PROTEOMIC AND FUNCTIONAL ANALYSIS SUGGESTS THAT 5-HT6 RECEPTORS COMPROMISE COGNITION BY RECRUITING MTOR

Abstracts 1

Departament de Biologia Animal, Facultat de Biologia, Universitat de Barcelona and Institut de Biomedicina de la Universitat de Barcelona (IBUB), Barcelona, Spain; 2Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Barcelona, Spain; 3Centre de Salut Mental de Lleida, Servei de Salut Mental i Addiccions, Hospital Santa Maria, Lleida, Spain; 4 Departament de Psicologia Clínica i de la Salut, Facultat de Psicologia, Universitat Autònoma, Barcelona, Spain; 5Servicio de Psiquiatría, Hospital Virgen Camino, Pamplona, Spain Background: Neurological Soft Signs(NSS) are subtle signs that indicate non-specific cerebral dysfunction(Dazan & Murray, 2002). It has been suggested that NSS might be markers for schizophrenia as studies have reported that NSS are more frequently present in schizophrenic patients compared to healthy controls (Heinrichs & Buchanan, 1988; Dazan & Murray, 2002). Furthermore, it has been demonstrated that neurological soft signs aggregate in relatives of schizophrenic patients (Chen et al., 2000; Egan et al., 2001 Gourion et al., 2003). The aim of this study was to investigate the influence of psychosis proneness factors, such as family history and Schizotypal personality, on the neurological soft signs observed in patients with recent diagnosis of functional psychosis and their first degree relatives. Methods: The sample consisted of 137 individuals from 38 nuclear families (probands with schizophrenic spectrum disorders and nonpsychotic parents and siblings). Patients had a mean age of 24.26 years (SD=3.99), a mean age of onset of 22.14 years (SD=3.92) and a mean duration of illness of 2.42 years (SD=2.11). Neurological Soft Signs were assessed, in all participants by the same rater, by means of the Spanish version of the Neurological Evaluation Scale (Buchanan & Heinrichs, 1989). This scale consists of 3 main subscales: sensory intergration(SI), motor coordination (MC) and sequencing of complex motor acts (SCMA). To test the genetic loading effect, subjects were classified into FH+ (at least one first or second degree relative with functional psychosis) or FH-, using Family Interview for Genetic Studies (FIGS, Maxwell 1992). Schizotypal personality was measured, in all family members, with the Schizotypal Personality Questionnaire-B (SPQ-B) (Raine & Benishay, 1995). This self-reported questionnaire consists of three factors: interpersonal, disorganized and cognitive-perceptual. ANOVA analyses were carried out to investigate differences in NSS between the status groups(patients, parents and siblings). Furthermore, linear regressions were carried out the investigate the influence of SPQ and family history on neurological soft signs. All analyses were corrected for age and gender and performed with SPSS 17.0. Results: The results from the ANOVA´s validated results from previous studies that found differences in NSS between patients, parents and siblings. In all three subscales parents differed significantly from patients (p-values ranging from 0.008 to 0.027). Patients differed from siblings in motor coordination and sequencing of complex motor acts. However, patients and siblings showed similar sensory integration. The regression analysis did not reveal any significant effect for family history, but did show an effect of Schizotypal personality (disorganized factor) on integrative sensory dysfunction in siblings (B = 0.370, SE = 0.140, p = 0.014). In parents and patients no relation was found between the SPQ and NSS. Discussion: These results suggest that overall patients have more NSS compared to their unaffected parents and siblings. However, the unaffected siblings do have equally elevated scores on the sensory integration subscale. This might indicate that this discussion is more related to genetic Background than specifically to schizophrenia. Furthermore, the elevated NSS in siblings may possibly be explained by the positive relation between the disorganized factor of the SPQ and integrative sensory dysfunction. This may suggest that using composite phenotypes is useful in elucidating genetic liability of schizophrenia spectrum disorders (Mechri et al., 2009). doi:10.1016/j.schres.2010.02.665

