WO2021010348A1 - TRANSFORMING GROWTH FACTOR β1 AS PREVENTIVE OR THERAPEUTIC AGENT FOR DEPRESSION OR SYMPTOMS OF DEPRESSION - Google Patents

Abstract

A purpose of the present invention is to provide a novel compound having preventive or therapeutic effects on depression or symptoms of depression. The present invention provides a preventive or therapeutic agent for depression or symptoms of depression, the agent containing transforming growth factor β1 or a salt thereof as an active ingredient.

Description

Transforming growth factor β1 as a prophylactic or therapeutic agent for depression or depressive symptoms

The present invention relates to a prophylactic or therapeutic agent for depression or depressive symptoms containing transforming growth factor β1 or a salt thereof.

The number of patients suffering from mental illness is increasing as a whole due to changes in social lifestyles and the aging of society.
Typical mental illnesses include depression and schizophrenia, and drug treatment is performed, but the drugs actually used in clinical practice are used for some patients and some symptoms. It is also known that there are patients who are effective but ineffective or so-called refractory to treatment.
Therefore, the development of new therapeutic agents for mental illness remains urgent.

Non-Patent Document 1 describes the relationship between major depression and TGF-β1, and discloses that transforming growth factor β1 (TGF-β1) decreases in major depression and increases after treatment.
In addition, Non-Patent Document 2 discloses that depressive symptoms are improved by inhibiting the signal transduction pathway of TGF-β1.
Furthermore, Non-Patent Documents 3 to 5 disclose that TGF-β1 is associated with schizophrenia, Parkinson's disease and Alzheimer's disease.

J Affect Disord. 2005 Oct; 88 (2): 167-173 FASEB J. 2019 Jan; 33 (1): 606-618 Psychoneuroendocrinology. 2019 Jan; 99: 265-270 J Cell Mol Med. 2019 Apr; 23 (4): 2568-2582 J Mol Neurosci. 2019 Jan; 67 (1): 142-149

However, in Non-Patent Document 1, no significant difference was observed in Table 2, and the results in Table 2 were improved after 8 weeks as a result of treatment with existing antidepressants and the like as compared with those before treatment. It only discloses the results. Further, the antidepressant effect disclosed in Non-Patent Document 2 is shown as an effect by inhibiting the signal transduction pathway of TGF-β1.

An object to be solved by the present invention is to provide a new compound having a preventive or therapeutic effect on depression or depressive symptoms.

The present inventor has made diligent studies to solve the above problems, found a new compound having a preventive or therapeutic effect on depression or depressive symptoms, and completed the present invention.

That is, the present invention is as follows.
(1)
A prophylactic or therapeutic agent for depression or depressive symptoms, which comprises transforming growth factor β1 or a salt thereof as an active ingredient.
(2)
A pharmaceutical composition for the prevention or treatment of depression or depressive symptoms, which comprises transforming growth factor β1 or a salt thereof as an active ingredient.
(3)
A method for screening a prophylactic or therapeutic agent for depression or depressive symptoms by enhancing the TGFβ-1 and TGFβ-1 receptor cascade.
(4)
The screening method according to (3), wherein the TGFβ-1 receptor is TGFBR1 or TGFBR2.
(5)
The screening method according to (3) or (4), wherein the enhancement of the TGFβ-1 and TGFβ-1 receptor cascade is confirmed by the increased expression of TGFβ-1.
(6)
The screening method according to (5), wherein the increase in mRNA expression is used as an index.
(7)
The screening method according to (5) or (6), wherein a behavioral test is further performed.

Transforming growth factor β1 (TGF-β1) is effective in the prevention or treatment of depression and depressive symptoms.

