NEUROPSYCHIATRY DISORDERS

FIELD OF THE INVENTION

[0001 ] The present invention relates to the use of pyrrolidineacetamide analogues in a method of treating dopamine-related neuropsychiatric disorders. "Dopamine-related neuropsychiatric disorders" is meant to refer to neurological disorders or mental illnesses resulting from abnormal dopamine neurotransmission

BACKGROUND OF THE INVENTION

[0002] Dopamine is a catecholamine neurotransmitter that activates the five types of dopamine receptors— Di, D ₂ , D ₃ , D ₄ , and D ₅ and their variants. There are a number of dopaminergic innervated pathways within the brain, including the mesocortical, mesolimbic, nigrostriatal, and tuberoinfundibular. As a neurotransmitter, dopamine functions as a mediator of glutamatergic and GABAergic neurotransmission, and plays important roles in mood, memory, learning, attention, reward, and addiction. Dopamine is also a neurohormone released by the hypothalamus. Its main function as a hormone is to inhibit the release of prolactin from the anterior lobe of the pituitary. Dopamine is found in a wide variety of animals, including both vertebrates and invertebrates.

[0003] Dopamine cannot cross the blood-brain barrier, and is therefore administered, for therapeutic purposes, as the precursor, L-DOPA.

[0004] Dopamine-related neuropsychiatric diseases and disorders, such as schizophrenia, bipolar disorder and autism spectrum disorder (ASD), are complex diseases that frequently affect an individual's performance in many physiological and cognitive functions. Currently, pharmaceutical drugs used to treat these mental and neurological disorders are dopamine D2 receptor antagonists which result in severe adverse side effects. Pharmaceutical agents currently used to treat these disorders are typically agents that help normalize the dopamine dysregulation characterizing these disorders. These include a range of dopamine receptor agonists and/or antagonists. However, complete stimulation or inhibition of these receptors can result in severe side effects such as movement and metabolic disorders.

[0005] Schizophrenia (SZ) is a serious mental disorder affecting 1 % of the population. Currently, there is no cure available for this devastating disorder, and current treatments are inadequate. SZ can manifest in various ways but it is often characterized by the appearance of positive symptoms (hallucinations, delusions, disorganized behaviour) and negative symptoms (alogia, affective blunting, avolition, anhedonia/associality) and cognitive dysfunction (impairment in attention, episodic and working memory, speed of information processing, and executive functioning). Its etiology involves multiple factors such as genetic predisposition, psychosocial factors, alterations in neurotransmitter systems, viral infections, and changes in neuroanatomical structures. Abnormalities in multiple neurotransmitter systems have been implicated in the etiology of schizophrenina, although dopaminergic dysregulation is the most widely accepted hypothesis.

[0006] Autism Spectrum Disorders (ASD) encompasses a wide range of behavioural and cognitive abnormalities which affect children at a young age. Symptoms often include impairments in social behaviour and cognitive function. Currently, there is no cure for ASD, although symptoms can be treated with a wide range of pharmacological agents that affect the dopaminergic system, such as antidepressants and antipsychotic drugs.

[0007] Bipolar disorder (BD) is a mood disorder that is characterized by a cycling between manic and depressive episodes. Features of manic episodes include elevated mood, hyperactivity and grandiose delusions. Antipsychotic drugs that affect dopaminergic neurotransmission, such as olanzapine and risperidone, are commonly prescribed for the treatment of BD.

[0008] Alzheimer's disease (AD) is characterized by the formation of neuronal plaques and tangles, and subsequent neurodegeneration. Patients exhibit severe, irreversible cognitive decline, including memory loss, impaired judgement, and decreased attention-span. Currently there is no cure for AD although some available treatments may slow the neurodegeneration. The dopamine system has been implicated as patients often display psychotic symptoms at later stages, and current treatments for this disorder influence dopamine release.

