Tropisetron sensitizes α7 containing nicotinic receptors to low levels of acetylcholine in vitro and improves memory-related task performance in young and aged animals

Highlights

• Tropisetron acts as a partial agonist at human α7 and α7β2 nAChRs in Xenopus oocytes.

• Tropisetron potentiates ACh-evoked currents demonstrating “co-agonist” effects.

• Tropisetron improves object recognition memory in young and aged rats.

• Tropisetron improves working/short-term memory in aged rhesus monkeys.

• Tropisetron plus donepezil improves memory performance of aged rhesus monkeys.

Abstract

Tropisetron, a 5-HT₃ receptor antagonist commonly prescribed for chemotherapy-induced nausea and vomiting also exhibits high affinity, partial agonist activity at α7 nicotinic acetylcholine receptors (α7 nAChRs). α7 nAChRs are considered viable therapeutic targets for neuropsychiatric disorders such as Alzheimer's disease (AD). Here we further explored the nAChR pharmacology of tropisetron to include the homomeric α7 nAChR and recently characterized heteromeric α7β2 nAChR (1:10 ratio) and we evaluated its cognitive effects in young and aged animals. Electrophysiological studies on human nAChRs expressed in Xenopus oocytes confirmed the partial agonist activity of tropisetron at α7 nAChRs (EC₅₀ ∼2.4 μM) with a similar effect at α7β2 nAChRs (EC₅₀ ∼1.5 μM). Moreover, currents evoked by irregular pulses of acetylcholine (40 μM) at α7 and α7β2 nAChRs were enhanced during sustained exposure to low concentrations of tropisetron (10 and 30 nM) indicative of a “priming” or co-agonist effect. Tropisetron (0.1–10 mg/kg) improved novel object recognition performance in young Sprague-Dawley rats and in aged Fischer rats. In aged male and female rhesus monkeys, tropisetron (0.03–1 mg/kg) produced a 17% increase from baseline levels in delayed match to sample long delay accuracy while combination of non-effective doses of donepezil (0.1 mg/kg) and tropisetron (0.03 and 0.1 mg/kg) produced a 24% change in accuracy. Collectively, these animal experiments indicate that tropisetron enhances cognition and has the ability to improve the effective dose range of currently prescribed AD therapy (donepezil). Moreover, these effects may be explained by tropisetron's ability to sensitize α7 containing nAChRs to low levels of acetylcholine.

Introduction

Tropisetron (Nabovan) is a potent 5-hydroxytryptamine type 3 (5-HT₃) receptor antagonist widely used outside the United States to treat patients with chemotherapy-induced nausea and vomiting (Simpson et al., 2000). In addition to its prominent effects at 5-HT₃ receptors (Barnes and Sharp, 1999), tropisetron is also a high affinity (Ki = 6.9 nM) partial agonist at α7 nicotinic acetylcholine receptors (α7 nAChRs) with an EC₅₀ of ∼800 nM (Macor et al., 2001, Papke et al., 2004). The homomeric α7 nAChR has been considered as an important therapeutic target in Alzheimer's disease (AD) and schizophrenia for several years due to deficits in α7 nAChR protein that have been observed in brains of patients who suffered from these disorders (for review see Bertrand et al., 2015). Neuronal α7 nAChRs are abundant in brain regions (e.g., prefrontal cortex and hippocampus) important for cognition where they modulate a number of calcium-dependent events including neurotransmitter release (Huang et al., 2014, Radcliffe et al., 1999), synaptic signaling (Berg and Conroy, 2002, Hefft et al., 1999) and neuroprotection (Bitner et al., 2009, Bitner et al., 2010, Roncarati et al., 2009). Moreover, α7 nAChR agonists (e.g., A-582941, ABT 107 and S24795) have been shown to increase the phosphorylation of ERK and CREB (signaling pathways linked to long term potentiation and memory formation) in rodent brain (Bitner et al., 2007, Bitner et al., 2010), to produce beneficial disease-modifying effects (e.g., reductions in tau hyperphosphorylation) and to improve cognitive function in animal models of AD (Bitner et al., 2010, Medeiros et al., 2014, Wang et al., 2009, Wang et al., 2010).

