AZD8529, a positive allosteric modulator at the mGluR2 receptor, does not improve symptoms in schizophrenia: A proof of principle study

SCHRES-06708; No of Pages 6 Schizophrenia Research xxx (2015) xxx–xxx

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AZD8529, a positive allosteric modulator at the mGluR2 receptor, does not improve symptoms in schizophrenia: A proof of principle study Robert E. Litman a,b, Mark A. Smith c,d, James J. Doherty c,e, Alan Cross c, Shane Raines c,f, Lev Gertsik g, Stephen R. Zukin c,h,i a

CBH Health, LLC, 9210 Corporate Blvd., Suite 110, Rockville, MD, United States Georgetown University, 3700 O St. NW, Washington, DC 20057, United States AstraZeneca Pharmaceuticals, 1800 Concord Pike, Wilmington, DE 19542, United States d Teva Pharmaceuticals, 41 Moores Rd., Frazer, PA 19355, United States e Sage Therapeutics, 215 First St., Cambridge, MA 02142, United States f 2b Analytics, LLC, 128 Westminster Dr., Wallingford, PA 19086, United States g California Clinical Trials Medical Group, 1560 East Chevy Chase Dr., Suite 140, Glendale, CA 91206, United States h Forest Research Institute, Jersey City, NJ, United States i Haborside Financial Center, Plaza V, Jersey City, NJ 07311, United States b c

a r t i c l e

i n f o

Article history: Received 28 October 2015 Received in revised form 27 January 2016 Accepted 1 February 2016 Available online xxxx

a b s t r a c t Introduction: Activation of metabotropic glutamate (mGluR2/3) receptors has been proposed as an alternative mechanism to dopaminergic-based antipsychotics to correct glutamatergic deficits hypothesized to underlie schizophrenia symptoms. This study investigates the efficacy and safety of AZD8529, a selective positive allosteric modulator (PAM) at the mGlu2 receptor, in symptomatic patients with schizophrenia. Methods: Patients were randomized to receive AZD8529 40 mg, risperidone 4 mg, or placebo as monotherapy. Treatment lasted for 28 days, and clinical efficacy was assessed using Positive and Negative Syndrome Scale (PANSS) and Clinical Global Impression (CGI) scores. Results: There were no significant differences between patients treated with AZD8529 versus placebo in change from baseline to endpoint in PANSS total, negative and positive symptom subscale, or CGI-S scores. In contrast, risperidone demonstrated significant efficacy relative to placebo. Conclusion: These results do not support a role for the mGluR-2 PAM AZD8529 as an antipsychotic and indicate that positive modulation of mGluR type 2 receptors alone is not sufficient for antipsychotic effects in acutely ill schizophrenia patients. © 2015 Published by Elsevier B.V.

1. Introduction Abnormal glutamatergic neurotransmission has been proposed as an important component of schizophrenia pathophysiology (Coyle, 2006; Lin et al., 2012; Javitt and Zukin, 1991). This hypothesis is based primarily on clinical evidence that antagonists of ionotropic, NMethyl-D-Aspartate (NMDA) glutamate receptors, such as phencyclidine (PCP) or ketamine, produce schizophrenia-like positive and negative symptoms in healthy volunteers, and exacerbate those symptoms in schizophrenia patients (Conn et al., 2009; Coyle et al., 2010; Moreno et al., 2009). Pre-clinical evidence suggests that the psychotomimetic effects of NMDA receptor antagonists such as PCP and ketamine may be due to disinhibition of neural circuits leading to increased excitatory glutamatergic activity in the prefrontal cortex (PFC) and increased extracellular release of glutamate. Combined with data from human neuroimaging and psychometrics, this evidence has led to the hypothesis that the effect of primary hypo-function in NMDA receptors on prefrontal cortical inhibitory neurons and the

imbalance of excitatory/inhibitory activity may contribute to core symptoms of schizophrenia. In addition to activity at ionotropic (e.g., NMDA) receptors, glutamate also acts at eight separate subtypes of G-protein coupled metabotropic glutamate receptors (mGluRs), whose activation modulates glutamatergic neurotransmission by regulating neuronal excitability, synaptic plasticity, and neurotransmitter release. Preclinical studies in rodents and non-human primates suggest that activation by glutamate or glutamate agonists of the mGluR receptor subtypes 2 and 3 reverses hypofunction of the NMDA receptor induced by PCP or ketamine (Conn et al., 2009; Coyle et al., 2010; Moreno et al., 2009; Vinson and Conn, 2012; Chaki, 2010). LY404039, an agonist of both mGluR2 and mGluR3 receptors, reverses behavioral effects induced by NMDA antagonists PCP and ketamine in animal models of psychosis, and also decreases glutamate release and excitatory synaptic neurotransmission (Vinson and Conn, 2012; Chaki, 2010). In addition, another agonist of both mGluR2 and mGluR3 receptors, LY354740, reverses some ketamine-induced cognitive deficits in non-human primates and in

