JP5222608B2 - Anti-anxiety agent - Google Patents

Description

The present invention relates to a novel anxiolytic agent comprising a component that indirectly or directly activates the DP ₁ receptor as an active ingredient and a novel pharmaceutical agent containing the anxiolytic agent.

In today's overstressed society, unnecessary anxiety can persist for a long time, causing many people to suffer. Symptoms such as sweating, motivation, headache, and insomnia caused by anxiety are considered to be a case of mental disorder called anxiety disorder. Conventionally, benzodiazepines have been prescribed as anti-anxiety agents for patients with such anxiety disorders.
However, benzodiazepines have problems that cause side effects such as movement disorders, sleepiness, and the development of dependence due to long-term use. Therefore, there is a need for a novel anxiolytic agent that replaces benzodiazepine drugs.

In recent years, it has been reported that a peptide composed of five amino acid residues having an anxiolytic action exists in a degradation product obtained by enzymatic degradation of β-conglycinin, which is a kind of protein contained in soybean (patent) Reference 1). The peptide has an amino acid sequence common to an opioid peptide consisting of seven amino acid residues that binds to an opioid receptor and exhibits an analgesic effect, but the mechanism of action that exerts an anxiolytic effect has not been clarified. .
JP 2007-91656 A

In the future, the number of patients with anxiety disorders is expected to increase further, and there is an increasing expectation for novel anti-anxiety agents that are completely different from conventional ones.
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a novel anxiolytic agent having sufficient anxiolytic activity and a novel pharmaceutical agent containing the anxiolytic agent.

In order to solve the above problems, the present inventors synthesized a peptide consisting of the amino acid sequence represented by Met-Arg-Trp and examined its effect on the central nervous function. Found to have. Furthermore, as a result of examining the action mechanism, it was found that the peptide exhibits an anxiolytic action by indirectly activating the DP ₁ receptor. From this, it was found that a component that indirectly or directly activates the DP ₁ receptor exhibits an anti-anxiety action, and the present invention has been completed. Component Here, the components that indirectly activates the DP ₁ receptor, which without directly interacting with DP ₁ receptor, refers to a component which activates this, directly activate the DP ₁ receptor The term refers to a component that directly interacts with and activates the DP ₁ receptor.

That is, in order to solve the problem,
The invention according to claim 1 is an anxiolytic agent comprising, as an active ingredient, a peptide consisting of an amino acid sequence represented by Met-Arg-Trp or a pharmaceutically acceptable salt thereof .
According to a second aspect of the invention, an anti-anxiety agent that prostaglandin D ₂ or a pharmaceutically acceptable salt thereof as an active ingredient.
The invention according to claim 3 is an anxiolytic agent comprising BW245C or a pharmaceutically acceptable salt thereof as an active ingredient .

  According to the present invention, a novel anxiolytic agent that can be easily taken orally and has a sufficient anxiolytic effect and a novel pharmaceutical agent containing the anxiolytic agent are obtained.

Hereinafter, the present invention will be described in detail.
The anxiolytic agent of the present invention comprises a component that activates the DP ₁ receptor indirectly or directly as an active ingredient.
Here, the components that indirectly activates the DP ₁ receptor, without directly interacting with DP ₁ receptor, refers to a component which activates it. In addition, the component that directly activate the DP ₁ receptor interacts directly with DP ₁ receptor, it refers to a component which activates it. The interaction refers to, for example, forming a bond between molecules by a covalent bond or the like, or forming a bond by an intermolecular attractive force such as a hydrogen bond, a hydrophobic bond, or an ionic bond.
Conventionally, it is known that the DP ₁ receptor is involved in the development of inflammation and allergies by binding, for example, prostaglandin D ₂ . In contrast, the present invention for the first time found that an anxiolytic action is manifested by indirectly or directly activating the DP ₁ receptor, and provides a completely new anxiolytic agent based on this finding. To do.

The anti-anxiety agent according to the first embodiment of the present invention comprises a component that indirectly activates the DP ₁ receptor (hereinafter sometimes abbreviated as active component 1) as an active component. The active component 1 may be any component that indirectly activates the DP ₁ receptor. For example, in vivo, by interacting with receptors other than the specific substance or DP ₁ receptor, increasing the concentration of a substance that directly interacts with DP ₁ receptor, to promote the interaction of the substance and the DP ₁ receptor Ingredients.
Such an anxiolytic agent may contain any component other than the active component 1 as long as the effects of the present invention are not hindered.

