The animal pain behavior results from the present study suggest that selective α4β2 nicotinic receptor agonist TC-2559 has analgesic property, and in vitro spinal cord slice patch clamp recording results indicate that this analgesic property is probably medicated by selective enhancement of inhibitory neurotransmission in the spinal dorsal horn via activation of α4β2 subtype of nicotinic receptors. This conclusion is based on the observation that: 1) In in vivo animal pain models, TC-2559 inhibited both intraplantar formalin injection induced biphasic nociceptive responses and CCI-induced mechanical allodynia; 2) In in vitro spinal cord slices, TC-2559 significantly enhanced inhibitory synaptic transmission, but has no effect on the excitatory synaptic transmission.
Bioavalaibility of TC-2559
Similar to what previously reported [
16], brain exposure data of TC-2559 was obtained first as a guidance for our later
in vivo and
in vitro studies. The present brain exposure result demonstrated that TC-2559 at 3 mg/kg (i.p.) produced an initial peak whole brain concentration of about ~4 μM (15 min after injection), which quickly declined to ~2 μM at the 30 min point. The elimination of TC-2559 was fast with a half life of ~20 min which was similar to that of given by i.v. shown previously [
16]. Since cerebral-spinal fluid is similar, we assumed that the spinal concentration of the TC-2559 after i.p. administration would be as the same as in brain. TC-2559 has been recently developed and characterised as a neuronal nAChR agonist [
13] and selective for α4β2 subtype both
in vitro [
14,
16] and
in vivo [
14,
16]. The EC
50 for TC-2559 to evoke excitation in VTA slice was found to be of 0.6 μM and TC-2559 is ineffective on α7 and α3β2 receptors up to 10 μM, and weakly effective on α2β4, α4β4 and α3β4 receptors, with EC
50's in the range of 10 - 30 μM [
14]. Thus, at the 3 mg/kg i.p. dose for rat CCI study and 2 μM dose for
in vitro brain slice experiment used in this study, TC-2559 is likely to be selective for the α4β2 nicotinic receptor subtype.
Antinociceptive effects of TC-2559 in neuropathic pain animal models
Pain initiated or caused by a primary lesion or dysfunction in the nervous system is called neuropathic pain. Several animal models of neuropathic pain based on the induction of neuropathic pain behaviors after induction of a controlled nerve injury have been developed [
20‐
22]. The formalin test is a model of injury-produced pain and is well established for its easier to perform and to standardize [
23,
24]. The intraplantar injection of formalin results in lifting, licking or other agitation behavior during an early phase, which resembles acute pain, followed by a late phase representative for neuropathic pain [
24] which is comparable to CCI model. In current study, we demonstrated that TC-2559 dose-dependently reduced intraplantar formalin injection induced both early and late phases of nociceptive behaviors in mice. The dose-dependent inhibition on the late phase especially suggested that TC-2559 exerts antinociception effects on neuropathic pain. Nicotine itself has been demonstrated to have dose dependent antinociceptive effects in both the early and late phases of the formalin test in mice [
25‐
27], and these effects were suggested to act via α4β2 subtype of nAChRs by using selective α4β2 nAChR antagonists. Thus, our results directly demonstrated for the first time that activation of α4β2 subtype of nAChRs by selective agonist could reduce the pain behavior induced by formalin in mice.
CCI model, which was first set up by Bennett in 1988, possesses the character of both peripheral and central sensitization [
20]. It shares many common way with the clinical disease of neuropathic pain, and thus has been widely used in the field of pain study [
28]. To further examine the antinociceptive action of TC-2559 on neuropathic pain, we then tested the effect of TC-2559 on CCI model in rat. In current study, in order to standardize the CCI model for our pharmacological study, the rats used for this study were selected initially by their pain threshold level during the pre-surgery pain behavioral test. Only those with a basal pain threshold between 6 and 10 g were chosen for CCI model surgery. As predicted, 4-7 days after CCI induction, 35% of the rats showed typical sensitized pain threshold under mechanical stimulation to be lower than 2 g in ipsilateral foot similar as previously reported [
19]. Under this condition, we demonstrated that TC-2559, at the dose known selective for α4β2 subtype of nAChRs
in vivo [
12,
13], reversed CCI-induced mechanical allodynia in all the rats tested. This result is the first time to show the analgesic role of α4β2 subtype of nAChRs in the rat CCI model. In addition, our data also showed that the analgesic effect of TC-2559 was quickly returning to the control level after injection, which is consistent to our ADME result of the property of TC-2559
in vivo. Our bioavailability study showed that TC-2559 was a compound with fast absorption and quick elimination properties. The
in vivo half life in both brain and serum for TC-2559 was only about 20 min, which was well reflected for its short analgesic action
in vivo in our current study. Thus, our
in vivo results demonstrated in two commonly used animal pain models and in addition, in two different species (mouse and rat) that TC-2559 is a potent antinociceptive compound.