FIG. 1A uses a social defeat stress model of depression to perform RNA-seq analysis of the frontal cortex 3 days after intraperitoneal administration (IP) of two ketamine isomer hydrochlorides (10 mg / kg). It is an explanatory diagram. FIG. 1B shows the results of RNA-seq analysis. After RNA-seq and GSEA (Gene Set Enrichment Analysis), a TGF-β signal group with a marked difference in gene expression between the two isomers was identified. In a social defeat stress model of depression, gene expression of TGF-β1, TGF-β2 and their respective receptors (TGFBR1, TGFBR2) in the frontal cortex and hippocampus was measured. Expression of TGF-β1 was significantly reduced in the depressive mouse prefrontal cortex and hippocampus and increased with R-ketamine hydrochloride (10 mg / kg), but with S-ketamine hydrochloride (10 mg / kg). It didn't improve. Expression of TGF-β2 was unchanged in the depressive mouse frontal cortex and hippocampus. Expression of TGF-β1 and TGF-β2 receptors was significantly reduced in the depressive mouse prefrontal cortex and hippocampus and increased with R-ketamine hydrochloride (10 mg / kg) administration, but with S-ketamine hydrochloride. Administration of salt (10 mg / kg) did not improve. The effect of the TGF-β1 receptor antagonist RepSox (Selleckchem) on the antidepressant effect of R-ketamine was investigated. Repsox (10 mg / kg) was intraperitoneally administered 30 minutes before administration of R-ketamine hydrochloride (10 mg / kg). The results of the locomotor activity test (LMT), tail suspension test (TST), forced swimming test (FST) and sucrose preference test with 1% sucrose (SPT) are shown respectively. Momentum was not different in all groups. Mice showing depressive symptoms to which saline was administered showed a significant increase in TST and FST immobility time as compared with normal mice, which was improved by administration of R-ketamine. Premedication with Repox abolished the antidepressant effect of R-ketamine. Mice showing depressive symptoms also showed a significant decrease in sucrose palatability (anhedonia), which was improved with R-ketamine. Premedication with Repox abolished the anti-anhedonia effect of R-ketamine on SPT. The effect of another TGF-β1 receptor antagonist SB431542 (Tocris Bioscience) on the antidepressant effect of R-ketamine was investigated. SB431542 (10 μM, 2 μL) was administered intraventricularly (ICV) 30 minutes before administration of R-ketamine hydrochloride (10 mg / kg). There was no difference in the amount of exercise in all groups. In TST and FST, pre-administration of SB431542 abolished the antidepressant effect of R-ketamine. In SPT, pre-administration of SB431542 abolished the anti-anhedonia effect of R-ketamine. The effect of the neutralizing antibody on TGF-β1 on the antidepressant effect of R-ketamine was investigated. Neutralizing antibody against TGF-β1 (R & D, Catalog No .: MAB1835-5001 μg / mL, 2 μL) was administered intracerebroventricularly 30 minutes before administration of R-ketamine hydrochloride (10 mg / kg). As a control, IgG antibody (R & D, Catalog No .: MAB002, 1 μg / mL, 2 μL) was administered intraventricularly. There was no difference in the amount of exercise in all groups. Premedication of neutralizing antibodies in TST and FST abolished the antidepressant effect of R-ketamine. In SPT, pre-administration of neutralizing antibody eliminated the anti-anhedonia effect of R-ketamine. On the other hand, administration of the control antibody did not antagonize the antidepressant and anti-anhedonia effects of R-ketamine. The results in FIGS. 1 to 5 suggest that TGF-β1 is involved in the antidepressant effect of R-ketamine. Therefore, recombinant mouse TGF-β1 (R & D, Catalog No .: 7666-MB-005, 10 ng / μL, 2 μL), R-ketamine (1 mg / mL, 2 μL), and physiological saline (2 μL) were intraventricularly administered. .. There was no difference in the amount of exercise in all groups. In TST, FST and SPT, TGF-β1 showed an antidepressant effect equivalent to that of R-ketamine, and the effect was observed even 7 days after administration. In socially defeated stress mice, recombinant mouse TGF-β2 (R & D, catalog number: 302-B2, 10 ng / μL, 2 μL), R-ketamine (1 mg / mL, 2 μL), saline (2 μL) was used in the brain. It was administered indoors. There was no difference in the amount of exercise in all groups. In TST, FST and SPT, R-ketamine showed antidepressant effect, but TGF-β2 did not show antidepressant effect. This is a test using a lipopolysaccharide (LPS) -induced inflammation model of depression. TGF-β1 (R & D, Catalog No .: 7666-MB-) in LPS-treated mice to which LPS (0.5 mg / kg) was intraperitoneally administered. It is a figure explaining the test plan which compared and examined the antidepressant effect after intracerebral administration of 005, 10 ng / μL, 2 μL) by FST. There was no difference in the amount of exercise between the three groups. A significant increase in akinesia time was observed in LPS-treated mice treated with saline as compared with normal mice. Intracerebroventricular administration of TGF-β1 significantly reduced the increased immobility time in LPS-treated mice. The vertical axis of the figure shows the immobility time (seconds) in FST. It is a figure explaining the test plan which compared and examined the antidepressant effect of TGF-β1 with the learned helplessness model of a rat. Learned helpless rats were administered TGF-β1 (R & D, Catalog No .: 7666-MB-005, 250 ng / side, 5 μL, bilateral) intracerebroventricularly and a conditional avoidance study was performed 5 days later. In the TGF-β1 administration group, a significant decrease in the number of failures and the latency was observed as compared with the saline solution. That is, intracerebroventricular administration of TGF-β1 showed antidepressant effect in a rat learned helplessness model. This is a test using an LPS-induced inflammation model of depression. It is a figure explaining the study plan which compared and examined the antidepressant effect after intranasal (intranasal; IN) administration of TGF-β1 by FST. There was no difference in the amount of exercise between the three groups. A significant increase in akinesia time was observed in LPS-treated mice treated with saline as compared with normal mice. Intranasal administration of TGF-β1 significantly reduced the increased immobility time in LPS-treated mice. The vertical axis of the figure shows the immobility time (seconds) in FST.