The active ingredient 1 may be either one derived from a living body or one not derived from a living body. Examples of the active ingredient 1 derived from a living body include a peptide having an amino acid sequence represented by Met-Arg-Trp (hereinafter sometimes abbreviated as peptide 1). As the active ingredient 1 not derived from a living body, an artificially synthesized substance, a chemically modified active ingredient 1 derived from the living body, or a biologically modified substance other than the active ingredient 1 derived from the living body is chemically modified. Among them, those that indirectly activate the DP ₁ receptor can be exemplified. Here, chemical modification refers to, for example, replacing an atom or group of a chemical substance or active component 1 with an atom or group different from these, or adding a new atom or group to the chemical substance or active component 1. Point to.

  The peptide 1 may be chemically synthesized, isolated from a living body, or obtained by decomposing a peptide or protein having an amino acid sequence represented by Met-Arg-Trp.

When peptide 1 is chemically synthesized, the synthesis method is not particularly limited, and either a liquid phase synthesis method or a solid phase synthesis method may be used, and it may be appropriately selected from known methods. Specifically, the Fmoc method using a 9-fluorenylmethyloxycarbonyl group (Fmoc group) as a protective group, if it is a method involving the introduction and deprotection of a protective group to an amino group during the formation of a peptide bond, Examples thereof include the Boc method using a tert-butoxycarbonyl group (Boc group).
Peptide 1 can be isolated by performing appropriate post-treatment and purification after the synthesis reaction. Alternatively, the mixture may be used as it is without isolation of peptide 1. Here, post-treatment and purification may be performed by known methods. Specifically, well-known processes such as pH adjustment, extraction, washing, centrifugation, filtration, concentration, drying, crystallization and column chromatography may be performed alone or in combination with a plurality of processes as necessary. For example, for concentration, examples include a method of distilling off the solvent by atmospheric concentration or vacuum concentration, and a method of removing water using a filtration membrane such as a reverse osmosis membrane, an ultrafiltration membrane, or a microfiltration membrane. it can. Examples of drying include heat drying and freeze drying. Examples of column chromatography include reverse phase chromatography, ion exchange chromatography, affinity chromatography, and molecular sieve chromatography using silica gel or various appropriate resins as fillers.
The amino acid sequence of the extracted peptide 1 can be identified by a known method such as Edman degradation.

  When peptide 1 or protein is decomposed to obtain peptide 1, decomposition conditions may be set according to the type of peptide or protein. Among them, it is preferable to perform hydrolysis using an enzyme. A hydrolase may be used individually by 1 type, and may use 2 or more types together. Alternatively, different types of enzymes may be used in stages. Specific examples of preferred methods include a method in which a hydrolase is allowed to act on ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO, hereinafter abbreviated as “rubisco”). In this case, it is preferable to further cause pancreatin to act after allowing pepsin to act as a hydrolase.

  It is preferable to appropriately adjust the conditions for the hydrolysis reaction with the enzyme according to the type of peptide or protein or enzyme to be subjected to the decomposition. In particular, the pH and reaction temperature are preferably set to the optimum pH and optimum temperature according to the type of enzyme. For example, as described above, when rubisco is hydrolyzed with pepsin and then with pancreatin, the pH of the reaction solution before addition of pepsin is preferably adjusted to 1 to 3. The reaction temperature is preferably 20 to 45 ° C, more preferably 30 to 40 ° C. The reaction time is preferably 2 to 10 hours, more preferably 3 to 7 hours. Moreover, when making pancreatine act next, it is preferable to make pH of the reaction liquid before pancreatine addition into 7-8. The reaction temperature and reaction time may be the same as in the hydrolysis reaction with pepsin.

  Peptide 1 can be isolated by performing appropriate post-treatment and purification after the completion of the decomposition reaction. Alternatively, the mixture may be used as it is without isolation of peptide 1. However, in this case, it is preferable to deactivate the hydrolase. Post-treatment, purification, and identification of peptide 1 may be performed in the same manner as in the case of the above-described chemical synthesis.

The anti-anxiety agent according to the second embodiment of the present invention comprises a component that directly activates the DP ₁ receptor (hereinafter sometimes abbreviated as active component 2) as an active component. The active ingredient 2 may be any ingredient that directly activates the DP ₁ receptor.
The anxiolytic agent may contain any component other than the active component 2 as long as the effect of the present invention is not hindered.