There is considerable preclinical and some clinical evidence that tropisetron (most likely via its effects at α7 nAChRs) could be repurposed as a therapeutic agent for AD and other neuropsychiatric disorders (e.g., mild cognitive impairment (MCI), Lewy-body dementia and schizophrenia). For example, in the preclinical literature, tropisetron exhibited neuroprotective effects in retinal ganglion cells against glutamate-induced excitotoxicity (Swartz et al., 2013) and it attenuated β-amyloid-induced inflammatory and apoptotic responses in the hippocampus of rats (Rahimian et al., 2013). Moreover, Spilman et al. (2014) demonstrated that tropisetron increased the ratio of the protective soluble amyloid precursor protein alpha to the neurotoxic Aβ1-42 peptide (i.e., the sAPPα/Aβ ratio) and improved spatial and working memory in J20 (PDAPP, huAPP Swe/Ind) AD mice. In these studies, tropisetron was efficacious during both the symptomatic, pre-plaque phase (5–6 months) and in the late plaque phase (14 months) in the J20 mice suggesting therapeutic utility of tropisetron throughout the various stages of AD. Tropisetron has also been shown to improve learning and memory (Hashimoto et al., 2006) and sensory gating deficits (Hashimoto et al., 2005, Kohnomi et al., 2010) in animal models of schizophrenia. These tropisetron-related improvements appeared to be mediated by α7 nAChRs since they were attenuated by the α7 nAChR antagonist methyllycaconitine (MLA; Nirogi et al., 2012). In clinical studies, tropisetron has been shown to improve auditory sensory gating (P50) deficits, cognitive impairments and negative symptoms in patients with schizophrenia (Noroozian et al., 2013, Shiina et al., 2010, Zhang et al., 2012).

The purpose of the present study was to explore the nAChR pharmacology of tropisetron in vitro and to evaluate its effects on cognition in both young and aged rats and aged nonhuman primates. To this end, we were particularly interested in characterizing the novel effects of tropisetron at the recently characterized heteromeric α7β2 nAChR (1:10 ratio) in vitro. This receptor is now known to be expressed in both rodent and human brain regions (e.g., basal forebrain, cortex and hippocampus) involved in learning and memory and may be involved in the pathogenesis of AD (Liu et al., 2009, Liu et al., 2012, Moretti et al., 2014, Thomsen et al., 2015). In the basal forebrain, it has been shown that α7 and β2 mRNAs are co-expressed on cholinergic neurons (i.e., neurons commonly damaged or lost in AD) (Azam et al., 2003). Moreover, nanomolar concentrations of the neurotoxic Aβ1-42 peptide blocked activation of α7β2 nAChR heteromers, suggesting that this receptor could also play a role in the pathology of AD (Liu et al., 2009, Liu et al., 2012). Additionally, we were interested in determining whether tropisetron produces “priming” (or co-agonist) effects at nAChRs (see Discussion for further details). Priming (as opposed to classical agonist or partial agonist) effects have been observed in vitro with the α7 nAChR ligands encenicline, FRM-17874 and RG 3487 (Prickaerts et al., 2012, Stoiljkovic et al., 2015, Wallace et al., 2010, Wallace et al., 2011a) at concentrations that were much more relevant to the doses (and consequent brain levels) that were associated with improved cognitive function in animal models. Lastly, we were interested in characterizing the effects of tropisetron in young and aged rats performing the object recognition task and the delayed-match-to-sample (DMTS) task in aged monkeys. We also sought to characterize the “adjunctive” therapeutic strategy of combining sub-effective doses of the AChEI donepezil and tropisetron in aged monkeys.