Evidence for positive allosteric modulation of cognitive-enhancing effects of nicotine in healthy human subjects.

RATIONALE: Cognitive benefits of nicotinic acetylcholine receptor (nAChR) agonists are well established but have generally been of small magnitude and uncertain clinical significance. A way of raising the effect size may be to facilitate agonist-induced responses by co-administering a nAChR positive allosteric modulator (PAM).
OBJECTIVE: The aim was to test whether galantamine, a PAM at several nAChR subtypes, can potentiate the cognitive-enhancing effects of nicotine.
METHODS: Twenty-six adult never-smokers were treated, in a double-blind counterbalanced sequence, with nicotine (7 mg/24 h, transdermally) and galantamine (4 mg, p.o.) combined, nicotine alone, galantamine alone, and double placebo. A low dose of galantamine was chosen to minimize acetylcholinesterase inhibition, which was verified in blood assays. In each condition, participants were tested with three cognitive tasks.
RESULTS: Nicotine significantly improved reaction time (RT) and signal detection in a visuospatial attention task and the Rapid Visual Information Processing Task. Galantamine did not modulate these effects. A trend toward RT reduction by galantamine correlated with acetylcholinesterase inhibition. In a change detection task, there were no effects of nicotine or galantamine alone on accuracy or RT. However, both drugs combined acted synergistically to reduce RT. This effect was not associated with acetylcholinesterase inhibition.
CONCLUSIONS: A pattern consistent with allosteric potentiation of nicotine effects by galantamine was observed on one of six performance measures. This may reflect specific nAChR subtype involvement, or additional pharmacological actions of galantamine may have overshadowed similar interactions on other measures. The finding suggests that allosteric potentiation of nAChR agonist-induced cognitive benefits is possible in principle.

RCT Entities:   Population Interventions Outcomes

Introduction

Several disease states marked by <span class="Disease">cognitive deficits, most prominently <span class="Disease">schizophrenia and <span class="Disease">Alzheimer’s disease, involve <span class="Gene">nicotinic acetylcholine receptor (<span class="Gene">nAChR) hypofunction (Adams and Stevens 2007; Hong et al. 2011; Kendziorra et al. 2011; Perry et al. 2000; Petrovsky et al. 2010; Wing et al. 2012) and may benefit from treatments that enhance <span class="Gene">nAChR activity (Levin and Rezvani 2002; Singh et al. 2004). Acute <span class="Disease">cognitive benefits of the prototypical non-selective <span class="Gene">nAChR agonist <span class="Chemical">nicotine are well established, particularly on attention but also on sensory information processing and mnemonic processes (Hahn 2015; Heishman et al. 2010; Newhouse et al. 2011), although the clinical benefit of chronic treatment with <span class="Chemical">nicotine is unclear. Drug development efforts have been invested into subtype-selective <span class="Gene">nAChR agonists for the above conditions. Effects with both α4β2- and α7-selective <span class="Gene">nAChR agonists have generally been in the expected direction, but tend to be of small magnitude and uncertain clinical significance (Haydar and Dunlop 2010; Radek et al. 2010; Wallace et al. 2011). Many compounds failed clinical trials due to limited efficacy (Haydar and Dunlop 2010; Hurst et al. 2013).

A way of raising the effect size ceiling may be to co-administer a <span class="Gene">nAChR agonist and a <span class="Gene">nAChR positive allosteric modulator (PAM). PAMs do not activate nAChRs on their own. Instead, PAMs bind to an allosteric site on the receptor and facilitate agonist-induced responses (<span class="Disease">Williams et al. 2011). Some, although not all (Gronlien et al. 2007), PAMs reverse desensitization of a fraction of nAChRs, specifically in the presence of low to intermediate agonist concentrations (<span class="Disease">Williams et al. 2011). Thus, through partial reversal of desensitization or other mechanisms, combined PAM and low-dose agonist treatment may enhance <span class="Gene">nAChR activity and associated behavioral effects to a greater degree than agonist treatment alone. Despite much discussion and interest in <span class="Gene">nAChR PAMs, the ability of a PAM to enhance cognitive effects of <span class="Chemical">nicotine or other <span class="Gene">nAChR agonists has never been tested in a systematic manner in either animals or <span class="Species">humans. Dual administration studies performed in <span class="Species">people with <span class="Disease">schizophrenia (Choueiry et al. 2019; Deutsch et al. 2013) were not designed to differentiate between the effects of the PAM, the agonist, or their combination.

To date, the only <span class="Gene">nAChR PAM commercially available for <span class="Species">human use is the <span class="Gene">acetylcholinesterase (<span class="Gene">AChE) inhibitor <span class="Chemical">galantamine, approved by the Food and Drug Administration for the treatment of mild to moderate <span class="Disease">Alzheimer’s disease. <span class="Chemical">Galantamine is a <span class="Gene">nAChR PAM at concentrations found in the <span class="Species">human brain after clinical doses (Coyle et al. 2007; Villarroya et al. 2007). It has been shown to potentiate α4β2, α3*, α6β4, and α7 <span class="Gene">nAChR currents induced by <span class="Chemical">acetylcholine, <span class="Chemical">nicotine, or <span class="Chemical">epibatidine, causing long-lasting increases in <span class="Gene">nAChR response amplitude and frequency (Dajas-Bailador et al. 2003; Samochocki et al. 2003; Santos et al. 2002). Thus, while <span class="Chemical">galantamine’s <span class="Gene">AChE inhibitory action increases concentrations of <span class="Chemical">acetylcholine in the synaptic cleft, its <span class="Gene">nAChR PAM action renders the <span class="Gene">nAChR more responsive to <span class="Chemical">acetylcholine and exogenous <span class="Gene">nAChR agonists (Albuquerque et al. 2009). Importantly, the concentration range for <span class="Chemical">galantamine’s PAM action appears to be slightly below that for <span class="Gene">AChE inhibition (Coyle et al. 2007). Thus, it appears that a bias toward its PAM action can be achieved by testing a small dose of <span class="Chemical">galantamine and minimizing <span class="Gene">AChE inhibition.