The active ingredient 2 may be either one derived from a living body or one not derived from a living body. The active ingredient 2 to be derived from an organism, prostaglandin D ₂ can be exemplified. As the active ingredient 2 not derived from the living body, an artificially synthesized substance, a chemical modification of the active ingredient 2 derived from the living body, or a biological modification substance other than the active ingredient 2 derived from the living body is chemically modified. Among them, those that directly activate the DP ₁ receptor can be exemplified. Here, chemical modification refers to, for example, replacing an atom or group of a chemical substance or active component 2 with an atom or group different from these, or adding a new atom or group to the chemical substance or active component 2. Point to. As the active ingredient 2 not derived from a living body, an agonist of DP ₁ receptor can be exemplified.

The agonist of DP ₁ receptor is not particularly limited. Specific examples of preferable ones include the following.
・ BW245C
(3- (3-Cyclohexyl-3-hydroxypropyl) -2,5-dioxo-4-imidazolidineheptanoic acid) ("Japan Chemical Dictionary web: http://nikkajiweb.jst.go.jp/nikkaji_web/pages/top. see html)
・ AS702224
("Brugger N et al, Pharmacological and Functional Characterization of Novel EP and DP Receptor Agonists: DP1 Receptor Mediates Penile Erection in Multiple Species., J Sex Med. 2008 Feb; 5 (2): 344-56. Epub 2007 Nov 28" reference)
・ TS-022
("Sugimoto M et al, The anti-pruritic efficacy of TS-022, a prostanoid DP1 receptor agonist, is dependent on the intrinsic prostaglandin D2 level in the skin of NC / Nga mice., Eur J Pharmacol. 2007 Jun 14; 564 (1-3): 196-203. Epub 2007 Feb 8 ”)
・ SQ27986
([1S- [1B, 2B (5Z), 3A (1E, 3S), 4B]] 7- [3- (3-cyclohexyl-3-hydroxy-1-propenyl) -7-oxabi-cyclo- [2.2. 1] hept-2-yl] 5-heptenoic acid) ("Sharif et al, Affinities, selectivities, potencies, and intrinsic activities of natural and synthetic prostanoids using intrinsic receptors: focus on DP class prostanoids., J Pharmacol Exp Ther. 2000 May; 293 (2): 321-8. "reference)
・ RS93520
((C3'S, 1R, 2R, 3S, 6R) -2-C3'-cyclohexyl-3'hydroxyprop-1-ynyl) -3-hydroxybicyclo [4.2.0] oct-7-ylidene) butyrate) ("Sharif et al , Affinities, selectivities, potencies, and intrinsic activities of natural and synthetic prostanoids using intrinsic receptors: focus on DP class prostanoids., J Pharmacol Exp Ther. 2000 May; 293 (2): 321-8.)
・ ZK118182
((5Z, 13E)-(9R, 11R, 15S) -9-chloro-15-cyclohexyl-11,15-dihydroxy-3-oxa-16,17,18,19,20-pentanor-5,13-prostadienoic acid) ("Sharif et al, Affinities, selectivities, potencies, and intrinsic activities of natural and synthetic prostanoids using intrinsic receptors: focus on DP class prostanoids., J Pharmacol Exp Ther. 2000 May; 293 (2): 321-8. "reference)
・ ZK110841
((5Z, 13E)-(9R, 11R, 15S) -9-chlor-15-cyclohexyl-11,15-dihydroxy-16,17,18,19,20-pentanor-5,13-prostadienoic acid) ("Sharif et al, Affinities, selectivities, potencies, and intrinsic activities of natural and synthetic prostanoids using intrinsic receptors: focus on DP class prostanoids., J Pharmacol Exp Ther. 2000 May; 293 (2): 321-8. "reference)