[0009] Lewy body disease is a type of dementia, which is characterized by the formation of protein aggregates in neuronal cells, resulting in cell loss, including the loss of dopaminergic neurons. Patients often exhibit psychotic-like hallucinations, and cognitive symptoms. There is currently no cure, although this disease can be treated with typical antipsychotic drugs. [0010] Drug Addictive Disorders (DADs) and Behaviour Addictive Disorders (BADs) are characterized by the chronic use of an agent or participation in an activity which may result in the development of tolerance, physical or psychological dependence, and finally behavioral changes to seek out the agent or activity. Addiction to a substance or behaviour is caused by exaggerated dopamine release in the nucleus accumbens in response to the stimulus. Addictive agents and activities include smoking, drug abuse (including but not limited to cocaine, amphetamine, phencyclidine, alcohol, cannabis, methylenedioxymethamphetamine (MDMA)), gambling, food consumption, shopping, sex, and other impulse control disturbances. More treatment options are needed to treat these disorders and facilitate the withdrawal from the addictive agent or behavior. Psychological factors such as impaired judgment and impulsivity are at the core of these disorders.

[001 1] Dopamine D2 receptor blocking agents (antipsychotic drugs) are used as a first measure in schizophrenia therapy; however, both typical D2 receptor blocking agents, e.g. haloperidol, as well as atypical blocking agents, e.g. clozapine, olanzapine, and risperidone, cause serious adverse effects such as extra-pyramidal side effects (movement disorders), weight gain, metabolic adverse effects, and development of type 2 diabetes.

[0012] In view of the foregoing, it would be desirable to develop an alternative treatment for one or more dopamine-related diseases or disorders.

SUMMARY OF THE INVENTION

[0013] It has now been determined that pyrrolidineacetamide analogues are useful in a method of treating a dopamine-related neuropsychiatric disorder in a mammal.

[0014] Accordingly, in one aspect a method of treating a dopamine-related neuropsychiatric disorder in a mammal is provided comprising administering to the mammal a therapeutically effective amount of a pyrrolidineacetamide analogue.

[0015] In another aspect, an article of manufacture is provided comprising packaging material and a composition, wherein the composition comprises a pyrrolidineacetamide analogue and a pharmaceutically acceptable carrier, and the packaging material indicates that the composition is effective to treat a dopamine-related neuropsychiatric disorder. [0016] These and other aspects of the invention are described by reference to the detailed description that follows and the following figures.

BRIEF DESCRIPTION OF THE FIGURES

[0017] FIGURE 1 A/B graphically illustrate prevention of the development of prepulse inhibition (a behavioural abnormality displayed by subjects with schizophrenia) by PAOPA in an amphetamine-induced preclinical animal model of schizophrenia (n=10/group);

[0018] FIGURE 2A/B graphically illustrate prevention and reversal of deficit in social interaction (negative symptom of schizophrenia) in an amphetamine-induced model of schizophrenia;

[0019] FIGURE 3 graphically illustrates prevention of hyperlocomotor activity (positive symptom of schizophrenia) by PAOPA;

[0020] FIGURE 4 A-E graphically illustrate prevention and reversal of deficit in social interaction (negative symptom of schizophrenia) in NMDA receptor blockade (MK801 ) model of schizophrenia;

[0021 ] FIGURE 5 graphically illustrates increased agonist (dopamine) induced- internalization of dopamine D2 receptor by PAOPA (a mechanism by which PAOPA prevents and reverses the schizophrenia-like behavioural abnormalities);

[0022] FIGURE 6 graphically illustrates increased agonist (quinpirole)-induced dopamine D2 receptor internalization by PAOPA; and

[0023] FIGURE 7 illustrates decreased abnormal involuntary movements (AIMS) with administration of PAOPA.

DETAILED DESCRIPTION OF THE INVENTION

[0024] A method of treating a dopamine-related neuropsychiatric disorders in a mammal is provided comprising administering to the mammal a therapeutically effective amount of a pyrrolidineacetamide analogue. [0025] The term "dopamine-related neuropsychiatry disorder" is meant to encompass neurological or mental disorders, e.g. a psychological or behavioural pattern generally associated with subjective distress or disability, associated with dysfunction of dopaminergic neurotransmission. Examples of dopamine-related neuropsychiatric disorders include, but are not limited to, schizophrenia, autism spectrum disorders (ASD), bipolar disorder, Alzheimer's disease, Lewy body disease, and drug and behavioural addictions.