The aim of the present study was to test whether performance-enhancing effects of a <span class="Gene">nAChR agonist can be potentiated by the co-administration of a <span class="Gene">nAChR PAM. For this proof-of-principle study, we tested the interaction of a low dose of <span class="Chemical">galantamine with the prototypical <span class="Gene">nAChR agonist <span class="Chemical">nicotine on a broad array of cognitive functions in healthy non-smoker. Non-smokers were selected to avoid potential confounds related to chronic <span class="Chemical">nicotine exposure, such as neuroadaptive changes and <span class="Chemical">nicotine withdrawal. Even at small doses, <span class="Chemical">galantamine may have additional pharmacological actions; however, any potentiation of <span class="Chemical">nicotine effects could not be explained by <span class="Gene">AChE inhibition but by allosteric potentiation of <span class="Gene">nAChR activity. Our specific predictions were no or little effect of <span class="Chemical">galantamine alone, but larger performance-enhancing effects of <span class="Chemical">nicotine in the presence of <span class="Chemical">galantamine than in its absence.

Methods

Participants

Out of 43 healthy non-smokers enrolled in the study, 27 completed it (16 females, 11 males; 10 African American, 2 Asian, 13 Caucasian, 2 Hispanic). Reasons for non-completion were adverse effects in 8 cases (7 cases of <span class="Disease">vomiting; 1 case of <span class="Disease">nausea, jitteriness, and palpitations), no longer meeting inclusion criteria in 2 cases, and withdrawal for personal reasons in 6 cases. Non-completers were replaced; our target was to have at least 24 completers based on power calculation indicating that an interaction of medium effect size could be detected with this sample size. One study completer was excluded from analyses of performance data because this subject’s performance in two of the three tasks was marked by a large proportion of no-response trials, suggesting a lack of task engagement.

The remaining 26 subjects who completed the study were 22–51 years of age (mean ± SD: 33.4 ± 10.4) with 13–22 years of education (16.3 ± 2.3). <span class="Species">Participants were recruited from the local community through internet advertising, flyers, and referrals, and gave written informed consent for a protocol approved by the University of Maryland Baltimore Institutional Review Board. <span class="Species">Participants had no more than 40 cigarettes in their lifetime and no <span class="Chemical">nicotine exposure in the last year. Use of centrally active medications, pregnancy, history of <span class="Disease">neurological or psychiatric disorders including <span class="Disease">drug abuse, significant <span class="Disease">liver or kidney impairment, heart problems, hyper- or <span class="Disease">hypotension, and <span class="Disease">learning disability were exclusion criteria.

Drugs

<span class="Chemical">Nicotine patches were over-the-counter Nicoderm CQ patches (GlaxoSmithKline, Brentford, Middlesex, UK) releasing 7 mg of <span class="Chemical">nicotine in 24 h, close to the lowest dose available. Placebo patches were generated using AquaHeal Hydrogel Bandages (Spenco Medical Corporation), cut to size and with identifying labeling removed. The hydrogel bandages closely resemble the <span class="Chemical">nicotine patch in color and consistency. The <span class="Chemical">nicotine or size-matched placebo patch was placed on the inside of an adhesive bandage on the day of the study and sealed in a small ziplock bag until application. The adhesive bandage with patch was applied by a study nurse not involved in any other study procedures.

<span class="Chemical">Galantamine HBr immediate release tablets (Patriot Pharmaceuticals, Horsham, PA) were ground up and packaged into capsules for p.o. administration at a dose of 4 mg per capsule by an in-house compounding pharmacist, who also produced matching placebo capsules containing microcrystalline cellulose. The low dose of <span class="Chemical">galantamine was chosen to induce a bias toward its PAM action (Coyle et al. 2007).

Study design and procedures

The study adopted a double-blind within-subject design. Each <span class="Species">participant was tested on four separate days. On each day, a skin patch was applied, and a capsule was administered. On one day, both the patch and the capsules were a placebo (placebo session). On another day, the patch was a <span class="Chemical">nicotine patch (7 mg/24 h) and the capsule was a placebo (<span class="Chemical">nicotine session); on another, the patch was a placebo and the capsule contained 4 mg of <span class="Chemical">galantamine (<span class="Chemical">galantamine session); and on another day, the patch was a <span class="Chemical">nicotine patch (7 mg/24 h) and the capsule contained 4 mg of <span class="Chemical">galantamine (<span class="Chemical">nicotine + <span class="Chemical">galantamine session). Thus, the four conditions followed a 2 × 2 factorial design (see Fig. 1) and were tested in a sequence that was counterbalanced across <span class="Species">participants to the degree possible.

Fig. 1

Experimental design

The study involved six total visits: one consent and screening visit, one training visit, and the four test sessions, the latter scheduled at least 1 week apart to ensure complete drug washout and normalization of any potential secondary drug effects between sessions. Screening included a medical history and physical exam, an electrocardiogram, blood and urine labs, a vision test, and tests for drug use, smoking, and pregnancy. During the training visit, <span class="Species">participants were given task instructions and performed a full-length version of each of the cognitive tasks described below, to minimize practice effects between test sessions.

Each test session took approximately 7 h. Upon arrival in the morning, <span class="Species">participants were tested for <span class="Disease">fever and recent <span class="Chemical">alcohol use or smoking, and a urine sample was tested for pregnancy and drug use, all of which had to be negative for the session to proceed. Resting blood pressure and heart rate measurements were taken, and <span class="Species">participants completed a side effect checklist, rating possible side effects of <span class="Chemical">nicotine and <span class="Chemical">galantamine (restlessness, <span class="Disease">weakness/<span class="Disease">fatigue, <span class="Disease">dizziness, <span class="Disease">headache, <span class="Disease">dry mouth, <span class="Disease">nausea, <span class="Disease">abdominal pain, sweating, palpitations, jitteriness, <span class="Disease">sleepiness, <span class="Disease">diarrhea, <span class="Disease">decreased appetite, stomach discomfort, difficulty urinating) as none (1), mild (2), moderate (3), or severe (4). <span class="Species">Participants then completed the Profile of Mood States (POMS), an adjective rating questionnaire considered a standardized subjective mood state inventory (McNair et al. 1971).