Rubisco is an enzyme contained in the chlorophyll of a green plant that performs photosynthesis, and produces bimolecular glyceric acid-3-phosphate from ribulose-1,5-diphosphate and carbon dioxide in the dark reaction of photosynthesis. By doing so, it is known to have a function of taking carbon dioxide into the Calvin circuit.
Until now, it has been disclosed that some bioactive peptides exist in the hydrolyzate of Rubisco (see, for example, JP-A Nos. 2001-213897 and 2003-300996). . For example, a peptide having an amino acid sequence represented by Met-Arg-Trp (ie, peptide 1) was isolated from a product obtained by stepwise hydrolysis of spinach-derived rubisco with pepsin or pepsin and pancreatin. Yes. Such a peptide is known to inhibit angiotensin I converting enzyme (ACE) and exhibit a blood pressure lowering action (see JP-A-2003-300996). In addition, for such peptides are known to prostaglandin D ₂ dependent manner showing the vasorelaxant ( "Zhao H et al, Met-Arg -Trp derived from Rubisco lowers blood pressure via prostaglandin D (2) -dependent vasorelaxation in spontaneously hypertensive rats., Peptides, 2008; 29 (3): 345-349 ").
Prostaglandins are a group of physiologically active substances synthesized from eicosapolyenoic acid such as arachidonic acid, and are known to express various physiological actions by binding to receptors present on cell membranes. Yes. Then, prostaglandin D _2, which is a type of prostaglandin, are known to have platelet aggregation inhibitory action and sleep inducing action.
Further, DP ₁ receptor and DP ₂ receptor are known as receptors specific for prostaglandin D ₂ .

However, any peptide having an amino acid sequence represented by Met-Arg-Trp, a peptide comprising the amino acid sequence (ie, peptide 1), and prostaglandin D ₂ has never been reported to have anxiolytic activity. It has not been reported at all that DP ₁ receptor and DP ₂ receptor are involved in anxiolytic action. That is, regarding the relationship between the activation of DP ₁ receptor or DP ₂ receptor and the development of an anxiolytic effect, it has been completely known whether the activation of these receptors is indirect or direct. Absent.

On the other hand, the anti-anxiety agent of the present invention is a completely novel one that exhibits an anxiolytic action by the action mechanism of indirectly or directly activating the DP ₁ receptor.
For example, peptide 1 exhibits anxiolytic action by increasing the synthesis of prostaglandin D ₂ by binding to an unknown receptor and activating DP ₁ receptor, as specifically described in the Examples. Has been suggested. Further, as described above, peptides 1, shows the prostaglandin D ₂ dependent manner vasorelaxant, as it is known to exhibit a hypotensive effect are described in the examples below, peptides 1 Shows an anxiolytic effect at a dose much lower than the dose that exhibits a blood pressure lowering effect. That is, Peptide 1 exhibits an anxiolytic action independent of the blood pressure lowering action. This is because the action mechanism of peptide 1 expressing the anxiolytic action is the action mechanism of developing the blood pressure lowering action. It is to support the fact that it is different and not known in the past.
Further, for example, prostaglandin _{D 2} and BW245C are all organic compounds having different skeleton together. And these show sufficient anti-anxiety action so that it may demonstrate concretely in an Example. On the other hand, a feature common to these organic compounds is that they directly activate the DP ₁ receptor. Therefore, these results support that the anxiolytic action is expressed by a novel mechanism of action that is not conventionally known, ie, direct activation of the DP ₁ receptor.

In the anxiolytic agent of the present invention, the active ingredients 1 and 2 may be pharmaceutically acceptable salts. The pharmaceutically acceptable salt is, for example, a salt formed by the action of an acid or a base, and is not particularly limited. Preferred salts formed by the action with an acid include inorganic acid salts such as hydrochloride, sulfate, nitrate and phosphate; organic acid salts such as acetate, citrate, lactate and tartrate Can be illustrated. Preferred salts formed by the action with a base include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; triethylamine salts and triethanolamine salts. An organic amine salt such as Alternatively, acidic amino acid salts such as aspartate and glutamate; basic amino acid salts such as lysine salt, arginine salt and histidine salt may be used.
As the pharmaceutically acceptable salt of the active ingredient, one kind may be used alone, or two or more kinds may be used in combination. When using 2 or more types together, the combination and ratio of a salt can be arbitrarily adjusted according to the objective.

The pharmaceutical product of the present invention contains the anxiolytic agent of the present invention.
The anti-anxiety agent contained in the pharmaceutical may be one kind or two or more kinds. In the case of two or more kinds, the combination and ratio of the anxiolytic agents can be arbitrarily adjusted according to the purpose.

