Compounds for treatment of schizophrenia

Abstract

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

Description

TECHNICAL FIELD

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

[0002] The Diagnostic and Statistical Manual of Mental Health Disorders (DSM) is the global authority on psychiatric diagnosis. The DSM-V, published in 2013, defines the symptoms of schizophrenia into three broad categories: (1) positive symptoms; (2) negative symptoms; and (3) cognitive symptoms.

[0003] Generally, the positive symptoms of schizophrenia include hallucinations, delusions, thought disorders, and movement disorders. The negative symptoms of schizophrenia include “flat affect,” lack of pleasure in daily life, lack of ability to start and sustain planned activities, and speaking very little, even when forced to interact. Finally, the cognitive symptoms of schizophrenia include poor “executive functioning;” difficulty concentrating, and problems with working memory.

[0004] Treatment of schizophrenia is generally with antipsychotic medications (typical and atypical antipsychotic medications), including aripiprazole, asenapine, brexpiprazole, cariprazine, clozapine, iloperidone, lurasidone, olanzapine, paliperidone, quetiapine, risperidone, ziprasidone, chloφromazine, fluphenazine, haloperidol, and perphenazine.

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

[0006] During periods of severe symptoms, patients are often hospitalized to ensure the safety of the patient, and in cases where the patient does not respond to pharmacological therapy, electroconvulsive therapy can be used.

[0007] The Positive and Negative Syndrome Scale (PANSS) is an assessment tool used to determine the total symptoms of schizophrenia. Based on two well-established psychiatric rating systems, the 30-item PANSS is considered to be a workable, drug-sensitive instrument that provides a balanced representation of positive and negative symptoms and measures their relationship to each other and to overall psychopathology.

[0008] In addition, schizophrenia symptoms purely associated with the negative domain can be assessed using the SANS rating scale. This measures the extent of negative symptoms and is divided into 5 subscales: (i) affective flattening or blunting; (ii) alogia; (iii) avolition / apathy; (iv) anhedonia / asociality; and (v) impairment of attention.

[0009] Cognitive symptoms of schizophrenia are often apparent at the first onset of psychosis. Existing antipsychotic drug treatments often fail to improve cognitive symptoms.

[0010] In 2006, the FDA stated that the current available drug treatments for schizophrenia were not satisfactory for the treatment of the negative symptoms of the disease (Laughren and Levin, 2006) as no drug addressing this problem had been approved at that time.

[0011] Antipsychotic drugs approved for the treatment of schizophrenia do not treat specific aspects of the disease, in particular they do not address the negative or cognitive symptoms of the disease. Therefore, more appropriate treatments, in particular for these symptoms, are needed.

[0012] The present application provides data from animal models to demonstrate that compounds of Formula I are useful in the treatment of schizophrenia. One study demonstrates that compounds of the application are useful in the treatment of the cognitive symptoms of schizophrenia. Further studies also demonstrate that compounds of the application are useful in the treatment of the negative symptoms of schizophrenia. SUMMARY

[0013] According to a first aspect of the application, there is provided a compound of Formula I for use in the treatment of schizophrenia.

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

[0015] More preferably, the treatment is of the negative symptoms of schizophrenia. Alternatively, the treatment is of 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 a liquid, a lozenge, a rapidly disintegrating tablet, a lyophilized formulation, a film, a spray, an aerosol, a sustained release tablet or capsule, a modified release tablet or capsule, a tablet, a capsule, a cream, an ointment, or a mucoadhesive.

[0018] Preferably, the compound of Formula I is administered in a single daily dose. Alternatively, the compound of Formula I is administered in multiple daily doses. Further, the compound of Formula I is administered twice, three times, four times, or five times daily.

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

[0020] In further embodiments, the compound of Formula I is administered with one or more additional pharmaceutical products.

[0021] According to a second aspect of the application, there is provided a method of treating schizophrenia in a subject in need thereof, 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 dosage of about 0.0001 mg to about 70 mg of the compound per kilogram of body weight per day. Dosage unit forms are prepared to provide about 0.01 mg, 0.1 mg, or 1 mg to about 500 mg, or about 1000 mg, of the active ingredient or combination of essential ingredients per dosage unit form, in some embodiments, about 10 mg to about 500 mg.

[0023] Exemplary dosages of the formulation include milligram or microgram amounts of active compound per kilogram of subject (e.g., about 1 microgram per kilogram to about 50 milligrams per kilogram, about 10 micrograms per kilogram to about 30 milligrams per kilogram, about 100 micrograms per kilogram to about 10 milligrams per kilogram, or about 100 micrograms per kilogram to about 5 milligrams per kilogram).

[0024] In certain embodiments, the same formulation provided herein can be administered repeatedly, and the administration can be separated by 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. BRIEF DESCRIPTION OF DRAWINGS

[0025] The application is described with reference to the following drawings:

[0026] Figure 1 The average exploration time for two identical objects in the acquisition trial of the acute phase NOR test is detailed.

[0027] Figure 2 Average exploration time to two identical objects in the acquisition trial of the NOR test during the acute phase is detailed.

[0028] Figure 3 Effect of acute treatment with test compound and risperidone on discrimination index (DI) in the NOR test is detailed.

[0029] Figure 4 Effect of 20 days treatment with test compound and risperidone on total number of crossing in the acquisition and retention trials of the NOR test is detailed.

[0030] Figure 5 Average exploration time to two identical objects in the acquisition trial of the NOR test during the 20 days treatment phase is detailed.

[0031] Figure 6 Average exploration time to familiar and novel objects in the 3 minutes retention trial of the NOR test during the 20 days treatment phase is detailed.

[0032] Figure 7 Effect of 20 days treatment with test compound and risperidone on discrimination index (DI) in the NOR test is detailed.

[0033] Figure 8 Effect of 20 days treatment with test compound and risperidone on total number of crossing in the acquisition and retention trials of the NOR test is detailed.

[0034] Figure 9 Effect of 21 days treatment with test compound and acute treatment with risperidone on sniffing duration in scPCP treated rats is detailed.