Next, the study patch was administered. Vital signs and the side effect checklist were obtained hourly thereafter. During the drug absorption period, <span class="Species">participants were permitted to read, watch movies, or use the internet. Three and a half hours after patch administration, <span class="Species">participants swallowed the study capsule, after which vital signs and the side effect checklist were obtained every 30 min. Five hours after patch application (1.5 h after capsule administration), the POMS was again completed, and cognitive testing began. This timing was based on peak drug concentrations after administration, as <span class="Chemical">nicotine plasma concentrations have been shown to reach asymptote by 5 h post-patch administration (Fant et al. 2000; Gupta et al. 1993), and <span class="Chemical">galantamine plasma concentrations reach tmax 1.2–1.6 h after p.o. administration of a 4-mg dose (Zhao et al. 2002). The order of the cognitive tasks always remained the same: first the Spatial Attentional Resource Allocation Task, then the Rapid Visual Information Processing Task, and last the Change Detection Task. Testing took approximately 1.5 h in total. Vital signs were measured after the first task. Immediately after cognitive testing, the POMS and side effect checklist were completed and vital signs were taken one last time, and a 5-ml blood sample was obtained from a forearm vein for analysis of <span class="Chemical">nicotine concentrations and <span class="Gene">AChE activity. The blood draw was performed approximately 3:15 h after <span class="Chemical">galantamine dosing.

Equipment

All tasks were performed on a 19-in. 5:4 <span class="Disease">IPS LCD monitor with a screen resolution of 1280 × 1024 and a refresh rate of 60 Hz. Responses were recorded using a Logitech F310 gamepad controller. Only the left and right bumper buttons were used. In tasks involving a single button, subjects responded with their dominant hand. All tasks were created and run in E-Prime version 2.0.

Task paradigms

Spatial Attentional Resource Allocation Task

The Spatial Attentional Resource Allocation Task (SARAT) is a visuospatial stimulus detection paradigm (Hahn et al. 2006), shown to be sensitive to the performance-enhancing effects of <span class="Chemical">nicotine (Hahn et al. 2013; Hahn et al. 2007). <span class="Species">Participants fixated on a quartered circle in the center of the screen (diameter approximately 2.6° of visual angle), black against a light gray (10% contrast; 130 cd/m2) background (Fig. 2a). They were instructed to respond as quickly as possible when detecting a 500-ms target stimulus appearing in one of four locations in the corners of the screen, marked by circular place holders (diameter 1.3° of visual angle), positioned at 10° of visual angle.

Fig. 2

Components of a trial in the Spatial Attentional Resource Allocation Task (a) and the Change Detection Task (b)

A cue in the central circle preceded the target signal by 400, 700, 1000, or 1300 ms, chosen randomly to make target onset temporally unpredictable and discourage anticipatory responding to the cue. Either one quarter turned black to indicate the location of the upcoming target (predictive cue), or all 4 quarters turned black (non-predictive cue). Predictive cue trials allowed for a narrow attentional focus, while non-predictive cue trials required attention to be spread widely to encompass the entire display. <span class="Species">Participants were asked to respond as quickly as possible with their dominant index finger upon detecting a target. Targets were presented in the continued presence of the cue and consisted of peripheral placeholders filling with a gray (40% contrast) and white checkerboard pattern of 3 × 3 pixels each. The cue persisted for 500 ms after target offset. Only task background was then displayed for an inter-trial interval (ITI) of 400, 700, 1000, or 1300 ms.

The task was presented in eight 5-min blocks of 60 trials each: 30 predictive cue trials, of which six had no target to discourage anticipatory responding to the cue, and 30 non-predictive cue trials, of which six had no target. To increase the temporal jitter of the task and augment stimulus detection demands, 30 additional 2.7-s periods during which only task background was presented were interspersed randomly between trials. The entire task took approximately 45 min to complete.

Rapid Visual Information Processing Task

The Rapid Visual Information Processing Task (RVIPT) requires the maintenance of intense rapid information processing and <span class="Disease">working memory demands over time. Performance therefore reflects processing speed, sustained attention, and working memory.

The RVIPT has been used extensively to evaluate the cognitive effects of cholinergic agents and is sensitive to the performance-enhancing effects of <span class="Chemical">nicotine (Foulds et al. 1996; Warburton and Mancuso 1998; Wesnes and Warburton 1984). The task consists of a string of digits (1 through 9), presented one at a time at a rate of 100/min. Each digit was presented for 600 ms, with no ITI. <span class="Species">Participants were instructed to respond with their dominant index finger when they identified three consecutive odd or even digits. Responses within an 1800-ms time window following the onset of the last digit of a target sequence were considered hits; all other responses were considered false alarms. On average, 8 target sequences were presented per minute. The number of digits separating targets ranged from 4 to 29. The task was performed in two blocks of 15 min each, with a break between to ensure feasibility.

Change Detection Task

The Change Detection Task (CDT) is a visual short-term memory task (Luck and Vogel 1997) and was included as a probe for potential mnemonic drug effects. It is relatively inconducive to verbal rehearsal. A 220-ms encoding array of either 1 or 5 colored squares was presented (Fig. 2b). Possible colors were red, magenta, purple, yellow, white, blue, cyan, green, <span class="Species">olive, and teal. Half of the trials showed 5 colored squares and the other half showed 1 colored square. After a 1100-ms retention interval, one square reappeared for 2000 ms, and <span class="Species">participants determined whether this square was of the same or a different color than the square previously displayed at this location. On half the trials, the color was the same, and on the other half, the color changed. <span class="Species">Participants responded “same” with a right button press, and “different” with a left button press. Trials were separated by a 1000-ms inter-trial interval. The task consisted of 180 total trials, presented over 5 blocks of 36 trials each, with short breaks between blocks. Total task duration was 13 min.

