Psychopharmacology DOI 10.1007/s00213-016-4506-4 ORIGINAL INVESTIGATION GABAA receptor occupancy by subtype selective GABAAα2,3 modulators: PET studies in humans Aurelija Jucaite1,2 & Zsolt Cselényi1,2 & Jaakko Lappalainen3,4 & Dennis J. McCarthy5,6 & Chi-Ming Lee5,7 & Svante Nyberg5,8 & Katarina Varnäs2 & Per Stenkrona2 & Christer Halldin2 & Alan Cross3 & Lars Farde 1,2 Received: 11 August 2016 /Accepted: 8 December 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract (5 to 40 mg). PET images were analyzed using a simplified Rationale Sedation, dependence, and abuse liability limit the reference tissue model, and regional binding potentials γ use of non-selective -aminobutyric acid (GABAA)receptor (BPND) were obtained. The relationship between plasma con- positive modulators for the treatment of anxiety. AZD7325 centration of AZD7325 or AZD6280 and GABAA receptor and AZD6280 are novel, subtype-selective GABAAα2,3 re- occupancy was described by hyperbolic function, and Ki,plasma ceptor positive modulators with limited sedative effects. (plasma concentration required for 50% receptor occupancy) Objectives The current study aimed to confirm target engage- was estimated. Assessments of safety and tolerability included ment at GABAA receptors by AZD7325 and AZD6280 in recording of adverse events, vital signs, electrocardiogram, humans and to determine the relationship between exposure, and laboratory tests. 11 GABAA receptor occupancy, and tolerability. Results The [ C]flumazenil binding was reduced in a dose- Method Two PET studies, using high-resolution research to- dependent, saturable manner by both agents. Maximum recep- mography (HRRT) and the radioligand [11C]flumazenil, were tor occupancy could be reached for both compounds without performed in 12 subjects at baseline and after administration causing sedation or cognitive impairment. The Ki,plasma esti- of single oral doses of AZD7325 (0.2 to 30 mg) and AZD6280 mates for AZD7325 and AZD6280 were 15 and 440 nmol/l, respectively. Electronic supplementary material The online version of this article Conclusion High GABAA receptor occupancy by AZD7325 (doi:10.1007/s00213-016-4506-4) contains supplementary material, and AZD6280 could be reached without clear sedative effects. which is available to authorized users. * Aurelija Jucaite Keywords GABAA receptors . PET . Anxiety . Agonist . [email protected] AZD6280 . AZD7325 . [11C]flumazenil 1 Department of Clinical Neuroscience, AstraZeneca PET Center, Karolinska Institutet, R5:02, SE-17176 Stockholm, Sweden Background 2 Department of Clinical Neuroscience, PET Centre, Karolinska Institutet, Stockholm, Sweden Benzodiazepines (BZs), discovered by serendipity in the 3 AstraZeneca Neuroscience Innovative Medicines, Cambridge, MA, USA 1950s (Sternbach 1979), are well-established medicines for the treatment of anxiety (Nemeroff 2003). Its anxiolytic ef- 4 Present address: Marinus Pharmaceuticals, Radnor, PA, USA fects and related agents are mediated by allosteric enhance- 5 AstraZeneca R&D, Södertälje, Sweden ment of the γ-aminobutyric acid, GABAA receptor complex 6 Present address: Independent Consultant, Newark, DE, USA thus potentiating inhibitory GABA-mediated neurotransmis- 7 Present address: Ever East Consultants Limited, Hong sion (Farb and Ratner 2014, review). This mechanism of ac- Kong, People’sRepublicofChina tion of BZs has led to hypotheses on a pivotal role for GABA 8 Present address: Department of Psychiatry, Karolinska University neurotransmission in the pathophysiology of anxiety (Nutt Hospital (Huddinge), Stockholm, Sweden et al. 1990; Tiihonen et al. 1997; Malizia et al. 1998). Psychopharmacology Although benzodiazepines are rapid onset highly effica- The binding of BZs at the GABAA receptor benzodiaze- cious drugs, significant adverse effects, such as sedation, am- pine binding site in humans has been examined extensively by nesia, ataxia, abuse, dependence, and withdrawal, limit their PET using the radioligand [11C]flumazenil (Persson et al. clinical usefulness in long-term treatment of anxiety disorders. 1985;Pikeetal.1993;Abadieetal.1996). Flumazenil is an Accordingly, they are now reserved for second-line treatment antagonist with high affinity for the GABAA α1, α2, α3, and (Baldwin et al. 2014). Building on an understanding of the α5subunits(Ki ∼1 nmol/l), whereas the affinity is lower for pharmacological profile of the first-generation benzodiaze- the α4 and α6 subunits (Ki ∼150 nmol/l) (Sieghart 1995). 