Compounds used in the treatment of schizophrenia

Abstract

The present invention relates to a compound for use in the treatment of schizophrenia, also known as CF3CN and referred to herein as the compound of formula I, 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazole-8-yl)benzonitrile. Preferably, the treatment of schizophrenia is the treatment of one or more symptom categories of schizophrenia. More preferably, the treatment is for the negative symptoms of schizophrenia. Alternatively, the treatment is for the cognitive symptoms of schizophrenia.

Description

Technical Field

[0001] Technical Field of the Invention The present invention relates to a compound for use in the treatment of schizophrenia, a compound known as CF3CN and also referred to herein as a compound of formula I, 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl)benzonitrile. Preferably, the treatment of schizophrenia is the treatment of one or more symptom categories of schizophrenia. More preferably, said treatment is for the negative symptoms of schizophrenia. Alternatively, said treatment is for the cognitive symptoms of schizophrenia.

Background Art

[0002] Background of the Invention The Diagnostic and Statistical Manual of Mental Disorders (DSM) serves as the universal authority for psychiatric diagnosis. The DSM-V, issued in 2013, defines the symptoms of schizophrenia into three broad categories: (1) positive symptoms; (2) negative symptoms; and (3) cognitive symptoms.

[0003] In general terms, the positive symptoms of schizophrenia include hallucinations, delusions, thought disorders, and movement disorders. The negative symptoms of schizophrenia include "flat affect", loss of joy in daily life, loss of the ability to initiate and maintain planned activities, and muteness (even when forced to communicate). Finally, the cognitive symptoms of schizophrenia include poor "executing function"; difficulty in concentration or distractibility, and problems with working memory.

[0004] The treatment of schizophrenia generally involves antipsychotics (typical and atypical antipsychotics), and examples of antipsychotics include aripiprazole, asenapine, brexpiprazole, cariprazine, clozapine, iloperidone, lurasidone, olanzapine, paliperidone, quetiapine, risperidone, ziprasidone, chlorpromazine, fluphenazine, haloperidol, and perphenazine.

[0005] Other interventions, including social interventions such as therapy, social skills training, vocational rehabilitation, and employment support, are often included in the treatment paradigm.

[0006] While symptoms are severe, patients are often hospitalized to ensure their safety, and if they do not respond to drug therapy, electroconvulsive therapy may be used on them.

[0007] The Positive and Negative Syndrome Scale (PANSS) is an assessment tool used to determine the symptomatics of schizophrenia. Based on two established psychiatric assessment systems, the 30-item PANSS is recognized as an operational, drug-sensitive tool that equally represents positive and negative symptoms, accurately measuring their relationships to each other and their relationship to overall psychopathology.

[0008] In addition, symptoms of schizophrenia that involve only the negative symptoms can be assessed using the SANS rating scale, which measures the degree of negative symptoms in five subscales: (i) flattening or blunting of emotions; (ii) allosis; (iii) apathy / indifference; (iv) anhedonia / antisociality; and (v) attention deficit.

[0009] The cognitive symptoms of schizophrenia are consistently evident at the time of the first episode of psychosis. Often, these cognitive symptoms do not improve with existing antipsychotic medications.

[0010] In 2006, the FDA indicated that the drug treatments for schizophrenia available at the time were insufficient to treat the negative symptoms of the disorder (Laughren and Levin, 2006), and since then, no approved drug has addressed this issue.

[0011] Antipsychotic medications approved for treating schizophrenia do not treat specific aspects of the disorder, particularly the negative or cognitive symptoms. Thus, there is a need for more appropriate treatments, especially for these symptoms.

[0012] This application provides data from animal models indicating that compounds of formula I may be useful in the treatment of schizophrenia. One study shows that the compounds of the present invention are useful in the treatment of cognitive symptoms of schizophrenia. Further studies additionally show that the compounds of the present invention are useful in the treatment of negative symptoms of schizophrenia. [Overview of the project]

[0013] In a first aspect of the present invention, a compound of formula I is provided for use in the treatment of schizophrenia.

[0014] Preferably, the treatment of schizophrenia involves treating one or more symptom categories of schizophrenia.

[0015] More preferably, the treatment is for the negative symptoms of schizophrenia, or for the cognitive symptoms of schizophrenia.

[0016] Preferably, the compound of formula I is administered with one or more pharmaceutically acceptable excipients.

[0017] Preferably, the compound of formula I is formulated into a dosage form selected from liquid, lozenge, rapidly disintegrating tablet, lyophilized preparation, film, spray, aerosol, sustained-release tablet or capsule, modified-release tablet or capsule, tablet, capsule, cream, ointment, or mucosal application formulation.

[0018] Preferably, the compound of formula I is administered as a single daily dose. Alternatively, the compound of formula I is administered as multiple daily doses. Furthermore, the compound of formula I is administered two, three, four, or five times a day.

[0019] Preferably, each dose contains at least 0.001 mg of the compound of formula I. More preferably, each dose contains about 0.001 mg to about 500 mg of the compound of formula I. Alternatively, each dose contains about 500 mg to about 1000 mg of the compound of formula I.

[0020] In further embodiments, the compound of formula I is administered together with one or more additional agents.

[0021] A second aspect of the present invention provides a method for treating schizophrenia in a patient requiring treatment, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I.

[0022] In other embodiments, the composition should provide a compound dose of about 0.0001 mg to about 70 mg per kg of body weight per day. Dosage units are prepared to provide doses from about 0.01 mg, 0.1 mg, or 1 mg to about 500 mg or about 1000 mg, and in some embodiments, each dosage unit is prepared to provide about 10 mg to about 500 mg of the active ingredient or combination of essential ingredients.

[0023] Examples of dosages for formulations include milligrams or micrograms of the active ingredient per kilogram of the target substance (for example, from approximately 1 μg to approximately 50 mg per kg, from approximately 10 μg to approximately 30 mg per kg, from approximately 100 μg to approximately 10 mg per kg, or from approximately 100 μg to approximately 5 mg per kg).

[0024] In some embodiments, the administration of the same formulation provided herein may be repeated, with intervals of at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months between administrations.

[0025] The present invention is described with reference to the following figures:

Brief Description of Drawings

[0026] [Figure 1] Details the average search time (seconds) of two identical objects in an acquisition trial.