The prophylactic or therapeutic agent for depression or depressive symptoms of the present invention contains transforming growth factor β1 (TGF-β1) or a salt thereof as an active ingredient.
Non-Patent Document 2 shows an action opposite to that of the present invention, and the views on TGF-β1 and antidepressant action do not agree with each other. However, in the present invention, as shown in the following examples, the antidepressant action In behavioral tests in multiple animal models known as evaluation models, the immediate antidepressant and sustained effects of TGF-β1 have been confirmed.

Transforming growth factor (TGF) is known as one of the growth factors that play an important role in tissue development, cell differentiation, embryonic development, etc., which is also called transforming growth factor.
TGF-β1 is produced in almost all cells such as kidney, bone marrow, and platelets, and there are five subtypes (β1 to β5).
TGF-β1 is secreted as a high molecular weight inactive form (latent form), and exerts its action by being activated in the vicinity of the target cell.
In the present invention, TGF-β1 means both the inactive form and the active form, but the active form TGF-β1 is preferable.
In addition, it may be administered as an inactive form and converted to the active form in vivo.

The protein is not particularly limited as long as it is a protein classified as TGF-β1, and conventionally known TGF-β1 may be used as the active ingredient in the present invention.
As TGF-β1, human-derived TGF-β1 can be preferably used, and it may be a protein having the same amino acid sequence as a natural protein, and is an artificial protein in which a part of the amino acid sequence is modified. May be.
Amino acid sequence modification means homology of 80% or more, preferably 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more in the amino acid sequence of a natural protein. It means that it is a protein having sex and has not lost its function as TGF-β1.
Further, the modification of the amino acid sequence means that one or more amino acids may be substituted, deleted or inserted in the amino acid sequence of the natural protein.

As TGF-β1 in the present invention, a part of a cohesive amino acid sequence is deleted in TGF-β1 having the same amino acid sequence as the natural amino acid sequence as long as the function as TGF-β1 is not lost. It may be a protein.

The TGF-β1 in the present invention may be produced by an in vivo reaction using cells or the like, or may be artificially produced, for example, by chemical synthesis.

The salt of TGF-β1 in the present invention is not particularly limited, and may be a salt in a form known as a protein salt, and may be selected from salts known as pharmacologically acceptable salts. Good.

In the present invention, the term "as an active ingredient" means that it is contained as a main active ingredient, and the content thereof is not particularly limited as long as it contains TGF-β1 or a salt thereof as a medicinal ingredient.

In the present invention, the term "prevention or treatment of depression or depressive symptoms" means to prevent the onset of depression or depressive symptoms, or to alleviate depressive symptoms in patients suffering from depression or exhibiting depressive symptoms. It means that it exerts a therapeutic effect such as depression and improvement.
Depression or depressive symptom, which is a kind of psychiatric disorder, is a illness that progresses over a long period of time (yearly). Therefore, it is possible to prevent the progression of the symptom by starting treatment at an early stage.
In addition, for patients with a genetic background who are potentially susceptible to depression or depressive symptoms, by administration before the symptoms of depression or depressive symptoms, depression or depressive symptoms It can also be used to prevent the onset.
The preventive agent for depression or depressive symptom in the present invention may be an agent for preventing the onset of depression or depressive symptom, or an agent for preventing the progression of symptom. In addition, the therapeutic agent for depression or depressive symptom in the present invention has a therapeutic effect such as preventing the progression of the symptom or alleviating or ameliorating the symptom.

In the present invention, "depression" is not particularly limited, but is a disease medically diagnosed as "depression". For example, it may be a disease that is classified as "depression" by the American Psychiatric Association's "Diagnosis and Statistics Manual for Mental Disorders, 5th Edition", or it may be a disease that is classified as "depression" by the international disease classification. Good. In addition, the disease may be regarded as "depression" according to the guidelines issued by the Japanese Society of Depression.
Depression includes major depressive illness (MDD), childhood depression and adult depression.

In the present invention, the "depressive symptom" means, but is not limited to, a symptom such as depression of mood, decreased motivation, anxiety and associated insomnia and loss of appetite. In addition, as depressive symptoms, even if the depression diagnosed as described above is not diagnosed, it is diagnosed that the symptoms such as mood depression, decreased motivation, anxiety and associated insomnia and loss of appetite are exhibited. Including the state to be done.
Diseases that present with depressive symptoms include bipolar disorder, autism spectrum disorder, attention deficit hyperactivity disorder (ADHD), generalized anxiety disorder, and obsessive disorder, in which depressive symptoms and their opposite symptoms, manic symptoms, are repeated. , Social anxiety disorder, post-traumatic stress disorder (PTSD), schizophrenia, feeding disorder, substance use disorder (including drug dependence) and the like.
The prophylactic or therapeutic agent for depressive symptoms in the present invention can be used for the prevention or treatment of depressive symptoms in these diseases.

Since "TGF-β1 or a salt thereof" used in "prevention or treatment of depression or depressive symptoms" in the present invention is considered to exhibit antidepressant effect of R-ketamine, depression or depressive symptoms in R-ketamine. It can be used in the same manner as the prevention or treatment of.
The prevention or treatment of depression or depressive symptoms in R-ketamine is as described in JP2015-78181.