Blood analyses

Immediately after the blood draw at the end of each test session, 0.2 ml of whole blood was pipetted off, and the rest of the sample was centrifuged to separate plasma from red blood cells. Whole blood and plasma samples were frozen at − 80 °C until analysis upon study completion. Only samples from study completers were analyzed.

Analyses of <span class="Chemical">nicotine and <span class="Chemical">cotinine plasma concentrations were performed by NMS Labs (Willow Grove, PA) by high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). Because smoking abstinence was considered sufficiently verified, only samples from the <span class="Chemical">nicotine session and <span class="Chemical">nicotine + <span class="Chemical">galantamine session were analyzed. The reporting limits were 2.5 ng/ml for <span class="Chemical">nicotine and 5 ng/ml for <span class="Chemical">cotinine.

Whole blood <span class="Gene">AChE activity was determined using a modification of the radiometric <span class="Gene">cholinesterase procedure (<span class="Disease">Johnson and Russell 1975). In brief, individual whole blood samples (45 μl) pretreated with the selective butyryl<span class="Gene">cholinesterase inhibitor <span class="Chemical">tetraisopropyl pyrophosphoramide (100 μM) were incubated with <span class="Chemical">acetylcholine (5 μl, 0.1 M ACh [<span class="Chemical">acetyl-3H (20 μCi/ml)]) at room temperature for 3 min. The reaction was stopped with a 100-μl aqueous solution of <span class="Chemical">chloroacetic acid (0.50 M), <span class="Chemical">sodium chloride (1 M), and <span class="Chemical">sodium hydroxide (0.25 M), and samples were clarified by centrifugation. The clarified samples (140 μl) were transferred to a scintillation fluor cocktail consisting of 90% (v/v) <span class="Chemical">toluene, 10% (v/v) <span class="Chemical">3-metyl-1-butanol, 0.03% (w/v) <span class="Chemical">1,4-bis(5-phenyl-2-oxazolyl), and 0.05% (w/v) 2,<span class="Chemical">5-diphenyloxazole (final reaction volume = 4 ml). The mix was vortexed for 60 s. The amount of tritiated <span class="Chemical">acetate in the organic phase was measured by liquid scintillation counting for 2 min (<span class="Chemical">Tri-Carb 2900TR, Perkin Elmer). Each sample was assayed in triplicate and counts were corrected for background by subtraction of counts obtained for whole blood samples devoid of <span class="Gene">cholinesterase activity.

Statistical analysis

Vital signs and each subjective state scale from the side effect checklist were analyzed by three-factor ANOVA with <span class="Chemical">nicotine, <span class="Chemical">galantamine, and time as within-subject factors, including only the last four measurement time points at which both <span class="Chemical">nicotine and <span class="Chemical">galantamine absorption had taken place (if administered).

Each of the seven POMS scales (tension/<span class="Disease">anxiety, <span class="Disease">depression, anger/hostility, vigor/activity, <span class="Disease">fatigue, confusion, total mood disturbance) was analyzed by three-factor ANOVA with <span class="Chemical">nicotine, <span class="Chemical">galantamine, and time (baseline, pre-test, post-test) as within-subject factors.

<span class="Chemical">Nicotine and <span class="Chemical">cotinine plasma concentrations were compared between the <span class="Chemical">nicotine session and the <span class="Chemical">nicotine + <span class="Chemical">galantamine session by paired samples t tests. <span class="Gene">AChE activity was compared between the four drug conditions by two-factor ANOVA for repeated measures with <span class="Chemical">nicotine (present vs. absent) and <span class="Chemical">galantamine (present vs. absent) as within-subject factors.

SARAT

Average RT and percentage of omission errors were analyzed by separate three-factor ANOVAs with <span class="Chemical">nicotine, <span class="Chemical">galantamine, and cue type (predictive vs. non-predictive) as within-subject factors.

RVIPT

Because of the large number of opportunities to make a false alarm, the false alarm rate was < 1% for all but one <span class="Species">participant. Consequently, the sensitivity index A’ (Grier 1971) yielded a virtually identical performance pattern across conditions as the hit rate. Expectably, analysis of the false alarm rate did not yield any significant effects; thus, analyses reported here focus on the hit rate (percentage of target detections out of all targets presented) and mean RT. These variables were analyzed by three-factor ANOVA with <span class="Chemical">nicotine, <span class="Chemical">galantamine, and time period (3 periods of 10 min each) as within-subject factors.

CDT

Trials without a response (mean ± SD 1.2 ± 2.9 trials) were excluded from analyses. Accuracy (percentage of correct responses out of all response trials) and mean RT were analyzed by separate three-factor ANOVAs with <span class="Chemical">nicotine, <span class="Chemical">galantamine, and set size (1 vs. 5) as within-subject factors.

<span class="Chemical">Nicotine × <span class="Chemical">galantamine interactions on any performance measure, which were of primary interest in this study, were Bonferroni-corrected for six analyzed performance measures. Any performance effects of <span class="Chemical">galantamine underwent Pearson’s correlation with <span class="Gene">AChE inhibition by <span class="Chemical">galantamine. Effect sizes are reported as partial eta squared (η2), with η2 = 0.06 generally considered a medium and η2 > 0.14 a large effect size (Cohen 1988). Significance testing was based on P < 0.05, two-sided.