[0027] [Figure 2] Details the average search time (seconds) of familiar and novel objects in a 3-minute retention trial of the NOR test in the acute phase.

[0028] [Figure 3] Details the effect of acute treatment with a test compound and risperidone on the discrimination index (DI) of the NOR test.

[0029] [Figure 4] Details the effect of acute treatment with a test compound and risperidone on the total number of line crossings in the acquisition and retention trials of the NOR test.

[0030] [Figure 5] Details the average search time (seconds) of two identical objects in an acquisition trial of the NOR test during a 20-day treatment phase.

[0031] [Figure 6] Details the average search time (seconds) of familiar and novel objects in a 3-minute retention trial of the NOR test during a 20-day treatment phase.

[0032] [[ID=四十一]] [Figure 7] Details the effect of treating with a test compound and risperidone for 20 days on the discrimination index (DI) of the NOR test.

[0033] [Figure 8] Details the effect of treating with a test compound and risperidone for 20 days on the total number of line crossings in the acquisition and retention trials of the NOR test.

[0034] [Figure 9] The effects of 21-day treatment with test compounds and acute treatment with risperidone on sniffing time in scPCP-treated rats are described in detail.

[0035] [Figure 10] The effects of 21-day treatment with test compounds and acute treatment with risperidone on object search time in scPCP-treated rats are described in detail.

[0036] [Figure 11] The effects of 21-day treatment with the test compound and acute treatment with risperidone on the number of line crossings in scPCP-treated rats are described in detail.

[0037] [Figure 12] This study details the effects of 21-day treatment with the test compound (TC) (10 and 30 mg / kg, oral administration) on the density (cells / mm2) of parvalbumin-mediated interneurons in subregions of the prefrontal cortex. Data are expressed as mean ± SEM (n=7-9 per group). *=p<0.05, **=p<0.01. Cg1=anterior cingulate cortex, PrL=panlimbic cortex, IL=inferior frontal cortex.

[0038] [Figure 13] This report details the effects of 21-day treatment with the test compound (TC) (10 and 30 mg / kg, oral administration) on the cell density (cells / mm2) of parvalbumin-mediated neurons in the prefrontal cortex. Data are expressed as mean ± SEM (n=7-9 per group). Compared to scPCP, *=p<0.05, **=p<0.01.

[0039] [Figure 14]This study details the effects of 21-day treatment with the test compound (TC) (10 and 30 mg / kg, oral administration) on the cell density (cells / mm2) of parvalbumin-mediated interneurons in subregions of the hippocampus. Data are expressed as mean ± SEM (n=7-9 per group). CA1 = Cornu Ammonis 1, CA2 / 3 = Cornu Ammonis 2 / 3, DG = Dentate gyrus.

[0040] [Figure 15] This study details the effects of 21-day treatment with the test compound (TC) (10 and 30 mg / kg, oral administration) on the density (cells / mm2) of parvalbumin-mediated neuronal cells in the hippocampus. Data are expressed as mean ± SEM (n=7-9 per group).

[0041] [Figure 16] This report details the effects of 21-day treatment (TC) with the test compound (10 and 30 mg / kg, oral administration) on BDNF levels (pg / ml) in the prefrontal cortex. Data are expressed as mean ± SEM (n=7-9 per group).

[0042] [Figure 17] This report details the effects of 21-day treatment with the test compound (TC) (10 and 30 mg / kg, oral administration) on BDNF levels (pg / ml) in the hippocampus. Data are expressed as mean ± SEM (n=9-11 per group).

[0043] [Figure 18] This report details the effects of 21-day treatment with the test compound (TC) (10 and 30 mg / kg, oral administration) on SNAP25 in the prefrontal cortex. Data are expressed as mean ± SEM (n=6-8 per group). Compared to scPCP, *=p<0.05.

[0044] [Figure 19]This report details the effects of 21-day treatment with the test compound (TC) (10 and 30 mg / kg, oral administration) on PSD95 in the prefrontal cortex. Data are expressed as mean ± SEM (n=7-9 per group).

[0045] [Figure 20] This paper details the effects of a 21-day treatment with the test compound on SCPCP-induced cognitive impairment in a set-shift task.

[0046] [Figure 21] The effects of a 21-day treatment with the test compound on PSD95 and SNAP25, neural markers in the prefrontal cortex, are described in detail.

[0047] [Figure 22] The effects of a 21-day treatment with the test compound on BDNF concentration in the hippocampus are described in detail. [Modes for carrying out the invention]

[0048] definition Throughout this book, various definitions are provided. Most terms have meanings that would belong to those skilled in the art. Terms specifically defined below or anywhere else in this book have meanings provided within the context of the entire invention and meanings that are generally understood by those skilled in the art.

[0049] "Subject," "individual," or "patient" are used interchangeably herein and refer to vertebrates, preferably mammals. Mammals include, but are not limited to, mice, rodents, apes, humans, domestic animals, sports animals, and pets.

[0050] In some embodiments, “treating” or “treatment” of any disease or disorder means alleviating that disease or disorder (i.e., preventing or reducing the onset of the disease or at least one clinical symptom). Treatment may be considered to include preemptive or prophylactic administration to alleviate, prevent, or inhibit the onset of the disease or at least one clinical symptom. Treatment may also mean reducing the severity and / or duration of one or more symptoms of the disease or disorder. In further features, the treatment provided is unlikely to have long-term side effects spanning multiple years. In other embodiments, “treating” or “treatment” means improving at least one physical parameter that is not recognizable to the patient. In yet another embodiment, “treating” or “treatment” means inhibiting the disease or disorder by physical means (e.g., stabilizing a recognizable symptom), physiological means (e.g., stabilizing a physical parameter), or both. In other embodiments, “treating” or “treatment” refers to slowing the progression of a disease or disorder.

[0051] The "therapeutic dose" refers to the amount of a compound that, when administered to a patient to treat a disease, is sufficient to have an effect on treating such a disease. The "therapeutic dose" will vary depending on the compound, the disease and its severity, as well as the age, weight, adsorption, distribution, metabolism, and excretion of the patient being treated.

