Original article Methyllycaconitine: A non-radiolabeled ligand for mapping α7 neuronal nicotinic acetylcholine receptors — In vivo target localization and biodistribution in rat brain Ramakrishna Nirogi ⁎, Vishwottam Kandikere, Gopinadh Bhyrapuneni, Ramanatha Saralaya, Nageswararao Muddana, Prashanth Komarneni Pharmacokinetics and Drug Metabolism, Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road No-5, Avenue-7, Banjara Hills, Hyderabad 500034, India abstract article info Article history: Received 14 April 2012 Accepted 8 May 2012 Keywords: ABBF (N-[(3R)-1-azabicyclo [2.2.2] oct-3-yl]-7-[2-(methoxy) phenyl]-1-benzofuran-2-carboxamide) α7 nAChRs LC–MS/MS Methyllycaconitine Nicotine PNU282987 (N-[(3R)-1-azabicyclo [2.2.2] oct-3-yl]-4-chlorobenzamide) Introduction: Reduction of cerebral cortical and hippocampal α7 neuronal nicotinic acetylcholine receptor (nAChR) density was observed in the Alzheimer's disease (AD) and other neurodegenerative diseases. Mapping the subtypes of nAChRs with selective ligand by viable, quick and consistent method in preclinical drug discovery may lead to rapid development of more effective therapeutic agents. The objective of this study was to evaluate the use of methyllycaconitine (MLA) in non-radiolabeled form for mapping α7 nAChRs in rat brain. Methods: MLA pharmacokinetic and brain penetration properties were assessed in male Wistar rats. The tracer properties of MLA were evaluated in rat brain by dose and time dependent differential regional distribution studies. Target specificity was validated after blocking with potent α7 nAChR agonists ABBF, PNU282987 and nicotine. High performance liquid chromatography combined with triple quad mass spectral detector (LC–MS/MS) was used to measure the plasma and brain tissue concentrations of MLA. Results: MLA has shown rapid brain uptake followed by a 3–5 fold higher specific binding in regions containing the α7 nAChRs (hypothalamus — 1.60 ng/g), when compared to non-specific regions (striatum — 0.53 ng/g, hippocampus — 0.46 ng/g, midbrain — 0.37 ng/g, frontal cortex — 0.35 ng/g and cerebellum — 0.30 ng/g). Pretreatment with potent α7 nAChR agonists significantly blocked the MLA uptake in hypothalamus. The non-radiolabeled MLA binding to brain region was comparable with the α7 mRNA localization and receptor distribution reported for [ 3 H] MLA in rat brain. Discussion: The rat pharmacokinetic, brain penetration and differential brain regional distribution features favor that MLA is suitable to use in preclinical stage for mapping α7 nAChRs. Hence, this approach can be employed as an essential tool for quicker development of novel selective ligand to map variation in the α7 receptor densities, as well as to evaluate potential new chemical entities targeting neurodegenerative diseases. © 2012 Elsevier Inc. All rights reserved. 1. Introduction Neuronal nicotinic acetylcholine receptors (nAChRs) are excitatory cationic ligand gated calcium channels comprised of α, β, γ, δ and ε subunits (Meyer et al., 1994; Sihver et al., 2000; Williams et al., 1994). In mammalian brain the abundant subunits of nAChRs are α4β2 and α7(Charpantier et al., 1998; Seguela et al., 1993). The sub- units alone have different functional and pharmacological properties (Meyer et al., 1994). Central α7 nAChRs are currently being evaluated as a potential therapeutic target for cognitive disorders associated in Alzheimer's disease (AD) and schizophrenia (Dajas & Wonnacott, 2004; Sihver et al., 2000). Several preclinical animal models demon- strated that systemic administration of α7 nAChR partial agonist showed improvement in several phases of cognition (Thomsen et al., 2010). High density of α7 nAChRs found in the hypothalamus, hippo- campus, and cortex and lower distribution in the striatum and cere- bellum of the rat brain (Davies et al., 1999). Radiolabeled methods are most widely employed to quantify brain receptor occupancy using labeled ligand measured by positron emission tomography (PET) or single photon emission computed to- mography (SPECT). These methods are time consuming, expensive and pollutant (Barth et al., 2006; Chernet et al., 2005; Need et al., 2007). High performance liquid chromatography combined with tan- dem mass spectrometric detector (LC–MS/MS) was successfully used in quantification of non-radiolabeled ligands (tracer) for the dopa- mine D2, serotonin 5HT 2A , NK1, opiate, histamine H 3 and α4β2 nACh receptor occupancy in preclinical species (Barth et al., 2006; Chernet et al., 2005; Need et al., 2007; Nirogi et al., 2012a,b). LC–MS/ MS based receptor occupancy is a high-throughput method, enables simultaneous measurement of receptor occupancy–drug exposure levels from a single animal and provide better correlation of exposure and receptor occupancy relationship (Barth et al., 2006; Chernet et al., 2005; Need et al., 2007; Nirogi et al., 2012a,b). Journal of Pharmacological and Toxicological Methods 66 (2012) 22–28 ⁎ Corresponding author at: Suven Life Sciences Ltd, Serene Chambers, Road No-5, Avenue-7, Banjara Hills, Hyderabad 500 034, India. Tel.: +91 40 23556038, +91 40 23541142; fax: + 91 40 23541152. E-mail address: nvsrk@suven.com (R. Nirogi). 1056-8719/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2012.05.003 Contents lists available at SciVerse ScienceDirect Journal of Pharmacological and Toxicological Methods journal homepage: www.elsevier.com/locate/jpharmtox