[0035] Figure 10 Effect of 21 days treatment with test compound and acute treatment with risperidone on object exploration duration in scPCP treated rats is detailed.

[0036] Figure 11 Effect of 21 days treatment with test compound and acute treatment with risperidone on number of crossing in scPCP treated rats is detailed.

[0037] Figure 12 Effect of 21 days treatment with test compound (TC) (10 and 30 mg / kg, p.o.) on cell density (cells / mm2) of parvalbumin interneurons in prefrontal cortex subregions. Data are expressed as mean ± s.e.m (n=7-9 per group). * = p<0.05, **=p<0.01. Cg1 = cingulate cortex, PrL = prelimbic cortex, IL = infralimbic cortex.

[0038] Figure 13 The effect of treatment with test compound (TC) (10 and 30 mg / kg, p.o.) for 21 days on parvalbumin interneuron cell density (cells / mm2) in the frontal cortex was detailed. Data are expressed as mean ± s.e.m (n=7-9 per group). * = p<0.05, ** = p<0.01 vs scPCP.

[0039] Figure 14 The effect of treatment with test compound (TC) (10 and 30 mg / kg, p.o.) for 21 days on parvalbumin interneuron cell density (cells / mm2) in the dorsal hippocampal subregion was detailed. Data are expressed as mean ± s.e.m (n=7-9 per group). CA1 = Cornu Ammonis 1, CA2 / 3 = Cornu Ammonis 2 / 3, DG = dentate gyrus.

[0040] Figure 15 The effect of treatment with test compound (TC) (10 and 30 mg / kg, p.o.) for 21 days on parvalbumin interneuron cell density (cells / mm2) in the dorsal hippocampus was detailed. Data are expressed as mean ± s.e.m (n=7-9 per group).

[0041] Figure 16 The effect of treatment with test compound (TC) (10 and 30 mg / kg, p.o.) for 21 days on BDNF levels (pg / ml) in the frontal cortex was detailed. Data are expressed as mean ± s.e.m (n=7-9 per group).

[0042] Figure 17 The effect of treatment with test compound (TC) (10 and 30 mg / kg, p.o.) for 21 days on BDNF levels (pg / ml) in the dorsal hippocampus was detailed. Data are expressed as mean ± s.e.m (n=9-11 per group).

[0043] Figure 18 The effect of treatment with test compound (TC) (10 and 30 mg / kg, p.o.) for 21 days on SNAP25 in the frontal cortex was detailed. Data are expressed as mean ± s.e.m (n=6-8 per group). * = p<0.05 vs scPCP.

[0044] Figure 19 The effect of treatment with test compound (TC) (10 and 30 mg / kg, p.o.) for 21 days on PSD95 in the frontal cortex was detailed. Data are expressed as mean ± s.e.m (n=7-9 per group).

[0045] Figure 20The effect of treatment with test compounds for 21 days on SCPCP-induced cognitive deficits in the set-shifting task is detailed.

[0046] Figure 21 The effect of treatment with test compounds for 21 days on synaptic markers PSD95 and SNAP25 in the prefrontal cortex is detailed.

[0047] Figure 22 The effect of treatment with test compounds for 21 days on BDNF concentration in the dorsal hippocampus is detailed.

[0048] Definitions

[0049] Various definitions are given in this document. Most of the words have the meaning that a person skilled in the art would attribute to these words. Words that are defined here below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and are the meaning that a person skilled in the art would generally understand.

[0050] "Subject", "individual" or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal. Mammals include, but are not limited to, murines, rodents, simians, humans, farm animals, sport animals, and pets.

[0051] In some embodiments, "treatment" of any disease or disorder refers to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one clinical symptom thereof). Treatment can also be considered to include prophylactic or preventative administration to improve, arrest or prevent the development of the disease or at least one clinical symptom. Treatment can also refer to reducing the severity and / or duration of one or more symptoms of the disease or disorder. In further features, the likelihood of long-term side effects of the administered treatment is low. In other embodiments, "treatment" refers to improving at least one physical parameter, which can not be perceptible to the patient. In further embodiments, "treatment" refers to inhibiting the disease or disorder, either physically (e.g., stabilization of a perceptible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In other embodiments, "treatment" refers to delaying the onset of the disease or disorder.

[0052] "Therapeutically effective amount" refers to the amount of a compound that, when administered to a patient for treating a disease, is sufficient to affect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity, and the age, weight, absorption, distribution, metabolism, and excretion of the patient to be treated.

[0053] "Vehicle" refers to a diluent, excipient, or carrier with which the compound is administered to a subject. In some embodiments, the vehicle is pharmaceutically acceptable.

[0054] “Active ingredient” or “active pharmaceutical ingredient” or “API” refers to the compound of the present invention.

[0055] “4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8-yl)benzonitrile”, also known as “CF3CN”, is referred to herein as the “Compound of Formula I”, 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 the structure defined below:

[0056] DETAILED DESCRIPTION

[0057] The compound tropoflavin, also known as 7,8-dihydroxyflavone (7,8-DHF), is a naturally occurring flavone found in the leaves of Godmania aesculifolio, Tridax procumbent, and Primula vulgaris. It is known to be a potent and selective agonist of the tropomyosin receptor kinase B (TrkB), the major signaling receptor for the neurotrophin brain-derived neurotrophic factor (BDNF).

[0058] Tropoflavin has been shown to have therapeutic efficacy in a variety of animal models, including depression, Alzheimer’s disease, cognitive deficits in schizophrenia, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, traumatic brain injury, cerebral ischemia, fragile X syndrome, and Rett syndrome.

[0059] A derivative of tropoflavin, 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8- d]imidazol-8-yl)benzonitrile, also known as CF3CN, is referred to herein as the Compound of Formula I, has been shown to be useful in the treatment of schizophrenia.

[0060] The following examples describe the effects of the Compound of Formula I in animal models, demonstrating the ability of the compound to treat symptoms of schizophrenia.

[0061] The novel object recognition (NOR) test is a two-trial cognitive paradigm that assesses visual recognition memory. Recognition memory tasks allow for a comparison between presented stimuli and previously stored information. Recognition memory is disrupted in a range of human conditions, and NOR is widely used in rodents to study deficits in animal models of various human conditions in which cognition is impaired.