[0052] "Vehicle" refers to a diluent, additive, or carrier administered to the patient along with the compound. In some embodiments, the vehicle is pharmaceutically acceptable.

[0053] "Active ingredient," "active pharmacological component," or "API" refers to the compound of the present invention.

[0054] "4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazole-8-yl)benzonitrile," also known as "CF3CN," is referred to herein as "the compound of formula I," and has the SMILES code N#CC1=CC=C(C2=CC(C3=CC=C4C(N=C(C(F)(F)F)N4)=C3O2)=O)C=C1, and has the structure defined as follows: [ka]

[0055] Detailed description of the present invention The compound tropoflavone, also known as 7,8-dihydroxyflavone (7,8-DHF), is a naturally occurring flavone found in the leaves of Godmania aesculifolio, daisy, and primrose trees. It is known to act as an effective and selective agonist of tropomyosin receptor kinase B (TrkB), the major signaling receptor for brain-derived neurotrophic factor (BDNF), a neurotrophic factor.

[0056] Toporoflavin has demonstrated therapeutic efficacy in various animal models, including depression, Alzheimer's disease, cognitive impairment in schizophrenia, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, traumatic brain injury, cerebral ischemia, fragile X syndrome, and Rett syndrome.

[0057] A derivative of toporoflavin, 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazole-8-yl)benzonitrile, also known as CF3CN and referred to herein as the compound of formula I, has been shown to be useful in the treatment of schizophrenia.

[0058] The following examples describe the effects of the compound of formula I in animal models and demonstrate the compound's ability to treat symptoms of schizophrenia.

[0059] Novel Object Recognition (NOR) tests are a cognitive paradigm of two tests that assess visual recognition memory. Recognition memory tasks allow for the comparison of presented stimuli with previously stored information. Recognition memory is impaired in a range of human impairments, and NOR is widely used in rodents to investigate impairments in various animal models of human pathologies with impaired cognition.

[0060] The social interaction test is a putative animal model for assessing social withdrawal, which is one aspect of the decreased motivation area in the negative symptoms of CNS disorders such as schizophrenia. The social interaction test is a 10-minute, single-trial task that assesses the behavioral interaction between a test rat and an unfamiliar, similarly weighted conspecific rat. This test is widely used in rodents to detect behavioral changes in social behavior and to investigate impairments in various animal models of human conditions in which social dysfunction (e.g., aspects of negative symptomatics associated with schizophrenia) is observed.

[0061] Example 1: Effect of test compound on subchronic PCP-induced impairment in recognition memory using a novel object recognition (NOR) paradigm in rats. The Novel Object Recognition (NOR) test is a two-trial cognitive paradigm that assesses visual recognition memory. Recognition memory tasks allow for the comparison of presented stimuli with previously stored information. Recognition memory is impaired in a range of human impairments, and NOR is widely used in rodents to investigate impairments in various animal models of human cognitive disorders.

[0062] The test consists of two trials, following initial acclimatization to an empty test area. In the first trial, rats are exposed to two identical objects in an open area (acquisition phase). In the second trial, after an interval (1 minute to 6 hours, depending on the type of memory and the target brain region), rats are exposed to two different objects (one familiar from the first trial and one novel) (retention phase). Object recognition can be measured as the difference in the time spent exploring the familiar and novel objects. Behavior is recorded and scored using a NOR scoring timer by a skilled experimenter blinded to the treatment group.

[0063] The total time spent exploring an object (defined as the length of time the animal spends licking, smelling, or touching the object, but not the time spent standing, sitting, or leaning on the object) is recorded for both familiar and novel objects in the learning and retention trials. The discrimination index (defined as the difference between the time spent exploring the novel object and the time spent exploring the familiar object, divided by the total time spent exploring both objects) and spontaneous kinetic energy (defined as the number of lines crossed in both trials) are also calculated. Rats were shown to spend more time exploring novel objects during the retention phase.

[0064] Treatment of rats with subchronic phencyclidine (scPCP) induces selective, long-term, and robust impairment in the non-reactive (NOR) paradigm. This study was conducted to evaluate the ability of compounds of formula I to reverse scPCP impairment in the NOR paradigm. The effect of scPCP in this paradigm may represent selective impairment in visual recognition memory, which is well known to be impaired in schizophrenia, and test compounds that demonstrate efficacy in this model may be used to treat cognitive symptoms of schizophrenia.

[0065] material and method Preparation and administration of drugs: The compound of formula I (5% DMSO / 0.5 methylcellulose 400 cp) was administered orally at doses of 10 and 30 mg / kg in a 5 ml / kg solution. This solution was prepared fresh daily and continuously stirred before administration. On behavioral days (D1 and D20), the compound of formula I was administered orally 120 minutes prior to the behavioral test.

[0066] Animal adaptations and living environment: Rats were housed in groups of 4-5 individuals in a standard laboratory environment, under a 12-hour light-dark cycle (lights turned on at 07:00).

[0067] The experiment was conducted during the lighting period. The animals had free access to food and water, except during the procedures of the behavioral test.

[0068] Throughout the experiment, the animals' weight was measured regularly.

[0069] All procedures were carried out in accordance with the Animal Scientific Procedures Act (UK, 1986) and were approved by the Animal Welfare and Ethical Review Body (AWERB) of the University of Manchester.

[0070] Novel Object Recognition (NOR) Protocol: A total of 66 female Lister Hooded rats were used in the NOR study. 56 rats were subchronically treated with PCP, and 10 were treated with a vehicle.

[0071] The PCP injection regimen involves administering 2 mg / kg (intraperitoneal) twice daily for 7 days, followed by at least 7 days of drug-free period. This regimen has been found to produce robust and long-lasting behavioral and pathological impairments within this paradigm (Neill et al., 2010; Cadinu et al, 2017).

[0072] After the scPCP drug regimen and before the behavioral test, the rats were treated as follows:

[0073] 1. Acute treatment using the test compound: Rats (n=36) were acutely treated with two doses of the compound of formula I (API) (10 mg / kg (n=18) and 30 mg / kg (n=18), administered orally, 120 minutes before the day 1 test), risperidone (0.1 mg / kg, administered intraperitoneally, 60 minutes before the test), or vehicle (5% DMSO / 0.5 methylcellulose 400 cp, administered orally, 120 minutes before the day 1 test). For PK analysis, brains (n=3) were collected from each API group immediately after the behavioral test.