[0026] The term "pyrrolidineacetamide analogue" refers to compounds having the following general formula (1):

wherein Rl , R2, R3, R4, R5, R6, R7 and R8 may be the same or different and may be H; OH; halogen; a -C1-C6 saturated or unsaturated alkyl group optionally substituted with one or more substituents selected from hydroxyl and halogen; Ci-C ₆ alkanol; and Ci-C ₆ alkoxy. An example of such an analogue includes, but is not limited to, 3(R)-[(2(S)- pyrrolidinylcarbonyl)amino]-2-oxo-l -pyrrolidineacetamide (PAOPA).

[0027] Pyrrolidineacetamide analogues in accordance with the invention may be synthesized using well-established techniques.

[0028] The present method encompasses the treatment of dopamine-related neuropsychiatric disorders in a mammal. The terms "treat", "treating" and "treatment" are used broadly herein to denote methods that favourably alter the targeted disorder, including those that moderate or reverse the progression of, reduce the severity of, prevent, or cure the disorder.

[0029] The term "mammal" is used herein to encompass both human and non-human mammals. [0030] In the present method, therapeutically effective dosages of a pyrrolidineacetamide analogue are administered to a mammal to treat a dopamine-related neuropsychiatric disorder. The term "therapeutically effective" as it is used herein with respect to dosages refers to a dosage that is effective to treat a psychological disorder without causing unacceptable adverse side effects. The term "administered" refers to any appropriate means of providing a pyrrolidineacetamide analogue to a mammal, and will depend on the dosage form being used as will be appreciated by one of skill in the art.

[0031] Therapeutically effective dosages according to the method, thus, may be in the range of 0.1-500 mg. The preferable dose is between 1-20 mg. The effective therapeutic dosage of a given pyrrolidineacetamide analogue will vary depending on many factors, including but not limited to, the type of disorder to be treated, the nature and severity of the disorder, the mammal to be treated, the symptoms of the mammal being treated, the compound used for the treatment, and the route of administration.

[0032] Pyrrolidineacetamide analogues may be administered by any number of routes including but not limited to oral, subcutaneous, intravenous, intraperitoneal, intranasal, enteral, topical, sublingual, intramuscular, intra-arterial, intramedullary, intrathecal, inhalation, ocular, transdermal, vaginal or rectal means. These analogues may be administered either alone or in a composition combined with a pharmaceutically acceptable carrier. The expression "pharmaceutically acceptable" means suitable for safe administration to mammals.. The selection of carrier depends on the intended mode of administration. In one embodiment of the invention, the compounds are formulated for administration by infusion or by injection either subcutaneously or intravenously, and are accordingly utilized as aqueous solutions in sterile and pyrogen-free form and optionally buffered or made isotonic. Thus, the compounds may be administered in distilled water or, more desirably, in saline, phosphate-buffered saline or 5% dextrose solution. Compositions for oral administration via tablet, capsule, lozenge, solution or suspension in an aqueous or nonaqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup are prepared using excipients including: sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof, including sodium carboxymethylcellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil and corn oil; polyols such as propylene glycol, glycerine, sorbital, mannitol and polyethylene glycol; agar; alginic acids; water; isotonic saline; and phosphate buffer solutions. Wetting agents, lubricants such as sodium lauryl sulfate, stabilizers, tableting agents, disintegrating agents, anti-oxidants, preservatives, colouring agents and flavouring agents may also be present. In another embodiment, the composition may be formulated for application topically as a cream, lotion or ointment. For such topical application, the composition may include an appropriate base such as a triglyceride base. Such creams, lotions and ointments may also contain a surface active agent and other cosmetic additives such as skin softeners and the like, as well as fragrance. Aerosol formulations, for example for nasal delivery, may also be prepared in which suitable propellant carriers are used. Compositions for mucosal administration are also encompassed, including but not limited to, oral, nasal, rectal or vaginal administration. Such compositions generally include one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax, a salicylate or other suitable carriers. Other carriers may also be added to the composition regardless of how it is to be administered which, for example, may aid to extend the shelf-life thereof.