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Please cite this article as: Litman, R.E., et al., AZD8529, a positive allosteric modulator at the mGluR2 receptor, does not improve symptoms in schizophrenia: A proof of principle ..., Schizophr. Res. (2015), http://dx.doi.org/10.1016/j.schres.2016.02.001

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R.E. Litman et al. / Schizophrenia Research xxx (2015) xxx–xxx

human volunteers, suggesting a potential therapeutic effect on cognitive and negative symptoms of schizophrenia (Krystal et al., 2005). Interestingly, an initial proof of concept study in schizophrenia patients showed that pomaglumetad methionil, or LY2140023, a pro-drug for the direct agonist for LY404039, significantly decreased both positive and negative symptoms when compared to placebo, without extrapyramidal symptoms or prolactin elevation (Patil et al., 2007). However, a subsequent double blind, placebo and olanzapine controlled Phase 2 study with LY2140023 failed to replicate these findings due to lack of statistical separation from placebo for both the treatment and active control arms (Kinon et al., 2011). In addition, LY2140023 showed no significant benefits in improving negative symptoms when administered as adjunctive treatment to second generation atypical antipsychotics (Stauffer et al., 2013). In the current study, we investigate the efficacy, tolerability and cognitive effects of AZD8529 (7-methyl-5-(3-piperazin-1-ylmethyl-[1,2,4] oxadiazol-5-yl)-2-(4-trifluoromethoxybenzyl)-2,3-dihydroisoindol-1one methanesulfonate), a selective positive allosteric modulator (PAM) of the mGlu2 receptor, in symptomatic patients with schizophrenia. Unlike direct orthosteric agonists such as LY404039, PAMs bind the receptor at a site which is topographically distinct and thereby enhance activation of the receptor by orthosteric agonists and endogenous glutamate. At excitatory synapses, this allows for potentiation of receptor activation in an activity-dependent manner, which may maintain efficacy while avoiding receptor desensitization and reducing side effects. In addition, AZD8529 is selective for the mGluR2 receptor (Cross, 2013), whose activation appears to be more significant than mGluR3 receptor activation in mediating antipsychotic effects of the mGluR2/3 agonists in mouse models (Moreno et al., 2009; Vinson and Conn, 2012). The pharmacokinetic properties of AZD8529 are sufficient to test the hypothesis that mGluR2 PAMs are efficacious in schizophrenia. Preclinical studies demonstrated that at a plasma concentration of 100 nM, AZD8529 completely inhibited PCP-induced glutamate release. Studies in normal volunteers indicated that AZD8529 is readily absorbed after oral administration. It has a relatively long plasma half-life of approximately 60 h. Although it is a weak substrate for p-glycoprotein transport, measurements in man indicated that CSF levels were 50% of the free plasma concentrations. In addition, AZD8529 was very well tolerated in normal volunteers with no dose-related adverse events (AstraZeneca, data on file). 2. Methods This was a double blind, double-dummy, placebo controlled, risperidone referenced, parallel groups, proof of concept clinical trial conducted in accordance with the Declaration of Helsinki at 3 US sites. IRB-approved informed consent was obtained from all subjects. Male and female inpatients and outpatients between the ages of 18 and 65, who met criteria for schizophrenia (paranoid, undifferentiated, disorganized, catatonic or residual subtypes) based on clinical psychiatric history and the Structured Clinical Interview for DSM Disorders (SCID) interview (Koback et al., 2008), had a Positive and Negative Syndrome Scale (PANSS) (Kay et al., 1987) total score of ≥70, were medically stable, had a history of clinically significant response to prior neuroleptic treatment, had no history of intolerance to olanzapine therapy, and did not meet criteria for substance abuse or substance dependence were eligible for inclusion. Female patients were either surgically sterile or post-menopausal. Eligibility for study was determined over a 28-day screening period, during which previous non-neuroleptic psychotropic pharmacotherapy was discontinued for at least two weeks. During the final phase of the screening period, patients were admitted to the clinical research unit for an inpatient washout period of a maximum of 7 days (for those patients with no prior antipsychotic therapy, washout period was 3 days). This inpatient period was also utilized as a placebo run-in period, during which patients were administered placebo to match AZD8529 in the