[0074] 2. Treatment with the test compound for 20 days: Rats (n=30) were administered either two doses of the compound of formula I (API) (10 mg / kg (n=15) and 30 mg / kg (n=15), orally, 120 minutes before the 20-day test), risperidone (0.1 mg / kg, intraperitoneally, 60 minutes before the test), or a vehicle (5% DMSO / 0.5 methylcellulose 400 cp, orally, 120 minutes before the 20-day test) for 20 days. For PK analysis, brains (n=3) were collected from each API group immediately after the behavioral test.

[0075] Statistical analysis: All data are expressed as mean ± SEM (standard error). Search time data for NOR in the acquisition and retention phases were analyzed separately using repeated measures of GLM with factors of drug and two objects (two identical objects in the acquisition phase, and a new object and a familiar object in the retention phase). Discriminant Index (DI) and spontaneous movement (total number of times the line was crossed in both phases) data were analyzed by one-way ANOVA. Post-hoc testing was performed using least significant difference (LSD) tests (for spontaneous movement and DI scores) after significant one-way ANOVA results.

[0076] Results - Acute treatment using the test compound: The effects of acute treatment using compound I (API) at doses of 10 and 30 mg / kg (oral administration), as well as risperidone (0.1 mg / kg, intraperitoneal administration) as a positive control group, on the total object search time (seconds) in acquisition and retention trials are shown in Table 1 and Figures 1-4 below. [Table 1]

[0077] As expected, in the trial learning phase, there was no difference in the time spent searching for two identical objects among the different processing groups (Figure 1).

[0078] In the retention portion of the trial, the positive control group and vehicle-treated animals showed a statistically significant increase in the time spent searching for novel objects (Figure 2). Untreated animals (PCP only) and animals acutely treated with the compound of formula I showed no difference in the time spent searching for novel or familiar objects.

[0079] As expected from the results shown in Figure 3, both the vehicle and the animals treated with risperidone had a higher discrimination index (DI) compared to the untreated animals and the animals treated with the test compound (Figure 3).

[0080] Figure 4 shows that there was no difference in the number of times the lines were crossed in the treatment groups, indicating that none of the treatments affected spontaneous movement.

[0081] Results - 20-day treatment of the test compound: The effects on the total object search time (seconds) in learning and retention trials of treatment with the compound of formula I (API) at doses of 10 and 30 mg / kg (oral administration) once daily for 20 days, as well as acute treatment with risperidone (0.1 mg / kg, intraperitoneal administration) as a positive control group, are shown in Table 2 and Figures 5-8 below. [Table 2]

[0082] As expected, in the trial learning phase, there was no difference in the time spent searching for two identical objects among the different processing groups (Figure 5).

[0083] In the retention portion of the trial, the positive control group and vehicle-treated animals showed a statistically significant increase in the time spent searching for novel objects (P<0.05) (Figure 6). Untreated animals (PCP only) showed no difference in the time spent searching for novel or familiar objects.

[0084] Figure 6 further shows a dose-dependent, statistically significant effect of the compound of formula I on the time animals treated with it spend exploring familiar and novel objects. ** (P<0.01). This effect was greater than that observed with risperidone in the positive control group.

[0085] Figure 7 details the discrimination index (DI) for all treatment groups. It can be seen that treatment with the compound of formula I at 30 mg / kg for 20 days resulted in a statistically significant increase in DI compared to untreated animals (P<0.015).

[0086] Figure 8 shows that there was no difference in the number of times the lines were crossed in the treatment groups, indicating that none of the treatments affected spontaneous movement.

[0087] conclusion NOR (Natural Orientation) trials have previously been shown to identify compounds that may be useful in treating visual recognition memory, which is known to be impaired in schizophrenia. The data shown in Example 1 demonstrate that risperidone, the positive control, was able to restore scPCP-induced impairment in rats when administered acutely, indicating that the trial was healthy.

[0088] Importantly, the data further demonstrate that administering the compound of formula I to animals for 20 days can dose-dependently restore scPCP impairment.

[0089] These findings provide evidence that the compound of formula I may be suitable for use in treating the cognitive symptoms of schizophrenia.

[0090] Example 2: Effect of the test compound on social interactions in rats with subchronic PCP-induced disorder The social interaction test is a putative animal model for assessing social withdrawal, which is one aspect of the decreased motivation area in the negative symptoms of CNS disorders such as schizophrenia. The social interaction (SI) test is a single 10-minute task that assesses the behavioral interaction between a test rat and an unfamiliar, similarly weighted, conspecific rat. This test is widely used in rodents to detect behavioral changes in social behavior and to investigate impairments in various animal models of human conditions in which social dysfunction (e.g., aspects of negative symptomatics associated with schizophrenia) is observed.

[0091] First, the animals are allowed to acclimate to an empty testing area, after which the experiment consists of one trial. In this trial, the test animals (i.e., scVeh, scPCP, scPCP+ treatments) are placed in the testing area with unfamiliar, similarly weighted, and of the same species.

[0092] The main actions that will be scored in this exam are as follows:

[0093] 1. Sniffing behavior

[0094] 2. Examine inanimate objects (control group).

[0095] 3. Crossing the line to measure spontaneous movement

[0096] The behavior of the treatment group is recorded and scored by a trained experimenter who is blinded to the treatment group.

[0097] The scPCP regimen has been shown to produce selective, long-lasting, and robust impairment in this task. Using the scPCP model in this study, we observe a significant reduction in prosocial behavior (i.e., sniffing) and no difference in exploring unfamiliar inanimate objects.

[0098] material and method A total of 54 female Lister Hooded rats were used for the SI study. 44 rats were subchronically treated with PCP, and 10 were treated with vehicle-based treatment. The PCP injection regimen consisted of 2 mg / kg (intraperitoneal administration) twice daily for 7 days, followed by at least 7 days of drug-free intervals. This regimen was found to produce robust and long-lasting behavioral and pathological impairments of this paradigm (Neill et al., 2010; Cadinu et al., 2017).

[0099] Following the scPCP administration regimen detailed in Example 1 above, and prior to the behavioral test, rats were treated with the test compound for 21 days.