In the present invention, the depressive symptom includes the symptom due to depression, but the prophylactic or therapeutic agent for depression or depressive symptom of the present invention is preferably a therapeutic agent for depression or depressive symptom. It is more preferable that it is a therapeutic agent for depression.

The prophylactic or therapeutic agents for depression or depressive symptoms of the present invention include depression, depressive symptoms, depressive symptoms, major depressive disorder, bipolar disorder, autism spectrum disorder, attention defect hyperactivity disorder (Attention-deficit). hyperactivity disorder (ADHD), generalized anxiety disorder, compulsive disorder, social anxiety disorder, post-traumatic stress disorder (PTSD), schizophrenia, eating disorder, substance use disorder (including drug dependence), suicide thought Can it be a prophylactic or therapeutic agent for suppression or for improvement in the subject in need of depression, decreased motivation, anxiety, insomnia, or loss of appetite, and is it a prophylactic or therapeutic agent for depression? , Preferably a prophylactic or therapeutic agent for depression, decreased motivation, anxiety, insomnia, or loss of appetite, and more preferably a prophylactic or therapeutic agent for depression.

The depressive symptom in the present invention may be a psychiatric disorder (mental disorder) other than the above-mentioned diseases or a depressive symptom in a neurodegenerative disease, and may be a mental disorder (mental disorder) such as cancer or cardiovascular disease. It may be a depressive symptom associated with other illnesses other than neurodegenerative illness.

The prophylactic or therapeutic agent, drug and pharmaceutical composition of depression or depressive symptoms in the present invention can be administered orally or parenterally.
For oral administration, known dosage forms such as tablets, capsules, coated tablets, troches, solutions such as solutions or suspensions can be used. Parenteral administration includes intravenous, intramuscular or subcutaneous administration by injection, transmucosal administration such as nasal cavity and oral cavity using powder, drops, spray or aerosol, and rectum using cream or suppository. Examples include administration, transdermal administration using patches, liniments or gels.
The route of administration is preferably nasal administration or intravenous administration by injection.

In addition to TGF-β1 or a salt thereof, the pharmaceutical composition in the present invention may contain other medicinal ingredients other than TGF-β1 used for treating or preventing depression or depressive symptoms as an active ingredient.
In addition to these medicinal ingredients, the pharmaceutical composition may appropriately contain an appropriate pharmaceutically acceptable carrier well known to those skilled in the art, depending on the dosage form and the like.
The pharmaceutically acceptable carrier is not particularly limited, and for example, an antioxidant, a stabilizer, a preservative, a flavoring agent, a colorant, a solubilizer, a solubilizer, a surfactant, an emulsifier, an antifoaming agent, etc. Examples include viscosity regulators, gelling agents, absorption promoters, dispersants, excipients and pH regulators.

When the prophylactic or therapeutic agent, drug and pharmaceutical composition of depression or depressive symptom in the present invention are prepared as an injectable preparation, the form of a solution or suspension preparation is preferable, and transmucosa such as the nasal cavity and oral cavity. When prepared as a preparation for administration, a powder, drop or aerosol preparation is preferable, and when prepared as a preparation for rectal administration, a semi-solid preparation such as a cream or suppository is used. Is preferable.
All of these formulations should be prepared by any method known to those skilled in the art of pharmaceutical technology, for example as described in Remington's Pharmaceutical Sciences (Mac Publishing Company, Easton, PA, 1970). Can be done.
As the carrier, the injectable preparation may use, for example, a plasma-derived protein such as albumin, an amino acid such as glycine, and a sugar such as mannitol, and may further use a buffer, a lysis aid, an isotonic agent, or the like. .. In addition, when used as a water-soluble preparation or a lyophilized preparation, surfactants such as Tween (registered trademark) 80 and Tween (registered trademark) 20 may be used in order to prevent aggregation.
Parenteral preparations other than injectable preparations use, for example, distilled water or saline, polyalkylene glycols such as polyethylene glycol, plant-derived oils, and hydrogenated naphthalene as carriers. You may. For example, a preparation for rectal administration such as a suppository may use, for example, polyacylene glycol, petrolatum, cacao oil or fat as an excipient. In the preparation for vaginal administration, an absorption enhancer such as bile salt, ethylenediamine salt, and citrate may be used as the carrier. The inhalation preparation may be a solid, and as an excipient, for example, lactose may be used. The drop for nasal administration may be a water or oil solution.