Results

Adverse effects by drug conditions

Out of the 8 <span class="Species">participants who were excluded due to greater-than-mild side effects (see above), 5 experienced side effects in the <span class="Chemical">nicotine session and 3 in the <span class="Chemical">nicotine + <span class="Chemical">galantamine session. Two of the latter 3 <span class="Species">participants dropped out before the capsule was administered. Thus, side effects were largely, if not entirely, related to <span class="Chemical">nicotine administration. The one <span class="Species">participant who got sick after combined <span class="Chemical">nicotine and <span class="Chemical">galantamine administration had previously completed the <span class="Chemical">nicotine session without side effects, suggesting that <span class="Chemical">galantamine may have potentiated adverse effects of <span class="Chemical">nicotine in this case. However, one <span class="Species">participant who experienced side effects in the <span class="Chemical">nicotine session had previously completed the <span class="Chemical">nicotine + <span class="Chemical">galantamine session without side effects, suggesting that day-to-day variation in other factors influenced the response to <span class="Chemical">nicotine.

In the 27 completers, vital signs and the subjective state variables of the side effect checklist were analyzed by three-factor ANOVA (<span class="Chemical">nicotine × <span class="Chemical">galantamine × time) including the last four measurement time points: 4.5 h after patch administration (= 1 h after capsule administration); 5 h post-patch (= 1.5 h post-capsule, start of testing); 5:45 h post-patch (= 2:15 h post-capsule, mid-testing); and 6:30 h post-patch (= 3 h post-capsule, post-testing). There were no main effects or interactions involving <span class="Chemical">galantamine. However, there was a significant main effect of <span class="Chemical">nicotine on systolic blood pressure, diastolic blood pressure, and heart rate [F(1,26) > 17.6, P < 0.001 in each case] reflecting increases in the presence of <span class="Chemical">nicotine (Fig. 3a). There were also main effects of <span class="Chemical">nicotine on <span class="Disease">nausea [F(1,26) = 5.90, P = 0.022] and <span class="Disease">weakness/<span class="Disease">fatigue [F(1,26) = 4.42, P = 0.045]. Six <span class="Species">participants reported <span class="Disease">nausea (all “mild”) at at least one of the time points analyzed: 4 in the <span class="Chemical">nicotine session and 2 in the <span class="Chemical">nicotine + <span class="Chemical">galantamine session. The effects of <span class="Chemical">nicotine on <span class="Disease">weakness/<span class="Disease">fatigue interacted with time [F(1,26) = 3.64, P = 0.016]: <span class="Disease">weakness/<span class="Disease">fatigue increased with cognitive testing, and <span class="Chemical">nicotine appeared to alleviate this increase (Fig. 3b).

Fig. 3

Effects of nicotine and galantamine on vital signs averaged over the last four measurement time points (a) and on self-report scales from the side effect checklist (b). Error bars reflect SEMs. Possible ratings on the self-report scales are 1 = none, 2 = mild, 3 = moderate, and 4 = severe

Effects of <span class="Chemical">nicotine and <span class="Chemical">galantamine on vital signs averaged over the last four measurement time points (a) and on self-report scales from the side effect checklist (b). Error bars reflect SEMs. Possible ratings on the self-report scales are 1 = none, 2 = mild, 3 = moderate, and 4 = severe

Subjective drug effects as measured by the POMS

There were no significant main effects of <span class="Chemical">nicotine or <span class="Chemical">galantamine on any of the seven POMS subscales, and no <span class="Chemical">nicotine × <span class="Chemical">galantamine interactions.

Nicotine and cotinine concentrations in blood plasma

For one <span class="Species">participant, we were unable to obtain a blood sample in the <span class="Chemical">nicotine session. In the other subjects, <span class="Chemical">nicotine concentrations averaged 6.04 ± 1.45 ng/ml (range 2.8–9.6) in the <span class="Chemical">nicotine session and 6.20 ± 1.71 ng/ml (range 2.7–9.2) in the <span class="Chemical">nicotine + <span class="Chemical">galantamine session [t(24) = 0.47, P = 0.65], comparable to the 5.9 ng/ml average plasma concentration observed with a <span class="Chemical">nicotine patch of the same dose in smokers (Gorsline et al. 1993). <span class="Chemical">Cotinine concentrations averaged 30.7 ± 9.7 ng/ml in the <span class="Chemical">nicotine session and 33.8 ± 11.5 ng/ml in the <span class="Chemical">nicotine + <span class="Chemical">galantamine session [t(24) = 1.54, P = 0.14].

Effects of galantamine on AChE activity in whole blood

A low dose of <span class="Chemical">galantamine (4 mg) was chosen with the purpose of minimizing <span class="Gene">AChE inhibition and associated behavioral effects. Figure 4 shows mean <span class="Gene">AChE activity in whole blood samples obtained at the end of each test session. <span class="Gene">AChE inhibition by <span class="Chemical">galantamine was modest (16.8%) but statistically significant [main effect of <span class="Chemical">galantamine: F(1,24) = 101.0, P < 0.001] and consistent with the 15% reduction in <span class="Gene">AChE activity reported by a previous study employing the same dose of <span class="Chemical">galantamine (Morasch et al. 2015). There was no main effect of <span class="Chemical">nicotine [F(1,24) = 0.00, P = 0.99] and no <span class="Chemical">galantamine × <span class="Chemical">nicotine interaction [F(1,24) = 1.25, P = 0.27].

Fig. 4

Average (± SEM) acetylcholinesterase (AChE) activity in whole blood in each drug condition. ***P < 0.001 in paired t tests comparing the galantamine session to the placebo session and the nicotine + galantamine session to the nicotine session

Average (± SEM) <span class="Gene">acetylcholinesterase (<span class="Gene">AChE) activity in whole blood in each drug condition. ***P < 0.001 in paired t tests comparing the <span class="Chemical">galantamine session to the placebo session and the <span class="Chemical">nicotine + <span class="Chemical">galantamine session to the <span class="Chemical">nicotine session

