JPET Fast Forward. Published on June 19, 2014 as DOI: 10.1124/jpet.114.213793 JPET ThisFast article Forward. has not been Published copyedited andon formatted.June 19, The 2014 final versionas DOI:10.1124/jpet.114.213793 may differ from this version. JPET #213793 1. Title Page Brexpiprazole I: In vitro and in vivo characterization of a novel serotonin-dopamine activity modulator Kenji Maeda, Haruhiko Sugino, Hitomi Akazawa, Naoki Amada, Jun Shimada, Takashi Downloaded from Futamura, Hiroshi Yamashita, Nobuaki Ito, Robert D. McQuade, Arne Mørk, Alan L. Pehrson, Morten Hentzer, Vibeke Nielsen, Christoffer Bundgaard, Jørn Arnt, Tine Bryan Stensbøl and jpet.aspetjournals.org Tetsuro Kikuchi Qs’ Research Institute, Otsuka Pharmaceutical Co., Ltd. Tokushima, Japan (K.M., H.S., H.A., at ASPET Journals on September 27, 2021 N.A., J.S., T.F., H.Y., N.I., T.K.), and Otsuka Pharmaceutical Development & Commercialization, Princeton, New Jersey (R.D.M.), Neuroscience Drug Discovery, H. Lundbeck A/S, Denmark (A.M., M.H., V.N., C.B., J.A., T.B.S.) & USA (A.L.P.) 1 Copyright 2014 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on June 19, 2014 as DOI: 10.1124/jpet.114.213793 This article has not been copyedited and formatted. The final version may differ from this version. JPET #213793 2. Running title page a) Running title: Brexpiprazole, a novel serotonin-dopamine activity modulator b) Corresponding author: Kenji Maeda, PhD 463-10 Kagasuno, Kawauchi-cho, Tokushima, 771-0192, Japan Phone: +81-88-665-2126 Fax: +81-88-666-4709 Downloaded from E-mail: [email protected] c) The number of text pages: 63 jpet.aspetjournals.org The number of tables: 6 The number of figures: 8 The number of references: 60 at ASPET Journals on September 27, 2021 The number of words in abstract: 250 The number of words in introduction: 655 The number of words in discussion: 1491 d) A list of nonstandard abbreviations 5-HT, serotonin 7-OH-DPAT, 7-hydroxy-2-(di-n-propylamino)tetralin 8-OH-DPAT, 2-dipropylamino-8-hydroxy-1,2,3,4- tetrahydronaphthalene [35S]GTPγS, guanosine 5’-O-(3-[35S]thio)-triphosphate aCSF, artificial cerebrospinal fluid ANOVA, analysis of variance CHO, Chinese hamster ovary 2 JPET Fast Forward. Published on June 19, 2014 as DOI: 10.1124/jpet.114.213793 This article has not been copyedited and formatted. The final version may differ from this version. JPET #213793 cIC50, corrected IC50 value CNS, central nervous system DHFR, dehydrofolate reductase D, dopamine D2L, long form of human D2 receptor DMSO, dimethyl sulfoxide DOI, 2,5-dimethoxy-4-iodoamphetamine Downloaded from DOPAC, 3,4-Dihydroxy-phenyl-acetic acid EPS, extrapyramidal symptoms jpet.aspetjournals.org HeLa, human cervical epithelial adenocarcinoma HPLC, high-performance liquid chromatography HTRF, homogeneous time resolved fluorescence at ASPET Journals on September 27, 2021 HVA, homovanillic acid IMDM, Iscove’s modified Dulbecco’s medium IP1, inositol monophosphate LSD, lysergic acid diethylamide MEM, minimum essential medium mPFC, medial prefrontal cortex MRM, multiple-reaction-monitoring MS/MS, tandem mass spectrometry NSD-1015, 3-hydroxybenzylhydrazine dihydrochloride PCR, polymerase chain reaction PET, positron emission tomography 3 JPET Fast Forward. Published on June 19, 2014 as DOI: 10.1124/jpet.114.213793 This article has not been copyedited and formatted. The final version may differ from this version. JPET #213793 SD, Sprague-Dawley e) A recommended section assignment: Neuropharmacology Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 4 JPET Fast Forward. Published on June 19, 2014 as DOI: 10.1124/jpet.114.213793 This article has not been copyedited and formatted. The final version may differ from this version. JPET #213793 3. Abstract Brexpiprazole (OPC-34712, 7-{4-[4-(1-benzothiophen-4-yl)piperazin-1-yl]butoxy}quinolin- 2(1H)-one) is a novel drug candidate in clinical development for psychiatric disorders with high affinity for serotonin, dopamine and noradrenaline receptors. In particular, it bound with high affinity (Ki <1 nM) to h5-HT1A, h5-HT2A, hD2L, hα1B and hα2C-adrenergic receptors. It displayed partial agonism at h5-HT1A and hD2 receptors in cloned receptor systems, and potent antagonism Downloaded from of h5-HT2A receptors and hα1B/2C-adrenoceptors. Brexpiprazole also had affinity (Ki <5 nM) for hD3, h5-HT2B, h5-HT7, hα1A and hα1D adrenergic receptors, moderate affinity for hH1 (Ki =19 nM), and low affinity for hM1 receptors (Ki >1000 nM). Brexpiprazole potently bound to rat jpet.aspetjournals.org 5-HT2A and D2 receptors in vivo, and ex vivo binding studies further confirmed high 5-HT1A receptor binding potency. Brexpiprazole inhibited DOI-induced head-twitches in rats, suggestive of 5-HT2A antagonism. Furthermore, in