The exact dosage and administration plan of a prophylactic or therapeutic agent, drug and pharmaceutical composition for depression or depressive symptoms in the present invention depends on the required amount, treatment method, illness or degree of necessity for each individual treatment subject. To be adjusted.
The dose can be specifically determined according to age, body weight, general health, gender, diet, administration time, administration method, excretion rate, drug combination, patient's medical condition, etc. It may be decided in consideration of the factors of.
When the prophylactic or therapeutic agent, drug and pharmaceutical composition of depression or depressive symptom in the present invention is administered to a patient having depression or depressive symptom, TGF-β1 or a salt thereof is effective for the symptom of each psychiatric disorder. It is preferable to include it in a large amount.
Like R-ketamine, TGF-β1 or a salt thereof may be safely used with few side effects, and the daily dose varies depending on the patient's condition, body weight, type of compound, route of administration, etc. However, in the case of parenteral administration, it is usually about 0.01 to 1000 mg / person / day, preferably 0.1 to 500 mg / person / day, and in the case of oral administration, it is usually about 0.01. It is ~ 500 mg / person / day, preferably 0.1-100 mg / person / day.
In the present invention, TGF-β1 or a salt thereof may be administered in a single dose, or may be administered once or twice a week.

In the present invention, it may be a method for preventing or treating depression or depressive symptoms, which comprises administering TGF-β1 or a salt thereof to a patient in need thereof.
The patient who needs it is not particularly limited, but is a mammal, preferably a human.
TGF-β1 or a salt thereof is preferably administered in a therapeutically effective amount.

The present invention also relates to a method for screening a compound that can be used as a prophylactic or therapeutic agent for depression or depressive symptoms.
As detailed in the examples below, it has been shown herein that the antidepressant effect of R-ketamine is associated with TGFβ-1 expression in depression model animals. Further, in the present specification, from the experimental results in depression model animals, it can be understood that the antidepressant effect of R-ketamine is exerted by the enhancement of the cascade via the TGFβ-1 receptor. Therefore, compounds that can be confirmed to enhance the TGFβ-1 and TGFβ-1 receptor cascades can be expected to exert antidepressant effects similar to the antidepressant effects of R-ketamine.
Here, enhancing the cascade of TGFβ-1 and TGFβ-1 receptors means a signal pathway in which TGFβ-1 binds to the TGFβ-1 receptor and is triggered.
Therefore, the present invention provides a method for screening a prophylactic or therapeutic agent for depression or depressive symptoms by enhancing the TGFβ-1 and TGFβ-1 receptor cascades.
Examples of the TGFβ-1 receptor include TGFBR1 or TGFBR2.

The enhancement of the TGFβ-1 and TGFβ-1 receptor cascade may be confirmed by increasing the expression of TGFβ-1, or a compound that binds to the TGFβ-1 receptor and has an agonistic action may be confirmed.
The present invention may be a method for screening a prophylactic or therapeutic agent for depression or depressive symptoms by confirming that it increases the expression of TGFβ-1.
In the present invention, an increase in the expression of TGFβ-1 can be confirmed by confirming an increase in the expression of the protein itself, but it is preferable to confirm by using an increase in the expression of TGFβ-1 mRNA as an index.
The increase in protein expression and the increase in mRNA expression can be carried out by a conventionally known method.
In confirming the increase in TGFβ-1 expression, TGFβ-1 expression was expressed using a compound known as a TGF-β1 receptor antagonist or an anti-TGF-β1 antibody that serves as a neutralizing antibody for TGF-β1. You may check the rise.
The present invention may be a method for screening a prophylactic or therapeutic agent for depression or depressive symptoms by identifying a compound that binds to the TGFβ-1 receptor and has an agonistic action.
In the present invention, a search for a compound that binds to the TGFβ-1 receptor and confirmation of whether the compound has an agonistic action can be carried out by a conventionally known method.

Further, in the screening method of the present invention, a behavioral test is further conducted on a compound capable of confirming an enhancement of the cascade of TGFβ-1 and TGFβ-1 receptors, and a method for screening a prophylactic or therapeutic agent for depression or depressive symptoms. It is preferable to do so.
In the present invention, a compound that can be confirmed to increase the expression of TGFβ-1 may be selected, and the compound may be administered to a model animal as a method for screening a prophylactic or therapeutic agent for depression or depressive symptoms by a behavioral test. ..
Behavioral tests include, but are not limited to, behavioral tests used as indicators for screening antidepressants, such as locomotor activity test (LMT), tail suspension test (TST), forced swimming test (FST), and show. The sugar preference test (SPT) and the like may be used alone or in combination.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples. In addition, various changes can be made without departing from the technical idea of the present invention.
All tests were conducted with the permission of the Chiba University Animal Experiment Committee.

Using a stress model mouse for social defeat of depression, RNA-seq analysis covers the difference in gene expression in the frontal cortex due to administration of (S) -ketamine or (R) -ketamine to the depression-like behavior of the model animal. Was analyzed.

1. 1. Materials and Methods (S) -Ketamine Hydrochloride and (R) -Ketamine Hydrochloride were prepared according to the method described in International Publication No. 2019/065900. The purity of these isomers is determined by high performance liquid chromatography (CHIRALPAK® IA, column size: 250 × 4.6 mm, mobile phase; n-hexane / dichloromethane / diethylamine (75/25 / 0.1), Daicel Corporation. ) Confirmed.