[0033] In accordance with the present method, a pyrrolidineacetamide analogue compound may be administered by any of a number of routes including but not limited to oral, subcutaneous, intravenous, intraperitoneal, intranasal, enteral, topical, sublingual, intramuscular, intra-arterial, intramedullary, intrathecal, inhalation, ocular, transdermal, vaginal or rectal means.

[0034] While not wishing to be limited to a particular mode of action, the present pyrrolidineacetamide compounds have been found to bind to the dopamine D2 receptor at an allosteric site rather than at the orthosteric site, or dopamine-binding site. This results in an increase in dopamine binding to the D2 receptor and maintains the receptor in an active state. In disease states with decreased dopaminergic neurotransmission, pyrrolidineacetamide analogues increase dopamine neurotransmission by increase dopamine D2 receptor stimulation, normalizing dopaminergic signalling. Conversely, in disease states with excessive dopaminergic neurotransmission, pyrrolidineacetamide analogues can attenuate dopaminergic signalling via the following mechanisms. The increase in the number of postsynaptic receptors in the active state induced by treatment with the present compounds causes internalization and down-regulation of the receptors, effectively decreasing the number of receptors available to transmit dopamine signals which compensates for the increase in dopamine. Furthermore, the increase in activation of pre-synaptic receptors induced by treatment limits dopamine synthesis and release, effectively decreasing the amount of dopaminergic neurotransmission. See data below for examples of the use pyrrolidineacetamide analogues to normalize dopamine-associated behavioural abnormalities in preclinical animal models.

[0035] The use of the present pyrrolidineacetamide analogues to treat neuropsychiatric disorders provide numerous advantages over current treatments. At the outset, due to the allosteric nature of the present compounds, the occurrence of adverse side effects, such as those that commonly occur in current treatments, e.g. extrapyramidal symptoms (EPS), drug- induced tardive dyskinesia, Parkinsonian-like symptoms, prolactin secretion, weight gain, elevated blood glucose, elevated blood insulin, increased triglyceride levels, and an increased risk of developing diabetes mellitus and heart disease, is minimized. In addition, due to the allosteric mode of action, the effect of the present compounds is receptor-limited minimizing the toxicity thereof. Further, the present compounds have been determined to be soluble in body fluids such as blood and cerebral spinal fluid, and thus, are effectively absorbed, distributed throughout the body on administration and eliminated. Their solubility also results in low effective doses, and lower production costs.

[0036] In another aspect of the present invention, an article of manufacture is provided. The article comprises packaging material and a composition. The composition comprises a pyrrolidineacetamide analogue and a pharmaceutically acceptable carrier. The packaging material includes an indication that the composition is effective to treat a dopamine-related psychiatric disorder.

[0037] Embodiments of the invention are described by reference to the following specific examples which are not to be construed as limiting.