In contrast to the antinociceptive action of TC-2559 on injured hindpaw at the doses of 1 and 3 mg/kg, TC2559 at the dose of 1 mg/kg had no effect on the paw withdrawal threshold in the contralateral hind paw. However, when the drug dose was increased to 3 mg/kg, TC-2559 significantly increased the paw withdrawal threshold in the contralateral hind paw, along with on ipsilateral hindpaw, compared to that of vehicle control. This effect suggests that over activation of α4β2 subtype of nAChR by using high dose of TC-2559 may suppress the basal sensation of spinal cord sensory transmission. It is consistent with the results obtained in a previous study, where a low dose of nicotine (10 nmol i.t.) or epibatidine (0.3 nmol i.t.) has no effect in sham animals but is antiallodynic in peripheral nerve injury mice [
11]. Both results suggest that the injured peripheral nerve is more sensitive to nicotinic receptor activation, which may indicate of an upregulation of nicotinic receptors after injury. This has given a therapeutical window to reduce sensitized pain threshold for nicotinic drugs such as TC-2559. Since the analgesic effect of TC-2559 on ipsilateral hindpaw was observed at the dose as low as 1 mg/kg, our current results indicated that the therapeutic window for TC-2559 on pain release might be likely between 1 and 3 mg/kg (i.p.) doses in rats. It suggests that the ligand acts as selective α4β2 subtype of nAChR agonist may be developed to have a clinically acceptable therapeutical window between pain signal sensitivity and normal sensory signal transmission.
DHβE, a widely used compound served as a selective α4β2 nAChR antagonist [
12,
13], reversed the antinociceptive action of TC-2559 in rat CCI model in this study, while DHβE applied alone had no significant effect on CCI-induced mechanical allodynia. These results suggest that the antinociceptive effect of TC-2559 on neuropathic pain is indeed mediated by activation of α4β2 subtype of nAChRs. In several previous studies in rats, intrathecal injection of DHβE alone had no effects while they blocked the nociceptive or antinociceptive effects of the nicotinic agonists [
29,
30]. These are consistent with our present observation. In addition, in our
in vitro studies in the spinal cord slices, DHβE alone also had no significant effect on sIPSCs, which is consistent with our
in vivo experiments.
However, the lack of effect of DHβE in rats seems contradictory with studies performed on mice. Rashied et al. demonstrated that bath application of DHβE induces a decrease in the frequency of IPSCs in two third of dorsal horn neurons in mice [
12]. Also, i.t. injection of DHβE induces mechanical allodynia in mice [
11,
12]. These differences in mice and rats are likely due to species differences. We hypothesis that the Ach release level in mice might be higher than that in rats. However, we choose to perform behavior experiments on different species of animals is because this study was designed to test whether the selective agonist of α4β2 subtype of nAChR (TC-2559) has antinociceptive effects and its possible mechanism, thus to test whether the agonist of α4β2 subtype of nAChR is capable to be developed as new common analgesic drug for treatment of neuropathic pain. It would be better to know whether TC-2559 could have analgesic effects on species known to have differences since finally it may be used on human beings. Therefore, we choose to perform behavior experiments (formalin and CCI) on different species of animals. Our results of formalin test and CCI demonstrated that TC-2559 has analgesic effects on both mice and rats, despite they have differences.
Thus, our current study is the first to demonstrate, by using selective α4β2 nAChR agonist (TC-2559), that direct stimulation of α4β2 subtype of nAChRs has strong antinociceptive effects in classic neuropathic pain animal models.
Spinal mechanisms of antinociceptive effects of TC-2559
The inhibitory interneurons in the spinal dorsal horn are crucial for maintaining normal input intergration and are localized throughout the spinal dorsal horn [
31,
32]. The neuronal pathways mediating allodynia/hyperalgesia in spinal dorsal horn are normally under strong inhibitory regulation [
33‐
35]. Since α4β2 subtype of nAChRs were reported to be preferentially expressed in the inhibitory interneurons in the spinal dorsal horn [
36], we hypothesized that activation of α4β2 subtype of nAChRs by TC-2559 would excite those inhibitory interneurons expressing α4β2 nAChRs. In turn, the pain input sensory pathway between primary afferent fibers and lamina I nociceptive specific neurons in spinal dorsal horn would receive more inhibitory control after activation of α4β2 subtype of nAChRs. Indeed, our results showed that TC-2559, a selective α4β2 nAChR partial agonist [
14], significantly enhanced the sIPSC frequency in majority of the spinal superficial dorsal horn neurons tested. This enhancement could be fully antagonized by a selective α4β2 nAChR antagonist (DHβE) indicated that TC-2559 was indeed activated α4β2 nAChRs in spinal dorsal horn. These results suggest that activation of α4β2 nAChRs would generally increase the inhibitory tone in the spinal dorsal horn, thus may regulate both the sensory input and signal integration in this region.
