KR102381026B1 - A Composition for Inducing Anxiety Disorder Comprising Clozapine as an Active Ingredient - Google Patents

Abstract

The present invention relates to a composition for inducing anxiety disorder in a mammal, and to a mammal in which an anxiety disorder is induced by using the same. The animal model of the present invention not only significantly increases anxiety behavior and grooming behavior, which are indicators of anxiety disorders, but also has responsiveness to the administration of therapeutic agents such as fluoxetine. It was confirmed that it is an excellent animal model that reflects the disease characteristics of the disorder with high reliability. The present invention can be usefully used as a means for research on the molecular and cyclical mechanisms of the development of anxiety disorders, as well as the development of therapeutic agents for anxiety disorders and discovery of antipsychotic drugs without side effects.

Description

A composition for inducing anxiety disorder comprising clozapine as an active ingredient {A Composition for Inducing Anxiety Disorder Comprising Clozapine as an Active Ingredient}

The present invention relates to a method of inducing an anxiety disorder including obsessive-compulsive disorder in a mammal by administering clozapine, and an animal model of anxiety disorder established through the method.

With the development of modern society, the social burden of diseases caused by mental and behavioral disorders is rapidly increasing, and accordingly, the size of the market for mental disorders such as antidepressants is also rapidly expanding. In Korea, the market for mental illness is also rapidly increasing, and considering that the suicide rate is the highest among OECD countries, early detection of mental illness and development of intensive treatment technology in the early stage of onset can improve social health and productivity and improve the national economy. can contribute to buoyancy.

Obsessive-Compulsive Disorder, a type of anxiety disorder, refers to a state in which a person repeatedly engages in certain thoughts or actions regardless of one's will. Obsessive compulsive disorder is divided into compulsions and compulsions, and if the compulsions cause anxiety or pain, the compulsions function to neutralize them. Because the cause of obsessive compulsive disorder is not clearly identified, it is difficult to develop a treatment and to establish a disease model for treatment. In order to construct an animal model that efficiently reproduces the characteristics and molecular mechanisms of the disease, first, the joint causes that cause obsessive-compulsive disorder-like symptoms must be identified so that these causes can be directly applied to animals, and second, obsessive-compulsive disorder observed in humans. Disorder-like symptoms must be observed at a level that can be identified and repeatedly observed in the animal, and third, it must respond to a drug applied as a treatment for obsessive compulsive disorder in humans.

Meanwhile, antiserotonergic second-generation antipsychotic drugs (SGA), such as clozapine, risperidone, and olanzapine, are widely used for the treatment of schizophrenia (schizophrenia). Unlike other SGAs that act primarily on dopamine receptors, they have prominent anti-serotonin properties [1].

Several clinical reports have reported that obsessive-compulsive disorder (OCS) often co-occurs in patients with schizophrenia [2,3], but it is unclear whether this effect is due to antiserotonergic SGA. It is difficult to distinguish whether cognitive or simply pre-existing OCS is manifested or worsened.

Numerous papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

Patent Document 1. US Registered Patent No. 9,415,030

The present inventors made intensive research efforts to develop an efficient animal model for an anxiety disorder, specifically, a pathological study of obsessive compulsive disorder, screening for therapeutic agents, and in vivo drug evaluation. As a result, when the compound represented by Formula 1 is administered to a mammal for a long time, not only anxiety behavior, which is a measure of anxiety disorder, but also grooming behavior, which is a major indicator of obsessive-compulsive disorder, is significantly increased, By observing that this cosmetic behavior is reduced again by administration of fluoxetine, a selective serotonin absorption inhibitor, it was confirmed that the pathological condition of anxiety disorder was successfully reproduced in mammals by the compound, thereby completing the present invention did it

Accordingly, an object of the present invention is to provide a composition for inducing anxiety disorder in a mammal and a mammal in which an anxiety disorder is induced by using the same.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a composition for inducing anxiety disorder in a mammal comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

Formula 1

In the above formula, X is halogen and R ₁ is C ₁ -C ₃ alkyl.