Example 1 - Treatment of Schizophrenia-like abnormalities

[0038] Male Sprague-Dawley rats weighing 250-300g (4 groups, n=20/group) were employed in this experimental work. All experiments were performed in accordance with the Canadian Council for Animal Care guide lines. Rats were housed individually in standard cages on 12-hour light-dark cycle in a room maintained at 22°C with 50% humidity with access to food and water ad libitum. [0039] Drug administration schedule: D-amphetamine was purchased from Sigma chemicals under the license issued by Health Canada, use of controlled substance for D- amphetamine research. All other chemicals were purchased from Sigma Chemicals. Amphetamine was injected according to the protocol described by Tenn et al., 2003 (Tenn et al, 2003). Prior to testing, animals were randomly divided into the following four groups (n=20/group) for drug sensitization: Saline, Amphetamine, PAOPA, and Amphetamine+PAOPA. General health and weight gain were monitored daily. Amphetamine was prepared as follows: 1 mg/mL for week one, 2 mg/mL for week two, 3 mg/mL for week three. PAOPA was synthesized by Dr. Rodney Johnson at the University of Minnesota, as previously described by Yu et al. (Yu et al, 1988) and prepared as a 1 mg/mL solution each week. For the Amphetamine+PAOPA group, escalating doses of D-amphetamine were prepared with 1 mg/mL PAOPA. Rats received 3 LP. injections each week (Monday, Wednesday, Friday) over 3 weeks, for a total of 9 injections. Saline rats were administered 0.9% saline (1 mL/kg). Amphetamine rats were administered escalating doses of D- amphetamine (1 mg/kg for week one, 2 mg/kg for week two, 3 mg/kg for week 3). PAOPA rats were administered 1 mg/kg of PAOPA. Amphetamine+PAOPA rats were administered the same doses of D-amphetamine as Amphetamine rats, as well as a 1 mg/kg dose of PAOPA. A 3 week drug withdrawal period followed the injections, during which the rats were not handled.

Prepulse inhibition test

[0040] PPI of the acoustic startle response refers to the reduction of a startle-eliciting "pulse" stimulus when it is shortly preceded by a weak "prepulse" stimulus. PPI is an unlearned phenomenon and can be shown using almost identical techniques and parameters, with similar sensitivity to stimulus parameters in mice, rats and humans (Tenn et al, 2003).

[0041 ] Startle responses were assessed using the SR-Lab Startle Response System (San Diego Instruments, and prepulse intensities were adapted from Tenn et al. (Tenn et al, 2003;Tenn et al, 2005). Briefly, startle reflexes were measured in four identical startle chambers. Each lighted, ventilated, sound-attenuated chamber contained a stabilimeter consisting of a non-restrictive Plexiglas cylinder mounted on a Plexiglas platform. Cylinder movements were detected by a piezoelectric accelerometer mounted under the Plexiglas platform and were digitized and stored by the interfacing computer assembly. Background noise, prepulse stimuli, and startle stimuli were provided by a speaker mounted above the animal. Movements were sampled each ms and startle amplitude was characterized as the peak accelerometer voltage that occurred during the first 100 ms after the onset of the startle stimulus. Stimuli presentation and data acquisition were controlled by a computer using SR- Lab software.

[0042] PPI testing began by bringing the animals to the testing room 1 hour before testing began. After this transition time, each rat was placed in the startle apparatus for a 5 min acclimatization period, with a 64 dB white noise present in the background. After this period, each mouse was presented with a series of five startle pulse-alone (HOdB) trials. This series of stimuli was followed by 64 randomized trials consisting of no pulse (0 dB, background noise present), a startle pulse (1 10 dB, 40 ms) or one of three prepulse intensities (67, 70, and 73 dB, 20 ms) presented alone or 100 ms preceding the startle pulse. Lastly, another series of five startle pulse-alone trials was presented. Intertrial times ranged from 10 to 20s to average 15s.

[0043] Results displayed in Figures 1A/B clearly show prevention and reversal of prepulse inhibition by PAOPA, suggesting PAOPA's usefulness and its therapeutic effect for the treatment of deficit in prepulse inhibition in patients with schizophrenia.

Locomotor activity test

[0044] Hyperlocomotor activity in rodents reflects positive symptoms of schizophrenia. Therefore, the effect of PAOPA was tested to determine its effectiveness in preventing positive symptoms induced by amphetamine.

[0045] All locomotor tests were performed as described by Dyck et al., 2009 during the dark period of the light/dark cycle since rats show maximum activity during these hours. AccuScan computerized cages (AccuScan Instruments, Columbus, OH) were utilized and multidirectional movements were recorded by a computerized system. A 15 min habituation period was allowed before beginning of recording. Total locomotor activity was monitored for a 30 min period.