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Poster 171 ALGORITHM-BASED TREATMENT OF FIRST-EPISODE SCHIZOPHRENIA: RESPONSE RATES OVER THREE SECOND-GENERATION ANTIPSYCHOTIC TRIALS Ofer Agid1, Tamara Arenovich2, Gautam Sajeev2, Robert B. Zipursky3, Shitij Kapur4, George Foussias1, Gary Remington1 1 University of Toronto, Toronto, Ontario, Canada; 2Centre for Addiction and Mental Health, Toronto, Ontario, Canada; 3McMaster University Hamilton, Ontario, Canada; 4Institute of Psychiatry, King's College London, England, United Kingdom Background: Early, effective treatment in first-episode schizophrenia is advocated although evidence based on a systematic approach over multiple antipsychotic trials is lacking. Employing a naturalistic design, we examined response rates over 3 circumscribed antipsychotic trials. Methods: Between June 2003 and December 2008, 244 individuals with first-episode schizophrenia were treated following an algorithm that moved them through 2 antipsychotic trials, followed by clozapine. For the first 2 trials, treatment consisted of risperidone followed by olanzapine, or vice versa; each consisted of 3 stages (low, full, or high-dose) lasting up to 4 weeks at each level and adjusted according to response/tolerability. Clinical response was defined as Clinical Global Impression Inventory-Improvement (CGI-I) = much or very much improved and/or Brief Psychiatric Rating Scale (BPRS) Thought Disorder subscale ≤6. Results: In trial 1, 74.5% of individuals responded with rates higher for olanzapine (82.1%, 115/140) versus risperidone (66.3%, 69/104), p = 0.005. With trial 2, response rate dropped dramatically to 16.6% but again was significantly greater with olanzapine (25.7%, 9/35) compared to risperidone (4.0%, 1/25), p = 0.04. Response rate climbed above 70% once more, specifically 74.5% (21/28), in those individuals who agreed to a third trial with clozapine. Discussion: Results confirm a high response rate (75%) to initial antipsychotic treatment in first-episode schizophrenia. A considerably lower response rate (<20%) occurs with a second antipsychotic trial. Results here were specific to olanzapine and risperdone, suggesting clinical differences (i.e. olanzapine more effective than risperidone). A subsequent trial with clozapine is clearly warranted, although it remains unclear whether outcome would be further enhanced if it was used earlier in the treatment algorithm. doi:10.1016/j.schres.2010.02.666

Poster 172 PROTEOMIC AND FUNCTIONAL ANALYSIS SUGGESTS THAT 5-HT6 RECEPTORS COMPROMISE COGNITION BY RECRUITING MTOR Julie Meffre2, Martial Séveno2, Mark J. Millan1, Clotilde Mannoury La Cour1, Anne Dekeyne1, Joel Bockaert2, Philippe Marin2 1 IDR SERVIER Croissy sur Seine, 78290, France; 2Institut de Génomique Fonctionnelle Montpellier, 34094, France Background: 5-HT6 receptors are mainly located in the CNS, predominantly in regions controlling mnemonic function and mood. Their blockade is associated with pro-cognitive, antidepressant and anxiolytic actions in rodents. Although 5-HT6 receptors signal through Gas-adenylyl cyclase, and possibly fyn, alternative coupling mechanisms remain poorly explored, an issue addressed by a proteomic approach for identifying proteins interacting with 5-HT6 receptors. Methods: Human, hemaglutine (HA)-tagged 5-HT6 receptors were expressed in HEK-293 cells and immunoprecipitated with a HA

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Abstracts

antibody. Co-immunoprecipitated proteins were separated by SDSPAGE, systematically digested by trypsin and generated peptides analyzed by Nano-LC-MS/MS with a Fourier transform tandem mass spectrometer (LTQ Orbitrap XL). Protein identification and validation were performed using Mascot and myProMS softwares, respectively. Activation of the mammalian target of rapamycin (mTOR) pathway was assessed by immunofluorescence staining of brain sections of mice using polyclonal antibodies against activated mTOR (phosphorylated at Ser2448) and the phosphorylated forms of eukaryotic translation initiation factor 4E-binding protein (elF4EBP, Ser65) and ribosomal protein S6 (Ser235/236 and Ser240/244), respectively. In the social recognition procedure, the same juvenile was presented to an adult rat for two consecutive 5-min sessions. Using a procedure of spontaneous loss of recognition, employing an inter-session interval of 120 min, WAY181,187, SB258,585 or scopolamine were administered just after the first session, and the specific mTOR inhibitor, rapamycin injected 30 min earlier. Results: A set of proteins interacting directly and/or indirectly with 5-HT6 receptors was identified. These included three members of the mTOR complex; phosphatidylinositol 3-kinase catalytic subunit type 3, neurofibromin and mTOR itself. This pathway modulates synaptic plasticity and cognition. In mice, the 5-HT6 receptor agonist, WAY181,187 (10.0 mg/kg, i.p., 30 min), increased phosphorylation of mTOR (Ser2448) in a sub-set of neurons of both the striatum and frontal cortex. Correspondingly, WAY181,187 increased phosphorylation of two downstream targets of mTOR, eIF4E-BP, Ser65 and S6 (Ser235/236 and Ser240/244). Phosphorylation of S6 by WAY181,187 was abolished by the 5 HT6 receptor antagonist, SB258,585 (10.0 mg/kg, i.p.), which itself did not affect phosphorylation. Further supporting engagement of the mTOR pathway upon 5-HT6 receptor activation, phosphorylation of S6 by WAY181,187 was markedly reduced by rapamycin (10 mg/kg, i.p.). Interestingly, rapamycin prevented the reduction of short-term memory evoked by WAY181,187 in a social recognition test without influencing the amnesic effect of the muscarinic antagonist, scopolamine. Further, rapamycin was inactive alone and did not blunt pro-cognitive effects of SB258,585. Discussion: Thus, activation of 5 HT6 receptors recruits mTOR signalling in brain including the frontal cortex, the site of modulation of social recognition by 5-HT6 receptors. These studies support the relevance of mTOR and 5-HT6 receptors to the etiology and control of the cognitive deficits of CNS disorders. doi:10.1016/j.schres.2010.02.667