[0062] The social interaction test is a well-accepted animal model to assess social withdrawal, which is an aspect of the anhedonia domain of negative symptoms in CNS disorders such as schizophrenia. The social interaction test is a 10-minute single-trial task designed to assess behavioral interactions of a test rat with an unfamiliar, body weight-matched conspecific. This test detects behavioral changes in social behavior and is widely used in rodents to study deficits in various human condition animal models in which social impairment is observed (e.g., multiple aspects of the negative total symptom associated with schizophrenia).

[0063] Example 1: Testing the effect of compounds on subchronic PCP-induced deficits in recognition memory using the novel object recognition (NOR) paradigm in rats

[0064] The novel object recognition test (NOR) is a two-trial cognitive paradigm that assesses visual recognition memory. Recognition memory tasks allow for a comparison between presented stimuli and previously stored information. Recognition memory is disrupted in a range of human disorders, and the NOR is widely used in rodents to study deficits in various human condition animal models in which cognition is impaired.

[0065] The test consists of two trials after an initial acclimation to the empty test arena. In the first trial, the rat is exposed to two identical objects in an open arena (acquisition phase). In the second trial, after a period of time (1 min - 6 h, depending on the type of memory and the brain region of interest), the rat is exposed to two different objects, one of which is the familiar object from the first trial and the other is a novel object (retention phase). Object recognition can be measured as the difference in time spent exploring the familiar and the novel object. Behavior is recorded and scored by a trained experimenter (blinded to treatment groups) using a NOR scoring timer.

[0066] In the acquisition and retention trials, the total object exploration time (defined as the duration the animal licks, sniffs, or touches the object, but not the time standing, sitting, or leaning on the object) is recorded for each familiar and novel object. In addition, the discrimination index (defined as the difference in time exploring the new and the familiar object divided by the total time exploring both objects) and locomotor activity (defined as movement, measured by the number of lines crossed during both trials) are calculated. It has been shown that rats spend more time exploring the novel object during the retention phase.

[0067] Treatment of rats with subchronic phenylcylidine (scPCP) induces selective, long-lasting and robust deficits in the NOR paradigm. This study was performed to assess the ability of the compound of formula I to reverse the scPCP deficits in the NOR paradigm. The effect of scPCP in this paradigm can represent a selective deficit in visual recognition memory, which is known to be impaired in schizophrenia, therefore, a test compound showing efficacy in this model can be useful for the treatment of the cognitive symptoms of schizophrenia.

[0068] Materials and methods

[0069] Drug preparation and administration:

[0070] The compound of formula I (5% DMSO in 0.5 methylcellulose 400 cp) was administered orally at 10 and 30 mg / kg, in a dose volume of 5 ml / kg. This was prepared fresh daily and constantly stirred prior to dosing. On the behavioural days (D1 and D20), the compound of formula I was administered orally 120 min prior to the behavioural test.

[0071] Animals - acclimatisation and housing conditions:

[0072] Rats were housed in groups of 4-5 under standard laboratory conditions, under a 12 h light:dark cycle (light starting at 07:00).

[0073] Testing was performed during the light phase. Except during the behavioural test procedure, animals had free access to food and water.

[0074] Animals were weighed regularly throughout the experiment.

[0075] All procedures were performed in accordance with the Animals (Scientific Procedures) Act (UK, 1986) and were approved by the AWERB (Animal Welfare and Ethical Review Body) of the University of Manchester.

[0076] New object recognition (NOR) protocol:

[0077] A total of 66 female Lister Hooded rats were used for the NOR study. 56 rats were treated subchronically with PCP and 10 rats were treated with vehicle.

[0078] The PCP injection protocol was 2 mg / kg i.p. twice daily for 7 days, followed by a drug-free period of at least 7 days. This protocol has been found to produce robust and long-lasting behavioural and pathological deficits in this paradigm (Neill et al., 2010; Cadinu et al., 2017).

[0079] Rats were treated as follows according to the scPCP dosing protocol prior to the behavioural test:

[0080] 1. Acute treatment with test compound:

[0081] 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), oral, 120 min prior to test on Day 1), risperidone (0.1 mg / kg, i.p. 60 min prior to test) or vehicle (5% DMSO in 0.5 methyl cellulose 400cp, oral, 120 min prior to test on Day 1). Immediately after the behavioral test, brains were removed from each API group (n=3) for PK analysis.

[0082] 2. Treatment with test compound for 20 days:

[0083] Rats (n=30) were acutely treated with two doses of the compound of Formula I (API) (10 mg / kg (n=15) and 30 mg / kg (n=15), oral, 120 min prior to test on Day 20), risperidone (0.1 mg / kg, i.p. 60 min prior to test) or vehicle (5% DMSO in 0.5 methyl cellulose 400cp, oral, 120 min prior to test on Day 20). Immediately after the behavioral test, brains were removed from each API group (n=3) for PK analysis.

[0084] Statistical analysis:

[0085] All data are expressed as mean ± s.e.m. (standard error of the mean). Exploration time data from NOR in both acquisition and retention phases were analyzed by repeated measures GLM, taking into account the factors drug and 2 objects (2 identical objects in acquisition phase and new and familiar objects in retention phase). Discrimination index (DI) and locomotor activity data (total number of crossings in both phases) were analyzed by one-way ANOVA. Post-hoc analysis was performed by least significant difference (LSD) test following significant one-way ANOVA (for locomotor activity and DI scores).

[0086] Results - Acute treatment with test compound:

[0087] The effects of acute treatment with the compound of Formula I (API) and the positive control risperidone (0.1 mg / kg, i.p.) at doses of 10 and 30 mg / kg oral on total object exploration time in acquisition and retention trials are described in Table 1 and Figures 1-4 below.

[0088] Table 1. Total time spent by rats in the two parts of the NOR test

[0089]

[0090] As expected, there were no differences in the amount of time spent exploring two identical objects in the acquisition portion of the test for any of the treatment groups Figure 1 ).