[0046] Results displayed in Figure 3 show prevention of hyperlocomotor activity in an amphetamine-induced model, suggesting PAOPA's usefulness and therapeutic effect in the treatment of positive symptoms of schizophrenia. Social interaction test

[0047] Social withdrawal in rodents represents negative symptoms of schizophrenia. Therefore, this test was performed to determine the effectiveness of PAOPA in preventing and reversing the social withdrawal following amphetamine injections.

[0048] The general design was adopted from that described in J Neurosci Methods 2: pp 219-238. Behaviour of the rats was monitored and recorded in an adjacent room via an overhead-mounted video camera in the observatory room. The room was lit with a 30W bulb and diffused to prevent shadows in the test arena. Two days before testing, the rats were familiarized with the arena. Each subject was given two 5 min trials to explore the apparatus. Between each test, the apparatus was wiped clean with the same cleaning solution.

[0049] The day before testing, rats were allocated to test partners on the basis of knockout, wild type or heterozygous genotype, with body weight differences between the two partners kept within lOg. Social behaviour was tested for a 5 min period, with no acclimation time allotted. The time spent in social interaction was recorded [in milliseconds (ms)], with interaction including sniffing, following, crawling under or over, grooming, and aggressive behaviour. Members of each pair were not familiar with each other, with each pair only used once per test.

[0050] Results displayed in Figure 2A/B show prevention and reversal of social withdrawal in amphetamine induced pre-clinical model of schizophrenia, suggesting the usefulness of PAOPA in the treatment of negative symptoms of schizophrenia.

Example 2 - Treatment in NMD A receptor blockade model of Schizophrenia

[0051 ] Antagonism of N-methyl D-Aspartate (NMDA) receptor by MK801 ((+)-5- methyl-10,1 l -dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate salt) results in development of social withdrawal in rodents representing negative symptoms of schizophrenia. Therefore, experiments were performed to determine whether PAOPA is effective in treating this behavioural abnormality in rodents.

[0052] Animals: Male Sprague Dawley rats (225-272g, Charles River Canada, St. Constant, QC, Canada) were used in accordance with the Canadian Council for Animal Care guidelines. Animals were housed individually in standard cages on a 12 hour light cycle in a room maintained at 22°C with 50% humidity with access to food and water ad libitum. [0053] Drugs and Administration schedule: (+)-MK-801 was purchased from Sigma- Aldrich (Oakville, ON, Canada). PAOPA was synthesized at the University of Minnesota as previously described (Yu et al, 1988). All drugs were dissolved in 0.9% saline. MK801 was injected at 0.5 mg/kg and PAOPA was injected at 1 mg kg. Four groups of rats were utilized and received daily injections intraperitoneally (LP.) for 7 days as follows: Group A (n=10) served as a control and was injected with saline; Group B (n=10) received MK801 ; Group C (n=10) received PAOPA; and Group D (n=10) received PAOPA followed 30 minutes later by MK801.

Determination of deficit in social interaction

[0054] Deficit in social interaction or social withdrawal represents negative symptoms of schizophrenia. PCP treatment has been shown to induce social withdrawal both in humans and rats, and prevention and reversal of deficit in social interaction was performed as previously described (Dyck et al, 2007;Dyck et al, 2009). This was measured in a 100cm x 100cm x 40cm open arena and recorded in an adjacent room via a video camera mounted on the ceiling of the room. Observations were analyzed by an experimenter unaware of the experimental treatments. The time spent in social interaction was scored for 5 minutes and separated into non-aggressive sniffing, following and grooming the partner, social play, and aggressive behaviour (kicking, boxing, aggressive grooming, biting). No two rats were paired together more than once.

[0055] The results displayed in Figures 4 A-E clearly show prevention of the development of social withdrawal by PAOPA in the NMDA receptor blockade, as well as implicating the validity of PAOPA across models. The present results, thus, indicate that PAOPA is useful to treat social withdrawal in neuropsychiatric disorders such as Alzheimer's, autism and bipolar disorder. These results also indicate that PAOPA is capable of preventing or treating negative symptoms developed either due to abnormal dopaminergic or glutamatergic neurotransmission. In the striatum, both dopaminergic and glutamatergic neurons converge on GABAergic neurons, therefore PAOPA influences functional connectivity of these two neurotransmitter pathways which are implicated in schizophrenia.