The pharmaceutical preparation form is not particularly limited. For example, oral preparations such as tablets, powders, granules, capsules, fine granules, and liquids; or parenteral preparations such as injections can be used.
These can be produced by known methods. For example, in the case of oral preparations, excipients, lubricants, surfactants, binders, disintegrants, stabilizers, flavoring agents, buffering agents, etc. that are usually used in the production of these preparations are used as anxiolytic agents. And can be produced according to a conventional method. Examples of excipients include lactose, crystalline cellulose, and starch. Examples of lubricants include magnesium stearate, talc and hydrogenated vegetable oil. Examples of the binder include hydroxypropyl cellulose, crystalline cellulose, and polyvinyl pyrrolidone. Examples of the disintegrant include crystalline cellulose, carboxymethyl cellulose, and carboxymethyl cellulose calcium salt.
In the case of a parenteral preparation, for example, an anxiolytic agent can be dissolved or suspended in distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, glycerin or the like, and can be produced according to a conventional method. Furthermore, you may mix | blend a buffering agent, antiseptic | preservative, antioxidant, etc. as needed.

The administration method of the anxiolytic agent or pharmaceutical agent of the present invention may be either oral administration or parenteral administration.
The dose of the anxiolytic agent or pharmaceutical agent varies depending on the age, sex, body weight, symptoms, etc. of the patient, but in the case of active ingredient 1 such as peptide 1, preferably 0.1 to 0.1 per adult day. In the case of active ingredient 2 such as an agonist of prostaglandin D ₂ or DP ₁ receptor, it is preferably 0.1 to 20 nmol / 60 kg per adult day. What is necessary is just to administer an anti-anxiety agent or a pharmaceutical once a day or in several times so that it may become this range.

The anti-anxiety agent and pharmaceutical agent of the present invention are novel and completely different from conventional ones and have a sufficient anxiolytic effect. Moreover, oral administration is also possible and it can be taken easily. Among the anti-anxiety agents, for example, prostaglandin D ₂ is already widely used for different applications and can be easily obtained or manufactured. Peptide 1 can be obtained, for example, by hydrolyzing rubisco with an enzyme, and rubisco is said to be the most abundant enzyme on earth, and peptide 1 can also be produced easily and easily. Moreover, since peptide 1 is a peptide consisting of three amino acid residues, production by chemical synthesis is simple and easy. That is, peptide 1 can be easily produced by any method. As described above, since the anxiolytic agent of the present invention is easily available and manufactured, the pharmaceutical agent of the present invention containing the anxiolytic agent can be easily and inexpensively manufactured. Therefore, the anxiolytic agents and pharmaceuticals of the present invention can be supplied in large quantities.

Hereinafter, the present invention will be described in more detail based on specific examples. However, the present invention is not limited to the following examples.
In the following Examples, “dosage (mg / kg)” of the active ingredient, whether oral or parenteral, indicates “dosage (mg) per kg body weight of the mouse” and “dosage “(Pmol / mouse)” means “dose per mouse (pmol)”.

Example 1
<Evaluation of anxiolytic activity by intraperitoneal administration of peptide 1>
The anti-anxiety action of peptide 1 was examined by the procedure shown below.
(A) Materials and Methods (A-1) Animals 5-week-old male ddY mice were obtained from Japan SLC. All mice were housed in a room set at 23 ° C. on a 12 hour light / dark cycle with lights on from 7 am to 7 pm. During breeding, all mice received food and water ad libitum.
(A-2) Peptide 1
Peptide 1 was chemically synthesized by the Fmoc method using a peptide synthesizer (PS-3, Protein Technologies), and after deprotection, an HPLC equipped with an ODS column (Cosmosil 5C18 ARII (20 × 250 mm), Nacalai Tests). After purification by lyophilization, it was obtained by lyophilization.
(A-3) Elevated Cross Maze Test The elevated plus maze test was performed according to a known method as described below.
As shown in FIG. 1, from two open arms (L: 24 cm × W: 5 cm × H: 0.5 cm), two closed arms (L: 24 cm × W: 5 cm × H: 13 cm) and the central platform An elevated plus-maze (EPM) was installed such that the floor surface was located 50 cm above the experimental bench.
The elevated cross maze has a high floor, but the closed arm has an enclosure so that the mouse can safely walk in the closed arm. On the other hand, since the periphery of the open arm is open and has no enclosure, the mouse feels uneasy about falling from a high position in the open arm. Therefore, as the residence time in the open arm of the mouse is longer, the anxiety of the mouse is alleviated. Therefore, the residence time in the open arm was used as an index for evaluating the anxiolytic activity.
The mice were then placed on the central platform and the test was conducted for 5 minutes. Number of times of entry into any of the four arms (hereinafter abbreviated as total entry number), number of times of entry into the open arm, or any of the four arms during the test time The residence time (hereinafter abbreviated as total entry time) and the residence time in the open arm were recorded. In the test, it was defined that “the mouse entered the arm” when all four limbs of the mouse entered the arm.
From the obtained records, (I) the ratio of the dwell time in the open arm with respect to the total approach time, and (II) the ratio of the number of times of entry into the open arm with respect to the total number of entries are calculated respectively. Along with the number of times, the following test groups were compared. In addition, all the values of the above (I) to (III) are shown as an average value ± standard error. Following analysis of variance (ANOVA), a Fisher's test was applied for multiple comparisons, and any were considered statistically significant if the p-value was less than 0.05.