[0091] In the retention portion of the test, the positive control and vehicle treated animals showed a statistically significant increase in the amount of time spent exploring the novel object Figure 2 The untreated animals (PCP only) and animals acutely treated with the compound of Formula I did not show differences in the amount of time spent exploring the novel or familiar objects.

[0092] As expected from the results shown in Figure 3 , the vehicle and risperidone treated animals had a higher discrimination index (DI) than the untreated and test compound treated animals Figure 3 .

[0093] Figure 4 It was demonstrated that any of the treatments had no effect on motor activity, since there were no differences in the number of crossings within the treatment groups.

[0094] Results - 20 days of treatment with the test compound:

[0095] The effect of 20 days of oral treatment with the compound of Formula I (API) at doses of 10 and 30 mg / kg once daily and acute treatment with the positive control risperidone (0.1 mg / kg, i.p.) on total object exploration time in the acquisition and retention tests is described in Table 2 and Figures 5-8 .

[0096] Table 2. Total time spent by rats in the two portions of the NOR test

[0097]

[0098] * P <0.0.5; significant increase in exploration compared to scPCP.

[0099] As expected, there were no expected differences in the amount of time spent exploring two identical objects in the acquisition portion of the test for any of the treatment groups Figure 5 .

[0100] In the retention portion of the test, the positive control and vehicle treated animals showed a statistically significant increase in the amount of time spent exploring the novel object (P <0.05) Figure 6 . The animals of the untreated group (PCP only) did not show differences in the amount of time spent exploring the novel or familiar objects.

[0101] Figure 6 Further, it was demonstrated that the amount of time spent exploring the familiar and the novel object in animals treated with the compound of Formula I was dose-dependently statistically significantly affected (P<0.01). This effect was greater than that of the positive control risperidone. ** P<0.01). This effect was greater than that of the positive control risperidone.

[0102] Figure 7 The discrimination index (DI) was detailed for all treatment groups. It can be seen that the effect of treatment with the compound of Formula I at 30 mg / kg for 20 days produced a statistically significant increase in DI compared to the non-treated animals (P<0.015).

[0103] Figure 8 It was demonstrated that none of the treatments had an effect on locomotor activity, as there was no difference in the number of crossings within the treatment groups.

[0104] CONCLUSION

[0105] The NOR test has previously been demonstrated to identify compounds that can be useful in the treatment of visual recognition memory, which is known to be impaired in schizophrenia. The data presented in Example 1 demonstrate that the acute administration of the positive control risperidone was able to rescue the deficit induced by scPCP in rats, demonstrating that the test is robust.

[0106] Importantly, the data also demonstrate that the administration of the compound of Formula I to the animals for 20 days was able to rescue the scPCP deficit in a dose-dependent manner.

[0107] This finding provides evidence that the compound of Formula I can be useful in the treatment of the cognitive symptoms of schizophrenia.

[0108] Example 2: Effect of test compound on social interaction in rats in a subchronic PCP-induced deficit

[0109] The social interaction test is a well-accepted animal model to assess social withdrawal, which is an aspect of the negative symptoms domain of CNS disorders such as schizophrenia. The social interaction (SI) test is a 10-minute single trial task designed to assess the behavioral interaction of a test rat with an unfamiliar, body weight-matched conspecific. This test detects behavioral changes in social behavior and is widely used in rodents to study deficits in animal models of various human conditions in which social impairment is observed (e.g., aspects of the negative total symptom domain associated with schizophrenia).

[0110] The test consists of one trial after an initial acclimation to the empty test arena. In this trial, the test animal (i.e., scVeh, scPCP, scPCP + treatment) is placed in the arena with one unfamiliar, body weight-matched conspecific.

[0111] The main behaviours scored in this test are:

[0112] 1. Sniffing behaviour

[0113] 2. Exploration of inanimate objects (control)

[0114] 3. Crossing the line to measure locomotor activity.

[0115] Behaviours were recorded and scored by trained experimenters (blind to treatment group).

[0116] The scPCP regimen has been shown to produce selective, long-lasting and robust deficits in this task. Using the scPCP model in this test, a significant reduction in prosocial behaviour (i.e. sniffing) was observed, while exploration of unfamiliar inanimate objects was not different.

[0117] Materials and Methods

[0118] A total of 54 female Lister Hooded rats were used for the SI study. Forty-four rats were subchronically treated with PCP and 10 rats were treated with vehicle. The PCP injection regimen was 2 mg / kg i.p. 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 behavioural and pathological deficits in this paradigm (Neill et al., 2010; Cadinu et al., 2017).

[0119] Rats were treated with test compounds for 21 days prior to behavioural testing, following the scPCP dosing regimen detailed above in Example 1.

[0120] Rats (n=24) were dosed with two doses of test compound for 21 days (10 mg / kg (n=12) and 30 mg / kg (n=12), p.o. 120 min prior to test on day 21), the positive control used was risperidone (0.1 mg / kg, i.p. 60 min prior to test) or vehicle (5% DMSO in 0.5 methylcellulose 400cp, p.o. 120 min prior to test on day 21). Immediately after the behavioural test, brains were removed from each test group (n=3) for PK analysis.

[0121] Statistical analysis:

[0122] All data will be expressed as mean ± s.e.m (n=10-12 per group) and analysed by one-way ANOVA, with further analysis by Dunnett’s post-hoc correction.

[0123] Results

[0124] Figures 9-11The results of the scPCP treated rats after 21 days of treatment with the compound of Formula I are detailed

[0125] Figure 9 It is shown that treatment with PCP significantly reduced the amount of time spent sniffing, demonstrating that treatment with PCP produced social impairment in the test animals.

[0126] Figure 9 It is continued that the compound of Formula I was able to rescue the social impairment deficits in scPCP treated rats in a dose dependent manner by increasing the amount of time spent sniffing. The higher dose of 30 mg / kg showed statistical significance (P < 0.05).

[0127] In addition, the positive control showed an increase in the amount of time spent sniffing by the rats to a similar level as observed with the higher dose of the test article.