Example 3 - Dopamine D2 receptor internalization experiments in cellular model

[0056] The mechanism of action that PAOPA utilizes to normalize the hyperdopaminergic state has been investigated using a robust cellular model. As mentioned previously, PAOPA causes an increase in the number of dopamine D2 receptors in the active state. Thus, it was hypothesized that such an increase in receptor activation would result in dopamine D2 receptor internalization as a compensatory mechanism, thereby normalizing the hyperdopaminergic state of schizophrenia. To test this hypothesis, human embryonic kidney (HEK293) cells were stably transfected with the DA D2 receptor, and the machinery required for internalization of the overexpressed receptor, including G-protein coupled receptor kinase-2 (GRK2), and arrestin-3.

Expression plasmid constructs and drug treatment for receptor internalization

[0057] A tetracycline-regulated expression line of HEK293 (T-Rex-293) cells was grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine calf serum and 5mg/mL blasticidin. Cells were incubated at 37°C and 5% C0 ₂ and passaged weekly. Bovine GRK2 was subcloned into pCDNA4/TO (Zeocin-resistant), rat arrestin 3 was subcloned into pcDNA5/TO (hygromycin-resistant) and dopamine D2S receptor cDNA with a FLAG epitope at the amino terminus was fused into pCIN4 (G418-resistant) with an enhanced yellow fluorescence protein (eYFP) tag. The constructs were serially transfected into the T-Rex-293 cells with Lipofectamine and resistant colonies were selected.

[0058] T-Rex-293 cells triply transfected with bovine GRK2, rat arrestin 3 and human D2S receptor were seeded onto poly (D)-lysine coated 24 well plates at a density of 2X10 ⁵ cells per well, and induced overnight with tetracycline (1 μg/mL). The following day, cells were incubated in the presence of DMEM with 0.2 mM sodium metabisulfite (assay buffer). Wells received either assay buffer only (control) or assay buffer with drug treatment. Treatments included just agonist (30 μΜ quinpirole or 10 μΜ dopamine) or agonist and PAOPA (1 μΜ or 10 μΜ), for 1 .5 hrs at 37° C.

[ ³ H]-sulpiride assays

[0059] [ ³ H]-sulpiride is a highly hydrophilic compound that only radioactively tags receptor remaining on the cell membrane, thus allowing for a quantification of percent receptor internalization. Plates of drug-treated cells were quickly cooled on ice and washed three times with ice-cold Earle's Balanced Salt Solution (EBSS). The cells were then incubated with 6nM [ ³ H]-sulpiride at 4°C for 3.5 hrs. Non-specific binding was determined in the presence of ImM cold sulpiride. Cells were washed three times with ice-cold EBSS to removed unbound radioactivity. Cells were lifted by adding 1 % Triton-X 100 with 5mM EDTA, and placed into a scintillation vial with scintillation cocktail. Radioactivity counts were measured 24 hours later.

[0060] Results displayed in Figures 5 and 6 clearly show that PAOPA significantly increased agonist-induced dopamine D2 receptor internalization. Co-treatment of cells with PAOPA and dopamine D2 agonist (quinpirole or dopamine) leads to increased receptor internalization in comparison to just agonist treatment. Thus, these results suggest PAOPA's mechanism of action to include increased receptor internalization, and implicates its usefulness as a therapeutic agent for the hyperdopaminergic-related disorders.

Example 4 - Effect of PAOPA on L-DOPA induced dyskinesia

[0061] L-DOPA induced abnormal involuntary movements (AIMs) are the common adverse effects in PD patients treated with L-DOPA. Although the mechanisms of L-DOPA induced dyskinesias remains unknown, supersensitization of striatal dopamine receptors have been implicated. It was investigated whether or not PAOPA could block the development of AIMS.

