Elsevier

Neuropharmacology

Volume 61, Issues 1–2, July–August 2011, Pages 161-171
Neuropharmacology

Pre-clinical validation of a novel alpha-7 nicotinic receptor radiotracer, [3H]AZ11637326: Target localization, biodistribution and ligand occupancy in the rat brain

https://doi.org/10.1016/j.neuropharm.2011.03.024Get rights and content

Abstract

The alpha-7 neuronal nicotinic receptor is a novel pharmacological target for psychiatric and cognitive disorders. Selective radiotracer tools for pre-clinical receptor occupancy can facilitate the interpretation of the biological actions of small molecules at a target receptor. We discovered a high affinity nicotinic alpha-7 subtype-selective ligand, AZ11637326, with physical–chemical and pharmacokinetic properties suitable for an in vivo radioligand tool. [3H]AZ11637326 synthesis by tritiodehalogenation of the corresponding tribromide precursor yielded a high specific activity radiotracer with high affinity alpha-7 receptor binding in the rat hippocampus determined by autoradiography (Kd = 0.2 nM). When [3H]AZ11637326 was administered to rats by intravenous bolus, rapid uptake was measured in the brain followed by a 3–4 fold greater specific binding in regions containing the alpha-7 receptor (frontal cortex, hippocampus, hypothalamus and midbrain) when compared to non-target regions (striatum and cerebellum). Systemic administration of the high affinity alpha-7 receptor antagonist, methyllycaconitine (MLA), or pretreatment with alpha-7 selective agonists (AR-R17779, PyrQTC, DBCO-4-POM, and DBCO-3-POM) significantly blocked the alpha-7 specific binding of [3H]AZ11637326 in the rat brain. The rank order of ligand ED50 values for in vivo alpha-7 receptor occupancy in rat hippocampus was: DBCO-4-POM > DBCO-3-POM ∼ MLA > PyrQTC > AR-R17779. The occupancy affinity shift was consistent with in vitro binding affinity in autoradiography. Our studies established the optimal conditions for [3H]AZ11637326 in vivo specific binding in the rat brain and support the use of [3H]AZ11637326 as a pre-clinical tool for assessment of novel alpha-7 compounds in drug discovery.

Highlights

► The alpha-7 neuronal nicotinic receptor is a drug target for CNS disorders. ► Pre-clinical receptor occupancy at a target receptor will aid drug discovery. ► We report a high affinity alpha-7 subtype-selective radiotracer, [3H]AZ11637326. ► CNS biodistribution and pharmacological selectivity in vivo is optimal in rat. ► Selective alpha-7 agonist occupancy in vivo matches functional data trends.

Introduction

The alpha-7 receptor is an abundant neuronal nicotinic receptor (NNR) in the mammalian brain. The alpha-7 NNR subtype is a ligand gated ion channel distinguished from other pentameric NNRs by subunit composition, functional activity, pharmacological ligands, and its low affinity for acetylcholine and high affinity for α-Bungarotoxin (α-Bgtx). Distributed in mesocorticolimbic and mesostriatal circuitry, the alpha-7 NNR has presynaptic, post-synaptic and perisynaptic localization on neurons primarily in the hippocampus, neocortex and limbic regions. Presynaptic alpha-7 receptors play a key role in neurotransmitter release, while post-synaptic receptors facilitate depolarization through cation entry and perisynaptic receptors modulate glutamergic and GABA-ergic neurotransmission directly and dopaminergic activity indirectly. (Albuquerque et al., 2009, Gotti et al., 2009).

Alpha-7 receptor agonists have generated interest as promising treatments for cognitive disorders in schizophrenia (Buccafusco and Terry, 2009, Buchanan et al., 2007, Olincy et al., 2006), inflammation (Conejero-Goldberga et al., 2008, de Jonge and Ulloa, 2007, Wang et al., 2003) and Alzheimer’s Disease (Carson et al., 2008, Kem, 2000, Parri and Dineley, 2010). Drugs targeting alpha-7 receptors, such as AR-R17779 (Levin, 2002) and AZD0328 (Sydserff et al., 2009, Castner et al., 2011) have shown enhancement of cognition and attention in animal models (reviewed by Leiser et al., 2009) and in humans (Olincy et al., 2006). Improvements seen in animal models have clear implications for the therapeutic potential in humans (Martin et al., 2004, Olincy and Stevens, 2007, Thomsen et al., 2010), providing scientists with both models and NNR tools with rich translational value.

In CNS drug discovery, selective radiotracer tools help us to interpret the biological actions of small molecules at a target receptor. Generally, radiotracers suitable for in vivo binding require high affinity and selectivity at the target receptor. Moderate values for radiotracer lipophilicity have been shown to improve brain penetration and reduce non-specific binding. Radiotracers targeting targets in the CNS require low efflux ratio and are not substrates for efflux pumps (Passchier et al., 2002). While NNR radiotracers for the α4β2 receptor are available for in vivo binding and PET imaging (Ding et al., 2004, Ogawa et al., 2009), fewer high affinity radiotracers selective for the alpha-7 receptor have been successfully developed. Radioligands that bind to the alpha-7 receptor with high affinity, such as the potent and selective antagonist [3H]MLA (Davies et al., 1999, Whiteaker et al., 1999) or the large polypeptide [125I]α-BTX have been useful for in vitro binding in rat (Clarke et al., 1985), monkey (Han et al., 2003) and human brain tissue (Breese et al., 1997), yet lack the physical and chemical properties to demonstrate in vivo occupancy. The in vitro binding profile and biodistribution of [125I]iodo-MLA in the rat brain (Navarro et al., 2000, Navarro et al., 2002) suggests it may provide a selective tool for alpha-7 receptor imaging, while recent autoradiography data showing high density [125I]iodo-MLA localization in the monkey thalamus (Kulak et al., 2006) challenges the cross-species translational value of this tool. One promising radiotracer is [3H]A-58553 (Anderson et al., 2008) which shows high affinity and low non-specific binding in rat and in human brain tissue using in vitro binding techniques.

