The use of nicotinic agonists for analgesia is limited by their unacceptable side effects. Sazetidine-A is a new partial agonist nicotinic ligand that has very high selectivity for beta2-containing nicotinic acetylcholine receptors. It potently and selectively desensitizes alpha4beta2 nicotinic acetylcholine receptors without measurable effects on alpha3beta4 receptors. The authors investigated the analgesic effects of Sazetidine-A using the formalin model of chronic inflammatory pain.
The formalin test was conducted after rats received intraperitoneal saline, Sazetidine-A (0.125, 0.25, 0.5, 1, 2 mg/kg), or subcutaneous epibatidine (2.5-5-10 mug/kg). In other experiments, Sazetidine-A was preceded by naloxone (0.5 mg/kg) or mecamylamine (10 mg). Effects of Sazetidine-A and epibatidine on locomotor were tested in an open field, and seizure activity was measured using the Racine scale. Locus coeruleus neuron extracellular single-unit spontaneous discharge was recorded in anesthetized animals after Sazetidine-A and epibatidine.
Higher doses of Sazetidine-A (0.5, 1, or 2 mg/kg) induced analgesia, with pain scores significantly lower than those seen after saline, lower doses of Sazetidine-A, and epibatidine (P < 0.001). Naloxone did not antagonize the effects of Sazetidine-A, and mecamylamine had partial, dose-dependent antagonistic effects. Epibatidine excited locus coeruleus neurons, whereas Sazetidine-A had no effect on these neurons. Epibatidine and Sazetidine-A affected animals' locomotor activity for the initial 20 min. While analgesic doses of epibatidine caused seizures, no seizure activity or other neurologic complications were seen in animals that received as much as four times the minimum analgesic dose of Sazetidine-A.
Sazetidine-A seems to be a potent analgesic without causing neurologic side effects.
THE analgesic properties of nicotine have been suspected for more than 75 yr and were well documented by the 1980s.1–4The discovery of epibatidine, a potent nicotinic acetylcholine receptor (nAChR) agonist5–7approximately 200 times greater than morphine, rekindled interest in nAChRs as targets for analgesics. Since then, other nicotinic ligands have been found to possess analgesic properties, and several have advanced to clinical trials.8,9However, an unacceptable level of side effects has limited their further development.
The α4β2 nAChR subtype is the most common heteromeric nAChR in rat brain,10,11and the presence of these receptors in pain pathways suggests that they may be involved in the analgesic effects of nicotinic ligands.1,12–15Studies with knock-out16and knock-in mice14strongly support this possibility. Thus, an effective and useful nicotinic analgesic agent might be expected to act preferentially at α4β2 nAChRs and have low affinity for receptor subtypes that are associated with side effects mediated by autonomic ganglia, such as the α3β4 subtype. Consistent with this, epibatidine cannot be used clinically because of severe neurologic and hemodynamic side effects,17probably because it has high affinity for virtually all heteromeric nAChR subtypes, including those in ganglia and brain stem autonomic centers.
Sazetidine-A is a new nicotinic ligand with very high affinity and selectivity for β2-containing nAChRs18in equilibrium binding assays. In previous studies, Sazetidine-A did not seem to act as either an agonist or a competitive antagonist in assays that measured 86Rb+efflux through the nAChR channel, but it did markedly and selectively desensitize α4β2 nAChRs in a time-dependent manner.18Since that report, however, others have found that Sazetidine-A does activate rat α4β2 nAChRs, possibly depending on the stoichiometry of the subunits.19Moreover, using patch clamp methods, we recently found that Sazetidine-A does elicit whole cell currents in transfected cells expressing α4β2 nAChRs but not in α3β4 nAChRs (Yingxian Xiao, Ph.D., Associate Professor; Robert Yasuda, Ph.D., Assistant Professor; Niaz Sahibzada, Ph.D., Associate Professor; Barry Wolfe, Ph.D., Professor; and Kenneth J. Kellar, Ph.D., Professor; Department of Pharmacology, Georgetown University School of Medicine, Washington, D.C.; unpublished data, May 2008). Those studies thus showed that Sazetidine-A is a highly selective partial agonist at α4β2 nAChRs.