serum and prefrontal cortex have been observed in schizophrenics, and multiple double blind, placebo control studies have demonstrated that d-serine, administered as an adjunct to atypical antipsychotics, improves positive, negative and cognitive symptoms of the disease. Inhibition of d-amino acid oxidase (DAO), a flavoenzyme that oxidatively deaminates small neutral amino acids including d-serine, is an example of an approach to elevate d-serine levels. A recent publication (Sparey et al., Bioorganic + Med Chem Lett (2009) 18(11)3386-91) describes Compound 1 (4H-furo[3,2] pyrrole-5-carboxylic acid), as a potent and selective DAO inhibitor. Methods: Compound 1's ability to inhibit human DAO was assessed both in a cellular assay using HEK293 cells stably expressing DAO, and in a purified enzyme assay, while its selectivity was determined in a cellular assay against the nearest neighbor of DAO, d-aspartate oxidase (DDO), which has 38% homology to DAO. A Biacore assay was utilized to demonstrate binding to the target. Following this determination, Compound 1 was evaluated behaviorally in several preclinical animal models with relevance to schizophrenia. Specifically, Compound 1's ability to attenuate an NMDA antagonist induced deficit in prepulse inhibition was determined, followed by characterization in both the rat and mouse conditioned avoidance response assays, evaluation of its ability to antagonize apomorphine induced climbing and stereotypy, and an assessment of its ability to reverse an NMDA antagonist induced deficit in social odor recognition. Results: Compound 1 has an IC50 of 33 nM against the purified hDAO enzyme, and an IC50 of 19 nM in a cellular assay, while exhibiting greater then 300 fold selectivity over hDDO, in the cellular assay. A similar potency (Kd = 9 nM) was observed in a Biacore binding assay, which demonstrated the compound's interaction with the target. Compound 1 completely reversed an MK-801 induced deficit in sensory gating as measured in the rat at a dose of 100 mg/kg i.p. suggestive of efficacy versus positive symptoms. In the conditioned avoidance response assay, a model predictive of antipsychotic efficacy, 100 mg/kg was calculated to be an IC50 for decreasing avoidance response in both rats and mice. Profiled in the mouse apomorphine-induced climbing and stereotypy model, Compound 1 (100 mg/kg i.p.) significantly attenuated climbing behavior without an effect on stereotypy, suggesting an atypical like profile, with a preferential effect of mesolimbic versus nigrostriatal dopamine pathways, although this decrease was modest. Finally, Compound 1 was tested in the mouse social odor recognition model, and was able to reverse an MK-801 induced deficit at 10 mg/kg i.p. Discussion: The profile that emerges of Compound 1 in these assays supports the idea of broad spectrum therapeutic utility of a DAO inhibitor for the treatment of schizophrenia.

Poster 173

doi:10.1016/j.schres.2010.02.668

COMPOUND 1, A POTENT AND SELECTIVE DAO INHIBITOR, DEMONSTRATES EFFICACY IN SEVERAL PRECLINICAL ANIMAL MODELS OF SCHIZOPHRENIA

Poster 174

Steven Grauer1, Paul Dollings1, Michael Popiolek2, Mark Pausch1, Joseph Zaccardi3, Julie Brennan1, Rachel Navarra1, Keith Pitts3, Girija Krishnamurthy3, Karen Marquis1, John Butera1, Nicholas Brandon2 1 Pfizer Princeton, NJ, USA; 2Pfizer Groton, CT, USA; 3Pfizer Pearl River, NY, USA Background: Multiple lines of evidence suggest that impaired Nmethyl-D-aspartic acid (NMDA) receptor function contributes to the pathophysiology of schizophrenia, and as such, multiple novel approaches are being pursued to counter this hypo-function. Increasing levels of d-serine, an obligatory co-agonist at the allosteric "Glycine Modulatory Site" (GMS) on the NR1 subunit of the NMDA receptor may have the benefit of avoiding neurotoxicity associated with direct agonists. Decreases in d-serine in the CSF,

A THREE ARM DOSE FINDING STUDY OF LURASIDONE: EFFICACY AND TOLERABILITY DATA Philip D. Harvey1, Mitsukuni Murasaki2, Josephine Cucchiaro3, Masaaki Ogasa3, Antony Loebel3 1 Emory University School of Medicine, Atlanta, GA, USA; 2Institute of CNS Pharmacology, Kanagawa, Japan; 3Dainippon Sumitomo America Fort Lee, NJ, USA Background: Lurasidone is a new atypical antipsychotic agent that has shown superiority to placebo in several registration trials. This compound has also shown a side effect profile notable for minimal metabolic effects and weight gain. Previous short-term, fixed dose studies have not examined daily doses below 40 mg and have been limited to inpatient samples. We report here an 8-week dose-