morning and placebo to match risperidone in the evening on a daily basis. On Day 1, patients were randomly assigned to one of three groups. Patients assigned to the active experimental drug group received an orally administered 40 mg AZD8529 capsule every second morning and an AZD8529-matching placebo capsule on alternate mornings; a risperidone-matching placebo capsule was administered each evening. Patients assigned to the placebo group received an AZD8529-matching placebo capsule each morning and a risperidone-matching placebo capsule each evening. Patients assigned to the active comparator group received an AZD8529-matching placebo capsule each morning and a 4 mg risperidone capsule each evening. The dose of 40 mg of AZD8529 every second day was selected based on tolerability data from prior healthy volunteer studies and preclinical toxicology studies indicating acceptable safety margins while maintaining human exposure corresponding to animal exposures that showed efficacy in schizophrenia models (data on file, Astra Zeneca). The rationale for the use of 4 mg of risperidone, used more recently as an active reference in Phase II antipsychotic efficacy trials, is based on evidence of its clinical efficacy without undue risk of unblinding due to extrapyramidal side effects (Patil et al., 2007). Assigned treatment for each subject was determined by a randomization schedule generated by the AstraZeneca Global Randomization (GRand™) group. AZD8529 oral capsules and placebo oral capsules, as well as risperidone and risperidone placebo capsules, were prepared by the site pharmacist and provided to the study site prior to dosing. All study personnel remained blind to the randomization code with the exception of site pharmacy personnel preparing and dispensing the study drug at the site and laboratory personnel analyzing pharmacokinetic samples. Patients received their randomized treatment regimen starting on the morning of Day 1 and continuing for 28 days. During this treatment period, patients abstained from all previous psychoactive treatments, including neuroleptic treatment and non-prescription, herbal or vitamin preparations starting 7 days prior to randomization, and from other psychoactive non-neuroleptic therapy, including any cytochrome P450 3A4 inducers or inhibitors, starting 14 days prior to randomization. Patients were allowed to receive lorazepam for agitation or anxiety (up to 8 mg daily), zolpidem tartrate for insomnia (up to 10 mg daily), and benztropine mesylate for extrapyramidal signs or symptoms (up to 2 mg per 8-hour period). Likewise, patients were allowed up to 3 g of acetaminophen for pain. Patients were continued on their usual doses of non-psychoactive oral medication for stable medical conditions such as essential hypertension, diabetes, or hypothyroidism. Clinical efficacy of AZD8529 was assessed utilizing the PANSS (Kay et al., 1987) and the Clinical Global Impression (CGI; Guy, 1976b) scales. The PANSS is an interview-based instrument designed to measure the severity of psychopathology in adult patients with psychotic disorders, especially schizophrenia, and consists of 30 items divided into three symptom subscales, including the Positive Symptom subscale (7 items), the Negative Symptoms subscale (7 items), and the General Psychopathology subscale (16 items). The PANSS was administered at baseline prior to randomization (Day − 1), and at weekly intervals (Day 8, Day 15, Day 22 and Day 28); all assessments were performed at the same time of day and at least 8 h after any instance of lorazepam administration. The PANSS total score was calculated as the sum of the 30 item scores. Change from baseline values were calculated for the PANSS total, positive subscale, and negative subscale scores. The primary outcome variable was the change from baseline to endpoint (Day 28) in the PANSS total score. The CGI is a 2-part, clinician-administered scale assessing global severity of illness and change (Guy, 1976b). The CGI Severity of Illness scale (CGI-S) measures the severity of illness on a 7-point scale ranging from a minimum score of 1 (“normal, not ill”) to a maximum score of 7 (“among the most extremely ill patients”). The CGI Global Improvement scale (CGI-I) measures improvement post treatment over time on a

Please cite this article as: Litman, R.E., et al., AZD8529, a positive allosteric modulator at the mGluR2 receptor, does not improve symptoms in schizophrenia: A proof of principle ..., Schizophr. Res. (2015), http://dx.doi.org/10.1016/j.schres.2016.02.001