  Peptide 1 solution in which peptide 1 was dissolved in physiological saline or physiological saline alone as a control was administered into the abdominal cavity of mice 30 minutes before the test, and each mouse was subjected to the elevated plus maze test. The peptide 1 solution was administered such that the doses of peptide 1 per kg body weight of the mouse were 0.03 mg / kg, 0.1 mg / kg, and 0.3 mg / kg, respectively. The number of mice subjected to the test was 17 in the 0.03 mg / kg administration group, 17 in the 0.1 mg / kg administration group, 17 in the 0.3 mg / kg administration group, and 18 in the control. The results are shown in FIG. In FIG. 2 (a), the values in () indicate the number of mice in each test group. 2A and 2B, regarding the p value in the Fisher test, “*” is p <0.05, “**” is p <0.01, “***” is p <0. .001.

(B) Evaluation results As shown in FIGS. 2 (a) and 2 (b), peptide 1 was administered intraperitoneally at a dose of 0.03-0.3 mg / kg, resulting in 0.1-0.3 mg / kg. (I) the ratio of residence time in the open arm and (II) the ratio of the number of times of entry into the open arm increased.
Multiple comparisons using the Fisher test showed that the anti-anxiety effect of Peptide 1 was significant at doses of 0.1 to 0.3 mg / kg with respect to (I) and (II).
On the other hand, as shown in FIG.2 (c), about the total approach frequency, there was no significant difference in any test group, and the total momentum did not change.

(Example 2)
<Evaluation of anxiolytic activity by oral administration of peptide 1>
(A) Materials and Methods Peptide 1 solution in which peptide 1 was dissolved in physiological saline or physiological saline alone as a control was orally administered to mice 30 minutes before the test, and each was subjected to the elevated plus maze test one by one. . The peptide 1 solution was administered so that the dose of peptide 1 per kg body weight of the mouse was 1.0 mg / kg, 3.0 mg / kg, and 10.0 mg / kg, respectively. The number of mice subjected to the test was 10 mg / kg administration group: 10 animals, 3.0 mg / kg administration group: 10 animals, 10.0 mg / kg administration group: 10 animals, and control: 10 animals. Other than this, the anxiolytic activity of peptide 1 was evaluated in the same manner as in Example 1. The results are shown in FIG. In FIG. 3 (a), the values in () indicate the number of mice in each test group. 3 (a) and 3 (b), regarding the p value in the Fisher test, “*” indicates p <0.05, and “**” indicates p <0.01.

(B) Evaluation results As shown in FIGS. 3A and 3B, peptide 1 was orally administered at a dose of 1.0 to 10.0 mg / kg, resulting in a dose of 3.0 to 10.0 mg / kg. (I) The ratio of the residence time in the open arm and (II) the ratio of the number of times of entry into the open arm increased.
According to multiple comparisons using the Fisher test, the anti-anxiety effect of Peptide 1 is significant at a dose of 3.0 to 10.0 mg / kg with respect to (I), and 1.0 with respect to (II). It was shown that the anxiolytic effect of peptide 1 was significant at a dose of ˜10.0 mg / kg.
On the other hand, as shown in FIG.3 (c), about the total approach frequency, there was no significant difference in any test group, and the total momentum did not change.

From the results of Examples 1 and 2, it was shown that Peptide 1 exhibits an anxiolytic action without changing the momentum. In particular, from the results of Example 2, peptide 1 showed an anxiolytic action even when administered orally at a dose as low as 1.0 mg / kg. On the other hand, in order for peptide 1 to exhibit blood pressure lowering activity, it was necessary to administer orally at a dose of 5 mg / kg or more.
From the above, it was determined that the anxiolytic action of peptide 1 was independent of the blood pressure lowering action.
Peptide 1 exhibits an anxiolytic action at a lower dose than the peptide having an anxiolytic action consisting of five amino acid residues described in JP-A-2007-91656 in both intraperitoneal administration and oral administration. It was shown to show.