vivo D2 partial agonist activity of brexpiprazole was at ASPET Journals on September 27, 2021 confirmed by its inhibitory effect on reserpine-induced DOPA accumulation in rats. In rat microdialysis studies, brexpiprazole slightly reduced extracellular dopamine in nucleus accumbens, but not in prefrontal cortex, while moderate increases of the dopamine metabolites, homovanillic acid and 3,4-dihydroxy-phenyl-acetic acid, in these areas, also suggested in vivo D2 partial agonist activity. In particular, based on a lower intrinsic activity at D2 receptors and higher binding affinities for 5-HT1A/2A receptors than aripiprazole, brexpiprazole would have a favourable antipsychotic potential without D2 receptor agonist- and antagonist-related adverse effects. In conclusion, brexpiprazole is a serotonin-dopamine activity modulator with a unique pharmacology, which may offer novel treatment options across a broad spectrum of central nervous system disorders. 5 JPET Fast Forward. Published on June 19, 2014 as DOI: 10.1124/jpet.114.213793 This article has not been copyedited and formatted. The final version may differ from this version. JPET #213793 4. Introduction The main strategy for treatment of schizophrenia is based on antagonizing dopamine D2 receptors. In addition, most second generation antipsychotics are antagonists of serotonin 5-HT2A receptors and α1-adrenoceptors, and individual compounds have a variety of effects on other monoamine receptors, such as 5-HT1A receptors. These broad target effects have the objective of either improving antipsychotic efficacy (with additional effects on affective symptoms or cognitive deficits) or mitigating adverse effects (e.g. extrapyramidal symptoms [EPS]) (Arnt and Downloaded from Skarsfeldt, 1998; Meltzer, 1999; Roth et al., 2004; Arnt et al., 2008; Wong et al., 2008; Newman-Tancredi, 2010; Newman-Tancredi and Kleven, 2011). jpet.aspetjournals.org However, due to tolerability issues, treatment with D2 receptor antagonists is not considered to be the optimal strategy to modulate dopaminergic activity, and the discovery and development of D2 receptor partial agonists has provided a well-tolerated treatment with stabilizing effects on at ASPET Journals on September 27, 2021 dopamine function (Stahl, 2001; Citrome, 2013). So far, only one D2 partial agonist, aripiprazole, with moderate D2 intrinsic activity, has reached the market (Burris et al., 2002; Potkin et al., 2003), while other compounds with higher D2 intrinsic activity are in development (Citrome, 2013) or have been discontinued during development, often because of lack of sufficient clinical efficacy, e.g. bifeprunox (Newman-Tancredi et al., 2007; Casey et al., 2008). A key issue for the D2 partial agonists is to ascertain how much intrinsic activity (or relative efficacy) is ideal in leading to optimal stabilization of dopaminergic transmission. If the D2 intrinsic activity is too high, this can lead to lack of robust antipsychotic activity as well as pronounced adverse effects related to increased D2 receptor tonus, e.g. nausea, vomiting, and motor side effects such as hyperkinesias and restlessness (Fleischhacker, 2005; Newman- Tancredi et al., 2007; Casey et al., 2008; Stip and Tourjman, 2010), while D2 antagonist activity 6 JPET Fast Forward. Published on June 19, 2014 as DOI: 10.1124/jpet.114.213793 This article has not been copyedited and formatted. The final version may differ from this version. JPET #213793 leads to an increased risk of EPS and increased prolactin secretion (Casey, 1996). Although aripiprazole has offered a new approach to stabilizing the dopaminergic system, an improvement could potentially be made by developing a novel compound that maintains significant partial agonist activity at D2 receptors, but with lower intrinsic activity. In addition to the issue of optimal D2 intrinsic activity, optimization of the pharmacological profile by a combination of additional target effects is a well-known strategy to improve the clinical efficacy and tolerability of antipsychotics. At the clinically equivalent dose range leading Downloaded from to 80–90% D2 receptor occupancy, aripiprazole modulates a limited number of additional target receptors, the primary effect being partial agonist activity at 5-HT1A receptors with lower jpet.aspetjournals.org potency than at D2 receptors (Mamo et al., 2007; Dahan et al., 2009). In addition, human 5-HT2A receptor occupancy is significantly lower at clinically relevant dosages (Mamo et al., 2007). Accordingly, an optimized target profile may lead to improvements in both clinical efficacy and at ASPET Journals on September 27, 2021 adverse effect profile in the treatment of schizophrenia. Furthermore, a broader pharmacological