[0100] Rats (n=24) were administered either two doses of the test compound (10 mg / kg (n=12) and 30 mg / kg (n=12), orally, 120 minutes before the 21st day of the study), a positive control group (using risperidone) (0.1 mg / kg, intraperitoneally, 60 minutes before the study), or a vehicle (5% DMSO / 0.5 methylcellulose 400 cp, orally, 120 minutes before the 21st day of the study) for 21 days. For PK analysis, brains (n=3) were collected from each test group immediately after the behavioral study.

[0101] Statistical analysis: All data are expressed as mean ± SEM (n=10-12 for each group), analyzed by one-way ANOVA, and further analyzed by Dunnett post hoc correction analysis.

[0102] result Figures 9-11 detail the results obtained after scPCP-treated rats were treated with the compound of formula I for 21 days.

[0103] Figure 9 shows that PCP treatment significantly reduces the time spent sniffing, and that PCP treatment causes social dysfunction in test animals.

[0104] Figure 9 details that the compound of formula I was able to dose-dependently restore social dysfunction in scPCP-treated rats, as evidenced by the increased sniffing time. The high dose of 30 mg / kg was shown to be statistically significant (P<0.05).

[0105] Furthermore, the positive control group was shown to increase the time rats spent sniffing to levels similar to those observed at high doses of the test substance.

[0106] Figure 10 shows that both the vehicle and the PCP-treated animals spent similar amounts of time searching for inanimate objects, which indicates the integrity of the experiment. Furthermore, there were no differences between the test substance and the positive control group, as expected.

[0107] Finally, there was no difference in line crossing, which indicates that the processing did not affect spontaneous movement (Figure 11).

[0108] conclusion Social interaction studies have previously demonstrated their potential to identify compounds that may be useful in treating negative symptoms of CNS disorders. The data presented in Example 2 show that risperidone in the positive control group, when acutely administered, was able to restore scPCP-induced social dysfunction in rats, indicating that the study was sound.

[0109] Importantly, the data further demonstrate that administering the compound of formula I to animals for 21 days can dose-dependently restore social dysfunction.

[0110] These findings provide evidence that the compound of formula I may be suitable for use in treating the negative symptoms of schizophrenia.

[0111] Example 3: Study on the effects of 21-day treatment with the test compound on pathological markers of the prefrontal cortex (parvalbumin interneurons, BDNF, SNAP25, and PSD95) and pathological markers of the dorsal hippocampus (parvalbumin interneurons and BDNF) in scPCP-treated rats. This example investigated the effects of treating two different brain regions associated with cognitive and negative symptoms of schizophrenia with the compound of formula I (10 and 30 mg / kg, orally) for 21 days on postmortem markers (BDNF, PVIs, and neuronal markers).

[0112] material and method Brain recovery Immediately after the behavioral analysis in the previous example, the brain was collected and prepared for post-mortem analysis. The carbon dioxide concentration was increased to induce lethality in the animals, and death was confirmed by cervical dislocation. The brain was removed, frozen with dry ice, and stored at -80°C until analysis.

[0113] Half of the brain (one hemisphere) was randomly selected, prepared for parvalbumin immunohistochemical staining (IHC), and quantified in the prefrontal cortex and hippocampus.

[0114] The remaining half of the brain (the remaining hemisphere) was processed for BDNF protein analysis using ELISA (quantified in the prefrontal cortex and hippocampus) and neuronal marker protein analysis using Simple Western blotting (quantified in the prefrontal cortex).

[0115] Immunohistochemical staining staining: Sections (30 μm thick) were washed three times with 1x PBS (5 minutes each time). The sections were treated with heat-induced antigen retrieval in sodium citrate buffer (0.294% (w / v) sodium citrate, 0.07% (v / v) tween-20, 500 ml distilled water, pH adjusted to 6.0) at 80°C for 30 minutes. Unless otherwise specified, samples were washed twice with 1x PBS between each step.

[0116] Parvalbumin (PV) sections were prepared for light microscopy. The samples were incubated in hydrogen peroxide solution (1.5% H2O2, Sigma; 0.4% Triton x-100, Sigma; 10% methanol, 88.1% 1x PBS) at room temperature (RT) for 30 minutes. The samples were then incubated in protein blocking reagent (5% normal horse serum, Vector Laboratories; 0.6% Triton x-100, Sigma; 94.4% 1x PBS) for 1 hour. Subsequently, without washing, the samples were incubated in parvalbumin (1:5000; 24 hours; 235, Swant) at 4°C. On day 2, the samples were incubated with secondary antibody (PV: horse anti-mouse; 1:200; BA-2000-15, Vector Laboratories) for 2 hours. Sections were incubated with the VECTASTAIN Elite ABC-HRP kit (PK-4000, Vector Laboratories) for 45 minutes and visualized using the DAB substrate kit (SK-4100, Vector Laboratories). Once sufficient discoloration was observed, the samples were transferred to distilled water to stop the peroxidase reaction. Sections were placed on Superfrost slides and assigned randomized codes. The slides were then dehydrated by increasing the ethanol concentration (70%, 90%, and 100% every 5 minutes) and washed in Histoclear (National Diagnostics) for 5 minutes. DPX embedding medium (Sigma) was used for the coverslips.

[0117] Image analysis: Images were acquired using a 3D-Histech Pannoramic-250 microscope slide scanner (using a 20x / 0.80 Plan Apochromat objective (Zeiss)). Snapshots of the slide scans were taken using CaseViewer software (3D-Histech). For PV images, the contour of the target region was drawn, and the number of detected cells was manually counted in the CaseViewer software. The density was calculated by dividing the number of positive cells detected by the area of ​​the target region.

[0118] Protein analysis Homogenization: From frozen brain tissue, the prefrontal cortex and hippocampus were excised and homogenized on ice in 10x volume (mg:μL) buffer solutions (10mM Trizma base, Sigma-Aldrich; 320μM sucrose, Sigma-Aldrich; 2mM EDTA, Sigma-Aldrich; and a protease- and phosphatase-inhibitor cocktail, Sigma-Aldrich) to pH 7.4. Phenylmethylsulfonyl fluoride (PMSF) and sodium orthovanadate were added to the solution at a concentration of 1% (v / v). The samples were centrifuged at 800xg at 40°C for 15 minutes. The supernatant was collected and centrifuged at 11,700×g for 20 minutes. The resulting supernatant was collected and stored at -20°C until analysis.