(Example 3)
<Estimation of action mechanism of anxiolytic action by peptide 1>
This peptide 1 to date, with respect to spontaneously hypertensive rats via the prostaglandin D ₂ dependent vasorelaxation, are known to exhibit a hypotensive effect. Therefore, evaluation mechanism of action of anxiolytic action of peptide 1, in order to prostaglandin D ₂ is discussed whether involved, the effect on the anxiolytic effects of BW A868C is an antagonist of DP ₁ receptor did.

(A) Material and Method Peptide 1 solution in which peptide 1 similar to that in Example 1 was dissolved in physiological saline, and BW A868C solution in which BW A868C (manufactured by Cayma Chemical Company) was dissolved in physiological saline were prepared. . And (i) peptide 1 solution only, (ii) BW A868C solution only, (iii) peptide 1 solution and BW A868C solution, and (iv) physiological saline only as a control, 30 minutes before the test, It was administered intraperitoneally. The peptide 1 solution was administered so that the dose of peptide 1 was 0.1 mg / kg. The BW A868C solution was administered so that the dose of BW A868C was 60 μg / kg. The number of mice subjected to the test was (i) administration group: 7 mice, (ii) administration group: 9 mice, (iii) administration group: 9 mice, and (iv) administration group: 8 mice. Other than this, the anxiolytic activity was evaluated in the same manner as in Example 1. The results are shown in FIG. In FIG. 4 (a), the values in () indicate the number of mice in each test group. 4A and 4B, regarding the p value in the Fisher test, “*” indicates p <0.05, and “**” indicates p <0.01.

(B) Evaluation results As shown in FIGS. 4 (a) and 4 (b), the anxiolytic action induced by peptide 1 is suppressed with respect to the above (I) and (II) by multiple comparison using a Fisher test. The effect of BW A868C was shown to be significant.
Furthermore, BW A868C itself has been shown not to cause anxiolytic effects.
On the other hand, as shown in FIG.4 (c), about the total approach frequency, there was no significant difference in any test group, and the total momentum did not change.
From the above, it was shown that the anxiolytic action of Peptide 1 is caused by the action mechanism different from the case of showing the blood pressure lowering action through the DP ₁ receptor.
Peptide 1 does not directly bind to the DP ₁ receptor (“Zhao H et al, Met-Arg-Trp derived from Rubisco lowers blood pressure via prostaglandin D (2) -dependent vasorelaxation in spontaneously hypertensive rats., Peptides, 2008 29 (3): 345-349), peptide 1 binds to an unknown receptor to enhance the synthesis of prostaglandin D ₂ and activates the DP ₁ receptor to exhibit anxiolytic activity. It has been suggested.

Example 4
<Evaluation of anxiolytic activity by intracerebroventricular administration of prostaglandin D _2>
As described above, the anxiolytic effects of Peptide 1, since it has been shown to be caused through the DP ₁ receptor, anti-anxiety effect was confirmed whether represented by prostaglandin D ₂ addition.

(A) Materials and Methods Prostaglandin D ₂ (manufactured by Cayma Chemical Company) was artificial cerebrospinal fluid (138.9 mM NaCl, 3.4 mM KCl, 1.3 mM CaCl ₂ , 4.0 mM NaHCO ₃ , 0.6 mM NaH ₂ PO _4, 5.6 mM glucose, as dissolved prostaglandin _{D 2} solution or control was the pH 7.4) only artificial cerebrospinal fluid, and administered to the brain compartment of the mouse 10 minutes before the test, one animal Each was subjected to an elevated plus maze test. The prostaglandin D ₂ solution was administered so that the dose of prostaglandin D ₂ per mouse was 1 pmol / mouse, 10 pmol / mouse, and 100 pmol / mouse, respectively. The number of mice used in the test was 1 pmol / mouse administration group: 6 animals, 10 pmol / mouse administration group: 5 animals, 100 pmol / mouse administration group: 6 animals, and control: 6 animals. Other evaluated the anxiolytic activity of Example 1 in the same manner as prostaglandin D _2. The results are shown in FIG. In FIG. 5 (a), the values in () indicate the number of mice in each test group. 5A and 5B, regarding the p value in the Fisher test, “*” indicates p <0.05, and “**” indicates p <0.01. Furthermore, in FIG. 5C, “PGD ₂ ” indicates prostaglandin D ₂ .