The stress model mouse for social defeat of depression is a C57 / B6 male mouse according to Golden SA, et al. A standardized protocol for repeated social defeat stress in mice. Nat Protocol. 2011; 6 (8): 1183-1191. It was created by applying a stress called "social defeat stress" in which ICR male mice (large and aggressive mice) were in contact with each other for 10 consecutive days. Depressive behavior was observed in mice subjected to social defeat stress. Specifically, the social defeat stress model mouse is a behavioral test used as an index for screening antidepressants, two tests, a tail suspension test (TST) and a forced swimming test (Force swimming test;). An increase in immobility time was observed in all of FST). In addition, in the sucrose preference test (SPT), the proportion of drinking sucrose water was significantly reduced, suggesting that depressive behavior was induced. On the other hand, in the locomotion test (LMT), which is an index of motor function, there was no difference in the amount of locomotor activity between the socially defeated stress mice and the normal mice.

The above-mentioned depression model animal was prepared and the drug was specifically administered as described below (Fig. 1A). As mice, male C57 / B6 mice (7 weeks old, Japan SLC) and ICR mice (9 weeks old, Japan SLC) were used. Mice were allowed to freely ingest water and feed. Social defeat stress was performed by cohabiting one C57 / B6 mouse and one ICR mouse for 10 days. On the 11th day, a social interaction test was conducted, mice exhibiting depressive symptoms were selected, and (S) -ketamine hydrochloride (10 mg / kg) or (R) -ketamine hydrochloride (10 mg / kg) was intraperitoneally administered. It was administered. Three days after administration, after isoflurane anesthesia, the brain was immediately removed, the frontal cortex was quickly divided, and the sample stored in a freezer at -80 ° C was sent to Takara Co., Ltd. for RNA-seq analysis. GSEA (gene set enrichment analysis) of the obtained data was carried out.

In the same manner as above, a social defeat stress model mouse was prepared, and 3 days after administration of (S) -ketamine hydrochloride (10 mg / kg) or (R) -ketamine hydrochloride (10 mg / kg), after isoflurane anesthesia. The brain was quickly removed, and the frontal cortex and hippocampus were quickly divided and stored in a freezer at -80 ° C (Fig. 2). Gene expression of TGF-β1, TGF-β2 and their respective receptors (TGFBR1, TGFBR2) in the frontal cortex and hippocampus was quantified using Applied Biosystems primers.

To examine the antidepressant effect of the drug, behavioral tests such as TST, FST, LMT, and SPT were performed (FIGS. 3 to 8). LMT and TST were performed on the day of administration and FST was performed the day after administration. SPT was performed 2 days, 4 days and 7 days after administration.
The TST was performed as follows. First, the mouse was removed from the cage, and a small piece of adhesive tape was attached to a portion approximately 2 cm from the tip of its tail. Small holes were made in the pieces and the mice were hung on hooks. The akinesia time of each mouse was recorded for 10 minutes. Only when the mouse was non-resistive and completely stationary was determined to be akinesia. Akinesia time increases in depression.
The FST was carried out as follows. First, cylinders (diameter: 23 cm, height: 31 cm) were filled with water up to 15 cm and maintained at 23 ± 1 ° C., and mice were placed in each cylinder. Mice were tested using SCANET MV-40 (Melquest) in an automatic forced swimming device. The immobility time was calculated using the analysis software of the device as a value obtained by subtracting the activation time from the total time. Cumulative akinesia time was recorded for 6 minutes during the test period.
The LMT was performed as follows. First, the mice were placed in an experimental cage (length x width x height: 560 x 560 x 330 mm). Spontaneous motor activity of mice was counted by SCANETMV-40 and cumulative movement was recorded for 60 minutes. The cage was washed between tests. Akinesia time increases in depression.
SPT was carried out by preparing ordinary drinking water and a 1% sucrose solution and ingesting them freely, and measuring the ratio of the consumption of the sucrose solution. In depression, the consumption of sucrose solution, which is a reward reaction, is reduced.

Using a social defeat stress model mouse, the effect of the TGF-β1 receptor antagonist RepSox (Selleckchem) on the antidepressant effect of R-ketamine was investigated (Fig. 3). Social defeat stress was performed for 10 days, the next day a social interaction test was performed, and only mice showing depressive symptoms were selected and used in the next experiment. Repox (10 mg / kg) or saline (10 mL / kg) was intraperitoneally administered 30 minutes prior to administration of R-ketamine hydrochloride (10 mg / kg) or saline (10 mL / kg). LMT was performed 1 hour after R-ketamine administration, and TST was performed 3 hours after R-ketamine administration. FST was performed the day after administration. SPT was performed 2 days after administration.