[0128] Figure 10 It is shown that the vehicle and PCP treated animals spent a similar amount of time exploring the inanimate object, demonstrating the robustness of the test. In addition, there was no difference between the test article and the positive control as expected.

[0129] Finally, there was no difference across the lines, demonstrating that the treatment had no effect on motor activity Figure 11 ).

[0130] Conclusion

[0131] It has been previously demonstrated that the social interaction test can identify compounds that can be useful in treating the negative symptoms of CNS disorders. The data presented in Example 2 demonstrate that the acute administration of the positive control, risperidone, was able to rescue the social impairment deficits induced by scPCP in rats, demonstrating that the test is robust.

[0132] Importantly, the data also demonstrate that the administration of the compound of Formula I to the animals for 21 days was able to rescue the social impairment in a dose dependent manner.

[0133] This finding provides evidence that the compound of Formula I can be useful in the treatment of the negative symptoms of schizophrenia.

[0134] Example 3: Investigation of the effect of 21 days of treatment with the test article on the pathological markers of the prefrontal cortex (parvalbumin interneurons, BDNF, SNAP25 and PSD95) and the dorsal hippocampus (parvalbumin interneurons and BDNF) of scPCP treated rats.

[0135] This example investigates the effect of 21 days of treatment with the compound of Formula I (10 and 30 mg / kg, p.o.) on post-mortem markers (BDNF, PVl and synaptic markers) in two different brain regions associated with the cognitive and negative symptoms of schizophrenia.

[0136] Materials and Methods

[0137] Brain Collection

[0138] Immediately after behavioral analysis of the preceding examples, brains were removed and prepared for necropsy analysis. Animals were euthanized by increasing carbon dioxide concentration and confirmed death by cervical dislocation. Brains were removed, frozen on dry ice, and stored at -80°C until analysis.

[0139] Randomly select ½ brain (one hemisphere) and process for quantification of astrocyte marker using IHC in the prefrontal cortex and dorsal hippocampus.

[0140] Process the remaining ½ brain (remaining hemisphere) for protein analysis of BDNF using ELISA (quantified in the prefrontal cortex and dorsal hippocampus) and analysis of synaptic markers using Simple Western (quantified in the prefrontal cortex).

[0141] Immunohistochemistry

[0142] Staining:

[0143] Wash sections (30 pm thickness) in lx PBS three times for five minutes each. Heat-induced antigen retrieval was performed on sections 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 min. Unless otherwise noted, samples were washed in lx PBS twice between each step.

[0144] Parvalbumin (PV) sections were examined by light microscopy. Samples were incubated in hydrogen peroxide solution (1.5% H2O2, Sigma; 0.4% Triton x-100, Sigma; 10% methanol, 88.1% lx PBS) for 30 min at room temperature (RT). Samples were incubated in protein block (5% normal horse serum, Vector Laboratories; 0.6% triton x-100, Sigma; 94.4% lx PBS) for one hour. Then, without washing, samples were incubated in parvalbumin (1:5000; 24 hours; 235, Swant) at 4°C. On day 2, samples were incubated with secondary antibody (PV: horse anti-mouse; 1:200; BA-2000-15, Vector Laboratories) for 2 hours. Sections were incubated with VECTASTAIN Elite ABC-HRP kit (PK-4000, Vector Laboratories) for 45 minutes and visualized with DAB substrate kit (SK-4100, Vector Laboratories). When sufficient color change was observed, samples were transferred to distilled water to stop the peroxidase reaction. Sections were mounted on Superfrost slides and randomly coded. Slides were then dehydrated in increasing concentrations of ethanol (70%, 90%, and 100% for 5 minutes each) and washed in Histoclear (National Diagnostics) for 5 minutes. Coverslips were applied with DPX mounting medium (Sigma).

[0145] Image analysis:

[0146] Images were acquired on a 3D-Histech Pannoramic-250 microscope slide scanner using a 20x / 0.80 planapochromat objective (Zeiss). Snapshots of the slide scans were taken using CaseViewer software (3D-Histech). For PV images, regions of interest were delineated and the number of cell detections were manually counted in the CaseViewer software. Density was calculated by dividing the number of positive cell detections by the area of the region of interest.

[0147] Protein analysis

[0148] Homogenization:

[0149] Prefrontal cortex and dorsal hippocampus were dissected from frozen brains and homogenized in 10 volumes (mg: pL) of buffer (10 mM Trizma base, Sigma-Aldrich; 320 mM sucrose, Sigma-Aldrich; 2 mM EDTA, Sigma-Aldrich; and protease and phosphatase inhibitor cocktail, Sigma-Aldrich) buffered to pH 7.4 on ice. Phenylmethylsulfonyl fluoride (PMSF) and sodium orthovanadate were added to the solution at a concentration of 1% (v / v). Samples were centrifuged at 800 x g for 15 minutes at 4°C. The supernatant was collected and centrifuged at 11,700 x g for 20 minutes, and the resulting supernatant was collected and stored at -20°C until analysis.

[0150] Bradford assay:

[0151] The Bradford assay was used to calculate the protein concentration of each sample. Samples were diluted into the protein assay dye reagent concentrate (BioRad) and compared to known concentrations (1.0-0.1 mg / ml protein) of bovine serum albumin (Sigma). A simple linear regression was used to create a standard curve. Triplicates were performed for both standards and samples. If the CV value was greater than 10%, outliers were removed from the triplicates.

[0152] ELISA (Brain-derived neurotrophic factor):

[0153] Rat Brain-derived Neurotrophic Factor (BDNF) ELISA Kit (Cat#EKU02787-96T, BIOMATIK) was used to quantitatively measure BDNF in brain tissue. Briefly, samples were diluted to 2.5 mg / mL protein. An 8-point, 2-fold standard curve (31.25-1000 pg / mL) was created, including a control with diluent only. Standards and samples were pipetted into a pre-coated 96-well plate and incubated at 37°C for 1 hour. The samples were removed from each well without washing. Next, detection reagent A was pipetted into the plate and incubated at 37°C for 1 hour. The samples were washed 8 times with wash buffer, and the remaining wash solution was removed from the plate by inverting the plate onto absorbent paper. Then, detection reagent B was added and incubated at 37°C for 30 minutes. The washing step was repeated. Finally, substrate solution was added and developed in the dark. After the addition of stop solution, the plate was read on a plate reader at 450 nm.

