The nicotinic acetylcholine receptor (nAChR), specifically nAChR containing α4β2* subunits, have been widely studied for their critical role in nicotine dependence and addiction [1]. Efforts surrounding α4β2 nAChR have yielded the development of Chantix (varenicline), an α4β2 nAChR* partial agonist, as a Food and Drug Administration-approved smoking cessation therapeutic. Although varenicline is a useful smoking cessation pharmacotherapy, the largest individual cause of preventable death continues to be cigarette smoking [1, 2]. Thus, new therapeutics to treat nicotine addiction are desperately needed, and developing compounds that target α4β2* nAChR remains a viable option for developing novel smoking cessation therapeutics [1, 3–5]. Drug discrimination has utility in elucidating receptor mechanisms, and training nicotine as a discriminative stimulus allows for both the interrogation of novel compounds for activity at α4β2* nAChR as well as the ability to identify additional nAChR subunits or additional receptors that mediate the in vivo effects of nicotine [1, 3–5]. Specifically, if a compound is able to substitute for nicotine, then there is a good likelihood that the compound shares similar receptor pharmacology as nicotine, and likely α4β2* nAChR agonist activity.

Up to 17% of the US population live with neuropathic pain, which is associated with quality of life impairments and produced from injury to the nervous system [6–8]. Many conditions that produce neuropathic pain are increasingly prevalent in tobacco-dependent individuals as a consequence of long-term tobacco use and include chemotherapy induced peripheral neuropathy and peripheral diabetic neuropathy [2, 7]. A hallmark neuropathic pain symptom is mechanical allodynia, or light touch mechanical sensitivity [6–8]. Neuropathic pain is often poorly treated by tricyclic antidepressants, serotonin and noradrenaline reuptake inhibitors, antiepileptic drugs, and opioids. Unfortunately, these treatments provide only temporary relief, are susceptible to breakthrough pain, and have significant side effect profiles [8–11].

Many nAChR agonists, including those that act on α4β2* and α7 produce analgesia [3, 12]. However, the potential of α4β2* nAChR agonists as a viable chronic neuropathic pain therapeutic has yet to be realized. Epibatidine is classified as an α4β2 nAChR agonist, and initially much interest surrounded this compound as both an analgesic as well as a treatment for nicotine dependence, but a low therapeutic window due to toxicity led to its therapeutic abandonment [13–15].

Epibatidine derivatives including 2′-fluoro-3'-(4-nitrophenyl)deschloroepibatidine (RTI-7527-102; i.e., RTI-102), 2′-fluorodeschloroepibatidine (RTI-7527-36; i.e., RTI-36), and 3'-(3″-dimethylaminophenyl)-epibatidine (RTI-7527-76; I, e., RTI-76) have been developed to selectively target α4β2* nAChR activity. In vitro each compound has been characterized to have α4β2* nAChR binding affinity, with a reported Ki value of ∼0.037 for RTI-36, ∼0.009 for RTI-76, and ∼0.009 for RTI-102 [16–19]. These compounds possess differing nAChR subtype affinities from one another, in addition to α4β2* nAChR activity, and have a longer duration of action than epibatidine. Previous work in mice and in transfected frog oocytes demonstrated RTI-36 has the highest in vivo agonist potency out of the three RTI compounds at α4β2* nAChR and has agonist activity at α7 nAChR [4, 19]. RTI-76 has been found to exhibit partial α4β2* nAChR agonist activity, as well as a functional α2 nAChR agonist [4, 19]. RTI-102 has been found to act as an α4β2* nAChR antagonist in vitro, and a α4β2* nAChR partial agonist in vivo [4, 19]. This difference in activity at α4β2* nAChR may be due to species differences between the in vitro studies, which used transfected human α4β2* nAChR and in vivo studies, which have previously used mice [4, 19]. Here we sought to examine the pharmacology of these compounds in the rat drug discrimination assay. One cohort of male and female Sprague-Dawley rats were trained on a fixed-ratio 10 schedule to discriminate nicotine (0.32 mg/kg base) from vehicle, and varenicline, RTI-102, RTI-36 and RTI-76 were substituted for the nicotine discriminative stimulus. The nAChR β2 subunit is critical for mediating the reinforcing effects of nicotine [20]. Thus, we also examined the relative contribution that the β2 subunit plays in the nicotine discriminative stimulus substitution effects obtained with each of the epibatidine derivatives via the use of the β2* nAChR antagonist, dihydro-β-erythroidine (DHβE). To confirm nAChR agonist activity we also utilized the non-selective nAChR antagonist mecamylamine.

