The alpha7 nicotinic acetylcholine receptor (α7-nAChR) is a homopentameric ligand-gated ion channel that is involved in the regulation of cognitive processes in normal conditions as well as is in the pathophysiology of some brain disorders. The receptor is composed of five identical α7 subunits, yielding an equal number of binding sites (Dani and Bertrand, 2007; Li et al., 2011). This receptor is widely distributed in the central nervous systems (CNS) and found with high densities in regions associated with cognitive functions (Tribollet et al., 2004; Wessler and Kirkpatrick, 2008). Of note, there is substantial evidence for the presence of heteromeric α7-nAChRs in mammalian CNS, where α7 subunits co-assemble with β2 subunits to form functional α7β2-nAChR (Wu et al., 2016). The exact implications of the existence of this receptor are unknown. The homopentameric α7-nAChR has been investigated as a potential therapeutic target for addressing cognitive impairments associated with neurological and psychiatric diseases, including Alzheimer's disease and schizophrenia (Wallace and Porter, 2011). Over the past 20 years, several compounds have been developed to selectively target the α7-nAChR, demonstrating promising effects in enhancing cognitive functions in animal models (Thomsen et al., 2010). However, a significant translational challenge persists, as these encouraging preclinical outcomes have not translated into corresponding benefits in human clinical trials (Lewis et al., 2017). Some notable examples are TC-5619 from Targacept (Figure 1A), encenicline (EVP-6124) from Forum Pharmaceuticals (Figure 1B), and SSR180711 from Sanofi (Figure 1C). In vitro, TC-5619 is a full and potent α7-nAChR agonist reported to be effective in rodent models of schizophrenia (Hauser et al., 2009). Initially, in an exploratory 12-week randomized clinical phase 2 trial involving 185 subjects with schizophrenia, TC-5619 showed promising effects on cognitive endpoints and negative symptoms compared to placebo (Lieberman et al., 2013). However, in a clinical phase 2 trial lasting 24 weeks, TC-5619 failed to meet the primary outcome measure of change from baseline on the Scale for the Assessment of Negative Symptoms (SANS) compared to placebo (Walling et al., 2016). Additionally, TC-5619 did not demonstrate improvement in the key secondary measures of cognitive function (ClinicalTrials.gov identifier: NCT01488929) and further development was stopped (Targacept, 2013). SSR180711 is a partial α7-nAChR agonist (Biton et al., 2007) that has been shown to improve long-term and short-term episodic memory and spatial working memory in rodents (Pichat et al., 2007). The compound was tested in a placebo-controlled phase 2 clinical trial, spanning 4 weeks and three different dosage regimens, in patients with mild Alzheimer's disease (NCT00602680). However, the trial was prematurely terminated in 2008 due to an inadequate risk-benefit ratio, as documented on clinicaltrials.gov. Encenicline is a partial α7-nAChR agonist in vitro and it reverses a scopolamine-induced memory deficit in vivo in rats (Prickaerts et al., 2012). Despite encenicline showing effects in a proof-of-concept, randomized trial in patients with schizophrenia (Preskorn et al., 2014) and in mild-to-moderate Alzheimer's disease patients on functional and cognitive skills compared to placebo (Deardorff et al., 2015), this outcome was not confirmed in two larger global clinical phase 3 trials as the trials were put on hold due to severe gastrointestinal adverse effects (Alzforum, 2016). Further, encenicline is so far the only compound targeting the α7-nAChR that has been evaluated in a large-scale clinical phase 3 trial in schizophrenia patients, but no effect could be seen (Brannon, 2019) [see review by Terry and Callahan (2020)]. The lack of success in clinical trials involving α7-nAChR ligands in Alzheimer's disease or schizophrenia has reduced the enthusiasm for this target and consequently, many pharmaceutical companies have discontinued their research efforts in this field (Bertrand and Terry, 2018).

However, before discarding α7-nAChR as a viable target, it is worth noting that despite several large clinical trials, little information regarding the compounds' blood-brain barrier permeability, target involvement and the level of α7-nAChR occupancy has been published. For these purposes, molecular brain imaging emerges as a powerful tool capable of providing invaluable insights into target engagement and occupancy. Several α7-nAChR positron emission tomography (PET) radioligands have so far been tested: ¹⁸F-ASEM was developed as an α7-nAChR antagonist with suitable binding properties (Horti et al., 2014) and tested in 21 healthy non-smoking volunteers and in 6 males with schizophrenia (Wong et al., 2014, 2018). We have also validated ¹¹C-NS14492 (Figure 1D) as a selective α7-nAChR agonist PET radioligand capable of measuring α7-nAChR occupancy of SSR180711 and unlabelled NS14492 in the pig brain (Ettrup et al., 2011b). This compound has been further described and validated as a tritiated in vitro radioligand (Magnussen et al., 2015). In our study, we chose pigs as experimental animal due to their physiological and anatomical similarities to humans, facilitating translational relevance. Furthermore, the presence of the nAChR in the pig brain has been well-documented, particularly validated through numerous PET experiments, including a recent study employing ¹⁸F-ASEM, confirming the comparability of nAChR expression patterns between humans and pigs (Donat et al., 2020). Here, we report the effect of TC-5619 and encenicline on ¹¹C-NS14492 binding in the pig brain using both in vitro autoradiography and in vivo PET imaging to elucidate the target occupancy for these molecules.