[0154] Samples for whole dorsal hippocampus BDNF analysis were run on two independent ELISA plates. A subset of samples from each group was run on both plates to investigate variability between assays. The data confirmed that this variability was <10% (Table 3).

[0155] Table 3. BDNF levels (pg / ml) on different ELISA plates. S1-S11 = samples from all four groups.

[0156]

[0157] Samples from both plates were combined for final analysis of BDNF in whole dorsal hippocampus. Figure 17 Specifically, plate A values were used where possible, and plate B values were used for samples where only plate B values were available.

[0158] Simple Western (synaptic markers)

[0159] WES plates were prepared according to the provided instructions (SM-W004, Protein Simple). Diluted samples were mixed with dithiothreitol and fluorescent master mix in a ratio of 8:1:1. Samples were denatured at 95°C for 5 minutes. Samples, antibody diluent, primary antibody, secondary antibody and luminol-peroxide mix were loaded into the appropriate wells. Also loaded were a biotinylated ladder, antibody diluent, streptavidin-HRP and luminol peroxide. After a short centrifugation of the plate, wash buffer was added. The plate was then placed in the WES machine, whereby loading separation matrix was drawn up the capillaries and stacking matrix was drawn into each capillary. The ladder and samples were then loaded and placed in running buffer. An electric current was applied to the samples and they were separated based on molecular weight. At the end of the separation, the samples were fixed by UV light, and cleavage buffer and non-protein components were removed. Then, the capillaries were drawn up blocking solution, primary and secondary antibody, with washing steps in between. Finally, chemiluminescent substrate was added to the capillaries and a camera detected chemiluminescence levels at 9 different time points (1-512 seconds). The software then calculated the high dynamic range (HDR) of the peaks, which is calculated from a single exposure and provides the maximum dynamic range of signal with the lowest background. The HDR peaks were then used to calculate the area under the curve, which is analogous to the amount of protein in the sample. The antibodies and diluents used are shown in Table 4 below.

[0160] Table 4. Antibody details for simple western analysis.

[0161]

[0162] For the purpose of normalizing the proteins, we used a total protein assay provided by protein simple (DM-TP01). The area under the curve for the protein of interest was divided by the area under the curve for total protein to obtain the normalized relative protein levels.

[0163] Statistical analysis

[0164] Analysis of parvalbumin density in the prefrontal cortex and dorsal hippocampus using repeated measures two-way ANOVA with region and treatment as fixed factors. Subsequent Fisher LSD post-hoc test was performed to find differences between treatment groups within each subregion.

[0165] Analysis of BDNF and synaptic markers using one-way ANOVA followed by planned post-hoc analysis to investigate differences between treatment groups in the prefrontal cortex (BDNF and synaptic markers) and dorsal hippocampus (BDNF), respectively.

[0166] Results

[0167] Effect of 21 days of test article treatment on parvalbumin interneurons in the prefrontal cortex

[0168] In the prefrontal cortex, treatment and region had significant main effects, with no interaction. PV cell density was significantly decreased in the cingulate gyrus and prelimbic cortex of scPCP animals (both p < 0.05 vs 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) (p < 0.01 vs scVeh). Figure 12 .

[0169] One-way ANOVA found a significant main effect of treatment in the whole prefrontal cortex (F(3,26) = 3.053; p = 0.0462). Planned post-hoc analysis found that PV cell density was decreased in scPCP animals (p < 0.05 vs scVeh).

[0170] Long-term treatment with the compound of Formula I at 30 mg / kg significantly increased PV cell density in the prefrontal cortex compared to the scPCP group (p < 0.01) (p < 0.01 vs scVeh). Figure 13 .

[0171] Effect of 21 days of test article treatment on parvalbumin interneurons in the dorsal hippocampus

[0172] In the dorsal hippocampus, treatment and region had significant main effects, with no interaction. Post-hoc analysis showed that there were no significant differences in PV cell density between groups in any region. Figure 14 .

[0173] One-way ANOVA of PV cell density in the whole dorsal hippocampus found no differences between groups (F(3,28) = 2.611, p = 0.0711) Figure 15 .

[0174] Effect of test article treatment for 21 days on BDNF levels in the prefrontal cortex

[0175] One-way ANOVA of BDNF levels in the prefrontal cortex found no difference between groups (F(3,27) = 0.7652, p = 0.5235) Figure 16 ).

[0176] Effect of test article treatment for 21 days on BDNF levels in the dorsal hippocampus

[0177] One-way ANOVA found a trend for a significant main effect of treatment on BDNF levels in the dorsal hippocampus as a whole (F(3,35) = 2.360; p = 0.0882). Planned post-hoc analysis found that BDNF levels were decreased in scPCP animals (p < 0.05 vs scVeh). Figure 17 ).

[0178] Effect of test article treatment for 21 days on synaptic markers in the prefrontal cortex

[0179] SNAP25:

[0180] One-way ANOVA of SNAP25 found a significant main effect of treatment on SNAP25 levels in the prefrontal cortex as a whole (F(3,25) = 4.416; p = 0.0127). Planned post-hoc analysis found that SNAP25 levels were decreased in scPCP animals (p < 0.05 vs scVeh). SNAP25 levels were significantly increased in long-term OT-003 10 mg / kg treatment (p < 0.05), but not in OT-003 30 mg / kg treatment (p = 0.5412) Figure 18 ).

[0181] PSD95:

[0182] One-way ANOVA of PSD95 in the prefrontal cortex as a whole found no significant difference between groups (F(3,28) = 1.827, p = 0.1651) Figure 19 ).

[0183] Conclusion

[0184] This example investigated the effect of treatment with a compound of Formula I (10 and 30 mg / kg, p.o.) for 21 days on post-mortem markers (i.e. BDNF, PVI and synaptic markers) in two different brain regions associated with cognitive and negative symptoms of schizophrenia.

