Kinase experiment:

Binding or activity of EVP-6124 is measured at 10 μM in a selectivity panel according to standard validated protocols under conditions defined by the contractor. For the 5-HT2A receptor binding assay, membranes are prepared from HEK293 cellsexpressing the human recombinant 5-HT2A receptor. For 5-HT2B and 5-HT2C receptor binding assays, membranes are prepared from CHO cells expressing the human recombinant 5-HT2B or 5-HT2C receptor. Affinity is determined by incubating different concentrations of EVP-6124 in binding buffer for 1 h. For 5-HT2A binding, the incubation is at 22℃ in the presence of 0.5 nM [3H]-ketanserin; for 5-HT2B, at 22℃ in the presence of 2 nM [3H]-mesulergine; and for 5-HT2C, at 37℃ in the presence of 1 nM [3H]-mesulergine. Nonspecific binding is determined in the presence of 1 μM ketanserin, 10 μM mesulergine, or 10 μM RS-102221 for 5-HT2A, 5-HT2B, or 5-HT2C, respectively. All measurements are performed in triplicate. EVP-6124 is also tested in the 5-HT2B rat gastric fundus tissue response assay. Briefly, inhibition of α-methyl serotonin-induced contraction is isometrically measured. All measurements are performed in duplicate[1].

Animal experiment:

Rats[1] Twenty-four 2.5-month-old male Wistar rats (average body weight: 329 g) are used. Before testing Encenicline, the effects of scopolamine alone at 0.03, 0.1, or 0.3 mg/kg, i.p. in the ORT are determined (n=8 per treatment). Scopolamine (0.1 mg/kg, i.p.) injected 30 min before T1 resulted in a robust deficit at T2 when a 1 h interval is used. The d2 index is not significantly different from the chance level of performance; and there are no changes in exploratory behavior for 0.1 mg/kg, i.p. of scopolamine compared with saline. Subsequently, the ability of Encenicline to reverse the memory impairment induced by 0.1 mg/kg of scopolamine is tested. First, scopolamine and then Encenicline (0.03, 0.1, 0.3, and 1.0 mg/kg, p.o.) are administered 30 min before T1. For the control treatments, animals received either deionized water (p.o.) plus saline (i.p.) or deionized water (p.o.) plus 0.1 mg/kg scopolamine (i.p.).Mice[2] Adult male mice (3-6 months old) are used throughout this study. Encenicline is injected i.p. (0.4 mg/kg) at Zeitgeber time (ZT0) in awake mice (9 mice total for this experiment), in the animal facility. Mice are then immediately returned to their home cage with their siblings and left undisturbed for 4 hr (ZT4). During this time, they are closely monitored to check for possible behavioral effects of Encenicline injection. All of the 9 injected mice nested and are immobile in the hour following the injection.

EVP-6124 is a partial agonist of α7 neuronal nicotinic acetylcholine receptors (nAChRs) that was proved high-affinity in vitro and in vivo. [1]

EVP-6124 was proved to show selectivity for α7 but low-affinity of α4β2 nAChRs in some binding and functional experiments. EVP-6124 had good brain penetration and an adequate exposure time. [1,2]

Co-administration of EVP-6124 and the selective nAChRs antagonist could contribute to the release of DA, ACH, and Glu. This could be used to treat cognitive impairment and possibly other dimensions of psychopathology. Co-administration of donepezil at 0.1 mg/kg, p.o. and EVP-6124 at 0.03 mg/kg proved to be fully restored memory while each of these did not improve memory in this task. [1] Pretreatment with the selective α7 nAChR antagonist, methyllycaconitine (MLA, 1.0 mg/kg), significantly blocked cortical DA and Glu efflux induced by EVP-6124 (0.1 mg/kg). [2,3]

References:
1. Jos Prickaerts, Nick P. van Goethem, et al. EVP-6124, a novel and selective α7 nicotinic acetylcholine receptor partial agonist, improves memory performance by potentiating the acetylcholine response of α7 nicotinic acetylcholine receptors. Neuropharmacology, 2012,62 (2): 1099-1110.
2. Mei Huang, Anna R. Felix, et al. Chaya Bhuvaneswaran, Dana Hilt, Gerhard K?nig, Herbert Y. Meltzer, The alpha-7 receptor agonist EVP-6124 increases dopamine and glutamate efflux in rat medial prefrontal cortex and nucleus accumbens. Biochemical Pharmacology, 2011, 82 (8): 1040.
3. Mei Huang, Anna R. Felix, et al. The novel α7 nicotinic acetylcholine receptor agonist EVP-6124 enhances dopamine, acetylcholine, and glutamate efflux in rat cortex and nucleus accumbens. Psychopharmacology, 2014, 231:4541-4551.