Based on its in vitro pharmacologic profile, we hypothesized that Sazetidine-A and drugs with similar potent and selective desensitizing actions at α4β2 nAChRs would produce some effects in vivo similar to those of nicotine and other potent nicotinic agonists, but with potentially fewer undesirable side effects.18In this study, we investigated the analgesic effects of Sazetidine-A using the formalin model of persistent pain in the rat.
Materials and Methods
Male Sprague-Dawley rats (250–300 g) were housed in pairs under a 12:12-h light/dark cycle with water and food available ad libitum . The protocols were in accordance with the animal care guidelines at the University of Pennsylvania and The Children’s Hospital of Philadelphia (Philadelphia, Pennsylvania) and followed the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health.
Sazetidine-A was synthesized by Alan P. Kozikowski, Ph.D. (Professor, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois), Sheela K. Chellappan, Ph.D., and Krishna Mohan Bajjuri, Ph.D. (Postdoctoral Fellows, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago), by previously described methods.18Epibatidine and mecamylamine were purchased from Sigma-Aldridge (St. Louis, MO).
To habituate animals to the formalin test environment, rats were placed singly in the test chamber for 10–15 min on each of 3 days. The testing room was maintained at 22°C, under normal lighting conditions. The formalin test was conducted in a 60 × 30 × 40-cm clear glass chamber with a mirror under the floor to allow a complete view of the animal and paws. To score, the behavior of each rat was rated for 60 min after the injection of formalin into a rear paw. Rats were scored every 20 s for pain response based on four mutually exclusive categories of behavior.20These consisted of the following: (1) normal behavior (equal weight bearing on both hind paws), (2) favoring (injected paw resting on the floor without pressure on the foot pad), (3) lifting (injected paw elevated without touching the floor), and (4) licking (injected paw licked or bitten).21,22The observer who evaluated the rats’ behavior was not blinded to the type of drug infused or concentration used.
Locomotor activity was measured in a 60-min session. Animals were placed in 70 × 70-cm black Plexiglas opaque platform with exterior walls 30 cm high. Immediately after the injection of the test drug(s), rats were placed in a corner of the open field, and behavior was videotaped for 1 h for later analysis with the Ethovision (Noldus, VA) video tracking system. The total distanced traveled inside the chamber was analyzed over a period of an hour using 10-min bins.
Seizure activity was measured in control as well as in animals that received epibatidine and Sazetidine-A. We used the standard Racine five-stage scale: stage 1, facial movements; stage 2, rhythmic head movements and head nodding; stage 3, unilateral forelimb clonus; stage 4, bilateral forelimb clonus and rearing; stage 5, falling and clonic convulsion.23
Recording from Locus Coeruleus Neurons
The firing rate of locus coeruleus (LC) neurons was measured by procedures similar to those described in Curtis et al. 24Rats were anesthetized with 2% halothane-in-air mixture administered through a nose cone. The anesthetic was maintained at 1.0–1.5% throughout the experiment, and body temperature was maintained at 37.5° by a feedback-controlled heating pad. Rats were positioned in a stereotaxic frame with the head oriented at an 11° angle to the horizontal plane (nose down). The skull was exposed, and a hole, centered at 1.2 mm lateral to the midline and 2.8–3.7 mm caudal to lambda, was drilled over the cerebellum to reach the LC. The dura over the cerebellum was carefully removed with fine iridectomy scissors and tweezers to facilitate introduction of the recording micropipette. Single-barreled glass micropipettes (2- to 4-μm-diameter tip, 4–7 MΩ) filled with 0.5 m sodium acetate buffer saturated with Pontamine sky blue dye were used to record single-unit LC discharge. Neuronal signals were amplified and filtered. Impulse activity was monitored with an oscilloscope and loudspeaker to aid in finding the LC. LC neurons were tentatively identified during recording by their spontaneous discharge rates (0.5–5 Hz), entirely positive, notched waveforms (2–3 ms duration), and biphasic excitation–inhibition responses to tail pinch. When stable, unitary action potentials were isolated, a window discriminator was used to convert the occurrence of a single action potential into a digital pulse, which was led into a Windows-based computer via a CED 1401 interface using Spike 2 software for on-line visualization and storage and off-line analysis (Cambridge Electronic Design, Cambridge, United Kingdom).