Using a social defeat stress model mouse, the effect of another TGF-β1 receptor antagonist SB431542 (Tocris Bioscience) on the antidepressant effect of R-ketamine was investigated (Fig. 4). Social defeat stress was performed for 10 days, the next day a social interaction test was performed, and only mice showing depressive symptoms were selected and used in the next experiment. SB431542 (10 μM, 2 μL) or saline (2 μL) was administered intracerebroventricularly 30 minutes prior to administration of R-ketamine hydrochloride (10 mg / kg) or saline (10 mL / kg). LMT was performed 1 hour after R-ketamine administration, and TST was performed 3 hours after R-ketamine administration. FST was performed the day after administration. SPT was performed 2 days after administration.

Using a social defeat stress model mouse, the effect of a neutralizing antibody on TGF-β1 on the antidepressant effect of R-ketamine was investigated (Fig. 5). Social defeat stress was performed for 10 days, the next day a social interaction test was performed, and only mice showing depressive symptoms were selected and used in the next experiment. Neutralizing antibody of TGF-β1 (R & D, Catalog No .: MAB1835-500, 1 μg / mL, 2 μL) or saline (2 μL) with R-ketamine hydrochloride (10 mg / kg) or saline (10 mL / kg) ) It was administered intraventricularly 30 minutes before administration. LMT was performed 1 hour after R-ketamine administration, and TST was performed 3 hours after R-ketamine administration. FST was performed the day after administration. SPT was performed 2 days after administration.

Using a social defeat stress model mouse, it was investigated whether TGF-β1 showed an antidepressant effect in the same manner as R-ketamine (Fig. 6). Social defeat stress was performed for 10 days, the next day a social interaction test was performed, and only mice showing depressive symptoms were selected and used in the next experiment. Saline (2 μL), R-ketamine (1 mg / mL, 2 μL) and recombinant mouse TGF-β1 (R & D, Catalog No .: 7666-MB-005, 10 ng / μL, 2 μL) were administered intraventricularly. LMT was performed 1 hour after R-ketamine administration, and TST was performed 3 hours after R-ketamine administration. FST was performed the day after administration. SPT was performed 2 days after administration, 4 days after administration, and 7 days after administration.

Using a social defeat stress model mouse, it was investigated whether TGF-β2 showed an antidepressant effect as well as R-ketamine (Fig. 7). Social defeat stress was performed for 10 days, the next day a social interaction test was performed, and only mice showing depressive symptoms were selected and used in the next experiment. Physiological saline (2 μL), R-ketamine (1 mg / mL, 2 μL), and recombinant mouse TGF-β2 (R & D, Catalog No .: 302-B2, 10 ng / μL, 2 μL) were administered intraventricularly. LMT was performed 1 hour after R-ketamine administration, and TST was performed 3 hours after R-ketamine administration. FST was performed the day after administration. SPT was performed 2 days after administration and 4 days after administration.

Inflammatory animal model mice for depression were prepared by administering lipopolysaccharide (hereinafter abbreviated as "LPS") during adulthood of the mice (Fig. 8). Depression-like behavior was observed in LPS-treated mice, suggesting that this mouse could be a novel animal model of depression. This model mouse was created by the inventor of the present application and its collaborators, and several papers have been reported (Zhang JC, et al. Antidepressant effects of TrkB ligands on depression-like behavior and dendritic changes in mice after inflammation. Int J Neuropsychopharmacol. 2014 Oct 31; 18 (4). Pii: pyu077, Ma M et al, Antidepressant effects of combination of brexpiprazole and fluoxetine on depression-like behavior and dendritic changes in mice after inflammation. Psychopharmacology (Berl). 2017 Feb; 234 (4): 525-533.). This model mouse showed an increase in immobility time in FST as compared with normal mice, while in LMT, there was no difference in locomotor activity between LPS-administered mice and normal mice. These results suggest that LPS administration induced depressive behavior in this model mouse.
LPS (0.5 mg / kg) or saline (10 mL / kg) was intraperitoneally administered, and 23 hours later, saline (2 μL) or recombinant mouse TGF-β1 (R & D, Catalog No .: 7666-MB-) 005, 10 ng / μL, 2 μL) was administered intraventricularly. Momentum was performed 1 hour after administration, and FST was performed 3 hours after administration.

Learned helplessness rat model and the antidepressant effects 1) Shirayama Y, et al. Alterations in brain-derived neurotrophic factor (BDNF) and its precursor proBDNF in the brain regions of a learned helplessness rat model and the antidepressant effects of a TrkB agonist and antagonist. Eur Neuropsychopharmacol. 2015 Dec; 25 (12): 2449-58., 2) Shirayama Y, et al. Effects of a single bilateral infusion of R-ketamine in the rat brain regions of a learned helplessness model of depression. Eur Arch Psychiatry Clin Neurosci. 2017 Mar; 267 (2): 177-182., 3) Shirayama Y, et al. Lack of Antidepressant Effects of (2R, 6R)-Hydroxynorketamine in a Rat Learned Helplessness Model: Comparison with (R)-Ketamine. Int J Neurops ychopharmacol. 2018 Jan 1; 21 (1): 84-88.).
Electroconvulsive stimulation was given on the 1st and 2nd days, the test was performed on the 3rd day, and only rats showing depressive symptoms were used for the next test (Fig. 9). Saline (5 μL) or recombinant mouse TGF-β1 (R & D, Catalog No .: 7666-MB-005, 250 ng / side, 5 μL, bilateral) was administered intraventricularly. A conditional escape study was performed 5 days after administration.