Hyperalgesia is mediated, in spinal cord level, by enhanced pain signal transmission and reduced local inhibition [
35,
37,
38]. However our data showed that TC-2559 by activation of α4β2 nAChR had no significant effect, in contrast to increase of inhibitory transmission, on the excitatory neurotransmission in spinal dorsal horn neurons. This result is consistent with a previous report that nicotine enhances excitatory synaptic transmission in the spinal dorsal horn by α7 [
39] but not by α4β2 nAChRs. This is further supported by the finding that α4β2 subtype of nAChRs expressed preferentially in the inhibitory interneurons in the spinal dorsal horn [
36]. Thus, as expected, activation of these receptors would have predominant effect on the inhibitory, rather than excitatory neurotransmitter release in this region. This result suggests that the antinociciptive effect of TC-2559, by activation of α4β2 nAChRs, is likely due to solely modulation of the inhibitory circuits within the spinal dorsal horn to enhance the basal inhibition, but not affecting either excitatory sensory input or excitatory interneuron function.
Although the methods that by patch clamp recording on visually identified spinal superficial dorsal horn neurons could not resolve cell identity, the high prevalence of enhancement on sIPSCs by TC-2559 (~85% of neurons with > 20%of increase on sIPSC frequency) suggests that this type of inhibitory modulation in spinal superficial dorsal horn might be common to multiple populations of the neurons in this region of the spinal cord. This is consistent with the action of nicotine with which GABAergic synaptic transmission was enhanced in 86% of neurons tested in the spinal dorsal horn [
40]. Thus it suggests that TC-2559 would enhance the general inhibition in the superficial dorsal horn neurons by activation of α4β2 subtype of nAChRs.
Although our results clearly demonstrated that TC-2559 by activation of α4β2 nAChRs to enhance the basal inhibition in spinal dorsal horn neurons which in turn, as expected, to reduce the pain related peripheral sensory input at the spinal cord level, we can not rule out the possibility of the involvement of the activation of α4β2 nAChRs localized in the regions other than spinal cord dorsal horn, while tested in in vivo pain animal models by systemic application of TC-2559. However, since spinal dorsal horn is an important pain signal primary integration region, we hypothesis that activation of α4β2 nAChRs localized in the spinal dorsal horn by TC-2559 to enhance the over all inhibitory tone is one of the major contributor for TC-2559's antinociciptive action.
Functional implication
Spinal dorsal horn neurons including those mediating allodynia/hyperalgesia signals are normally under strong inhibitory regulation [
33‐
35]. And any mechanism to release those inhibition on the pain signaling pathway in the spinal dorsal horn opens the gate to pathological pain sensory input to spinal dorsal horn neurons [
33,
35,
41], which would cause the chronic pain hypersensitivity [
11]. Reports have shown that disruption of this inhibition by blockade of α4β2 subtype of nAChRs induced thermal and mechanical hyperalgesia in both normal rats [
29,
30] and naive mice [
12], which indicates that α4β2 subtype of nAChRs play a crucial modulatory role to maintain the tonic inhibition in spinal dorsal horn for sensory and/or pain signal input. The data from our current study were indeed showing that direct activation of α4β2 subtype of nAChRs by agonist of TC-2559 did exert antinociceptive effects in pathological pain animal models of both formalin test and CCI possibly by enhancing the inhibitory neurotransmission in spinal dorsal horn. In addition, we showed that TC-2559, acting as a selective α4β2 subtype of nAChR agonist, deferentially enhanced inhibitory neurotransmission in spinal dorsal horn neurons but not affecting excitatory neurotransmission. This property gives the hope that α4β2 nAChRs agonist based analgesic drug might have fewer side effects than that of currently marketed pain relieves, in terms of the spinal cord level. However, when given intracerebroventricularly (i.c.v.) and intracisternally (i.c.), TC-2559 has been reported to induce an increase in blood pressure and renal nerve activity [
42]. What's more, we have also previously demonstrated that TC-2559, while given intravenously, activates dopaminergic neurons in the VTA and suppress hippocampal long term potentiation [
29,
30] in anaesthetized rats. Thus TC-2559 itself is therefore likely to have numerous physiological functions beside analgesia. Since TC-2559 is an selective partial α4β2 nAChR agonist [
29,
30], developing more potent agonist selective for α4β2 nAChRs may be one of the directions for developing nicotinic receptor based analgesic drug.