Extracellular single-unit LC spontaneous discharge was recorded until it became stable (3–5 min). Study drugs were then injected. At the end of the experiment, the site of recording was labeled by iontophoresis (−15 μA, 25 min) of Pontamine sky blue, and the rats were killed with halothane overdose. Brains were removed and frozen, after which 30-μm-thick coronal sections were cut on a cryostat, mounted on glass slides, and stained with neutral red for localization of the Pontamine sky blue mark. Data presented are from neurons that were histologically identified, under the microscope, as being inside the LC.
Experimental Design Formalin Test
Formalin test. Eight groups of rats received intraperitoneal saline (control group, n = 7); 0.125, 0.25, 0.5, 1, or 2 mg/kg Sazetidine-A (n = 7 per group); or 2.5 and 5 μg/kg subcutaneous epibatidine (n = 9). Five minutes later, 5% formalin (50 μl) was injected subcutaneously into the plantar surface of one rear paw.
Antagonists experiments. Six additional group of rats received intraperitoneal mecamylamine followed by saline, naloxone, or mecamylamine 5 min before the intraperitoneal administration of Sazetidine-A. In the naloxone group, rats received 0.5 mg/kg naloxone followed by either 0.5 (n = 5) or 2 mg/kg (n = 5) Sazetidine-A (the lowest and highest tested analgesic doses, respectively). In the mecamylamine group, animals received 10 mg/kg mecamylamine alone (n = 5) or 10 mg/kg mecamylamine followed by 0.5 (n = 6) or 2 mg/kg (n = 6) Sazetidine-A. Five minutes after the administration of the drugs, 5% formalin (50 μl) was injected subcutaneously into the plantar surface of one rear paw.
Locomotor activity. Four different groups of animals received intraperitoneal saline (control group, n = 8), 2.5 μg/kg epibatidine (n = 8), or 0.5–2 mg/kg Sazetidine-A (n = 8 per group). We chose a dose of 2.5 μg/kg epibatidine because higher doses have been shown to cause significant neurologic impairments that could affect the data interpretation.
Seizure activity. Three groups of animals received intraperitoneal Sazetidine-A (2 mg/kg; n = 5) or subcutaneous epibatidine (2.5–5–10 μg/kg; n = 5 per group).
LC neuron recording. After identification of an LC neuron, three groups of rats received a subcutaneous injection of saline (n = 7); 0.5 or 2 mg/kg intraperitoneal Sazetidine-A (n = 4 and 3, respectively); or 10 μg/kg subcutaneous epibatidine (n = 5).
The pain behavioral data from each rat were analyzed from a composite pain score. Behavior was rated for 1 h calculating the time spent in four mutually exclusive categories of behavior. The timed behaviors were those originally described by Dubuisson and Dennis25: 0 = normal weight bearing on the injected paw; 1 = limping during locomotion or resting the paw lightly on the floor; 2 = elevation of the injected paw so that at most the nails touch the floor; and 3 = licking, biting, or grooming the injected paw. The scores were binned into 5-min epochs, and for each epoch a pain score was calculated by the amount of time an animal spent in each behavioral category by the category weight (1, 2, or 3), summed, and then divided by the test session period.
The pain scores were compared among groups at different time points using one-way analysis of variance for repeated measures to examine responses over time, followed by the Tukey multiple-comparison test when a difference between mean values was observed. The area under the curve was calculated as a measure of global pain score. Paired data were analyzed with a t test. Statistical analyses were performed with Stata (StataCorp LP, College Station, TX).
Effects of Sazetidine-A in the Formalin Test
Intraplantar injection of formalin induces a biphasic pattern of pain-related behavior, with an early acute period (phase 1; 0–9 min), which corresponds to an acute pain response, a brief quiescent period, and a second phase of sustained “tonic” pain behavior (phase 2; 10–60 min), which represents a chronic inflammatory condition.26
As shown in figure 1A, control rats, which received an injection of saline, exhibited the typical biphasic time course. The pain behavior decreased after the initial 5-min peak (phase 1) but then increased again after approximately 10 min and reached a second peak at 25–30 min (phase 2). The two lower doses of Sazetidine-A (0.125 and 0.25 mg/kg) resulted in pain scores similar to that of the control rats. In contrast, the three higher doses of Sazetidine-A (0.5, 1, and 2 mg/kg) (P < 0.0001) and both doses of epibatidine (2.5 and 5 μg/kg) (P < 0.001) (fig. 1B) resulted in significantly lower initial pain scores in phase 1.