[0119] Bradford Assay: The protein concentration of each sample was calculated using the Bradford assay. Samples were diluted in the protein assay dye concentrate (BioRad) and compared to bovine serum albumin (Sigma) at known concentrations (1.0–0.1 mg / ml protein). A standard curve was constructed using linear simple regression analysis. The standard and samples were analyzed in three replicates (triplicate). Outliers from the three replicates were excluded if their CV value was greater than 10%.

[0120] ELISA (brain-derived neurotrophic factor): BDNF in brain tissue was quantified using the rat brain-derived neurotrophic factor (BDNF) ELISA Kit (Cat#EKU02787-96T, BIOMATIK). In short, samples were diluted to 2.5 mg / mL of protein. Eight 2x standard curves were created, including a control group with only the diluent (31.25–1000 pg / mL). Standards and samples were pipetted into pre-coated 96-well plates and incubated at 37°C for 1 hour. Samples were removed from each well without washing. Next, detection reagent A was incubated on the plate and incubated at 37°C for 1 hour. Samples were washed eight times with washing buffer, and the plate was inverted onto absorbent paper to remove any remaining washing solution. Then, detection reagent B was added and incubated at 37°C for 30 minutes. The washing process was repeated. Finally, substrate solution was added, and expression was carried out in the dark. Stop solution was added, and the plate was read using a microplate reader (450 nm).

[0121] Samples for BDNF analysis from the total dorsal hippocampus were analyzed using two separate ELISA plates. To investigate inter-assay variability, a subset of samples from each group was analyzed in both plates. Data confirmed that this variability was less than 10% (Table 3). [Table 3]

[0122] For the final analysis of BDNF in the entire hippocampus, samples from both plates were combined (Figure 17). Specifically, values ​​from plate A were used where available, and for samples with only values ​​from plate B, these were used instead.

[0123] Simple Western (neuromarkers) A WES plate was prepared according to the provided instructions (SM-W004, ProteinSimple). Diluted samples were mixed with dithiothreitol and fluorescent master mix in an 8:1:1 ratio. The samples were denatured at 95°C for 5 minutes. The samples, antibody diluent, primary antibody, secondary antibody, and luminol-peroxide mix were loaded into the appropriate wells. Biotinylated ladders, antibody diluent, streptavidin-HRP, and luminol peroxide were also loaded. After briefly centrifugating the plate, wash buffer was added. The plate was then placed in a WES instrument, the loaded separation matrix was aspirated, and the stacking matrix was aspirated into each capillary. The ladders and samples were then loaded and placed in electrophoresis buffer. The samples were passed through an electric current and separated based on molecular weight. At the end of the separation, the samples were fixed with UV light to remove lysate buffer and non-protein components. The capillaries were then aspirated with blocking solution, primary antibody, and secondary antibody, and sandwiched between washes. Finally, the chemiluminescent substrate was added to the capillary, and the chemiluminescence level was detected by camera at nine different time points (1–512 seconds). The software then calculated the high dynamic range (HDR) of the peaks, which is derived from individual exposures and provides the maximum dynamic range of the signal with minimal background. The area under the curve was then calculated using the HDR peaks, which is analogous to the amount of protein in the sample. The antibodies and dilutions used are shown in Table 4 below. [Table 4]

[0124] To normalize the target protein, use the total protein assay provided by protein simple (DM-TP01). The normalized relative protein level is found by dividing the area under the curve for the target protein by the area under the curve for the total protein.

[0125] statistical analysis Parvalbumin densities in the prefrontal cortex and hippocampus were analyzed using two-way ANOVA repeated measures with region and treatment as fixed factors. Subsequently, Fisher's LSD post-hoc test was used to identify differences between treatment groups in each subregion.

[0126] BDNF and neural markers were analyzed using ANOVA, and then planned post-hoc tests were performed to examine differences between treatment groups in the prefrontal cortex (BDNF and neural markers) and hippocampus (BDNF).

[0127] result Effects of 21-day treatment of the test substance on parvalbumin interneurons in the prefrontal cortex. In the prefrontal cortex, there were significant main effects on treatment and region, with no interaction observed. PV cell density in scPCP-treated animals was significantly reduced in both the anterior cingulate cortex and inferior limbic cortex (both p<0.05 compared to scVeh). Long-term treatment with the compound of formula I at 30 mg / kg significantly increased PV cell density in scPCP-treated rats in all three regions (Cg1 and IL-p<0.05; PrL-p<0.01) (Figure 12).

[0128] One-way ANOVA revealed a significant main effect of processing across the entire prefrontal cortex (F(3,26)=3.053; p=0.0462). Planned post-hoc tests showed a decrease in PV cell density in scPCP animals (p<0.05 compared to scVeh).

[0129] Long-term treatment with the compound of formula I at 30 mg / kg significantly increased the density of PV cells in the prefrontal cortex compared to the scPCP group (p<0.01) (Figure 13).

[0130] Effects of 21-day treatment of the test substance on parvalbumin interneurons in the hippocampus In the hippocampus, significant main effects were observed for treatment and region, with no significant interactions. Post-hoc testing revealed no significant differences in PV cell density among any of the region groups (Figure 14).

[0131] One-way ANOVA of PV cell density across the hippocampus revealed no significant differences between the groups (F(3,28)=2.611, p=0.0711) (Figure 15).

[0132] Effect of 21-day treatment with the test substance on BDNF levels in the prefrontal cortex One-way ANOVA of BDNF levels in the prefrontal cortex revealed no significant differences between the groups (F(3,27)=0.7652, p=0.5235) (Figure 16).

[0133] The effect of 21-day treatment with the test substance on hippocampal BDNF levels. One-way ANOVA revealed a significant main effect trend across the entire hippocampus (F(3,35)=2.360; p=0.0882). Planned post-hoc tests showed decreased BDNF levels in scPCP animals (p<0.05 compared to scVeh). (Figure 17).