[0185] The results of these studies emphasize that long-term administration of a compound of Formula I can reverse some of the molecular changes underlying the behavioral deficits, in particular the restoration of PVI and SNAP25 in the PFC.

[0186] Thus, chronic administration of a compound of Formula I can be effective in the treatment of the cognitive and negative deficits observed in schizophrenic patients.

[0187] Example 4: Effect of test compounds in reversing subchronic PCP-induced deficits in the attentional set shifting task (ASST)

[0188] In this example, the ability of treatment with a compound of Formula I for 21 days to alleviate disruption of complex cognitive tasks induced by subchronic PCP (scPCP) was tested.

[0189] The effect of scPCP in attentional set shifting is thought to mimic the cognitive deficit symptoms of schizophrenia and we have shown that the atypical antipsychotic drugs clozapine, risperidone and the a7 nicotinic receptor positive allosteric modulator (McLean et al., 2008, 2012) can alleviate this deficit, whereas the typical antipsychotic drug haloperidol does not.

[0190] The attentional set shifting task in perception investigates the ability of rats to learn rules and form a set within the same categorisation class (intra-dimensional shift, IDS, as well as to shift set between different categorisation classes (extra-dimensional shift, EDS; Birrell and Brown, 2000). It represents the rat analogue of the human CANTAB ID / ED task (Downes et al., 1989) in which schizophrenic patients show impaired set shifting and reduced cognitive flexibility (Berg et al., 1948; Pantelis et al., 1999; Tyson et al., 2004).

[0191] Female rats treated subchronically with PCP show a selective deficit in the extra-dimensional shift in this task, which is reversed by treatment with clozapine, risperidone and the a7 nicotinic receptor positive modulator for 7 days, but not by treatment with haloperidol (McLean et al., 2008, 2012).

[0192] Materials and methods

[0193] Set shifting task

[0194] Apparatus:

[0195] The test box is a specially designed cage, measuring 59 cm x 35 cm x 24 cm, made of transparent plastic. A fixed 1 cm thick Plexiglas panel permanently divides 1 / 3 of the length of the box into two separate compartments (choice areas). These two choice areas can be further isolated from each other by means of two movable panels. A digging bowl is placed in each choice area and the rat is given the opportunity to enter by lifting the (one or more) dividers, which can also be used to deny access to the bowl, for example when an incorrect choice is made.

[0196] Acclimatization and training:

[0197] Immediately after the end of the 7-day drug washout period, acclimatization to specific aspects of the test paradigm was started. For three consecutive days prior to training, acclimatization to the test box lasted 1 h. The same two smooth ceramic round digging pots (9 cm in diameter, 5 cm in internal depth) used during the test phase were placed in each cage. Each bowl was filled with 5 grades of sawdust (also covering the floor of the cage) and baited with multiple ¼ honey nut rings (Kellogg's, UK) as food reward.

[0198] During this acclimatization period, the digging bowls were constantly re-baited and left in the cage throughout the study.

[0199] After acclimatization, all rats had to successfully complete the entire training protocol to be tested. First, rats were trained to dig quickly and reliably in both bowls by gradually covering the single food reward with layers of progressively thicker digging medium in each trial. Once the rat repeatedly demonstrated that it had learned the training procedure, the divider was introduced. Digging was defined as a vigorous movement of the forepaws to displace the digging medium and gain access to the food reward buried 2-2.5 cm below the horizontal. If the rat failed to complete the digging within 15 min, training for that session was terminated. The rat was retrained the next day.

[0200] The second phase of training introduced the concept of simple discrimination (SD) between two media. The rats were presented with the same digging bowls, with two different digging media in the bowls, only one of which was baited with food reward. By means of an adjusted pseudo-random Gellerman schedule, the bowls were randomly placed in the left or right compartment. In the first four trials, the rats were allowed to explore both bowls, regardless of which one they dug first, thus having the opportunity to associate the food reward with the positive predictor. Subsequent incorrect choices ended the trial without the opportunity to explore the correct bowl. The criterion for successful learning of each discrimination was 6 consecutive correct trials.

[0201] Test protocol:

[0202] In all cases, rats were subjected to a shift in attentional set 24 hours after training. The test began with the raising of two dividers, allowing the rats to access two digging bowls, only one of which contained the lure. The first phase was simple discrimination, identical to the simple discrimination of the previous day's training session. Testing continued until the rats reached a criterion of six consecutive correct responses.

[0203] In the test session, rats were subjected to a series of discriminations (see Table 5). For complex discrimination (CD), a second dimension (odor) was introduced, but the relevant dimension (digging medium) remained the same. For reversal, the sample and relevant dimension remained the same (medium or odor), but the rats had to recognize that the previous lure dimension was now incorrect, and the other dimension was now correct. New samples were used for ID and ED shifts. For ED shifts, the previously irrelevant dimension (i.e., odor) was now relevant. It has been shown that rats find the difficulty of each dimension change (i.e., medium to odor or odor to medium) to be equivalent. In simple discrimination, the digging medium was chosen as the relevant dimension for all rats.

[0204] Table 5: Overall order of discriminations performed by rats receiving the shift in attentional set task

[0205]

[0206] Summary:

[0207] • In simple discrimination, only one dimension was relevant and introduced (i.e., digging medium; M1 / M2)

[0208] • In complex discrimination, a second dimension was introduced (i.e., odor; O1 / O2), while the relevant dimension remained the same (i.e., digging medium)

[0209] • In reversal 1, the rewarding medium was swapped from M1 to M2.

[0210] • In IDS, new samples were introduced (M3 / M4 / O3 / O4), while the relevant dimension remained the same (i.e., digging medium)

[0211] • In reversal 2, the rewarding medium was swapped from M3 to M4.

[0212] • In EDS, new samples were introduced (M5 / M6 O5 / O6), and the relevant dimension was switched from medium to odor

[0213] • In reversal 3, the rewarding odor was swapped from O5 to O6.