Here, we present in vitro receptor autoradiography and in vivo ¹¹C-NS14492 PET data on α7-nAChR ligands in the pig brain. The in vitro autoradiography revealed laminar binding in cortical layers with a clear discrimination between gray and white matter binding, consistent with previous studies (Gotti et al., 2006). The three α7-nAChR ligands TC-5619, encenicline, and SSR180711 were all able to block ¹¹C-NS14492 binding in vitro, supporting their effectiveness as competitive ligands for the α7-nAChR orthosteric site.

With in vivo PET imaging we find that 3 mg/kg TC-5619 given i.v. results in 40% occupancy at the α7-nAChR whereas the same dose of encenicline results in negligible occupancy. By using an in vitro homogenate binding assay with ³H-NS14492, we previously found K_i values for TC-5619 and encenicline of 0.063 nM and 0.194 nM respectively (Magnussen et al., 2015). That is, given the observed occupancy of TC-5619, one could expect to find that a dose of 3 mg/kg encenicline would result in 16% α7-nAChR occupancy.

Several factors could explain the low in vivo occupancy of encenicline in the pig brain. If encenicline displayed a very rapid drug clearance it may have insufficient time to be taken up by the brain. We know from rats that encenicline has good brain penetration after oral administration (0.3 mg/kg), with brain-to-plasma ratios of approximately 2 between 1 and 4 h and 5 at 8 h (Prickaerts et al., 2012), indicating that the plasma clearance of encenicline is considerably faster than the brain uptake and receptor equilibration. In humans, and after oral administration, encenicline has a long plasma half-life of 50–65 h (Barbier et al., 2015), but pharmacokinetic differences between humans and pigs could be a factor as well as differences between oral and intravenous administration. Another explanation could lie with the known interspecies differences in blood-brain barrier permeability for compounds like encenicline and TC-5619 among rats, pigs, and potentially humans (Syvanen et al., 2009; Deo et al., 2013; Stanimirovic et al., 2015). This theory could partially explain the reason for encenicline's clinical trial failure. Furthermore, an extensive review of the existing literature and public domain resources has revealed no documented evidence demonstrating the ability of encenicline to cross the blood-brain barrier in humans.

Our investigation in the pig model reveals that TC-5619 effectively penetrates the blood-brain barrier and exhibits significant binding to the α7-nAChR with a reasonable occupancy. However, it is noteworthy that no prior studies have definitively established an optimal range of α7-nAChR occupancy necessary for eliciting therapeutic effects. This lack of established benchmarks raises the possibility that the failure of TC-5619 to produce significant pro-cognitive effects in clinical trials may stem from the selection of suboptimal dosages. Interestingly, previous research on selective α7-nAChR agonists, including encenicline (Keefe et al., 2015), TC-5619 (Hauser et al., 2009), as well as other compounds such as AZD0328 (Castner et al., 2011), DMXB-A (Olincy et al., 2006), and PHA543613 (Yang et al., 2013) has indicated a trend where the pro-cognitive effects peak at lower doses, following an inverted U-shaped dose-response curve. This phenomenon suggests that while lower doses may elicit optimal cognitive enhancement, escalating doses beyond this threshold could lead to diminishing effect or even receptor desensitization, thereby limiting further cognitive improvement. The implications of this dose-response pattern are profound, particularly in the context of human clinical trials. The absence of a clear understanding of optimal dosing presents a considerable challenge, underscoring the need for robust pre-clinical tools for dose finding.

Our study is not without limitations. First, it was not possible to measure drug concentrations during the PET studies so an eventual ultra-fast drug metabolism of encenicline cannot be excluded. As discussed above, this could potentially result in an underestimation of its occupancy. Secondly, only 4 animals were used in total limiting the statistical power of the study. However, each animal served as its own control, and we measured five distinct occupancy regions in each animal. Thirdly, we administered both TC-5619 and encenicline at 3 mg/kg but cannot exclude that an even higher dose of encenicline could have returned a larger occupancy.

In conclusion, we find that the two α7-nAChR ligands TC-5619 and encenicline, when given at equal doses, display different α7-nAChR occupancy in vivo in the pig brain. Our findings underscore the significance of utilizing PET radiotracers in CNS pre-clinical drug development in assessing crucial factors such as blood-brain barrier permeability and target engagement, as well as in aiding dose finding for potential therapeutic agents. By proposing the establishment of target occupancy through PET experiments prior to embarking on clinical trials, we advocate for a more precise dosing strategy for emerging α7-nAChR selective drug candidates. This proactive approach has the potential to mitigate uncertainties surrounding dosing regimens, thereby enhancing the likelihood of therapeutic success while minimizing adverse effects.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal studies were approved by the Danish Council for Animal Ethics. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

JHM: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. AE: Formal analysis, Methodology, Writing – review & editing. SL: Formal analysis, Software, Writing – review & editing. DP: Writing – review & editing. AD: Data curation, Formal Analysis, Writing – review & editing. MT: Methodology, Writing – review & editing. JDM: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Resources, Supervision, Writing – review & editing. GK: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Resources, Supervision, Writing – review & editing.