We next examined the analgesic effects of these novel epibatidine derivatives in a separate rat cohort that had undergone the chronic constriction injury (CCI) of the sciatic nerve neuropathic pain model and were tested for CCI-induced mechanical allodynia via the von Frey test [8]. The variety of nAChR subtype activity produced by these epibatidine derivatives, in addition to α4β2*, provides a plethora of drug development targets.

Here we show that RTI-36, RTI-102, and RTI-76 dose-dependently reversed CCI-induced mechanical allodynia. These three compounds, RTI-36, RTI-102, and RTI-76, also fully substituted for 0.32 mg/kg nicotine (0.32 mg/kg) in the nicotine drug discrimination assay. Varenicline, a commonly prescribed drug for nicotine addiction, produced full nicotine substitution. We examined the relative contribution of the β2 subunit with DHβE pretreatment. DHβE (3.2 mg/kg, s.c.) antagonized the discriminative stimulus effects of nicotine but not varenicline. However, relative to antagonism of nicotine, DHβE produced less antagonism of RTI-102 and RTI-76 and greater antagonism of RTI-36. To confirm that the full discriminative stimulus of these compounds was due to nAChR activity beyond the β2 subunit, we examined these compounds in the presence of the non-selective nicotinic receptor antagonist mecamylamine. Mecamylamine (0.56 mg/kg, s.c.) pretreatment abolished nicotine-paired lever responding for all compounds.

The nicotine discrimination assay was pharmacologically selective, as morphine only produced a ∼13% nicotine substitution. The ED₅₀ values for nicotine-like discriminative stimulus effects were 0.12 mg/kg for nicotine, 0.001 mg/kg for RTI- 36, 0.12 mg/kg for RTI-102, and 0.2 mg/kg for RTI- 76, The relative potency of epibatidine derivates reported here is supportive of previous in vitro work demonstrating RTI-36 and RTI-102 have higher binding affinity at αβ-containing nAChR subtypes than RTI-76 [16–18]. In mice discriminating nicotine (1 mg/kg, s.c.) RTI-102 failed to fully substitute for the nicotine discriminative stimulus [4]. Here, in rats RTI-102 produced full substitution for a lower nicotine (0.32 mg/kg, s.c.) training dose. These observed differences in the substitution of RTI-102 for nicotine in the drug discrimination assay may be due to a species difference between rats and mice. Additionally, it is well established that the dose of the training drug influences the ability of various test compounds to substitute for the training drug, with higher training doses having greater discriminative stimulus receptor specificity. The rank order potency of nicotine and the epibatidine derivatives to be antagonized by DHβE was RTI-36 > nicotine > RTI-76 = RTI-102. The relative potency of epibatidine derivates reported here is supportive of previous in vitro work that demonstrated RTI-36 and RTI-102 have higher binding affinity at αβ-containing nAChR subtypes than RTI-76 [16–18], and that RTI-102 has lower efficacy at α4β2 nAChR than nicotine [23–25]. Differences in α4β2 nAChR efficacy may underlie the observed potency of each compound to substitute for nicotine discriminative stimulus effects. However, the failure of DHβE to antagonize the discriminative stimulus effects of varenicline, which fully substituted for the nicotine discriminative stimulus has similarly been demonstrated in mice trained to discriminate nicotine [4, 5, 26]. Thus, with regard to the in vivo activity of both RTI-102 and varenicline, these findings suggest an alternative conclusion. In vitro, both RTI-102 and varenicline have less efficacy at the α4β2 nAChR than nicotine [23–25]. DHβE is assumed to be a competitive β2 nAChR antagonist. Varenicline is a full agonist at the α7 subunit [27] and it appears that nAChRs containing the α5 subunit may mediate at least some of the physiological effects of varenicline [28, 29]. Therefore, other nAChR receptor subunits in addition to α4β2 subunits could have a contribution to the nicotine-related discriminative stimulus effects and the nAChR agonists used in this study, including both varenicline and RTI-102, which produced nicotine substitution [30].