Several novel in vivo imaging agents have been developed for the alpha-7 receptor subtype NNRs. For example, [11C](R)-MeQAA, a moderate affinity (41 nM) alpha-7 ligand, showed high brain uptake that was reduced by pretreatment with MLA in biodistribution studies in mice, yet PET imaging studies in the monkey showed only modest selectivity for the alpha-7 receptor (Ogawa et al., 2009, Ogawa et al., 2010). The radiotracer [125I]I-TSA, showed high affinity (0.54 nM) binding at alpha-7 receptors in the brain, however, it showed high non-specific binding in bioavailability studies in the mouse (Ogawa et al., 2006). Other groups have examined bioactive NNR compounds such as analogs of SSR108711 (Hashimoto et al., 2008, Toyohara et al., 2009, Tanibuchi et al., 2010) and quinuclidine derivatives (Pomper et al., 2005) with moderate to high affinity for the alpha-7 receptor in order to develop a tool for PET or SPECT imaging. 11C-Chiba-1001 (11C-Methyl-SSR 180711) uptake and pharmacological selectivity studies conducted in the conscious monkey concluded that this was a alpha-7 selective radiotracer (Hashimoto et al., 2008). Other findings suggest that [3H]Chiba-1001 has low affinity for the rat and human alpha-7 receptor in vitro (Tanibuchi et al., 2010) and poor in vivo regional and pharmacological selectivity in the rodent brain (personal communication, Min Ding), presenting challenges for using this tool for translational studies. Specific quinuclidine radiotracers provided an acceptable radiotracer yield, yet, in rodent biodistribution studies these compounds showed only modest selectivity in the hippocampus, the target region for alpha-7 binding (Pomper et al., 2005).

In light of these findings, a radiotracer selective for the alpha-7 receptor with high affinity for the receptor, and that shows brain uptake and target receptor localization after systemic administration would provide an essential in vivo translational tool for the development of novel therapeutics. AZ11637326, a spirofuropyridine, is a NNR alpha-7 subtype-selective compound shown to compete with [125I]α-BTX at sub-nanomolar affinity. [3H]AZ11637326 high affinity binding in rodent hippocampal membranes (Kd = 0.40 nM) and in HEK-cells transfected with the human alpha-7 receptor (Kd = 0.18 nM) is blocked competitively by compounds with high selectivity for the alpha-7 receptor (Gordon et al., 2010). Here, we add to these and other previous findings (Ding et al., 2007, Elmore et al., 2008) to describe the in vitro autoradiography, the in vivo biodistribution and the pharmacological properties of [3H]AZ11637326 to further demonstrate the usefulness of this pre-clinical radiotracer for comparing novel compounds using alpha-7 receptor occupancy.

Section snippets

Subjects

Male Sprague Dawley adult rats (150–170 g) were purchased from Charles River, USA. Rats were group housed in the vivarium on a 50:50 light:dark cycle and received ad libitum food and water. All procedures were approved by the Institutional Animal Care and Use Committee in accordance with The Guide for the Care and Use of Laboratory Animals. All procedures were conducted in a manner to minimize animal suffering, to reduce the number of animals used and to utilize alternative techniques where

Properties of AZ11637326

AZ11637326 has a molecular weight of 310.37. The LogD of 2.1 at pH 7.4 is a lipophilicity value that suggests low potential for non-specific binding. The ClogP of 3.3, clearance measured in human microsomes (hClint = 7 μl/min/mg) and Efflux ratio of 0.9 (permeability of 46.4), indicate adequate brain penetration. The functional potency of AZ11637326 at the human (hu) alpha-7 receptor was determined to be 0.2 μM in Xenopus oocyte electrophysiology, see Fig. 13. For chemical structures see Fig. 1

Discussion

The high affinity alpha-7 radiotracer, [3H]AZ11637326, shows high specific binding to the alpha-7 receptor in the rodent brain, using both in vitro and in vivo binding techniques. In vitro binding studies in tissue sections prepared for autoradiography support the properties of high affinity, target localization and pharmacological selectivity for [3H]AZ11637326, making it a likely candidate for in vivo characterization. When administered to rats by bolus intravenous injection, [3H]AZ11637326

Acknowledgments

The authors would like to acknowledge the following individuals for their contribution to this work: Amy Hehman & Lynn India Neveras for jugular cannulation surgery, Eifion Phillips for synthesis of AZ11637326, J. Richard Heys for the original radiochemical synthesis of [3H]AZ11637326, Ladislav Mrzljak, Kaoru Kondo for the generous and rigorous experimental support she provided to the in vitro autoradiography work, Mark Pietras for mining the data base and Kathy Neilson for her tireless DMPK

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