Drug effects on task performance

Responses were slower and there were more omission errors for non-predictive than predictive cue trials [main effect of cue type on RT: F(1,25) = 66.8, P < 0.001, η2 = 0.73; omissions: F(1,25) = 4.84, P < 0.037, η2 = 0.16]. <span class="Chemical">Nicotine shortened RT [F(1,25) = 5.40, P = 0.028, η2 = 0.18] and reduced omission errors overall [F(1,25) = 5.78, P = 0.024, η2 = 0.19]. There were no main effects of <span class="Chemical">galantamine [RT: F(1,25) = 0.54, P = 0.47, η2 = 0.02; omissions: F(1,25) = 0.07, P = 0.80, η2 = 0.003] and no <span class="Chemical">nicotine × <span class="Chemical">galantamine interactions [RT: F(1,25) = 1.22, P = 0.28, η2 = 0.05; omissions: F(1,25) = 1.20, P = 0.29, η2 = 0.05]. However, on RT, the effects of cue type interacted with both <span class="Chemical">nicotine [F(1,25) = 11.3, P = 0.002, η2 = 0.31] and <span class="Chemical">galantamine [F(1,25) = 11.5, P = 0.002, η2 = 0.31]. Figure 5 suggests that both drugs reduced RT in non-predictive more than in predictive cue trials. Paired t tests comparing <span class="Chemical">nicotine and placebo collapsed over levels of <span class="Chemical">galantamine confirmed that <span class="Chemical">nicotine reduced RT in non-predictive cue trials [t(25) = 3.17, P = 0.004, η2 = 0.29; P = 0.016 after Bonferroni correction for 4 comparisons] but not in predictive cue trials [t(25) = 1.23, P = 0.21, η2 = 0.06]. t tests comparing <span class="Chemical">galantamine and placebo collapsed over levels of <span class="Chemical">nicotine found no significant effect in either predictive [t(25) = 0.004, P > 0.99, η2 = 0.00] or non-predictive cue trials [t(25) = 1.42, P = 0.17, η2 = 0.07].

Fig. 5

Effects of nicotine and galantamine on reaction time in the Spatial Attentional Resource Allocation Task. Bars reflect the mean performance in each drug condition. Error bars reflect SEMs, adjusted to remove between-subject variability in the average performance across dose levels (Cousineau 2007; Morey 2008) to yield variability related to interindividual differences in drug effect. **P < 0.01 in paired t test comparing performance after nicotine to performance after vehicle

Effects of <span class="Chemical">nicotine and <span class="Chemical">galantamine on reaction time in the Spatial Attentional Resource Allocation Task. Bars reflect the mean performance in each drug condition. Error bars reflect SEMs, adjusted to remove between-subject variability in the average performance across dose levels (Cousineau 2007; Morey 2008) to yield variability related to interindividual differences in drug effect. **P < 0.01 in paired t test comparing performance after <span class="Chemical">nicotine to performance after vehicle

To test whether the trend for <span class="Chemical">galantamine to reduce RT in non-predictive cue trials reflected residual <span class="Gene">AChE inhibition, individual <span class="Species">participants’ RT and <span class="Gene">AChE activity, averaged over non-<span class="Chemical">galantamine test days (i.e., the placebo and <span class="Chemical">nicotine sessions), was subtracted from values averaged over <span class="Chemical">galantamine test days (the <span class="Chemical">galantamine and <span class="Chemical">nicotine + <span class="Chemical">galantamine sessions) and underwent Pearson’s correlation. There was a significant correlation between the effects of <span class="Chemical">galantamine on <span class="Gene">AChE activity and on RT (R = 0.39, P = 0.045), reflecting greater RT reduction in <span class="Species">participants with greater <span class="Gene">AChE inhibition (Fig. 6).

Fig. 6

Correlation between the effect of galantamine on AChE activity and on SARAT reaction time across cue types. For both variables, difference (Δ) values were derived by subtracting the average value across the two no-galantamine sessions (placebo session and nicotine session) from the average value across the two sessions involving galantamine administration (galantamine session and nicotine + galantamine session)

Correlation between the effect of <span class="Chemical">galantamine on <span class="Gene">AChE activity and on SARAT reaction time across cue types. For both variables, difference (Δ) values were derived by subtracting the average value across the two no-<span class="Chemical">galantamine sessions (placebo session and <span class="Chemical">nicotine session) from the average value across the two sessions involving <span class="Chemical">galantamine administration (<span class="Chemical">galantamine session and <span class="Chemical">nicotine + <span class="Chemical">galantamine session)

Figure 7a shows that <span class="Chemical">nicotine increased hit rate [main effect: F(1,25) = 13.1, P < 0.001, η2 = 0.34] and reduced RT [F(1,25) = 6.94, P = 0.014, η2 = 0.14]. There was no significant main effect of <span class="Chemical">galantamine on hit rate [F(1,25) = 0.004, P = 0.95, η2 = 0.00] or RT [F(1,25) = 0.000, P > 0.99, η2 = 0.00]. The effects of <span class="Chemical">nicotine appeared larger in the presence of <span class="Chemical">galantamine, in part because <span class="Chemical">galantamine alone appeared to slow RT; however, the <span class="Chemical">nicotine × <span class="Chemical">galantamine interaction was not significant on RT [F(1,25) = 2.27, P = 0.15, η2 = 0.08] or hit rate [F(1,25) = 0.53, P = 0.47, η2 = 0.02]. There was a significant main effect of time period on both hit rate [F(1,25) = 12.4, P < 0.001; η2 = 0.33] and RT [F(1,25) = 3.52, P = 0.037; η2 = 0.12], reflecting performance decrement with time on task in both cases. The only significant interaction involving time period was with <span class="Chemical">galantamine on hit rate [F(1,25) = 3.43, P = 0.04; η2 = 0.12; all other P > 0.3], which was based on <span class="Chemical">galantamine attenuating the decrement over time (Fig. 7b).