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7-point scale ranging from a minimum score of 1 (“very much improved”) to a maximum score of 7 (“very much worse”). CGI-I scores greater than 4 indicate worsening, while CGI-I scores less than 4 indicate clinical improvement. The CGI was performed at baseline (Day −1) and weekly on the same visit days as the PANSS. Change from baseline values were calculated for CGI-S scores, and dichotomized improvement values (improvement defined as “much” or “very much” improved) at endpoint were calculated for CGI-I scores (Guy, 1976b). Safety was assessed by the severity and incidence of adverse events, as well as the results of vital signs, physical examinations, fasting clinical laboratory parameters and electrocardiograms, done prior to randomization, throughout the treatment period, and at endpoint (Day 28). In addition, assessment of extrapyramidal symptoms was performed using the Simpson Angus Scale (SAS) to assess Parkinson-like extrapyramidal symptoms (EPS) (Simpson and Angus, 1970), the Barnes Akathisia Scale (BAS) to measure akathisia (Barnes, 1989), and the Abnormal Involuntary Movement Scale (AIMS) to measure dyskinetic movements (Guy, 1976a), prior to treatment, throughout the treatment period, and at endpoint. Finally, the CogState (Pietrzak et al., 2009), a computerized neuropsychological test battery, was utilized as for assessing any adverse cognitive effects of AZD8529. The CogState battery was comprised of three tasks to assess psychomotor speed (Detection Task), attention (Identification Task) and working memory (Continuous Paired Associate Learning (CPAL) Task). Subjects performed the CogState four times prior to treatment (once for training purposes and thrice to establish baseline performance), and then once at Day 15 and once at endpoint (Day 28). Timed venous blood sampling was collected pre- and post-dose on Days 1 and 28 and pre-dose on Days 9, 17, 23 and 25 to obtain plasma levels of AZD8529 for calculation of pharmacokinetic measures including maximum plasma concentration (Cmax), time to Cmax (Tmax), the terminal half-life from plasma (T1/2), and the area under the plasmaconcentration time curve from 0 to 24 h (AUC0–24). Plasma concentrations of AZD8529 were measured by a fully validated bioanalytical method (Covance Laboratories, Inc., Madison, WI), using liquid chromatography mass spectrometry (LC MS/MS). The method includes a standard curve ranged from 1.00 to 1000 ng/mL using a plasma sample volume of 0.0500 mL. 2.1. Data analysis A sample size of 50 evaluable subjects per arm was selected as it would provide 80% power (assuming a two-sided alpha level of 0.20) to detect an effect size of 0.435, corresponding to a treatment difference between AZD8529 and placebo of 10 points (SD = 23) in the change from baseline in PANSS total score. A risperidone active control arm was included as a reference for gauging assay sensitivity and was not included in the formal statistical tests. Using a 2:2:1 randomization ratio, 125 evaluable patients were required. Evaluable patients were defined as those that received at least one dose of study medication and had both a baseline and at least one post-baseline efficacy assessment. These patients comprised the full analysis set with their data used for all efficacy analyses. The change from baseline in PANSS total score was analyzed using mixed model repeated measures (MMRM) methods. The model included protocol scheduled assessment, treatment group, and treatment-by-assessment interaction as fixed factors, center as a random factor, and baseline PANSS total score as a covariate. Robust variance estimates for the fixed effects were used for testing treatment differences. Within subject variability was modeled using an unstructured (UN) covariance pattern. The comparison of interest was the difference between the AZD8529 and placebo groups at Day 28. Similar analyses were performed for continuous secondary outcome variables, including change from baseline in PANSS positive, negative, and general psychopathology subscale scores, as well as change from baseline in CGI-S score. For each secondary analysis, the comparison of

