from a living organism not exposed to acute stress.

[0058] Anxiolytic agent C can be produced by collecting neural tissue (such as the brain) from a living body that has not been exposed to acute stress, and then isolating neural cells from the neural tissue by enzymatic digestion and mechanical disruption, or by using a commonly available neural cell line.

[0059] The nerve cells or nerve cell line are modified to forcibly express the specific microRNA using a vector or the like, thereby increasing its production amount.

[0060] Brain-derived exosomes can be collected from the culture supernatant of the modified neurons or neuronal cell line by methods such as ultracentrifugation, affinity purification, size exclusion chromatography, antibody capture, polymer precipitation, or a microfluidic system, and then formulated as an anxiolytic agent. The formulation methods are as described in the respective experimental examples.

[0061] Although some of the anxiolytic agents in the various embodiments described above are produced using body fluids or nerve cells collected from a living organism exposed to acute stress or a living organism not exposed to acute stress, it is preferable that the living organism is an animal other than a human. However, if there are no ethical or legal issues with using a substance derived from a human, or if it is otherwise acceptable, the anxiolytic agent can also be produced using human body fluids or nerve cells.

Claims

1. An anxiety-relieving agent characterized by containing brain-derived exosomes collected from the blood, serum or plasma of mice exposed to one hour of water immersion restraint stress as acute stress, and / or specific microRNAs, including at least miR-99b-3p, miR-140-5p, and miR-199a-3p.

2. The anxiety-relieving agent described in Claim 1, characterized in that the specific microRNA further includes at least one selected from a group of microRNAs including miR-136-5p, miR-339-3p, miR-376a-3p and miR-466f-3p.

3. An anxiolytic agent comprising at least miR-99b-3p, miR-140-5p, and miR-199a-3p as specific microRNAs, Brain-derived exosomes collected from the body fluids of a living organism not exposed to acute stress and encapsulating the artificially synthesized specific microRNA; Brain-derived exosomes collected from living neurons that have not been exposed to acute stress and encapsulating the specific artificially synthesized microRNA; An anxiety-relieving agent comprising one or more brain-derived exosomes selected from brain-derived exosomes collected from a neuronal cell line and artificially synthesized, each of which encapsulates the specific microRNA.

4. An anxiolytic agent comprising at least miR-99b-3p, miR-140-5p, and miR-199a-3p as specific microRNAs, Brain-derived exosomes collected from neurons that have been modified to forcibly express the specific microRNA and collected from a living organism that has not been exposed to acute stress; An anxiety-relieving agent comprising one or both of brain-derived exosomes collected from a neural cell line modified to forcibly express the specific microRNA.

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