The present inventors made intensive research efforts to develop an efficient animal model for an anxiety disorder, specifically, a pathological study of obsessive compulsive disorder, screening for therapeutic agents, and in vivo drug evaluation. As a result, when the compound represented by Formula 1 is administered to a mammal for a long time, not only anxiety behavior, which is a measure of anxiety disorder, but also grooming behavior, which is a major indicator of obsessive-compulsive disorder, is significantly increased, By observing that this cosmetic behavior was again reduced by administration of fluoxetine, a selective serotonin uptake inhibitor, it was found that the pathological condition of anxiety disorder was successfully reproduced in mammals by the compound.

As used herein, the term “alkyl” refers to a straight-chain or branched saturated hydrocarbon group, and includes, for example, methyl, ethyl, propyl, isopropyl, and the like. C ₁ -C ₃ alkyl refers to an alkyl group having an alkyl unit having 1 to 3 carbon atoms, and when C ₁ -C ₃ alkyl is substituted, the carbon number of the substituent is not included.

As used herein, the term “halogen” refers to a halogen element, and includes, for example, fluoro, chloro, bromo and iodo.

According to a specific embodiment of the present invention, X is Cl and R ₁ is C ₁ alkyl.

The compound of Formula 1 wherein X is Cl and R ₁ is C ₁ alkyl(methyl) is clozapine. Clozapine is an atypical antipsychotic drug used for the treatment of schizophrenia. It is mainly used for the treatment of drug-resistant schizophrenia patients or patients with schizophrenia causing severe extrapyramidal disorders. The present inventors found that when clozapine is administered to non-human mammals, particularly rodents such as mice, for a certain period of time, obsessive-compulsive disorder-like behavior is efficiently induced and these symptoms respond by selective serotonin uptake inhibitors. The correlation between clozapine and obsessive-compulsive disorder in animals of

As used herein, the term “pharmaceutically acceptable salt” refers to a salt derived from a pharmaceutically acceptable inorganic acid, organic acid, or base. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methane sulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, and ammonium and the like. Specifically, the pharmaceutically acceptable salt used in the present invention is a sodium salt.

As used herein, the term “mammal” refers to all animals other than humans as animals belonging to mammals, for example, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons, or red including, but not limited to, woolly monkeys. Specifically, the mammal is a rodent.

As used herein, the term “animal model” refers to a non-human animal that has been artificially modified directly or indirectly to have the symptoms or phenotype of an anxiety disorder, and specifically, acquired through the administration of the compound of Formula 1, acquired and non-genetically anxious It refers to an animal that has symptoms of a disorder and reflects the overall biological process that progresses in an anxiety disorder state.

As used herein, the term “anxiety disorder” is a mental disorder that causes disturbances in daily life by excessive and widespread manifestation of various types of abnormal and pathological anxiety and fear, including panic disorder, social phobia, Post traumatic stress disorder (PTSD), Generalized anxiety disorder, Acute stress disorder and Obsessive-Compulsive Disorder (OCS).

According to a specific embodiment of the present invention, the anxiety disorder is Obsessive-Compulsive Disorder (OCS).

According to another aspect of the present invention, there is provided a method for preparing an animal in which anxiety disorder is induced, comprising administering to the mammal the composition of the present invention as described above.

Since the compound of Formula 1 used in the present invention, the anxiety disorder induced in the present invention, and the target mammal have already been described above, description thereof will be omitted to avoid excessive duplication.

According to a specific embodiment of the present invention, the method is performed by administering the composition so that 0.5 - 1.5 mg/kg is released in the mammal daily. More specifically, it is 0.7 - 1.3 mg/kg, and most specifically, it is 0.9 - 1.1 mg/kg.

According to the present invention, the composition of the present invention can induce anxiety disorders including obsessive-compulsive disorder by long-term administration of a certain amount to a target mammal. The method of long-term administration of a certain amount may be achieved by regularly repeating administration of a predetermined dose, or may be mounted on a sustained-release formulation to induce long-term release. Specifically, the method of the present invention is carried out by loading and administering the composition of the present invention in a sustained release formulation.

As used herein, the term “sustained release formulation” means that the formulation containing the active ingredient is immediately disintegrated in the body and released so that the drug can be maintained for a desired time without temporarily releasing the drug.

More specifically, it is carried out by administering such that the composition is released within the mammal for 50 to 200 days.

