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BackgroundNeuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels. The 42 subtype of nAChRs plays an important role in the mediation of pain and several nicotine-evoked responses. Agonists and partial agonists of 42 nAChRs show efficacy in animal pain models. In addition, the antinociceptive properties of nicotine, a non-selective nAChR agonist with a high affinity for 42 nAChRs, is well-known. There is a growing body of evidence pointing to allosteric modulation of nAChRs as an alternative treatment strategy in experimental pain. Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) at 42 nAChRs that enhances agonist responses without activating receptors. We hypothesized that dFBr may enhance nicotine-induced antinociception. MethodsThe present study investigated whether dFBr could attenuate mouse chronic constriction injury (CCI)-induced neuropathic pain by increasing endogenous cholinergic tone or potentiating the nicotine-evoked antiallodynic response. ResultsWe found that subcutaneous administration of dFBr failed to reduce pain behaviour on its own. However, the combination of dFBr with nicotine significantly reversed neuropathic pain behaviour dose- and time-dependently without motor impairment. Our data revealed that this effect was mediated by the 42 nAChRs by using competitive 42 antagonist dihydro--erythroidine. In addition, dFBr failed to potentiate the antiallodynic effect of morphine, which shows the effect of dFBr is unique to 42 nAChRs. ConclusionsThe present results suggest that allosteric modulation of 42 nAChR may provide new strategies in chronic neuropathic pain. Significance42 nAChRs are involved in pain modulation. dFBr, a PAM at 42 nAChRs, potentiates the nicotine response dose-dependently in neuropathic pain. Thus, the present results suggest that allosteric modulation of 42* nAChR may provide new strategies in chronic neuropathic pain. United States Department of Health & Human Services National Institutes of Health (NIH) - USA (R01-CA206028) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Cancer Institute (NCI) (R01CA206028)

Background Preclinical assays of affective and sensorial aspects of nociception play a key role in research on both the neurobiology of pain and the development of novel analgesics. Therefore, we investigated the effects of nicotine and alpha-7 nicotinic acetylcholine receptor (nAChR) modulators in the negative affective and sensory components of visceral pain in mice. Methods and results Intraperitoneal acetic acid (AA) administration resulted in a robust stretching behaviour and conditioned place aversion (CPA) in mice. We observed a dose-dependent reduction in AA-induced stretching and CPA by the nonselective nAChRs agonist nicotine. Mecamylamine, a nonselective nAChRs agonist, was able to block its effects; however, hexamethonium, a peripherally restricted nonselective nicotinic antagonist, was able to block nicotine's effect on stretching behaviour but not on CPA. In addition, systemic administration of α7 nAChR full agonists PHA543613 and PNU282987 was failed to block stretching and CPA behaviour induced by AA. However, the α7 nAChR-positive allosteric modulator PNU120596 blocked AA-induced CPA in a dose-dependent manner without reducing stretching behaviours. Conclusions Our data revealed that while nonselective nAChR activation induces antinociceptive properties on the sensorial and affective signs of visceral pain in mice, α7 nAChRS activation has no effect on these responses. In addition, nonselective nAChR activation-induced antinociceptive effect on stretching behaviour was mediated by central and peripheral mechanisms. However, the effect of nonselective nAChR activation on CPA was mediated centrally. Furthermore, our data suggest a pivotal role of allosteric modulation of α7 nAChRS in the negative affective, but not sensory, component of visceral pain. Significance The present results suggest that allosteric modulation of α7 nAChR may provide new strategies in affective aspects of nociception.

The pharmacology of synthetic D- and L-epibatidine, an alkaloid originally characterized from frog skin, were studied in different behavioral tests in mice and rats. The two enantiomers have potent antinociceptive activity in mice using the tail-flick test, with an ED50 of 6.1 and 6.6 micrograms/kg for L- and D-epibatidine respectively. Epibatidine enantiomers were 200 x more potent than L-nicotine as an antinociceptive agent in mice after s.c. administration. Their analgesic effect was blocked by mecamylamine but not naloxone, an opiate antagonist. Both D- and L-epibatidine have high affinity (Ki 54.7 and 55.0 pM, respectively) for [3H]nicotine binding site in rat brain. In addition, they reduced mice locomotor activity and body temperature in a dose-dependent manner. In rats trained with nicotine (0.4 mg/kg), epibatidine enantiomers engendered nicotine-like responding in a dose-related manner with an ED50 of 1.00 and 0.93 micrograms/kg for D and L, respectively. The discriminative effect of L- and D-epibatidine in rats was blocked by mecamylamine but not by hexamethonium. As in binding results, there was no significant enantioselectivity for these effects in our study.

5-(1'-Phenyl-4'-piperazinomethyl)-2-amino-2-oxazoline (1; COR3224), a derivative of 2-amino-2-oxazolines with antidepressant properties in rats, was assayed in plasma by high-performance liquid chromatography. After back extraction, 1 and a ortho-O-methyl derivative of 1 as the internal standard were separated by reversed-phase liquid chromatography and measured by UV detection (235 nm). The method is rapid and specific: the detection was linear in the range 125-1000 micrograms.L-1, with a detection limit of 50 micrograms.L-1. This method allowed the determination of pharmacokinetic parameters in six beagle dogs after intravenous and oral administration of 14C-labeled 1 ([14C]1) in a crossover study. The comparison of the results obtained from total radioactivity counting and unchanged product evaluated by HPLC-UV suggest the presence of metabolites.

The influence of a calcium agonist (BAYK 8644) and several calcium channel blockers on nicotine-induced antinociception was investigated in mice. The effect of nicotine was sharply increased by BAYK 8644. This potentitation by BAYK 8644 was blocked by pretreating the animals with nifedipine at 2 and 10 mg/kg. The calcium channel antagonists, nifedipine and nimodipine at doses that had no effect on tail-flick time, reduced significantly the antinociception induced by nicotine (1.5 mg/kg s.c.). However, the effect of verapamil on nicotine was not significant. These results indicate that DHP calcium channels (type L-channel) play a role in some of the pharmacological effects of nicotine, particularly, locomotor activity and antinociception.

A reversed-phase (CN as stationary phase) liquid chromatographic method with electrochemical detection is described for the quantitation of COR 3224, a new 2-amino-2-oxazoline in plasma and brain tissue of the rat. Extraction was performed with dichloromethane and detection was achieved at a working electrode potential of +0.85 V versus an Ag/AgCl reference electrode. The recovery of the method is about 80 and 60% for plasma and brain, respectively. The limit of detection was less than 10 ng/ml for both plasma and brain, five times lower than that with ultraviolet detection.