11 pines, effective, safer, and tolerable GABAA receptor modu- [ C]flumazenil continues to be used as a tool in research on lators are now being sought (Skolnick 2012). the pathophysiology of neuropsychiatric disorders as well as There is potentially a great diversity in the GABAA recep- for examination of GABAA receptor occupancy by classical tor system at a detailed molecular level, as these ionotropic and novel receptor modulators. receptors are pentamers made up from 19 known subunits We aim to confirm target GABAA receptor engagement in (α1–6, β1–3, γ1–3, δ, ε, θ, π,andρ1–3). In the human brain, humans by the modulators AZD7325 and AZD6280 and ex- the majority of combinations consist of two α,twoβ, and one amine the relationship to sedation and previously published γ subunit (Olsen and Sieghart 2009). Benzodiazepine-like pharmacodynamics effects. Twelve subjects were examined modulators of GABAA receptors bind to a site, located on using high-resolution research tomography (HRRT) and the the α/γ subunit interface (Trincavelli et al. 2012, review), radioligand [11C]flumazenil. For dose finding purposes, we and pharmacological responses to BZ site activators vary with examined the relationships between dose, plasma concentra- specific subunit composition. Studies with transgenic mice tion, and receptor occupancy for both compounds. with introduced point mutations in murine α1 subunit gene have led to the association of anxiolytic-like activity with the GABAA receptor α2and/orα3 subunits, sedation with α1 Method subunit, and some aspects of cognition to the GABAA recep- tor α5 subunit (Rudolph et al. 1999). Thus, it has been hy- Study design and subjects pothesized that benzodiazepine site modulators which selec- tively potentiate activity of GABAA receptors containing α2/ Two separate open-label phase I PET studies were conducted α3 receptor subunits would produce a greater separation be- in 2008–2009. In the first study (study 1), we examined re- tween therapeutic and side effects than non-selective com- ceptor occupancy at GABAA receptors after administration of pounds. In parallel, it has been hypothesized that side effects AZD7325 and in the second study (study 2) after administra- of BZs may be related to the intrinsic activity of modulators tion of AZD6280. (Haefely et al. 1990;Puiaetal.1992); thus, partial agonism The studies were approved by the Medical Products may also allow for differentiation of anxiolytic effects from Agency of Sweden, the Regional Ethical Review Board in unfavorable CNS effects such as sedation. Stockholm, and the Radiation Safety Committee at the Based on these concepts, a series of subunit selective Karolinska University Hospital (KUH), Stockholm, Sweden. GABAAα2,3 partial receptor modulators, including The studies were performed in accordance with the AZD7325 and AZD6280, were developed by AstraZeneca Declaration of Helsinki and International Conference on (Suppl. Fig. 1,Alhambraetal.2011). Both compounds Harmonization/Good Clinical Practice Guidelines. Written in- exert their function selectively via GABAA receptor sub- formed consent was obtained from all subjects prior to the units as characterized by high in vitro affinity to the α1, α2, initiation of the study. and α3 subunits and low affinity to the α5 subunit (Suppl. Subjects were enrolled and remained at the AstraZeneca Table 1) and are partial BZ site modulators with efficacy Clinical Pharmacology Unit, KUH, Huddinge, for the dura- selective for α2β3γ2orα3β3γ2 subunits, i.e., AZD6280 tion of the study. Magnetic resonance imaging (MRI) and PET produces 32–34% and AZD7325 produces 15% of maximal examinations were performed at KUH, Solna. Four men, 23– diazepam response, respectively (Chen et al. 2014, 2015, 34 years of age, underwent repeated PET examinations with Suppl. Table 1). In preclinical models, these compounds [11C]flumazenil in study 1, and eight men 21–32 years of age have potent anxiolytic-like effects without sedation and in- in study 2. The subjects were healthy according to medical duce a distinct pharmacoEEG signature (Alhambra et al. history, physical examination, blood and urine analyses, and 2011; Christian et al. 2015). Examination of pharmacody- brain MRI. None of the subjects discontinued the study. namic effects in phase I clinical trials has shown a novel The initial study design included two sequential panels, pattern of effects on EEG (reduction in delta and theta with two subjects in each. The first panel was intended to bands) by AZD7325 and AZD6280, as well as effects on obtain initial receptor occupancy values, to be used for dose saccadic peak velocity, a suggested marker of anxiolysis selection in the second panel. Each of the subjects was (Chen et al. 2014, 2015). planned to participate in four PET examinations with Psychopharmacology [11C]flumazenil, including one baseline assessment and sub- Metabolism, Pharmacokinetics, and Bioanalysis in sequent weekly examinations at the approximate time of max- Wilmington, DE,