The nonselective nAChR antagonist mecamylamine (0.56 mg/kg) attenuated the discriminative effects of nicotine, as well as the substitution effects of varenicline and the three epibatidine derivatives, reinforcing previous studies that conclude nicotine exerts discriminative stimulus effects through nAChRs. It should be noted that DHβE-induced antagonism of nicotine, RTI-36, RTI-102, and RTI-76 substitution in the nicotine drug discrimination assay was surmounted at drug doses that did not disrupt schedule-controlled responding. However, mecamylamine-induced antagonism of nicotine drug discrimination was not surmountable, at least regarding nicotine, up to doses (i.e., 1.78 and 3.2 mg/kg nicotine) that produced significant operant rate decreases. Likewise, mecamylamine-induced antagonism of RTI-36, RTI-102, and RTI-76 nicotine substitution was not surmountable at any dose of the epibatidine derivatives tested. Due to limited compound amounts, the doses of epibatidine derivatives were not studied to the observational point of operant rate decreases. Here we found that in the presence of mecamylamine, nicotine response rates were significantly decreased at higher drug doses (i.e., 1.78 and 3.2 mg/kg) with only partial nicotine stimulus discrimination, an effect not seen when nicotine was administered alone at partial stimulus discrimination doses (i.e., 0.01 and 0.0178 mg/kg). One interpretation of this finding is that at higher doses (i.e., 1.78 and 3.2 mg/kg) nicotine has activity at receptors other than nAChR [28]. Taken together, the current studies support the idea that each of the epibatidine derivatives possess differing nAChR subunit activity and differing α4β2 nAChR potency, which could produce useful analgesic profiles.

Nicotine-related analgesic effects have been established since 1932 [31]. Activation of nAChR in the spinal cord leads to the release of neurotransmitters believed to play a role in pain modulation, including acetylcholine, noradrenaline, serotonin, gamma-aminobutyric acid, and opioid peptides [32–34]. Due to the modest analgesic potency of nicotine and its substantial safety liabilities the use of nicotine as a clinical analgesic has not materialized [35]. In preclinical models nAChR agonists have shown activity in mitigating pain-related behaviors from numerous different etiologies, including acute nociceptive, inflammatory, and neuropathic pain [3, 12, 36]. Considerable issues regarding the development of nAChR analgesics generally include a relatively narrow therapeutic dosing window where beneficial effects are separated from toxic effects, as well as sex-related potency and efficacy differences [37]. Epibatidine is 200 times more potent than morphine to produce analgesic effects [38]. However, epibatidine is extremely toxic, causing hypertension, respiratory paralysis, and seizures, with death occurring at doses not much higher than those required for antinociception [39, 40].

Although not studied in this current report, in mice epibatidine produced significant hypothermia and nicotine substitution in mice trained to discriminate nicotine [4]. From this study, the ED₅₀ for epibatidine to produce nicotine (1 mg/kg base, s.c.) substitution was 0.002 mg/kg, while the ED₅₀ for epibatidine to produce hypothermia was 0.005 mg/kg [4]. This was compared to the doses of each epibatidine analog studied in the current report. Indeed, in mice, it was found that RTI-36, RTI-102, and RTI-76 produced significant hypothermia, with reported 0.07, 3.0 and 4.3 mg/kg ED₅₀ values, respectively [4]. The reported ED₅₀ values (in mg/kg) for these compounds to produce nicotine cross-substitution were 0.023, no substitution, and 2.29 for RTI-36, RTI-102, and RTI-76, respectively [4]. A limitation of the current study is that Sprague Dawley rats are less sensitive to nicotine-induced hypothermia than other rat strains (i.e., Flinders Sensitive Line rats), and hypothermia was not measured in the current study [41]. In future studies, the use of hypothermia sensitive rat strains would lead to a better characterization of therapeutic vs. hypothermic (i.e., toxic) effects. In previous mouse studies these epibatidine derivatives were found to display limited acute antinociceptive activity in mice subjected to the hot plate nociception assay [24].

Although epibatidine may be of little clinical use, epibatidine derivatives may be useful analgesics if they display analgesic-like effects at doses that do not display toxicity or other side effects. In our hands RTI-36, RTI-102, and RTI-76 produced full reversal of CCI-induced mechanical allodynia in both male and female rats. Although there were potency differences, doses that reversed mechanical allodynia did not produce lethality. However, each compound produced full mechanical allodynia reversal at doses that fully substituted for nicotine, and indeed each compound was more potent in the nicotine drug discrimination assay than the von Frey assay. As nicotine is highly addictive, the abuse and dependence potential of these compounds should be further explored before these derivatives are considered as viable potential novel analgesics. Taken together, these findings suggest more work on the receptor selectivity and binding activity of the scaffolds of these compounds may yield better therapeutic leads.

Here we show that each epibatidine derivative dose-dependently reversed CCI-induced mechanical allodynia and fully substituted for 0.32 mg/kg nicotine (0.32 mg/kg) in the nicotine drug discrimination assay. Activity in both assays was likely due to differences in potency at α4β2* nAChRs as well as effects on different nAChR subunits. In addition, the contribution of receptor activity beyond nAChRs cannot be ruled out. However, these studies support the idea that neuronal nAChR agonists could serve the dual purpose of a smoking cessation aid and analgesic. The utility of epibatidine derived compounds for both therapeutic indications could be realized if a compound lacks significant abuse and dependence liabilities as well as achieves a suitable therapeutic window.