[0214] Odors and mediums used:

[0215] • O1: Salted caramel and vanilla

[0216] • 02: Mango

[0217] • 03: Watermelon

[0218] • 04: White musk

[0219] • 05: Coconut

[0220] • 06: Basil and thyme

[0221] • M1 : Cat litter

[0222] • M2: Paper for bedding

[0223] • M3: Vermiculite

[0224] • M4: Wooden pellets

[0225] • M5: Sawdust

[0226] • M6: Chopped straw.

[0227] Summary of ASST protocol

[0228] Six groups of rats (total 80) were tested in the attentional set shifting task (rats will receive subchronic treatment with PCP, 2 mg / kg, i.p. (n=66) or vehicle, i.p. (n=14), twice daily, followed by at least 7 days of drug-free period). This protocol has been found to produce strong and long-lasting behavioural and pathological deficits in this paradigm (Neill et al., 2010; Cadinu et al., 2017).

[0229] Treatment with test compounds for 21 days:

[0230] Rats were tested in the task after the scPCP treatment protocol and 21 days of treatment with the test substances alone (3, 10 and 30 mg / kg, p.o.), vehicle (5% DMSO in 0.5 methylcellulose 400cp) or the positive experimental control risperidone (0.1 mg / kg, i.p.).

[0231] Statistical analysis

[0232] Data will be expressed as mean ± s.e.m. (mean) and analysed by repeated measures GLM (set shifting phase x treatment group) followed by post-hoc analysis using the least significant difference (LSD) test at each phase.

[0233] Experimental groups

[0234]

[0235] Preparation of samples for pharmacokinetic analysis after ASST study

[0236] Immediately after the ASST test at D21, plasma and brain samples were collected (groups 1-5).

[0237] Trunk blood was obtained and stored in EDTA-coated tubes on ice before centrifugation. Blood was centrifuged at 5,000 RPM for 10 min at a temperature of 4°C. Plasma was transferred to Eppendorf vials (2 ml) and stored at -80°C until analysis. The brain was divided into two hemispheres, one of which (randomly selected) was post-fixed in 4% PFA for 24 hours. The sample was then transferred to 30% sucrose, snap-frozen using isopentane, and stored at -80°C for immunohistochemistry (IHC) analysis. The other hemisphere was stored directly at -80°C for protein analysis (e.g. ELISA and westerns). Three hemispheres (randomly selected) were shipped for PK analysis.

[0238] Results

[0239] Figure 20 The effect of treatment with the compound of Formula I for 21 days on scPCP-induced cognitive deficits in the attentional set shifting task is detailed. Following treatment with scPCP, the number of trials to criterion was significantly increased (P < 0.01) in the extra-dimensional shift (EDS) stage of the attentional set shifting task compared to rats treated with vehicle.

[0240] Following treatment with the compound of Formula I at 30 mg / kg for 21 days, the number of trials to criterion in the EDS stage was significantly decreased (P < 0.05) compared to the scPCP group (P < 0.01), and a near significant decrease was observed in animals treated at 10 mg / kg (P = 0.053).

[0241] No significant decrease was observed in animals treated at 3 mg / kg.

[0242] Risperidone significantly (P < 0.01) reversed the scPCP-induced deficit in the EDS stage of the study.

[0243] Figure 21 The relative protein levels of synaptic markers PSD95 and SNAP25 in the brains of animals following treatment with the compound of Formula I for 21 days are detailed. It can be seen that the compound of Formula I at doses of 3 and 10 mg / kg significantly attenuated the loss of both markers induced by scPCP, but the compound of Formula I at a dose of 30 mg / kg did not.

[0244] Figure 22The concentration of BDNF in the dorsal hippocampus was measured after 21 days of treatment with the compound of formula I at two different protein concentrations. It can be seen that treatment with scPCP did not significantly decrease the levels of BDNF in the rat brain. However, the compound of formula I showed an increase in the levels of BDNF back to the levels of the animals treated with vehicle, thus demonstrating that the treatment was effective in restoring the levels to normal.

[0245] Conclusions

[0246] The data described in this example demonstrate the ability of the compound of formula I to reverse the cognitive deficits in scPCP animals. This cognitive deficit is a model of the cognitive symptoms of schizophrenia, and thus these data provide evidence that the compound of formula I is useful in the treatment of the cognitive symptoms of schizophrenia.

Claims

1. A compound of Formula I for use in the treatment of schizophrenia.

2. The compound of Formula I according to claim 1, wherein the treatment of schizophrenia is the treatment of one or more symptoms associated with schizophrenia.

3. The compound of Formula I according to claim 2, wherein the symptom associated with schizophrenia is a cognitive symptom.

4. The compound of Formula I according to claim 2, wherein the symptom associated with schizophrenia is a negative symptom.

5. The compound of Formula I according to claim 1, wherein the compound of Formula I is administered with one or more pharmaceutically acceptable excipients.

6. The compound of Formula I according to claim 1, wherein the compound of Formula I is formulated into a dosage form selected from a liquid, a lozenge, a rapidly disintegrating tablet, a lyophilized formulation, a film, a spray, an aerosol, a sustained release tablet or capsule, a modified release tablet or capsule, a tablet, a capsule, a cream, an ointment, or a mucoadhesive.

7. The compound of Formula I according to claim 1, wherein the compound of Formula I is administered in a single daily dose.

8. The compound of Formula I according to claim 1, wherein the compound of Formula I is administered in multiple daily doses.

9. The compound of Formula I according to claim 8, wherein the compound of Formula I is administered two, three, four, or five times daily.

10. The compound of Formula I according to any one of claims 7 to 9, wherein each dose comprises at least 0.001 mg of the compound of Formula I.

11. The compound of Formula I according to any one of claims 7 to 9, wherein each dose comprises about 0.001 mg to about 500 mg of the compound of Formula I.

12. The compound of Formula I according to any one of claims 7 to 9, wherein each dose comprises about 500 mg to about 1000 mg of the compound of Formula I.

13. The compound of Formula I according to claim 1, wherein the compound of Formula I is administered with one or more additional pharmaceutical products.

14. A method of treating schizophrenia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula I.

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