Preclinical characterization of A-582941: a novel alpha7 neuronal nicotinic receptor agonist with broad spectrum cognition-enhancing properties

Abstract

Among the diverse sets of nicotinic acetylcholine receptors (nAChRs), the alpha7 subtype is highly expressed in the hippocampus and cortex and is thought to play important roles in a variety of cognitive processes. In this review, we describe the properties of a novel biaryl diamine alpha7 nAChR agonist, A-582941. A-582941 was found to exhibit high-affinity binding and partial agonism at alpha7 nAChRs, with acceptable pharmacokinetic properties and excellent distribution to the central nervous system (CNS). In vitro and in vivo studies indicated that A-582941 activates signaling pathways known to be involved in cognitive function such as ERK1/2 and CREB phosphorylation. A-582941 enhanced cognitive performance in behavioral models that capture domains of working memory, short-term recognition memory, memory consolidation, and sensory gating deficit. A-582941 exhibited a benign secondary pharmacodynamic and tolerability profile as assessed in a battery of assays of cardiovascular, gastrointestinal, and CNS function. The studies summarized in this review collectively provide preclinical validation that alpha7 nAChR agonism offers a mechanism with potential to improve cognitive deficits associated with various neurodegenerative and psychiatric disorders.

Figure 1

Synthetic route for preparation of A‐582941 on multigram scale.

Figure 2

(A) Enhancement of A‐582941‐evoked responses by PNU‐120596. Shown are responses to varying concentrations of A‐582941 in the presence or absence of PNU‐120596 (3 μM) in Xenopus oocytes expressing α7 nAChR. Data are normalized to that evoked by 1 mmol ACh. (B) Effect of A‐582941 on GABAergic spontaneous inhibitory postsynaptic potentials (IPSCs) recorded from dentate gyrus granule cells. Experiments were performed where the chloride content of the internal solution was reduced to 13 mM (methanesulfonate substitution) so that the GABA_A chloride current reversal potential was −60 mV, while the glutamate receptor cation current reversed near 0 mV. To record IPSCs, the holding potential was set to 0 mV, minimizing glutamatergic currents and revealing GABAergic IPSCs as positive (upward) deflections. Traces are from one cell exposed to 10 μM PNU‐120596 alone (control) and subsequently 100 nM A‐582941 in the presence of PNU‐120596. Similar results were obtained in 7 other cells and recorded using pClamp9 (Axon Instruments, Union City, CA, USA) in 5‐min epochs before (control), during, and after (wash) exposure to A‐582941 by bath perfusion. IPSC events during each epoch were detected off‐line using MiniAnalysis software (Synaptosoft, Inc., Fort Lee, NJ, USA). (C) Shows the number of events detected in each epoch plotted according to amplitude bin, demonstrating an increase in events across essentially all amplitudes coincident with the administration of A‐582941.

Figure 3

Microdialysis studies with A‐582941. (A) Effect of A‐582941 on acetylcholine release in the medial prefrontal cortex of freely moving rats. Five animals per treatment group received A‐582941 (3 μmol/kg, i.p.) repeated for 3 days, once daily, whereas control animals received only sterile water. After drug administration, microdialysate samples were analyzed for extracellular levels of acetylcholine by HPLC with electrochemical detection. Shown is mean ± S.E.M. area under the curve (AUC in arbitrary units, 0–180 min) after administration of A‐582941 collapsed from days 1–3; n= 5 per group. *P < 0.05 A‐582941 (3 μmol/kg) versus vehicle‐treated animals. (B) Recovery of A‐582941 from a microdialysis probe in the medial prefrontal cortex of freely moving Sprague‐Dawley rats (n= 5) following systemic administration of drug (3 μmol/kg, i.p.). Extracellular brain levels are extrapolated from the dialysate levels using an estimated probe in vitro recovery of 5.9–8.4% for A‐582941.

Figure 4

CNS levels of A‐582941 in rodent. Brain:plasma distribution of A‐582941 in mouse following a 1‐μmol/kg (i.p.) dose (n= 3). The plasma C_max of 23 ng/mL is achieved within 15 min after dosing and the brain C_max (285 ng/g) is reached at by the first time point (20 min). The brain:plasma ratio is maintained at ∼10 for the 9‐h duration of the experiment.

Figure 5

(A) Time course for in vitro metabolism of [³H]A‐582941 in liver hepatocytes. The starting concentration of A‐582941 was 1.0 μM. (B) Turnover of A‐582941 across a panel of CYP and FMO supersomes.

Figure 6

Metabolism of A‐582941. Oxidation to the N‐oxide, 6, is the predominant route for metabolism in microsomes and hepatocytes (all species). The formation of tritiated water from [³H]A‐582941 in vivo suggests that N‐demethylation to 7 may occur. The secondary amine could undergo rapid conjugation and/or elimination.

Figure 7

Procognitive efficacy of A‐582941. (A) A‐582941 is efficacious across different species and models at similar plasma exposures: (i) lowered trial2:trial1 investigation ratio indicating enhanced efficacy in the rat social recognition model; (ii) increased correct response rate at the long delay interval for monkeys in the DMTS task; and (iii) increased crossover latency in the 24‐h test trial of the mouse inhibitory avoidance test, a measure of memory consolidation. Data are replotted from Buccafusco et al. (2007) (ii) and Bitner et al. (2007) (i and iii) with plasma exposure (extrapolated from dosing in a satellite group of animals) representing the C_max at the corresponding dose. The left‐hand segment of each graph is on the same horizontal scale. (B) Efficacy of A‐582941 in social recognition model following acute and sustained 7‐day exposure. Recognition ratio was measured following acute treatment with A‐582941 (i.p.) or after sustained exposure via s.c. osmotic minipumps for 7 days. The plasma level extrapolated from an i.p. dose of 1 μmol/kg in satellite mice corresponds to 4.7 ng/mL, whereas minipump exposure maintained steady‐state plasma levels at 3 ng/mL. (C) Effect of A‐582941 on the performance of spontaneously‐hypertensive rat pups in the 5‐trial inhibitory avoidance model. A trend toward efficacy is observed in the dose range 0.01–1.0 μmol/kg, s.c., but full efficacy is not achieved. Ciproxifan (cpx, 3 mg/kg) is the positive control in this assay.

Figure 8

Lack of effect of A‐582941 on cell growth and proliferation on keratinocytes in vitro and in vivo. (A) In vitro effect of A‐582941 (100 and 10,000 nM) on proliferation of cultured keratinocytes in basic medium: (i) without growth factors and (ii) in complete medium containing growth factors. Cells were exposed to A‐582941 for 9 days, with medium refreshed every 3–4 days. (B) Histologic sections of skin from SENCAR mice sacrificed after treatment with BrdU reagent (Zymed^® Laboratories, San Francisco, CA, USA; 1 mL/100 g body weight, i.p., 2 h before sacrifice): (i) vehicle‐treated mice; (ii) mice treated with TPA (12‐O‐tetradecanoylphorbol‐13‐acetate, 1.7 nmol) as positive control. Note the thickening of the epidermis (epidermal hyperplasia) and the increase in the number of BrdU‐labeled basal keratinocytes; (iii) A‐582941‐treated mice (1000 nmol) showing no evidence of epidermal changes and few BrdU‐labeled basal keratinocytes. BrdU immunohistochemistry with hematoxylin counterstain (magnification 200×).