During phase 2 of the reaction to the formalin injection, the pain scores of the two lower doses Sazetidine-A were still similar to that of saline. But again, the three higher doses of Sazetidine-A markedly attenuated the pain scores through the remaining 50-min observation period (P < 0.0001) (fig. 1A). Figure 1Balso shows pain scores from rats that were injected with epibatidine (2.5–5 μg/kg). Treatment with epibatidine significantly reduced the pain score compared with animals treated with saline or the two lowest doses of Sazetidine-A (P < 0.001). However, epibatidine did not reduce the pain score to the same degree as the three highest doses of Sazetidine-A (0.5–2 mg/kg) (P < 0.01).
The area under the curve was calculated as a measure of the global pain score over time and to assess the ability of drugs to lower that score. The global pain score was reduced by approximately 75% in rats that received the three highest doses of Sazetidine-A compared with that observed in control rats or rats that received the two lower doses of Sazetidine-A (*P < 0.0002; fig. 2). The analgesic effects of 2.5 and 5 μg/kg epibatidine were similar, each reducing the overall pain score by approximately 45% (fig. 2). Epibatidine (2.5–5 μg/kg) reduced the pain score significantly compared with saline or the two lowest doses of Sazetidine-A (P < 0.003), but it did not reduce the pain score to the same extent as the three higher doses of Sazetidine-A (P < 0.006). We did not check pain scores after 10 μg/kg epibatidine because of significant seizure activity.27
Effects of Antagonists on the Sazetidine-A Response
The noncompetitive nAChR antagonist mecamylamine (10 mg/kg, intraperitoneal) did not itself significantly affect the pain response to formalin, but pretreatment with it 5 min before injection of Sazetidine-A nearly completely blocked the analgesic response to 0.5 mg/kg Sazetidine-A and partially blocked the response to the 2-mg/kg dose of Sazetidine-A (fig. 3). In contrast, pretreatment with the opiate antagonist naloxone (0.5 mg/kg, intraperitoneal) did not significantly affect the analgesic response to either dose of Sazetidine-A (fig. 3).
Open Field Activity
As shown in figure 4, locomotor activity during the first 20 min after injection was significantly decreased by analgesic doses of both epibatidine and Sazetidine-A, compared with saline (P < 0.007), but the locomotor activity during the following 40 min was similar in the treatment and saline groups. There were no significant differences in locomotion between the three treatment groups at any time after the administration of the drugs.
Animals that received analgesic doses of Sazetidine-A did not develop any observable seizure activity (fig. 5). Similarly, epibatidine at 2.5 μg/kg did not show seizure activity (data not shown). In contrast, higher doses of epibatidine did induce seizures. Thus, 60% of rats that received epibatidine at a dose of 5 μg/kg reached a stage 1 seizure within 2 min after the administration of the drug, but seizure activity disappeared within 16 min (fig. 5). After epibatidine at a dose of 10 μg/kg, 66% of animals reached stage 5 seizure scores within 4 min (fig. 4), and 20% of these animals died. Seizure activity disappeared in the surviving animals within 26 min from the administration of epibatidine (fig. 5).
Locus Coeruleus Recording
The subcutaneous administration of Sazetidine-A (2 mg/kg) slightly reduced neuronal discharge rate of LC neurons, though this did not reach statistical significance (fig. 6and table 1). In contrast, the administration of subcutaneous epibatidine (10 μg/kg) resulted in a significant increase in the discharge of LC neurons from 1.4 ± 0.6 to 5.3 ± 3.9 Hz (fig. 6and table 1).
Although the nAChR subtypes involved in the analgesic activity of nicotinic agonists are not known with certainty, there is very good evidence for the involvement of α4β2 subtypes.14Nicotinic agonists have dual actions on nAChRs: first to activate them and then to desensitize them.28It is widely believed that activation of nAChRs plays the essential role in initiating analgesic effects of nicotinic agonists, but the role of receptor desensitization in sustaining these effects is unknown.