(B) Evaluation results As shown in FIGS. 5 (a) and 5 (b), prostaglandin D2 was administered into the ventricle at a dose of ₁ to 100 pmol / mouse. As a result, it was administered at a dose of 10 to 100 pmol / mouse. In this case, (I) the ratio of the residence time in the open arm and (II) the ratio of the number of times of entry into the open arm increased.
Multiple comparison according to the Fisher test, the terms (I) and (II), at a dose of 10～100Pmol / mouse, anxiolytic effects of the prostaglandin _{D 2} was shown to be significant.
On the other hand, as shown in FIG.5 (c), about the total approach frequency, there was no significant difference in any test group, and the total momentum did not change.
From the above, prostaglandin D _2, without changing the momentum, it has been shown to cause an anti-anxiety effect.

(Example 5)
<Evaluation of anxiolytic activity by intraventricular administration of BW245C>
In order to confirm whether an anxiolytic effect was exhibited by the DP ₁ receptor agonist, the effect of BW245C on the anxiolytic effect was evaluated.
(A) Materials and Methods BW245C (manufactured by Cayma Chemical Company), a DP ₁ receptor agonist, was dissolved in artificial cerebrospinal fluid or only artificial cerebrospinal fluid as a control, and the brain of the mouse 10 minutes before the test. The animals were administered indoors and were subjected to the elevated plus maze test one by one. The artificial cerebrospinal fluid was the same as in Example 4. The BW245C solution was administered so that the dose of BW245C per mouse was 0.1 pmol / mouse and 1 pmol / mouse, respectively. The number of mice subjected to the test was 0.1 pmol / mouse administration group: 8 mice, 1 pmol / mouse administration group: 8 mice, and control: 8 mice. Other than this, the anxiolytic activity of BW245C was evaluated in the same manner as in Example 1. The results are shown in FIG. In FIG. 6 (a), the values in () indicate the number of mice in each test group. In FIGS. 6A and 6B, “**” indicates p <0.01 with respect to the p value in the Fisher test.

(B) Evaluation Results As shown in FIGS. 6A and 6B, when BW245C was administered into the ventricle at a dose of 0.1 to 1 pmol / mouse, when administered at a dose of 1 pmol / mouse, (I ) The ratio of residence time in the open arm and (II) the ratio of the number of times of entry into the open arm increased.
Multiple comparisons applying the Fisher test showed that the anxiolytic effect of BW245C was significant at a dose of 1 pmol / mouse for the above (I) and (II).
On the other hand, as shown in FIG.6 (c), about the total approach frequency, there was no significant difference in any test group, and the total momentum did not change.
From the above, BW245C, like prostaglandin D _2, without changing the momentum, it has been shown to cause an anti-anxiety effect.

  The present invention can be used as a therapeutic agent for anxiety disorder patients.

It is a figure explaining the elevated cross maze used in the Example. (A) a graph showing “ratio of dwell time in the open arm with respect to the total approach time” in Example 1, (b) graph showing “ratio of the number of times of entry into the open arm with respect to the total number of entries”, (c ) A graph showing the “total number of times of entry”. (A) a graph showing “ratio of residence time in the open arm with respect to the total approach time” in Example 2, (b) graph showing “ratio of the number of times of entry into the open arm with respect to the total number of entries”, (c ) A graph showing the “total number of times of entry”. (A) a graph showing “ratio of residence time in the open arm with respect to the total approach time” in Example 3, (b) graph showing “ratio of the number of times of entry into the open arm with respect to the total number of entries”, (c ) A graph showing the “total number of times of entry”. (A) a graph showing “ratio of residence time in the open arm with respect to the total approach time” in Example 4, (b) graph showing “ratio of the number of times of entry into the open arm with respect to the total number of entries”, (c ) A graph showing the “total number of times of entry”. (A) a graph showing “ratio of residence time in the open arm with respect to the total approach time” in Example 5, (b) graph showing “ratio of the number of times of entry into the open arm with respect to the total number of entries”, (c ) A graph showing the “total number of times of entry”.

Claims (3)

An anti-anxiety agent comprising a peptide having an amino acid sequence represented by Met-Arg-Trp or a pharmaceutically acceptable salt thereof as an active ingredient. Prostaglandin D ₂ or anti-anxiety agent to a pharmaceutically acceptable salt thereof as an active ingredient. An anxiolytic agent comprising BW245C or a pharmaceutically acceptable salt thereof as an active ingredient.

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