Fig. 7

a Effects of nicotine and galantamine on hit rate and reaction time in the Rapid Visual Information Processing Task. Bars reflect the mean performance in each drug condition. *P < 0.05, **P < 0.01 in paired t tests comparing performance after nicotine to performance after vehicle. b Effects of galantamine, averaged over the presence and absence of nicotine, on hit rate in each of three 10-min time periods. Error bars in a and b reflect SEMs, adjusted to remove between-subject variability in the average performance across dose levels (Cousineau 2007; Morey 2008)

a Effects of <span class="Chemical">nicotine and <span class="Chemical">galantamine on hit rate and reaction time in the Rapid Visual Information Processing Task. Bars reflect the mean performance in each drug condition. *P < 0.05, **P < 0.01 in paired t tests comparing performance after <span class="Chemical">nicotine to performance after vehicle. b Effects of <span class="Chemical">galantamine, averaged over the presence and absence of <span class="Chemical">nicotine, on hit rate in each of three 10-min time periods. Error bars in a and b reflect SEMs, adjusted to remove between-subject variability in the average performance across dose levels (Cousineau 2007; Morey 2008)

Responses were less accurate and slower for set size 5 than set size 1, as supported by significant main effects of set size for accuracy [F(1,25) = 286.7, P < 0.001, η2 = 0.92] and RT [F(1,25) = 85.8, Ps < 0.001, η2 = 0.77]. For accuracy, no other main effects or interactions were significant. However, there was a significant <span class="Chemical">nicotine × <span class="Chemical">galantamine interaction on RT [F(1,25) = 8.40, P < 0.008, η2 = 0.25], which remained significant even after Bonferroni correction for six analyzed performance measures. Figure 8 illustrates that the combined administration of <span class="Chemical">nicotine and <span class="Chemical">galantamine reduced RT relative to all other conditions, while the other conditions did not differ from each other. Thus, while neither <span class="Chemical">nicotine nor <span class="Chemical">galantamine alone had any effects by themselves, their combination acted synergistically to speed responding. This interaction did not depend on set size [<span class="Chemical">nicotine × <span class="Chemical">galantamine × set size interaction: F(1,25) = 0.02, P = 0.90, η2 = 0.00].

Fig. 8

Effects of nicotine and galantamine on reaction time in the Change Detection Task, averaged over set sizes 1 and 5. Bars reflect the mean performance in each drug condition. Error bars reflect SEMs, adjusted to remove between-subject variability in the average performance across dose levels (Cousineau 2007; Morey 2008). *P < 0.05, **P < 0.01 in paired t tests

Effects of <span class="Chemical">nicotine and <span class="Chemical">galantamine on reaction time in the Change Detection Task, averaged over set sizes 1 and 5. Bars reflect the mean performance in each drug condition. Error bars reflect SEMs, adjusted to remove between-subject variability in the average performance across dose levels (Cousineau 2007; Morey 2008). *P < 0.05, **P < 0.01 in paired t tests

To test whether RT reduction by the combined administration of <span class="Chemical">nicotine and <span class="Chemical">galantamine was related to <span class="Gene">AChE inhibition, RT and <span class="Gene">AChE activity values in the placebo session were subtracted from values in the <span class="Chemical">nicotine + <span class="Chemical">galantamine session, and the difference values underwent Pearson’s correlation. There was no significant correlation (R = − 0.29, P = 0.15), and the direction of the trend would be consistent with less <span class="Gene">AChE inhibition being associated with greater RT reduction.

Discussion

The purpose of the present proof-of-concept study was to test whether a <span class="Gene">nAChR PAM could in principle augment the performance-enhancing effects of a <span class="Gene">nAChR agonist, employing <span class="Chemical">nicotine and <span class="Chemical">galantamine as pharmacological probes. <span class="Chemical">Nicotine is a non-selective <span class="Gene">nAChR agonist, and similarly, no subtype selectivity of <span class="Chemical">galantamine’s <span class="Gene">nAChR PAM action has been shown (Dajas-Bailador et al. 2003; Samochocki et al. 2003; Santos et al. 2002). A broad array of cognitive functions was probed, including stimulus detection under conditions of narrow focusing (SARAT predictive cue trials) and broad monitoring (non-predictive cue trials); rapid information processing, sustained attention, and working memory (RVIPT); and short-term memory (CDT). It was only on response time in the context of the CDT (a task in which response speed is not emphasized) that synergistic effects of <span class="Chemical">nicotine and <span class="Chemical">galantamine were observed, specifically, a reduction in RT by both drugs combined, but no effect of either drug alone.

A possible explanation for why only one of six performance measures showed the hypothesized interaction is that cognitive functions differ in their performance-optimal tone at nAChRs, or critical subtypes thereof. This would suggest that optimal doses of <span class="Gene">nAChR agonists and/or PAMs depend on the specific therapeutic effect desired. Performance of both SARAT and RVIPT improved with <span class="Chemical">nicotine, and <span class="Chemical">galantamine did not significantly increase these benefits, suggesting that <span class="Gene">nAChR tone at the critical subtypes may have been close to optimal for these attention-demanding tasks after <span class="Chemical">nicotine administration. Thus, it is possible that <span class="Chemical">galantamine would have potentiated effects of an even lower dose of <span class="Chemical">nicotine on these measures; further research could test this possibility. In contrast, performance of the CDT was insensitive to <span class="Chemical">nicotine alone, and RT benefits were seen only with the addition of <span class="Chemical">galantamine. This suggests that cognitive functions reflected by this measure benefit from only a large increase in <span class="Gene">nAChR tone, and/or involve specific <span class="Gene">nAChR subtypes that either do not respond to concentrations of <span class="Chemical">nicotine achieved here or are prone to desensitization by <span class="Chemical">nicotine and benefited from a reversal of desensitization by <span class="Chemical">galantamine’s PAM action.