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interest was the difference between the AZD8529 and placebo groups at Day 28. Safety and tolerability variables were summarized using descriptive statistics with no formal hypothesis tests. Finally, raw data scores for the CogState Test Battery were analyzed using ANCOVA methods with averaged baseline score as a covariate, session as a fixed factor, and subject as a random factor. 3. Results 3.1. Patient characteristics Out of 152 patients randomized, 8 were screen failed patients who were allocated a randomization number but never received study drug. The remaining 144 patients received at least one dose of study drug and were included in the safety analysis set used to assess safety and tolerability endpoints. Demographic and clinical characteristics, including baseline symptoms ratings, were generally similar across treatment groups (Table 1). Overall, 104 patients (72.2%) completed the 4-week treatment trial with the highest completion rate seen for patients receiving risperidone (83.9%), and the lowest for patients receiving AZD8529 (65.5%). The most common reason for withdrawal was lack of efficacy. Similar rates of withdrawal for this reason were observed in patients treated with placebo (14.5%) and AZD8529 (12.1%); no patient receiving risperidone withdrew due to lack of efficacy. The mean time receiving treatment was 24.6 days for the entire patient sample, with the shortest time (23.6 days) for patients receiving AZD8529 and the longest time for patients receiving risperidone (25.4 days) (Table 1). 3.2. Efficacy Of those patients treated with randomized study drug, 137 had at least one post-baseline efficacy assessment and were included in the full analysis set (FAS) used to assess all efficacy endpoints. There were no significant differences in patients treated with AZD8529 versus placebo on change in PANSS total score, positive score, and negative score, or on change in the CGI-S score (Fig. 1, Table 2). Likewise there were no effects on any cognitive endpoints including working memory. For risperidone-treated patients, improvements in PANSS total, positive subscale, negative subscale, and the CGI-S scores differed significantly from placebo (Table 2). However, there was no effect for on any cognitive endpoints for risperidone-treated patients. Of 15 patients who discontinued early due to lack of therapeutic response, 8 were on placebo and 7 were on AZD8529; none were randomized to risperidone. 3.3. Safety and tolerability Treatment with AZD8529 was generally well tolerated. While the frequency of reported adverse events for AZD8529 was moderately high (62%), most of these were mild to moderate in severity, and similar in frequency and severity to those reported for placebo treated patients (54%). Adverse events occurring more frequently in AZD8529-treated than in placebo-treated patients included headache (12% vs. 10%), akathisia (5% vs 2%), sedation (5% vs 2%), anxiety (5% vs. 2%), and increased appetite (5% vs 0%). Patients treated with risperidone had the highest frequency of adverse events (65%), with the most common being vomiting (16% vs. 9% on placebo), nasal congestion (16% vs. 2% on placebo), and pruritus (13% vs. 2% on placebo). Risperidone patients experienced the highest weight gain (1.5 ± 2.7 kg; mean ± SD) compared to placebo (0.9 ± 4.4 kg), and AZD8529 (0.5 ± 3.8 kg). Extrapyramidal symptoms including Parkinson-like symptoms, akathisia, and dyskinesia were essentially unchanged from baseline to end of treatment for all groups. There were no clinically important changes on safety laboratory measures with the exception of elevated levels of prolactin in risperidone treated patients which averaged 43 ± 5 ng/ml compared to 16 ± 6 in the placebo group and 10 ± 1 in the AZD8529

Please cite this article as: Litman, R.E., et al., AZD8529, a positive allosteric modulator at the mGluR2 receptor, does not improve symptoms in schizophrenia: A proof of principle ..., Schizophr. Res. (2015), http://dx.doi.org/10.1016/j.schres.2016.02.001

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Table 1 Demographics, baseline clinical characteristics, and patient disposition (safety analysis set, N = 144).

Demographics Men, n (%) Age in years, mean (range) Ethnicity, n (%) White Black Asian Other Patient characteristics, baseline PANSS total score, mean (range) PANSS positive subscale score PANSS negative subscale score CGI severity, n (%) Moderately ill Markedly ill Severely ill Patient disposition Patients who received treatment Patients who completed treatment Number of days receiving treatment Patients who discontinued treatment Adverse event Lack of therapeutic response Voluntary discontinuation Severe non-compliance to Protocol

AZD8529 (N = 58)

Placebo (N = 55)

Risperidone (N = 31)

Total (N = 144)

45 (77.6) 38.8 (19–58)

44 (80.0) 36.9 (18–56)

26 (83.9) 39.0 (23–56)

115 (79.9) 38.3 (18–58)

8 (13.8) 43 (74.1) 3 (5.2) 4 (6.9)

7 (12.7) 45 (81.8) 0 (0.0) 3 (5.5)

5 (16.1) 21 (67.7) 3 (9.7) 2 (6.5)

92.9 (71–133) 23.9 (16–36) 23.3 (15–39)

93.6 (72–124) 24.2 (18–38) 23.4 (15–40)

91.0 (76–122) 23.6 (16–34) 22.6 (11–32)

20 (13.9) 109 (75.7) 6 (4.2) 9 (6.3)

92.7 (71–133) 24.0 (16–38) 23.2 (11–40)

30 (51.7) 26 (44.8) 2 (3.4)

26 (47.3) 29 (52.7) 0 (0.0)

15 (48.4) 16 (51.6) 0 (0.0)

71 (49.3) 71 (49.3) 2 (1.4)

58 (100.0) 38 (65.5) 23.6 (7.9) 20 (34.5) 4 (6.9) 7 (12.1) 9 (15.5) 0 (0.0)