[0134] Effect of 21-day treatment with test substance on prefrontal cortex neuronal markers SNAP25: One-way ANOVA of SNAP25 revealed a significant main effect of treatment across the entire prefrontal cortex (F(3,25)=4.416; p=0.0127). Planned post-hoc tests showed a decrease in SNAP25 levels in scPCP animals (p<0.05 compared to scVeh). This increase was significantly increased by long-term treatment with OT-003 at 10 mg / kg (p<0.05), but not significantly increased by treatment with OT-003 at 30 mg / kg (p=0.5412) (Figure 18).

[0135] PSD95: One-way ANOVA of PSD95A across the entire prefrontal cortex revealed no significant differences between the groups (F(3,28)=1.827, p=0.1651) (Figure 19).

[0136] conclusion In this example, the effects of treating two different brain regions associated with cognitive and negative symptoms of schizophrenia with the compound of formula I (10 and 30 mg / kg, orally administered) for 21 days on postmortem markers (i.e., BDNF, PVIs, and neuronal markers) were investigated.

[0137] These research findings highlight that long-term administration of the compound of formula I can restore the degeneration of molecules causing behavioral disorders, particularly the repair of PVIs and SNAP25 in the PFC.

[0138] Therefore, long-term administration of the compound of formula I may be effective in treating the cognitive and negative symptoms observed in patients with schizophrenia.

[0139] Example 4: Effect of test compound on restoring subchronic PCP-induced disorder in the attentional set-shifting task (ASST) This embodiment tested the ability of treatment with the compound of formula I for 21 days to attenuate disruption of complex cognitive tasks induced by subchronic PCP (scPCP).

[0140] The effects of scPCP on attentional set shift are thought to contribute to the formation of cognitive impairment symptoms in schizophrenia. Atypical antipsychotics, clozapine, and risperidone and α7 nicotinic receptor positive allosteric modulators (McLean et al., 2008, 2012) have been shown to attenuate the impairment, while typical antipsychotic haloperidol was ineffective.

[0141] The attentional set-shifting task examines rats' ability to learn rules and form attentional sets within the same classification category (intradimensional shift (IDS) and extradimensional shift (EDS), the ability to switch attentional sets between different classification categories; Birrell and Brown, 2000). This represents a rat analog of the CANTAB ID / ED task in humans (Downes et al., 1989), and schizophrenic patients exhibit impaired set-shifting and reduced cognitive flexibility (Berg et al., 1948; Pantelis et al., 1999; Tyson et al., 2004).

[0142] Female rats subchronically treated with PCP exhibited selective impairment in out-of-dimensional shift on this task, which was reversible with 7 days of treatment with clozapine, risperidone, and an α7 nicotinic receptor positive modulator, but not with haloperidol (McLean et al., 2008, 2012).

[0143] material and method Setshift Challenge Device: The test box is a custom-made cage made of clear plastic, measuring 59cm x 35cm x 24cm. A fixed 1cm thick plexiglass panel permanently divides the box into two separate compartments (selection areas) for one-third of its length. These two selection areas can be further separated from each other with the help of two removable panels. Digging bowls are placed in each selection area, and rats can access them by lifting the partition(s), although the partitions can also be used to deny access to the bowls, for example, when an incorrect selection is made.

[0144] Habituation and training: Habituation to a specific aspect of the experimental paradigm begins immediately after the 7-day drug washout period. Habituation to the test box is performed for 1 hour for 3 consecutive days prior to training. Two smooth ceramic circular digging pots (9 cm in diameter and 5 cm deep), identical to those intended for use in the experimental phase, were introduced into each home cage. Each bowl was filled with Grade 5 sawdust (which also covered the floor of the home cage) and several quarter-piece honey nut loops (Kellogg's, UK) were placed in it as a food reward.

[0145] During this acclimatization period, the digging bowl was continuously replenished with food and left as is in the home cage throughout the study period.

[0146] After acclimatization, all rats must successfully complete the entire training regime before proceeding to the test. Initially, rats are trained to dig quickly and reliably in both bowls, with a single food reward for each test gradually covered by a progressively thicker layer of digging medium. Once it is repeatedly demonstrated that the rats have mastered the training procedure, a partition panel is introduced. Digging itself is defined as vigorously moving the forelegs to remove the digging medium and retrieve the supplement food buried 2-2.5 cm below the surface. If the rat is unable to dig within 15 minutes, the training session is terminated. The rat is trained again the following day.

[0147] In the second stage of training, the concept of simple discrimination (SD) is introduced to two media. Rats are presented with identical digging bowls, each containing two different digging media, with only one containing a food reward. The placement of the bowl in the left or right compartment is randomized with the aid of an adapted imitation Gellerman schedule. In the first four trials, regardless of which bowl is dug first, the rats are allowed to explore both bowls, thereby acting as an opportunity to associate the food reward with the positive predictor. Subsequent incorrect selections result in termination of the trial without an opportunity to explore the correct bowl. Successful learning of each discrimination is defined as six consecutive correct selections.

[0148] Test protocol: In all cases, the rats are tested 24 hours after the test in an attention set shift procedure. The test begins by raising both partitions to allow access to both digging bowls (only one of which contains food). The first stage is simple discrimination, identical to the simple discrimination in the training session the previous day. The test continues until the rats reach a criterion of six consecutive correct responses.

[0149] In the test session, rats perform a series of discriminations (see, for example, Table 5). In compound discrimination (CD), a second dimension (odor) is introduced, but the dimension associated with correct and incorrect answers remains the same (excavation medium). In reversal, the sample and associated dimension remain the same (medium or odor), but the rat must learn that the dimension where the food was previously located is now incorrect, and the other dimension is correct. New samples are used in ID and ED shifts. In the ED shift, a dimension that was previously unrelated (i.e., odor) is now related. It has been shown that rats can find the difficulty of each dimension change (i.e., medium to odor, or odor to medium, equivalent). In simple discrimination, the excavation medium was selected as a parameter relevant to all rats. [Table 5] summary: In simple discrimination, only one dimension is relevant and introduced (i.e., drilling medium; M1 / M2). • In compound discrimination, a second dimension is introduced (i.e., odor; O1 / O2), while the relevant dimension remains the same (i.e., drilling medium). • In Reversal 1, reward-bearing media are exchanged from M1 to M2. • In IDS, samples are introduced (M3 / M4 / O3 / O4), while the related dimensions remain the same (i.e., drilling medium). • In Reversal 2, reward-bearing media are exchanged from M3 to M4. • In EDS, new samples are introduced (M5 / M6O5 / O6), and the associated dimensions are switched from media to scent. • In Reversal 3, the scent that provides a reward is exchanged from O5 to O6. Scents and media used: • O1: Salted caramel and vanilla • O2: Mango • O3: Watermelon • O4: White Musk • O5: Coconut O6: Basil and Thyme · M1: Cat litter M2: Bedding paper M3 Vermiculite • M4 Wood Pellets M5 sawdust M6 Shredded straw