According to another aspect of the present invention, there is provided a mammal induced with anxiety disorder prepared by the method of the present invention as described above.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for inducing anxiety disorder in a mammal and a mammal in which an anxiety disorder is induced by using the same.

(b) the animal model of the present invention not only significantly increases anxiety behavior and grooming behavior, which are indicators of anxiety disorders, but also has responsiveness to the administration of therapeutic agents such as fluoxetine for anxiety disorders It was confirmed that it is an excellent animal model that reflects the disease characteristics of

(c) The present invention can be usefully used as a means for research on molecular and cyclical mechanisms of the development of anxiety disorders, as well as development of therapeutic agents for anxiety disorders and discovery of antipsychotic drugs without side effects.

1 is a diagram showing that clozapine-treated mice exhibit cosmetic behavior. 1A is a schematic diagram of an experimental process. Wild-type and Sapap3+/- adult mice were administered clozapine or placebo for 28 weeks. 1B is a diagram showing an increase in repetitive cosmetic behavior in clozapine-treated wild-type mice. Clozapine or placebo pellets were injected into 12-week-old mice every 60 days. Mouse behavior at 12, 15, 20, 30 and 40 weeks of age was recorded and analyzed for 2 hours (two-way repeated measures ANOVA, main effect of dosing; F(1,16) = 9.161, p = 0.008, main effect of time. ; F(3,48) = 5.525, p = 0.002, treatment x time interaction; F(3,48) = 2.981, p = 0.04, ** p < 0.01) (left). A cosmetic behavior lasting more than 3 seconds was recorded once (two-way repeated measures ANOVA, main effect of dosing; F(1,16) = 6.073, p = 0.025, main effect of time; F(3,48) = 3.491 , p = 0.023, treatment x time interaction; F(3,48) = 2.674, p = 0.058, * p < 0.05) (right) n = 8-10 per group. 1C is a diagram showing that clozapine-treated Sapap3+/- mice display an increase in repetitive cosmetic behaviors earlier than clozapine-treated wild-type mice. Clozapine or placebo pellets were injected into 12-week-old Sapap3+/- mice every 60 days. Mice behaviors at 12, 15, 20, 30 and 40 weeks of age were recorded and analyzed for 2 hours. Grooming time was measured via video recordings taken over a period of 2 hours. Cosmetic time was increased in Sapap3+/- mice 8 weeks after clozapine treatment (two-way repeated measures ANOVA, main effect of dosing; F(1,12) = 16.99, p = 0.01, main effect of time; F(3) ,36) = 10.12, p < 0.001, treatment x time interaction; F(3,36) = 3.348, p = 0.03, * p < 0.05, ** p < 0.01) (left). The number of cosmetic behaviors was measured through video recordings taken for 2 hours. The number of embellishments was increased in Sapap3+/- mice 18 weeks after clozapine treatment (two-way repeated measures ANOVA, main effect of administration; F(1,12) = 14.16, p = 0.003, main effect of time; F(3) ,36) = 11.17, p < 0.001, treatment x time interaction; F(3,36) = 0.095, p = 0.013, ** p < 0.01) (right) n = 7. per group. is a figure showing that clozapine-treated wild-type mice ameliorated cosmetic behavior (two-way analysis of variance, main effect of fluoxetine administration; F(1,6) = 21.75, p = 0.003, main effect of clozapine administration; F(1, 6) = 9.27, p = 0.023, fluoxetine treatment x clozapine treatment interaction; F(1,6) = 7.071, p = 0.038, * p < 0.05, *** p < 0.001) n = 4. per group. All data is expressed as mean ± standard error.
Figure 2a is a diagram showing the plasma clozapine concentration 10 days after clozapine pellet injection. Age-matched mice injected with placebo were used as controls (Student's t test, ** p < 0.01). n = 4. per group FIG. 2B is a diagram showing the change in body weight in wild-type and Sapap3+/- mice treated with clozapine or placebo. There were no significant differences between clozapine- and placebo-treated mice. n = 5-6 per group. 2C shows survival curves for skin wound development in Sapap3-/- mice (left). Sapap3-/- mice had scars on the neck and facial skin (white arrows). n = 7-12 per group. All data are presented as mean ± standard error.
3 is a figure showing the results of an Elevated plus maze experiment for mice administered with clozapine and placebo, respectively, and the time to stay in the center zone (Student's t test, * p < 0.05) ( Figure 3a), time on the open arm (Student's t test, * p < 0.05) (Figure 3b) and time on the closed arm (Student's t test, * p < 0.05) (Figure 3c) ), respectively. n = 6. per group