A test of intranasal administration of TGF-β1 was conducted using an inflammatory animal model mouse of depression (Fig. 10). LPS (0.5 mg / kg) or saline (10 mL / kg) was intraperitoneally administered, and 23 hours later, saline (2 μL) or recombinant mouse TGF-β1 (R & D, Catalog No .: 7666-MB-) 005, 10 ng / μL, 2 μL) was administered intranasally. Momentum was performed 1 hour after administration, and FST was performed 3 hours after administration.

Statistical analysis of the results of gene expression in the social defeat stress model was performed by one-way ANOVA followed by a minimum significant difference test. Data are expressed as mean ± standard error (n = 6 mice / group). Statistical analysis of the results of the social defeat stress model and LPS dosing model was performed by one-way ANOVA followed by a minimal significant difference test. Data are expressed as mean ± standard error (n = 8 mice / group). * P <0.05, ** p <0.01, *** p <0.001 show a significant difference compared with the group of socially defeated stress mice treated with saline. * P <0.05 and ** p <0.01 show significant differences as compared with the LPS-administered mouse group to which physiological saline was administered. Statistical analysis of the results of the rat learned helplessness model was performed by performing a t-test. Data are expressed as mean ± standard error (n = 5 rats / saline administration group, n = 6 rats / TGF-β1 administration group).

2. 2. Results Three days after administration of both ketamine isomers, the frontal cortex was removed, and RNA-seq analysis and GSEA were performed. As a result, a TGF-β signal showing a large difference in gene expression between the two isomers was identified (Fig.). 1B).

In a social defeat stress model, TGF-β1 expression was significantly reduced in the depressive mouse prefrontal cortex and hippocampus and increased with R-ketamine hydrochloride (10 mg / kg) administration, but with S-ketamine hydrochloride. Administration of (10 mg / kg) did not improve. Expression of TGF-β2 was unchanged in the depressive mouse frontal cortex and hippocampus. Expression of TGF-β1 and TGF-β2 receptors was significantly reduced in the depressive mouse frontal cortex and hippocampus and increased with R-ketamine hydrochloride (10 mg / kg) administration, but with S-ketamine hydrochloride. Administration of salt (10 mg / kg) did not improve (Fig. 2).

In a social defeat stress model, pre-administration of the TGF-β1 receptor antagonist RepSox antagonized the antidepressant and anti-anhedonia effects of R-ketamine (Fig. 3).

In a social defeat stress model, pre-administration of the TGF-β1 receptor antagonist SB431542 antagonized the antidepressant and anti-anhedonia effects of R-ketamine (Fig. 4).

In the social defeat stress model, pre-administration of TGF-β1 neutralizing antibody antagonized the antidepressant and anti-anhedonia effects of R-ketamine (Fig. 5).

In the social defeat stress model, intracerebroventricular administration of recombinant mouse TGF-β1 showed an immediate antidepressant effect and a sustained effect equivalent to those of R-ketamine (Fig. 6).

In the social defeat stress model, intracerebroventricular administration of recombinant mouse TGF-β2 did not show antidepressant effect unlike R-ketamine (Fig. 7).

In the LPS treatment model of depression, intracerebroventricular administration of recombinant mouse TGF-β1 showed antidepressant effect (Fig. 8).

In a rat learned helpless model of depression, intracerebroventricular administration of recombinant mouse TGF-β1 showed antidepressant effect 5 days after administration (Fig. 9).

In the LPS treatment model of depression, intranasal administration of recombinant mouse TGF-β1 showed antidepressant effect (Fig. 10).

In the present invention, a prophylactic or therapeutic agent for depression or depressive symptoms containing transforming growth factor β1 or a salt thereof as an active ingredient is useful as a novel drug.

Claims (7)

A prophylactic or therapeutic agent for depression or depressive symptoms, which comprises transforming growth factor β1 or a salt thereof as an active ingredient. A pharmaceutical composition for the prevention or treatment of depression or depressive symptoms, which comprises transforming growth factor β1 or a salt thereof as an active ingredient. A method for screening a prophylactic or therapeutic agent for depression or depressive symptoms by enhancing the TGFβ-1 and TGFβ-1 receptor cascade. The screening method according to claim 3, wherein the TGFβ-1 receptor is TGFBR1 or TGFBR2. The screening method according to claim 3 or 4, wherein the enhancement of the TGFβ-1 and TGFβ-1 receptor cascade is confirmed by the increased expression of TGFβ-1. The screening method according to claim 5, wherein the increased expression of mRNA is used as an index. Further, the screening method according to any one of claims 3 to 6, wherein a behavioral test is performed.

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