55 (100.0) 40 (72.7) 25.1 (6.1) 15 (27.3) 4 (7.3) 8 (14.5) 1 (1.8) 2 (3.6)

31 (100.0) 26 (83.9) 25.4 (6.8) 5 (16.1) 3 (9.7) 0 (0.0) 2 (6.5) 0 (0.0)

144 (100.0) 104 (72.2) 24.6 (7.0) 40 (27.8) 11 (7.6) 15 (10.4) 12 (8.3) 2 (1.4)

group at the end of treatment. There were no adverse effects on cognitive endpoints for any treatment group (Table 3). Plasma exposure of AZD8529 averaged 73 ng/ml at Cmax and 38 ng/ml at Cmin in the current study where 40 mg AZD8529 was administered every other. This is similar to previous Cmax/min and other Pk measures obtained in healthy volunteers (AstraZeneca data on file).

4. Discussion Results of the current clinical trial do not support a role for AZD8529 as an antipsychotic monotherapy for the treatment of acute schizophrenia. AZD8529 was no more efficacious than placebo as measured by changes in PANSS total CGI, and PANSS positive and negative symptom subscale scores. In contrast, risperidone was superior to placebo in all of these measures. These results are also inconsistent with experimental

Fig. 1. Change from baseline in PANSS (Positive and Negative Syndrome Scale) total score across 4-week treatment period — MMRM analysis (full analysis set).

Please cite this article as: Litman, R.E., et al., AZD8529, a positive allosteric modulator at the mGluR2 receptor, does not improve symptoms in schizophrenia: A proof of principle ..., Schizophr. Res. (2015), http://dx.doi.org/10.1016/j.schres.2016.02.001

R.E. Litman et al. / Schizophrenia Research xxx (2015) xxx–xxx Table 2 PANSS total, positive subscale, negative subscale change from baseline to Day 28 — MMRM analysis (full analysis set, N = 137). Efficacy measure PANSS total Change Difference vs. placebo

PANSS positive Change Difference vs. placebo PANSS negative Change Difference vs. placebo CGI-S Change Difference vs. placebo

AZD8529 (N = 54)

Placebo (N = 53)

Risperidone (N = 30)

LS mean (SE) LS mean 95% CI

2.75 (1.51)

0.99 (1.53)

−8.78 (1.18)

p-Value

0.414

LS mean (SE) LS mean 95% CI p-Value

0.32 (0.48)

LS mean (SE) LS mean 95% CI p-Value

0.78 (0.55)

LS mean (SE) LS mean 95% CI p-Value

−0.11 (0.06) −0.02 −0.18, 0.14 0.799

−9.76 −13.59, −5.94 b0.001

1.77 −2.50, 6.03

−0.22 (0.45)

−2.75 (0.53) −2.53 −3.91, −1.14 b0.001

0.54 −0.78, 1.86 0.421 0.33 (0.61)

−2.98 (0.51) −3.31 −4.79, −1.83 b0.001

0.45 −1.15, 2.06 0.578 −0.09 (0.05)

−0.37 (0.08) −0.29 −0.47, −0.11 0.002

Table 3 Adverse events (safety analysis set, N = 144). Common AEs (≥5%)

AZD8529 (N = 58)

Placebo (N = 55)

Risperidone (N = 31)

Total (N = 144)

Patients with any AE, n (%) Headache Dyspepsia Back pain Vomiting Constipation Myalgia Nasal congestion Akathisia Cough Sedation Decreased appetite Nausea Pruritus Hypotension Lethargy Sinus tachycardia Tachycardia Abdominal pain Anxiety Diarrhea Dizziness Insomnia Oropharyngeal pain Weight increased Dystonia Ear pain Increased appetite Somnolence Asthma Extrapyramidal disorder Galactorrhoea Pharyngitis

36 (62.1)

30 (54.5)

20 (64.5)

86 (59.7)

7 (12.1) 5 (8.6) 4 (6.9) 1 (1.7) 3 (5.2) 2 (3.4) 2 (3.4) 3 (5.2) 2 (3.4) 3 (5.2) 3 (5.2) 1 (1.7) 1 (1.7) 2 (3.4) 0 1 (1.7) 3 (5.2) 1 (1.7) 3 (5.2) 0 0 2 (3.4) 3 (5.2) 0 1 (1.7) 0 3 (5.2) 1 (1.7) 0 0