[0062] AIMS were induced in 6-OHDA lesioned Sprague Dawley rats, by injecting (LP.) L-DOPA methyl ester 6 mg/kg combined with peripheral DOPA-decarboxylase inhibitor, benserazide (15 mg/kg, Sigma) for 3 weeks. The AIMS were evaluated by previously published rating scales, (Lundblad et al., 2002). AIMS were classified according to topographic distribution, forelimb, orolingual, axial, and locomotive behaviour. Forelimb and orolingual dyskinesias occur predominantly and hyperkinesias, and the axial dyskinesia occurs predominantly as dystonia. Locomotor dyskinesia was expressed as circling movements away from the lesioned side. Scores from 0 to 4 were used to indicate the severity of AIMS subtypes (0: absent, 1 : occasional, 2: present less than 50% of the time, 3: continuous but interrupted by strong sensory stimuli, and 4: continuous).

[0063] The results displayed in Figure 7 shows the blockade of the development of L- DOPA induced abnormal involuntary movements by PAOPA, suggesting PAOPA's usefulness and therapeutic effects for the treatment of L-DOPA induced dyskinesias. Distribution of PAOPA in Various Brain Regions

[0064] In order to determine the relative distribution of PAOPA, [ ³ H] -labeled PAOPA was injected into rats and the radioactivity was determined by the scintillation counter. Results as set out below in Table 1 clearly demonstrate that the highest concentrations of PAOPA enter the striatum, the region which is involved in movement and mental disorders. Similarly, the prefrontal cortex displayed the next highest concentrations of PAOPA, a region which is involved in cognitive functions. These data provide further evidence for the functional effects of PAOPA in neurological and mental disorders.

Table 1 : Distribution of [ ³ H] PAOPA upon intraperitoneal administration ( 100,000 DPMs were injected).

Striatum > Frontal Cortex > Hypothalamus > Cerebellum

Toxicological Studies Reveal No Toxic Effects of PAOPA

[0065] To determine whether PAOPA causes toxic effects in preclinical models, PAOPA was injected into rats at various doses for 28 days. The results set out below in Table 2 clearly show no toxic effects and animals did not display any abnormal movements or cognitive behaviours.

Table 2: Drug Toxicity Profile of PAOPA in Preclinical Animal Model - Lack of Extrapyrimidal Side Effects

Each group consisted n=20 rats group; except lOOmg/kg (n=5) Effects of PAOPA on metabolic parameters

[0066] In order to establish whether PAOPA induces any adverse effects on glucose and insulin, its effects were compared with the commonly prescribed antipsychotic drug, olanzapine. Results set out in Table 2 above show no adverse effects on glucose and insulin levels.

Reference List

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Dyck BA, Skoblenick K J, Castellano J M, Ki K, Thomas N and Mishra R K (2009) Behavioral Abnormalities in Synapsin II Knockout Mice Implicate a Causal Factor in Schizophrenia. Synapse 63: pp 662-672.

Lundblad M, Andersson M, Winkler C, Kirik D, Wierup N and Cenci M A (2002) Pharmacological Validation of Behavioural Measures of Akinesia and Dyskinesia in a Rat Model of Parkinson's Disease. Eur J Neurosci 15: pp 120-132.

Tenn CC, Fletcher P J and Kapur S (2003) Amphetamine-Sensitized Animals Show a Sensorimotor Gating and Neurochemical Abnormality Similar to That of Schizophrenia. Schizophr Res 64: pp 103- 1 14.

Tenn CC, Kapur S and Fletcher P J (2005) Sensitization to Amphetamine, but Not Phencyclidine, Disrupts Prepulse Inhibition and Latent Inhibition. Psychopharmacology (Berl) 180: pp 366-376.

Yu KL, Rajakumar G, Srivastava L K, Mishra R K and Johnson R L (1988) Dopamine Receptor Modulation by Conformational ly Constrained Analogues of Pro-Leu-Gly-NH2. JMe Chem 31 : pp 1430-1436.

The relevant portions of references referred to herein are incorporated by reference.