The lack of effect of <span class="Chemical">nicotine alone in the CDT which, contrary to SARAT and RVIPT, does not pose any significant challenge on attentional functions is consistent with the view that <span class="Chemical">nicotine’s performance-enhancing effects are most robust in paradigms of attention (Hahn 2015; Newhouse et al. 2004; Stolerman et al. 1995). The present synergistic action with <span class="Chemical">galantamine suggests that this lack of effect of <span class="Chemical">nicotine alone may reflect an inability to achieve the right tone at the right <span class="Gene">nAChR subtype(s), rather than a general lack of <span class="Gene">nAChR involvement or reticence to <span class="Gene">nAChR-based modulation. The specific neuronal systems and <span class="Gene">nAChR subtypes that may be differentially involved in the cognitive functions probed by the CDT could only be speculated upon at this time. Data to date indicate that both α7 and α4β2* nAChRs, the most abundantly expressed <span class="Gene">nAChR subtypes throughout the brain (Gotti et al. 2009), mediate effects of <span class="Gene">nAChR agonists on attentional functions (e.g., Grottick et al. 2003; Hahn et al. 2011; Haydar and Dunlop 2010; Howe et al. 2010) and short-term memory (Levin et al. 2002; Rushforth et al. 2010). However, these functions may still be differentially influenced by a change in tone at these and other <span class="Gene">nAChR subtypes.

An alternative explanation for why only one of six performance measures showed the hypothesized interaction would be that additional pharmacological actions of <span class="Chemical">galantamine confounded the results. A trend suggested that <span class="Chemical">galantamine shortened SARAT RT independent of the presence or absence of <span class="Chemical">nicotine, and correlation analysis indicated that this reflected residual <span class="Gene">AChE inhibition. Furthermore, <span class="Chemical">galantamine alleviated the RVIPT performance decrement over time independent of the presence or absence of <span class="Chemical">nicotine, consistent with a previous study testing a larger dose of <span class="Chemical">galantamine (8 mg), whose effects presumably were dominated by <span class="Gene">AChE inhibition (Sofuoglu et al. 2012). An even smaller dose of <span class="Chemical">galantamine may have achieved better separation of <span class="Gene">AChE inhibition from <span class="Gene">nAChR PAM effects. In this regard, it is of interest that the hypothesized <span class="Chemical">nicotine × <span class="Chemical">galantamine interaction was seen only in the task that was administered last, when <span class="Chemical">galantamine blood levels were past their peak (Zhao et al. 2002). Clearly, a pure <span class="Gene">nAChR PAM would be a preferential tool to demonstrate positive allosteric potentiation of <span class="Gene">nAChR agonist effects, but no such compound is commercially available to date.

Despite <span class="Chemical">galantamine’s additional mechanism(s) of actions, the synergistic effects with <span class="Chemical">nicotine on CDT RT are informative. First, the nature of the interaction is conceptually explainable by <span class="Chemical">galantamine’s PAM action facilitating <span class="Chemical">nicotine effects, but not by <span class="Gene">AChE inhibition because a greater concentration of <span class="Chemical">acetylcholine in the synaptic cleft would compete with <span class="Chemical">nicotine for the same binding sites. This would reduce, not enhance, effects of <span class="Chemical">nicotine, as seen with an 8-mg dose of <span class="Chemical">galantamine (Sofuoglu et al. 2012). Second, the effect was not associated with <span class="Gene">AChE inhibition by <span class="Chemical">galantamine as measured in blood, with even a trend association in the opposite direction. Thus, this finding strongly suggests that positive allosteric potentiation of cognitive-enhancing <span class="Gene">nAChR agonist effects is possible in principle.

While the present study was a proof of principle performed in healthy individuals, the ultimate target populations would be those with <span class="Gene">nAChR hypofunction such as <span class="Species">people with <span class="Disease">schizophrenia, mild <span class="Disease">cognitive impairment, or <span class="Disease">Alzheimer’s disease, for which the <span class="Gene">nAChR agonist-PAM combination may be particularly beneficial. Combining a low-dose <span class="Gene">nAChR agonist and a PAM may represent a <span class="Gene">nAChR modulation strategy that is more fine-tuned and more sparing of native circuit dynamics than larger doses of agonist alone. <span class="Gene">nAChR subtype selectivity can be achieved with both <span class="Gene">nAChR agonists and PAMs; thus, a combination approach may achieve greater flexibility when targeting a critical subset of <span class="Gene">nAChR subtypes. For example, sub-threshold doses of a <span class="Gene">nAChR agonist selective for one group of <span class="Gene">nAChR subtypes may be combined with sub-threshold doses of a PAM selective for another group but overlapping with the first group on the critical subtype(s). Thus, the targeted co-administration of a <span class="Gene">nAChR PAM may enable the use of very small doses of <span class="Gene">nAChR agonist and achieve a narrower effects profile.

Limitations of the present study include a moderate sample size, a lack of verification of treatment blind fidelity, and the use of a single dose of both <span class="Chemical">nicotine and <span class="Chemical">galantamine. While additional test sessions would have made <span class="Species">participant retention challenging, inclusion of multiple (and even smaller) doses of <span class="Chemical">galantamine may have achieved better separation of <span class="Gene">AChE inhibition from <span class="Gene">nAChR PAM effects. Furthermore, given the absence of an in vivo marker of <span class="Chemical">galantamine’s <span class="Gene">nAChR PAM action, we attributed its observed potentiation of <span class="Chemical">nicotine effects to positive allosteric modulation based on the absence of an association with <span class="Gene">AChE inhibition. Given that <span class="Gene">AChE inhibition and <span class="Gene">nAChR positive allosteric modulation to date are recognized as the primary mechanisms of action of <span class="Chemical">galantamine, positive allosteric modulation would appear a likely mediator of the effects of <span class="Chemical">galantamine reported here, but our inability to measure it directly is a limitation. Finally, neither <span class="Chemical">nicotine’s agonist effects nor <span class="Chemical">galantamine’s PAM effects are selective for any specific subtypes of the <span class="Gene">nAChR. Thus, the present findings provide a proof of principle, but do not advance the more targeted strategies outlined in the previous paragraph.

In summary, the present findings suggest that positive allosteric potentiation of cognitive effects of exogenous <span class="Gene">nAChR agonists is possible in principle and encourage further study of this mechanism with even smaller doses of <span class="Gene">nAChR agonist and with novel <span class="Gene">nAChR PAMs devoid of additional pharmacological actions.