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Production of sustained-release clozapine pellets

The present inventors tried to determine whether clozapine can induce OCD-like symptoms in de novo using normal mice (B6J). The present inventors assumed that long-term administration of clozapine would cause OCS, and designed clozapine pellets with a special composition so that clozapine could be continuously released slowly for 60 days. Clozapine pellets consist of a composition such as cholesterol, cellulose, lactose, phosphate, and stearate along with the clozapine drug. The composition was used to maintain the model of the drug and to continuously release the drug without affecting the drug's efficacy.

Administration of Clozapine Pellets and Observation of Cosmetic Behavior

Clozapine was administered for 240 days by transplanting the first clozapine pellet into a 12-week-old mouse (B6J), and follow-up injections were performed at intervals of 60 days ( FIG. 1 ). When the blood concentration of clozapine was measured 10 days after the injection, it was found that the plasma concentration of clozapine was continuously maintained (FIG. 2a). In 15, 20, 30 and 40 week old mice, grooming behavior was recorded with a video camera for 2 hours. In two independent experiments, the length of the dressing and the number of cosmetic actions were scored. As a result, it was found that clozapine increased the grooming time in wild-type mice from 30 weeks of age (18 weeks after the first injection of clozapine pellets) ( FIG. 1b , left).

The present inventors also found that mice administered with clozapine pellets for 40 weeks had a higher number of embellishments (Fig. 1b, right). No difference in body weight was observed between clozapine- and placebo-treated mice ( FIG. 2B ).

Administration of Clozapine to Sapap3 Knockout Mice

In humans, the genetic interaction between SAPAP3 and SLC1A1 gene variants is a predictor of antiserotonergic SGA-induced OCS symptoms. Homozygous deletion of Sapap3 in mice induces cosmetic behavior sufficient to develop facial and neck skin wounds [5]. Accordingly, the present inventors investigated the effect of clozapine on OCD-like behavior in Sapap3 knockout mice. In homozygous mice, the development of skin wounds began to be observed from 20 weeks of age, and 100% penetration was reached at 40-60 weeks of age. Clozapine did not accelerate skin wounding in Sapap3 homozygous KO mice, probably because cosmetic behavior had already reached severe (Fig. 2c). Sapap3 heterozygous KO mice, on the other hand, did not show an increase in cosmetic behavior or skin scarring [5]. Interestingly, we found that clozapine induced decorative behavior faster in Sapap3 heterozygous KO mice than in wild-type mice (Fig. 1c).

Assessment of fluoxetine reactivity

Next, the present inventors investigated whether fluoxetine, a selective serotonin uptake inhibitor, could reduce the cosmetic behavior induced by long-term clozapine treatment. As shown in FIG. 1D, when fluoxetine was intraperitoneally injected into mice every day for 10 days at the 30th week, the decoration time was significantly reduced. This suggests that 5-HT signaling is implicated in the pathogenesis of clozapine-induced OCS, and that OCD-like behavior in mice responds to therapeutics typically used in humans.

crisscross high maze experiment

The cross-shaped elevated maze (Elevated plus maze) is an animal behavioral experiment to analyze the anxiety level of rodents. A maze of open and closed branches is used at a height of about 50 cm from the floor, and the higher the time spent in the closed branches, the higher the level of anxiety is interpreted. In order to further investigate the effect of clozapine on anxiety behavior, the present inventors performed a cruciform high maze experiment on 40-week-old mice that received clozapine for 28 weeks and mice that received placebo. As a result, as shown in FIG. 3 , it was confirmed that mice administered with clozapine exhibited higher anxiety behavior than mice administered with placebo. In the cross-shaped high maze, mice receiving clozapine significantly reduced the time spent in the center zone compared to mice receiving placebo (Student's t test, * p < 0.05) (Fig. 3a), and open branches (open branches) arm) was also significantly reduced (Student's t test, * p < 0.05) (Fig. 3b). In addition, the mice receiving clozapine significantly increased the time to stay on the closed arm compared to the mice receiving the placebo (Student's t test, * p < 0.05) (Fig. 3c). n = 6. per group These results confirmed the fact that clozapine efficiently induces anxiety behavior in mice from various angles.