[0150] Overview of the ASST Protocol A total of 80 rats in 6 groups were tested on an attention set-shift task (rats received subchronic PCP treatment (2 mg / kg, oral administration (n=66)) or vehicle treatment (intraperitoneal administration (n=14)) twice daily, after at least 7 days of drug-free treatment). This regimen was found to induce robust, long-lasting behavioral and pathological disorders within this paradigm (Neill et al., 2010; Cadinu et al., 2017).

[0151] 21-day treatment of the test compound Rats were treated for 21 days with either an scPCP treatment regimen and three doses of the test substance (3, 10, and 30 mg / kg, orally), a vehicle (5% DMSO / 0.5 methylcellulose 400 cp), or risperidone (0.1 mg / kg, intraperitoneally), which served as the positive control group for the experiment, before their performance on tasks was tested.

[0152] statistical analysis The data were expressed as mean ± SEM (standard error), analyzed using repeated measures GLM (set shift stage × treatment group), and then post-hoc tests were performed for each stage using the least significant difference (LSD) test. [Table 6]

[0153] Sample preparation for pharmacokinetic analysis after ASST studies Plasma and brain samples (groups 1-5) were collected immediately after the ASST trial on day 21.

[0154] Trunk blood was obtained and stored in EDTA-coated tubes on ice before centrifugation. The blood was centrifuged at 5,000 RPM at 4°C for 10 minutes. Plasma was transferred to Eppendorf vials (2 ml) and stored at -80°C until analysis. The brain was separated into hemispheres, and one hemisphere (randomly selected) was post-fixed in 4% PFA for 24 hours. The sample was then transferred to 30% sucrose, flash-frozen with isopentane, and stored at -80°C for immunohistochemical (IHC) analysis. The other hemisphere was stored directly at -80°C for protein (e.g., ELISA and Westerns) analysis. Three hemispheres (randomly selected) were sent for PK analysis.

[0155] result Figure 20 details the effect of treating rats with the compound of formula I for 21 days on scPCP-induced cognitive impairment in an attentional set-shift task. After treatment with scPCP, the number of attempts to reach the baseline in the out-of-dimensional shift (EDS) stage of the attentional set-shift task was significantly increased compared to vehicle-treated rats (P<0.01).

[0156] After treatment with the compound of formula I at 30 mg / kg (P<0.01) for 21 days, the number of trials required to reach the baseline in the EDS stage was significantly reduced compared to the scPCP group (P<0.05), and an approximate significant reduction was observed in animals treated with 10 mg / kg (P=0.053).

[0157] No significant reduction was observed in animals treated with 3 mg / kg.

[0158] Risperidone significantly restores scPCP-induced impairment in the EDS phase of the study (P<0.01).

[0159] Figure 21 details the relative protein levels of the neuronal markers PSD95 and SNAP25 in animal brains after 21 days of treatment with the compound of formula I. As can be seen, the compound of formula I significantly reduced the scPCP-induced loss of both markers at 3 and 10 mg / kg, but not at 30 mg / kg.

[0160] Figure 22 details the BDNF concentration in the hippocampus at two different protein concentrations after treatment with the compound of formula I for 21 days. As can be seen, scPCP treatment did not significantly reduce BDNF levels in the rat brain. However, the compound of formula I was shown to increase BDNF levels, returning them to the same levels as those in vehicle-treated animals, thus indicating that the treatment was effective in restoring BDNF levels to normal.

[0161] conclusion The data described in this embodiment demonstrate the ability of the compound of formula I to restore cognitive impairment in scPCP-treated animals. Such cognitive impairment is a model of the cognitive symptoms of schizophrenia, and therefore these data provide evidence that the compound of formula I may be useful in treating the cognitive symptoms of schizophrenia.

Claims

1. A compound of formula I used for the treatment of schizophrenia.

2. A compound of formula I for use according to claim 1, wherein the treatment of schizophrenia is the treatment of one or more symptoms associated with schizophrenia.

3. A compound of formula I for use according to claim 2, wherein the symptoms associated with schizophrenia are cognitive symptoms.

4. A compound of formula I for use according to claim 2, wherein the symptoms associated with schizophrenia are negative symptoms.

5. A compound of formula I for use according to claim 1, wherein the compound of formula I is administered together with one or more pharmaceutically acceptable excipients.

6. A compound of formula I for use according to claim 1, wherein the compound of formula I is formulated into a dosage form selected from liquid, lozenge, rapidly disintegrating tablet, lyophilized preparation, film, spray, aerosol, sustained-release tablet or capsule, modified-release tablet or capsule, tablet, capsule, cream, ointment, or mucosal adhesive.

7. A compound of formula I for use according to claim 1, wherein the compound of formula I is administered once daily.

8. A compound of formula I for use according to claim 1, wherein the compound of formula I is administered multiple times a day.

9. A compound of formula I for use according to claim 8, wherein the compound of formula I is administered two, three, four, or five times a day.

10. A compound of formula I for use according to claims 7 to 9, wherein each dose contains at least 0.001 mg of the compound of formula I.

11. A compound of formula I for use according to claims 7 to 9, wherein each dose contains about 0.001 mg to about 500 mg of the compound of formula I.

12. A compound of formula I for use according to claims 7 to 9, wherein each dose contains about 500 mg to about 1000 mg of the compound of formula I.

13. A compound of formula I for use according to claim 1, wherein the compound of formula I is administered together with one or more additional agents.

14. A method for treating schizophrenia in a person requiring treatment, the method comprising administering to the person a therapeutically effective amount of a compound of formula I.

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