6 (10.9) 6 (10.9) 6 (10.9) 5 (9.1) 5 (9.1) 4 (7.3) 1 (1.8) 1 (1.8) 3 (5.5) 1 (1.8) 3 (5.5) 4 (7.3) 1 (1.8) 0 3 (5.5) 3 (5.5) 2 (3.6) 3 (5.5) 1 (1.8) 2 (3.6) 3 (5.5) 0 0 2 (3.6) 0 1 (1.8) 0 0 0 0

1 (3.2) 1 (3.2) 1 (3.2) 5 (16.1) 2 (6.5) 3 (9.7) 5 (16.1) 3 (9.7) 2 (6.5) 3 (9.7) 0 1 (3.2) 4 (12.9) 3 (9.7) 2 (6.5) 1 (3.2) 0 0 0 2 (6.5) 1 (3.2) 2 (6.5) 1 (3.2) 2 (6.5) 2 (6.5) 2 (6.5) 0 2 (6.5) 2 (6.5) 2 (6.5)

14 (9.7) 12 (8.3) 11 (7.6) 11 (7.6) 10 (6.9) 9 (6.3) 8 (5.6) 7 (4.9) 7 (4.9) 7 (4.9) 6 (4.2) 6 (4.2) 6 (4.2) 5 (3.5) 5 (3.5) 5 (3.5) 5 (3.5) 4 (2.8) 4 (2.8) 4 (2.8) 4 (2.8) 4 (2.8) 4 (2.8) 4 (2.8) 3 (2.1) 3 (2.1) 3 (2.1) 3 (2.1) 2 (1.4) 2 (1.4)

0 0

0 0

2 (6.5) 2 (6.5)

2 (1.4) 2 (1.4)

5

evidence to date supporting a role for mGluR2 receptor activation in antipsychotic therapy, including one clinical trial utilizing a direct orthosteric agonist of mGlu2/3 receptors shown to be efficacious for both positive and negative symptoms in schizophrenia patients (Patil et al., 2007). More recent studies with LY2140023 did not demonstrate significant antipsychotic effects (Kinon et al., 2011; Stauffer et al., 2013; Li et al., 2015). Compared to placebo, AZD8529 was well tolerated by schizophrenia subjects, and while the frequency of adverse events was high, most adverse events were mild in severity, resolved quickly, and were not a reason for early discontinuation of treatment. In contrast, treatment with risperidone was associated with clinically significant levels of akathisia, nasal congestion and vomiting. Also, unlike many antipsychotic agents, AZD8529 had no extrapyramidal motor side effects. No abnormal levels of reproductive hormones or of prolactin were reported in patients receiving AZD8529, whereas risperidone was associated with an increase in prolactin levels. It is possible that the dose of AZD8529, 40 mg every other day, which was selected based on tolerability data from prior healthy volunteer studies and prior preclinical toxicology was too low to provide sufficient activation of the mGluR2 receptor to have a detectable effect on psychotic symptoms. However, average plasma exposure at Cmax was 73 ng/ml or about 140 nM which is consistent with in-vitro effects and greater than the concentrations needed to block PCP-induced glutamate release and MK801 induced disruption of cortical activity in preclinical models (Cross, 2013). It is also possible that despite adequate plasma levels, brain exposure of AZD8529 was too low to activate central mGluR2 receptors. However, evidence from two fMRI studies completed after the current Phase 2 study indicate AZD8529 affected the BOLD signal in brain regions of interest at similar exposures in normal healthy volunteers (Krystal, 2013) and in patients with schizophrenia (Wolf et al., 2013). Although the likelihood of Type II error might have been increased due to the use of a less symptomatic patient sample, (i.e., both outpatients and inpatients on prior neuroleptic treatment could be enrolled as long as the PANSS total score was N70), we observed clinically significant improvements in patients randomized to risperidone suggesting that the patient sample was sensitive enough for the detection of response to an established antipsychotic. Moreover, the baseline PANSS total score observed in this study for all patients randomized was similar to that in previous studies on mGluR2/3 agonist, where a significant benefit was observed. These findings highlight an emerging discrepancy between preclinical and healthy volunteer data (Salih et al., 2015) showing reduced NMDAR activity in models of schizophrenia and the inability of agents designed to correct this imbalance to improve symptoms in patients. Inconsistencies have been noted not just in agents acting at group II mGluRs, but also in compounds acting at the NMDAR coagonist (glycine) site and with indirect NMDA activators including inhibitors of the GlyT1 reuptake transporter (Bugarski-Kirola et al., 2014; Umbricht et al., 2014). It is clear that chronic treatment with D2 antagonists or 5HT2a antagonists can produce significant changes in glutamatergic systems (Gill et al., 2014). It is possible that patients stabilized on SGAs and then transiently withdrawn may be less responsive to glutamatergic therapies, but retain responsiveness to established antipsychotics. Indeed, chronic treatment with atypical antipsychotics has been reported to downregulate mGluR2 expression (Kurita et al., 2012). It is also possible that discrepant clinical efficacy data can be explained by evidence that NMDAR hypofunction is age-dependent, i.e., more likely to manifest in prodromal or first-episode schizophrenia patients, versus chronically ill patients (de la Fuente-Sandoval et al., 2013a, 2013b). Recent data also suggest that patients may differ in their response to dopamine-based antipsychotic treatment depending on the degree of cingulate cortex glutamate, those patients with normal dopamine function but elevated glutamate being resistant to established antipsychotics (Demjaha et al., 2013). Thus, it could be