Sazetidine-A has very high affinity and selectivity for α4β2 compared with α3β4 nAChRs.18Interestingly, in measurements of receptor function with [86Rb] ion efflux assays, Sazetidine-A does not display measurable agonist or antagonist activity at α4β2 nAChRs, but it does potently and selectively desensitize these receptors for sustained periods of time, resulting in markedly diminished responses.18Similarly, in patch clamp studies, when Sazetidine-A is introduced to cells via the slow bath application method, it shows very little if any agonist activity at concentrations that completely desensitize α4β2 receptors (Drs. Yingxian Xiao, Robert Yasuda, Niaz Sahibzada, Barry Wolfe, and Kenneth J. Kellar, Department of Pharmacology, Georgetown University School of Medicine; unpublished data, May 2008). In contrast, however, when Sazetidine-A is introduced to α4β2 receptors via rapid application methods using Y-tubing or a picospritzer, it shows clear partial agonist activity in patch clamp studies.
In the current studies, Sazetidine-A produced marked and sustained analgesia in the formalin test, a well-established model of persistent chemical pain. Because the α4β2 nAChRs in the central nervous system would probably be desensitized for all but a few seconds after systemically administered Sazetidine-A, the observation of sustained analgesia raises the interesting possibility that desensitization of α4β2 nAChRs plays the crucial role in the analgesic effects of nicotinic drugs.
The analgesic effects of Sazetidine-A are attenuated by pretreatment with mecamylamine, a broad spectrum nAChR noncompetitive antagonist. This suggests that even the partial agonist activity of Sazetidine-A is important to its analgesic activity; indeed, it may be required to initiate the desensitization of the receptors. In contrast, pretreatment with naloxone, a μ-opioid competitive antagonist, did not inhibit the analgesic effects of Sazetidine-A, suggesting that antinociceptive effects of Sazetidine-A do not require activation of μ receptors.
Sazetidine-A produced a higher level of analgesia than epibatidine, as indicated by significantly lower pain scores after effective doses of Sazetidine-A compared with the highest tested dose of epibatidine used here that did not result in overt seizure activity. In fact, Sazetidine-A did not cause obvious seizure activity at doses at least four times higher than its effective analgesic dose. This is in marked contrast to the seizures observed after epibatidine in this study and to what has been reported previously.27,29The difference in the side effect profile between Sazetidine-A and previously tested nicotinic agonists may be related to its very high selectivity for α4β2 receptors compared with α3β4 receptors, which are thought to mediate several important side effects of nicotinic agonists.9
Both Sazetidine-A and epibatidine reduced spontaneous locomotor activity of rats during the first 20 min after injection. The mechanism related to these decreased locomotor effects after Sazetidine-A is unclear, although it resembles other nicotinic agonists in this respect, and a brief sedative-like effect similar to that of opioids cannot be excluded.
Early research on the mechanisms mediating the analgesic effects of nicotinic agonists suggested a potentially important role of midbrain nuclei.30In particular, the monoaminergic nucleus raphe magnus, dorsal raphe, LC, and A5 and A7 nuclear groups seem to be important components of the antinociceptive systems originating from midbrain nuclei.31,32Stimulation of these nuclei results in activation of neurons that project to the spinal cord and may inhibit pain pathways at the level of the dorsal horn. However, the involvement of these midbrain nuclei in the marked analgesia produced by Sazetidine-A is unclear. For example, both epibatidine and ABT-594, a more selective α4β2 nAChR agonist, have been shown to, at least initially, activate midbrain nuclei located in descending pathways associated with analgesia,21,22,33and a similar effect of systemically administered epibatidine on LC neurons was seen in the current study. In contrast, the systemic administration of Sazetidine-A did not excite these neurons and may have actually caused a modest inhibition of their firing rate, though this did not reach statistical significance.
After systemic administration, drugs enter the central nervous system in a manner that is probably much more similar to slow bath application than to rapid application via Y-tubing or picospritzer. Under these conditions, Sazetidine-A potently and selectively induces sustained desensitization of α4β2 nAChRs.18Therefore, the sustained analgesic effects of Sazetidine-A, and indeed possibly of all nicotinic agonists, are likely due much more to desensitization of α4β2 nAChRs than to their activation.
In conclusion, this study shows that Sazetidine-A is a potent and efficacious analgesic in a rat model of formalin-induced pain. Sazetidine-A is highly selective for β2-containing nAChRs and, in these early in vivo studies, seems to have a much better analgesic and side effect profile compared with epibatidine.
The authors thank Alan P. Kozikowski, Ph.D. (Professor, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois), and Sheela K. Chellappan, Ph.D., and Krishna Mohan Bajjuri, Ph.D. (Postdoctoral Fellows, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago), for synthesizing and supplying Sazetidine-A.