Discussion

We provided experimental evidence for the long-standing question of whether clozapine induces de novo OCS even under normal conditions, demonstrating for the first time that long-term clozapine administration can induce OCD-like behavior in mice. The animal model of the present invention can be a useful research tool for OCS for the following reasons: First, it takes a long time for OCD-like behavior to be induced in mice after administration of clozapine. Second, this animal model satisfies all validity criteria of an animal model of mental illness [7]; The mouse model exhibits OCD-like behavior (surface area validity), induced by clozapine (constitutive validity), and responsive to fluoxetine (predictive validity). Finally, the present inventors confirmed a clear correlation between clozapine and OCS-related genes in Sapap3 KO mice.

Although clozapine is the only antipsychotic drug that can be used for drug-resistant schizophrenia [1], additional dysfunction and poor prognosis of the disease may occur after initiation of clozapine administration. The animal model of the present invention can be usefully used for the development of effective antipsychotic drugs that do not have OCS as a side effect, and can be easily used for research on molecular and cyclic mechanisms of OCS pathogenesis. The therapeutic effect of fluoxetine confirmed in the present invention suggests that 5-HT receptors (5-HT1A, 5-HT2A and 5-HT2C), which were considered targets of clozapine, are involved [1].

The association of the SLC1A1 polymorphism with symptom development suggests a possible role for glutamate neurotransmission [7]. On the other hand, it is possible that long-term clozapine administration reconstitutes OCD-related neural matrix, such as the cortico-striato-thalamo-cortical (CSTC) circulatory pathway [8, 9].

As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, these specific descriptions are only preferred embodiments, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

references

1. Meltzer HY: Update on typical and atypical antipsychotic drugs. Annu Rev Med 2013, 64:393-406.

2. Baker RW, et al: Emergence of obsessive compulsive symptoms during treatment with clozapine. J Clin Psychiatry 1992, 53:439-442.

3. de Haan L, Linszen DH, Gorsira R: Clozapine and obsessions in patients with recent-onset schizophrenia and other psychotic disorders. J Clin Psychiatry 1999, 60:364-365.

4. Poyurovsky M, Weizman A, Weizman R: Obsessive-compulsive disorder in schizophrenia: clinical characteristics and treatment. CNS Drugs 2004, 18:989-1010.

5. Welch JM, et al: Cortico-striatal synaptic defects and OCD-like behaviors in Sapap3-mutant mice. Nature 2007, 448:894-900.

6. Nestler EJ, Hyman SE: Animal models of neuropsychiatric disorders. Nat Neurosci 2010, 13:1161-1169.

7. Kwon JS, et al: Association of the glutamate transporter gene SLC1A1 with atypicalantipsychotics-induced obsessive-compulsive symptoms. Arch Gen Psychiatry 2009, 66:1233-1241.

8. Ahmari SE, et al: Repeated cortico-striatal stimulation generates persistent OCD-like behavior. Science 2013, 340:1234-1239.

9. Ahmari SE, Dougherty DD: Dissecting OCD Circuits: From Animal Models to Targeted Treatments. Depress Anxiety 2015, 32:550-562.

Claims (11)

A composition for inducing obsessive-compulsive disorder (OCD) in normal rodents comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
Formula 1

In the above formula, X is halogen and R ₁ is C ₁ -C ₃ alkyl.
delete The composition of claim 1, wherein X is Cl and R ₁ is C ₁ alkyl.
delete A method for producing an obsessive-compulsive disorder (OCD) induced rodent comprising administering the composition of claim 1 or 3 to a normal rodent.
6. The method according to claim 5, wherein said method is carried out by administering said composition to be released in said rodent at a daily release rate of 0.5 - 1.5 mg/kg.
The method according to claim 6, wherein the method is carried out by loading the composition in a sustained-release dosage form and administering it.
7. The method according to claim 6, wherein said method is carried out by administering said composition to be released in said rodent for 50 to 200 days.
delete A rodent induced by obsessive-compulsive disorder (OCS) prepared by the method of claim 5.
delete

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