Please cite this article as: Litman, R.E., et al., AZD8529, a positive allosteric modulator at the mGluR2 receptor, does not improve symptoms in schizophrenia: A proof of principle ..., Schizophr. Res. (2015), http://dx.doi.org/10.1016/j.schres.2016.02.001

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R.E. Litman et al. / Schizophrenia Research xxx (2015) xxx–xxx

hypothesized that pharmacologic interventions to reverse NMDAR hypofunction will be effective only early in the course of illness prior to illness progression whereas these interventions in chronically ill patients stabilized on antipsychotics might be less effective. It may well be the case that further directions for therapeutic development of mGluR2/3 allosteric potentiators should include investigation of these approaches in a patient population with a predominant glutamatergic phenotype. Contributors Robert E. Litman: Conducted the study protocol as the primary principle investigator, enrolled and randomized the majority of the patients, organized, analyzed and verified the study data and results, organized study figures and tables, was the principal author of all sections of the manuscript, and organized the contributions and reviews of the other authors, and in charge of manuscript submission. Mark A. Smith: Designed the study, co-wrote the study protocol, provided key assistance and overall supervision of the study project, including administration, approvals, and study management for the sponsor, contributed to the data analysis including initial internal presentation of the data, organized and verified study data, and contributed to key parts of the manuscript, including the discussion. James J. Doherty: Invented AZD5829, including lead responsibility for its preclinical development of AZD5829 including preclinical evidence regarding mechanism of action and its putative utility as an antipsychotic, toxicology and other aspects of its pharmacology, and on this basis contributed to the design of the study, writing and approval of the protocol, especially concerning dosing, safety and toxicology in humans. Alan Cross: reviewed the literature, reviewed compound preclinical data, co-designed the study protocol, wrote the majority of the discussion. Shane Raines Organized, tabulated and performed proper statistical analysis. Wrote section on statistical methods and analysis. Reviewed and verified analyzable data and manuscript results. Lev Gertsik: conducted the study protocol as a principle investigator, enrolled second highest number of patients, provided critique and review of the manuscript, contributed to selected portions of the manuscript. Stephen R. Zukin: Reviewed the literature, reviewed preclinical data for the compound, designed the study, co-wrote the protocol, administered, organized, got approvals for and managed the study as the medical project lead, was the medical monitor for the study organized the data, contributed to key parts of the manuscript and provided overall review and critique of the contents of the manuscript. Conflict of interest Robert Litman owns CBH Health LLC, and has taught at Georgetown University. He is a speaker for Otsuka Pharmaceutical and Janssen Pharmaceutica. Mark A. Smith has worked for Astra Zeneca Pharmaceuticals, and Teva Pharmaceuticals. James Doherty has worked for Astra Zeneca Pharmaceuticals and Sage Therapeutics. Alan Cross has worked at Astra Zeneca Pharmaceuticals. Shane Raines has worked for Astra Zeneca Pharmaceuticals. Lev Gertsik has consulted with California Clinical Trials Medical Group. Stephen Zukin has worked for Astra Zeneca Pharmaceuticals and Forest Research Institute. Acknowledgments Risperidone is produced Janssen. The study described in this publication was fully funded by AstraZeneca.

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Please cite this article as: Litman, R.E., et al., AZD8529, a positive allosteric modulator at the mGluR2 receptor, does not improve symptoms in schizophrenia: A proof of principle ..., Schizophr. Res. (2015), http://dx.doi.org/10.1016/j.schres.2016.02.001